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Albert  Fl.  Mann  Library 
Cornell  University 


Dr.  Roger  a.  Morse 


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http://www.archive.org/details/cu31924090242698 


SUREAU  OF  ENTOMOWay. 

L.  O.  HowABD,  Entomologist  and  Ohief  of  Bureau. 

C.  L.  Mablatt,  Entomologist  and  Acting  Chief  in  absence  Of  Chief. 

R.  S.  Ci.ift6n,  Chief  Clerk. 

F.  H.  Chittenden,  in  charge  of  breeding  experiments.^ 

A.  D.  Hopkins,  in  charge  of  forest,  insect  investigation^. 

W.  D.  IIuNTEB,  in  charge  of  cotton  holl  weevil  investigations. 

F.  M.  Webster,  in  charge  of  cereal  and  forage-plant  ijisect  investigations. 

A.  L.  QuAiNfANCE,  in  charge  of  deciduous-fruit  insect  investigations. 

J)., M.  UooERS,  in  charge  of  gipsy  and  hrown-tail  moth  work. 

A.  W.  MoERiix,  engaged  in  white  fly  investigations. 
E.  S.  G.  Titus,  in  charg^  of  gipsy  moth  laboratory. 
C.  J.  GiLTJss,  engaged  in  silk  Investigations. 

R.  P.  CuKEiE ,  assistant  in  charge  of,  editorial^  work. 
Mabel  Colcoed,  librarian. 

Apictjltueal  Investigations.  . 

Feank  Benton,  in  charge  (absent). 

B.  F.  Phillips,  acting  in  charge.  ' 

J.  M.  Rankin,  in  charge  of  apicultural  station,  Chico,  Cfit. 
Jessie  E.  ^abks,  apicultural  clerk. 


Technical  Series,  No.  14. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

L.  0.   HOWARD,  Entomologist  and  Chief  of  Bureau. 


THE 

BACTERIA  OF  THE  APIARY, 

WITH  SPECIAL  REFEREI^CE  TO 
BEE  DISEASES. 


GERSHOM  FRANKLIN  WHITE,  Ph.  D., 

Expert  in  Animal  Bacteriology,  Biochemic  Division,  Bureau  of  Animal  Industry. 


Issued  November  6,  1906. 


WASHINGTON: 

GOVERNMENT   PRINTING   OFFICE. 
1906. 


LEHER  OF  transmittal; 


U.  S.  Department  or  Agricultttee, 

Bureau  of  Entomology, 
Washington,  D.  G.,  September  2Ji.,  1906. 

Sir:  I  have  the  honor  to  transmit  the  manuscript  of  a  paper  on 
the  bacteria  of  the  apiary,  with  special  reference  to  bee  diseases,  by 
Dr.  G.  F.  ^Vhite,  expert  in  animal  bacteriology  in  the  Biochemic 
Division  of  the  Bureau  of  Animal  Industry.  This  paper  was  pre- 
pared by  Doctor  "White  as  a  thesis  in  part  fulfilment  of  the  require- 
ments for  the  degree  of  doctor  of  philosophy,  at  Cornell  University, 
in  June,  1905.  The  Bureau  of  Entomolo^  considers  itself  fortu- 
nate in  obtaining  it  for  publication,  since  in  this  way  a  wider  distri- 
bution can  be  made  than  would  be  possible  were  it  published  in  a 
journal  devoted  exclusively  to  bacteriological  investigations.  It  is 
hoped  that  the  publication  of  these  facts  may  help  to  clear  up  the 
confusion  which  now  exists  concerning  the  causes  of  the  two  most 
common  diseases  of  the  brood  of  bees.  I  recommend  that  the  manu- 
script be  published  as  Technical  Series,  No.  14,  of  this  Bureau. 

Doctor  White  wishes  to  acknowledge  his  indebtedness  to  Dr. 
Veranus  A.  Moore,  professor  of  comparative  pathology  and  bac- 
teriology of  Cornell  University,  under  whose  direction  this  work 
was  done;  to  Dr.  E.  F.  Phillips,  acting  in  charge  of  apiculture, 
Bureau  of  Entomology,  United  States  Department  of  Agriculture, 
for  encouragement  and  assistance  in  the  preparation  of  this  manu- 
script; and  to  Messrs.  Mortimer  Stevens,  Charles  Stewart,  N.  D. 
West,  and  W.  D.  Wright,  bee  inspectors  of  the  State  of  New  York, 
for  their  interest  in  the  work. 

EespectfuUy,  L.  O.  Howard, 

Entomologist  and  Chief  of  Bureau. 

Hon.  James  Wilson, 

Secretary  of  Agriculture. 


PREFACE 


The  spread  of  diseases  of  the  brood  of  bees  is  to-day  a  great  menace 
to  the  bee-keeping  industry  of  the  United  States.  It  is  therefore  of 
great  importance  that  all  phases  of  these  diseases  should  be  investi- 
gated as  thoroly  as  possible,  and  this  paper,  it  is  believed,  will  help 
in  clearing  up  some  disputed  points  in  regard  to  the  cause  of  the  two 
most  serious  brood  diseases. 

Dr.  G.  F.  White  has  offered  this  paper  for  publication  as  a  bulletin 
in  the  Bureau  of  Entomology  because  in  that  way  the  statements 
herein  contained  may  become  more  widely  known  than  would  be  the 
case  were  it  published  in  some  journal  devoted  exclusively  to  bacteri- 
ological investigations.  Obviously  there  are  many  points  still  un- 
settled, and  it  is  hoped  that  some  of  these  may  be  taken  up  for  in- 
vestigation in  the  near  future,  but  the  results  so  far  obtained  should 
by  all  means  be  made  known  to  the  persons  practically  engaged  in 
bee  keeping. 

The  necessity  for  the  study  of  nonpathogenic  bacteria  found  in 
the  apiary  may  not  be  at  first  evident  to  the  ordinary  reader.  When 
it  is  seen,  however,  that  some  of  the  investigators  of  bee  diseases  have 
apparently  mistaken  Bacillus  A  or  some  closely  allied  species  for 
Bacillus  alvei  it  will  be  evident  that  a  study  of  nonpathogenic  germs 
is  necessary  to  a  thoro  investigation  of  the  cause  of  these  diseases  and 
a  full  understanding  of  the  confusion  which  has  existed. 

The  names  which  should  be  used  for  the  diseased  conditions  of 
brood  was  a  matter  which  arose  after  this  paper  was  offered  for  pub- 
lication. It  was  desired  that  out  of  the  chaos  of  names  in  use  cer- 
tain ones  be  chosen  which  would  be  distinctive  and  still  clear  to  the 
bee  keepers  who  are  interested  in  work  of  this  nature.  Unfortu- 
nately, after  a  short  investigation.  Dr.  W.  K.  Howard,  of  Fort 
Worth,  Tex.,  gave  the  name  "  New  York  bee  disease,"  or  "  black 
brood,"  to  a  disease  which  Cheshire  and  Cheyne  described  in  1885  as 
"  foul  brood."  Since  this  is  the  disease  in  which  Bacillus  alvei  is 
present,  we  can  not  drop  the  name  "  foul  brood,"  and  the  word 
"  European  "  is  used  to  distinguish  it  from  the  other  disease.  The  bee 
keepers  of  the  United  States  have  been  taught  that  the  type  of  brood 
disease  characterized  by  ropiness  of  the  dead  brood  is  true  foul  brood, 

3 


4  PREFACE. 

but  since  Bacillus  alvei  is  not  found  in  this  disease  it  obviously  is  not 
the  same  disease  as  that  described  by  Cheyne.  It  would  be  well-nigh 
impossible,  however,  to  change  the  name  of  this  disease,  and  any  effort 
in  that  direction  would  merely  result  in  complicating  laws  now  in  force 
which  control  the  infectious  diseases  of  bees  and  would  serve  no  good 
purpose.  This  disease  is  here  designated  "American  foul  brood." 
These  names  have  been  chosen  only  after  consultation  with  some  of 
the  leading  bee  keepers  of  the  United  States,  and  these  distinguishing 
terms  were  chosen  by  the  majority  of  those  consulted  as  indicating 
the  place  in  which  the  diseases  were  first  investigated  in  a  thoroly 
scientific  manner.  Both  diseases  are  found  in  Europe,  as  well  as  in 
America,  so  that  the  names  indicate  nothing  concerning  the  geo- 
graphical distribution  of  the  maladies. 

Strangely  enough,  certain  writers  for  our  American  apicultural 
papers  have  seen  fit  to  take  exception  to  some  of  the  statements  made 
in  this  paper  without  having  first  found  out  the  reasons  for  the  de- 
cisions herein  published.  Apiculture  will  not  be  advanced  to  any 
appreciable  extent  by  such  eagerness  to  rush  into  print,  especially 
when  there  is  not  a  semblance  of  scientific  investigation  back  of  the 
criticism. 

E.  F.  Phillips, 
Acting  in  Charge  of  Apiculture. 


CONTENTS. 


Page. 

Introduction    7 

Technique    7 

Obtaining  material  for  study 7 

Obtnining^  cultures 7 

Differentiation  and  identification  of  bacteria 9 

Tbe  cultures  which  are  described 9 

Morphology,  staining  properties,  and  oxygen  requirements,  with  sug- 
gestions on  variations 9 

Media  employed  and  suggestions  as  to  the  description  of  cultures 10 

PART   I.      BACTERIA   OF   THE    NORMAL   APIARY. 

Bacteria  from  the  combs 13 

Bacteria  from  pollen 15 

Bacteria  in  honey  and  normal  larvae 16 

Bacteria  upon  the  adult  bees 16 

Bacteria  of  the  intestine  of  the  healthy  honey  bee 18 

Saccharomyces  and  fungi 25 

Tabulation  of  micro-organisms  normally  present  in  the  apiary 28 

Summary  to  Part  I 29 

Bibliography  to  Part  I 29 

PART   II.    THE   DISEASES   OF   BEES. 

Brief  history ^ 30 

The  term  "foul  brood"  as  hitherto  applied 31 

European  foul  brood  (foul  brood  of  Cheyne) 32 

Symptoms    32 

Confusion  regarding  foul  brood  in  America 33 

The  present  investigation 34 

'   Bacillus    alvei 36 

Inoculation    experiments 37 

Distribution  of  Bacillus  alvei  in  infected  hives 38 

Experiments  with  formaldehyde  gas . 39 

American  foul  brood 40 

Symptoms 40 

The  present  investigation 41 

Bacillus    larval 42 

The  so-called  "  picljle  brood" 43 

The  so-called  "  blacic  brood" 43 

Palsy  or  paralysis 44 

Summary  to  Part  II 44 

Conclusions     45 

Bibliography  to  Part. II 46 

Index    47 

6 


THE  BACTERIA  OF  THE  APIARY  WITH  SPECIAL 
REFERENCE  TO  BEE  DISEASES. 


INTBODTJCTION. 

Since  bacteriology  is  one  of  the  youngest  of  the  sciences,  it  is  only 
natural  that  there  should  be  many  problems  concerning  which  there 
is  much  confusion,  and  many  others  concerning  which  nothing  is 
known.  In  a  study  of  the  saprophytic  bacteria  this  is  especially 
true;  the  exploration  of  this  jungle  of  micro-organisms  is  scarcely 
begun.  Comparatively  few  species  have  been  studied  and  named, 
and  a  much  less  number  can  be  identified.  From  studies  that  have 
been  made  one  is  led  to  believe  that  the  species  which  might  be 
classed  under  bacteria  outnumber  by  far  all  the  macroscopic  plants 
known.  Comparatively  little  is  as  yet  known  concerning  the  dis- 
tribution of  these  minute  organisms  in  nature,  their  needs  for  multi- 
plication and  growth,  their  power  of  endurance,  their  relations  the 
one  to  the  other,  their  relations  to  man  and  industries,  and  their 
relation  to  pathogenic  species.  Both  from  the  standpoint  of  scien- 
tific interest  and  from  the  standpoint  of  practical  economy  these 
problems  call  for  further  investigation. 

By  far  the  greatest  amount  of  work  which  has  been  done  in  the 
science  of  bacteriology  has  been  prompted  by  the  direct  or  indirect 
economic  importance  of  the  question.  This  is  largely  true  of  the 
present  investigation,  since  honey  bees  suffer  from  a  number  of 
diseases,  some  of  which  are  considered  in  Part  II. 

TECHNIQUE. 
Obtaining  Material  for  Study. 

If  necessary,  bees  may  be  conveniently  shipped  alive  by  mail  in 
cages  constructed  for  that  purpose.  Combs  also  may  be  sent  by  mail 
in  small  boxes.  If  combs,  honey,  pollen,  or  larvae  are  desired,  the  hive 
must  be  entered.  In  case  older  adult  bees  are  wanted  it  is  not  difficult 
to  supply  the  needs  from  the  entrance  to  the  hive.  To  capture  them 
one  may  stand  at  the  entrance  and  catch  the  unwary  toiler  as  she 

7 
9583— No.  14—06  m 2 


8  THE    BACTEEIA    OF    THE    APIAEY. 

comes  in  loaded  with  pollen  and  honey.  After  the  victim  alights  on 
the  entrance  board,  by  the  aid  of  a  pair  of  forceps,  before  she  disap- 
pears within,  one  can  easily  lodge  her  safely  in  a  petri  dish.  It  is, 
however,  an  advantage  to  study  the  young  adult  bees  as  well  as  the 
older  ones,  and  if  young  ones  are  desired  they  may  be  taken  from 
the  combs  or  from  the  front  of  the  hive,  near  the  entrance. 

Obtaining  Cultures. 

(a)  From  combs. — With  sterile  forceps  small  pieces  of  the  comb 
are  put  directly  into  gelatin  or  agar  for  plates  or  incubated  in  bouil- 
lon for  24  hours  and  then  plated.  Growing  in  bouillon  and  plat- 
ing on  gelatin  is  usually  preferable. 

{h)  From  pollen. — The  same  technique  is  used  as  for  combs,  but 
the  direct  inoculation  of  gelatin  tubes  for  plates  is  generally  pre- 
ferable. 

(c)  From  honey. — With  sterile  loops  honey  is  taken  from  uncapped 
and  capped  cells.  The  caps  are  removed  with  sterile  forceps  and  the 
honey  is  plated  directly  on  gelatin  or  agar.  Bouillon  tubes  are  in- 
oculated also  with  varying  quantities  of  the  honey. 

{d)  From  larvm. — The  larva  is  carefully  removed  to  a  sterile  dish, 
and  with  sterile  scissors  the  body  is  opened  and  the  contents  plated 
directly,  or  bouillon  cultures  are  first  made  and  later  plated,  if  a 
growth  appears. 

(e)  From  parts  of  the  adult  hee. — In  studying  the  adult  bee,  a 
small  piece  of  blotting  paper  wet  with  chloroform  is  slipt  under 
the  cover  of  the  petri  dish  in  which  the  insects  have  been  placed,  and 
in  a  short  time  the  bees  are  under  the  influence  of  the  anesthetic. 
Then  with  sterile  scissors  a  leg,  a  wing,  the  head,  the  thorax,  or  the 
abdomen,  the  intestine  being  removed,  is  placed  in  bouillon  and,  after 
24  hours  incubation,  plated,  preferably  on  gelatin. 

When  it  is  desired  to  make  a  study  of  the  bacteria  of  the  intestine, 
the  intestinal  tract  is  removed  and  studied  as  follows:  The  bee  is 
flamed  and  held  in  sterile  forceps.  With  another  sterile  pair  of  for- 
ceps the  tip. of  the  abdomen  is  seized  and,  by  pulling  gently,  the  tip 
and  the  entire  intestine  are  easily  removed.  This  can  then  be  plated 
directly.  If  gelatin,  which  is  preferable,  is  used,  the  intestine  itself 
must  not  be  left  in  the  gelatin  or  the  medium  will  become  liquefied 
by  the  presence  of  the  tissue.  If  one  desires  to  obtain  cultures  of  the 
anaerobe,  which  is  quite  common  in  the  intestine,  it  is  most  easily 
obtained  in  pure  culture  by  the  use  of  the  deep  glucose  agar  (Liborius's 
method).  Cover  glass  preparations  made  direct  from  the  walls  of 
the  intestine  or  its  contents  give  one  some  idea  of  the  great  number  of 
bacteria  frequently  present. 


MORPHOLOGY,  STAINING   PKOPEETIES,  ETC.  9 

Differentiation  and  rdentification  of  Bacteria. 

These  very  low  forms  of  plant  life  show  a  marked  susceptibility  to 
environmental  conditions  and  those  desirous  of  speculating  on  prob- 
lems in  evolution  may  find  here  food  for  thought  and  experimenta- 
tion. On  account  of  this  susceptibility,  various  cultures  which  belong 
to  the  same  species  may  possess  slight  variations  in  some  one  or  more 
specific  characters.  Consequently  one  can  not  say  that  a  species  must 
possess  certain  definite  characters  and  no  others.  It  is  convenient, 
then,  to  think  of  a  species  as  more  or  less  of  a  group  of  individuals 
whose  characters  approximate  each  other  very  closely. 

In  this  paper  are  described  a  number  of  species  each  of  which,  in 
fact,  represents  a  group,  the  individual  cultures  of  which  approxi- 
mate each  other  so  closely  in  character  that  the  differences  may  be 
easily  attributed  to  environmental  conditions  which  are  more  or  less 
recent. 

Concerning  the  identification  of  species,  the  conditions  have  been 
well  summed  up  by  Chester.     He  says: 

Probably  nine-teuths  of  tbe  forms  of  bacteria  already  described  might  as  well 
be  forgotten  or  be  given  a  respectful  burial.  This  will  then  leave  comparatively 
few  well-defined  species  to  form  the  nuclei  of  groups  In  one  or  another  of  which 
we  shall  be  able  to  place  all  new  sufficiently  described  forms. 

The  variations  which  occur  and  the  very  incomplete  descriptions 
which  can  be  found  make  it  impossible  to  identify  many  species  even 
to  a  more  or  less  restricted  group.  For  these  reasons  some  of  the 
cultures  are  not  identified  or  named,  but  letters  are  used  for  conven- 
ience in  this  paper  to  represent  the  specific  part.  Migula's  classifica- 
tion has  been  used. 

The  Cultures  Which  are  Described. 

Plate  cultures  were  observed  for  some  weeks,  the  different  kinds  of 
colonies  which  appeared  being  especially  noted.  Subcultures  were 
then  made  in  bouillon,  and  after  24  hours  the  subculture  was  re- 
plated.  Subculturing  and  replating  were  then  repeated.  From  this 
last  plate  the  pure  culture  was  made  on  agar  for  study.  These  were 
not  studied  culturally,  as  a  rule,  for  some  weeks,  thus  allowing  time 
for  the  organism  to  eliminate  any  character  due  to  recent  environ- 
mental conditions  (1)." 

Morphology,    Staining   Properties,   and   Oxygen   Bequirements,   with   Sug- 
gestions on  Variations. 

(a)  Size.— The  length  and  thickness  of  a  micro-organism  often 
varies  so  much  with  its  environmental  conditions  that  certain  re- 

o  Numbers  in  parentheses  refer  to  papers  in  the  bibliography  at  the  end  of 
Part  I  or  that  at  the  end  of  Part  II. 


10  THE    BACTEEIA    OF    THE    APIAEY. 

corded  dimensions  should  always  be  accompanied  by  facts  concerning 
the  medium,  age,  and  temperature  of  incubation.  The  measure- 
ments recorded  in  this  paper  were  all  taken  of  organisms  in  prepara- 
tions made  from  a  24-hour  agar  culture  stained  with  carbol-fuchsin. 
The  involution  forms  are  not  reckoned  in  the  results. 

(5)  Spores. — The  presence  of  spores  was  determined  in  each  case 
by  staining  the  various  cultures  at  different  ages.  A  check  was  made 
on  their  presence  by  means  of  the  thermal  death  point. 

(c)  Flagella. — Loeffler's  method,  as  modified  by  Johnson  and 
Mack,  was  used  for  staining  the  flagella  (2). 

{d)  Motility. — Motility  may  be  present  in  cultures  when  first  iso- 
lated, but  after  artificial  cultivation  appear  to  be  entirely  lost.  The 
reverse  of  this  also  may  be  noted.  No  cultures  should  be  recorded 
as  nonmotile  until  cultures  on  various  media  at  different  temperatures 
and  of  different  ages  shall  have  been  studied.  Hanging-drop  prepar- 
tions  were  made  from  cultures  on  agar  and  bouillon,  both  incubated 
and  not  incubated,  and  on  gelatin. 

(e)  Staining  froperties. — Basic  carbol-fuchsin  was  the  stain  used 
almost  exclusively.  In  the  use  of  Gram's  staining  method,  carbolic 
gentian  violet  (5  per  cent  carbolic  acid  20  parts,  saturated  alcoholic 
solution  gential  violet  2  parts)  was  applied  to  a  cover-glass  prepara- 
tion from  a  24-hour  culture  on  agar  for  5  minutes,  placed  in  Lugol's 
solution  2  minutes,  and  placed,  without  rinsing,  in  95  per  cent  alcohol 
for  15  minutes,  removed,  washt  in  water,  and  allowed  to  dry. 

(/)  Oxygen  requirements. — Determinations  were  made  by  ob- 
serving whether  a  growth  took  place  in  the  closed  or  open  arm  or 
both,  of  the  fermentation  tube  containing  glucose  bouillon. 

Media  Employed  and  Suggestions  as  to  tlie  Description  of  Cultures. 

{a)  Bouillon. — All  bouillon  used  was  made  from  beef  (meat  1 
part,  water  2  parts) ,  to  which  infusion  1  per  cent  Witte's  peptonum 
siccum  and  one-half  per  cent  sodium  chlorid  were  added.  The  re- 
action of  the  solution  was  then  determined  by  titrating,  and  made 
-j-1.5  to  phenolphthalein. 

In  describing  a  culture  growing  in  bouillon  as  a  medium,  there 
is  usually  a  more  extended  description  given  than  in  the  case  of 
sugar  and  sugar-free  bouillons,  since  cultures  in  these  media  do  not 
differ  materially  in  gross  appearance  from  those  observed  in  the 
plain  bouillon. 

(6)  Sugar-free  houillon. — This  bouillon  is  made  free  from  sugar 
by  the  use  of  B.  coli  communis,  after  which  peptone  and  sodium 
chlorid  (NaCl)  were  added  as  in  bouillon. 

(c)  Sugar  bouillons. — Five  different  sugars — glucose,  lactose,  sac- 
charose, levulose,  and  maltose,  as  well  as  mannite — were  used  in  the 
study.    If  a  1-per-cent  solution  of  glucose  in  plain  bouillon  Avas  fer- 


MEDIA   EMPLOYED,  ETC.  11 

merited  with  the  production  of  gas,  fermentation  tubes  were  used 
for  all  the  sugars  and  mannite.  If  no  gas  was  formed  in  the  glucose, 
the  straight  tubes  were  inoculated.  The  sugars  and  mannite  were 
used  in  a  1-per-cent  solution  in  sugar-free  bouillon. 

{d)  Rcaetion  of  media. — The  reaction  of  cultures  is  determined 
as  it  appears  on  the  fifth  day  in  the  different  media,  unless  otherwise 
stated.  The  medium  in  the  open  arm  is  used  to  determine  the  re- 
action in  the  fermentation  tube.  Beginning  with  a  reaction  of  -|-1.5 
to  phenolphthalein,  or  slightly  alkaline  to  litmus,  the  detection  of  an 
increase  in  acidity  is  not  difficult.  But  inasmuch  as  the  production 
of  an  alkali  is  very  frequently  small  in  degree,  cultures  are  often  in 
this  paper  recorded  alkaline  in  reaction  when  probably  the  reaction 
has  not  changed. 

(e)  Fermentation  with  the  production  of  gas. — Gas  may  be  formed 
in  such  small  quantities  as  not  to  be  observed  as  such,  but  to  be  en- 
tirely absorbed  by  the  medium.  Whenever  gas  formation  is  men- 
tioned as  a  character,  visible  gas  is  meant.  The  analysis  of  the  gas 
was  made  in  the  usual  manner  by  absorbing  a  portion  with  potassium 
hydrate  (KOH)  and  testing  the  remainder  with  the  flame.  The 
amount  absorbed  by  potassium  hydrate  (KOH)  is  referred  to  as 
carbon  dioxid  (CO,)  and  the  remainder,  if  an  explosion  is  obtained, 
as  hydrogen  (H).  This  is,  naturally,  only  approximately  correct. 
Since  the  gas  formula  may  vary  from  day  to  day,  too  much  value 
must  not  be  given  to  the  exact  proportion.  It  is  well  to  observe 
whether  the  proportion  of  hydrogen  to  carbon  dioxid  is  greater  or 
less  than  1. 

(/)  Agar. — One  per  cent  agar  is  used.  The  description  of  the 
growth  on  this  medium  is  made  from  the  appearance  as  seen  on  the 
surface  of  an  agar  slant.  The  description  is  usually  very  brief,  since 
it  has,  as  a  rule,  little  differential  value. 

{g)  Acid  agar. — This  medium  is  made  acid  by  titrating  to  +3  to 
phenolphthalein.  The  absence  or  presence,  as  well  as  the  degree  of 
growth,  is  noted. 

(A)  Serum. — The  serum  used  is  taken  from  the  horse,  sterilized  at 
55°  C.  and  congealed  at  80°  C.  Deep  inoculations  are  made,  and  the 
surface  of  slanted  serum  is  also  inoculated.  The  degree  of  growth  is 
usually  noted.  Cultures  are  observed  for  6  weeks  to  2  months.  The 
presence  or  absence  of  liquefaction  is  the  chief  character  sought  for. 
Since  room  temperature  varies  so  greatly,  the  time  at  which  liquefac- 
tion begins  varies,  and  little  differential  value,  therefore,  can  be  given 
to  the  exact  time  of  this  phenomenon. 

(«')  Potato. — The  composition  of  potato  varies  so  markedly  that  a 
description  of  a  culture  on  this  medium  may  differ  materially  from 
that  which  is  observed  on  another  tube  of  the  same  medium.  It  is  the 
aim  to  omit  for  the  most  part  the  observed  variations  due  to  the 
composition  of  the  different  potatoes. 


12  THE    BACTEKIA    OF    THE   APIAEY. 

(j)  Potato  water.— To  potatoes  sliced  very  thin  is  added  an  equal 
amount  of  water  by  weight  and  the  mixture  is  then  boiled.  This  is 
btrained  and  distributed  in  straight  and  fermentation  tubes.  The 
reaction  of  the  solution  was  made  +1.5  to  phenolphthalein.  If  any 
of  the  micro-organisms  ferment  glucose  with  the  production  of  gas, 
fermentation  tubes  are  inoculated  to  test  the  fermentation  of  starch ; 
if  not,  straight  tubes  are  inoculated. 

(k)  Milk.— If  a  micro-organism  breaks  up  glucose  with  the  forma- 
tion of  gas,  a  fermentation  tube  of  milk  is  inoculated  with  the 
culture;  if  not,  straight  tubes  are  used.  Separator  milk  is  used. 
The  coagulation  of  the  casein  with  or  without  liquefaction  is  the 
chief  character  noted.  Very  little  stress  is  laid  upon  the  time  ele- 
ment in  the  coagulation  of  the  casein  and  the  other  phenomena 
which  are  to  be  observed  in  milk.  Different  samples  of  milk  and 
the  different  environmental  conditions  are  factors  which  vary  the 
length  of  time  at  which  the  different  phenomena  appear. 

(1)  Litmus  milk. — The  reaction  as  shown  by  the  litmus  and  the  dis- 
charging of  the  color  are  the  chief  points  observed. 

(m)  Gelatin. — The  color,  degree  of  growth,  the  presence  or 
absence  of  liquefaction,  and  the  form  of  liquefaction  are  the  chief 
points  observed.  The  cultures  are  kept  under  observation  2  months 
or  longer  and,  as  in  serum,  the  time  given  at  which  liquefaction  takes 
place  is  only  approximate. 

(w)  Indol. — The  cultures  are  allowed  to  grow  in  sugar-free  pep- 
tonized bouillon  for  3  to  5  days,  and  are  tested  with  potassium  nitrite 
(KNOj)  and  sulfuric  acid  (H,S04)  after  the  ring  method.  Too 
much  stress  may  be  placed  upon  the  ability  of  an  organism  to  form 
indol.  This  character  has  been  shown  to  be  a  somewhat  transient 
one  (3). 

{o)  Reduction  of  nitrates  to  nitrites. — Cultures  are  cultivated  7 
days  in  a  solution  of  1  gram  of  Witte's  peptonum  siccum  and  one- 
fifth  gram  of  sodium  nitrate  in  1,000  c.  c.  of  tap  water.  To  such  a 
culture  and  to  a  control  tube  are  added  a  mixture  of  naphthylamine 
and  sulfanilic  acid  (napthylamine,  1  part;  distilled  water,  1,000 
parts:  sulfanilic  acid,  one-half  gram,  dissolved  in  dilute  acetic  acid 
in  the  proportion  of  1  part  of  acid  to  16  parts  of  water) .  If  nitrate 
is  reduced  to  nitrite,  a  pink  color  develops.  The  control  tube  should 
remain  clear,  or  slightly  pink — owing  to  the  absorption  of  a  trace  of 
nitrite  from  the  atmosphere. 

PART  I.  BACTERIA  OF  THE  NORMAL  APIARY. 

Before  studying  the  cause  of  a  disease  it  is  necessary  that  we 
know  what  bacteria  are  normally  present,  so  that  later,  in  studying 
diseased  conditions,  a  consideration  of  these  nonpathogenic  species 
may  be  eliminated.     In  view  of  this  necessity  a  bacteriological  study 


BACTEEIA   PROM   THE   COMBS,  13 

of  the  hives,  combs,  honey,  pollen,  larvae,  and  adult  bees  was  begun, 
to  determine  the  bacteria  normally  preseftt.  It  was  not  hoped  that 
all  the  species  isolated  could  be  easily  identified,  or  that  all  would 
merit  a  careful  description,  but  it  was  hoped  that  those  species  which 
seemed  to  be  localized  in  any  part  of  the  apiary,  or  upon  or  within  the 
bees,  might  be  studied  and  described  with  sufficient  care  to  guarantee 
their  identification  upon  being  isolated  again.  The  chance  of  varia- 
tion in  morphology,  pathogenesis,  and  cultural  characters  due  to 
environmental  conditions  to  which  these  micro-organisms  were  being 
subjected  at  the  time,  or  to  which  they  had  been  subjected  before 
isolation  or  study,  has  been  carefully  borne  in  mind. 

BACTERIA  PBOM  THE  COMBS. 

One  might  naturally  suppose  that  very  many  species  of  bacteria 
would  be  present  on  combs,  since  these  are  exposed  more  or  less  to  the 
contaminating  influence  of  the  air.  The  reverse,  however,  seems  to 
be  true.  The  number  of  different  species  isolated  is  comparatively 
small.  Those  which  appear  most  often  are  described  below.  Some 
other  species  mentioned  in  this  paper  are  found  on  combs,  but  inas- 
much as  they  appear  most  frequently  from  other  sources  they  are 
described  there.  One  species  of  Saccharomyces  from  the  comb,  also, 
is  described  under  the  heading  "  Saccharomyces  and  fungi." 

Bacillus  A. 
{B.  mesentericus?) 

Occurrence. — Found  very  frequently  on  combs,  on  scrapings  from  hives,  and 
on  the  bodies  of  bees,  both  diseased  and  healthy. 

Oelatin  colonies. — Very  young  colonies  show  irregular  edges,  but  very  soon 
liquefaction  takes  place  and  the  colony  gives  rise  to  a  circular  liquefied  area, 
covered  with  a  gray  membrane,  which  later  turns  brown. 

Agar  colonies. — Superficial  colonies  present  a  very  irregular  margin  consist- 
ing of  outgrowths  taking  place  in  curves.  Deep  colonies  show  a  filamentous 
growth  having  a  moss-like  appearance. 

Morphology. — In  the  living  condition  the  bacilli  appear  clear  and  often  grauu 
lar,  arranged  singly,  in  pairs,  and  in  chains.  The  flagella  are  distributed  over 
the  body.  The  rods  measure  from  Sn  to  4/i  in  length,  and  from  0.9/4  to  L2|U 
in  thickness. 

Motility. — The  bacUli  are  only  moderately  motile. 

Spores. — Spores  are  formed  in  the  middle  of  the  rod. 

Gram's  stain. — The  bacilli  take  Gram's  stain. 

Oxygen  requirements. — Aerobic  and  facultatively  anaerobic. 

Bouillon. — Luxuriant  growth  in  24  hours,  with  cloudiness  of  medium ;  a  gray 
flocculent  membrane  is  present.  Later,  the  membrane  sinks  and  the  medium 
clears,  leaving  a  heavy,  white,  flocculent  sediment,  with  a  growth  of  the  organ- 
isms adhering  to  the  glass  at  the  surface  of  the  medium.     Reaction  alkaline. 

Glucose. — Luxuriant  growth  takes  place  in  the  bulb,  with  a  moderate,  floccu- 
lent growth  in  closed  arm.    The  gradual  settling  of  the  organisms  causes  a 


14  THE   BACTEEIA   OF   THE   APIARY. 

heavy  white  sediment  to  form  in  the  bend  of  the  tube.  The  reaction  is  at  first 
slightly  acid,  but  subsequently  becomes  alkaline.     No  gas  is  formed. 

Lactose. — Reaction  alkaline. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  acid. 

Maltose. — Reaction  acid. 

Mannite. — Reaction  alkaline. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — A  luxuriant  growth  takes  place  on  this  medium.  The  growth 
gradually  increases  to  a  moist,  glistening  one,  being  then  friable  and  of  a  grayish 
brown  color. 

Serum. — A  luxuriant,  brownish,  glistening,  friable  growth  spreads  over  the 
entire  surface.     No  liquefaction  is  observed. 

Potato. — An  abundant  fleshy  growth  of  a  brown  color  spreads  over  the  entire 
surface.     The  water  supports  a  heavy  growth.     The  potato  is  slightly  discolored. 

Milk. — Precipitation  takes  place  rapidly,  followed  by  a  gradual  digestion  of 
the  casein,  the  medium  changing  from  the  top  downward  to  a  translucent 
liquid,  becoming  at  last  semi-transparent  and  viscid. 

Litmus  milk. — Precipitation  of  the  casein  takes  place  usually  within  24  hours, 
followed  by  a  gradual  peptonization.  Reduction  of  the  litmus  occurs  rapidly, 
leaving  the  medium  slightly  brown  ;  later  the  blue  color  will  return  on  exposing 
the  milk  to  the  air  by  shaking.     Reaction  alkaline. 

Gelatin. — An  abundant  growth  takes  place  with  rapid,  infundibuliform  lique- 
faction. A  heavy,  white,  friable  membrane  is  formed  on  the  surface  of  the 
liquefied  medium.  A  flocculent  sediment  lies  at  the  bottom  of  the  clear  lique- 
fied portion. 

Acid  agar. — Growth  takes  place. 

Indol. — None  has  been  observed. 

Nitrate. — Reduction  to  nitrite  is  positive. 

Bacterium  acidiformans.     (Sternberg,  1892.) 

Occurrence. — Isolated  from  the  scraping  of  propolis  and  wax  from  the  hives 
and  frames  of  healthy  colonies. 

Gelatin  colonies. — The  superficial  colonies  are  friable,  convex,  opaque,  and 
white  with  even  border ;  when  magnified  they  are  finely  granular,  sometimes 
radiately  marked.  They  are  from  1  to  4  millimeters  in  diameter.  The  deep 
colonies  are  spherical  or  oblong  and  entire. 

Morphology. — When  taken  from  an  agar  slant  24  hours  old,  the  rods  are 
short,  with  rounded  ends,  singly  and  in  pairs.  Length  about  1.6|ti,  thickness 
O.Sfi.  They  stain  uniformly  with  carbol-fuchsin.  Flagella  are  apparently  ab- 
sent. 

Motility. — No  motility  has  been  observed  in  any  medium. 

Spores. — Spores  are  apparently  absent. 

Gram's  stain. — The  bacteria  are  decolorized  by  Gram's  method. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — The  medium  becomes  slightly  clouded  with  a  feeble  ring  of  growth 
on  the  glass  at  the  surface  of  the  liquid.  A  moderate  amount  of  white  friable 
sediment  is  formed.     Reaction  alkaline. 

Glucose. — Uniformly  and  slightly  clouded.  No  gas  is  formed.  Reaction 
acid. 

Lactose. — Reaction  acid. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  acid. 


BAOTEEIA  FROM  POLLEN.  '       15 

Maltose. — Reaction  acid. 

Mannite. — Reaction  acid. 

Potato  water. — Reaction  acid. 

Agar  slant. — A  moderate,  gray,  glistening  growth,  confined  to  tlie  area  Inocu- 
lated with  the  loop,  is  formed  on  the  inclined  surface. 

Serum. — A  feeble  gray  growth  Is  formed  only  on  the  inoculated  surface.  No 
liquefaction  taljes  place. 

Potato. — A  gray  growth  covers  the  inoculated  surface. 

Milk. — Heat  causes  a  ready  coagulation  of  the  casein.    Reaction  acid. 

I/itmus  milk. — Coagulation  of  casein  occurs  promptly  on  boiling  a  culture  2 
weeks  old.    Reaction  acid. 

Gelatin. — Growth  of  spherical  colonies  appears  along  the  line  of  inocula- 
tion, the  surface  growth  being  grayish  and  spreading  slowly.  No  liquefaction 
takes  place. 

Acid  agar. — Growth  takes  place. 

Indol. — A  trace  was  observed. 

Nitrate. — No  reduction  to  nitrite  could  be  observed. 

BACTERIA  PROM  POLLEN. 

As  in  the  case  of  the  examination  of  the  combs,  the  number  of  spe- 
cies of  bacteria  found  in  pollen  is  comparatively  small.  The  follow- 
ing are  often  found  to  be  present.  Other  species  have  been  isolated, 
but  their  distribution  in  the  pollen  is  not  at  all  constant. 

Bacillus  B. 

Occurrence. — Found  frequently  in  pollen  and  in  the  intestine  of  healthy 
honey  bees. 

dclatin  colonies. — The  colonies  are  egg-yellow  with  even  border.  Liquefac- 
tion takes  place  slowly.  Surface  colonies  are  about  1.5  millimeters  in  diameter, 
have  coarsely  granular  center,  finely  granular  margin,  and  clear  and  sharply 
defined  border.    A  peculiar  toruloid  growth  is  often  observed. 

Morphology. — The  organisms  are  short  rods  with  rounded  ends,  which  stain 
uniformly  with  carbol-fuchsln,  and  are  1/i  to  2|H  in  length.  Few  short  involu- 
tion forms  occur. 

Motility. — The  bacilli  are  actively  motile  in  young  cultures. 

Spores. — No  spores  have  been  observed. 

Oram's  stam.— .The  bacilli  are  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — This  medium  becomes  uniformly  clouded,  frequently  with  a  scanty, 
friable  membrane.  Sometimes  the  organisms  settle,  clearing  the  medium  and 
forming  a  viscid  sediment.  A  growth  of  the  culture  adheres  to  the  glass  at  the 
surface  of  the  liquid.  This,  together  with  the  membrane,  is  of  a  light  egg-yellow 
color,  which  deepens  somewhat  with  age.    Reaction  alkaline. 

Glucose. — At  first  both  arms  of  the  fermentation  tube  are  clouded  slightly,  and 
the  cloudiness  later  Increases.  Sometimes  a  stronger  growth  occurs  in  the 
closed  arm  than  in  the  open  one.  Reaction  Is  at  first  acid,  but  slowly  changes  to 
alkaline. 

Lactose. — Reaction  alkaline. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  alkaline. 

Maltose. — Reaction  slightly  acid. 
9583— No.   14—06  m 3 


16  THE    BACTERIA   OF    THE   APIAKY. 

Mannite. — Reaction  slightly  acid,  later  alkaline. 

Agar  slant. — A  moderate,  slightly  yellow,  nonviscid  glistening  gi:pwth  appears 
along  the  inoculated  surface.  This  growth  gradually  spreads  and  deepens  in 
color  to  an  egg-yellow. 

Potato. — A  moderate,  egg-yellow,  nonviscid,  glistening  growth  spreads  over 
the  entire  surface.     The  potato  Is  slightly  discolored. 

Milk. — The  milk  is  covered  by  a  yellow  growth  of  the  culture,  resembling 
cream.     Coagulation  takes  place  on  boiling. 

Litmus  milk. — Reaction  alkaline. 

Gelatin. — Growth  takes  place  along  the  line  of  inoculation.  Deep  in  the 
medium  the  colonies  are  white  and  spherical ;  the  surface  growth  is  yellow. 
After  a  few  days  liquefaction  begins,  and  at  the  end  of  2  weeks  one-half  the 
tube  is  liquefied.  The  liquefaction  is  infundibuliform.  Liquefied  gelatin  is  sur- 
mounted by  a  friable,  egg-yellow  pellicle.  The  growth  in  the  liquefied  portion 
is  flocculent,  which,  on  settling,  forms  a  yellow  sediment  at  the  apex. 

Indol. — None  could  be  observed. 

Nitrates. — No  reduction  to  nitrites  occurs. 

BACTEEIA  IN  HONEY  AND  NORMAL  IiAIlV.ai. 

Comb  honey  from  a  large  number  of  sources  has  been  examined 
and  found  to  be  quite  uniformly  sterile.  The  healthy  larvae  likewise 
are  usually  sterile. 

BACTERIA  UPON  THE  ADULT  BEES. 

On  the  external  part  of  the  bee  we  again  find  only  a  few  different 
species.  Bacillus  A,  described  as  found  upon  the  combs,  is  fre- 
quently isolated  from  the  bee.  Other  species  which  are  found  fre- 
quently are  described  below. 

Bacterium  cyaneus  (Micrococcus  cyaneus). 

Occurrence. — Isolated  from  the  body  of  a  healthy  honey  bee  and  from  pollen. 

Gelatin  colonies. — The  colonies  are  lemon-yellow,  with  entire  border,  growth 
taking  place  readily  on  this  medium.  The  superficial  colonies,  having  well- 
defined  border,  are  finely  granular,  and  liquefy  the  medium  within  3  to  6  days. 

Morphology. — Short  oval  rods  0.8/n  to  l.T/j,  in  length,  O.Y/i  to  0.8|U  in  thickness. 
Short  involution  forms  are  present.  The  rods  occur  singly,  paired,  and  in 
clumps.     No  flagella  have  been  demonstrated. 

Motility. — No  motion  has  been  demonstrated. 

Spores. — No  spores  have  been  demonstrated. 
'     Gram's  stain. — The  bacterium  takes  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — At  first  a  slight  cloudiness  appears,  the  medium  becoming  turbid 
in  old  cultures.  A  heavy  yellowish-white,  slightly  viscid  ring  forms  on  the 
tube  at  the  surface  of  the  medium.  The  sediment,  and  sometimes  the  medium, 
show  marked  viscidity.     Reaction  alkaline. 

Glucose. — ^The  growth  of  the  culture  is  confined  entirely  to  the  open  bulb,  in 
which  the  medium  becomes  turbid.    No  gas  is  formed.    Reaction  alkaline. 

Lactose. — Reaction  alkaline. 

Saccharose. — Reaction  alkaline. 

Levulose. — Reaction  alkaline. 


BACTEEIA   UPON   THE   ADULT   BEES.  17 

Maltose. — Reaction  allialino. 

Mannitc. — Reaction  allialine. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — On  the  surface  of  the  agar  there  takes  place  an  abundant  growth, 
which  is  confined  to  the  surface  inoculated  with  the  loop.  The  culture  is 
tleshy,  nonviscid,  and  lemon-yellow.  It  produces  a  soluble  pigment  that  dif- 
fuses thru  the  agar,  giving  it  a  dark-pink  color. 

Scniiii. — Luxuriant  growth  takes  place,  lurompanled  by  liquefaction. 

Potato. — A  lemon-yellow,  fieshy,  glistening  growth  spreads  over  the  inclined 
surface  of  the  potato. 

Milk. — Precipitation  followed  by  slow  liquefaction  of  the  casein  occurs ;  later 
the  medium  becomes  alkaline  and  very  viscid. 

Litmus  iiiillc. — The  litmus  is  discharged  and  the  casein  is  liquefied.  Reaction 
alkaline. 

Gelatin. — Infundibuliform  liquefaction  soon  begins,  which  is  followed  by 
stratiform  liquefaction.     The  liquefied  gelatin  is  turbid  and  viscid. 

Acid  agar. — On  this  mediimi  a  moderate  lemon-yellow  growth  is  observed. 

Indol. — None  could  be  observed. 

Xitrates. — No  reduction  of  nitrates  could  be  observed. 

Micrococcus  C. 

Occurrence. — Isolated  from  the  body  of  a  healthy  honey  bee. 

Gelatin  colonics. — The  surface  colonies  are  round  and  slightly  yellow. 
Liquefaction,  begins  in  from  2  to  4  days.  The  magnified  colonies  are  finely 
granular,  with  sharply  defined,  entire  border. 

Morphology. — Cocci,  about  0.8|U  in  diameter,  occur  in  pairs  and  in  small 
clusters. 

Motility. — Nonmotile. 

Spores. — Spores  are  apparently  absent. 

Grants  stain. — The  coccus  takes  the  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — ^This  medium  becomes  uniformly  clouded  in  24  hours  after  in- 
oculation, growth  increases,  and  friable  sediment  forms.  The  liquid  clears 
somewThat  on  standing.  Reaction  at  first  slightly  acid ;  later  returns  to 
neutral. 

Glucose. — The  medium  in  the  bulb  becomes  cloudy,  while  that  in  the  closed 
arm  remains  clear.  White  friable  sediment  forms  in  bend  of  tube.  Reaction 
acid.     No  gas  is  formed. 

Lactose. — Reaction  slowly  becomes  acid. 

Saccharose. — Reaction  acid. 

Levulose. — Reaction  acid. 

Maltose. — Reaction  acid. 

Mannite. — Reaction  acid. 

Potato  water. — Reaction  acid. 

Agar  slant. — A  grayish  white,  fleshy,  nonviscid,  glistening  growth  takes 
place  along  the  inoculated  surface.  It  does  not  spread,  and  retains  a  dis- 
tinct boundary. 

Serum. — A  spreading  growth  takes  place,  accompanied  by  liquefaction. 

Potato. — A  gray,  fleshy,  glistening,  nonviscid  growth  forms  over  the  entire 
cut  surface  of  the  potato.     The  potato  is  slightly  discolored. 

Milk. — This  medium  becomes  firmly  coagulated  and  later  the  casein  liquifies 
with  the  formation  of  a  milky  serum. 


18  THE    BACTEEIA    OF    THE   APIARY. 

Litmus  milJc. — In  this  medium  coagulation  takes  place,  accompanied  bj 
reduction  of  the  litmus.     Reaction  slightly  acid. 

Gelatin. — After  a  day  or  two  infundibuliform  liquefaction  occurs,  being 
followed  by  stratiform  liquefaction;  the  liquefied. gelatin  is  turbid.  Growth 
below  this  portion  Is  in  the  form  of  small  spherical  colonies. 

Acid  agar. — A  white,  fleshy,  nonviscid  growth  is  observed. 

Indol. — A  trace  was  observed. 

y  Urates. — Reduced  to  nitrites. 

BACTERIA  OF  THE  INTESTINE  OF  THE  HEALTHY  HONEY  BEE. 

A  great  many  investigations  have  been  made  in  recent  years  on  the 
bacteria  found  present  in  the  intestines  of  vertebrates  (4,  5,  6,  Y,  8, 
9),  and  striking  similarities  are  noticed  in  the  species  found  in  many 
of  them.  In  this  investigation  the  intestinal  contents  of  about  150 
bees,  mostly  from  one  apiary,  have  been  studied  more  or  less  thoroly. 
Several  species  which  are  found  to  be  constant  in  many  of  the  verte- 
brates are  found  in  the  intestine  of  the  honey  bee.  Since  the  tem- 
perature of  the  bee  approximates  much  of  the  time,  especially  when 
in  the  hive,  that  of  the  warm-blooded  animals,  many  of  the  same 
species  of  bacteria  inhabit  the  intestine  of  this  insect  as  are  found 
thriving  in  the  same  locality  in  man  and  other^  animals.  A  stained 
cover-glass  preparation  made  directly  from  a  healthy  adult  field  bee 
reveals,  almost  without  exception,  a  multitude  of  bacteria. 

In  a  study  of  the  bacterial  flora  stress  has  been  placed  upon  the 
different  species  which  were  found  to  be  more  or  less  constant,  rather 
than  upon  the  actual  number  of  bacteria  oi-  species  in  any  quantity 
of  material  from  a  single  bee.  From  the  observations  which  have 
been  made,  it  appears  that  the  number  of  species  in  any  individual 
is  comparatively  small,  but  the  number  of  bacteria  is  in  many  cases 
very  large.  Sometimes,  however,  the  plates  show  very  few  colonies, 
while  cover-glass  preparations  show  a  very  large  number  of  bacteria. 
These  organisms  are  probably  the  anaerobe,  which  is  quite  constant, 
as  shown  by  cultures  made  direct  from  the  intestine  into  glucose  agar 
(Liborius's  method). 

When  a  loopful  of  the  material  from  the  intestine  was  used  for  the 
inoculation,  the  following  data  give  the  approximate  findings : 

Bee  No.  1, 300  to  400  yellow  colonies,  probably  alilie. 

Bee  No.  2,  a  few  colonies  of  fungi  only. 

Bee  No.  3,  500  colonies,  mostly  yeast. 

Bee  No.  4,  100  or  more  colon-like  colonies. 

Bee  No.  5,  2,000  or  more,  mostly  yellow. 

Bee  No.  6,  20  or  more  colonies,  mostly  yeasts. 

Bee  No.  8,  400  or  more  yellow  colonies. 

Bee  No.  9,  30  yeasts  with  a  few  fungi. 

Bee  No.  10,  50  yeast  colonies  with  a  few  fungi. 

Bee  No.  11,  no  growth. 

Bee  No.  12,  300  colonies,  slightly  yellow. 


BACTERIA   OP   THE   INTESTINE.  19 

Bee  No.  13,  2,000  or  more  gray  colonies. 

Bee  No.  14,  yeast  colonies  and  a  few  colonies  of  bacteria  showing  ground- 
glass  appearance. 
Bee  No.  15,  2,000  or  more  colon-like  colonies  {B.  cloaca;). 

The  following  are  the  species  which  have  been  found  to  be  most 
constant.  The  reader  is  referred  also  to  the  description  of  the  yeast 
plant  found  very  frequently  in  the  intestine  of  the  normal  honey  bee, 
described  under  "  Saccharomyces  and  fungi." 

Bacterium  S. 

Occurrence. — Frequent  in  the  intestine  of  the  healthy  honey  bee. 

Agar  colony. — Deep  colonies  when  magnified  are  coarsely  granular,  showing  a 
dark  brown  center,  with  a  thin  and  ill-deflned  border. 

Morphology. — A  preparation  made  from  a  young  culture  taken  from  a  glu- 
cose fermentation  tube  shows  rods  with  rounded  ends,  occurring  singly  and  in 
pairs',  staining  easily  and  uniformly  with  carbol-fuchsin,  and  measuring  0.7^  to 
1.5/»  in  length  and  0'.5/i  to  0.7/t  in  thickness. 

Motility. — No  motility  could  be  observed. 

Spores. — No  spores  could  be  demonstrated  in  young  cultures.  In  old  cultures 
their  presence  is  questionable. 

Oxygen  requirements. — Strictly  anaerobic. 

Bouillon. — In  straight  tubes  no  growth  occurs. 

Olucose. — A  moderate  cloudiness  can  be, seen  in  the  closed  arm,  while  the 
open  bulb  remains  clear.     No  gas  is  produced.     Reaction  about  neutral. 

Glucose  agar  (Liborius's  method). — Growth  is  rather  slow.  After  3  days  a 
moderate  growth  may  be  observed;  later,  if  cultures  have  recently  been  iso- 
lated from  the  bee's  intestine,  the  growth  imparts  to  the  medium  a  diffused 
haziness  or  cloudiness.     After  many  generations  the  culture  loses  this  property. 

Glucose  gelatin  (Liborius's  method). — Very  slow  growtji  occurs  in  the  depth 
of  the  mediiuu.    No  liquefaction  takes  place.    ' 

Bacillus  cloacae. 

Occurrence. — Found  in  the  intestine  of  a  large  number  of  healthy  honey  bees. 

Oelatin  colonies. — Superficial  colonies  are  thin  and  blue  to  gray  In  color ;  deep 
colonies,  brown,  regular,  granular,  and  spherical  to  lenticular. 

Agar  colonies. — Superficial  colonies  are  partially  opaque,  brown,  finely  granu- 
lar, with  well-defined  margin ;  deep  colonies  are  regular,  spherical,  or  lenticular, 
with  well-defined  margin. 

Morphology. — The  rods  from  24-hour  agar  cultures  have  rounded  ends,  vary- 
ing in  length  from  V  to  2  it  and  in  width  from  0.7/i  to  0.9 /i».  They  are  usually 
found  singly  or  in  pairs.  Involution  forms  are  not  uncommon.  With  carbol- 
fuchsin  they  stain  uniformly.    This  species  possesses  a  few  peritrichic  flagella. 

Motility. — Active  motility  is  observed  In  young  cultures. 

Spores. — No  spores  are  formed. 

Gram's  stain. — ^The  bacillus  does  not  take  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — A  uniform  cloudiness  appears  in  24  hours.  Growth  continues  until 
the  medium  becomes  heavily  clouded,  followed  by  a  gradual  settling  of  many  of 
the  organisms,  forming  a  viscid  grayish-white  sediment.  A  gray  friable  mem- 
brane, which  adheres  to  the  sides  of  the  tube  at  the  surface  of  the  medium,  is 
sometimes  produced.    Upon  agitation  this  membrane  breaks  up  and  sinks  to  the 


20  THE    BACTERIA   OF    THE   APIAEY. 

bottom,  leaving  a  gray  ring  of  the  growtli  adhering  to  the  glass.  Reaction 
alkaline. 

Glucose.— The  medium  in  the  bulb  becomes  turbid,  while  that  in  the  closed 
arm  is  uniformly  cloudy.  A  heavy  grayish-white  sediment  is  formed.  The 
reaction  is  at  first  slightly  acid,  but  in  a  few  days  becomes  alkaline.  Abundant 
and  rapid  gas  formation  takes  place,  filling  usually  from  one-half  to  nine-tenths 
of  the  closed  arm.  The  ratio  of  hydrogen  to  carbon  dioxid  is  approximately 
1  to  2 ;  that  is,  the  ratio  of  hydrogen  to  carbon  dioxid  is  less  than  1. 

Lactose. — In  this  medium  gas  formation  takes  place  more  slowly  than  in 
glucose.  At  the  end  of  8  days  one-fourth  of  the  closed  arm  is  filled  with  gas. 
The  ratio  of  hydrogen  to  carbon  dioxid  is  greater  than  1.     Reaction  acid. 

Saccharose. — Gas  is  formed  abundantly  and  rapidly;  more  than  one-half  of 
the  tube  is  usually  filled  with  gas.  The  ratio  of  hydrogen  to  carbon  dioxid  is 
less  than  1.     Reaction  alkaline. 

Levulose. — A  rapid  fermentation  takes  place ;  more  than  one-half  of  the  closed 
arm  is  filled  with  gas.  The  ratio  of  hydrogen  to  carbon  dioxid  is  approximately 
1  to  5 ;  that  is,  less  than  1.  A  slight  formation  of  acid  takes  place  at  first,  but 
the  reaction  rapidly  becomes  alkaline. 

Maltose. — Formation  of  gas  takes  place  with  the  result  that  at  the  end  of  5 
days  approximately  one-half  of  the  tube  is  filled.  The  ratio  of  hydrogen  to 
carbon  dioxid  will  approximate  that  of  1  to  1.    Reaction  acid. 

Mamnite. — Gas  is  formed  rapidly  and  abundantly ;  at  the  end  of  5  days  the 
closed  arm  is  usually  much  more  than  half  filled  with  the  gas.  The  reaction  is 
at  first  slightly  acid,  but  soon  becomes  alkaline.  The  ratio  of  hydrogen  to  car- 
bon dioxid  is  approximately  1  to  2 ;  that  is,  less  than  1. 

Potato  loater. — Gas  forms  rapidly  and  fills  half  the  closed  arm.  The  ratio  of 
hydrogen  to  carbon  dioxid  is  as  1  to  2  ;  that  is,  less  than  1. 

Agar  slant. — A  moderate,  grayish-white,  glistening,  friable  growth  appears 
along  the  line  of  inoculation,  which  usually  spreads  to  the  sides  of  the  tube. 

Serum. — Moderate  gray  growth  appears,  which  is  confined  quite  closely  to  the 
line  of  inoculation.    Liquefaction  takes  place  slowly  after  .3  weeks. 

Potato. — A  moderate  amount  of  gray  fleshy  growth  covers  the  slope.  The 
potato  is  slightly  discolored. 

MilJc. — Coagulation  takes  place  after  4  days'  growth.    Gas  is  formed. 

Litmus,  milk. — A  marked  production  of  acid  takes  place,  followed  by  firm 
coagulation. 

Gelatin. — A  heavy  white  growth  takes  place  along  the  line  of  inoculation ;  the 
surface  growth  is  flat,  bluish-white,  and  spreads  with  an  uneven  margin.  Slow 
infundibuliform  liquefaction  takes  place  after  2  weeks. 

Acid  agar. — A  growth  takes  place. 

Indol. — A  trace  is  sometimes  produced. 

Nitrates. — Reduction  to  nitrites  is  positive. 

B.  coli  communis. 

Occurrence. — Pound  in  the  intestine  of  healthy  honey  bees. 

Gelatin  colonies. — The  superficial  colonies  are  blue,  lobate-lobulate,  and 
slightly  spreading;  when  magnified  they  are  brownish  yellow  in  the  center 
and  more  transparent  toward  the  margin;  the  deep  colonies  are  spherical  to 
lenticular  and  brownish  yellow,  with  well-defined  borders. 

Morphology.— The  short  rods  with  rounded  ends  measure  1.5^  to  2^1  in  length 
and  0.7/t  to  O.S/j.  in  thickness.  They  occur  singly  or  in  pairs,  stain  uniformly, 
and  are  motile  by  means  of  a  few  peritriehie  flagella. 


BACTERIA   OP   THE   INTESTINE.  21  ■ 

Motility. — ^The  bacilli  are  actively  motile  from  some  cultures. 

Spores. — No  spores  are  formed. 

Oi-am's  stain. — The  bacillus  is  decolorized  by  Gram's  method. 

Oxygen  requirements. — It  is  a  facultative  anaerobe. 

Bouillon. — The  medium  becomes  uniformly  clouded  in  24  hours,  with  a  slight 
acid  reaction ;  the  medium  later  becomes  allialine,  ^yitll  a  gray  and  friable 
sediment.  A  feeble  pellicle  is  formed  and  u  growth  of  the,  organism  often 
adheres  to  the  glass  at  the  surface  of  the  liquid. 

Glucose. — Both  branches  of  the  fermentation  tube  become  clouded.  The 
sugar  splits  by  fermentation  into  gas  and  acid,  one-half  or  more  of  the  closed 
arm  being  filled.     The  ratio  of  hydrogen  to  carbon  dioxid  is  2  to  1. 

Lactose. — Gas  fills  one-fourth  of  the  closed  tube.     Reaction  acid. 

Saccharose. — Gas  fills  one-sixth  of  the  closed  tube.     Reaction  acid. 

Levulose. — Gas  fills  one-half  of  the  closed  tube.  The  value  of  hydrogen  to 
carbon  dJoxid  is  2  to  1.     Reaction  acid.  ^ 

Maltose. — One-sixth  of  the  closed  arm  is  filled  with  gas.     Reaction  acid. 

Mannite. — One-half  of  the  closed  tube  is  filled  with  gas.     Reaction  acid. 

Potato  tratei: — Reaction  acid. 

Agar  slant. — A  moderate,  gray,  nonviscid,  spreading  growth  takes  place  on  the 
surface  of  the  inclined  agar. 

Serum. — A  gray,  glistening,  nonspreading  growth  is  observed  on  the  inclined 
serum.     No  liquefaction  takes  place. 

Potato. — A  moderate,  fleshy,  glistening  growth  spreads  over  the  inoculated 
surface.     Potato  slightly  discolored. 

Milk. — Coagulation  of  the  casein  takes  place  in  about  4  days.  A  small  quan- 
tity of  gas  is  produced. 

Litmus  milk. — Coagulation  occurs.     Reaction  strongly  acid. 

Gelatin. — ^A  moderate  growth  occurs  along  the  line  of  inoculation ;  the  growth 
is  spreading  with  an  irregular  margin  on  the  surface.     No  liquefication  occurs. 

Acid  agar. — A  moderate  grayish  growth  occurs  on  surface. 

Indol. — A  trace  was  obtained  in  some  cultures. 

Nitrates. — Reduced  to  nitrites. 

B.  cliolerse  suis. 

Occurrence. — Isolated  from  the  intestine  of  healthy  honey  bees. 

Gelatin  colonies. — Colonies  are  translucent  by  transmitted  light;  bluish  to 
gray  by  reflected,  the  border  being  uneven  and  well  defined.  When  the  colonies 
are  magnified  they  appear  brownish  and  finely  granular. 

Morphology. — ^The  rods  are  short,  with  rounded  ends,  occurring  singly  and 
in  pairs,  and  staining  uniformly  with  carbol-fuchsin,  1  to  2.8^  in  length,  and 
0.6/1  to  0.8/1  in  thickness.    A  few  peritrichic  flagella  are  present. 

Motility. — ^Usually  only  jl  few  are  motile  at  a  time  in  the  field,  and  these 
present  a  rapid  whirling  motion. 

Spores. — ^No  spores  are  formed. 

Gram's  stain. — ^The  bacteria  are  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — A  uniform,  moderate  cloudiness  arises  in  this  medium  in  24 
hours;  later  a  grayish-white  membrane  is  formed  which,  upon  shaidng  the 
tube,  sinks  to  the  bottom,  forming  a  gray  sediment.  The  reaction  is  at  first 
slightly  acid,  but  later  becomes  alkaline. 

Glucose. — The  medium  becomes  clouded  in  both  arms  of  the  fermentation 
tube,  with  the  production  of  a  small  amount  of  gas.    Reaction  acid. 


22  THE    BACTERIA    OE    THE    APIARY. 

Lactose.— Growth  takes  place  in  both  arms  of  the  tube,  but  the  sugar  Is  not 
split  into  either  acid  or  gas. 

Saccharose.— Giowth  occurs  in  both  arms  of  the  tube,  neither  acid  nor  gas 
being  formed. 

f  Levulose.— Growth  takes  place  in  both  arms  with  the  production  of  gas  and 
acid;  one-third  of  the  closed  arm  is  filled.  The  ratio  of  hydrogen  to  carbon 
dioxid  is  about  3  to  1 — that  is,  greater  than  1. 

Maltose. — The  medium  in  both  arms  of  the  tube  becomes  clouded.  Fermenta- 
tion results  in  the  production  of  gas  sufficient  to  fill  about  one-fifth  of  the 
tube.  Only  a  small  portion  of  the  gas  is  absorbed  by  sodium  hydroxid,  leaving 
behind  an  explosive  gas. 

Mannite. — The  medium  in  both  branches  of  the  tube  becomes  clouded;  gas 
is  not  formed.     Reaction  alkaline. 

Potato  water. — ^About  one-fifth  of  the  closed  arm  is  filled  with  gas.  Reaction 
acid. 

Agar  slant. — A  moderate,  grayish-white,  glistening,  nonspreading  growth  is 
formed  along  the  surface  inoculated  with  the  loop. 

Serum. — A  moderate,  gray,  glistening,  nonspreading  growth  takes  place  on 
the  inclined  surface.     No  liquefaction  occurs. 

Potato. — A  feeble,  grayish  growth  is  observed.  The  potato  becomes  slightly 
discolored. 

Millc. — No  coagulation  occurs,  and  no  gas  is  produced.     Reaction  alkaline. 

Litmus  milk. — The  medium  slowly  becomes  more  and  more  alkaline. 

Gelatin. — A  moderate,  white  growth  takes  place  along  the  line  of  inocula- 
tion.    On  the  surface  it  spreads  with  irregular  margin.     No  liquefaction  occurs. 

Acid  agar. — ^A  moderate  growth  appears. 

Indol. — Indol  is  produced. 

Nitrates. — Reduction  to  nitrites  (?). 

Bacillus  E. 

Occurrence. — Isolated  from  the  intestine  of  healthy  honey  bees. 

Oelatin  colonies. — The  colonies  are  lemon-yellow.  Surface  colonies  are  con- 
vex, smooth,  with  entire  margin ;  when  magnified '  they  are  finely  granular. 
Deep  colonies,  when  magnified,  are  lenticular,  finely  granular,  and  may  appear 
dark  green.    Liquefaction  takes  place  slowly. 

Morphology. — The  rods  are  short,  with  rounded  ends,  and  usually  occur  singly. 
The  bacilli  are  l.5/i  to  2/<  in  length  and  0.7ju.  in  thickness.  This  species  pos- 
sesses a  few  peritrichic  flagella. 

Motility. — ^The  bacteria  are  actively  motile. 

Spores. — No  spores  are  present. 

Gram.'s  stain. — They  stain  with  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — The  medium  becomes  uniformly  clouded  in  24  hours.  Later  a 
tough,  yellowish-white  membrane  is  formed,  which  sinks  upon  shaking.  The 
medium  is  very  viscid  in  old  cultures.     Reaction  alkaline. 

Glucose. — Growth  is  confined  to  the  open  bulb.  No  gas  formation  occurs. 
Reaction  slightly  acid. 

Lactose.— There  is  a  marked  mucous-like  appearance  in  the  medium.  Reac- 
tion alkaline. 

Saccharose. — Reaction  acid. 

Levulose. — Reaction  alkaline. 

Maltose. — Reaction  alkaline. 

Mannite. — Reaction  slightly  acid. 


BACTERIA    OF    THE    INTESTINE.  28 

Potato  iratcr.— Reaction  iilkaline. 

Agar  slant.— A  moderiite,  yellowish-gi-iiy,  iioiivisoid  growth  takes  place  on  the 
surface. 

Serum.— A  strong  growth  takes  place  and  the  inediuiii  is  liquefiea. 

Potato. — A  yellowish-gray,  noiuMscid  growth  is  observed  over  the  entire 
inclined  surface. 

ilfiifc.— Precipitation  of  casein  takes  place  with  very  slight  digestion  (V). 

Litvius  milh: — Precipitation  of  the  casein  occurs.     Kenctioii  alkaline. 

Oelatin.—A  white  growth  forms  along  the  line  of  inoculation,  which  becomes 
slowly  liquefied  from  above. 

Acid  agar. — A  moderate,  slightly  yellow  growth  is  observed. 

Indol. — None  demonstrated. 

Nitrates. — No  reduction  to  nitrites  occurs. 

Bacillus  subgastricus. 

Occurrence. — Isolated  from  the  Intestine  of  a  healthy  honey  bee. 

Qelatin  colony. — The  colon-like,  superficial  colonies  are  thin,  blue,  spreading, 
and  lobate-lobulate.  When  magnified  they  are  finely  granular,  with  brown 
center.     Deep  colonies  are  spherical  and  yellow. 

Mwphology. — Short  rods,  singly  and  in  pairs,  are  from  1.5/i  to  2.5/n  long  and 
from  0.6/1  to  0.8^  thick.     They  stain  uniformly  with  carbol-fuchsin. 

Motility. — Marked  whirling  motion  from  gelatin  cultures. 

Spores. — No  spores  could  be  demonstrated. 

Oram's  stain. — The  bacilli  are  decolorized  with  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — This  medium  becomes  clouded  in  24  hours.  A  slight  band  of 
growth  is  formed  on  the  glass  at  the  surface  of  the  liquid.  Later  a  feeble 
pellicle  Is  sometimes  formed.  Reaction  at  first  slightly  acid,  later  becomes 
alkaline. 

Glucose. — The  medium  in  both  branches  of  the  tube  becomes  clouded.  Gas 
is  readily  formed  until  about  one-fourth  of  the  closed  branch  is  filled.  The 
ratio  of  hydrogen  to  carbon  dioxid  is  2  to  l — that  is,  greater  than  1.  Reaction 
strongly  acid. 

Lactose. — Gas  formation  occurs.  About  one-sixth  of  the  tube  is  filled  with 
gas,  part  of  which  is  absorbed  by  sodium  hydroxid  and  another  part  is  explo- 
sive.    Reaction  acid. 

Saccharose. — This  sugar  is  fermented  to  the  point  of  formation  of  acid,  but 
no  gas  is  formed. 

Levulose. — This   sugar   splits   in   the  process  of  fermentation   to   form   acid 
and  gas,  the  gas  filling  about  one-sixth  of  the  tube.     A  pgrtion  of  the  gas  is 
absorbed  by  sodium  hydroxid,  the  remainder  being  explosive. 
I      Maltose. — Fermentation  takes  place  with  the  formation  of  acid.     No  gas  is 
produced. 

Mannitc. — One-fifth  of  the  closed  arm  is  filled  with  gas.  A  portion  of  the  gas 
is  absorbed  by  sodium  hydroxid  and  a  portion  is  explosive.     Reaction  acid. 

Potato  water. — Reaction  alkaline. 

Agar  slant. — A  moderate,  translucent,  gray,  nonviscid  and  glistening  growth 
spreads  slowly  from  the  surface  inoculated  with  the  loop. 

Serum. — A  moderate,  glistening  growth  appears  along  the  surface  inoculated. 
No  liquefaction  occurs. 

Potato. — A  grayish  growth  takes  place  on  the  sloped  surface. 

Milk. — Firm  coagulation  of  the  milk  takes  place  with  the  formation  of  a 
small  amount  of  clear  serum.    A  small  amount  of  gas  is  produced, 
9583— No.  14—06  m i 


24  THE    BACTERIA    OF    THE    APIAEY. 

Litmus  milk. — Reaction  .strongly  acid.     Coagulation  occurs  in  about  six  days. 

Gelatin. — Wtiite,  spherical  colonies  appear  along  the  line  of  inoculation.  The 
surface  growth  is  grayish  blue  and  spreading,  with  irregular  margin.  Slow 
liquefaction  takes  place,  beginning  usually  in  2  weeks. 

Acid  agar. — A  growth  takes  place. 

Indol. — None  could  be  demonstrated. 

Nitrates. — No  reduction  to  nitrites  occurs. 

Bacterium  mycoides. 

Occiirrrnec. — Isolated  from  the  intestine  of  a  healthy  honey  bee. 

Gelatin  coJouiea. — A  rapid  growth  of  root-like  colonies  appears  in  24  hours. 
In  macroscopic  appearance  it  somewhat  resembles  cotton  fibers ;  when  magni- 
fied these  appear  thick  and  somewhat  felted  in  the  center,  while  toward  the 
margin  they  are  beautifully  filamentous.  After  a  day  or  two  the  gelatin  begins 
to  liquefy. 

ilorpliologii. — The  rods  are  large,  scarcely  rounded  at  the  ends,  and  frequently 
in  chains.  They  measure  from  2.;V  to  5.5/n  long  and  1.5/(.  thick.  No  flagella 
have  bpen  demonstrated. 

Motility. — No  motility  could  be  demonstrated. 

Spores. — Spores  are  present. 

Gram's  stain. — The  bacteria  are  not  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Facultatively  anaerobic. 

Bouillon. — A  decided  fieecy  gi'owth  with  heavy,  cotton-like  sediment  occurs. 

Glucose. — No  gas  is  formed.     Reaction  acid. 

Lactose. — Reaction  acid. 

Saccharose. — Reaction  acid. 

Levulose. — Reaction  acid. 

Maltose. — Reaction  acid. 

Mannitc. — Reaction  acid. 

Potato  tcatcr. — Reaction  alkaline. 

Agar  slant. — A  luxuriant  growth  that  appears  root-like  takes  place  on  this 
medium.  This  growth  tends  to  extend  into  the  agar,  which  causes  it  to  adhere 
to  the  medium. 

Serum. — A  luxuriant  growth  is  formed,  accompanied  by  liquefaction. 

Potato.— A  thick,  gray,  moist  growth  is  found,  the  potato  not  being  discolored. 

3IUJc. — Coagulation  occurs  promptly,  with  formation  of  a  clear  serum. 

Litmus  milk. — The  color  is  discharged  in  48  hours. 

Ge/aM».— Hair-like  outgi-owths  occur  along  the  line  of  inoculation.  Lique- 
faction begins  at  the  surface  and  proceeds  along  the  needle  tract.  In  a  few  days 
the  entire  medium  is  liquefied. 

Indol. — No  indol  isproduced. 

^"itrates. — Reduction  to  nitrites  is  positive. 

Pseudomonas  fluorescens  liquefacierLs. 

Occurrence. — Isolated  from  the  Intestine  of  the  healthy  honey  bee. 

Gelatin  colonies.— Betore  liquefaction,  the  superficial  colonies,  when  magni- 
fied, are  finely  granular,  Avith  regular  margin;  deep  colonies  are  spherical, 
brown,  with  regular  margin.  Liquefaction  takes  place  rapidly.  The  surface 
of  liquefied  gelatin  is  covered  by  a  friable  membrane.  Later  the  liquefied  gela- 
tin takes  on  a  green  fluorescence. 

Morphology.— The  bacteria  are  short  rods,  varying  from  l/i  to  2^^  in  length 
and  from  0.5/t  to  0.7/1  in  thickness.  They  stain  uniformly  with  carbol-fuchsin 
and  are  motile  by  means  of  one  or  more  polar  fiagella. 


SAOCHAROMYCES   AND   FUNGI.  25 

Spores. — No  spores  could  be  demonstrated. 

Oram's  stain.— The  bacteria  do  not  take  Gram's  stain. 

Oxygen  rc(iitirciiients. — Aerobic 

Teiiiiii'ratiire  require luents. — Culture  must  be  grown  at  room  temperature. 

Bouillon. — Tbe  medium  becomes  clouded  in  48  hours,  forming  a  moderately 
tough  pellicle.  A  greenish-yellow  fluorescence  begins  at  the  surface,  which 
gradually  increases  until  the  entire  medium  talcos  on  that  appearance.  Rfcac- 
tion  alkaline. 

Gliico!se. — A  cloudiness  is  formed  in  the  open  arm,  but  the  closed  arm  is  clear. 
Reaction  ailjaline. 

Lactose. — Iteaction  allialine. 

Saccharose. — Reaction  allcaiine. 

Levulose. — Reaction  alkaline. 

Maltose. — Reaction  alkaline. 

ilannitc. — Reaction  alkaline. 

Agar  slant. — At  first  a  gray  friable  growth  is  formed  confined  to  the  surface 
inoculated,  which  later  takes  on  a  brown  hue.  Greenish-yellow  fluorescence  is 
observable  in  the  medium.  * 

Serum. — A  slow  liquefaction  occurs. 

Potato. — Very  scanty  growth  occurs  with  slight  discoloration. 

MiVc. — Rapid  liquefaction  of  the  casein  takes  place. 

Litmus  milk. — Rapid  liquefaction  of  the  casein  takes  place.     Reaction  alkaline. 

Gelatin. — Infundibuliform  liquefaction  takes  place  rapidly. 

Acid  agar. — No  growth  occurs. 

Indol. — No  indol  observed. 

Nitrates. — No  reduction  to  nitrites  occurs. 

SACCHAROMYCES  AND   rtTNGI. 

The  first  yeast  plant  described  below  is  of  very  frequent  occurrence 
in  the  intestine  of  the  normal  bee.  Saccharomyces  roseus  can^be  iso- 
lated from  the  comb.  A  large  number  of  common  fungi  wei-e  found 
in  the  flora  of  the  intestines  and  in  cultures  from  the  pollen  and 
combs. 

In  addition  to  the  above  the  third  Saccharomyces  here  described 
was  found  in  two  samples  of  brood  apparently  diseased,  which  could 
not  be  diagnosed  as  any  disease  commonly  known. 

Saccharomyces  P. 

Occurrence. — Very  common  in  the  intestine  of  healthy  honey  bees. 

Oelatin  colonies. — Colonies  form  slowly;  the  superficial  colonies  are  white, 
glistening,  convex,  capitate,  and  about  1  to  2  millimeters  In  diameter.  When 
magnified  they  are  finely  granular,  brownish  yellow,  with  entire  margin.  Deep 
colonies  are  finely  granular,  with  uniform  margin,  spherical  to  lenticular,  and 
brownish  green. 

Morphology. — The  cells  are  oval  and  on  agar  in  24  hours  .attain  their  full 
size  of  4.5/tt  in  length  and  3.5(1.  in  thickness.  They  stain  uniformly  with  carbol 
fuchsin. 

Motility. — The  yeast  is  not  motile. 

Oram's  stain. — The  cells  take  the  Gram's  stain. 

Oxygen  requirements. — Aerobic 


26  THE    BACTEEIA   OF    THE    APIAEY. 

Bouillon.— This  medium  remains  flour,  with  tbe  formation  of  a  friable  white 
sediment.     Reaction  neutral. 

Glucose.— The  closed  arm  remains  clear.    No  gas  is  formed.    Reaction  acid. 

Lactose. — Reaction  neutral. 

Saccharose. — Reaction  neutral. 

Levulose. — Reaction  neutral. 

Maltose. — Reaction  neutral. 

Mannite. — Reaction  neutral. 

Agar. — A  white,  nonspreading  growth  occurs. 

Serum.— ^yhite,  moderate,  nonviscid,  nonspreading  growth  occurs  along  the 
surface  inoculated.    No  liquefaction  takes  place. 

Potato  water. — Reaction  neutral. 

Potato. — Gray,  luxuriant,  fleshy  growth  occurs. 

Alilk. — No  change  occurs. 

Litmus  milk. — No  change  occurs. 

Oelatim. — A  moderate  growth  is  formed,  accompanied  by  no  liquefaction. 

Acid  agar. — Moderate  growth  takes  place. 

Indol. — Negative. 

Nitrates. — Reduced  to  nitrites. 

Saccharomyces  roseus. 

Occurrence. — Isolated  from  comb  of  healthy  hive. 

Gelatin  colonies. — Superficial  colonies' are  pink,  convex,  capitate,  with  lobate- 
lobulate  margin ;  when  magnified,  the  deep  colonies  are  irregular,  brownish- 
yellow,  and  finely  granular. 

Morpliology. — This  cell  is  oval,  attaining  about  6.5,14  in  length  and  3.5/i  in 
thickness.     The  cells  stain  uniformly. 

Motility. — No  motility  occurs. 

Grain's  stain. — The  cells  are  not  decolorized  by  Gram's  stain. 

Oxygen  requirements. — Aerobic. 

Bouillon. — This  medium  remains  clear,  forming  a  pink,  friable  sediment.  A 
pink  band  forms  at  the  surface  of  the  medium  and  adheres  to  the  glass. 

Glucose.— ^The  closed  arm  remains  clear.     No  gas  is  formed.     Reaction  acid. 

Lactose. — Reaction  neutral. 

Saccharose. — Reaction  neutral.         , 

Levulose. — Reaction  slightly  acid. 

Maltose. — Reaction  slightly  acid. 

Mannite. — Reaction  neutral. 

Potato  water. — Reaction  acid. 

Glucose  agar. — Luxuriant,  red  growth  forms  on  the  surface. 

Serum. — A  pink,  fleshy,  nonspreading  growth  is  formed,  accompanied  by  no 
liquefaction. 

Potato. — ^A  thick,  nonspreading,  red  growth  occurs. 

Millc. — No  apparent  change  takes  place.     The  milk  coagulates  on  boiling. 

■Litmus  milk. — Reaction  alkaline. 

Gelatin. — Moderate  pink  growth  is  formed,  accompanied  by  no  liquefaction. 

Aoid  agar. — Slow  growth  occurs. 

Indol. — Negative. 

Nitrates. — Reduction  to  nitrites  is  positive. 

Saccharomyces  G. 

Occurrence. — Found  in  the  dead  larvse  of  diseased  adult  bees. 
Morphology.— Thej   appear    in    hanging-drop   preparation   in    large   clusters 


SACCHABOMYCES   AND   PUNGT.  27 

stain  uniformly  witli  carbol-fuclisin  and  are  oval,  nearly  spherical,  attaining 
(he  length  of  i.ofi  and  thickness  of  3.5/1. 

Gram's  stain. — The  cells  are  not  decolorized  by  Gram's  stain. 

Oxygen  reqii iromeii ts. — Aerobic. 

BoHiUon. — A  slight,  friable,  white  sediment  is  formed,  with  a  clear  medium 
above.     Reaction  slightly  acid. 

Glucose. — The  medium  in  the  closed  ai-m  remains  practically  clear  and  about 
one-fifth  of  the  closed  arm  Is  filled  with  gas.     Reaction  acid. 

Lactose. — Reaction  neutral. 

Saccharose. — Reaction  neutral. 

Levulqse. — Reaction  slightly  acid. 

Maltose. — Reaction  slightly  acid. 

ilannitc. — Reaction  neutral. 

Potato  water. — Reaction  acid. 

Agar. — A  moderate,  white  growth  is  formed. 

Serum. — Very  feeble  growth  occurs,  accompanied  by  no  liquefaction. 

Potato. — A  luxuriant,  moist,  white  growth  occurs. 

Milk. — No  appreciable  change  taiies  place. 

Litmus  milk. — No  appreciable  change  takes  place. 

Gelatin. — A  moderate,  white  growth  occurs  along  needle  tract  and  on  the 
surface.     No  liquefaction  results. 

Acid  agar. — A  feeble  white  growth  occurs. 

Indol. — None  could  be  demonstrated. 

Nitrates. — No  reduction  to  nitrites  occurs. 

Glucose  agar. — A  thick,  white,  fleshy  growth  occurs. 


28 


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BIBLIOGRAPHY    TO    PART   I.  29 

SUMMARY  TO  PART  I. 

The  results  of  the  study  of  the  bacteria  found  normally  in  the 
apiary  may  be  briefly  summarized  as  follows : 

(1)  The  temperature  of  the  hive  approximates  that  of  warm- 
blooded animals. 

(2)  Upon  adult  bees  and  upon  the  comb  there  occurs  quite  con- 
stantly a  species  of  bacteria  which  we  refer  to  in  this  paper  as 
BaciUiis  A,  and  which,  it  is  believed,  is  the  organism  that  some 
workers  have  confused  with  Bacillus  alvei,  the  cause  of  European 
foul  brood  (p.  33). 

(3)  There  occurs  very  constantly  in  the  pollen  and  intestine  of 
adult  bees  a  species  here  referred  to  as  Bacillus  B. 

(4)  From  the  combs  Bacterium  cyaneus,  Saccharomyces  roseus, 
and  a  Micrococcus  referred  to  here  as  Micrococcus  C,  have  been  iso- 
lated and  studied. 

(5)  Honey  from  a  healthy  hive  is,  as  a  rule,  sterile. 

(6)  The  normal  larvae  are,  as  a  rule,  sterile. 

(7)  There  is  an  anaerobe  found  quite  constantly  in  the  intestine  of 
the  healthy  honey  bee.    It  is  referred  to  in  this  paper  as  Bacterium  D. 

(8)  From  the  intestine  there  have  been  isolated  and  studied  the 
following  micro-organisms:  Bacillus  cloacw,  Bacillus  coli  communis. 
Bacillus  cholercp  suis,  Bacillus  subgastricus.  Bacterium  mycoides, 
Pseudomonas  fluorescens  liquefaciens,  and  two  referred  to  as  Bacillus 
E,  and  Saccha/romyces  F.  Others  less  frequently  present  have  been 
isolated,  but  not  studied. 

(9)  In  two  samples  of  brood  with  unknown  disease  there  was 
found  a  species  of  yeast  plant  here  referred  to  as  Saccharomyces  G. 

BIBLIOGRAPHY  TO  PART  I. 

1.  Fuller,  Geo.  W.,  and  Johnson,  Geo.  A.     On  the  Differentiation  and  Distribu- 

tion of  Water  Bacteria.     <Jour.  of  Exper.  Medicine,  Vol.  IV,  p.  609,  1899. 

2.  Johnson,  0.  P.,  and  Mack,  W.  B.     A  Modification  of  Existing  Methods  for 

StainiQg  Flagella.     <American  Medicine,  Vol.  VII,  p.  754,  1904. 

3.  Peckham,  Adelaide  W.     The  influence  of  environment  upon  the  biological  pro- 

cesses of  the  various  members  of  the  colon  group  of  bacilli.     <Jour.  of  Exp. 
Medicine,  Vol.  II,  No.  5,  p.  549,  1897. 

4.  Ford,  Wm.  W.     The  Classification  and  Distribution  of  the  Intestinal  Bacteria  in 

Man.     <Studies  from  the  Royal  Victoria  Hospital,  Montreal,  Vol.  I,  No.  5, 
1903. 

5.  King,  W.  E.     A  Study  of  the  Bacterial  Flora  of  the  Intestinal  Mucosa  and  Eye 

of  the  Common  Fowl.     <Thesis,  Cornell  University  Library,  1905. 

6.  BuLLARD,  M.  J.     A  Study  of  the  Bacterial  Flora  of  the  Intestinal  Mucosa  of  the 

Normal  Babbit.     <American  Medicine,  Vol.  IV,  No.  14,  pp.  546-548,  1902. 

7.  Dyar,  Harrison  G.,  and  Kieth,  Simon  C,  jr.     Notes  on  the  Normal  Intestinal 

Bacilli  of'  the  Horse  and  of  other  Domesticated  Animals.     <Technological 
Quarterly,  Vol.  VI,  No.  3,  1893.  ' 


30  THE    BACTEEIA    OF    THE    APIABY. 

8.  MooEE,  V.  A.,  and  Wright,  F.  R.     Observations  of  Bacillus  coli  communis 

from  certain  species  of  Domesticated  Animals.     <American  Medicine,  Vol. 
Ill,  No.  13,  p.  504,  1902. 

9.  LoEBEE,  E.     A  Bacteriological  Study  of  the  Intestine  of  the  Fish.     <Am.  Bled., 

Vol.  VII,  No.  4,  p.  152,  1904. 

10.  Matzuschita,  T.     Bacteriologische  Diagnostik,  1902. 

11.  Chester,  F.  D.     A  Manual  of  Determinative  Bacteriology,  1901. 

PART  II.— THE  DISEASES  OF  BEES. 

The  bee  industry  in  this  country,  and  other  countries  as  well,  is 
suffering  large  losses  from  various  diseases  among  bees.  Those  which 
are  most  destructive  attack  the  brood  and  weaken  the  colony  by  kill- 
ing off  large  numbers  of  the  young  larvae  which  would  otherwise 
mature.  There  are  other  diseases  which  attack  the  adults  and  so 
decrease  the  strength  of  the  colony  in  that  way. 

In  order  to  combat  a  disease  to  the  best  advantage  it  is  clear  that 
its  cause  must  be  known,  as  well  as  the  means  by  which  the  infection 
is  transmitted  and  the  environmental  conditions  which  are  favorable 
for  the  breaking  out  of  an  epidemic.  The  brood  diseases  among  bees 
are  on  the  increase.  The  custom  of  selling  and  shipping  the  honey, 
which  is  now  carried  on  more  extensively  than  formerly,  the  manner 
in  which  the  products  of  the  apiary  are  handled,  and  the  absence  of 
a  general  knowledge  by  the  mass  of  bee  keepers  of  the  nature  of  the 
diseases  are  conditions  which  must  be  met  before  the  spread  of  these 
diseases  can  be  checked.  When  a  colony  is  diseased,  very  little  or  no 
profit  is  realized  from  it;  consequently  the  wealth  and  comfort  of  a 
very  large  number  of  people  are  greatly  endangered  by  the  existence 
of  bee  diseases.  This  suggests  the  importance,  from  an  economic 
standpoint,  of  a  thoro  knowledge  of  these  disorders. 

BRIEF   HISTORY. 

The  attention  of  investigators  has  been  attracted  by  these  diseased 
conditions,  not  only  from  the  economic  interests  attached  thereto, 
but  from  the  scientific  point  of  view  as  Avell.  The  writings  of  Aris- 
totle (12)  contain  an  account  of  certain  disorders  which  were  then 
prevalent  among  bees ;  at  that  time  it  was  thought  that  the  blight  of 
flowers  bore  a  relation  to  bee  diseases.  In  1769  Schirach  (13)  gave 
the  name  foul  brood  to  a  diseased  condition  of  the  brood  of  bees; 
he  attributed  the  cause  to  (a)  unwholesome  food,  and  (h)  the  placing 
of  the  larvae  with  head  inward  in  the  cell.  Leuckhart  (14)  thought 
the  cause  to  be  a  fungus,  related  to  the  cause  {Panhistophyton  ova- 
turn)  of  the  disease  of  the  silkworm.  Muhlfeld  (15),  in  1868, 
thought  the  trouble  to  be  of  two  kinds — infectious  and  noninfec- 
tious— and  that  the  cause  of  the  infectious  one  is  the  larva  of  a  para- 
sitic fly  (Ichneumon  apium  fneUificarium)  feeding  upon  the  larvse  of 
the  bee.     In  1868  Preuss   (16)   exprest  the  view  that  the  cause  of" 


BBIEF   HISXOBY  OF  BEE   DISEASES.  31 

foul  brood  is  a  fermenting  fungus  belonging  to  the  genus  Cryptococ- 
cus.  Geilen  (17),  in  1868,  thought  that  when  bees  alight  on  the 
remains  of  animal  bodies  the  putrefying  matter  thus  carried  with 
them  may  cause  foul  brood.  The  fermentation  of  bee  bread  was 
thought  by  Lambrecht  (18)  to  be  a  suiBcient  cause  of  the  disease; 
while  Hallier  (19)  thought  that  various  fungi  could  prpduce  the 
disorder.  On  the  contrary,  Cornallia  (20),  in  1870,  exprest  the 
opinion  that  a  fungus  {Cryptococcus  alvearis)  is  the  specific  cause  of 
the  trouble.  Fischer  (21),  in  1871,  supposed  that  a  predisposing 
factor  of  foul  brood  is  to  be  found  in  insufficient  nourishment.  In 
1874  Cohn  and  Eidem  received  from  Schonfeld  samples  of  foul  brood 
and,  upon  examination,  they  found  spores  and  rods.  In  1885  Chesh- 
ire and  Cheyne  (22)  determined  the  cause  and  named  the  germ 
Bacilhis  alvei.  Dickel  (23)  claimed  that  a  number  of  different 
species  might  be  the  cause  of  foul  brood.  In  1900  Harrison  (24) 
writes  on  foul  brood  and  Bacillus  alvei,  its  cause.  Doctor  Lambotte 
(25),  in  1902,  made  some  interesting  studies  concerning  the  relation 
of  Bacillus  alvei  and  Bacillus  mesentericus  vulgatus. 

Since  so  many  conflicting  views  have  been  held  as  to  the  cause  of 
foul  brood,  one  might  conclude  that  the  term  "  foul  brood  "  has  been 
applied  incorrectly  to  a  number  of  different  disorders.  In  the  light 
of  more  recent  work  this  supposition  is  strengthened. 

In  June,  1902,  the  author,  under  the  direction  of  Dr.  Veranus  A. 
Moore,  began  an  investigation  of  bee  diseases,  especially  as  they  ex- 
isted in  New  York  State.  There  were  recognized  at  that  time  by 
bee  inspectors  of  that  State  a  number  of  distinct  diseases  which 
attacked  the  brood.  Those  which  caused  the  greatest  loss  to  the 
apiarists  were  known  to  the  bee  experts  as  "  black  brood,"  "  foul 
brood,"  and  "  pickle  brood."  The  results  of  the  investigations  of 
1902  (26),  1903  (27),  and  1904  (28)  on  these  disorders,  and  on  palsy 
or  paralysis,  are  embodied  in  the  following  pages. 

THE  TERM  "  EOUL  BROOD  "  AS  HITHERTO  APPtlED. 

In  the  discussion  of  foul  brood  of  bees  it  must  be  remembered  that 
\mtil  recent  years  the  name  has  been  applied  to  what  is  now  known  to 
be  two  distinct  diseases. 

Schirach,  in  1769,  gave  the  name  foul  brood  to  a  diseased  condition 
in  the  brood  of  bees,  but  it  is  impossible  to  know  to  which  of  the  two 
he  referred.  It  may  be  that  both  diseases  existed  then  as  now  and 
that  he  did  not  observe  the  fact  that  the  two  were  different.  "We 
have  reason  to  think  that  there  are,  at  the  present  time  in  Europe, 
two  distinct  diseases  to  which  the  name  foul  brood  is  being  applied. 
It  is  definitely  known  that  such  is  the  case  in  America. 

It  becomes  necessary,  then,  to  have  two  names  to  designate  these 


32  THE   BACTERIA   OP    THE    APIAEY. 

two  diseased  conditions  in  the  brood  of  bees.  For  reasons  given  by 
Dr.  E.  F.  Phillips,  in  the  preface  to  this  paper,  it  has  been  considered 
advisable  to  retain  the  name  foul  brood  and  to  use  a  qualifying  word 
to  distinguish  the  two  diseases.  "European  foul  brood'  and 
'•American  foul  brood  "  are  the  names  by  which  these  two  diseased 
conditions  are  to  be  designated. 

In  1885  Cheyne  (22)  in  England  (Europe)  found  present  in  the 
decayed  larvae  suffering  from  a  diseased  condition  known  as  "  foul 
brood  "  a  new  bacillus,  which  he  named  Bacillus  alvei  and  to  which 
he  ascribed  the  cause  of  the  disease.  The  diseased  condition  which 
contains  Bacillus  alvei  is  to  be  called  "  European  foul  brood,"  because 
this  fact  was  first  observed  by  an  investigator  working  in  Europe 
(England).  In  1903  (27)  the  author  observed  that  there  was  con- 
stantly present  in  the  other  diseased  condition  known  as  "  foul  brood  " 
another  bacillus  which  was  new,  and  to  which  the  name  Bacillus 
larvce  is  given.  In  view  of  the  fact  that  Bacillus  larvm  was  con- 
stantly found  to  be  present  in  the  larvae  suffering  from  this  disorder 
in  the  brood  of  bees,  by  investigations  carried  on  in  New  York  State 
(America)  (27)  (28),  this  diseased  condition  is  to  be  called  "Ameri- 
can foul  brood."  From  a  scientific  standpoint  this  choice  of  names 
for  two  distinct  diseases  might  be  easily  criticized,  but  from  the 
standpoint  of  the  apiarist  the  selection  of  these  names  as  the  common 
ones  for  these  two  distinct  disorders  seemed  almost  necessary,  or  at 
least  advisable. 

EtTROPEAN  EOTJL  BROOD  (EOTJL  BROOD  OF  CHEYNE). 

The  first  scientific  investigation  of  this  disease  bacteriologically 
was  performed  by  Cheyne  in  1885  (22).  At  this  time  he  isolated  a 
new  bacillus  from  the  dead  larvae.  It  was  described  by  him  and 
given  the  name  Bacillus  alvei  (literally,  hive  bacillus) .  This  afforded, 
then,  a  means  for  a  positive  diagnosis  of  this  diseased  condition. 

Symptoms. 

The  symptoms  of  European  foul  brood,  as  given  by  Dr.  E.  F. 
Phillips  in  Circular  No.  79,  Bureau  of  Entomology,  are  as  follows: 

Adult  bees  in  infected  colonies  are  not  very  active,  but  do  succeed  in  cleaning 
ovit  some  of  the  dried  scales.  This  disease  attacks  larvse  earlier  than  does 
American  foul  brood,  and  a  comparatively  small  percentage  of  the  diseased 
brood  is  ever  capped ;  the  diseased  larvse  vphich  are  capped  over  have  sunken 
and  perforated  cappings.  The  larvse  when  first  attacked  shovy  a  small  yellow 
spot  on  the  body  near  the  head  and  move  uneasily  in  the  cell ;  when  death 
occurs  they  turn  yellow,  then  brown,  and  finally  almost  black.  Decaying  larvse 
which  have  died  of  this  disease  do  not  usually  stretch  out  in  a  long  thread 
when  a  small  stick  is  inserted  and  slowly  removed ;  occasionally  there  is  a  very 
slight  "  ropiness,"  but  this  is  never  very  marked.  The  thoroly  dried  larvse  form 
irregular  scales  which  are  not  strongly  adherent  to  the  lower  side  wall  of  the 


CONFUSION   EEGABDING   FOUL  BBOOD   IN   AMEHICA.  33 

cell.  There  is  very  little  odor  from  decaying  larvas  which  have  died  from 
this  disease,  and  when  an  odor  is  noticeable  it  is  not  the  "glue  pot"  odor  of 
American  foul  brood,  but  more  nearly  resembles  that  of  soured  dead  brood. 
This  disease  attacks  drone  and  queen  Inrvic  very  soon  after  the  colony  is 
infected.  It  is,  as  a  rule,  much  more  infectious  than  American  foul  brood  and 
spreads  more  rapidly.  On  the  other  hand,  it  sometimes  happens  that  the 
disease  will  disappear  of  its  own  accord,  a  thing  which  the  author  never  knew 
to  occur  In  a  genuine  case  of  American  foul  brood.  European  foul  brood  is 
most  destructive  during  the  spring  and  early  summer,  often  almost  disap- 
pearing in  late  summer  and  autumn. 

Confusion  Begarding  Foul  Brood  in  America. 

Prof.  J.  J.  Mackenzie  in  1882  made  what  seems  to  have  been  a 
short  study  of  a  bee  disease  as  it  appeared  in  Ontario,  Canada,  which 
was  known  to  the  apiarists  of  that  Province  as  foul  brood.  He  says 
very  little  of  the  character  of  the  species  of  bacteria  with  which  he 
was  working,  but  he  supposed  that  they  were  Bacillus  alvei  of 
Cheyne.  The  author  has  examined  samples  of  brood  from  Ontario 
which  have  what,  in  the  opinion  of  bee  experts,  is  the  most  prevalent 
disease,  and  has  not  found  Bacillus  alvei  present  in  any  one.  The 
bacteriological  findings  and  the  experience  of  bee-disease  experts 
show  that  American  foul  brood  is  the  prevalent  disease  in  that  Prov- 
ince. As  the  bee  experts  see  the  disease  in  the  light  of  recent  studies, 
there  is  no  authentic  report  of  which  we  are  aware  that  European 
foul  brood  exists  in  Ontario.  We  can  safely  say,  then,  that  Bacillus 
alvei  can  not  be  isolated  from  larvae  taken  from  the  prevalent  disease 
in  the  above-named  Province.  No  difficulty  is  exprest  on  the  part 
of  Professor  Mackenzie  in  the  isolation  of  Bacillus  alvei  from  any 
sample.  The  author  is  inclined  to  think,  therefore,  that  this  investi- 
gator was  in  error  as  to  the  identity  of  his  culture,  and  therefore  his 
conclusion  can  have  little  weight. 

The  foul  brood  of  bees  received  some  attention  also  from  Prof. 
F.  C.  Harrison,  of  Ontario.  In  a  paper  of  some  length  he  gives  a 
description  of  a  species  of  bacteria  which  he  identified  as  Bacillus 
alvei.  The  description  which  he  gives  and  the  accompanying  photo- 
micrographs (another  plate  which  was  given  being  after  Cheyne 
and  correct  for  Bacillus  alvei)  might  easily  be  that  of  a  member  of 
a  group  represented  by  and  described  as  Bacillus  "A"  in  Part  I  of 
this  paper.  He  also  says  that  he  has  isolated  Bacillus  alvei  from 
diseased  larvae  from  13  States  of  the  Union,  ranging  from  New 
York  to  California  and  from  Michigan  to  Florida.  European  foul 
brood  has  had  a  very  limited  geographical  distribution,  spreading 
only  recently  from  New  York  to  adjoining  States.  In  Professor 
Harrison's  work,  too,  there  seems  to  have  been  no  difficulty  in  iso- 
lating Bacillus  alvei  from  diseased  brood  diagnosed  by  bee  inspectors 


34  THE   BACTEEIA   OP    THE   APIAKY. 

as  foul  brood  thruout  the  United  States  and  Canada.  In  the  experi- 
ence of  the  author  it  has  not  been  possible  to  obtain  Bacillus  alvei 
from  diseased  brood  which  the  inspectors  in  most  of  the  States  and 
in  Canada  have  been  calling  foul  brood.  For  the  above  reasons  the 
author  believes  that  Harrison,  too,  has  made  a  serious  error  in  the 
identity  of  his  culture  and  therefore  was  not  working  with  Bacillus 
alvei  at  all.  The  author  considers  himself  unfortunate  in  that  he 
was  unable  to  obtain  a  culture  of  Bacillus  alvei  for  study  and  identi- 
fication from  Professor  Harrison. 

■  Dr.  William  E.  Howard,  of  Fort  Worth,  Tex.,  also  studied  foul 
brood  somewhat,  and  gave  a  description  of  Bacillus  alvei  as  he  found 
it.  From  his  description  and  from  the  fact  that  he,  too,  worked  with 
a  diseased  condition  which  does  not  contain  Bacillus  alvei,  and  ex- 
prest  no  difficulty  in  obtaining  his  cultures  from  any  samples,  the 
author  believes  that  this  investigator  made  an  error  in  the  identifica- 
tion of  the  culture  with  which  he  was  working. 

\  Some  writers — Cowan,  Bertrand,  and  others— have  attempted  the 
positive  diagnosis  of  foul  brood  with  the  microscope  alone  from  a 
preparation  made  direct  from  the  dead  larvae.  If  the  reader  will 
remember  that  with  the  microscope  alone  it  would  be  impossible  to 
distinguish  between  Bacillus  larvce  and  Bacillus  alvei,  the  verdict  of 
these  men  can  have  no  weight.  As  shown  later  in  this  paper  under 
black  brood  (pp.  43^4),  the  Doctor  Howard,  of  Fort  Worth,  Tex.,  re- 
ferred to  above,  made  an  error  in  supposing  that  the  European  foul 
brood  was  a  new  disease  and  naming  it  "  New  York  bee  disease  "  or 
"  black  brood." 

'  It  is  very  unfortunate  for  the  apiarist  that  these  men  should  have 
fallen  into  error  as  to  the  identity  of  their  culture  with  Bacillus  alvei, 
as  it  has  caused  great  confusion  in  the  names  of  bee  diseases.  This 
confusion  in  the  identity  of  cultures  may  be  excused  to  a  certain  ex- 
lent  by  the  fact  that  European  foul  brood  did  not  appear  in  this 
country,  or  at  least  did  not  attract  much  attention,  until  after  Mac- 
kenzie, Harrison,  and  William  R.  Howard  had  done  their  work  on 
foul  brood. 

The  Present  Investigation. 

When  the  author's  investigations  were  begun  in  1902  there  were 
two  especially  troublesome  diseases  in  this  country,  which  were  then 
known  to  the  bee  experts  as  "  black  brood  "  and  "  foul  brood." 

The  following  summary  and  table  shows  the  results  of  the  exami- 
nation of  a  number  of  samples  of  diseased  brood  from  different 
apiaries,  sent  by  the  New  York  State  bee  inspectors  during  the  sum- 
mer of  the  year  1902 : 


THE   PRESENT   INVESTIGATION   OF   EUROPEAN   TOUL   BROOD.       35 

Tahle  showing   the  results   of  examinations   of  European   foul   irooA.     (The 
samples  were  called  "  hldck  hrood  "  by  the  apiarists  at  that  time. ) 


Brood  sent  by— 

Date. 

Bacteriological  findings. 

W.D.Wright 

W.D.Wright 

N.D.West 

N.D.West 

N.D.West 

June  12 

N.D.West 

June  12  . . .               .            ... 

Bodllus  ctlvei. 

N.D.West 

June  12 

Bacillus  oXvei. 

N.D.West 

N.D.West 

W.D.Wright 

Oct.  8 

It  can  be  seen  clearly  from  the  above  table  that  the  diseased  condi- 
tion which  the  apiarists  were  calling  "  black  brood  "  is  really  the 
disease  "  foul  brood  "  of  Cheshire  and  Cheyne,  because  of  the  con- 
stant presence  of  Bacillus  alvei. 

The  work  upon  European  foul  brood  was  continued  during'  the 
year  1903.  The  following  table  gives  the  results  of  the  examination 
of  specimens  received  during  that  year.  The  samples  were  taken 
from  different  apiaries. 

Table  giving  a  summary  of  the  examination  of  specimens  of  European  foul  brood 

("black  brood"). 


Brood  sent  by- 


Date. 


Sources  of  brood  in  New  York. 


Bacteriological 
findings. 


W.  D.  Wright 
W.  D.  Wright 
N.D.West... 
N.D.West... 
N.D.West... 
N.  D.  West. . . 
N.D.West... 
N.  D.  West... 
N.D.West... 
N.D.West... 
N.D.West... 
N.D.West... 
N.D.West..., 
N.  D.West.... 
N.D.West.... 
N.D.West..., 
N.D.West... 
N.D.West..., 
N.D.West... 
N.D.West... 
N.D.West..., 
N.D.West... 
N.D.West..., 
N.  D.  West. . . , 
N.D.West..., 
N.D.West..., 


May  1 
May  1 
June  25 
June  29 
June  29 
June  29 
June  29 
July  6 
July  6 
July  6 
July  10 
July  10 
July  10 
July  10 
July  15 
July  15 
July  22 
,Tuly  22 
July  22 
July  30 
July  30 
July  30 
July  30 
July  30 
Aug.  20 
Aug.  20 


Columbia  County 

Albany  County 

Schoharie  County 

,  Schoharie  County 

Schoharie  County 

Schoharie  County 

Schoharie  County 

Schoharie  County 

Schoharie  County 

Schoharie  County 

Montgomery  County . . . 

Schoharie  County 

Schoharie  County  j 

Schoharie  County 

Schoharie  County 

Montgomery  County . . . 

Schoharie  County #. 

Schoharie  County 

Schoharie  County 

Schoharie  County 

Schoharie  County 

Greene  County 

Albany  County 

Greene  County 

Greene  County 

Greene  County 


BadUus 
BaHUus 
Bae-iMiM 
BacUlue 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
Badllus 
BadUus 
Bacillus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 
BadUus 


dl/vei. 
alvei. 
alvd. 
alvd. 
alvd. 
alvd. 
atad. 
alvei. 
alvd. 
alvei. 
alniei. 
alvei. 
alvd. 
alvei. 
alvd. 
alvei. 
alvd. 
alvei. 
alvd. 
alvei. 


alvd. 
alvd. 
alvd. 
alvei. 


The  above  table  shows  that  Bacilliis  alvei  was  present  in  each  speci- 
men of  European  foul  brood  received.  Frequently  pure  cultures 
of  this  species  were  obtained  from  dead  larvae,  but  with  it  sometimes 
were  associated  other  rod-shaped  bacteria  of  different  species. 

In  1904  the  work  upon  bee  diseases  was  confined  principally  to  the 
diagnosis  of  the  diseased  brood  sent  in  and  a  further  study  of  the 
organisms  found.    Bacillus  alvei  was  found  in  a  large  number  of 


36  THE   BACTERIA   OF    THE    APIARY. 

samples  received  from  New  York  State  and  in  some  received  from 
Pennsylvania. 

Bacillus  alvei. 

Occurrence— This  bacillus  was  fouud  in  all  samples  of  European  foul  brood 
examined. 

Morphotoffy.— The  bacillus  is  a  motile,  rod-shaped  organism,  occurring  singly 
and  in  pairs,  and  varying  wben  taken  from  the  surface  of  agar  from  1.2» 
to  3.9/1  in  length,  and  from  0.5/i  to  0.7/i  in  width.  Involution  forms  are  some- 
times present.  Spores  are  produced -and  occupy  an  intermediate  position  in 
the  organism.  They  are  oval  and  vary  from  1.5/i  to  2/x  in  length  and  from 
0.7m  to  l/x  in  breadth;  they  exhibit  polar  germination.  The  few  flagella  are 
arranged  peritrichic. 

Oxygen  requirements. — This  bacillus  is  a  facultative  anaerobe  which  grows  at 
room  temperature,  but  better  at  37°  C. 

Bouillon. — The  medium  becomes  uniformly  clouded  in  24  hours;  later  it 
shows  a  tendency  to  clear  by  a  settling  of  the  organisms.  A  somewhat  viscid 
sediment  is  thus  formed  in  the  bottom  of  the  tube.  In  older  cultures  a 
slightly  gray  band  of  growth  adheres  to  the  glass  at  the  surface  of  the  me- 
dium. The  acidity  is  at  first  slightly  increased,  and  a  pellicle  is  sometimes 
formed. 

Glucose. — The  medium  in  both  branches  of  the  fermentation  tube  becomes 
uniformly  clouded.     Gas  is  not  formed.     Keaction  acid. 

Lactose. — The  medium  becomes  uniformly  clouded  in  both  branches  of  the 
fermentation  tube,  but  the  cloudiness  is  not  so  marked  as  when  glucose  is  used. 
The  acidity  is  slightly  increased,  as  shown  by  phenolphthalein.  No  gas  is 
formed. 

Saccharose. — The  bouillon  in  this  case  also  becomes  clouded  in  both  arms. 
A  heavier  growth  is  observed  than  when  lactose  is  used,  but  less  than  when 
glucose  is  used.     Acidity  is  slightly  increased.     Gas  is  not  formed. 

Agar  plates. — Small,  grayish,  circular  colonies  form  in  24  hours.  When  many 
are  on  the  plate,  they  do  not  exceed  2  millimeters  in  diameter.  Under  low 
magnification  they  appear  granular,  with  no  definite  margin.  When  fewer 
colonies  are  on  the  plate,  the  granular  center  of  the  colony  is  surrounded  by 
numerous  smaller  but  similar  growths.  The  organism  has  a  tendency  to  grow 
into  the  medium  rather  than  upon  the  surface.  Sometimes,  however,  when 
there  are  but  a  few  colonies  on  the  plate  a  thin,  transparent  growth  spreads 
ragidly  over  the  surface.     Later  it  takes  on  a  brown  tint. 

Agar  slant. — A  gray  layer  spreads  over  the  surface  in  24  hours,  which  later 
takes  on  a  slightly  brown  color.  A  strong,  slightly  viscid  growth  occurs  in  the 
condensation  water. 

Add  agar. — Growth  takes  place  with  the  reactions  varying  from  neutral  to 
-|-3.5  to  phenolphthalein. 

Serum. — A  slightly  raised  growth  which  is  confined  quite  closely  to  the  line 
of  inoculation  appears  on  the  surface  of  solidified  serum. 

Potato. — On  this  medium  the  bacillus  grows  rather  slowly  at  first,  but  after 
3  or  4  days  a  milky  growth  is  observed,  which  increases  until  a  luxuriant  growth 
is  formed,  which  varies  from  a  lemon-yellow  to  a  gray  color,  and  which  later 
becomes  tinted  with  brown. 

Milh. — Acidity  is  increased  after  inoculation.  Coagulation  ♦usually  takes 
place  after  the  third  day. 

Litmus  milk. — Much  of  the  blue  color  is  discharged,  leaving  the  coagulated 
milk  of  a  light  brown. 


INOCULATION   EXPEEIMENTS   WITH   BACILLUS  ALVEI.  37 

Oelatin  colonies.— GelnUn  is  a  medium  in  which  it  develops  slowly.  The  col- 
ony becomes  very  irregular  in  outline,  owing  to  thread-iilte  outgrowths  which 
take  place  In  curves  from  its  border.  Growth  Is  better  when  5  per  cent  glycerin 
is  added.  From  the  small,  white,  spherical  colonies  which  form  along  the  line 
of  puncture  gray,  thread-like  growths  shoot  out  thru  the  medium.  In  about  2 
months  the  gelatin  is  changed  to  a  thick  liquid,  holding  gray  flocculent  masses 
of  organisms  which  gradually  settle,  forming  a  strong,  slightly  viscid  sediment. 

Indol. — In  old  cultures  a  decided  Indol  reaction  is  obtained. 

Power  to  resist  disinfectants. — Preliminary  observations  give  the  following 
results:  The  spore  form  resists  drying  for  a  considerable  time.  Spores  which 
have  been  drying  for  1  year  germinate  promptly  when  introduced  into  bouillon. 
The  vegetative  form:  One  per  cent  carbolic  acid  kills  in  10  minutes;  3  per 
cent  carbolic  acid  kills  in  2  minutes;  mercuric  chlorid  solution,  1  to  1,000. 
kills  in  1  minute ;  mercuric  chlorid  solution,  1  to  2,000,  kills  in  2  minutes. 

8pm-e  form. — Mercuric  chlorid,  1  to  1,000,  kills  in  30  minutes. 

Pathogenesis  in  vertelirates. — Inoculations  Into  guinea  pigs  and  frogs  have 
not  proven  this  organism  to  be  pathogenic  to  these  animals. 

Inoculation  Experiments. 

That  part  of  the  investigation  which  involves  the  producing  of  the 
disease  experimentally  by  inoculating  with  pure  cultures  of  the 
organism  is  usually  the  most  difficult  one.  Very  rarely  indeed  is  one 
able  to  produce  the  disease  with  symptoms  closely  simulating  those 
found  in  nature.  The  experimental  production  of  a  disease  involves 
many  variable  factors,  such  as  attenuation  of  the  organism,  methods 
of  inoculation,  resistance  of  the  host,  and  the  immediate  environment. 

On  August  4,  1902,  we  inoculated  a  hive  containing  nothing  but 
healthy  brood,  free  from  bacteria,  by  feeding  with  sirup  (sugar  and 
water  in  equal  parts)  to  which  was  added  the  growth  from  the  sur- 
face of  the  plate  cultures  containing  spores  and  bouillon  cultures  of 
Bacillus  alvei.  Similar  feedings  were  given  to  these  bees  from  one 
to  three  times  a  week  until  September  28,  but  symptoms  of  foul 
brood  did  not  develop.  On  August  6  cultures  were  made  from  a 
few  of  the  hive  larvae.    They  were  found  to  contain  the  bacilli. 

Inoculation  experiments  were  again  made  in  1903.  Because  of  a 
failure  to  produce  a  diseased  condition  with  cultures  of  Bacillus  alvei 
in  the  experiment  of  1902,  the  variable  factors  above  mentioned  were 
carefully  considered  in  the  experiment  of  this  year.  The  inocula- 
tions were  made  when  climatic  conditions  were  such  as  seemed  to 
favor  the  ravages  of  the  disease  in  the  apiaries;  namely,  low  tem- 
perature, dampness,  and  cloudiness.  A  colony  of  black  bees  was 
used,  as  they  were  almost  universally  considered  more  susceptible. 
Cultures  of  Bacillus  alvei  were  freshly  isolated  from  foul-brood 
specimens  and  kept  in  stock  on  bee-larvae  agar  (described  under 
American  foul  brood,  pp.  41-42).  All  cultures  were  incubated  at  34° 
C,  which  temperature  is  observed  to  be  slightly  below  that  of  the 
hive.    The  spore  form  of  Bacillus  alvei  was  used. 

Inoculations  were  made  in  different  ways.    A  diseased  condition 


38  THE   BACTBEIA   OF    THE   APIAKY. 

appeared  in  the  hive  when  the  following  method  was  used :  The  agar 
from  plates  on  which  the  culture  was  grown  was  finely  crusht  and 
mixt  with  sterile  sirup.  A  jelly  glass,  in  the  lid  of  which  holes  had 
been  punctured,  was  filled  and  inverted  on  strips  of  wood  inside  the 
hive.  In  this  way  the  bees  take  up  the  culture  with  the  sirup  as 
rapidly  as  it  flows  out  of  the  glasses.  A  colony  having  brood  free 
from  Bacillus  alvei  was  fed  in  the  above  manner  on  August  8,  with 
repeated  feedings  on  the  9th,  10th,  12th,  13th,  15th,  and  I7th.  On 
the  12th  Bacillus  alvei  was  found  in  the  living  larvse  and  on  the  17th 
many  larvae  were  dead  under  cappings  and  some  were  dead  which 
were  not  capped;  all  were  soft  and  of  a  dull  color.  Many  of  the 
capped  cells  containing  dead  larvse  had  their  capping  freshly  punc- 
tured. Bacillus  alvei  was  usually  obtained  from  these  larvse  in  pure 
cultures.  In  no  cell  examined  where  the  cell  capping  was  punctured 
did  we  find  gas-producing  organisms;  this  fact  would  suggest  the 
conclusion  that  these  punctures  which  are  found  in  the  capping  in 
foul  brood  are  made  by  the  bees  and  not  by  gas-producing  organ- 
isms. During  this  series  of  inoculations  the  days  were  quite  cool 
and  sometimes  cloudy  and  damp.  On  the  20th  of  August  the  tem- 
perature was  much  higher,  the  bees  were  more  active,  and  much  of 
the  dead  brood  had  been  cleaned  out  by  the  bees.  On  the  22d  no 
dead  brood  was  noticed  by  casually  looking  over  the  brood  nest.  On 
the  24th  of  the  same  month  a  careful  search  was  made  by  uncapping 
all  the  cells  of  one  brood  frame,  and  12  decaying  larvaj  of  a  brown 
color  were  found.  At  this  time  the  larvse  were  not  viscid.  All  the 
remaining  dead  brood  had  evidently  been  cleaned  out  by  the  bees. 
A  condition  similar  to  this,  where  only  a  few  scattered  about  in  the 
brood  nests  contain  dead  larvse,  occurs  sometimes  in  affected  apiaries. 
Two  other  colonies  which  were  near  by  but  not  inoculated  gave  no 
signs  of  disease. 

Mr.  N.  D.  West  reports  that  the  climatic  conditions  seem  to  have 
something  to  do  with  the  extent  of  the  ravages  of  European  foul 
brood,  since  the  disease  is  much  more  destructive  in  cool,  damp 
weather.  This  seems  to  be  a  very  plausible  idea.  The  larvaj  at  such 
times  may  receive  more  infected  food  than  when  fresh  is  being 
rapidly  gathered;  the  resistance  of  the  body  of  the  larvse  to  the 
growth  of  Bacillus  alvei  is  at  such  times  much  lessened;  and  the 
adult  bees  being  less  active,  the  dead  larvse  are  not  cleaned  out  of  the 
combs  so  rapidly.  The  results  of  the  experimental  work  seem  to 
confirm  this  theory. 

Distribution  of  Bacillus  alvei  in  Infected  Hives. 

In  order  to  combat  this  disease  it  is  well  to  know  where  these  patho- 
genic bacteria  may  be  found.  The  following  is  a  summary  of  the 
results  of  the  investigation  along  this  line : 


EXPERIMENTS   WITH   FOBMALDEHYDE   GAS.  39 

1.  The  greatest  number  of  infecting  germs  are  found  in  the  bodies 
of  dead  larvfe. 

2.  The  pollen  stored  in  the  cells  of  the  foul-brood  combs  contains 
many  of  these  infecting  organisms. 

3.  The  honey  stored  in  brood  combs  infected  with  this  disease  has 
been  found  to  contain  Bacillus  alvei  in  small  numbers. 

4.  The  surface  of  the  combs,  frames,  and  hives  may  be  contami- 
nated. 

5.  The  wings,  legs,  head,  thorax,  abdomen,  and  intestinal  contents 
of  adult  bees  are  found  to  be  contaminated  with  Bacillus  alvei. 

6.  Cheshire  (29),  Mackenzie  (30),  and  others  have  found  Bacillus 
alvei  in  the  ovary  of  the  queen.  This  has  suggested  a  means  of  in- 
fection. From  a  bacteriological  examination  of  queens  from  three 
badly  infected  hives  we  were  able  to  isolate  Bacillus  alvei  in  small 
numbers  in  two  cases.  Since  a  very  large  number  of  this  species  of 
bacteria  may  be  found  in  the  intestinal  tract  and  upon  all  parts  of 
the  body,  it  is  very  probable  that  such  findings  are  the  results  of  con- 
tamination in  making  cultures  and  have  no  special  significance. 

Experiments  with.  Formaldehyde  Gas. 

Within  the  last  few  years  several  articles  have  appeared  in  the  bee 
journals  entertaining  great  hopes  that  a  cure  for  foul  brood  has  been 
found  in  the  use  of  formaldehyde  gas.  The  methods  described  for  its 
use  have  been  tested  by  the  apiarists  and  bee  experts  in  New  York 
State,  with  the  result  that  the  disease  sometimes  breaks  out  anew  In 
colonies  so  treated. 

In  order  to  test  the  value  of  formaldehyde  gas  as  a  disinfectant 
when  used  in  foul-brood  combs  a  number  of  experiments  were  made 
in  the  laboratory.  A  common  frame  hive  was  first  used,  in  which 
were  placed  specimens  of  foul  brood.  The  hive  was  charged  with 
gas  by  heating  formalin  in  a  closed  vessel  which  was  in  communica- 
tion with  the  hive;  15  c.  c.  was  used  each  time  and  evaporated  to 
dryness.  The  charging  of  the  hive  with  gas  was  repeated  in  this 
way  at  the  end  of  2,  4,  6,  and  20  hours.  Before  each  charging  and 
at  the  end  of  24  hours  after  the  first  application  of  gas,  cultures  were 
made.  Of  all  the  tubes  inoculated  90  per  cent  showed  Bacillus  alvei 
to  be  present.  There  was  no  decrease  in  the  number  of  tubes  in 
which  Bacillus  alvei  appeared  following  the  several  applications  of 
formaldehyde  gas. 

The  examination  of  specimens  of  foul  brood  which  had  been  treated 
with  the  gas  by  an  apiarist  gave  the  following  results : 

Thirty  tubes  which  were  inoculated  from  larvae,  capped  and  un- 
capped, showed  the  presence  of  Bacillus  alvei  in  21. 

Thirty  tubes  which  were  inoculated  with  pollen  in  cells  gave 
Bacillus  alvei  in  28. 


40  THE   BACTEEIA   OF    THE    APIAEY. 

Four  series  of  agar  plates  showed  apparently  no  diminution  in 
the  number  of  bacteria  present. 

Further  experiments  were  made  by  using  Novy's  anaerobic  jar 
(a  very  tight  chamber)  as  a  chamber  in  which  to  put  the  diseased 
brood  combs  and  cultures.  This  vessel  will  retain  the  gas  much  more 
perfectly  than  the  devices  made  for  practical  use  in  the  apiary. 
Treatment  of  brood  in  this  jar  by  recharging  with  the  gas  resulted 
usually  in  complete  disinfection  after  2  days.  Agar  plates  con- 
taining spores  and  cheese  cloth  on  which  cultures  were  spread  and 
dried  were  disinfected  after  a  short  length  of  time  by  the  applica- 
tion of  formaldehyde  gas. 

From  the  experiments  made  the  conclusion  can  be  drawn  that 
formaldehyde  gas  is  a  good  disinfectant,  but  that  it  penetrates  very 
slowly  and  that  24  hours'  application  of  the  gas  to  the  combs,  as 
usually  applied,  is  not  sufficient  to  kill  all  the  spores  in  the  decayed 
larvae  (27). 

AMERICAN  POUL  BROOD. 

The  diseased  condition  which  we  shall  call  American  foul  brdod 
and  the  micro-organism  found  constantly  present  in  the  diseased  and 
dead  larvae,  which  we  shall  call  Bacillus  larvae,  were,  for  convenience, 
referred  to,  respectively,  as  "  X  Brood  "  and  Bacillus  "X "  in  a 
former  report  (27).  This  disease  has  been  called  "  foul  brood  "  by 
many  bee  keepers  in  this  country  and  in  other  countries  as  well.  It 
is  the  diseased  condition  with  which  Mackenzie,  Harrison,  and 
William  R.  Howard  were  working  largely,  if  not  altogether,  in  their 
investigations  of  foul  brood.  The  disorder  is,  as  a  rule,  dreaded  less 
than  European  foul  brood  by  the  apiarist,  yet  in  the  aggregate  the 
bee  industry  suffers  enormous  losses  from  the  trouble.  The  general 
character  of  the  diseased  brood  is  so  much  like  that  of  foul  brood 
that  the  two  may  be  easily  confused  by  those  unfamiliar  with  the 
variety  of  appearances  which  one  finds  in  each  disease  and  a  few 
characters  which  are  differential.  Therefore  it  is  not  strange  that 
the  mistaken  diagnosis  should  be  made  from  the  symptoms  mani- 
fested by  these  two  diseases.  When,  however,  European  foul  brood 
and  American  foul  brood  are  subjected  to  a  bacteriological  exami- 
nation, the  diagnosis  is  easy.  Experts  when  comparing  specimens 
of  the  two  diseased  conditions  are  able  to  see  a  difference  in  the 
gross  appearance. 

Symptoms. 

The  symptoms  are  given  by  Dr.  E.  F.  Phillips  in  Circular  No.  79, 
Bureau  of  Entomology,  as  follows : 

The  adult  bees  of  an  infected  colony  are  usually  rather  inactive  and  do  little 
toward  cleaning  out  infected  material.  When  the  larvas  are  first  affected  they 
turn  to  a  light  chocolate  color,  and  in  the  advanced  stages  of  decay  they  become 


THE   PEESENT   INVESTIGATION   OF   AMERICAN   FOUL   BKOOD.       41 

darker,  resembling  roasted  coffee  in  color.  Usually  the  larvse  are  attacked  at 
about  the  time  of  capping,  and  most  of  the  cells  containing  infected  larvae  are 
capped.  As  decay  proceeds  these  cuppings  become  sunken  and  perforated,  and, 
as  the  healthy  brood  emerges,  the  comb  shows  the  scattered  cells  containing 
larv®  which  have  died  of  disease  still  capped.  Tlie  most  noticeable  charac- 
teristic of  this  infection  is  the  fact  that  when  a  small  stick  is  inserted  In  a 
larva  which  has  died  of  the  disease,  and  slowly  removed,  the  broken-down 
tissues  adhere  to  it  and  will  often  stretch  out  for  several  inches  before  break- 
ing. When  the  larva  dries  it  forms  a  tightly  adhering  scale  of  very  dark 
brown  color,  which  can  best  be  observed  when  the  comb  is  held  so  that  a  bright 
light  strikes  the  lower  side  wall.  Decaying  larvas  which  have  died  of  this  disease 
have  a  very  characteristic  odor,  which  resembles  a  poor  quality  of  glue.  This 
disease  seldom  attacks  drone  or  queen  larvse.  It  appears  to  be  much  more 
virulent  in  the  western  part  of  the  United  States  than  in  the  East. 

A  microscopic  preparation  from  the  diseased,  but  not  dead  larvae, 
or  from  larvae  recently  dead,  at  first  shows  a  iew  comparatively  long 
slender  rods;  later  these  increase  rapidly  in  number,  and  spores  also 
are  seen.  In  the  later  stages  of  decay  in  the  ropy  mass  and  the  dried 
scales  spores  only  are  found;  these  occur  in  very  large  numbers. 
When  this  investigation  was  begun,  in  1902,  it  was  observed  (26) 
that  in  the  dried  dead  larvae  there  are  very  large  numbers  of  spores, 
but  these,  when  inoculated  into  the  media  commonly  used  in  the 
laboratory,  fail  to  grow.  The  cultures  were  sterile,  except  for  an  oc- 
casional contamination. 

The  Present  Investigation. 

The  following  samples  from  different  sources  were  examined  in 
1902: 

Results  of  examination  of  specimens  of  American  foul  irood  diagnosed  by  the 
experts  at  that  time  simply  as  "  foul  hrood." 


Brood  sent  by— 

Date. 

Source. 

Bacteriological 
findings. 

June  12 

Sept.  19 

Oct.)9 

Nov.ll 

No  growth. 

2  unjjdenjtified  ba- 

W. D.  Wright 

Wisconsin           

W.  D.  Wiight 

Canacta .- 

cilli. 
No  groMjHi. 
No  growth;  4  sam- 

W.  D.  Wrieht 

Wisconsin 

Bles. 

Inasmuch  as  Bacillus  alvei  was  absent,  it  is  evident  that  this  condi- 
tioji  is  not  European  foul  brood  (26). 

In  1903  the  investigations  were  continued.  Several  media  were 
devised  in  which  it  was  hoped  that  it  would  be  possible  to  obtain  a 
germination  of  the  spores  which  were  observed  the  year  before  and 
which  failed  to  grow  on  our  ordinary  media.  The  one  which  proved 
successful  was  prepared  as  follows :  Larvae  are  picked  from  the  brood 
combs  of  a  number  of  frames  of  healthy  brood  and  a  bouillon  (bee- 
larvae  bouillon)  is  made  from  them  following  the  same  directions  as 
when  bouillon  is  made  from  meat.    Our  first  growth  from  these 


42 


THE    BACTEKIA   OF    THE   APIAKY. 


spores  was  secured  in  an  agar  (bee-larvae  agar)  made  from  this  special 
bouillon  when  Liborius's  method  for  cultivating  anaerobes  was  used. 

The  technique  for  making  cultures  successfully  from  the  diseased 
material  is  not  difficult  if  the  following  method  is  used:  Place  a 
loopful  of  the  decayed  tissue  of  the  larvse  into  a  tube  of  bouillon; 
heat  to  65°  C.  for  10  minutes  to  kill  any  vegetative  forms  which  might 
be  present;  incubate  for  12  hours,  and  heat  again  to  65°  C.  for  10 
minutes.  This  is  usually  sufficient,  but  it  may  be  necessary  to  repeat 
the  same  process.  Liquefied  bee-larvse  agar  in  a  test  tube  is  then  in- 
oculated and  incubated.  The  successive  heating  will  destroy  the  veg- 
etative stage  of  any  spore-producing  species  which  is  common  about 
the  apiary,  e.  g.^  members  of  the  group  represented  by  Bacillus  A,  as 
described  on  pp.  13-14  of  this  paper.  Agar  slant  and  bouillon,  when 
inoculated  from  this  source,  remain  sterile ;  but  when  bee-larvse  agar 
is  used  a  slow  but  abundant  growth  takes  place.  Under  certain  con- 
ditions the  growth  appears  very  near  or  at  the  surface  when  cultures 
are  made  in  the  above  manner.  A  surface  growth  can  be  obtained 
after  a  few  generations  by  reinoculating  slant  agar  of  this  same 
medium. 

The  above  method  was  used  successfully  in  diagnosing  the  follow- 
ing samples  from  different  apiaries: 

Results  of  examination  of  specimens  of  American  foul  irood,  formerly  called 

simply  "  foul  brood." 


Brood  Bent  by— 

Date. 

Source. 

Bacteriological 
findings. 

W.  D.  Wright 

Oct.    19,1902 
Nov.  11,1902 
Nov.  11,1902 
July  24,1903 
Aug.    3,1903 
Aug.    3,1903 
Aug.    3,1903 

W  D.  Wright 

Wisconsin 

BaciUus  larvse. 

W.  D.  Wright 

C.  H.  W.  Weber 

Ohio..-'- 

Bacillus  larvae. 

N.  D.  West 

Broome  Countv  N.  Y 

N.  D.West 

N.  D.West 

Chenango  County,  N.  Y. 

The  results  of  these  examinations  show  that  BaciUus  larvae-  was 
present  in  all  the  specimens  examined,  which  suggests  that  it  very 
probably  figures  as  an  etiological  factor  in  this  disease.  Other  bac- 
teria of  different  species  are  occasions^^lly  found  associated  with  this 
bacillus. 

Baoillus  larvse. 

Occurrence. — Constantly  present  in  diseased  brood  from  colonies  affected  with 
American  foul  brood. 

Gelatin. — ^There  is  no  growth. 

Morphology. — It  is  a  slender  rod;  having  a  tendency  to  form  In  chains.  This 
is  especially  true  when  grown  in  bee-larvae  bouillon. 

Motility. — The  bacillus  is  rather  sluggishly  motile. 

Spores. — Spore  formation  talies  place.  This  can  be  observed  best  in  the  dif- 
ferent stages  of  the  disease  and  decay  of  the  larvse. 

Oxygen  requirements. — When  Liborius's  method  is  used,  the  best  growth 
usually  appears  near  to  but  not  on  the  surface.  After  a  few  generations  a 
surface  growth  may  be  obtained. 


THE   SO-CALLED  " BLACK  BROOD."  43 

Bouillon. — There  is  no  growth. 

Glucose  iouillon. — There  is  no  growth. 

Lactose. — There  is  no  growth. 

Saccharose. — There  is  no  growth. 

Agar  plate. — There  is  no  growth. 

Bee-larvw  agar. — The  inoculations  must  be  made  with  the  medium  liquefied. 
The  growth  takes  place  near  to  but  rarely  on  the  surface.  Cultures  must 
pass  thru  a  few  generations  before  a  satisfactory  surface  growth  can  be 
secured. 

Bee-larva;  agar  slant. — On  the  surface  of  this  medium  a  thin,  gray,  nonviscid 
growth  takes  place. 

Olucose  agar. — Slight  growth  has  been  observed  in  the  medium.  No  gas  is 
produced. 

Potato. — There  is  no  growth. 

MilJc. — There  is  no  growth. 

Litmus  inilk. — There  is  no  growth. 

Fermentation. — In  bee-larvje  bouillon  no  gas  is  produced. 

Indol. — ^There  is  no  growth  in  sugar-free  bouillon. 

THE  SO-CALLED  "  PICKLE  BROOD." 

The  name  "  pickle  brood  "  was  given  by  Dr.  William  R.  Howard,  of 
Fort  Worth,  Tex.,  to  a  disorder  found  in  the  brood  of  bees.  He 
stated  that  the  cause  of  the  disease  was  a  specific  fungus  which  he 
called  Aspergillus  pollinis.  His  results  have  not  been  confirmed  by 
other  investigators. 

The  bee  keepers  are  sustaining  a  loss  from  a  diseased  condition  in 
their  apiaries  which  they  are  diagnosing  as  "  pickle  brood."  The 
larvae  usually  die  late  in  the  larval  stage.  Most  of  them  are  found 
on  end  in  the  cell,  the  head  frequently  blackened  and  the  body  of  a 
watery,  granular  consistency. 

The  following  table  gives  a  summary  of  the  results  of  an  examina- 
tion of  specimens  received  labeled  "  pickle  brood :  " 

Results  of  examination  of  specimens  of  so-called  "  pickle  brood." 


Brood  sent  by— 

Date. 

Bacteriological  findings. 

W.  D.Wright 

June  17, 1902 

W.  D.  Wright      

July31,1902 

No  growth. 

W.  D.  Wright 

Aug.  4, 1902 

Aug.  20, 1902 

Unidentified  bacilli. 

W,  D.  Wright                     ... 

Sept.  2, 1902 

Unidentified  bacilli. 

W.  D.  Wright 

June  24, 1903 

N.  D.  West 

Aug.  5, 1903 

No  growth. 

M.  Stevens 

Aug.  20, 1903                            

No  growth. 

The  results  of  the  examinations  show  that  Aspergillus  pollinis  was 
not  found.  Further  investigations  must  be  made  before  any  conclu- 
sion can  be  drawn  as  to  the  real  cause  of  this  trouble. 


THE  SO-CALLED  "  BLACK  BROOD." 


In  1890  some  specimens  of  diseased  brood  were  sent  from  New 
York  State  to  Dr.  William  E.  Howard,  of  Fort  Worth,  Tex.,  and 
unfortunately,  after  a  short  and  inadequate  study  of  the  disease,  he 


44  THE   BACTERIA   OF   THE   APIARY. 

reported  it  to  be  a  new  disease  and  called  it  "  New  York  bee  disease  " 
or  "  black  brood."  He  described  as  its  cause  a  species  of  bacteria 
which  he  called  Bacillus  millii  (31). 

In  our  investigations  of  this  diseased  condition,  which  have  covered 
five  years,  we  have  not  found  an  organism  corresponding  to  Bacillus 
millii  in  any  of  the  specimens  that  we  have  received;  but  we  have 
found  Bacillus  alvei,  the  supposed  cause  of  foul  brood,  to  be  present 
constantly  in  samples  of  brood  which  the  bee  experts  of  New  York 
State  say  are  samples  of  the  same  diseased  condition  as  that  received 
by  Howard. 

From  this  we  conclude  that  the  diseased  brood  that  has  received 
the  name  of  "  New  York  bee  disease  "  or  "  black  brood  "  is  really 
genuine  European  foul  brood. 

PALSY  OR  PARALYSIS. 

The  disease  known  to  the  apiarists  as  palsy  or  paralysis  attacks 
the  adult  bees.  The  name  is  suggestive  of  the  symptoms  manifested 
by  the  diseased  bees.  A  number  of  bees  affected  were  received  from 
Messrs.  W.  D.  Wright  and  Charles  Stewart,  taken  from  apiaries  in 
New  York  State.  In  1903  bacteriological  examinations  were  made  of 
a  number  of  bees  so  affected.  Several  species  of  bacteria  were  isolated 
and  some  experimental  inoculations  made,  but  no  conclusions  could  be 
drawn  from  the  results  obtained  as  to  the  cause  of  the  disorder. 

From  a  study  of  the  normal  flora  of  the  bee  it  was  soon  found 
that  AA'e  had  here  quite  a  number  of  species  of  bacteria  present. 
This  fact  stimulated  a  study  of  the  normal  flora,  the  results  of  which 
are  recorded  in  Part  I.  From  this  point  the  work  can  be  carried 
on  with  the  hope  that,  if  the  disease  has  a  bacterium  as  an  etiological 
factor,  it  may  be  found.  It  is  believed  by  some  bee  keepers  that 
Bacilhis  gaytoni  of  Cheshire  is  the  cause  of  paralysis,  but  this  is  not 
claimed  by  Cheshire,  and  the  belief  is  not  grounded  on  bacteriological 
findings. 

SUMMARY  TO  PART  II. 

Following  is  a  brief  summary  of  the  results  of  the  present  investi- 
gation of  bee  diseases : 

(1)  There  are  a  number  of  diseased  conditions  which  affect  the 
apiary. 

(2)  The  disease  which  seems  to  cause  the  most  rapid  loss  to  the 
apiarist  is  European  foul  brood,  in  which  is  found  Bacillus  alvei — 
first  isolated,  studied,  and  named  by  Cheshire  and  Cheyne  in  1885. 

(3)  The  distribution  of  Bacillus  alvei  in  the  infected  hive  is  as 
follows : 

{a)  The  greatest  number  of  infecting  germs  are  found  in  the 
bodies  of  dead  larvse. 

(&)  The  pollen  stored  in  the  cells  of  the  foul-brood  combs  contains 
many  of  these  infecting  organisms. 


CONCLUSIONS.  45 

(c)  The  honey  stored  in  brood  oombs  infected  with  this  disease 
has  been  found  to  contain  a  few  bacilli  of  this  species. 

(d)  The  surface  of  oombs,  frames,  and  hives  may  be  contaminated. 

(e)  The  wings,  head,  legs,  thorax,  abdomen,  and  intestinal  con- 
tents of  adult  bees  were  found  to  be  contaminated  with  Bacillus  alvei. 

(f)  Bacillus  alpei  may  appear  in  cultures  made  from  the  ovary  of 
queens  from  European  foul-brood  colonies,  but  the  presence  of  this 
species  suggests  contamination  from  th«  body  of  the  queen  while  the 
cultures  are  being  made  and  has  no  special  significance. 

(4)  The  disease  which  seems  to  be  most  widespread  in  the  United 
States  we  have  called  American  foul  brood,  and  the  organism  which 
has  been  found  constantly  present  in  the  disease  we  have  called 
Bacillus  larva'.  This  disorder  was  thought  by  many  in  this  country 
and  other  countries  as  well  to  be  the  foul  brood  described  by  Cheshire 
and  Cheyne,  but  such  is  not  the  case. 

(5)  From  the  nature  of  American  foul  brood  it  is  thought  that  the 
organism  has  a  similar  distribution  to  that  of  Bacillus  alvei. 

(6)  It  appears  that  European  foul  brood  was  erroneously  called 
"  New  York  bee  disease  "  or  "  black  brood  "  by  Dr.  Wm.  R.  Howard 
in  1900. 

(7)  There  is  a  diseased  condition  affecting  the  brood  of  bees  which 
is  being  called  by  the  bee  keepers  "  pickle  brood."  No  conclusion  can 
be  drawn  from  the  investigation  so  far  as  to  the  cause  of  the  disease. 

(8)  Aspergillus  pollinis,  ascribed  by  Dr.  William  R.  Howard  as 
the  cause  of  pickle  brood,  has  not  been  found  in  this  investigation 
and  is  not  believed  by  the  author  to  have  any  etiological  relation  to 
the  so-called  "  pickle  brood." 

(9)  Palsy  or  paralysis  is  a  diseased  condition  of  the  adult  bees. 
No  conclusion  can  yet  be  drawn  as  to  its  cause. 

(10)  Formaldehyde  gas  as  ordinarily  used  in  the  apiaries  is  insuffi- 
cient to  insure  complete  disinfection. 

CONCLTTSIONS. 

In  a  paragraph  the  author  wishes,  if  possible,  to  present  the  status 
of  the  bee  diseases  in  this  country.  It  should  be  remembered,  firstly, 
that  "  black  brood  "  can  now  be  dropt  from  our  vocabulary,  and 
probably  does  not  exist ;  secondly,  that  the  term  "  foul  brood  "  was 
being  applied  to  two  distinct  diseases.  One  of  these  diseases  we  now 
refer  to  as  European  foul  brood,  because  it  first  received  a  scientific 
study  from  a  European  investigator.  We  refer  to  the  other  disease 
as  American  foul  brood,  because  it  was  first  studied  scientifically  in 
America.  There  is  one  more  disorder  in  the  brood  of  bees  which  has 
attracted  considerable  attention — the  so-called  "  pickle  brood." 
There  are,  then,  these  three  principal  diseases :  European  foul  brood, 
American  foul  broody  and  the  so-called  "  pickle  brood." 


46  THE   BACTEEIA   OP   THE   APIAEY. 

BIBLIOGRAPHY  TO  PABT  II. 

12.  Ahistotelbs.     <Hi8toria  Animalium,  Book  IX,  Ch.  27. 

13.  ScHiKACH.     <Histoire  des  Abeilles,  Ch.  3,  p.  56,  1769. 

14.  Leuckhart.     <Binen-zeitung.     Eichstadt,  p.  232,  1860. 

15.  MuHLFBLD.     <Bienen-zeitung.     Eichstadt,  p.  232,  1868. 

16.  Preuss.     <Bienen-zeitung,  p.  95,  1868. 

17.  Geilen.     <Bienen-2eitung,  Nos.  21  and  22,  1868. 

18.  Lambrecht.     <Bienen-zeitung,  No.  2,  1870. 

19.  Hallier.     <Bienen-zeitung,  No.  2,  1870. 

20.  Cornallia.     <Bienen-zeitung,  No.  5,  1870. 

21.  Fischer.     <Bienen-zeitung,  p.  105,  1871. 

22.  Cheshire  and  Cheyne.     The  pathogenic  history  and  history  under  cultivation 

of  a  new  bacillus  {B.  alvei)  the  cause  of  a  disease  of  hive  bees  hitherto  known 
as  foul  brood.     <Jour.  Roy.  Mic.  Soc,  Vol.  V.,  p.  581,  1885. 

23.  Dickel.     <Bienen-zeitung,  p.  124,  1888.  ' 

24.  Harrison,  F.  C.     The  foul  brood  of  bees.     <Bulletin  No.  112,  Ontario  Agric. 

College.     Also  in  Centralblatt  fiir  Bakteriologie,  Parasitenkunde  und  Infek- 
tionskrankheiten,  Zweite  Abtheilung,  VI  Band,  1900. 

25.  Lambotte.     Recherches  sur  le  Microbe  de  la  "Loque,"  maladie  des  abeilles. 

<Annales  de  I'Institut  Pasteur,  Vol.  XVI,  p.  694,  1902. 

26.  Moore,  V.  A.,  and  White,  G.  Franklin.     A  report  on  the  investigation  of  an 

infectious  bee  disease.     <New  York  State  department  of  agriculture,  Jan., 
1903. 

27.  White,  G.  Franklin.     A  report  of  the  further  investigation  of  bee  diseases  of 

the  State  affecting  the  apiaries  of  the  State  of  New  York.     <New  York  State 
department  of  agriculture,  Jan. ,  1904. 

28.  White,  G.  Franklin.     A  report  of  the  work  on  bee  diseases  for  1904.     <New 

York  State  Department  of  Agriculture,  Jan.,  1905. 

29.  Cheshire.     <Bees  and  bee  keeping.     Vol.  II,  London.     1885. 

30.  Mackenzie.     Ontario  Agricultural  College  Report,  1893. 

31.  Howard,  Wm.  R.     New  York  Bee  Disease,  or  Black  Brood.     <Gleanings  in 

Bee  Culture,  Feb.  15,  1900. 

32.  Benton.     <Bulletin  of  Apiculture,  No.  4,  1886. 

33.  Smith,  W.  G.     <British  Bee  Journal,  Vol.  XIV,  p.  1225,  1886. 

34.  Jones,  S.  A.     Foul  Brood,  its  management  and  cure.     <Beeton,  Canada,  1886. 

35.  McLean.     <Department  of  Agriculture  Report.     Washington,  p.  584,  1886. 

36.  Ward,  F.  F.     <British  Bee  Journal,  p.  396,  1887. 

37.  Schreuter.     <Bienen-zeitung,  1887. 

38.  Klamann.     <Bienenwirtschaftliches  Centralblatt.     Hanover,  No.  18,  1888. 

39.  Reports  of  the  bee  keepers'  association  of  the  Province  of  Ontario,  1890. 

40.  Planta.     <Schweizerische  Bienen-zeitung,  1893. 

41.  Howard,   W.  R.     Foul   Brood;    Its  natural  history   and    rational    treatment. 

<Chicago,  1894. 

42.  McEvoy.     Foul  Brood,  its  cause  and  cure.     <Trenton,  N.  J.,  1895. 

43.  Root,  A.  I.     <Gleanings  in  Bee  Culture,  Vol.  XXIV,  p.  853,  1896. 

44.  Cowan.     <British  Bee  Journal,  Vol.  XII,  p.  128. 

45.  GovAN.     <British  Bee  Journal,  Vol.  XXIII,  p.  434. 

46.  Formalin  as  a  cure  for  foul  brood.     <Gleanings  in  Bee  Culture  Vol  XXX  No 

13,  p.  544,  1902.  ' 

47.  Weber,  C.  H.  W.     Formalin  gas  as  a  cure  for  foul  brood.     <Cincinnati  Ohio, 

1903. 

48.  BuRRi,  R.     Bakteriologische  Forschungen  uber  die  Faulbrut.     <Schweizerische 

Bienen-zeitung,  Nos.  10  and  11,  1904. 

49.  Reidenbach.     1st  das  Vernichten  der  Faulbrautstocke  das  ficherste  Mittel  zur 

Bekampfung  der  Faulbrut?    <Leipziger  Bienen-zeitung,  January,  1903. 

50.  Neumann.     Zur  Klarung  der  Faulbrutfrage.     <Ibid.,  1904. 


INDEX. 


Page. 

Acknowledgments  of  author 2 

Apiary,  diseases 30-46 

normal,  bacteria  found 12-30 

tabulation  of  micro-organisms 28 

technique  for  study  of  bacteria 7-13 

Aspergillus  poUinis,  probably  not  cause  of  ' '  pickle  brood  " 43, 45 

BaciUus  A  {B.  mesentmcus?),  description 13-14 

mistaken  for  BodKitgoZm —  3,29,33 

on  combs  in  normal  apiary 13,29 

on  healthy  adult  honeybees IB 

cUvei,  confusion  with  Bacillus  A 3,29,33 

description 36-37 

discovery ,      31 

distribution  in  infected  hives 38-39,44-45 

European  foul  brood  produced  experimentally  by  inoculation. .  37-38 

name  wrongly  given  to  other  bacteria 33-84 

not  present  in  American  foul  brood 4,32 

present  in  European  foul  brood 3, 32, 35, 44 

so-called  "New  York  bee  disease"  or  "black  brood"         3 

relation  with  Bacillus  mesmtericus  vulgatus 31 

B,  description 15-16 

in  pollen  and  intestine  of  healthy  honeybees 15, 29 

cholerse  mis,  description 21-22 

in  intestine  of  healthy  honeybee 21,29 

doacse,  description 19-20 

in  intestine  of  healthy  honeybee 19,29 

colt  communis,  description 20-21 

in  intestine  of  healthy  honeybee 20, 29 

j;,  description 22-23 

in  intestine  of  healthy  honeybee 22,29 

gaytoni,  considered  by  some  as  cause  of  paralysis  of  bees 44 

larvse,  description  ..., 42-43 

formerly  termed  Bacillus  X. 40 

present  in  American  foul  brood 32,40,42,45 

mesentericus  f    (See  BadUus  A. ) 

mesentericus  vulgatus,  relation  with  Bacillus  alvei 31 

milUi,  not  found  in  so-called  "black  brood" 44 

mbgastricus,  description 23-24 

in  intestine  of  healthy  honeybee 23,29 

X=Bacillualarvss.... 40 

47 


48  INDEX. 

Page. 

Bacteria,  from  combs  of  normal  apiary 13-15 

pollen  of  normal  apiary '■^  -^^ 

in  healthy  larvae  not  usual lb, /9 

honey  from  normal  apiary  not  usual 16,29 

intestine  of  healthy  honeybee 18-25 

of  apiary,  cultures,  how  obtained ^ 

suggestions  for  description 10-12 

which  are  described  in  paper 9 

differentiation  and  identification 9 

material  for  study  how  obtained 7-8 

media  employed  for  cultures 10-12 

oxygen  requirements 10 

staining  properties 10 

technique  in  study 7-13 

variations  in  size 9-10 

of  normal  apiary 12-30 

on  healthy  adult  honeybees ■- 16-18 

Bacterium  acidiformans,  description 14-15 

on  combs  in  normal  apiary 14 

cyaneus  {Micrococcus  cyaneus),  description 1 16-17 

on  combs  of  normal  apiary 29 

on  healthy  adult  bees  and  pollen 16 

D,  description 19 

in  intestine  of  healthy  honeybee 19-29 

mycoides,  description 24 

in  intestine  of  healthy  honeybee 24,29 

Bee  bread,  fermentation  considered  cause  of  foul  brood  formerly 31 

diseases 30-46 

history 30-32 

modified  names  necessary 3-4 

theories  as  to  cause 30-31 

Bees,  diseased  adult,  parts  of  body  infected  by  Bacillus  alvei 39, 45 

healthy  adult,  bacteria  found  externally 16-18 

in  intestine 15, 18, 25, 29 

Saccharomyces  F  in  intestine 25,  29 

ovary  of  queen.  Bacillus  alvei  present  accidentally 39, 45 

Bibliography  to  Part  I 29-30 

11 46 

"Black  brood"  =  European  foul  brood,  i 44,45 

=  foul  brood  of  Cheshire  and  Cheyne 3 

occurrence  of  Bacillus  alvei 35 

origin  of  term 31,43-44 

term  may  be  discarded 45 

Blight  of  flowers,  supposed  by  ancients  related  to  bee  diseases 30 

Brood,  diseased,  occurrence  of  Saccharomyces  0 26-27, 29 

Climatic  conditions,  as  affecting  European  foul  brood 38 

Combs,  of  healthy  apiary,  occurrence  of  bacteria 13-15 

fungi 25 

Saccharomyces  roseus 25, 26 

diseased  apiary,  occurrence  of  Bacillus  alvei 39  45 

Oryptococcus  alvearis,  formerly  considered  cause  of  foul  brood 31 

formerly  considered  cause  of  foul  brood 31 

Cultures,  of  bacteria  of  apiary,  how  obtained - S 


INDEX.  49 

Fagei 

Cultures,  of  bacteria  of  apiary,  media  employed 10-12 

suggestions  for  description 10-12 

those  described 9 

Differentiation  of  bacteria  of  apiary 9 

Formaldehyde  gas,  iusutBcient  disinfectant  against  European  foul  brood  as 

ordinarily  uwed 39-40 

Foul  brood,  American,  application  of  term 45 

author's  investigations 41-42 

Bacillus  iili'ei  not  present 4, 32 

larvx  present 32,42,45 

confusion  with  foul  brood  of  Cheshire  and  Cheyne. .  4, 40, 45 

symptoms 40-41 

confusion  regarding  diseases 33 

disease  of  Cheshire  and  Cheyne  renamed  European  foul  brood.  3, 32, 44 

European,  application  of  term 45 

author's  investigations 34-36 

Bacillus  alvei  present 4, 32, 35, 44 

=  foul  brood  of  Cheshire  and  Cheyne 3,  32, 44 

formaldehyde  gas  insuflficient  disinfectant  as  ordinarily 

used 39-40 

more  destructive  in  cool,  damp  weather 38 

produced  by  experimental  inoculation  with  Bacillus 

alvei 38 

symptoms 32-33 

of  Cheyne,  named  European  foul  brood 32 

term  applied  to  two  distinct  diseases 31-32, 45 

use  of  term  in  New  York  State 31 

Frames,  in  diseased  apiaries,  occurrence  of  Bacillus  alvei 39, 45 

Fungi,  formerly  considered  cause  of  foul  brood 31 

in  intestines  of  healthy  honeybees 25 

gollen  and  combs  of  normal  apiaries 25 

Fungus.     {See  Aspergillus  pollinis,  Cryptococcus,  Oryptococcus  alvearis,  a.nd  P(m- 
higtophyion  ovatum. ) 

Hives,  of  diseased  apiaries,  occurrence  of  Bacillus  alvei 39, 45 

temperature  approximates  that  of  warm-blooded  animals 29 

Honey,  from  foul-brood  combs,  occurrence  of  Bacillus  alvei 39, 45 

healthy  hives,  quite  uniformly  sterile 16, 29 

Ichneumon  apium  mellificarium,  formerly  supposed  cause  of  infectious  bee  dis- 
ease           30 

Identification  of  bacteria  of  apiary 9 

Intestine  of  healthy  honeybee,  occurrence  of  bacteria 18-25 

fungi 25 

Saccharomyces  F 25,29 

Larvse  of  honeybee  dead  from  disease,  occurrence  of  Bacillus  alvei 39, 44 

healthy  honeybee,  usually  sterile 16,  29 

Micrococcus  C,  description 17-18 

on  combs  of  healthy  honeybees 29 

healthy  adult  honeybees ■. 17 

cyaneus,     (See  Bacterium  cyaneus.) 

Micro-organisms  normally  present  in  the  apiary,  tabulation 28 

Morphology  of  bacteria  of  apiary 9-10 

''New  York  bee  disease."     {See  "Black  brood.") 


50  INDEX. 

Page. 

Nonpathogenic  bacteria  of  honeybees,  necessity  for  study 3 

Oxygen  requirements,  of  bacteria  of  apiary ^^ 

Palsy.     (&e  Paralysis. ) 

Panhistophyton  ovatum,  a  related  fungus,  formerly  supposed  cause  of  bee  disease .  30 

Paralysis,  of  honeybees,  cause  unknown 44,45 

"Pickle  brood,"  Aspergillus pollinis -pTohahly  not  cause 43,45 

bacteriological  findings  from  author's  examinations 43 

disease  of  bees 31,43,44,45 

Pollen,  in  foul-brood  combs,  occurrence  of  Bacillus  alvei 39, 44 

healthy  combs,  occurrence  of  bacteria 15, 16 

fungi - 25 

PropoUs.     {See  Combs. ) 

Pseudomonas  fluoresceins  liquefaciens,  description 24-25 

in  intestine  of  healthy  adult  honeybee  .  - .  24, 29 

Saccharomyces  F,  description 25-26 

in  intestine  of  healthy  adult  honeybee 25, 29 

G,  description 26-27 

in  dead  larvae  of  diseased  honeybees 26, 29 

in  normal  apiary 25-27 

roseus,  description : 26 

in  comb  of  normal  apiary , 25,29 

Staining  properties  of  bacteria  of  apiary 10 

Summary  to  Part  I 29 

II..... - 44-45 

Technique  in  study  of  bacteria  of  apiary 7-13 

Variations  in  size  of  bacteria  of  apiary 9-10 

Wax.     {See  Combs. ) 

"X  Brood "  =  American  foul  brood 40 


UNITED  STATES  DEPARTMENT  OF  AGRICULTURE 
BULLETIN  No.  810 

Contrlbntlon  from  the  Barean  of  Entomolosy 
L.  O.  HOWARD.  Chief 


Washington,  D.G. 


PROFESSIONAL  PAPER 


February  26, 1920 


EUROPEAN  FOULBROOD 


By 


G.  F.  WHITE,  Specialist  in  Insect  Diseases 


CONTENTS 


Pago 

Introdnctlon 1 

Name  of  the  Disease 2 

Healthy  Larvae  of  the  Age  at  which  they 

Dieof  EnropeanFonlbrood     ....  3 

Symptoms 4 

Etlolocy 7 

Technique IS 

Theimal  Death  Point  of  Baciilas  pioton  17 

Resistance  of  Barillas  piston  to  Drying  .  17 
Resistance  of  Bacillus  piuton  when  Dry 

to  Direct  Sunlight 19 

Resistance  of  BacIUns  pinton  in  Water  to 

Direct  Sunlight  ....  ^  ...  .  20 
Resistance  of  BaeHlns  pinton  !n  Honey  to 

Direct  Sunlight 21 


Page 
Resistance  of  Baciiiua  pinton  to  Fermen- 
tation    21 

Resistance  of  Bacillus  piuton  to  Putrefac- 
tion      22 

Viability  of  Bacillus  pinton  in  Honey  .    .  23 

Viability  of  Bacillus  pinton  in  Pollen  .    .  24 
Resistance  of  Baciilas  pinton  to  Carbolic 

Acid    '. 24 

Eifect  of  Drugs  on  European  Foulbrood  29 

Transmission  of  European  Foulbrood     .  26 

Diagnosis 28 

Prognosis 31 

Summary  and  Conclusions 81 

Literature  Cited 84 

Explanation  of  Platea 87 


WASHINGTON 
GOTERNMENT  PBlNTINa  OVFICB 

1920 


UNITED  STATES  DEPARTMENT  OF  AGRICULTURE 


BULLETIN  No.  810 


Contribution  from  tlie  Bureau  of  Entomology 
L.  O.  HOWARD,  Chief 


Washington,  D.  C. 


PROFESSIONAL  PAPER 


February  26, 1920 


EUROPEAN  FOULBROOD 

By  G.  F.  White 
Specialist  in  Insect  Diseases 


CONTENTS 


Page 

Introduction 1 

Name  of  tlie  disease 2 

Healthy  larvse  of  the  age  at  which 

they  die  of  European  foulhrood 3 

Symptoms 4 

Etiology 7 

Technique 13 

Thermal    death    point    of    Bacillus 

pluton 17 

Resistance     of    Bamllua    pluton    to 

drying 17 

Besistance  of  Bacillus  pluton  when 

dry  to  direct  sunlight 19 

Besistance    of    Baolllus    plutoi    in 

water  to  direct  sunlight 20 

Besistance    of    Baeillus    pluton    in 

honey  to  direct  sunlight 21 


Page 
Besistance  of  Bacillus  pluton  to  fer- 
mentation   21 

Besistance    of    Badttus    pluton    to 

jlutrefaction 22 

Viability  of  Bamllus  pluton  in  honey_-  23 
Viability  of  Ba^llus  pluton  in  pollen_.  24 
Besistance  of  Baolllus  pluton  to  car- 
bolic   acid 24 

Effect    of  drugs   on    European   foul- 
brood 26 

Transmission  of  European  foulbrood-  26 

Diagnosis 28 

Prognosis 31 

Summary  and  conclusions 31 

Literature    cited 34 

Explanation   of   plates — . 37 


INTRODUCTION 

European  foulbrood  is  an  infectious  disease  of  the  brood  of  bees 
caused  by  BaciUus  pluton.  It  is  characterized  by  the  death  of  brood 
during  its  uncapped  stage  and  by  the  absence  of  any  marked  odor. 
The  disorder  has  a  wide  distribution  and  is  fairly  well  known  to  bee- 
keepers. The  losses  sustained  by  the  infected  apiary  vary  from  a 
slight  weakening  of  the  colonies  in  some  instances  to  the  destruction 
of  all  of  them  in  others. 

Practical  apiarists  have  determined  mucn  concerning  the  disorder 
while  pursuing  their  profession.  The  writer  in  an  earlier  paper  (15)  ^ 
referred  to  the  nature  and  extent  of  the  progress  that  had  been  made 
in  the  study  of  the  disease  from  the  laboratory  point  of  view.  The 
present  paper  deals  with  results  which  have  been  obtained  from  a  con- 
tinuation of  the  work.    Among  the  problems  considered  are :  The  re- 


132817° 


'  Figures  in  parentheses  refer  to 
-20— Bull.  810 1 


'  Literature  cited,"  p.  34. 


2  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGEICULTTJRE. 

sistance  of  Badllus  pluton  to  heat,  drying,  sunlight,  fermentation, 
and  disinfectants ;  the  effect  of  the  disease  on  the  colony  and  on  the 
apiary ;  and  the  transmission,  diagnosis,  and  prognosis  of  the  disease. 
Work  directly  on  the  treatment  of  the  disease  has  not  been  attempted 
by  the  writer.  Naturally,  however,  any  treatment  that  is  devised,  if  it 
is  to  be  elBcient  and  at  the  same  time  economical,  must  be  based  upon 
results  obtained  from  the  solution  of  such  problems  as  those  which 
have  received  attention  in  these  studies. 

Eesults  obtained  from  a  study  of  the  disease  in  the  laboratory  and 
in  the  experimental  apiary  form  the  basis  of  the  discussions  contained 
in  the  present  paper.  Since  the  disease  encountered  in  nature  is  very 
similar  to  the  one  produced  by  artificial  inoculation,  the  importance 
of  the  studies  is  at  once  evident. 

The  paper  ^  will  be  of  interest,  it  is  believed,  not  only  to  the  apiarist 
who  may  wish  to  apply  the  facts  here  determined  in  the  pursuit  of 
his  profession,  but  also  to  the  investigator  whose  desire  primarily  is 
a  further  study  of  the  disease. 

NAME  OF  THE  DISEASE 

The  term  "  foulbrood  "  was  quite  generally  used  in  the  past,  as  it 
still  frequently  is,  for  the  two  infectious  diseases  now  known  in 
America  as  European  foulbrood  and  American  foulbrood.  In  1885 
when  Cheshire  and  Cheyne  (4)  in  England  made  their  studies  on 
foulbrood  and  described  BacUlus  alvei,  evidently  they  were  not  con- 
vinced that  there  were  two  distinct  diseases  that  were  being  called 
by  the  one  name  foulbrood.  The  disease  studied  by  them  is  the  one 
which  is  the  subject  of  discussion  in  the  present  paper.  In  the  names 
for  the  two  diseases  it  will  be  observed  that  the  word  "  foulbrood  "  is 
retained  in  both  instances.  To  this  "  European "  is  added  for  the 
disease  on  which  early  laboratory  studies  were  made  by  these  Euro- 
peans (Cheshire  and  Cheyne). 

Dr.  William  K.  Howard  (6),  of  Texas,  in  1900,  worked  for  a  brief 
period  with  this  disease,  reached  the  conclusion  that  it  was  a  new  one, 
and  referred  to  it  by  the  names  "  New  York  bee  disease,"  or  "  black 
brood."  Work  by  Moore  and  White  (11)  in  1902  showed  that  the 
disease  was  not  new,  but  was  the  foulbrood  studied  by  Cheshire  and 
Cheyne  (4).  The  names  "  New  York  bee  disease,"  or  "  black  brood," 
therefore,  were  superfluous,  and  as  their  use  would  have  added  to  the 
confusion  that  already  existed  they  were  discarded.  Beekeepers,  ento- 
mologists, and  pathologists,  as  a  rule,  are  more  or  less  familiar  with 
the  terms  "  foulbrood  "  and  "  Bacillus  alvei."  Usually,  however,  the 
ropy  foulbrood— American  foulbrood— is  the  one  that  is  thought  of, 

iThe  present  studies  are  similar  to  those  made  by  the  writer  on  sacbrood  (17)  Nosema- 
dlsease  (18),  and  American  foulbrood  (19).  A  reference  to  these  papers  may 'be  found 
helpful  where  the  discussions  in  the  present  one  are  especially  brief.  The  investlKatlons 
were  completed   in   September,    1916.   and  the  paper   was  submitted   for   publication   in 


EUROPEAN  FOULBROOD.  3 

and  the  one  (hat  frequently  has  been  associated  in  the  literature  with 
Bacillus  (dvei.  This  is  unfortunate.  While  5.  aZrez  is  not  the  cause 
of  any  bee  disease,  it  occurs  very  frequently  with  European  foul- 
brood  and  is  found  only  seldom  in  the  ropy  disease.  In  using  the 
names  European  foulbrood  and  American  foulbrood  it  is  possible, 
however,  to  avoid  confusion  by  bearing  well  in  mind  the  history  of 
the  disease. 

HEALTHY  LARV^  OF  THE  AGE  AT  WHICH  THEY  DIE  OF 
EUROPEAN  FOULBROOD 

Bees  dying  of  European  foulbrood  do  so  during  the  larval  stage.^ 
Death  may  take  place  at  any  time  from  the  fourth  day  of  larval  life  to 
pupation.  For  convenience  of  description  the  brood  of  the  age  at 
which  death  from  European  foulbrood  occurs  is  placed  here  in  three 
groups.  Groups  1  and  2  include  the  uncapped  and  group  3  the  capped 
larvae. 

GROUP  1 

The  youngest  larva  (PI.  II,  D,  G)  that  dies  of  European  foulbrood 
practically  covers  the  bottom  of  the  cell.  It  lies  either  on  its  right  or 
its  left  side,  with  its  dorsal  portion  extending  to  the  lateral  walls  of 
the  cell.  Its  form  is  C  shaped  with  the  anterior  and  posterior  ex- 
tremities almost  together.  Its  color  is  bluish  white  with  a  glistening 
surface,  presenting  a  pearly  appearance.  The  body  is  more  or  less 
opaque,  due  largely  to  the  adipose  tissue.  Folds  and  furrows  divide 
the  surface  into  segments.  In  health  these  are  quite  prominent  and 
the  entire  larva  is  turgid  in  appearance. 

With  the  unaided  eye  spiracles  and  tracheae  can  be  seen  with  diffi- 
culty, but  by  slight  magnification  they  are  readily  observed.  Most 
of  the  tracheae,  appearing  as  white  lines,  extend  either  dorsally  or 
ventrally  on  the  lateral  side  of  the  larva,  but  a  distinct  chain  con- 
necting them  will  be  observed  to  extend  at  right  angles  to  these. 

GROUP  2 

Healthy  larvae  (PI.  Ill,  D,  G)  slightly  older  than  those  described  in 
Group  1  constitute  Group  2.  The  larva  now  completely  fills  the 
bottom  of  the  cell.  The  dorsal  side  pressing  against  the  lateral  side 
walls  of  the  cell  causes  the  contour  of  the  body  to  be  in  general 
hexagonal.  The  tracheae  are  seen  less  easily  than  in  younger  larvae, 
while  the  color,  glistening  appearance,  prominence  of  segments,  and 
turgidity  are  similar  to  those  of  the  younger  larvae  described  in 
Group  1. 

By  turning  the  larva  so  that  its  dorsal  surface  may  be  brought  into 
view  (PI.  Ill,  A)  there  is  observed  a  more  or  less  transparent  narrow 

•  The  term  larvse  as  used  in  the  present  paper  applies  to  the  prepupae  as  well  as  to 
earlier  stages  of  the  brood. 


4  BULLETIN   810,   U.   S.   DEPAETJIEXT   OF   AGKICTJLTITKE. 

area  along  the  dorsal  median  line  extending  nearly  the  length  of  the 
body.  The  contents  of  the  stomach  may  be  seen  through  this  area. 
The  color  of  the  mass  is  due  chiefly  to  the  presence  of  pollen.  It  is 
usually  some  shade  of  yellow.  The  median  area  presents  in  its 
appearance  a  sharp  contrast  to  the  bluish-white,  opaque  portions  on 
either  side  of  it.  Similar  appearances  are  to  be  noted  in  the  larvae  of 
Group  1. 

The  larva  removed  from  the  cell  performs  only  slight  movements, 
lies  partly  coiled,  and  is  more  or  less  turgid.  The  segments  are  promi- 
nent. When  the  body  wall  is  torn  there  flows  from  the  ruptured  wall 
the  clear  larval  blood,  in  which  are  suspended  often  fat  and  other 
tissue  cells  which  give  to  it  a  somewhat  milky  appearance.  The 
stomach,  a  transparent  tube  easily  torn  into  segments,  contains  a 
mass  of  partially  digested  food,  pollen  constituting  usually  a  con- 
spicuous portion  of  it. 

GROUP  3 

Group  3  consists  of  capped  larvae.  These  are,  therefore,  larger  than 
those  described  in  Groups  1  and  2.  In  the  group  are  included  the 
larvae  which  have  spun  a  cocoon  as  well  as  those  which  have  not.  An 
endwise  position  in  the  cell  may  or  may  not  have  been  assumed.  The 
larvae  are  seen  in  various  positions.  Not  infrequently  some  portion 
of  the  dorsal  surface  is  turned  toward  the  observer,  the  narrow,  me- 
dian, transparent  area  being  in  evidence  as  iix  younger  larvae.  Healthy 
larvae  occupying  an  endwise  position  are  described  in  papers  on  sac- 
brood  and  American  foulbrood  (17,  19)  and  will  not  be  referred  to 
further  at  this  time. 

SYMPTOMS 

In  European  foulbrood,  as  in  other  brood  diseases,  the  colony  as 
a  whole  and  not  the  individual  bee  should  be  considered  as  the  unit  in 
the  discussion  of  the  symptoms  of  the  disease.  The  description  of  the 
symptoms  recorded  in  the  present  paper  is  based  chiefly  upon  observa- 
tions made  on  the  disease  produced  through  artificial  inoculations. 
In  making  the  studies  in  the  experimental  apiary  observations  made 
by  beekeepers  have  been  duplicated  and  new  facts  determined.  It 
has  been  possible  also  to  locate  errors  which  have  been  made  in 
discussions  of  symptoms  of  the  disease. 

GENERAL    SYMPTOMS   FROM  A   CASUAL   EXAMINATION 

Death  of  brood  during  the  feeding  stage,  in  uncapped  cells,  is  a 
characteristic  of  European  foulbrood.  The  brood  nest  in  the  disease 
usually  presents  an  irregular  appearance,  capped  cells  and  uncapped 
ones  being  found  scattered  irregularly  over  the  brood  frames,  giving 
to  them  the  "  pepper  box  "  appearance  (PI.  I)  often  referred  to  by 


EUROPEAN   FOULBROOD.  5 

beekeepers,  a  condition  noticeable  when  the  disease  is  fairly  well 
advanced  in  the  colony. 

The  dead  larviv  lose  their  pearly  whiteness  and  assume  a  yellowish 
color,  later  becoming  brownish.  This  deepens  often  to  a  dark  brown. 
The  decaying  remains  are  not  characteristically  ropy,  as  in  American 
foulbroocl.  Marked  viscidity  is  usually  absent.  When  it  is  present 
the  decaying  mass  can  be  drawn  into  threads  but  to  a  less  extent  than 
in  the  ropy  disease.  In  advanced  cases  the  disease  may  be  accom- 
panied by  an  odor,  but  in  the  writer's  experience  this  never  has  been 
marked  and  never  offensive. 

As  the  disease  in  the  colony  advances,  weakness  becomes  a  symp- 
toni.  In  severe  cases  queenlessness  may  result  from  the  infection. 
This,  however,  is  by  no  means  the  rule. 

SYMPTOMS  MANIFESTED   BY  INDIVIDUAL  LARV.S:   SICK   OR  DEAD   OP  EUROPEAN 

FOULBROOD 

Evidences  of  European  foulbrood  in  the  individual  larvse  appear 
before  and  after  death.  The  colony  symptoms  used  most  frequently 
in  the  diagnosis  of  the  disease  are  largely  post-mortem  appearances 
of  larvae.  Of  much  interest  and  frequently  of  considerable  diagnos- 
tic value  are  the  symptoms  manifest  by  larvae  sick  but  not  dead  of 
the  disease.  For  convenience  in  the  description  of  the  appearances 
of  the  sick  or  dead  larvae,  the  grouping  used  in  describing  the 
healthy  larvae  (p.  3)  is  followed.  The  appearances  of  affected 
larvae  both  living  and  dead  are,  of  course,  changing  constantly.  A 
description  which  is  correct  for  one  day  or  hour,  it  should  be 
realized,  is  not  likely  to  be  entirely  correct  for  the  next. 


The  youngest  larvae  manifesting  symptoms  of  European  foul- 
brood  are  approximately  4  days  old  (PI.  II,  A,  B,  C,  E,  F,  H,  I).  In 
many  cases  at  this  stage  of  the  disease  a  peristalsis-like  movement  of 
the  body  is  marked  and  is  readily  observed  by  the  unaided  eye,  but 
in  others  no  such  bodily  movements  are  observed.  The  diseased 
larvae  at  the  time  may  be  more  transparent  (PL  II,  B,  H)  than 
healthy  ones  of  the  same  size.  In  such  larvae  the  tracheae  are  quite 
prominent  and  more  readily  seen  than  in  healthy  ones.  Occasionally 
numerous  minute  opaque  areas  are  observed  in  these  more  transpar- 
ent larvae,  giving  to  them  a  punctate  appearance.  Very  often,  how- 
ever, this  sign  is  not  present.  In  many  instances,  indeed,  no  distinct 
symptom  is  observed  until  the  larva  approaches  death.     (PI.  II,  A). 

Larvae  (PI.  II,  A,  B,  C,  E,  H,  I)  of  this  group  dying  or  just  dead 
of  the  disease  lose  their  marked  glistening  appearance ;  their  pearly 
whiteness  gives  way  to  a  yellowish  tint ;  the  turgidity  seen  in  healthy 
larvae  is  diminished  in  the  sick;  and  the  folds  and  furrows  indicat- 


6  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGRICULTURE. 

ing  the  segments  of  the  body  become  less  prominent.  As  the  process 
of  decay  advances  the  yellowish  hue  changes,  the  color  assuming  a 
brownish  tone.  The  segmental  markings  are  less  prominent,  while 
the  tracheae  often  become  quite  distinct,  appearing  as  white  lines 
contrasted  with  the  darker  color  of  the  larval  remains  (PI.  II,  B) . 
Not  infrequently  at  this  time  there  will  be  seen  a  chitinous  envelope 
containing  a  watery-looking  fluid  in  which  is  the  larva  proper  (PI. 
II,  C ;  PI.  IV,  A).  The  decay  proceeds  and  the  drying  becomes  evi- 
dent. The  larval  mass  settling  upon  the  concave  bottom  of  the  cell 
causes  the  upper  surface  of  the  mass  to  be  depressed  about  the  cen- 
ter. At  this  stage  the  tracheae  not  infrequently  are  seen  distinctly  in 
the  drying  mass.  When  the  larval  remains  become  dry  they  are 
known  as  the  scale  (PI.  II,  F).  The  scales  do  not  adhere  closely  to 
the  cell  and  when  removed  are  found  to  be  thin  and  more  or  less 
circular  in  outline.  They  are  convex  and  smooth  on  the  side  which 
was  in  contact  with  the  bottom  of  the  cell  while  the  opposite  surface — 
the  one  which,  while  in  the  cell,  was  toward  the  observer— is  slightly 
roughened  and  concave. 

GEOtrP  2 

Larvae  (PI.  Ill)  showing  symptoms  of  European  foulbrood  and 
classed  in  this  group  have  reached  a  sufficient  size  to  fill  the  deepest 
third  or  more  of  the  cell.  The  yellowish  tint  appears  in  contrast  to  the 
bluish  white  of  the  healthy  larva  (PI.  Ill,  D,  G) .  Increased  movement 
may  or  may  not  be  observed.  Before  and  after  death  the  remains 
may  assume  one  of  a  number  of  positions  in  the  cell.  Not  infre- 
quently a  portion  of  the  dorsal  surface  is  turned  toward  the  observer 
(PI.  Ill,  B).  Usually  through  the  transparent  area  along  the  me- 
dian dorsal  line  a  whitish  or  yellowish- white  mass  is  to  be  observed. 
This  mass  is  within  the  stomach  of  the  larva  and  contains  a  large 
amount  of  bacterial  growth  (PI.  VIII,  a,  b,  c)  consisting  very  largely 
of  Bacill'us  flniton.  Often  before  death  this  mass  is  seen  to  move 
within  the  stomach  in  response  to  the  peristalsis-like  movements  of 
the  body  of  the  larva. 

At  the  time  of  death  the  larva  usually  occupies  some  unnatural 
position,  being  more  or  less  curled  up  and  lying  upon  the  floor  of  the 
cell  (PI.  Ill,  C,  E,  F,  H,  I).  Lessened  turgidity,  a  relative  dullness 
of  the  surface  appearance,  and  a  yellowish  tint  are  present.  Not  in- 
frequently the  two  ends  of  the  larva  are  directed  more  or  less 
toward  the  bottom  of  the  cell  and  some  portion  of  the  dorsal  surface 
is  toward  the  opening  of  it  (PI.  Ill,  E,  H,  I).  Among  the  dead 
larvae  will  be  found  some  with  one  end  directed  toward  the  bot- 
tom, and  the  other  toward  the  mouth  of  the  cell,  the  body  occupying 
a  more  or  less  spiral  position  against  the  side  walls  and  floor  of  the 
cell  (PI.  Ill,  F). 


EUROPEAN   FOULBROOD.  7 

Later  the  dead  larval  remains  assume  a  brownish  tint  which 
deepens  to  varying  shades  as  decay  continues  and  drying  takes  place. 
During  the  early  part  of  the  decay,  the  firmness  of  the  body  wall  per- 
mits the  removal  of  the  larva  intact  from  the  cell.  Later,  however,  it 
offers  but  little  resistance  and  is  easily  ruptured.  The  decaying  mass 
before  di7ing  often  attains  a  certain  amount  of  viscidity.  Sometimes 
it  is  of  a  doughy  consistency,  at  other  times  it  is  purulent  or  sputum- 
like, while  at  times  it  assumes  a  viscidity  that  will  permit  of  its  being 
drawn  out  to  the  extent  of  an  inch  or  more.  When  the  larval  mass 
becomes  dry  it  forms  an  irregular  scale,  usually  brown  in  color,  lying 
on  the  floor  or  side  wall  of  the  cell  or  both,  but  not  adhering  closely 
to  them. 

GBOUP    3 

A  lar^a  dying  of  European  foulbrood  after  being  capped  may  be 
found  occupying  one  of  many  positions  within  the  cell  (PI.  IV, 
C,  D,  E ;  PI.  V,  D,  E,  F,  G,  H).  Dying  before  the  two-day  quiescent 
period  that  precedes  pupation,  the  remains  during  decay  and  as  a 
scale  resemble  in  manj'  respects  those  of  larvaj  described  in  group 
2.  The  dry  scales  occupy  usually  an  irregular  position  on  the  floor 
of  the  cell  (PI.  IV,  F,  G).  Dying  during  the  two-day  quiescent 
period,  however,  the  scales  (PI.  V,  F,  I)  resemble  very  much  those 
of  larvae  dying  at  the  same  age  of  American  foulbrood.  The  larval 
mass  assumes  the  brownish  hue  which  deepens  as  the  decay  advances, 
reaching  a  dark  brown.  Viscidity  is  present  in  the  decaying  larval 
mass,  but  the  extent  to  which  the  decaying  material  may  be  drawn 
out  is  less  than  in  American  foulbrood.  The  scale  is  less  brittle  and 
more  rubberlike. 

At  no  time  has  the  writer  observed  pupse  dead  of  European  foul- 
brood.   If  they  die  of  the  disease  it  is  a  rare  occurrence. 

The  removal  of  larvae  sick  or  dead  of  the  disease  is  accomplished 
to  a  greater  or  less  degree  by  adult  workers.  The  larvae  are  either 
partially  or  entirely  removed.  This  is  usually  done  piecemeal.  In 
an  infected  colony  will  be  found,  therefore,  the  remains  of  larvae  of 
different  ages  (PI.  IV,  B)  and  (PI.  V,  A)  in  varying  numbers. 

ETIOLOGY 

PREDISPOSING  CAUSES 

Age. — Infection  in  European  foulbrood  takes  place  during  the 
feeding  stage  and  at  some  time  after  the  first  day  of  larval  life, 
the  larvae  being  more  often  2  days  of  age,  or  older.  Death  takes 
place  somewhat  more  than  2  days  from  the  time  of  infection.  As 
a  rule,  therefore,  a  larva  has  passed  its  fourth  day  of  larval  life 
before  death  from  European  foulbrood  occurs.  From  this  age  to 
pupation  larvae  may  die  of  the  disease.    The  writer  has  not  encoun- 


8  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGRICULTURE. 

tered  death  among  brood  which  has  reached  the  pupal  stage.  Adult 
bees  are  not  susceptible  to  infection. 

8ex. — ^Worker,  drone,  and  queen  larvae  are  all  susceptible  to  in- 
fection with  Bacillus  pluton  and  any  of  these  may  die  of  European 
foulbrood. 

Eace. — Complete  immunity  from  European  foulbrood  has  not 
been  found  among  the  races  of  bees  studied.  Experimental  work 
recorded  in  the  present  paper  involved  the  use  of  at  least  five 
colonies  of  "tested  Italians,"  two  of  "tested  Carniolans,"  and  two 
of'"  tested  Caucasians."  For  the  most  part  the  bees  used  were  "  un- 
tested Italians,"  but  among  the  colonies  were  a  few  common  blacks. 
In  all  these  strains  the  disease  was  readily  produced  through  experi- 
mental inoculation.  The  examination  of  numerous  samples  of  dis- 
eased brood  received  from  beekeepers  throughout  the  United  States 
suggests  that  all  races  conunonly  kept  by  American  beekepeers  are 
susceptible  to  European  foulbrood.  The  relative  immunity  of  the 
different  races  has  not  been  demonstrated  by  the  studies.  These 
facts,  however,  do  not  dispute  the  observation  by  practical  bee- 
keepers that  some  strains  of  bees  show  a  greater  colony  resistance 
than  others.  • 

Climate. — From  reports  of  studies  made  in  Austria  by  Muck  (12), 
in  Denmark  by  Bahr  (1),  in  England  by  Cheshire  and  Cheyne  (4), 
in  Germany  by  Zander  (20),  and  in  Switzerland  by  Burri  (3),  it  is 
clearly  evident  that  the  disease  discussed  in  the  present  paper  occurs 
in  these  different  countries.  It  has  been  encountered  also  in  many 
sections  of  the  United  States  and  Canada.  This  distribution  shows 
that  the  infection  can  exist  under  a  variety  of  climatic  conditions. 
The  practical  import  of  the  fact  is  that  the  presence  of  European 
foulbrood  in  any  locality  can  not  be  attributed  entirely  to  the  climate 
of  the  region. 

Season. — Beekeepers  have  observed  that  European  foulbrood  oc- 
curs with  greatest  severity  before  midsummer  rather  than  later  in 
the  season.  The  disease,  it  has  been  shown  experimentally,  can  be 
produced,  however,  at  any  season  of  the  year  at  which  brood  is  bein.t^ 
reared.  Its  severity  at  any  given  season  is  to  be  attributed,  there- 
fore, to  environmental  conditions  rather  than  to  the  difference  in 
the  susceptibility  of  larvae  during  the  different  seasons. 

Food. — As  in  American  foulbrood  it  is  found  that  the  cause  of  the 
disease  in  the  colony  is  governed  very  little  if  at  all  by  the  quality 
of  food  gathered  by  bees.  Indirectly,  however,  the  quantity  present 
in  the  hive  or  obtainable  often  does  influence  its  course  materially. 

EXCITING  CAUSE 

That  BaciUus  alvei  may  be  present  in  large  numbers  in  brood  dead 
of  foulbrood  was  demonstrated  by  Cheshire  and  Cheyne  (4)  in  1885. 


EUROPEAN   FOTJLBROOD.  9 

For  a  decade  and  a  half  following  the  observation  the  belief  was 
quite  general  that  this  bacterium  was  the  exciting  cause  of  a  bee  dis- 
ease. The  view  was  then  seriously  challenged.  In  1906  the  only 
positive  conclusion  in  regard  to  the  relation  between  European  foul- 
brood  and  Bacillus  aivei  that  could  be  drawn  by  the  writer  (13)  was 
that  this  species  occurs  in  brood  dead  of  the  disease. 

William  E.  Howard  (6),  of  Texas,  after  a  brief  study  of  the  dis- 
ease reported  in  1900  the  presence  of  an  organism  which  he  called 
Bacillus  miUli.  He  cultivated  the  species  apparently  with  ease.  In 
1904  Bahr  (1)  in  Denmark  found  a  small  oval  bacterium  in  a  brood 
disease  in  which  larvai  dying  in  uncapped  cells  are  yellowish  in  color 
and  not  ropy  in  consistency.  Burri  (3)  in  1906  encountered  in  his 
studies  on  the  brood  diseases  a  small  bacterium  which  he  referred  to 
as  guntheri-iorms,.  The  species  was  cultured  and  compared  with 
Baeterium  guntheri  and  found  to  be  somewhat  different.  In  1907 
Maassen  (7)  obtained  from  brood  material  cultures  of  a  species 
which  he  named  Streptococcus  apis.  White  (14)  in  1908  reported 
the  presence  of  a  small  organism  in  European  foulbrood  which  had 
refused  to  grow  on  artificial  media.  The  species  was  not  the  one, 
therefore,  with  which  the  investigators  just  referred  to  had  worked. 
That  this  organism  might  be  the  exciting  cause  of  the  disease  was 
noted.  Pending  more  information  regarding  it,  the  species  was  not 
given  a  name  biit  was  referred  to  as  bacillus  "  Y."  That  this  species 
bears  a  direct  etiological  relation  to  the  disease  was  demonstrated  in 
1912  by  the  writer  (15)  and  the  name  Baoilhis  pluton  was  then  given 
to  it. 

As  the  cultivation  of  Bacillus  pluton  on  artificial  media  had  not 
been  accomplished  the  conclusion  that  it  is  the  exciting  cause  of 
European  foulbrood  was  arrived  at  by  eliminating  all  other  possible 
agencies.  The  observations  furnishing  the  proof  appear  in  an  earlier 
paper  (15).  By  demonstrating  Bacillus  pluton  to  be  the  cause  of 
the  disease,  Bacillus  alvei,  Streptococcus  apis,  Bacterium  eu/rydice, 
and  Bacillus  orpheus,  and  still  other  species  occasionally  encountered, 
were  thereby  proven,  to  be  secondary  invaders. 

To  eliminate  the  possibility  of  a  filterable  virus  in  European  foul- 
brood 10  colonies  were  inoculated  with  filtrates  obtained  from  aque- 
ous suspensions  of  brood  sick  and  dead  of  the  disease.  In  six 
instances  the  Berkefeld  N  filter  was  used  and  in  four  the  Pasteur- 
Chamberland  F  was  employed.  In  no  case  was  the  disease  produced. 
Studies  recorded  in  the  present  paper  on  the  resistance  of  Bacilhos 
pluton  to  heating,  drying,  fermentation,  and  disinfectants  show  that 
when  the  virus  of  the  disease  is  not  destroyed  this  species  is  still  alive. 
This  fact  is  further  evidence  in  support  of  the  conclusion  that  the 
species  Bacillus  pluton  is  the  virus  of  the  disease. 
132817°— 20— Bull.  810 2 


10  BXILLETIN   810,   V.   S.   DEPAKTMENT   OF   AGEICXJLTTJEE. 

BACILLBS   PLUTON 

An  artificial  medium  for  the  cultivation  of  Bacillm  piston  has  not 
yet  been  devised.  To  accomplish  this  may  or  may  not  be  a  particu- 
larly difficult  task.  The  media  ordinarily  used  m  the  laboratory  are 
not  suitable.  Bee-larv^  agar,  brood-filtrate  media,  egg-yolk-sus- 
pension agar  (19),  and  combinations  of  these  have  not  thus  tar 
proved  sufficient  for  the  purpose.  The  species  is  an  unusual  one. 
The  generic  classification  has  not  been  determined  definitely  and  this 
may  not  be  possible  until  the  proper  condition  for  the  artificial  culti- 
vation of  the  species  has  been  supplied. 

The  morphology  of  Bacillus  pluton  is  somewhat  variable,    in  very 

early  infection  its  form  is  that  of  a  short  rod  in  pairs  or  in  chains,  or 

possibly  of  a  coccus  with  the  individuals  similarly  arranged  (fag.  1; 

PI  VII  B)     The  length  is  then  equal  to  or  somewhat  greater  than 

'  the    breadth.      In    slightly    later 

stages  of  infection  the  predomi- 
nating form  is  that  of  a  lancet- 
shaped  coccus  (fig.  1;  PI.  VII, 
A),  and  in  late  stages  this  form 
is  present  almost  exclusively. 
The  lancet  form  occurs  singly, 
varying  greatly  in  size  and  hav- 
ing a  length  which  approximates 
twice  the  width.  The  length  is 
more  often  less  than  1  [^  than 
greater.  The  organism  colors 
uniformly  with  the  aniline  stains, 
FIG.  i.-BaciJius  pluton.  ^tains  with  iron  hematoxylin,  and 

is  gram-positive.  It  does  not 
form  spores.  This  is  evidenced  by  the  microscopic  appearance  and 
also  by  the  thermal  death  point  of  the  species.  Its  resistance  to  dry- 
ing, disinfectants,  and  other  environments  is  discussed  later  in  the 
present  paper. 

Seven  rabbits  inoculated,  six  subcutaneously  and  one  intraperi- 
toneally,  with  a  suspension  of  larvae  dead  of  European  foulbrood 
proved  to  be  refractory.  Only  a  slight  rise  of  temperature  followed 
the  inoculations  and  the  weight  was  not  materially  affected.  Six 
guinea  pigs  inoculated  subcutaneously  with  similar  material  proved 
not  to  be  susceptible  to  infection  with  the  species.  Four  pigeons 
inoculated  in  the  pectoral  muscles  and  two  white  rats  inoculated  sub- 
cutaneously also  proved  refractory.  In  none  of  these  inoculated  ani- 
mals were  there  any  lesions  of  particular  note  produced. 

Growth  of  Bacillus  pluton  in  the  infected  larva  begins  close  to  the 
surface  of  the  peritrophic  membrane  (PI.  VII,  I)  in  contact  with  the 
food  of  the  larva.  As  growth  continues  the  bacterial  mass  extends 
toward  the  center  of  the  lumen  of  the  peritrophic  sac  (PI.  VII,  K), 


EUROPEAN  FOULBROOD. 


11 


finally  filling  it  more  or  less  completely  (PI.  VII,  J) .  The  growth  does 
not  always  take  place  uniformly  along  the  peritrophic  membrane  (PI. 
VII,  J) ,  nor  does  it  extend  beyond  it  (PI.  VII,  I,  J,  K) ,  but  is  inclosed 
withm  the  sac,  the  tissues  of  the  larvae  not  being  reached.  The  mul- 
tiplication of  the  organism  after  the  death  of  the  host,  if,  indeed,  it 
takes  place  at  all,  is  limited. 

Secondary  invaders,  chiefly  Bacillus  alvei,  BacteHwm  emydice, 
Streptococcus  apis,  and  occasionally  Baaillus  orpheus,  and  a  few 
others,  are  encountered  at  various  stages  of  the  disease  and  during  the 
decay  of  the  lar\'a.  During  the  life  of  the  larva  these  species  also 
remain  within  the  peritrophic  sac. 


BACILLUS    ALVEI 

Bacillus  alvei  (fig.  2;  PL  VII,  D,  F)  is  present  very  frequently  and 
in  very  large  numbers  in  larvae  dead  of  European  foulbrood.  The 
species  was  well  described  by 
Cheyne  (4).  Descriptions  maybe 
found  elsewhere  also  (11,  13).  It 
is  readily  recognized  and  may  be 
differentiated  easily  from  other 
spore  -  producing  species  occa- 
sionally encountered  in  the  dis- 
eased brood. 

Bacillus  alvei  is  not  the  active 
cause  of  any  bee  disease.  It 
seems  probable,  however,  that  it 
plays  a  role  in  European  foul- 
brood,  but  the  extent  is  not  fully 
known.  The  species  is  present 
usually,  if  not  invariably,  in  large 
numbers  in  the  rubber  like  scales  (PI.  V,  F,  I),  which  resemble 
so  much  those  of  American  foulbrood.  The  decayed  larval  mass, 
which  forms  the  scale,  before  becoming  dry  is  ropy  in  consistency 
similar  to  that  of  American  foulbrood  but  to  a  less  degree.  It 
seems  probable  that  this  ropiness  is  due  more  or  less  directly  to 
Bacillus  alvei.  On  account  of  this  viscidity  the  decaying  mass,  as 
well  as  the  scales,  are  removed  with  greater  difficulty  than  ai'e  most 
larvae  dead  of  European  foulbrood.  The  result,  as  often  observed,  is 
that  these  brown  viscid  decaying  larvae  or  the  rubberlike  scales  result- 
ing from  them  are  the  only  evidence  that  European  foulbrood  is 
present  in  the  colony. 

While  Bacillus  pluton  in  such  larval  masses  and  scales  is  often  diffi- 
cult to  detect  microscopically,  its  presence  can  be  demonstrated 
through  the  experimental  inoculation  of  healthy  larva.  Inasmuch  as 
Bacillus  pluton  will  live  for  a  considerable  period  in  the  scales,  it 


Fig.  2. — Baeillus  alvei.     Spores  free  from 
and  others  within  rods. 


12  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGBICULTUEE. 

seems  quite  probable  that  the  disease  might  in  some  cases  be  carried 
over  for  months  or  even  over  winter  through  the  medium  of  these 
rubberlike  scales. 

It  is  of  interest  to  know  that  the  amount  of  disease  resulting  imme- 
diately from  inoculations  in  which  scale  material  is  used  is  much  less 
than  when  larvae  recently  dead  of  the  disease  are  used.  This  is  true 
also  of  dead  larvffi  stored  in  Petri  dishes  compared  with  smears  allowed 
to  dry  immediately  from  larvae  recently  dead  of  the  disease.  These 
facts  indicate  a  possible  deleterious  effect  on  Bacillus  pluton  of  the  sec- 
ondary invaders  multiplying  in  the  decaying  larvae. 

STREPTOCOCCUS     APIS 

It  is  most  probable  that  Streptococcus  apis  is  the  species  that  was 
isolated  from  diseased  brood  by  Burri  (3)  and  referred  to  by  him 

in  1906  as  "  guntheri-iorms."  Maas- 
sen  described  it  in  1908  (8).  The 
organism  grows  well  at  incubator, 
room,  and  refrigerator  tempera- 
tures in  most  of  the  media  ordi- 
narily used  in  the  laboratory. 
Its  cultural  characteristics  suggest 
the  micrococci  rather  than  the 
streptococci.  Confusion  in  some 
of  the  earlier  investigations  was 
due  evidently  to  the  resemblance 
of  Streptococcus  apis  and  Bacillus 
pluton  morphologically.  To  this 
FIG.  z.-streptoooccu.  avis.  ^^ct  is  due  the  chicf  interest  in  the 

species  Streptococcus  apis.  Wlien 
encountered  in  larvae  dead  of  European  f oulbrood  it  can  be  identified 
readily  by  culturing.  The  generic  position  of  this  species  should  be 
considered  as  being  not  altogether  certain. 

Occurrence. — Streptococcus  apis  is  occasionally  encountered  in  larvse  dead  oC 
European  foulbrood  and  often  is  present  in  large  numbers. 

Morphology. — It  is  more  or  less  spherical  (fig.  3 ;  PI.  VII,  E),  occurring  singly 

and  in  pairs  with  occasionally  a  chain  of  2  or  more  pairs  when  grown  in  liquid 

media.    In  larval  remains  not  infrequently  the  ends  may  be  somewhat  pointed. 

Staining  properties. — It  colors  uniformly  and  readily  witli  the  common  stains, 

and  retains  the  stain  after  Gram's  method. 

Glucose  agar  plate. — ^AYlthin  a  day  growth  is  visible.  Colonies  never  become 
large.  Surface  colonies  are  usually  less  than  2  mm.  They  are  circular  with 
uniform  outline  and  a  well-defined  border,  are  grayish  by  reflected  and  bluish 
by  transmitted  light,  are  smooth  and  convex,  are  moist  and  glistening  in  ap- 
pearance, and  are  friable  in  consistency.  When  magnified  the  surface  colonies 
appear  light  brown  in  color,  and  granular  in  structure,  the  density  decreasing 
from  the  center  to  the  periphery.  Deep  colonies  appear  dense,  dark  brown,  and 
coarsely  granular.  They  are  in  general  lenticular  to  oval  but  are  sometimes 
almost  spherical  in  form. 


EUROPEAN   FOULBROOD.  13 

Glucose  gelatin  plate.— A.t  refrigerator  temperature  and  within  3  days,  the 
surface  colonies  begin  to  liquefy  the  gelatin,  each  liquefied  area  appearins 
somewhat  as  a  minute  drop  of  water. 

Agar  slant.~la  one  day  numerous  gray  colonies  cover  the  Inoculated  surface. 

Bouillon.— Within  a  day  the  medium  is  uniformly  and  moderately  clouded. 

Fermentation.— In  glucose,  lactose,  saccharose,  levulose,  maltose,  and  man- 
nite  bouillons,  a  uniform  clouding  of  the  media  occurs.  The  growth  takes  place 
in  both  arms  of  the  tube,  but  is  heavier  in  the  open  one.  Considerable  acidity, 
but  no  gas,  is  produced. 

Milk. — Milk  is  rapidly  coagulated.  Disestion  of  the  coagulum  follows.  In 
from  3  to  5  days  more  than  one-half  has  been  changed.  Within  24  hours  the 
color  is  discharged  in  litmus  milk,  except  at  the  top  of  the  medium.  In  other 
respects  it  is  like  the  plain  milk. 

Potato.— No  visible  growth.  That  growth  in  the  potato  water  takes  place  is 
confirmed  by  microscopic  examination. 

Gelatin  stab. — Liquefaction  along  the  line  of  puncture  is  appreciable  after  one 
day.  In  four  days  a  cylinder  of  liquefied  gelatin  1  cm.  in  diameter  surrounds 
the  original  line  of  puncture  and  soon  extends  to  the  walls  of  the  tube. 

Pathogenesis. — No  disease  results  when 
the  brood  of  bees  is  fed  cultures  of 
Streptococcus  apis  either  by  the  direct 
or  indirect  method.  A  rabbit  and  two 
guinea  pigs  inoculated  with  a  pure  cul- 
ture of  Streptococcus  apis  were  not  sus- 
ceptible to  infection  with  the  species. 

BACTEKIUM   EURTDIOE 

The  presence  of  this  species  in 
European  foulbrood  was  pointed 
out  by  the  writer  in  an  earlier  pub- 
lication (15).  Among  the  second- 
ary invaders  in  larvae  infected  with 
Bacillus  pliMon,  Bacterium  eury-  ^^°-  ^■—Baeteriam  euryaiee. 

dice  is  one  of  the  earliest  to  be  found.  It  is  often  present  in  consid- 
erable numbers.  In  plating  for  the  species  the  stomach  contents  from 
larvse  sick,  but  not  dead,  of  the  disease  should  be  used.  In  studying 
this  species  cultures  were  isolated  which  in  some  respects  differed 
from  it.  Whether  these  are  different  species  or  belong  to  a  group  of 
which  Bacteriwn  eurydice  is  a  representative  has  not  been  definitely 
determined. 

To  isolate  Bacterium  eurydice  the  plating  has  been  done  with  glu- 
cose agar.  Incubation  must  be'  carried  out  at  room  temperature. 
Growth  of  the  species  is  always  slow  and  never  luxuriant.  Under 
favorable  conditions  colonies  are  visible  after  one  day.  To  preserve 
cultures  they  must  be  renewed  frequently. 

Occurrence. — Bacterium  eurydice  is  frequently  present  in  larvse  sick  or 
recently  dead  of  European  foulbrood. 

Glucose  agar  plate. — To  the  naked  eye  the  surface  colonies  are  slightly 
convex,  smooth,  and  glistening.    They  are  from  1  to  2  mm.  in  diameter,  cir- 


14  BVLLETIN   810,   U.   S.   DEPAET-MENT   OF   AGRICULTURE. 

cular  and  uniform  in  outline.  The  color  is  bluisli  by  transmitted  and  grayish 
by  reflected  light.  Under  a  two-thirds  objective  they  are  a  light  brown,  and  are 
finely  granular  near  the  periphery,  but  more  coarsely  granular  near  the 
center. 

Morphology.— The  rod  (fig.  4;  PI.  VII,  C)  is  smaU  and  slender  with  slightly 
rounded  ends,  occurring  usually  in  pairs  or  singly.  It  is  nonmotile  and  no 
spores  are  produced. 

Staining  properties.— It  is  stained  easily  and  uniformly  with  the  ordinary 
aniline  stains  and  is  Gram-negative. 

Oxygen  requrenveiits— Growth  is  better  in  the  presence  of  air  than  in  anaero- 
bic conditions. 

Bouillon.— Grov^th  takes  place  slowly,  producing  a  uniform  cloudiness  with 
no  pellicle.     After  a  week  or  more  a  somewhat  viscid  sediment  is  present. 

Sugars. — Growth  in  the  sugar  media  is  slow,  variable,  and  never  luxuriant. 
Both  arms  may  be  clouded.  Glucose  or  levulose  when  added  improves  a 
medium.  Fermentation  with  gas  does  not  take  place  in  any  of  the  sugars. 
A  noticeable  amount  of  acid  is  formed  when  glucose  and  levulose  are  used, 
the  other  sugars  being  less  affected.  A  1  per  cent  honey  solution  supports 
a  moderate  growth.     Brood  filtrate  as  a  rule  improves  media. 

Milk. — In  plain  and  litmus  milk  no  changes  are  visible. 

Potato. — Growth  on  potato  is  slow.  When  present,  the  culture  is  for  the  most 
part  grayish  in  color. 

Gelatin  stab. — A  bluish  gray  growth  appears  slowly  along  the  line  of  inocula- 
tion.   No  liquefaction  follows. 

Pathogenesis. — No  ill  results  are  ob- 
served when  cultures  of  Bacterium  eury- 
dice  are  fed  to  healthy  colonies  of  bees. 
A  rabbit  inoculated  subcutaneously  with 
a  pure  culture  proved  to  be  refractory. 

BACILLUS    ORPHEUS 

The  name  Bacillus  orpheios  was 
given  to  an  interesting  species  occa- 
sionally encountered  in  European 
foulbrood  (15).  In  one  instance 
the  species  was  found  very  widely 
distributed  in  an  apiary  in  which 
FIG.  s.—Baoiiius  orphens:  Spore  for-  heavy  losses  Were  being  sustained 
™^*i°''-  from  the  disease.    In  this  case  the 

dead  larvae  when  dry  were  stonelike  in  character,  the  petrified  re- 
mains breaking  like  so  much  marble.  Usually  the  species  is  met  with 
in  a  less  number  of  the  affected  larvae.  It  can  be  readily  identified 
from  its  morphology  and  cultural  characteristics.  A  description  of 
the  species  has  been  made  by  McCray  (9).  An  organism  similar  to 
B.  orpheus  in  many  respects  has  been  described  by  Laubach  (5)  and 
named  Bacillus  laterosporus. 

The  organism  is  a  motile  spore-bearing  rod  with  a  few  peritrichic  flagella. 
Spore  formation  begins  in  a  few  hours  on  the  surface  of  the  agar  at  incubator 
temperature,  the  rod  swelling  toward  the  center  and  becoming  fusiform.  Soon, 
as  determined  from  stained  preparations,  the  spore  is  seen  occupying  one  side 


EUROPEAN   FOULBEOOD.  15 

of  the  rod  with  the  protoplasm  distributed  along  the  opposite  side  and  the  two 
ends  (flg.  5;  PI.  VII,  H).  The  rod,  together  with  the  spore  within  It,  measures 
about  2.4|U  In  length  and  1.2^  In  width.  This  relation  of  spore  and  rod  persists 
in  cultures  on  a  solid  medium  for  a  long  period,  especially  at  room  temperature. 
Good  growth,  no  gas,  and  only  slight  changes  in  reaction  occur  In  the  sugar 
media.  A  slight  coagulum  forms  In  the  milli  which  is  slowly  digested.  Gelatin 
Is  rapidly  fluidified. 

Bacillus  orplieus  is  not  pathogenic  for  the  brood  of  bees  when  Inoculated  by 
feeding  either  by  the  direct  or  indirect  method.  Silkworm  larvsa  succumb  fol- 
lowing inoculation  by  feeding  and  also  by  puncture. 

TECHNIQUE' 

Artificial  conditions  for  the  successful  cultivation  of  Bacillus  pluton 
have  not  yet  been  obtained.  That  this  can  be  achieved  by  further 
study  is  not  at  all  improbable.  Without  having  accomplished  this, 
it  has  been  possible,  however,  to  make  the  studies  on  the  biology  of 
the  parasite  that  were  most  desired..  This  was  done  through  ex- 
perimental work,  using  the  larvae  of  bees.  The  inoculations  were 
made  by  feeding  a  suspension  of  the  organism  in  sugar  sirup. 

Two  methods  were  employed  in  making  the  feedings,  which  will 
be  referred  to  here  as  (a)  the  indirect  method,  in  which  the  colony 
is  inoculated,  and  (&)  the  direct  method,  in  which  only  a  few 
larvae  are  inoculated.  Cane  sugar  and  water  were  used  in  preparing 
the  sirup  in  the  proportion  approximately  of  3  to  2.  This  solution 
was  then  brought  to  the  boiling  point. 

From  5  to  10  diseased  larvae  furnish  sufficient  infective  material 
when  the  indirect  method  is  followed.  These,  after  being  picked 
from  the  brood  frame,  are  thoroughly  crushed,  added  to  about  300 
c.  c.  of  the  cooled  sirup,  and  fed  to  a  colony.^  When  the  suspension 
contains  the  living  virus,  symptoms  of  European  foulbrood  appear 
in  3  days  following  the  inoculation.  The  earliest  evidence  of  disease 
is  manifested  by  sick  rather  than  dead  larvae  (p.  5).  Often  frag- 
ments of  larvae  (PI.  IV,  B)  are  found  upon  examination  of  the 
brood  nest. 

In  the  direct  method  BacUlios  phuton  is  taken  from  the  stomachs 
of  infected  bees.  Sick  rather  than  dead  larvae  are  preferred  for  ob- 
taining the  virus  free  from  the  body  tissues.  By  the  use  of  dissect- 
ing needles  and  with  a  little  care  the  stomach  contents  (PI.  VIII) 
can  be  pulled  out  of  the  blind  end  of  the  organ  (15).  The  virus- 
containing  material  thus  obtained  is  triturated  with  water  and  the 
aqueous  suspension  is  added  to  sirup.  The  suspension  of  Bacilhis 
phiton  in  a  thin  sirup  is  used  in  making  the  inoculation.  Larvae 
about  2  days  old  are  especially  desirable  for  the  direct  method.  The 
inoculation  is  made  by  adding  a  small  amount  of  the  suspension  to 

1  The  technique  in  general  which  was  found  to  be  satisfactory  for  hee-disease  studies 
is  detailed  to  some  extent  in  the  sacbrood  paper  (17). 
'The  experimental  colony  is  described  in  earlier  papers  (17,  18). 


16 


BULLETIN   810,   U.   S.   DEPAETMENT   OF   AGEICTJLTUKE. 


the  food  of  the  larvae  by  means  of  a  capillary  pipette  made  from 
glass  tubing  of  small  bore.  Care  must  be  exercised  in  thus  feeding 
the  larvae.  Too  much  of  the  suspension  will  often  float  the  larva. 
There  is  danger  also  that  it  will  be  changed  in  position  mechanically 
by  means  of  the  feeding  pipette.  In  either  event  the  chances  are 
that  such  lars'ae  will  be  removed  subsequently  by  the  bees.  Consider- 
able larval  food  already  in  the  cell  is  advantageous.  This  method 
has  proved  to  be  especially  useful  in  much  of  the  experimental  work 
recorded  in  the  present  paper.  It  has  the  advantage  of  being  both 
economical  as  to  the  number  of  colonies  needed,  and  definite.  Dur- 
ing the  third  day  following  the 
hour  of  inoculation  symptoms 
of  European  f  oulbrood  will  be 
observed  if  infection  is  pro- 
duced. By  the  fourth  day  fre- 
quently all  of  the  infected  lar- 
vae will  have  been  removed  by 
the  bees.  Symptoms  of  Euro- 
pean f oulbrood  infection  mani- 
fested by  larvae  sick  rather  than 
dead  have  proved  to  be  espe- 
cially useful  for  experimental 
purposes  in  these  studies. 

During  most  of  the  time  that 
experimental  studies  are  being 
made  it  is  necessary  to  have 
fresh     diseased     material     at 
hand.    A  supply  can  be  main- 
tained by  using  one  or  more 
colonies  for  this  purpose.    Re- 
peated inoculations  of  the  col- 
ony usually  must  be  made  at  intervals  of  a  few  days  or  after  longer 
periods,  depending  on  its  condition  and  the  need  for  the  virus.    The 
indirect  method  is  especially  indicated  in  inoculating  these  colonies. 

Frequently  colonies  which  have  been  employed  in  European  foul- 
brood  experiments  can  be  used  again  for  further  experiments  on  the 
disease.  This  must  be  done  with  some  care,  however.  The  condition 
of  the  brood  always  should  be  noted  before  an  inoculation  is  made. 
European  foulbrood  colonies  serve  very  well  the  purpose  of  experi- 
mental colonies  for  the  other  brood  diseases  and  for  Nosema-disease. 
In  fact,  not  infrequently  during  these  studies  experiments  on  two  or 
more  of  the  diseases  were  in  progress  in  a  colony  at  the  same  time. 

The  apiary  (PI.  VI)  used  in  the  experimental  work  with  European 
foulbrood  was  the  same  as  the  one  employed  in  the  study  of  sac- 
brood  ( ir ) ,  Nosema-disease  ( 18 ) ,  and  American  foulbrood  ( 19 ) .  The 
hive  (fig.  6)  and  the  experimental  colonies,  where  they  were  not  the 


Pig.  6. — Experimental  hive,  having  4  Hoffman 
frames,  a  division  board,  Petri  dishes  as  feed- 
ers, the  entrance  nearly  closed  with  wire 
cloth,  and  the  opening  on  the  side  of  the 
hive  body  occupied  by  the  frames.  (Author's 
illustration.) 


EUROPEAN   FOULBROOD. 


17 


same,  were  similar  to  those  used  in  the  other  studies.  The  method 
of  making  the  inoculations  was  also  similar.  The  colonies  were,  there- 
fore, in  the  open  and  the  bees  had  free  access  of  flight.  The  same  pre- 
cautions taken  to  minimize  robbing,  swarming,  absconding,  and  drift- 
ing of  bees  were  observed  in  the  experiments  with  this  disease  as  with 
the  other  diseases.  All  hives  which  had  housed  European  foulbrood 
colonies  were  flamed  before  they  were  used  again  to  be  certain  that 
there  would  be  no  infection  from  such  a  source.  Whether  the  queens 
used  had  been  in  diseased  colonies  need  not  give  one  any  concern. 
Further  reference  to  the  technique  followed  in  the  present  studies  will 
be  made  as  the  experiments  are  discussed. 

THERMAL  DEATH  POINT  OF  BACILLUS  PLUTON 

The  result  of  the  experiments  recorded  by  the  writer  in  an  earlier 
paper  (16)  shows  that  when  suspended  in  water  the  thermal  death 
point  of  Bacillus  pluton  is  approximately  63°  C,  the  period  of  ap- 
plication being  10  minutes.  Further  experiments  have  been  con- 
ducted in  which  the  organism  was  suspended  in  honey  and  heated. 
After  being  heated,  healthy  larvae  are  inoculated  by  feeding,  using 
the  direct  or  pipette  method.  Table  I  summarizes  the  experiments 
made: 

Table  I. — Resistance  to  heat  of  Bacillus  pluton  suspended  in  honey 


Bate  of  inoculation. 

Temperature. 

Period  of 
heating. 

Results  of  inoculation. 

1915. 

'C. 
67 
70 
75 
76 
78 
79 
80 
80 
81 
85 
90 

'F. 
153 

158 
167 
169 
172 
174 
176 
176 
178 
186 
194 

MimUes. 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 

European  foulbrood  produced. 

Do 

Do.                               

Do. 

June  22 

Do. 

Do -• 

Do. 

Sept.22.         .                    

No  disease  produced. 

Do. 

Do. 

Sept.27..                            

Do. 

Do. 

Do 

Do. 

The  results  given  in  the  foregoing  table  show  that  the  thermal 
death  point  of  Bacillus  pluton  suspended  in  honey  is  approximately 
79°  C,  maintained  for  10  minutes. 

RESISTANCE  OF  BACILLUS  PLUTON  TO  DRYING 

In  conducting  experiments  relative  to  the  effect  of  drying  on 
Bacillus  pluton  the  stomach  contents  (PI.  VIII)  of  larvae  sick  or 
recently  dead  of  European  foulbrood  are  spread  in  a  thin  layer  in 
Petri  dishes  or  on  slides.  From  time  to  time  after  the  films  are  made 
healthy  larvae  are  inoculated  by  feeding  a  suspension  of  the  drying 
larval  material  suspended  in  a  weak  sirup  solution.  When  no  in- 
132817°— 20— BuU.  810 3 


18 


BULLETIN   810,   U.   S.   DEPAETMEXT   OF   AGRICULTURE. 


fection  results  the  germ  is  considered  as  having  been  destroyed. 
Observations  have  been  made  on  the  virus  kept  at  incubator,  room, 
outdoor,  and  refrigerator  temperatures  and  shielded  from  the  light 
in  each  instance.  The  experiments  conducted  with  Bacillus  plwton 
in  these  environments  are  summarized  in  Tables  II,  III,  IV,  and 
V  which  follow : 

Table  II. — Resistance  of  Bacillus  plvton  to  drying  at  incubator  temperature 


July  3 . . . 
July  10.. 
July  17.. 
July  25.. 
Aug.  3... 
Aug.  15.. 
Sept.  1 . . 
Sept.  16. 
Sept.  29. 


June  29.. 
July  13 . . 
JulyQ... 
Sept.  20. 
Sept.  17. 


Sept.  8. 


Date  of  inoculation. 


Period  of  exposure. 


Months. 


Results  of  inoculation. 


JSuropean  foulbrood  produced. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 


Do. 
Do. 
No  disease  produced. 
Do. 
Do. 


Do. 


Table  III. — Resistance  of  Bacillus  pluton  to  drying  at  room  temperature 


Date  of  inoculation. 

Period  of  drying. 

Results  of  inoculation. 

July25,1914 

Months. 

1 

1 

2 

2 

3 

3 

9 

12 

11 

11 

11 

12 

14 

14 

24 

36 

Bays. 

1 
21 

8 
21 

0 
14 
10 

6 
13 
IS 
18 

2 
10 
18 

0 

0 

European  foulbrood  produced. 

Sept.  16,1914 

Sept.  1, 1914 

Do. 

Sept.  28, 1914 

Sept.  29, 1914 

Do. 
■  Do. 
Do. 
Do. 
No  disease  produced. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 

Oct.  6, 1914 

Jan.  29, 1915 

July  9, 1915 

Aug.  3, 1914 

June  22, 1915 

June  25, 1915 

Aug.  9, 1915 

Sept.  17, 1915 

Sept.20,1915 

Sept.  8,1916 

Do 

Table  l\. —Resistance  of  Bacillus  pluton  to  drying  at  outdoor  temperature 


Date  of  inoculation. 

Period  of  drying. 

Results  of  inoculation. 

Sept.  2, 1914 

M&rWis. 

Bays. 
33 
47 
74 
21 
18 
17 
3 
17 

European  foulbrood  produced. 

Do! 
Do. 
Do. 
Do. 
No  disease  produced. 
Do. 
Do. 

Sept.  16,1914 

Oct.  13,1914 

May  26, 1915 

9 
10 
13 
12 
12 
23 

June  19, 1915 

May  17, 1915 

Aug.  3, 1915 

Aug.  17, 1915 

June  23, 1916 

EUROPEAN   FOULBKOOD.  19 

Table  V. — KcKistniirc  of  Baciltiis  ijIiiIuu  to  clri/iiii/  at  ivfrigcrator  ttniperaturc 


Dato  of  inoculation. 

Period  ot  drying. 

Results  ofinoculation. 

Oct.  17,  1915 

Months. 

Days. 

26 

28 

12 

0 

2 

0 

7 

18 

European  foullirood  produced. 

Sept.  18, 1916 

3 
6 
8 
8 
9 
10 
10 

May  3, 1916 

Do. 

June  23, 1916 

Do, 

May  26, 1916 

Do. 

June  23, 1916 

Do. 

July  31 ,  1916 

Do. 

Sept.  IS,  1916 

Do. 

From  Tuble  II  it  will  be  observed  that  Bacillus  pluton  in  a  dry 
film  made  from  the  contents  of  infected  larvaj  resisted  drying  at 
incubator  temperature  for  approximately  one  year.  Table  III  shows 
that  at  room  temperature,  other  conditions  being  similar,  the  re- 
sistance is  approximately  equal  to  that  at  incubator  temperature. 
At  outdoor  temperature,  as  shown  by  Table  IV,  the  resistance  is 
again  approximately  the  same.  At  refrigerator  temperature,  Table 
V,  the  experiments  do  not  include  the  period  at  which  Bacillus  pluton 
is  destroj^ed.  In  10  months  the  organism  was  still  viable  and  the 
results  of  the  inoculations  indicate  from  the  character  of  the  infec- 
tion produced  after  such  a  period  that  at  refrigerator  temperature 
Bacillus  pluton  will  remain  alive  for  a  longer  period  than  at  the 
other  temperatures  studied. 

RESISTANCE  OF  BACILLUS  PLUTON  WHEN  DRY  TO  DIRECT 

SUNLIGHT 

In  experiments  relative  to  the  resistance  of  Bacillus  pluton,  when 
dry,  to  the  direct  rays  of  the  sun,  smears  are  made  of  the  contents  of 
stomachs  of  European  foulbrood  larvae  in  Petri  dishes  or  on  slides, 
and  after  becoming  dry  are  exposed  to  the  direct  rays  of  the  sun. 
After  intervals  reckoned  in  hours  inoculations  are  made  by  feeding, 
using  the  direct  method.  Infection  resulting  from  such  inoculations 
shows  that  the  drying  has  not  killed  the  organism.  In  Table  VI 
experiments  performed  in  this  connection  are  summarized : 

Table  VI. — Re-iiiUs  of  inoculation  mth  Bacillus  pluton  in  a  dry  film  exposed  to 

direct  sunlight 


Date  of  inoculations. 


July  21, 1914. 
Sept.  18, 1913. 
July  31, 1914. 
Sept.  22, 1915. 
July  21, 1914. 
Sept.  27, 1915. 
Aug.  7,1914.. 
July  21, 1914. 
Aug.  7, 1914.- 
Sept.  10,1915. 
Sept.  25, 1915. 
July  22, 1914. 
Sept.  8,1915.. 


Period 

of 

exposure. 

Results  of  inoculations. 

Hours. 
3 
6 
7 
8 

10 
14 
15 
16 
20 
20 
21 
23 
27 

European  fouIlDrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

20  BULLETIN   810,   V.   S.   DEPAETMEKT   OF   AGEICULTXJEE. 

Table  \1. — Results  of  inoculation  icith  Banlliis  pluton,  etc. — Continued 


Date  of  inoculations. 


Sept.  20, 1915. 
Sept.  24, 1916. 
Sept.  14, 1915. 
Aug.  16,1915. 
Sept.  14,1915. 
Sept.  13,1915. 
Sept.  20, 1915. 
Aug.  3, 1915.. 
Sept.  14, 1915. 
Aug.  16, 1915. 
Aug.  23, 1915. 
Sept.  14, 1915. 


Period 

of 

Results  of  inoculation. 

exposure. 

Hours. 

21 

No  disease  produced. 

23 

Do. 

24 

Do. 

26 

Do. 

26 

Do. 

31 

Do. 

38 

Do. 

40 

Do. 

44 

Do. 

46 

Do. 

63 

Do. 

95 

Do. 

Observations  lecorcled  in  Table  VI  show  that  Bacillus  pluton  in  a 
dry  film  made  from  the  contents  of  the  stomachs  of  larvae  sick  or 
recently  dead  of  European  foulbrood  resists  the  direct  rays  of  the  sun 
for  from  21  to  31  hours. 

RESISTANCE  OF  BACILLUS  PLUTON  IN  WATER  TO  DIRECT 

SUNLIGHT 

In  performing  the  experiments  relative  to  the  effect  of  direct  sun- 
light on  Bacillus  pluton  suspended  in  water,  an  aqueous  suspension 
of  the  contents  of  stomachs  of  infected  larvae  is  exposed,  in  a  Petri 
dish  with  the  top  removed,  to  the  direct  rays  of  the  sun.  After  in- 
tervals reckoned  in  hours  inoculations  of  healthy  larvae  are  made  to 
determine  whether  the  organism  is  viable.  The  direct  method  is 
used.  Experiments  made  in  this  connection  are  summarized  in 
Table  VII: 


Table  VII. — Resistance  of  Bacillus  pluton  suspended  in  water  exposed  to  the 

direct  rays  of  the  sun 


Date  of  inoculation. 


Results  of  Inoculation. 


1915. 
Aug.  24 

Do 

Aug.  16 

Aug.8 

Aug.  9 

Aug.  16 

Aug.  24 

Sept.  13 

Aug.  18 

Aug.  16 

Sept.14 

Aug.  17 

Aug.  20 

Sept.l4 

Do 

Do 

July  28 

Aug.  20 


European  foulbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


Table  VII  shows  that  Bacillus  pluton,  when  suspended  in  water 
and  exposed  to  the  direct  rays  of  the  sun,  was  destroyed  in  from 
6  to  6  hours. 


EUROPEAN   FOULBROOD. 


21 


RESISTANCE  OF  BACILLUS  PLUTON  IN  HONEY  TO  DIRECT 

SUNLIGHT 

Experiments  were  made  to  determine  the  resistance  of  Bacillus 
pluton  when  suspended  in  honey  to  the  direct  rays  of  the  sun. 
In  these  experiments  a  honey  suspension  of  the  organism  obtained 
from  the  stomachs  of  infected  bees  is  exposed  to  the  sun  in  a 
Petri  dish  with  the  top  removed.  After  intervals,  reckoned  in  hours, 
inoculation  tests  are  made  using  healthy  larvae  and  the  direct 
method.  *  Table  VTII  contains  a  summary  of  the  experiments  per- 
formed : 


Table  VIII. — Resistance  of  Bacillus  pluton  suspended  in  honey  and  exposed 

to  direct  sunlight 


Date  of  inoculation. 


Period 

of 

exposure, 


Results  of  inoculation. 


1915. 
Aug.  24 

Do!!;!!;;;:;;;:;;;!;;;;:;;;: 
Aug.3 

Aug.  20 

Sept.  13 

Sept.l9 

Aug.  20 

Sept.14 

Do 

Sept.  11 

Sept.14 

Do 

Sept.  28 


HOUTS. 

1 

2 
3 


European  foulbrood  produced. 

Do: 
No  disease  produced. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 
Do. 


It  is  shown  by  the  experiments  recorded  in  Table  VIII  that  in 
direct  sunlight  Bacillus  pluton  was  destroyed  in  from  3  to  4  hours. 

The  results  obtained  by  the  experiments  summarized  in  the  la,st 
three  tables  above,  it  will  be  noted,  show  that  Bacillus  pluton  is  sus- 
ceptible to  the  destructive  effects  of  the  direct  rays  of  the  sun ;  that 
the  resistance  of  the  organism  suspended  in  honey  is  about  equal  to  its 
resistance  when  suspended  in  water;  and  when  dry  the  resistance  is 
considerably  greater  than  when  suspended  in  either  water  or  honey. 
It  is  to  be  expected  that  the  period  required  for  the  destruction  of  the 
organism  by  the  rays  of  the  sun  will  vary  with  the  intensity  of  the 
rays  at  the  time  of  the  exposure.  In  the  foregoing  experiments  clear 
days  were  chosen  and  preference  was  given  to  the  middle  of  the  day 
for  the  exposures. 

RESISTANCE  OF  BACILLUS  PLUTON  TO  FERMENTATION 

In  obtaining  data  relative  to  the  resistance  of  Bacillus  pluton  to 
fermentation,  the  stomach  contents  of  larvae  sick  or  recently  dead  of 
European  foulbrood  were  suspended  in  a  10  per  cent  sugar  (saccha- 


22 


BULLETIN   810,   U.   S.   DEPAKTMENT   OF   AGRICULTUEE. 


rose)  solution.  A  bit  of  soil  was  added  to  inoculate  it  further. 
Records  were  made  on  suspensions  fermenting  at  incubator  and  room 
temperatures,  respectively.  Tables  IX  and  X  which  follow  sum- 
marize experiments  made : 

Table  IX. — Bacillus  pluton  in  a  10  per  cent  sugar  solution  fermenting  at  incu- 
bator temperature 


Date  of  inoculation. 


Period  of 
fermen- 
tation. 


Results  of  inoculation. 


Aug.  12, 1916 
June  26, 1916. 
Sept.  2, 1916- 
Sept.  7, 1915. 
Aug.  9, 1915. 
June  30, 1916. 
Julys,  1915.. 
Aug.  24, 1915. 


Days. 
3 
5 
7 
8 
10 
15 
15 
24 


European  foulbrood  produced. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


Table  X. — Bacillus  pluton  in  a  .10  per  cent  sugar  solution  fermenting  at  room 

temperature 


Date  of  inoculation. 


Period  of 
fermen- 
tation. 


Results  of  inociilation. 


June  30, 1916. 
July  17, 1915. 
Sept.  8, 1915.. 
July  21, 1915. 
Aug.  25, 1916. 
Julys,  1916.. 
Sept.  10,  1916 
July  5, 1916. . 
Aug.  26, 1916. 
Aug.  3,  1915.. 
Aug.  9,  1915.. 
Aug.  25,  1915. 


Days. 
9 
ID 
10 
14 
16 
17 
U 
14 
21 
27 
32 
49 


European  foulbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 


The  experimental  results  contained  in  Tables  IX  and  X  show  that 
Bacillus  pluton  is  destroyed  in  a  fermenting  solution.  At  incubator 
temperature  the  virus  was  destroyed  in  from  3  to  5  days,  and  at  room 
temperature  it  was  killed  in  from  11  to  21  days. 

Similar  experiments  were  made  in  which  suspensions  in  20  per 
cent  honey  solutions  were  allowed  to  ferment  at  outdoor  temperature. 
The  records  obtained  show  that  Bacillus  pluton  in  this  environment 
was  still  alive  and  virulent  after  one  month. 

RESISTANCE  OF  BACILLUS  PLUTON  TO  PUTREFACTION 

Suspensions  of  the  contents  of  stomachs  from  larvaj  sick  or  dead 
of  European  foulbrood  were  made  in  a  1  per  cent  peptone  solution. 
Soil  was  added  to  inoculate  it  further.  Putrefactive  changes  were 
allowed  to  take  place  at  incubator  and  room  temperatures,  respec- 


EUROPEAN   FOXJLBROOD. 


23 


tively.     In  Tables  XI  and  XII.  which  follow,  are  summarized  the 
experiments  performed : 

Tablm  XI. — Bacillus  [ilutoii  in  the  presence  of  putrefactive  processes  at  incubator 

tciiiperatiirc 


T>i\io  of  inoculation. 


June  30, 1916. 
Sept.  2, 1916.. 
Aug.  15, 1916. 
Sept.  7, 1915.. 
Julys,  1916.. 
Sept.  10, 1915, 
Julys,  1916... 
Aug.  23, 1915. 
Aug.  30, 1915. 


Period  of 
putre- 
faction. 

Eosults  of  inoculation. 

Bays. 

9 

7 

8 

13 

15 

16 

18 

19 

28 

ISuropoan  toulbrood  produced. 
No  disease  produced. 
Do. 

Do 

Do 

Do. 

Do 

Do. 

Do 

Table  XII. — Bac-illioi  pluton  in  titc  presence  of  putrefactive  processes  at  room 

temperature 


Date  of  inoculation. 


Period  of 
putrefac- 
tion. 


Results  of  inoculation. 


Aug.  4, 1914. 
July  17, 1915. 
July  5, 1916.. 
July  23, 1915. 
Aug.  14, 1914. 
Sept.  17, 1915 
Aug.  25, 1916. 
Sept.  2, 1916. 
Aug.  3, 1915.. 
Aug.  28, 1916. 
Sept.  1,1914. 
Sept.  16, 1914 
Aug.  12, 1916. 


Days. 


European  foulbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 


As  shown  by  Tables  XI  and  XII  Bacillus  pluton  is  destroyed  in 
the  presence  of  putrefactive  processes.  At  incubator  temperature 
it  resisted  the*  effects  of  these  processes  for  from  7  to  13  days  and 
at  room  temperature  for  from  21  to  35  days. 

During  August  and  September,  1916,  preliminary  experiments 
were  made  testing  the  resistance  of  Bacillus  flvion  to  putrefaction 
at  outdoor  temperature.  The  parasite  was  alive  and  virulent  after 
40  days.  The  maximum  period  during  which  it  will  remain  so  has 
not  been  determined. 


VIABILITY   OF  BACILLUS  PLUTON  IN  HONEY 

Honey  suspensions  of  Bacillus  pluton  from  the  stomach  contents 
of  larvse  sick  or  recently  dead  of  European  foulbrood  were  made  and 
distributed  in  flasks  each  containing  about  300  c.  c.  These  were 
allowed  to  stand  at  room  temperature  shielded  from  the  light.  At 
intervals  thereafter  colonies  fre6  from  the  disease  were  inoculated 


24 


BULLETIN   810,   U.   S.   DEPAETMENT   OF   AGEICtJLTURE. 


each,  with  the  contents  of  a  single  flask.     A  summary  of  the  exi^eri- 
ments  is  contained  in  Table  XIII : 

Table  XIIL — Resistance  of  Bacilhis  phiton  in  lionen  at  room  temperature 


Bate  of  inoculation. 


Period  in  honey. 


Results  of  inoculation. 


May  22, 1915.. 
June  12,1915.. 
July  23, 1915.. 
June  25, 1915.. 
Aug.  23, 1915.. 
Aug.  3, 1915... 
July  12, 1915... 
Aug.  23, 1915.. 
Sept.  10, 1915.. 
Aug.  16, 1916.. 
May  19,1916.. 
May  4, 1915... 
June  7, 1913... 
June  13, 1913.. 
May  13, 1915... 
May  14, 1915.. 
May  22, 1915.. 
May  24, 1915.. 
July  31, 1916. - 
May  15, 1916.. 


Months. 


Days. 
4 
25 
6 
11 
15 
17 
25 
7 
25 
0 
0 
17 
0 
0 
25 
26 
5 
S 
11 


European  foulbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


Experimental  evidence  recorded  in  Table  XIII  shows  that  the 
virus  of  European  foulbrood  when  suspended  in  honey  at  room  tem- 
perature ceased  to  be  virulent  in  from  3  to  7  months. 

VIABILITY  OF  BACILLUS  PLUTON  IN  POLLEN 

Preliminary  experiments  were  made  to  determine  the  viability  of 
Bacillus  pluton  in  pollen.  Pollen  is  removed  from  brood-comb,  and 
an  aqueous  suspension  of  the  organism  obtained  from  the  stomachs 
of  larvae  sick  or  recently  dead  of  the  disease  is  added  to  it  until  a 
moderately  thick,  pastelike  mass  is  obtained.  This  is  distributed  in 
Petri  dishes  and  allowed  to  stand  at  room  and  refrigerator  tempera- 
tures, respectively.  After  different  intervals  of  time  Jhe  contents  of 
a  single  dish,  after  being  suspended  in  water,  are  added  to  about 
300  c.  c.  of  sirup  and  the  suspension  is  fed  to  a  colony,  using  the 
indirect  method.  The  results  show  that  Bacillus  pluton  was  viru- 
lent after  7  months  at  room  temperature  and  for  more  than  10 
months  in  the  refrigerator.  The  maximum  period  during  which  the 
organism  will  remain  alive  in  these  two  environments  has  not  been 
determined. 

RESISTANCE  OF  BACILLUS  PLUTON  TO  CARBOLIC  ACID 

Preliminary  experiments  were  made  to  determine  the  effect  of 
carbolic  acid  on  the  virus  of  European  foulbrood.  An  aqueous  sus- 
pension of  the  contents  of  the  stomachs  of  larvte  sick  or  dead  of  the 
disease  is"  first  made.  A  measured  quantity  of  this  suspension  is 
added  to  an  equal  quantity  of  an  aqueous  suspension  of  carbolic  acid 


EUROPEAN   rOULBKOOD. 


25 


of  a  strength  twice  that  dewired  in  the  experiment.  After  shaking, 
it  is  allowed  to  stand  at  room  temperature.  At  intervals  brood  free 
from  the  disease  is  fed  a  bit  of  this  suspension,  using  the  direct 
method.    Table  XIV  summarizes  the  experiments  performed : 


Table  XIV. — Effect  of  carbolic  acid  on  Bacillus  pluton 


Bate  of  inoculation. 


Aug.  22, 1914 
Aug.  M,  1914 
Julys,  1915.. 
Aug.  21, 1914 
Sept.  4, 1914. 
June  29, 1915 
Julys,  1915.. 
June  29, 1915 
Aug.  22, 1914 
Aug.  14, 1914 
Aug.  17, 1914 
Aug.  25,  1914 
Aug.  22,  1914 
June  29, 1915 


Strength 

of 
solution. 


Per  cent, 
i 

1 


Period 
of  sus- 
pension. 


Days. 

i 
1 

4 
8 

18 

15 
4 

'5 
'18 
1 
4 
4 
9 

'5i 


Results  of  inoculation. 


European  foulbrood  produced. 

Do! 

Do. 
No  disease  produced. 
European  ioulbrood  produced. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


1  Hours. 


The  experiments  outlined  in  Table  XIV  show  that  Bacillus  pluton 
withstood  a  one-half  per  cent  solution  of  carbolic  acid  for  8  days 
but  not  for  18  days;  that  it  withstood  1  per  cent  for  5  hours  but 
not  for  4  days ;  and  that  it  was  destroyed  by  2  and  4  per  cent  solu- 
tions, respectively,  in  less  than  6  hours.  Probably  it  is  destroyed 
by  these  latter  strengths  in  considerably- less  time  than  this. 

It  is  seen  by  these  preliminary  experiments  that  Bacillus  pluton 
is  destroyed  easily  by  carbolic  acid  as  a  disinfectant.  As  a  drug, 
however,  less  can  be  expected  of  it,  inasmuch  as  a  strength  twice 
that  which  the  bees  will  accept  in  honey  (Table  XV)  requires  days 
to  destroy  the  germ.  While  the  fact  does  not  furnish  conclusive 
proof  of  the  value  of  carbolic  acid  as  a  drug,  it  indicates  what 
might  be  expected  of  it  in  the  treatment  of  the  disease. 

In  using  the  results  recorded  on  the  foregoing  pages  for  the 
purpose  of  destroying  the  virus  of  European  foulbrood  and  con- 
trolling the  disease  in  practical  apiculture,  it  must  be  borne  in 
mind,  as  has  been  urged  in  the  discussions  on  the  other  bee  diseases, 
that  due  allowance  must  be  made  by  the  beekeeper  for  variations 
which  always  occur.  These,  however,  are  relatively  slight  and  can 
lye  met  readily.  In  the  destruction  of  the  virus  through  heating, 
for  example,  the  temperature  can  be  raised  a  few  degrees  above  that 
which  is  found  to  be  the  minimum  required,  or  the  time  can  be 
extended  somewhat.  Similarly  for  the  other  destructive  agencies 
the  effectiveness  of  the  process  can  be  increased. 


26  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGRICULTUEB. 

EFFECT  OF  DRUGS  ON  EUROPEAN  FOULBROOD 

rreliminary  experiments  have  been  made  to  obtain  data  relative 
to  the  effect  of  drugs  on  Bacilhis  pinion.  In  conducting  the  experi- 
ments a  suspension  of  the  stomach  contents  of  larvae  sick  or  recently 
dead  of  European  f  oulbrood  is  made  in  an  aqueous  solution  of  the  drug. 
This  is  added  to  diluted  honey  and  healthy  brood  is  fed  this  sus- 
pension. In  some  instances  the  direct  and  in  others  the  indirect 
method  was  followed.  In  Table  XY  are  summarized  the  experiments 
which  were  performed : 

Table  XV. — The  effect  of  drugs  on  European  foulbrood 


Date  of  experiment. 


July  11.. 
May  31.. 
June  T.- 
July 11. . 
June  21. 

Do.. 
May  31.. 
July  11.. 
June  7-- 
July  11. . 

Do.. 
May  31. . 
June  7. . 
July  11.. 
May  31.. 
June  7.  - 
July  11. . 
May  31.. 
June  7. . 


Drugs. 


Betanaphthol. 
-...do 


do 

Carbolic  acid. 
do 


do 

Oil  of  eucalyptus. . 

do 

Formic  acid 

do 

Salicylic  acid 


.do. 


do. 
Salol... 
..-.do. 
.do. 


Quinin. . 

do.. 

do-. 


Strength. 


1:2000 
1:1000 
2:1000 
1:2000 
1:1000 
2:1000 
4:1000 
4:1000 
1:1000 
3:1000 
1:2000 
1:1000 
2:1000 
1:2000 
1:1000 
2:1000 
2:1000 
4:1000 
10:1000 


Hesults  of  inoculation. 


European  foulbrood  produced. 


It  will  be  observed  from  Table  X^'  that  European  foulbrood  was 
produced  in  all  cases  in  which  larvae  were  fed  a  suspension  of 
Bacillus  pluton  in  sirup  medicated  with  betanaphthol,  carbolic  acid, 
eucalyptus,  formic  acid,  salicylic  acid,  salol,  and  quinin  (bisulphate 
of  quinin) ,  respectively,  in  the  proportions  noted. 

The  strongest  solutions  of  the  drugs  used  in  the  experiments  are 
in  most  instances  approximately  the  maximum  proportion  of  the 
chemical  in  honey  that  will  be  taken  by  the  bees.  These  prelimi- 
nary results  indicate  that  drugs  should  not  be  depended  upon,  for 
the  present  at  least,  in  the  treatment  of  European  foulbrood,  and 
emphasize  the  fact  that  beekeepers  should  make  sure  that  the  value 
of  a  drug  has  been  demonstrated  fully  before  it  is  used. 

TRANSMISSION  OF  EUROPEAN  FOULBROOD 

^Vhile  there  is  yet  much  to  be  learned  concerning  the  transmission 
of  European  foulbrood,  the  data  at  hand  relative  to  this  important 
phase  in  the  study  of  the  disease  justify  certain  statements  in  regard 
to  it.  The  disease  can  be  produced  experimentally  by  feeding  a 
healthy  colony  the  crushed  larva  sick  or  dead  of  the  disease,  sug- 
gesting that  infection  takes  place  by  way  of  the  alimentary  tract. 


^EnjRDPE7nr~F0ULBR00D.  27 

Through  the  study  of  microtome  sections  of  such  larvae,  it  has  been 
conclusively  proved  that  infection  takes  place  in  this  way.  The  fact 
is  naturally  one  of  special  moment  in  the  solution  of  the  transmission 
of  the  disease.  There  is  a  tendency  on  the  part  of  adult  bees  to 
remove  sick  and  dead  larvffi  from  the  brood  comb.  This  is  done 
largely  at  least  in  a  piecemeal  manner.  Were  the  fate  of  the  frag- 
ments removed  known  definitely  the  solution  of  the  problem  natu- 
rally would  be  aided  greatly. 

If  infective  material  thus  removed  were  fed  to  susceptible 
healthy  larvae,  disease  would  result.  On  the  other  hand  should  the 
fragments  of  diseased  larvae  be  stored  with  the  honey  of  the  hive 
or  with  the  pollen,  or  consumed  by  the  adult  bees,  or  by  larvae 
later  in  the  feeding  stage,  the  chances  that  such  material  would 
ever  reach  susceptible  larvae  to  cause  infection  are  very  much  re- 
duced. Stored  in  honey  the  virus  remains  virulent  only  a  few 
months  (p.  24) ;  in  pollen,  however,  it  remains  virulent  much  longer 
(p.  24).  Drying  within  the  hive  Bacillus  pluton  would  probably 
remain  alive  more  than  a  year  (p.  19). 

The  chances  that  any  portion  of  the  infectious  material  of  any 
given  fragment,  if  it  is  removed  entirely  from  the  hive  by  the  bees 
of  the  colony,  and  released  from  them,  will  be  taken  up  by  other 
bees  and  carried  to  healthy  brood  and  cause  infection  are  compara- 
tively slight.  If  thus  removed  and  exposed  to  the  direct  rays  of 
the  sun,  the  virus  will  be  destroyed  within  a  few  hours  (p.  19) ; 
or  if  subjected  to  fermentative  or  putrefactive  processes  it  will  be 
destroyed  in  a  few  weeks  (p.  23).  If  BaeilJms  pluton  is  present  in 
honey  extracted  from  diseased  colonies  it  will  be  destroyed  within 
a  few  months  while  in  storage  (p.  24).  It  is  seen,  therefore,  that  in 
nature  there  are  many  means  that  destroy  the  virus  of  European 
foulbrood  and  thus  limit  the  spread  of  the  disease. 

All  of  the  colonies  of  the  experimental  apiary  used  in  making  the 
inoculations  cited  in  the  present  paper  had  free  access  to  the  fields 
and  there  was  no  evidence  at  anj-  time  of  the  transmission  of  the 
disease  from  infected  to  healthy  colonies.  This  fact  supports  the 
conclusion  that  the  disease  is  not  spread  by  way  of  flowers  visited 
by  bees  from  healthy  colonies  which  had  been  visited  previously  by 
bees  from  diseased  ones.  The  fact  further  indicates  that  if  the  dis- 
ease is  transmitted  at  all  by  way  of  the  water  supply  of  the  bees,  it 
takes  place  to  a  limited  extent  only.  The  fact  still  further  indicates 
that  if  drones  or  straying  or  drifting  workers  transmit  European 
foulbrood  they  do  so  to  a  slight  extent  only.  If  these  observations 
are  at  variance  with  the  experience  of  the  practical  beekeepers, 
as  the  writer  has  been  informed  that  they  are,  they  will  probably  be 
of  particular  interest. 


28  BULLETIN   810,   p.    S.    DEPARTMENT   OF  AGKICULTURE. 

Observations  made  during  the  present  studies  indicate  that  queens 
from  European  foulbrood  colonies  are  not  likely  to  transmit  the 
disease  when  introduced  into  healthy  colonies.  The  experiences  fur- 
ther show,  and  the  facts  in  general  regarding  the  disease  support  the 
conclusions,  that  the  infection  will  not  be  transmitted  by  the  hands 
or  clothing  of  the  beekeeper,  or  by  visitors  to  the  apiary  when  the 
manipulations  ordinarily  practiced  are  followed.  Tools  and  equip- 
ment used  about  the  apiary  are  not  to  be  feared  unless  they  supply  a 
source  for  robbing.  Hives  which  have  housed  infected  colonies  are 
not  likely  to  be  a  medium  for  the  spread  of  the  disease. 

Eobbing  of  infected  colonies  is  the  most  fruitful  source  of  infec- 
tion. A  colony  weakened  by  disease  (p.  5)  becomes  a  prey  for  other 
bees.  Infectious  material  is  carried  to  other  colonies,  thereby  trans- 
mitting the  infection.  Manipulations  in  the  apiary,  whereby  brood 
combs  from  diseased  colonies  are  placed  in  healthy  ones,  are  another 
fruitful  source  for  the  transmission  of  the  disease.  Preliminary 
Avork^  indicates  that  stored  brood  combs  from  European  foulbrood 
colonies  may  transmit  the  disease  after  a  considerable  period. 

The  disease,  it  would  seem,  might  be  spread  through  the  medium 
of  honey  from  infected  colonies.  The  danger  from  this  source,  how- 
ever, probably  has  been  overestimated  at  times  (p.  23).  That  pollen 
stored  in  the  comb  would  serve  as  a  protection  to  Bacillus  fluton,  if 
the  parasite  were  lodged  with  it,  has  been  determined  (p.  24). 

DIAGNOSIS 

The  diagnosis  of  European  foulbrood  offers  more  difficulty  than 
does  that  of  either  American  foulbrood  or  sacbrood.  It  can  usually 
be  made,  however,  from  the  symptoms  alone.  Inasmuch  as  these 
symptoms  (p.  4)  are  rather  varied,  much  care  should  be  exercised  in 
diagnosing  the  disease. 

The  appearance  of  the  adult  bees  does  not  aid  in  the  diagnosis. 
A  weak  colony  should  arouse  suspicion.  Increased  suspicion  is  jus- 
tified when  no  other  readily  discernible  cause  for  the  weakness  is  to 
be  observed.  The  disease  may  be  present,  however,  in  a  strong  colony. 
Such  a  case  may  be  one  of  recent  infection  or  one  which  late  in  the 
recovery  from  the  disease  has  gained  in  strength.  It  may  be,  how- 
ever, a  colony  which  has  suffered  only  a  slight  attack  of  the  disease. 

The  following  outstanding  gross  characters  are  often  sufficient  for 
a  diagnosis :  The  dying  of  the  brood  before  the  time  for  capping  (Pis. 

1  Brood  combs  were  removed  from  European  foulbrood  colonies  in  October,  1914,  and 
stored  in  the  laboratory.  In  May,  1915,  one  frame  of  brood  comb  was  placed  In  each  of 
two  colonies  with  the  result  that  European  foulbrood  was  produced  In  both,  instances. 
When  a  frame  of  the  comb  was  placed  In  the  colony  in  May,  1916,  no  disease  resulted. 
After  6  months  tjie  combs  were  still  able  to  transmit  the  disease ;  after  18  months  they 
did  not.  These  experiments  are  not  sufficient  to  Justify  definite  conclusions  but  are 
suggestive. 


EUROPEAN  rOULBROOD.  29 

II,  III,  IV),  the  yellow  hue  of  the  larvae  more  recently  dead,  and 
the  brown  shade  of  those  longer  dead,  the  irregidarity  of  the  brood 
(PL  I),  and  the  absence  of  a  disagreeable  odor. 

Not  infrequently,  however,  the  diagnosis  is  not  so  simple.  During 
recovery  from  the  disease  scales  (PI.  V,  F,  I)  of  larvae  dying  in 
capped  cells  may  be  the  only  remains  of  diseased  brood  to  be  found, 
all  of  the  younger  larvaj  having  been  removed  by  the  bees.  These 
scales^  are,  as  a  rule,  comparatively  few  in  number  and  resemble 
somwhat  those  of  American  foulbrood,  but  would  rarely  be  mistaken 
for  those  of  sacbrood.  In  these  cases  a  diagnosis  can  be  made  fre- 
quently by  a  microscopic  examination  alone.  Cultures,  however,  are 
needed  in  some  instances. 

Special  attention  is  needed  in  cases  of  early  infection  and  in  other 
instances  where  only  a  small  amount  of  diseased  brood  in  uncapped 
cells  is  present  (PI.  I,  A).  The  symptoms  manifested  by  larva  sick 
or  only  recently  dead  of  the  disease  furnish  often  the  readiest  and 
most  conclusive  evidence  of  the  presence  of  the  disease.  Larvae  of 
the  age  at  which  they  comfortably  fill  the  bottom  of  the  cell  exhibit- 
ing increased  peristalsis-like  movements  of  the  body  suggest  European 
foulbrood.  Increased  transparency  of  larvae  of  this  age  (PI.  II,  B) 
is  also  suggestive.  The  presence  of  a  white  or  yellowish-white  mass 
within  the  stomach  (midgut)  as  seen  through  the  dorsal  median  line 
of  the  body  is  strong  evidence  of  the  presence  of  the  disease.  If 
on  puncturing  the  body  of  larvae  nearly  dead  or  only  recently  dead  the 
contents  of  the  stomach  flows  out  as  a  fluid  and  more  or  less  finely 
granular  mass,  the  fact  furnishes  further  evidence  of  European  foul- 
brood. 

A  symptom  which  is  pathognomonic  of  the  disease  is  to  be  seen  in 
larvae  that  have  been  infected  somewhat  more  than  two  days,  but 
wherein  the  disease  has  not  reached  an  advanced  stage.  The  test 
(15)  involves  the  removal  of  the  stomach  contents,  which  con- 
sist of  a  bacterial  mass,  together  with  a  small  amount  of  larval  food 
and  a  clear  envelope  (PI.  VIII,  a,  b,  c).  The  slight  tension  necessary 
to  remove  the  contents  stretches  the  envelope  and  breaks  the  whitish 
bacterial  mass  into  a  number  of  fragments. 

1  The  number  of  larrse  that  die  of  European  foulbrood  In  capped  cells  after  assuming 
the  endwise  position  represents  a  very  small  percentage  of  the  brood  that  dies  of  the 
disease.  These  remains  may  be  found  in  practically  all  colonies  in  which  the  disease  has 
been  present  for  a  suflaclently  long  period  and  in  which  a  considerable  amount  of  dead 
brood  has  resulted.  Before  becoming  dry  they  are  somewhat  viscid  and  are  less  easily 
removed  than  are  those  of  larvae  dying  at  an  earlier  age.  These  and  the  scales  resulting 
from  them  are  used  In  diagnosis  principally  (1)  when  the  younger  larvse  sick  or  dead  of 
the  disease  have  been  removed,  (2)  when  a  demonstration  of  the  presence  of  Bacillus  alvei 
Is  desired,  and  (3)  when  both  European  foulbrood  and  American  foulbrood  infection  is 
suspected.  Such  a  double  infection  has  been  encountered  in  the  writer's  experience  very 
rarely.  In  making  diagnoses,  therefore,  after  European  foulbrood  has  been  found  in 
the  sample  American  foulbrood  Is  seldom  looked  for. 


30  BUIXETIN   810,   U.   S.   DEPARTMENT   OF   AGRICULTURE. 

By  one  or  more  of  these  colony  symptoms  manifested  by  larvae 
sick  or  only  recently  dead  of  the  disease  the  experienced  can  diagnose 
European  foulbrood  definitely  without  a  microscopic  examination. 
The  methods  not  only  give  definite  results,  but  are  also  easy  of 
application.  They  have  been  indispensable  in  much  of  the  writer's 
experimental  work  and  it  is  believed  that  the  beekeeper  will  find 
them  to  be  valuable  in  practical  apiculture  where  other  gross  meth- 
ods fail. 

BACTERIOLOGICAL   EXAMINATION 

The  findings  from  microscopic  examinations  and  from  cultures 
liave  been  set  forth  in  an  earlier  publication  (10).  These  are  always 
adequate  for  a  definite  diagnosis  when  a  suitable  sample  is  at  hand. 
Baoillus  alvei  (p.  11)  (fig.  2;  PI.  VII,  D,  F)  frequently  overshadows 
all  other  species.  In  larvje  sick  of  the  disease  Bacilkis  pluion  (PL 
Yll,  A,  B)  overshadows  all  others.  With  experience  one  learns  to 
recognize  this  species  in  stained  preparations.  The  individuals  are 
seen  frequently  in  groups.  They  are  more  or  less  lancet  shaped,  and 
a  variation  in  size  is  often  sufficient  to  be  noticeable  (fig.  1).^  In 
larvae  nearly  dead  and  in  those  only  recently  dead  Bacterium,  eurydice 
(p.  13)  (fig.  4;  PI.  VII,  C)  is  frequently  encoimtered.  Streptococ- 
cus apis  (p.  12)  (fig.  3;  PI.  VII,  E)  occurs  in  a  small  number  of 
cases.  Bacillus  orpheus  (p.  14)  (fig.  5;  PI.  VII,  H),  B.  vulgatvis, 
and  B.  mesentencus  are  occasionally  encountered.  While  B.  pluton 
is  present  in  all  cases  of  European  foulbrood,  not  infrequently  in 
routine  examinations  it  is  so  masked  by  the  secondary  invaders  that 
the  microscopic  examination  fails  to  reveal  it.  In  many  cases  B. 
alvei  and  B.  orpheus  are  recognized  microscopically.  Cultures  are 
necessary  for  the  differentiation  of  B.  vulgatus  and  B.  mesentericus. 
In  many  cases  cultures  are  needed  to  differentiate  Strep,  apis  and 
B.  pluton.  Strep,  apis  grows  on  the  ordinary  media,  B.  pluton 
does  not. 

DIFFERENTIAL  DIAGNOSIS 


AMEMOAN   rOUIBEOOD 


American  foulbrood  is  recognized  by  the  death  of  larvae  in  capped 
cells  and  of  pupse  soon  after  transformation,  the  viscidity  of  the  decay- 
ing remains  of  the  brood,  and  the  "  foulbrood  "  odor  which  is  fre- 
quently present.  The  presence  of  the  spores  of  Bacillus  larvae  in 
large  numbers  and  the  absence  of  other  species  is  conclusive  proof 
of  American  foulbrood. 

1  Smears  made  from  laryse  sick  of  European  foulbrood  and  quite  early  In  the  course  of 
tlie  disease  were  selected  in  malsing  a  study  of  the  morphology  of  B.  pluten.  These  were 
stained  with  iron  hematoxylin.  In  smears  made  from  dead  larrse  and  stained  with  carhol 
fuchsin,  as  is  usually  done,  the  pointed  ends  and  the  more  or  less  rod-shaped  forms  are 
less  prominent  than  illustrated  in  figure  1. 


EUROPEAN  FOULBROOD.  31 


S&CBBOOn 


Sacbrood  is  recognized  by  the  death  of  larvae  after  capping,  by  the 
saclike  appearance,  the  watery  granular  consistency  of  the  larval 
remains,  and  the  absence  of  viscidity.  The  absence  of  microorgan- 
isms characterizes  the  microscopic  picture  in  sacbrood. 


OTHER  CONDITIONS 


Conditions  referred  to  as  chilled  brood,  overheated  brood,  and 
starved  brood  must  be  differentiated  from  European  foulbrood.  This 
can  usually  be  done  with  little  diiRculty  by  a  comparison  of  the  symp- 
toms present  with  those  of  European  foulbrood.  The  history  of 
the  case  is  of  much  value.  Brood  dying  after  being  removed  from 
the  hive  and  before  examination  is  made  shows  often  an  interest- 
ing similarity  to  European  foulbrood.  B.  alvei  and  B.  flmton  are  not 
found  in  these  conditions.  The  absence  of  bacteria,  or  their  presence 
in  small  numbers  only,  and  a  lack  of  uniformity  of  the  species  when 
present,  characterize  the  bacteriological  findings  in  these  cases. 

PROGNOSIS 

There  is  no  uniformity  in  the  prognosis  in  European  foulbrood. 
The  diseased  colony  may  recover  completely  from  the  infection,  suf- 
fering only  a  slight  loss  in  strength  as  a  result  of  it ;  the  colony  may 
recover  but  sustain  considerable  loss ;  or  it  may  die  out  entirely,  as  a 
result  of  the  disease.  The  infection  may  spread  only  slightly  to  other 
colonies  of  the  apiary  or  the  entire  apiary  may  become  infected.  The 
losses  sustained  vary  from  slight  to  total.  The  tendency  for  Euro- 
pean foulbrood  to  disappear  is  greater  after  midsummer  than  before. 

Whether  a  larva  once  infected  ever  recovers  from  this  disease  is 
not  known,  but  the  evidence  at  hand  indicates  that  it  may.  This 
seems  to  be  especially  probable  when  the  infection  takes  place  during 
the  latter  part  of  the  feeding  period  of  the  larva.  Queen  larvse  are 
susceptible  to  infection,  but  sufficient  data  are  wanting  from  which 
to  estimate  the  extent  to  which  queenlessness  may  result  from  the 
disease.  In  experimental  colonies  queens  have  been  reared  in  the 
presence  of  a  considerable  amount  of  European  foulbrood  infection. 

The  prognosis  for  the  colony  in  the  case  of  European  foulbrood 
may  be  said,  therefore,  to  vary  from  very  good  to  very  grave,  many 
recovering  entirely  from  the  infection  without  treatment  and  without 
appreciable  losses,  while  others  rapidly  decline  and  finally  die  out. 

SUMMARY  AND  CONCLUSIONS 

The  following  is  a  brief  summary  of  facts  regarding  European 
foulbrood,  together  with  some  conclusions  based  upon  them: 
1.  European  foulbrood  is  an  infectious  brood  disease  of  bees  caused 
by  Bacillus  pluton. 


32  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGKICtTLTURE. 

2.  All  larvae — worker,  drone,  and  queen — are  susceptible  to  the  dis- 

ease; adult  bees  are  not. 

3.  Man  evidently  is  not  susceptible  to  infection  with  Bacillus  pluton 

nor  are  the  experimental  animals. 

4.  As  far  as  is  known  insects  other  than  bees  are  not  susceptible. 

6.  Brood  can  be  infected  by  feeding  the  colony  a  suspension  of 
crushed  larvae  sick  or  dead  of  the  disease.  This  is  described  in 
the  present  paper  as  the  indirect  method. 

6.  The  virus  contained  in  a  single  larva  recently  dead  of  European 

foulbrood  will  produce  a  considerable  amount  of  disease  when 
fed  to  a  colony. 

7.  The  larvae  can  be  infected  also  by  a  more  direct  method.     A 

fraction  of  a  drop  of  a  suspension  of  the  stomach  contents  of 
a  larva  sick  of  the  disease,  added  with  a  capillary  pipette 
directly  to  the  food  surrounding  the  larva  to  be  inoculated 
will  result  in  infection. 

8.  BaciUus  pluton  gains  entrance  to  the  larva  by  way  of  the  mouth. 

The  growth   and  multiplication  of  the  parasite  take  place 
within  the  stomach  (mid-intestine)  of  the  larva  and  do  not, 
during  the  life  of  the  larva,  get  beyond  the  peritrophic  mem- 
brane.   The  tissues,  therefore,  are  not  invaded  by  it. 

9.  The  secondary 'invaders  in  European  foulbrood.  Bacillus  alvei, 

Streptococcus  apis,  Bacterium,  eurydice,  and  Bacillus  orpheus, 
rarely,  if  ever,  invade  the  tissues  until  the  larva  is  dead  or 
nearly  so.  In  a  few  instances  in  microtome  sections  rod  fonns 
have  been  encountered  in  the  act  of  invading  the  tissues  of 
living  larvae.  The  species,  however,  was  not  determined  defi- 
nitely. 

10.  The  period  of  incubation  is  slightly  less  than  3  days. 

11.  Brood  is  susceptible  to  infection  at  all  seasons  of  the  year. 

12.  More  brood  die  of  the  disease  during  the  first  half  of  the  brood- 

rearing  season  than  during  the  second  half. 

13.  The  writer  has  examined  samples  of  the  disease  from  Canada 

and  the  United  States.  From  written  reports  it  seems  quite 
certain  that  it  occurs  also  at  least  in  Denmark,  England,  Ger- 
many, France,  and  Switzerland. 

14.  Occurring  as  it  does  in  this  somewhat  wide  range  of  climatic 

conditions,  the  presence  of  the  disease  in  any  particular  locality 
can  not  be  attributed  entirely  to  the  prevailing  climatic  con- 
ditions. 

15.  The  quality  of  food  obtained  by  the  bees  does  not  affect  greatly, 

if  at  all,  the  course  of  the  disease  in  the  colony,  although  the 
quantity  may  affect  it  to  a  variable  extent. 


EUROPEAN  FOULBEOOD.  33 

16.  Experimental  colonies  may  be  inoculated  and  kept  in  the  apiary 

without  transmitting  the  disease  to  others.  This  fact  is  of 
special  importance,  not  only  in  connection  with  the  technique 
of  making  studies  on  the  disease,  but  also  in  the  control  of  the 
malady. 

17.  The  thermal  death  point  of  Baoillm  pluton  suspended  in  water 

is  approximately  63°  C.  maintained  for  10  minutes. 

18.  ^Vhen  suspended  in  honey  Baoillus  pluton  is  destroyed  in  10 

minutes  at  approximately  79°  C. 

19.  Drying  at  room  or  incubator  temperature  Bacillus  phiton  re- 

mains alive  and  virulent  for  approximately  one  year. 

20.  Wlien  dry,  Bacillus  pluton  resisted  the  direct  rays  of  the  sun  for 

from  21  to  31  hours. 

21.  When  suspended  in  water  Bacillus  pluton  was  destroyed  by  the 

direct  rays  of  the  sun  in  from  5  to  6  hours. 

22.  "When  suspended  in  honey  and  exposed  to  the  direct  rays  of  the 

sun  Bacillus  pluton  was  destroyed  in  from  3  to  4  hours. 

23.  In  the  presence  of  fermentative  processes  in  a  10  per  cent  sugar 

solution  Bacillus  plvion  was  destroyed  in  from  3  to  6  days  at 
incubator  temperature  and  in  from  11  to  21  days  at  room 
temperature. 
2-1.  In  a  fermenting  honey  solution  outdoors  Bacillus  pluton  was 
still  alive  and  virulent  after  one  month. 

25.  In  the  presence  of  putrefactive  processes  at  incubator  tempera- 

ture Bacillus  pluton  was  destroyed  in  from  7  to  13  days  and  at 
room  temperature  in  from  21  to  35  days. 

26.  In  a  putrefying  medium  at  outdoor  temperature  Bacillus  pluton 

remained  alive  and  virulent  for  more  than  40  days.  The  maxi- 
mimi  period  has  not  been  determined. 

27.  In  honey  at  room  temperature  Bacillus  pluton  ceased  to  be  viru- 

lent in  from  3  to  7  months. 

28.  Mixed  with  pollen.  Bacillus  pluton  remained  alive  and  virulent 

for  more  than  7  months  at  room  temperature  and  more  than 
10  months  at  refrigerator  temperature,  the  maximum  time  not 
being  determined. 

29.  In  one-half  per  cent  carbolic  acid  solution  Bacillus  pluton  was 

destroyed  in  from  8  to  18  days ;  in  1  per  cent  it  was  destroyed  in 
from  5  hours  to  4  days,  and  in  2  and  4  per  cent  ip  less  than 
6  hours.  The  probability  is  that  at  these  higher  strengths  of 
the  solution  minutes  rather  than  hours  are  sufficient  for  the 
destruction  of  the  virus. 

30.  Experimental  evidence  indicates  that  at  the  present  time  drugs 

should  not  be  depended  upon  in  the  treatment  of  European 
foulbrood. 


34  BULLETIN   810,   U.   S.   DEPARTMENT   OF   AGEICXJLTUKE. 

31.  Robbing  from  diseased  colonies  of  the  apiary  or  from  neigh- 

boring apiaries  is  the  most  likely  manner  in  which  European 
foulbrood  is  transmitted  in  nature. 

32.  Brood-combs  containing  diseased  brood,  if  given  to  a  healthy 

colony,  serve  as  a  medium  for  the  transmission  of  the  disease. 

33.  European  foulbrood  is  not  likely  to  be  transmitted  by  queens  or 

drones.    Whether  they  ever  do  so  has  not  been  demonstrated. 

34.  As  a  rule  a  hive  which  has  housed  a  European  foulbrood  colony 

should  not  be  considered  as  a  fruitful  source  of  infection.  The 
facts  indicate  that  often  such  hives  could  be  used  with  im- 
punity for  housing  colonies  without  treatment.  Flaming 
them  inside  certainly  removes  all  danger. 

35.  The  transmission  of  European  foulbrood  by  way  of  flowers, 

visited  by  bees  from  diseased  colonies  and  subsequently  by 
those  from  healthy  ones,  is  not  to  be  considered  as  a  likely 
source  of  infection.  Whether  the  water  supply  is  ever  a  source 
of  danger  is  not  known.    It  is  evidently  not  a  fruitful  source. 

36.  The  disease  is  not  likely  to  be  transmitted  through  the  medium  of 

the  clothing  or  hands  of  the  apiarist. 

37.  Tools  and  bee  supplies  in  general  do  not  serve  as  means  for  the 

transmission  of  the  disease  in  the  absence  of  robbing  from  such 
sources. 

38.  It  is  usually  possible  to  diagnose  European  foulbrood  from  the 

symptoms  alone.  A  definite  diagnosis  can  be  made  from  suit- 
able samples  by  bacteriological  methods. 

39.  The  prognosis  in  European  foulbrood  varies  from  very  good  to 

exceedingly  grave.  The  tendency  for  a  colony  to  recover  en- 
tirely from  the  disease  is  much  greater  than  in  American 
foulbrood. 

40.  Considered  from  the  technical  point  of  view,  much  is  yet  to  be 

learned  concerning  European  foulbrood.  For  practical  pur- 
poses, however,  it  can  be  said  that  sufficient  knowledge  has 
been  gained  to  make  it  possible  for  the  beekeeper  to  devise  a 
treatment  which  will  be  logical,  efficient,  and  at  the  same  time 
economical. 

LITERATURE  CITED 

(1)  Bahb,  Louis. 

1904.  "Vore  bisygdomme.  Foredrag  holdt  ved  DBF's  Diskusslonsm0de  i 
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(2)  

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Kgl.  Veterinser-og  Landboh^jskoles  Serumlaboratorinm 
XXXVII.    109  p.,  11  fig. 


EUROPEAN   FOULBROOD.  35 

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(8)  

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1918.  The  diagnosis  of  bee  diseases  by  laboratory  methods.    U.  S.  Dept. 

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(13)  White,  G.  F. 

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(14)  

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(15)  

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(16)  

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(17)  White,  G.  F. — Continued. 

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ruary 9. 

(18)  

1918.  Nosema-dlsease.     U.  S.  Dept.  Agr.  Bui.  780.     59  p.,  7  fig.,   4  pi. 

(Professional  paper.)    June  12. 

(19)  

1920.  American  foulbrood.    U.  S.  Dept.  Agr.  Bui.  809.    46  p.,  9  fig.,  8  pi. 
March  10,  1920. 

(20)  Zandee,  Enoch. 

1910.  Die  Faulbrut  und  ihre  Bekampfung.     32  p.,  8  fig.,  4  pi.     Stutt- 
gart.    (Handbueh  der  Bienenkunde  I.) 


EXPLANATION  OF  PLATES 


Plate  I 


Brood-combs  containing  larvse  that  are  sick  and  others  that  are  dead  of  Euro- 
pean foulbrood,  showing  the  Irregular  appearance  of  the  brood.  About  one-half 
natural  size. 

A. — The  dead  larv£e  have  all  been  removed.  Some  of  the  remaining  larvse 
are  sick,  others  are  not  infected.  The  disease  was  produced  by  experimental 
inoculation. 

B. — Many  of  the  dead  larvce  have  not  been  removed.  The  comb  had  been 
out  of  the  colony  for  a  considerable  period.  The  larvae  that  are  quite  young 
showing  abnormal  position  and  appearance  are  not  sick  or  dead  of  European 
foulbrood,  but  are  so  as  a  result  of  the  comb  being  away  from  the  colony. 
Disease  was  produced  by  experimental  Inoculation. 

C. — ^The  comb  was  taken  from  a  colony  in  which  the  disease  had  appeared 
in  nature  and  not  as  the  result  of  artificial  inoculation.  Before  being  photo- 
graphed the  brood-comb  had  been  out  of  the  hive  for  a  few  days.  Aside  from 
the  larvse  which  are  dead  of  European  foulbrood,  other  larvse  present  are  dead 
from  lack  of  attention  by  adult  bees — starvation,  exposure,  and  other  causes. 

Plate  II 

A. — Live  larva  showing  first  symptoms  of  European  foulbrood.  The  tur- 
gidity  is  slightly  less  than  in  a  healthy  larva  (D). 

B. — ^Live  larva  showing  early  symptoms  of  European  foulbrood.  The  body 
is  more  transparent  than  that  of  a  healthy  larva  (D).  Small  opaque  areas  give 
it  a  punctate  appearance. 

C. — ^Larva  dead  of  European  foulbrood  contained  within  a  chitinous  envelope 
iilled  with  a  watery-appearing  fluid. 

D. — ^Healthy  larva  of  the  earliest  age  at  which  larvse  die  of  European  foul- 
brood.   Turgidity  marked. 

E. — European  foulbrood  larva  which  may  or  may  not  be  dead.  Surface  less 
glistening  than  in  healthy  larvse.  Marked  turgidity  lost.  Prominence  of  tracheae 
not  increased. 

P. — Scale  formed  by  drying  of  larvae  dead  at  early  age.  Prominence  of 
tracheae  marked. 

G. — View  of  healthy  larva  in  normal  position  with  roof  of  cell  removed. 
Larva  turgid.     Surface  glistening. 

H. — ^Larva  sick  with  European  foulbrood.  Lack  of  turgidity  and  increased 
prominence  of  tracheae  observed. 

I. — European  foulbrood  larva  which  may  or  may  not  be  dead.    Less  turgidity, 

a  relative  dullness  in  the  surface  appearance,  and  punctate  condition  present. 

Similar  to  B. 

Plate  III 

A. — Healthy  lai;va  immediately  preceding  the  age  at  which  the  capping  of  the 
cell  is  done.  Dorsal  surface  turned  toward  the  observer.  The  narrow  trans- 
parent area  along  the  dorsal  median  line  is  prominent. 

B. — ^Larva  dead  of  European  foulbrood  of  the  same  age  as  A.  The  turgidity, 
glistening  surface,  and  transparent  area  are  less  marked. 

0. — ^Larva  dead  of  European  foulbrood  partly  coiled  and  partly  endwise  in 

cell. 

37 


38  BULLETIN   810,   U.   S.   DEPAETMENT   OF  AGRICtrLTTJRE. 

D. — ^Healthy  larva  near  the  age  at  which  capping  takes  place. 

E.— Dorsolateral  view  of  a  larva  dead  of  European  foulbrood.  The  ends  are 
directed  toward  the  bottom  of  the  cell. 

F.— Larva  dead  of  European  foulbrood.  The  body  occupies  a  spiral  position 
in  the  cell. 

G.— Healthy  larva  approaching  the  age  at  which  capping  takes  place. 

H. — Lateral  view  of  larva  dead  of  European  foulbrood  seen  with  the  roof  of 
the  cell  removed.  The  ends  are  directed  toward  the  bottom  and  the  dorsal 
surface  toward  the  mouth  of  the  cell. 

L — Dead  larva  similar  to  H  but  having  been  dead  somewhat  longer. 

Plate  IV 

A. — ^Toung  larva  dead  of  European  foulbrood.  The  chitinous  capsule  and 
tracheae  are  prominent. 

B. — Fragments  of  young  larva  dead  of  European  foulbrood,  a  portion  having 
l)een  removed  by  adult  bees  after  its  death. 

C. — Lateral  view  of  larva  dead  of  European  foulbrood,  the  roof  of  the  cell 
having  been  removed.  The  ends  in  this  instance  are  directed  more  or  less  to- 
ward the  mouth  of  the  cell. 

D. — Lateroventral  view  of  larva  dead  of  European  foulbrood.  The  body  lies 
with  the  dorsal  portion  against  the  floor  of  the  cell. 

E. — Larva  dead  of  European  foulbrood  lying  on  the  floor  of  the  cell  in  some- 
what lengthwise  position. 

F. — Scale  of  European  foulbrood  larva  which  had  occupied  a  somewhat  spiral 
position  in  the  cell. 

G. — Scale  of  a  European  foulbrood  larva  which  had  occupied  a  position  some- 
what as  shown  in  D.  This  scale  and  the  one  shown  in  F  can  be  removed  intact 
rather  easily  and  without  tearing  the  waU  of  the  cell. 

Plate  V 

Larvaa  (prepupse)  of  bees  dead  of  European  foulbrood  which  had  already 
assumed  before  death  a  lengthwise  position  in  the  cell. 

A. — Fragment  of  European  foulbrood  soon  after  death.  A  portion  of  the 
larva  has  been  removed  by  the  adult  bee. 

B. — Entire  cap  of  ceU  containing  larva  dead  of  European  foulbrood. 

C. — Punctured  cap  of  cell  containing  the  remains  of  a  larva  dead  of  Euro- 
pean foulbrood. 

D. — End  view  of  larva  dead  of  European  foulbrood. 

E. — End  view  of  larva  dead  of  European  foulbrood,  lying  with  its  dorsal 
surface  against  the  floor  of  the  cell.  Considerable  drying  of  the  remains  has 
taken  place. 

F. — End  view  of  scale  of  European  foulbrood  larva  which  had  reached  be- 
fore death  the  age  at  which  the  endwise  position  in  the  cell  is  assumed. 

G. — Ventral  view  of  European  foulbrood  larva.  Stage  similar  to  D.  Turgid- 
ity  is  lost  to  a  large  extent  and  the  segmented  markings  are  less  distinct  than 
in  healthy  larvae. 

H.— Larva  which  has  been  dead  of  European  foulbrood  for  a  longer  period 
than  illustrated  in  G.  The  ridge  and  furrows  indicating  the  segments  of  the 
body  are  not  marked. 

I.— Scale  of  European  foulbrood  similar  to  F.  The  larva  before  death  had 
reached  the  endwise  position  in  the  cell.  These  scales  resemble  very  much 
those  of  American  foulbrood.  They  are  more  easily  removed,  however,  do 
not  adhere  so  closely  to  the  floor  of  the  cell,  and  are  more  rubberlike  in  'con- 
sistency, breaking  less  readily  than  those  of  American  foulbrood. 


EUROPEAN  POULBROOD.  39 

Plate  VI 
A  view  of  the  exptMiiuental  tiiiinry  of  54  colonies  iu  which  tlie  inoculation  ex- 
periments made  during  the  sunnner  of  1915  were  conducted. 

Plate  VII 

Photomicrosniphs  illustrutinn  the  wore  commonly  encountered  bacteria 
in  European  foulbrood. 

A. — Bacillus  phuton:  A  smear  from  tlie  siomacli  of  a  larva  sick  with  Euro- 
pean foulbrood.  Note  the  paired  forms  and  short  chains.  These  forms  are 
numerous  In  a  recent  infection,  suggesting  the  organism  in  the  process  of  mul- 
tiplication. The  lancet-shaped  form  is  by  far  the  predominant  one  in  all  later 
stages  of  the  disease.     X  1000. 

B. — Bacillus  phiton:  A  smear  from  a  larva  quite  recently  infected.  The 
multiplying  paired  forms  are  at  this  stage  present  almost  exclusively.     X  1000. 

C. — Bacterium  curydkc:  Stained  preparation  from  a  pure  culture  on  the 
surface  of  agar.     X  1000. 

D. — Bacillus  alvci:  Stained  preparation  showing  spores  and  spore  forma- 
tion.    X  800. 

E. — Streptococcus  apis:  Stained  preparation  from  a  pure  culture.     X    800. 

P. — Ba-cillus  alrci:  The  peculiar  arrangement  of  the  spores  as  sometime.s 
seen.  From  a  pure  culture,  the  smear  having  been  made  by  suspending  the 
culture  on  the  slide  in  normal  salt  solution.     X  1000. 

G. — Bacillus  orpheus:  Stained  preparation  made  from  a  pure  culture  only 
a  few  hours  old.     Grown  on  the  surface  of  agar.     X  1000. 

H. — Bacillus  orpheus:  Stained  preparation  showing  spore  formation.  Note 
the  stained  portion  along  one  side  and  about  both  ends  of  the  spore.  The 
stage  is  soon  reached  in  a  culture  at  incubator  temperature.  At  room  tempera- 
ture it  remains  in  this  stage  for  a  considerable  period.     X  800. 

I. — Longisection  of  a  young  larva  showing  early  infection  in  European 
foulbrood.  The  bacterial  growth  is  seen  as  a  narrow  black  area  just  within 
the  peritrophic  membrane  on  one  side  of  the  food  mass. 

J. — ^Longisection  of  larva  sick  of  European  foulbrood,  showing  a  later  stage 
of  infection  than  that  present  in  I.  The  dark  area  in  the  food  mass  shows 
the  bacterial  growth.  Note  that  the  growth  mass  does  not  extend  beyond 
the  peritrophic  membrane  and  that  it  does  not  extend  uniformly  along  this 
membrane  and  throughout  the  food  mass. 

K. — Transverse  section  of  larva  about  the  time  of  its  death  from  European 
foulbrood  infection.  Note  the  bacterial  mass  along  the  peritrophic  mem- 
brane and  extending  from  the  membrane  into  the  food  mass.  As  seen  within 
the  living  larva  this  bacterial  mass  in  the  sick  larva  is  practically  white,  but  is 
more  or  less  yellowish  white  when  present  with  larval  food  material.  The 
gelatinous-like  envelope  outside  the  peritrophic  membrane  and  inside  the  stom- 
ach epithelium  in  healthy  larvje  thins  out  as  the  disease  advances. 

Plate  Vni 
The  stomach  contents  of  larviB  sick  of  European  foulbrood  removed  from 
the  organ.  The  anterior  end  of  the  larva  is  shown.  Fairly  early  stage 
of  infection  (a)  showing  the  white  bacterial  mass  broken  into  fragments 
as  a  result  of  the  tension  produced  in  removing  the  stomach  contents  from 
the  organ.  A  somewhat  later  stage  (b)  in  the  course  of  the  disease,  show- 
ing the  bacterial  growth  contained  in  the  stomach  fragmented,  also  the 
mucous  or  gelatinous  envelope  surrounding  the  petritrophic  membrane.  The 
stomach  contents  removed  from  a  European  foulbrood  larva  (c)  about  the 
time  of  its  death.  The  bacterial  growth  at  this  time  is  surrounded  by  very 
little  other  than  the  peritrophic  membrane.  When  this  membrane  is  ruptured 
the  contents  flow  out  as  a  thin  yellowish-white  mass.    • 


Plate  I. 


fill 

^ 

istfai«fyjigg&^ 

v't-v-.>"^V 

:  -^  -  cr-'^:-^-  T:.-cr«r-'  i^  i 

yiS^i 

^Si^^^^rT^jE^^j^^HlHl^li^na^Si 

MiffliPi^EMi 

HkJflSU<^JMKr  jB^H 

European  Foulbrood. 


Bui.  810,  U.  S.  Dept.  of  Agriculture. 


A 


B 


Plate  II. 


D 


H 
European  Foulbrood. 


Bui,  810,  U.  S.   Dept.  of  Agriculture. 


PLATE  III. 


B 


D 


European  Foulbrood. 


Bui.  810,   U.  S.   Dept.  of  Agriculture. 


PLATE  IV. 


European  Foulbrood. 


Bui.  810,  U.  S.  Dept.  of  Agriculture. 


PLATE  V. 


H 


European  Foulbrood. 


Bui.  810,  U.  S.   Dept.  of  Agriculture. 


Plate  VI. 


Bui,  810,  U.  S.   Dept.  of  Agriculture. 


PLATE  VII. 


European  Foulbrooo. 


Bui.  810,  U.  S.  Dept.  of  Agriculture. 


PLATE  VIII, 


European  Foulbrood. 


UNITED  STATES  DEPARTMENT  OF  AGRICULTURE 
BULLETIN  No.  431 


Oonlrtbntlon  IVom  the  Bureau  of  Entomologr 
t.  O.  HOWARD.  CUef 


Washington,  D.  C. 


PROFESSIONAL  PAPER 


Febraary  9, 1917 


SACBROGD 


G.  F.WHITE 
Expert,  Engaged  in  the  Investigation  of  Bee  Diseases 


CONTENTS 


Blitoiical  Aceouit  ..•....•. 

Nwne  of  the  Disease 

AReennce  of  Healthy  Breed  at  the  Ace 
MlindchitDleBefSacbnMd    .    .    .    . 
ItoaiBof  Sacbrood   ....... 

e«rSacbroad  . 

I  Effect  of  Sacbrood  Upon  a 

i&luinnt  of  Tiros  Bevdred  to  Produce 
■  )ie  Dbease.  and  the  Bapidtty  of  its 

in  Making  Ezperimentai 


Page 

I 
2 

4 

6 
10 
24 

SO 


31 

_      _  32 

sDeMmefienof  the  Tims  of 

_-^  to  Destroy  Sacbrood 

iSnapended  In  Water     .    .     34 

IjMuired  to  Destroy  Saclwoad 
"^"■Saqeaded  in  Glycerine   .     35 
^        aired  to  Itestroy  Sacbrood 
TlQuinien  Suspended  in  Honey    .    .     36 
Rcdstsnee  of  Sacbrood  Tins  to  Oifiac 
atBoantltaBpantua.  ...  ._.  .  .     37 


Bcststaoo)  of  Sacbrood  Ttrha  to  Direct 
Snnllrin  When  Dry ........ 

BeslBtanee  of  Sacbrood  Tims  to  Direct 
Sunlight  When  Suspended  in  Water   . 

BeslBtanee  of  Sacbrood  Tirni  to  Direct 
Sunlight  When  Sumended  in  Honey   . 

length  of  Time  that  Sacbrood  Tiroa  Be> 
mains  Tiralent  in  Honey 

Besistance  of  SaCbrood  Tims  to  thePraS' 
eilceofPennentatiTePiocesaea  .    .   . 

Resistance  of  Sacbrood  Tints  to  Fer- 
mentation in  Diluted  Honey  at  Out. 
door  Temperature   .   .  .  '.   .   .   .   . 

Besistance  of  Sacbrood  Tims  to  the  Frea. 
enceof  Pnirefacttire  ProcesseO   .   •„. 

Resistance  of  Sacbrood  Tims  to  Carboue 
Acld^. .   .   .   .   . 

Modes  of  Transmission  of  Sacbrood    .    . 

Dhigno^  of  Sacbrood 

Prognosis   .......••.•. 

Belation  ofTfaese  Studies  to  the  Treat* 


Page 
38 


otSacMtiod   .  .  . 
Coachisfonji. 


Summary  and  . 
UtetatuM  Cited 


39 
40 


40 
41 


42 
43 

44 

46 
48 
49 

80 
82 
63 


WASHINGTON 

GOVERNMENT  PBtNTINa  OFilO 

1917 


UNITED  STATES  DEPARTMENT  OF  AGRICULTURE 


BULLETIN  No.  431 


Contribution  from  the  Bureau  of  Entomology 
L.  O.  HOWARD,  Chief 


Washington,  D.  C. 


PROFESSIONAL  PAPER 


February  9, 1917 


SACBROOD. 

By  G.  P.  White, 
Expert,  Engaged  in  the  Investigation  of  Bee  Diseases. 


CONTENTS. 


Introduction 1 

Historical  aoootmt 2 

Name  of  the  disease * 

Appearanceof healthy  brood  attheageat which 

it  dies  of  sacbrood 6 

Symptoms  of  sacbrood 10 

Cause  of  sacbrood 24 

Weakening  effect  of  sacbrood  ujMn  a  colony. . .    30 
Amount  of  virus  required  to  produce  the  dis- 
ease, and  the  rapidity  of  its  increase 31 

Methods  used  in  maldng  experimental  inocula- 
tions     32 

Means  for  the  destruction  of  the  virus  of  sac- 
brood      34 

Heating  required  to  destroy  sacbrood  virus 

when  suspended  in  water 34 

Heating  required  to  destroy  sacbrood  virus 

when  suspended  in  glycerine 35 

Heating  required  to  destroy  sacbrood  virus 

when  suspended  in  honey 36 

Kesistance  of  sacbrood  virus  to  drying  at  robm 
temperature ■ ''' 


Resistance  of  sacbrood  virus  to  direct  sunlight 

when  dry 38 

Resistance  of  sacbrood  virus  to  direct  sunlight 

when  suspended  in  water 39 

Resistance  of  sacbrood  virus  to  direct  sunlight 

when  suspended  in  honey 40 

Length  of  time  that  sacbrood  virus  remains 

virulent  in  honey 40 

Resistance  of  sacbrood  virus  to  the  presence  of 

fermentative  processes 41 

Resistance  of  sacbrood  virus  to  fermentation  in 

diluted  honey  at  outdoor  temperature 42 

Resistance  of  sacbrood  virus  to  the  presence  of 

putrefactive  processes 43 

Resistance  of  sacbrood  virus  to  carbolic  acid . . .    44 

Modes  of  transmission  of  sacbrood 46 

Diagnosis  of  sacbrood 48 

Prognosis *9 

Relation  of  these  studies  to  the  treatment  of 

sacbrood 50 

'summary  and  conclusions 52 

1  Literature  cited 53 


INTRODUCTION. 

Sacbrood  is  an  infectiotis  disease  of  the  brood  of  bees.  It  is  fre- 
quently encountered  and  has  often  been  the  cause  of  fear  on  the  part 
of  beekeepers  through  a  suspicion  that  one  of  the  more  serious 
maladies— the  foulbroods— was  present. 

The  disease  is  more  benign  than  mahgnant.  It  is  insidious  m  its 
nature  and  somewhat  transient  in  its  character.  The  number  of 
colonies  that  die  as  a  direct  result  of  sacbrood  is  comparatively  small; 
the  loss  of  individual  bees  from  it,  however,  in  the  aggregate  is 
enormous.  The  loss  tends  naturally  to  weaken  the  colony  m  which 
the  disease  is  present,  a  fact  whicli  makes  the  disease  one  of  great 
economic  importance. 

5S574°-BuU.  431—17 1 


2  BULLETIN  431,  U.  S.  DEPARTMENT   OF   AGRICULTURE. 

Until  recently  no  laboratory  study  has  been  made  of  this  disease. 
Circular  No.  169,  Bureau  of  Entomology,  is  a  preliminary  report  on 
recent  studies  made  by  the  writer.  The  present  buUetm  represents 
the  results  obtained  from  a  contiauation  of  these  studies.  In  it  are 
included  only  such  results  as  it  is  beheved  can  be  applied  by  the 
beekeeper  directly  to  his  needs  or  as  will  be  otherwise  of  particular 
interest  to  him. 

HISTORICAL  ACCOUNT. 

There  are  a  nmnber  of  references  in  beekeeping  literature  to  a  dis- 
order of  the  brood  of  bees  which  had  been  recognized  by  the  presence 
of  dead  brood  that  was  different  from  that  dead  of  "foulbrood." 
It  will  be  profitable  to  cite  here  a  few  of  these  articles: 

Langstroth  (1857)  writes  as  follows: 

There  are  two  kinds  of  foul-brood,  one  of  which  the  Germans  call  the  dry  and  the 
other  the  moist  or  fcetU.  The  dry  appears  to  be  only  partial  in  its  effects  and  not 
contagious,  the  brood  simply  dying  and  drying  up  in  certain  parts  of  the  combs.  The 
moist  differs  from  the  dry  in  this  that  the  brood  dies  and  speedily  rots  and  softens, 
diffusing  a  noisome  stench  through  the  hive. 

In  this  statement  it  will  be  seen  that  beekeepers  had  already 
recognized  differences  in  the  brood  diseases  which  caused  Langstroth 
to  write  that  there  were  two  kinds  of ' '  foulbrood."  The  ktad  referred 
to  as  "dry"  foulbrood  might  easily  have  been  sacbrood. 

Doolittle  (1881),  following  a  description  of  "foulbrood,"  writes: 

We  have  been  thus  particular  in  describing  the  disease  [foulbrood]  so  none  can 
mistake  it;  and  also  because  there  is  another  disease  similar,  called  foul  brood,  which 
is  not  foul  brood.  With  this  last-named,  the  caps  to  the  cells  have  very  much  the 
same  appearance  as  in  the  genuine,  but  the  dead  larva  is  of  a  grayish  color,  and  instead 
of  being  stretched  out  at  full  length  in  the  cell,  it  is  drawn  up  in  a  more  compact  shape. 
After  a  time  it  so  dries  up  that  the  bees  remove  it,  and  no  harm  seems  to  arise  .from  it, 
only  as  there  are  a  few  larvae  that  die  here  and  there  through  the  combs  at  different 
periods;  sometimes  never  to  appear  again,  and  sometimes  appearing  with  the  next 
season;    *    *    *. 

Doohttle,  therefore,  as  early  as  1881,  had  also  observed  a  brood 
disease  which  he  says  is  similar  to  foulbrood  and  called  foulbrood,  but 
which  is  different  from  the  genuine  foulbrood.  From  his  description 
one  can  readily  beheve  that  the  disease  which  he  says  was  not  foul- 
brood was  sacbrood. 

Jones  (1883),  of  Beeton,  Ontario,  Canada,  writes  the  following: 

There  is  also  another  disease  of  the  larvse  which  is  sometimes  found  both  in  Europe 
and  America,  which  is  more  like  foul  brood  than  any  of  the  above  [chilled,  starved, 
or  neglected  brood]  and  which  frequently  deceives  those  who  we  might  claim  should 
be  good  judges,  but  which,  however,  is  not  the  genuine  article.  It  is  a  dying  of  the 
brood  both  before  and  after  it  has  been  capped  over.  The  appearance  of  this  and  the 
genuine  is  much  the  same  during  the  earlifer  stages  of  their  existence,  but  the  former 
is  usually  removed  by  the  bees  and  no  further  trouble  ensues. 


SACBBOOD.  3 

It  will  be  noted  that  Jones  also  recognized  that  there  was  a  disease 
that  resembled  somewhat  the  genuine  foul  brood,  but  was  different 
from  it,  and  that  it  was  also  different  from  chilled,  starved,  or 
neglected  brood.  Most  likely  the  disorder  referred  to  in  his  article 
was  sacbrood. 

Sunmins  (1887),  writing  from  Kottingdean,  England,  points  out 
the  difference  between  "deadbrood' '  and  foulbrood: 

That  loul  brood  is  ol'lon  confused  with  eimplo  dead  brood  I  am  well  aware.  *  *  * 
But  that  every  beo  keeper  may  decide  for  himself  without  the  aid  of  a  microscope, 
which  is  the  genuine  foulbrood  and  which  is  not,  I  will  show  how  I  have  always  been 
able  to  detect  the  difference.  With  simple  deadbrood,  while  some  may  appear  like 
the  foul  disease,  much  of  the  older  brood  dries  up  to  a  white  cinder,  in  many  cases 
retaining  its  original  form,  which  I  have  never  found  to  occur  when  genuine  foul- 
brood is  present.  Chilled  brood  can  be  distinguished  from  the  more  serious  malady 
in  like  manner. 

In  addition  to  emphasizing  the  difference  between  "deadbrood" 
and  "foulbrood,"  Simmias  says  that  these  two  diseases  are  in  turn  to 
be  differentiated  from  chiUed  brood.  He  adds  the  additional  £act 
also  that  Cheshire  had  examuied  this  "deadbrood"  and  failed  to  find 
any  microscopic  evidence  of  disease. 

Cook  (1904),  under  the  heading  "New  Bee  Disease,"  writes  as 
follows: 

In  California  and  some  other  sections  the  brood  dies  without  losing  its  form.  We 
use  the  pin-head,  and  we  draw  forth  a  larva  much  discolored,  often  black,  but  not  at 
all  like  the  salvy  mass  that  we  see  in  foulbrood. 

.  From  his  description,  and  from  the  fact  that  the  disease  is  quite 
prevalent  ia  California,  it  is  very  probable  that  the  disorder  men- 
tioned by  Cook  is  sacbrood. 

A  study  of  this  "dead  brood"  recognized  by  the  beekeepers  as  being 
different  from  foulbrood  was  begun  by  the  writer  in  New  York  State 
in  1902,  under  the  direction  of  Dr.  V.  A.  Moore.  In  a  brief  report 
on  the  work  (1904)  the  following  is  found: 

The  beekeepers  are  sustaining  a  loss  from  a  diseased  condition  in  their  apiaries 
which  they  are  diagnosing  as  "pickled  brood."  The  larvse  usually  die  late  in  the 
larval  stage.  The  most  of  them  are  found  on  end  in  the  cell,  the  head  frequently 
blackened  and  the  body  of  a  watery  granular  consistency.    *    *    * 

The  results  of  the  examinations  showed  that  Aspergillus  pollinis  was  not  found. 
Further  investigations  must  be  made  before  any  conclusion  can  be  drawn  as  to  the 
real  cause  of  this  trouble. 

It  will  be  observed  from,  this  quotation  that  the  so-called  pickled 
brood  did  not  conform  to  the  description  of  pickled  brood  and  could 
not  therefore  be  the  condition  which  had  called  forth  the- description 
of  and  the  name,  "pickled  brood"  (see  p.  4). 

Burri  (1906),  of  Switzerland,  writes: 

Dead  brood,  said  to  have  been  black  brood,  I  have  occasionally  met  with  in  my 
investigations.    It  occiured  in  the  older  larvae,  and  showed  a  gray  to  blackish  colora- 


4  BULLETIN  431,  XT.   S.  DEPAETMENT   OF   AGEICULTTJKE. 

tion,  pai-tially  drying  the  lai-vse  until  mummified.  Tliese  larvss  of  the  black-brood 
type  gave  a  negative  result  both  in  microscopic  examination  and  in  the  usual  bac- 
teriological culture  experiments.  Bacteria  seem  to  take  no  part  in  this  disease,  and 
so  far  as  I  have  come  in  contact  with  black  brood,  I  have  been  able  to  reach  no  certain 
opinion  as  to  its  cause.     [Translation.] 

It  is  very  probable  that  the  disorder  encountered  by  Burri,  which 
was  free  from  bacteria,  was  sacbrood.  Out  of  25  samples  examined 
between  1903  and  1905,  he  found  four  samples  containing  this  dis- 
ease alone,  while  in  a  few  of  the  samples  the  disorder  was  accom- 
panied by  one  of  the  other  brood  diseases. 

Kursteiner  (1910),  of  Switzerland,  gives  a  summary  of  all  samples 
exammed  by  Burri  and  hunself  from  1903  to  1909.  Out  of  360 
samples  of  suspected  disease  examined,  94  were  diagnosed  as  "  dead 
brood  free  from  bacteria."  These  were  probably  samples  of  sac- 
brood.  As  shown  by  his  later  reports,  Km^teiner  has  continued  to 
find  this  disease  in  the  examination  of  suspected  samples. 

The  foregoing  references  to  the  literature  show  that  beekeepers  in 
different  countries  had  been  observing  dead  brood  in  their  apiaries 
which  was  imhke  brood  dead  of  "foulbrood."  On  this  point  all  of 
the  observers  practically  agreed.  No  name  had  been  given  to  the 
disorder. 

NAME  OF  THE  DISEASE. 

Before  1912,  very  httle  definite  information  concerning  this 
somewhat  mysterious  disorder  of  the  brood  had  been  obtained. 
After  discovering  its  cause  and  determining  its  true  nature,  the 
writer  (1913)  used  the  name  "sacbrood"  to  designate  it.  The 
name  was  coined  to  suggest  the  saclike  appearance  of  the  dead  larvae 
in  this  disease  at  the  time  they  are  most  frequently  seen  by  the  bee- 
keeper. 

The  fact  should  here  be  emphasized  that  sacbrood  is  not  a  new 
disease.  It  is  only  the  knowledge  concerning  the  disease  and  its  name 
that  is  of  recent  origin.  It  is  far  better,  and  in  aU  probabiHty  much 
more  accurate,  to  think  of  sacbrood  as  a  disease  which  has  affected 
bees  longer  than  history  records  the  keeping  of  bees  by  man.  The 
disease,  therefore,  has  been  collecting  its  toll  of  death  for  centuries, 
often  \mawares  to  the  beekeeper.  Simply  knowing  that  there  is  such 
a  disease  should  not  be  the  cause  of  any  additional  anxiety  concern- 
ing its  losses.  On  the  other  hand,  less  fear  should  be  experienced, 
since  by  knowiag  of  it  hope  may  be  entertaiaed  that  the  losses  resulting 
from  it  may  be  reduced. 

PICKLED  BROOD. 

The  term  "pickled  brood"  was  introduced  into  beekeeping  litera- 
ture 20  years  ago  (1896),  by  WUliam  E.  Howard  of  Texas.  The 
condition  which  he  described  imder  this  term  he  declared  was  caused 


SACBEOOD.  5 

by  a  fungus  to  which  he  yavd  tho\  iiuine  Aapergillus  polUni.  In  a 
second  article  (1898)  ho  writes  that  pupaj  and  adult  bees,  as  well  as 
the  larvae,  are  attacked  by  the  disease,  stating  his  behef  that  the 
disease  in  adult  bees  had  been  diagnosed  as  paralysis.  Technically, 
therefore,  the  term  "piclded  brood"  refers  to  an  infectious  disorder 
of  bees  affectmg  both  the  brood  and  adult  bees  and  caused  by  a 
specific  fungus,  Aspergillus  pollini. 

It  was  particularly  unfortunate  that  these  articles  on  pickled 
brood  should  have  appeared  at  the  time  they  did,  as  through  them 
some  beekeepei-s  have  been  led  to  the  mistaken  belief  that  the  brood 
disease,  which  they  had  so  long  observed  as  being  similar  to  "foul- 
brood,"  but  differing  from  it,  had  been  described  in  his  articles  as 
pickled  brood. 

Whether  such  a  disease  (pickled  brood)  does  exist,  can  not  be  defi- 
nitely stated.  It  may  be  said,  however,  that  it  probably  does  not. 
The  writer  has  not  encountered  such  a  disorder  during  his  study  on  the 
bee  diseases.  He  believes  that  if  the  condition  is  present  it  cer- 
tainly has  not  attracted  the  attention  of  beekeepers  to  any  great 
extent.  It  can  safely  be  advised,  therefore,  that  all  fear  of  losses 
from  such  a  possible  condition  should  be  dispelled,  at  least  until  the 
disease  is  met  with  again. 

It  would  seem  that  the  name  "pickled  brood"  is  being  used  among 
beekeepers  at  present  in  a  very  general  sense.     Root  (1913)  writes: 

The  name  pickled  brood  has  been  applied  to  almost  any  form  of  dead  brood  that  was 
not  foul  brood.  In  a  rather  general  way,  it  seems  to  cover,  then,  any  form  of  brood 
that  is  dead  from  some  natural  causes  not  related  to  disease  of  any  sort. 

This  quotation  suggests  that  a  number  of  conditions  are  most 
Hkely  included  under  the  term  "pickled  brood"  as  it  is  popularly 
used.  Brood  dead  of  starvation  and  that  foimd  dead  before  capping 
and  not  dead  of  an  infectious  disease  seem  to  be  referred  to  especially 
by  the  name. 

Beekeepers  sending  samples  of  disease  to  the  laboratory  have  been 
asked  the  question :  "  What  disease  do  you  suspect  V  In  the  replies 
received  more  than  one  disease  was  sometimes  suggested  as  being 
suspected.  Out  of  189  repHes  received  from  beekeepers  sending 
samples  of  sacbrood,  European  foulbrood  was  suggested  in  55  repHes, 
pickled  brood  in  39,  foulbrood  in  19,  blackbrood  in  15,  poisoned  brood 
in  7,  chilled  brood  in  5,  starved  brood  in  6,  American  foulbrood  in  13, 
dead  brood  in  3,  neglected  brood  in  1,  scalded  brood  in  1,  suffocated 
brood  in  1,  and  in  24  cases  the  reply  was:  "Don't  know."  These 
replies  show  that  beekeepers  generally  had  not  learned  to  recognize 
the  disorder  which  is  now  called  sacbrood  by  any  one  name. 

It  is  natiiral  to  suppose  that  sacbrood  would  have  been  one  of  the 
conditions  occasionally  referred  to  under  the  term  "pickled  brood." 


6  BULLETIN  431,  TJ.  S.  DEPAETMENT   OF   AGKICULTtJBE. 

As  sacbrood  has  been  proved,  ho\\-(:'ver,  to  be  a  disUiift  disease  and 
different  from  all  other  disorders,  iiaturaUy  it  js  incorrect  to  use  the 
terms  "sacbrood"  and  "pickled  brood"  synonymously,  either  m  the 
popular  or  in  the  technical  sense.^ 

APPEARANCE  OF  HEALTHY  BROOD  AT  THE  AGE  AT  WHICH  IT  DIES  OF 

SACBROOD. 

By  comparing  the  appearance  of  healthy  brood  with  that  of  brood 
dead  of  a  disease,  both  the  description  and  the  recognition  of  the 
symptoms  of  the  disease  are  often  materially  aided.  Before  discuss- 
ing the  symptoms  of  sacbrood,  therefore,  a  description  of  the  healthy 

brood  at  the  age  at  which  it  dies  of  sac- 
brood wiU be  given.  In  this  description 
the  same  method  will  be  used  and  simi- 
lar terms  employed  as  will  be  found  in 
the  description  of  the  symptoms  of  the 
disease. 

It  will  be  recalled  by  those  who  are 
at  all  familiar  with  healthy  comb  in 
which  brood  is  being  reared  that  the 
brood  is  arranged  in  such  a  way  that 
capped  and  uncapped  areas  occur  alter- 
nately and  in  more  or  less  semicircular 
fashion .  Practically  aU  cells  in  the  un- 
capped areas  will  be  without  caps  while 
practically  all  in  the  capped  areas  wiU 
be  capped. 

Since  the  brood  that  dies  of  sac- 
brood, with  but  few  exceptions,  does 
so  in  capped  cells,  a  description  of  such  brood  involves  the  form,  size, 
and  position  of  these  cells. 

A  cell  (figs.  1  and  2)  may  be  described  as  having  six  side  walls,  a 
bottom  or  base,  andacap.  (The  cap  has  been  removed  by  the  bees  from 
the  cells  from  which  these  figures  were  drawn.)  In  general  the  six  side 
walls  are  rectangular  and  equal.  These  walls  form  six  equal  obtuse 
angles  within  the  cell  (fig.  1 ) .  The  angle  which  is  uppermost  in  the  cell 
(Ai)  is  formed  by  two  sides  which  together  may  be  termed  the  roof  of 
the  cell.  The  angle  which  is  lowermost  (figs.  1  and  2,  A^)  is  formed  by 
two  sides  which  with  equal  propriety  may  together  be  termed  the 
floor  of  the  cell  (fig.  2,  F).     When  a  cell  is  cut  along  its  long  axis 

1  For  the  purpose  of  an  explanation  for  those  who  may  have  learned  to  refer  to  sacbrood  by  the  term 
"pickled  brood,"  it  might  be  felt  advisable  by  some  to  continue  for  a  while  in  some  way  a  reference  to  the 
latter  term.  In  such  an  event,  the  expression  "so-called  pickled  brood"  is  suggested  as  being  more  nearly 
accurate  than  the  term  "pickled  brood." 


Fig.  1. — Looking  into  an  empty  worker  cell 
uncapped  by  bees.  The  uppermost  angle 
(Ai),the  lowermost  angle  (As),  the  lateral 
wall  (L),  and  the  wrinkling  of  the  inner  sur- 
face of  the  cell  near  the  opening,  indicating 
the  presence  of  a  mass  of  cocoons  (C),  are 
shown.  Enlarged  about  8  diameters. 
(Original.) 


SACBROOD. 


the  cut  surface  of  the  older  ones  shows  the  presence  of  a  varying  num- 
ber of  old  cocoons  (fig.  2,  C).  Near  the  mouth  of  the  ceU  on  the  side 
walls  (figs.  1  and  2,  C)  will  often  be  noted  a  wrinkling  of  the  surface. 
This  wrinkling  is  caused  by  the  presence  of  old  cocoons.  The  two 
remaining  walls  are  parallel  and  wiU  be  referred  to  as  the  lateral 
walls  (fig.  1,  L).     The  bottom  is  concave  on  the  inside.     The  cap 


Fig.  3.— End  view  oJ  cell  capped.  Tlie  cap  is 
convex,  being  recently  constructed.  (Origi- 
nal.) 


is  also  concave  on  the  inside,  making 
it  convex  on  the  outside. 

When  freshly  constructed  the  sur- 
face of  the  cap  (fig.  3)  is  smooth  and 
and  entire  and  shows  considerable 
convexity.  Later,  not  infrequently 
it  is  found  to  be  less  convex  and 
somewhat  irregular.  The  cap  should 
remain  normally  for  the  most  part 
entire  (fig.  8) .  While  this  is  the  rule, 
there  are  exceptions  to  it.  The  bee- 
keeper is  familiar  with  the  appear- 
ance which  suggests  that  it  had  not  been  entirely  completed  (fig.  11 ; 

PI.  n,  I).  X  ,.       „ 

The  long  axis  of  the  cell  is  nearly  horizontal,  the  bottom  of  the  cell 
being  normally  only  shghtly  lower  than  the  mouth.  The  long  axis 
measures  approximately  one-half  inch,  while  the  perpendicular  dis- 
tance between  any  two  diametrically  opposite  side  walls  is  approx- 
imately one-fifth  of  an  inch.  The  side  walls  are  each  approximately 
one-tenth  of  an  inch  wide.  It  is  in  such  a  cell,  then,  that  the  brood 
of  the  age  at  which  it  rHes  of  sacbrood  is  found. 


Fig.  2. — Empty  worker  cell  cut  in  half  along 
the  long  axis  ol  the  cell,  showing  cocoons  (C) 
at  the  base  and  near  the  mouth  of  the  cell, 
and  the  lowermost  angle  (As)  formed  by  the 
two  walls  which  constitute  the  floor  (F)  of 
the  cell.  Enlarged  about  8  diameters. 
(Original.) 


8  BULLETIN  431,  TJ.  S.  DEPAETMENT   OF   AGEICULTUBE. 

APPEARANCE  OF  A  HEALTHY  LARVA  AT  THE  AGE  AT  WHICH  IT  DIES   OF  SACBROOD. 

The  symptoms  which  differentiate  sacbrood  from  the  other  brood 
diseases  are  to  be  found  primarily  in  the  post-mortem  appearances 
of  the  larvffi  dead  of  the  disease.  As  an  aid  in  interpreting  the 
description  of  these  appearances  a  description  of  the  healthy  larvae 
is  &st  made. 

Larvse '  that  die  of  sacbrood  do  so  almost  invariably  after  capping 
and  at  some  time  dm-ing  the  four  days  just  preceding  the  change  in 
form  of  the  maturing  bee  to  that  of  a  true  pupa. 

During  the  jBrst  two  days  of  this  prepupal  period  the  larva  moves 
about  more  or  less  in  the  cell  and  spins  a  cocoon.  It  is  then  com- 
paratively quiet  for  about  two  days,  lying  on  its  dorsal  side  and  ex- 


FiG.  4,— Lateral  view  of  healthy  worker  larva  showing  the  normal  position  within  the  cell.  For  conven- 
ience of  description  the  length  is  divided  into  thirds— anterior  third  (AT),  middle  third  (MT)  and 
posterior  third  (PT).    Enlarged  about  8  diameters.    (Original.) 

tended  lengthwise  in  the  cell.  At  the  close  of  this  two-day  period  of 
rest,  as  a  result  of  the  metamorphosis  going  on,  the  larva  changes 
very  rapidly  to  a  true  pupa,  assuming  the  outward  form  of  an  adult 
bee. 

Although  many  larvae  die  of  sacbrood  during  the  first  two  days 
or  active  period,  of  the  4-day  prepupal  period,  by  far  the  greater 
number  of  deaths  occur  during  the  last  two  days,  the  period  of  rest. 
A  healthy  larva  at  this  resting  period  of  its  development  is  chosen, 
therefore,  for  description.  As  dead  worker  larvae  are  the  ones  usually 
encountered  in  sacbrood  and  the  ones  almost  invariably  chosen  in 
discussing  the  symptoms  of  the  disease,  the  worker  larva  is  here 
described. 

The  normal  larva  lies  extended  in  the  cell  (fig.  4)  on  its  dorsal 
side,  motionless,  and  with  its  head  pointing  toward  the  mouth  of  the 
cell.     Its  posterior  or  caudal  end  lies  upon  the  bottom  of  the  cell, 

i  As  beekeepers  usually  refer  to  the  brood  at  this  age  as  "larvae,"  the  term  is  used  here  to  designate  the 
developing  bee  at  this  stage  of  its  growth. 


SAOBKOOD. 


9 


while  Its  extreme  anterior  or  cephalic  end  extends  almost  to  the  cap 
and  roof.  The  length  of  the  larva  is  approximately  one-half  inch, 
being  nearly  that  of  the  cell.  Its  two  lateral  sides  cover  about  one- 
half  each  of  the  two  lateral  walls.  The  width  of  the  larva  is  approxi- 
mately one-fifth  of  an  inch,  being  the  distance  between  the  two 
lateral  walls  of  the  cell. 

The  dorsal  portion  of  the  larva  lies  against  the  floor  of  the  cell, 
being  more  or  less  convex  from  side  to  side  and  also  from  end  to  end. 
Its  ventral  surface  is  convex  from  side  to  side,  and  is,  generally  speak- 
ing, concave  from  end  to  end.  Considerable  empty  space  is  found 
between  the  larva  and  the  roof  of  the  cell.  The  spiracles  are  visible. 
The  glistening  appearance,  characteristic  of  a  larva  before  capping, 
very  largely  disappears  after  capping.  Although  larvae  at  this 
age  might  be  thought  of  as  white,  they 
are  in  fact  more  or  less  bluish  white  in 
color.  It  is  possible  to  remove  a  healthy 
larva  at  this  age  from  the  cell  without 
rupturing  the  body  wall,  but  care  is 
required  in  doing  so. 

For  purposes  of  description  it  is  con- 
venient to  divide  the  length  of  the  larva 
into  three  parts.  These  may  be  denom- 
inated the  anterior  (AT),  middle  (MT), 
and  posterior  thiMs  (PT). 

Anterior  third. — On  removing  the  cap 
from  a  cell  the  anterior  cone-shaped 
portion  of  the  larva  is  seen  (fig.  5;  PI. 
II,  g).  The  apex  of  this  cone-shaped 
third  is  directed  upward  toward  the 
angle  in  the  roof  of  the  cell,  but  is  not  in  contact  with  the  roof  or  the 
cap.  Transverse  segmental  markings  are  to  be  seen.  Along  a  por- 
tion of  the  median  dorsal  line  there  is  frequently  to  be  observed  a 
narrow  transparent  area.  A  cross  section  of  this  third  is  circular  in 
outline.  The  anterior  third  passes  rather  abruptly  into  the  middle 
third.  At  their  juncture  on  each  lateral  side,  owing  to  a  rapid  increase 
in  the  width  of  the  larva  at  this  point,  there  is  presented  the  appear- 
ance of  a  "shoulder." 

Middle  third. — This  third  (figs.  6  and  4 ;  PI.  II,  m)  lies  with  its  dorsal 
portion  upon  the  floor  of  the  cell,  its  axis  being  nearly  horizontal. 
The  ventral  surface  is  convex  from  side  to  side,  and  is  considerably 
below  the  roof  of  the  cell.  This  upper  surface  is  crossed  from  side  to 
side  by  well-marked  furrows  and  ridges  representing  ^segments  of 
the  larva.  These  furrows  and  ridges  produce  a  deeply  notched 
appearance  at  the  lateral  m  argins.  In  some  of  the  segments  a  trans- 
verse trachea  may  be  seen  appearing  as  a  very  fine,  scarcely  per- 
58574°— Bull.  431—17 2 


Fig.  5.— End  view  of  healthy  worker  larva 
in  normal  position  in  the  cell.  Cap 
torn  and  turned  aside  with  forceps.  En- 
larged about  8  diameters.    (Original.) 


10  BULLETIN  431,  XT.  S.  DEPABTMENT    OF    AGEICTJLTUKE. 

ceptible,  white  line.  Sometimes  there  may  be  seen  a  narrow  area 
along  the  median  line  of  the  ventral  sm-f ace  that  is  more  nearly  trans- 
parent than  the  remaining  portion  of  the  surface.  This  area  may 
extend  slightly  into  the  anterior  and  posterior  thirds.  It  is  sunilar 
in  appearance  to  the  one  on  the  dorsal  side,  but  less  distinct.  A  cross 
section  of  this  third  is  slightly  eUiptical  in  outline.  The  middle  third 
passes  more  or  less  gradually  into  the  posterior  third.  The  ]uncture 
on  the  ventral  surface  is  indicated  by  a  wide  angle  formed  by  the 

ventral  surfaces  of  these  two  thirds. 
Posterior  third.— In  form  the  pos- 
terior third  (figs.  6  and  4)  is  an  im- 
perfect cone,  the  axis  of  which  is 
directed  somewhat  upward  from 
the  horizontal.  This  third  occupies 
the  bottom  portion  of  the  cavity  of 
the  cell.  Its  dorsal  surf  ace  lies  upon 
the  bottom  wall,  with  the  extreme 
caudal  end  of  the  larva  extending  to 
the  roof  of  the  cell  (fig.  4).  The 
third  is  marked  off  into  segments 
by  ridges  and  furrows  similar  to, 
but  less  regular  than,  those  of  the 
middle  third. 

TISSUKS  OF  A  HEALTHY  LABVA  AT  THE  AGE 
AT  WHICH  IT  DIES  OP  SACBROOD. 

Upon  removing  a  larva  in  the  late 
larval  stage  and  pimcturing  its  body 
wall  lightly,  a  clear  fluid  almost 
water-like  m  appearance  flows  out. 
This  fluid  consists  chiefly  of  larval 
blood.  By  heating  it,  or  by  treat- 
ing it  with  any  one  of  a  number 
of  different  reagents,  a  coagulum  is 
formed  in  it.  Upon  rupturing  the 
body  wall  sufficiently,  the  tissues  of  the  larva  flow  out  as  a  semiliquid 
mass.  The  more  nearly  solid  portion  of  the  mass  appears  almost 
white.  This  portion  is  suspended  in  a  thin  liquid,  chiefly  blood  of  the 
larva.  A  microscopic  examination  shows  that  the  cellular  elements 
of  the  mass  are  chiefly  fat  cells.  Many  fat  globules  suspended  in  the 
liquid  tend  to  give  it  a  milky  appearance. 

SYMPTOMS  OF  SACBROOD. 

The  condition  of  a  colony  depends  naturally  upon  the  condition  of 
the  individual  bees  of  which  it  is  composed.  In  the  matter  of  diseases 
in  practical  apiculture  the  beekeeper  is  interested  piimarily  in  the 


Fig.  6.— Healthy  larva  and  cell  viewed  from 
above  and  at  an  angle.    (Original.) 


SACBKOOD. 


11 


colony  as  a  whole,  and  not  in  individual  bees.  Therefore,  in  describ- 
ing the  symptoms  of  a  bee  disease,  the  colony  as  a  whole  should  be 
considered  as  the  unit  for  description,  and  not  the  individual  bee. 
\  symptom  of  tUsease  manifested  by  an  individual  bee,  broadly  con- 
sidered, is,  in  fact,  also  a  colony  symptom.  The  symptoms  of  sacbrood 
as  described  in  this  paper  are,  therefore,  those  evidences  of  disease 
that  are  manifested  by  a  colony  affected  by  the  disease. 

It  has  been  found  that  sacbrood  can  be  produced  in  a  healthy  colony 
by  feeding  it  a  suspension  in  sirup  of  crushed  larvae  dead  of  the  disease. 
With  sacbrood  thus  produced  in  ex- 
perimental colonies  the  symptoms  of 
the  disease  have  been  studied,  and  the 
desci'iption  of  these  symptoms  given 
here  is  based  chiefly  upon  observations 
made  in  these  experimental  studies. 
The  facts  thus  obtained  are  in  accord 
with  those  observed  in  numerous  sam- 
ples of  the  disease  sent  by  beekeepers 
from  various  localities  in  the  United 
States  for  diagnosis.  They  are  in  ac- 
cord, furthermore,  with  the  symptoms 
as  they  have  been  observed  in  colonies 
in  which  the  disease  has  appeared,  not 
through  experimental  inoculation  but 
naturally. 

The  symptoms  of  sacbrood  which 
would  ordinarily  be  observed  through 
a  more  or  less  casual  examination  of 
the  disease  will  first  be  considered.  It 
must  be  remembered  that  the  brood  is 
susceptible  to  the  disease,  but  that  the 
adult  bees  are  not. 


SYMPTOMS  AS  OBSEKVED  FROM  A  CASUAL 
EXAMINATION. 


Fig.  7.— Larva  dead  of  sacbrood  lying  in  tlie 
cell  as  viewed  from  above  and  at  an  angle. 
It  may  have  been  dead  a  month.  Cap  of 
cell  removed  by  bees.  Enlarged  about  8 
diameters.    (Original.) 


The  presence  of  dead  brood  is  usually 
the  first  symptom  observed.  An  irreg- 
ularity in  the  appearance  of  the  brood 
nest  (PI.  I,  figs.  1  and  2;  PI.  IV)  frequently  attracts  attention  early 
in  the  examination.  The  strength  of  a  colony  in  which  the  disease 
is  present  is  often  not  noticeably  diminished.  Should  a  large 
amount  of  the  brood  become  affected,  however,  the  colony 
naturaUy  becomes  weakened  thereby,  the  loss  in  strength  soon 
becoming  appreciable.  Brood  that  dies  of  the  disease  does  so 
almost     invariably     in     capped     cells,     but     before     the     pupal 


12 


BULLETIN  431,  U.  S.  DEPAETMENT   OF   AGBICULTUEE. 


Fig.  8. — End  view  of  capped  cell  wliich  con- 
tains a  larva  dead  of  sacbrood,  being  simi- 
lar to  the  one  shown  in  figure  9.  The  cap 
here  is  not  difierent  from  a  cap  of  the  same 
age  over  a  healthy  larva.    (Original.) 


stage  is  reached.  It  is  rare  to  find  a  pupa  dead  of  sacbrood  (PL  II, 
zz).  The  larv^  that  die  (fig.  7)  are  found  lying  extended  lengthwise 
with  the  dorsal  side  on  the  floor  of  the  cell.     They  may  be  found  in 

capped  (fig.  8)  cells  or  iu  cells  which 
have  been  ^mcapped  (fig.  9),  as  bees 
often  remove  the  caps  from  cells 
containing  dead  larvae.  Caps  that 
are  not  removed  are  more  often  en- 
tire, yet  not  infrequently  they  are 
foimd  to  have  been  pxmctured  by 
the  bees.  Usually  only  one  ptmcture 
is  found  in  a  cap  (PI.  II,  d),  but 
there  may  be  two  (fig.  10)  or  even 
more  (PI.  H,/).  The  punctures  vary 
in  size,  sometimes  approximating 
that  of  a  pinhead,  although  usually 
smaller,  and  are  often  irregular  in 
outline.  Sometimes  a  cap  (fig.  11, 
PL  II,  h)  has  a  hole  through  it  which 
suggests  by  its  position  and  uniform 
circumference  that  it  has  never  been 
completed.  Through  such  an  opening  (fig.  11;  PL  II,  e)  or  through 
one  of  the  larger  punctiires  the  dead  larva  may  be  seen  within  the  cell. 
A  larva  recently  dead  of  sacbrood  is  slightly  yellow.  The  color  in  a 
few  days  changes  to  brown.  The  shade 
deepens  as  the  process  of  decay  con- 
tinues, imtil  it  appears  in  some  in- 
stances almost  black.  Occasionally  for 
a  time  during  the  process  of  decay 
the  remains  present  a  grayish  appear- 
ance. 

In  sacbrood,  during  the  process  of 
decay,  the  body  wall  of  the  dead  larva 
(figs.  7  and  9)  toughens,  permit- 
ting the  easy  removal  of  the  re- 
mains intact  from  the  cell.  The 
content  of  the  sachke  remains,  dur- 
ing a  certain  period  of  its  decay,  is 
watery  and  granidar  in  appearance. 
Much  of  the  time  the  form  of  the 
remains  is  quite  similar  to  that  of  a 
healthy  larva.  If  the  dead  larva  is  not  removed,  its  surface 
through  evaporation  of  its  watery  content,  becomes  wrinkled,  dis- 
torting its  form.     Further  drying  results  in  the  formation  of  the 


Fig.  9.— Looking  into  a  cell  containing  a 
larva  dead  of  sacbrood.  The  stage  of 
decay  is  about  the  same  as  in  figure  8. 
(Original.) 


Bui.  431,  U.  S.  Dept.  of  AgricuHuro. 


Plate 


v_WS««V5fy  O  '^  >%  > 


■JW*V  J^if^ft^    f^^»5^?i^  r»  ->  '^  >  •^<&(i|0' 


If  -  . 


d-^^aa- 


■%m 


Fig.  1.— Marked  Sacbrood  Infection.    Size  Slightly  Less  than   Natural. 

(Original.) 


mm^m^^^0iijp^^^^^ 


:-A^^ 


^K'iskv^m^^ 


iwiilam.  ^m^mm^m 


FiQ.  2.— Heavy  Sacbrood  Infection,  Showing  a  Number  of  Different  Stages 
OF  Decay  of  Larv/e.  Eggs,  Young  Larvae  in  Different  Stages  of  Develop- 
ment, and  Diseased  Larv/E  in  Same  Area.    Natural  Size.    (Original.) 

SACBROOD    PRODUCED    BY    EXPERIMENTAL    INOCULATION. 


Bui.  431,  U.  S.  Dept.  of  Agriculture. 


Plate  II. 


oo 


y 


k 


□  Q 


s 


t 


u- 


l> 


r"^""n 


W  X 


y 


z 


WW 


JCX 


!/¥ 


zz 


Comparison  of  a  Healthy  Larva  and  the   Remains  of   Larv/e  Dead  of 

Sacbrood. 

a,  A  cap  of  a  healthy  larva;  &,  c,  d,  c,  and  /,  caps  over  larvos  in  first,  second,  third,  fourth,  and 
fifth  stages  of  decay,  respectively;  g,  a  healthy  larva,  end  view;  h,  i,  j,  k,  and  I,  an  end  view 
of  the  five  stages  of  decay;  m,  a  healthy  larva  viewed  from  above;  n,  o,  p,  q,  and  r,  cor- 
responding view  of  the  five  stages  of  decay;  s  and  y,  healthy  larva  removed  from  the 
cell;  t,  u,  V,  w.  and  s,  larval  remains  in  different  stages  of  decay  removed  from  the  cell; 
WW,  a  larva  recently  dead  of  sacbrood  with  the  anterior  third  removed  by  the  bees;  x,  a 
scale  removed  from  the  cell;  xx,  larval  remains  from  which  a  small  portion  has  been 
removed  by  bees:  i/.v.  almost  a  pupa;  cz,  a  pupa  dead  of  sacbrood  which  had  only  recently 
transformed.    (Onglnal.) 


SACBROOD. 


13 


"scale"  (tigs.  22,  23;  PL  II,  I,  r,  and  x).     This  scale  is  not  adherent 
to  the  cell  wall. 

In  sacbrood  the  brood  combs  may  be  said  to  have  no  odor.  Larvae 
midergoing  later  stages  of  decay  in  the  disease,  however,  when 
crushed  in  a  mass  and  held  close  to 
the  nostrils  are  found  to  possess  a 
disagreeable  odor. 

From  a  superficial  or  casual  ex- 
amination alone  of  a  case  of  sac- 
brood  it  may  be  mistaken  for  some 
other  abnormal  condition  of  the 
brood.  A  careful  study  of  the  post- 
mortem appearances  of  larvae  dead 
of  the  disease,  however,  will  make  it 
possible  to  avoid  any  such  confusion. 
A  more  carefid  study  of  the  dead 
larvse  is  therefore  justified. 


Fig.  10. — Cap  of  cell  contaming  the  remains 
of  a  larva  dead  of  sacbrood.  The  cap  is 
slightly  suTLken  and  bears  two  perforations 
made  by  the  bees.     (Original.) 


APPEARANCE  OF  LARV^  DEAD  OF  SACBROOD. 

No  signs  in  a  larva   dying  of  sac- 
brood have  yet  been   discovered  by 
which  the  exact  time  of  death  may  be  determined.     As  the  larvse  in 
this  disease  usually  die  during  the  time  when  they  are  motionless,  lack 

of  movement  can  not  be  used  as  an 
early  sign  of  death.  I-n  this  descrip- 
tion it  is  assumed  that  the  larva  is 
dead  if  it  shows  a  change  in  color 
from  bluish-white  to  yellowish  or 
indications  of  a  change  from  the 
normal  turgidity  to  a  condition  of 
flaccidity. 

The  appearance  of  a  larva  dead 
of  sacbrood  varies  from  day  to  day, 
changing  gradually  from  that  of  a 
living  healthy  larva  to  that  of  the 
dried     residue — the    scale.       A    de- 

FiG.  11.— End  view  of  cell  containing  a  laiva      scrip  tion    that    WOuld    be    COrrect    for 
deadofsacbrood.withacapwhichhasthe  _■       ,      ^avva      nn      r,r\P     flnv     there- 

appearance  of  never  having  been  com-     ^    dead    larva    On    One    uay,   inere 
pieted.  (Original.)  forc,   may    and    probably   Would    be 

incorrect  for  the  same  larva  on  the  following  day.  Moreover,  all 
larvro  dead  of  the  disease  do  not  undergo  the  same  change  in  appear- 
ance, causing  another  considerable  range  of  variation.  For  con- 
venience of  description,  this  gradual  and  contmual  change  in  appear- 
ance is  here  considered  in  five  more  or  less  arbitrary  stages.    As  the 


14 


BULLETIN"  431,  U.  S.  DEPAETMENT  OF   AGEICULTTJEE . 


same  plan  will  be  followed  and  similar  terms  will  be  used  in  describing 
these  stages  as  were  employed  in  tbe  description  of  a  healthy  larva 
of  the  same  age,  the  interpretation  of  the  description  wiU  be  aided 
if  the  appearance  of  a  healthy  larva  as  described  above  is  borne  in 

mind. 

FiEST  Stage. 

Uncapping  a  larva  showing  the  first  symptoms  of  the  disease,  it 
win  be  observed  that  it  has  assumed  a  slightly  yellowish  appearance. 


Fig.  12.— First  stage:  Larva  showing  first 
symptoms  of  sacbrood  and  presenting  tlie 
dorsal  view  of  the  anterior  third.  Cap 
removed  artificially.    (Original.) 


This  shade  deepens  somewhat  during 
the  stage,  but  does  not  become  a  deep 
yellow. 

Anterior  third. — The  lateral  margins 
and  extreme  cephahc  end  of  the  an- 
terior third  (fig.  12;  PI.  II,  &,  Ti)  may 
have  assmned,  and  frequently  do  as- 
sume, a  more  or  less  transparent  ap- 
pearance (represented  in  the  figure  by 
shading).  The  position  and  the  sur- 
face markings  of  the  anterior  third  are 
approximately  those  of  the  normal  larva.  When  a  change  in  the 
position  is  observed,  however,  the  extreme  anterior  end  of  the  larva — 
the  apex  of  this  cone-like  third — having  settled  somewhat,  does  not 
approach  so  near  the  roof  of  the  cell  as  does  that  of  a  healthy  larva. 
It  is  sometimes  found  also  that  this  cone-hke  third  is  deflected  more 
or  less  to  one  side  or  the  other. 

Middle  and  posterior  thirds. — The  changes  from  the  normal  that 
have  taken  place  in  these  two  thirds  are  similar  and  can,  therefore,  be 
described  together.  The  yellowish  tint  is  here  observed.  The  trans- 
verse ridges  and  furrows  are  still  well  marked  (fig.  13).     The  trans- 


FiG.  13. — First  stage:  Ventral  view  of  larva 
dead  of  sacbrood  as  seen  from  above  and  at 
an  angle,  giving  a  ventral  view  of  all  three 
thirds.    Cap  torn  across.    (Original.) 


SACBROOD. 


15 


verse  trachess  under  slight  magnification  may  be  distinctly  seen. 
The  narrow,  somewhat  transparent  area  present  along  the  ventral 
median  line  of  the  healthy  larva  is  still  to  be  seen  in  this  stage  of  the 
decay.  The  lateral  and  posterior  margins  are  stiU  deeply  notched 
and  are  frequently  found  to  appear  quite  transparent.  This  appear- 
ance is  due  to  a  watery  looking  fluid  beneath  the  cuticular  portion  of 
the  body  wall. 

Sometimes  only  the  remnant  of  a  larva  (fig.  14;  PI.  II,  ww)  dead 
of  sacbrood  is  found  in  the  cell.    Such  remnants  vary  in  size.     The 


'  Fig.  15. — Second  stage:  Dorsal  view  of  an- 
terior third  of  a  larva  dead  of  sacbrood. 
(Original.) 

surface  left  from  the  removal  of  tissues 
is  somewhat  roughened,  indicating  that 
the  removed  portion  has  been  taken 
away  piecemeal,  and  is  more  or  less 
transverse  to  the  larva. 

Consistency  of  the  larva  in  the  first 
stage. — The  cuticular  portion  of  the 
body  wall,  which  chiefly  constitutes  the 
sac  that  characterizes  the  disease  sac- 
brood, is  less  easily  broken  at  this  time 
than  in  the  healthy  larva.  When  the 
body  wall  is  broken  the  tissues  of  the 
larva,  which  constitute  the  contents  of 
the  sac,  flow  out.  This  fluid  tissue  mass  is  less  milky  in  appearance 
than  that  from  a,  normal  larva.  The  granular  character  of  the  con- 
tents of  the  sac  which  is  marked  in  later  stages  of  decay  is  already  m 
evidence.  By  microscopic  exammation  the  granular  appearance  is 
found  to  be  due  chiefly  to  fat  cells. 

Condition  of  the  virus  in  the  -first  stage.— When  larvae  of  this  stage 
are  crushed,  suspended  in  sirup,  and  fed  to  healthy  bees,  a  large 


Fig.  14.— First  stage:  Portion  of  a  larva 
dead  of  sacbrood,  showing  a  more  or  less 
transverse  roughened  surface  from  which 
the  bees  have  removed  a  portion  of  the 
larva  piecemeal.    (Original.) 


16  BULLETIN  431,  V.  S.  DEPAETMENT  OF   AGKICULTUEE. 

amount  of  sacbrood  is  readily  produced,  showing  that  the  larval  re- 
mains in  this  stage  are  particularly  infectious.  This  is  an  important 
fact,  as  it  is  the  stage  of  decay  at  which  the  larva  is  frequently  re- 
moved piecemeal  from  the  cell. 

Second  Stage. 

The  color  of  the  decaying  larva  has  changed  from  the  yellowish  hue 
of  the  first  stage  to  a  brownish  tint.     The  yellow,  however,  has  not 


Fig.  17.— Third  stage:  Dorsal  view  of  an- 
terior tliird  of  larva  dead  of  sacbrood. 
(Original.) 

yet  in  all  cases  entirely  disappeared. 
Anterior  third. — The  shade  of 
brown  is  deeper  in  the  anterior  third 
(fig.  15;  PI.  II,  i)  as  a  rule  than  in  the 
other  two  thirds.  On  the  ventral 
surface  of  the  anterior  third  there  are 
sometimes  present  minute,  very 
dark,  nearly  black  areas,  appearing 
httle  more  than  mere  points.  Upon 
dissecting  away  the  molt  skin,  these 
areas  are  found  to  be  associated  with  the  developing  head  and  thoracic 
appendages  of  the  bee.  The  position  of  the  anterior  third  in  this 
stage  has  changed  only  shghtly  from  that  observed  in  the  preceding 
one.  The  apex  is  farther  from  the  roof  of  the  cell  (PL  II,  i).  The 
deflection  is  more  marked  and  is  seen  ia  a  greater  number  of  larvae. 
The  surface  markings  have  not  changed  materially. 

Middle  and  posterior  thirds. — ^The  changes  that  have  occurred  in 
each  of  these  two  thirds  are  still  similar  and  can,  therefore,  again  be 
described  together. 


Fig.  16. — Second  stage:  Larva  dead  of  sacbrood, 
ventral  view.    tOriginal.) 


SAOBEOOD. 


17 


The  ventral  surface  of  these  two  thirds  (fig.  16,  PI.  II,  o)  is  less  con- 
vex from  side  to  side.  The  ridges  and  furrows,  representing  the  seg- 
ments, are  less  pronounced.  The  lateral  margins  are  stiU  deeply 
notched.  The  prominent  angle  seen  on  the  ventral  side  of  a  healthy 
larva,  at  the  jimcture  of  the  middle  and  posterior  thirds,  has  given 
place  to  a  wider  one  in  this  stage  of  decay.  The  clear  subcuticular 
fluid  frequently  observed  at  the  lateral  and  posterior  margins  of  lar- 
vae dead  of  this  disease  is  here  increased  in  quantity. 

Consistency  of  the  contents  of  the  sac. — The  cuticular  sac  is  now 
more  readily  observed  and  less  easily 
broken.  The  decaying  contents  con- 
sist of  a  more  or  less  granular-appear- 
ing mass  suspended  in  a  watery  ap- 
pearing fluid,  the  mass  possessing  a 
slightly  brownish  hue.  The  micro- 
scopic examination  shows  that  the 
granular  appearance  is  due  to  the 
presence  of  decaying  tissue  cells, 
chiefly  fat  cells,  which  are  changing 
slowly  as  the  decay  of  the  larva  goes 
on. 

Condition  of  the  virus. — The  results 
of  inoculations  show  that  the  remains 
of  larvae  at  this  stage  of  decay  are 
still  in  some  instances  infectious.  The 
amount  of  infection  produced  when 
such  larvae  are  used  in  making  in- 
oculations is  very  much  less,  how- 
ever, than  when  larvae  in  the  first 
stage  are  used. 


Third  Stage. 


Fig.  18. — Third  stage:  Larva  dead  of  sacbrood, 
ventral  view.    (Original.) 


The  color  of  the  dead  larva  of  this 
stage  is  quite  brown,  that"  of  the  an- 
terior third  being  a  deeper  shade  than 
that  of  the  other  two  thirds.  An  indication  that  the  remains  are 
drying  is  observed  in  the  wrinkling  of  the  surface  that  is  beginning  to 
be  in  evidence. 

Anterior  third. — The  color  of  the  anterior  third  is  a  deep  brolwn. 
This  third  still  preserves  its  coneHke  form  (figs.  17  and  9;  PI.  II,  j), 
the  distance  of  the  apex  from  the  roof  of  the  ceU  being  still  further 
increased.  This  may  equal  one-fourth  or  more  of  the  diameter  of  the 
mouth  of  the  cell.  The  surface  markings  are  still  quite  similar  to 
those  of  a  healthy  larva  with  the  exception  that  evidences  of  drying 
are  present. 

58574°— BuU.  431—17 3 


18 


BULLETIN  431,  V.  S.  DEPARTMENT  OF   AGEICULTUEE. 


Middle  third.— While  the  color  of  the  middle  third  is  similar  to 
and  often  approaches  in  its  shade  that  of  the  anterior,  very  frequently 
it  is  considerably  Hghter.  The  ventral  surface  of  this  third  (figs.  18 
and  7)  is  less  convex  from  side  to  side  than  ui  the  preceding  stage, 
and  the  segmental  markings,  while  stiU  plainly  visible,  are  less  pro- 
nounced. The  notches  along  the  lateral  margins  are*  also  less  pro- 
nounced. 

Posterior  third. — ^The  color  of  the  posterior  third  (figs.  18  and  7; 
PI.  II,  p)  equals  or  exceeds  in  depth  of  shade  that  of  the  middle 
third  and  sometimes  equals  that  of  the  anterior  third.  The  surface 
markings  are  stUl  pronounced  and  much  resemble  those  of  the 
normal  larva. 

That  the  watery  content  of  the  sac  is  being  lessened  through  evapo- 
ration is  evidenced  by  the  diminution  of  the  quantity  of  the  watery- 


FiG.  19.— Third  stage:  Larva  dead  of  sacbrood,  lateral  view.    (Original.) 

appearing  substance  seen  at  the  lateral  margins  of  the  middle  and 
posterior  thirds  and  by  the  wrinkling  of  the  cuticular  sac.  These 
wrinkles  are  small  and  numerous. 

The  lateral  view  of  the  larva  in  the  third  stage  (fig.  19)  shows  that  it 
stni  maintains,  in  a  general  way,  the  form  and  markings  of  the  normal 
larva  (fig.  4).  The  turgidity  is  gone,  although  the  position  in  the 
ceU  is  very  much  as  it  is  in  the  healthy  larva. 

Consistency  of  the  sac  and  its  contents. — It  is  the  appearance  of  the 
remains  of  the  larva  in  the  third  stage  of  the  decay  that  best  character- 
izes the  disease,  sacbrood.  The  cuticular  sac  is  now  quite  tough, 
permitting  the  removal  of  the  larva  from  the  cell  with  considerable 
ease  and  with  httle  danger  of  its  being  torn.  The  content  of  the  sac  is 
a  granular  mass,  brownish  in  color  and  suspended  in  a  comparatively 
small  quantity  of  a  more  or  less  clear  watery-appearing  fluid.  Upon 
microscopic  examination  the  mass  is  found  to  consist  of  decaying 
tissues,  chiefly  fat  cells. 

Condition  of  the  virus  in  the  third  stage. — When  the  larval  remains 
in  this  stage  of  decay  are  crushed  and  fed  in  sirup  to  healthy  colonies 
no  sacbrood  is  produced,  indicating  that  the  dead  larvae  at  this  stage 


SACBBOOD. 


19 


are  not  infectious.  The  status  of  the  virus  in  this  stage  is  not  defi- 
nitely known,  but  the  facts  thus  far  obtained  indicate  that  it  is 
probably  dead. 

Fourth  Stage. 

The  brown  color  of  the  larval  remains  has  further  deepened,  the 
anterior  thh-d  being  much  darker  as  a  rule  than  the  other  two-thirds. 
The  marked  evidence  of  drying  now  present  might  be  said  to  charac- 
terize this  stage. 

Anterior  third.— The  color  is  a  very  deep  brown,  often  appearing 
almost  black.     As  a  result  of  drying,  the  apex  of  this  conehke  third 


Fig.  20.— Fourth  stage:  Remains  of  larva 
dead  of  saebrood.    (Original.) 

is  often  nearer  the  roof  of  the  cell  in 
this  stage  than  in  the  preceding  one. 
As  a  result  it  has  also  been  drawn 
inward  from  the  mouth  of  the  cell. 
The  surface  markings  seen  in  the 
normal  larva  are  in  this  stage  (fig. 
20;  PI.  II,  Ic)  of  decay  almost  obhter- 
ated  through  the  wrinkling  of  the 
surface,  due  to  drying. 

Middle  third. — This  third  is  de- 
cidedly brown,  but  lighter  in  shade 
than  the  anterior  third.  The  ventral  surface  (fig.  21;  PI.  II,  q)  is 
slightly  concave  from  side  to  side.  The  segmental  markings  are  still 
to  be  seen,  but  are  not  at  all  prominent.  The  notched  lateral  mar- 
gins extend  upon  the  side  walls  of  the  cell.  The  subcuticular  fluid 
so  noticeable  in  some  of  the  earlier  stages  has  disappeared  through 
evaporation.  The  effect  of  drying  is  very  noticeable,  causing  a 
marked  wrinkling  of  the  surface. 

Posterior  third. — The  posterior  third  (PI.  II,  q)  may  or  may  not  be 
darker  than  the  middle  third,  but  it  is  not  darker  than  the  anterior 


Fig.  21. — Fourtli  stage:  Remains  of  larva  dead 
of  saebrood,  ventral  view.    ( Original.) 


20  BULLETIN  431,  U.  S.  DEPAHTMENT   OF   AGEICULTUEE. 

third.  The  effect  of  the  drying  on  this  third  is  quite  perceptible  also. 
The  surface  markings  and  notched  margin  of  the  normal  larva  are 
still  indicated  in  the  decaying  remams,  but  are  much  less  pronounced. 
The  subcuticular  fluid  is  no  longer  in  evidence. 

Consistency  of  the  contents  of  the  sac— Upon  tearing  thesac,  the 
contents  are  found  to  be  less  fluid  than  in  preceding  stages.  The 
decaying  tissue  mass  is  stiU  granular  in  appearance.     As  the  drying 


TiQ.  22.— Fifth  stage:  Scale,  or  larval  re- 
mains, in  sacbrood  as  seen  on  looking 
into  the  cell.    (Original.) 

proceeds  further  the  contents  of  the 
sac  become  pastelike  in  consistency. 
Condition  of  the  virus  in  the  fourth 
stage. — As  in  the  preceding  stage,  the 
larval  remains  in  the  fom-th  stage  do 
not  seem  to  be  infectious. 

Fifth  Stage. 


Fig.  23.— Filth  stage:  Scale,  or  larval  remains, 
in  sacbrood  viewed  at  an  angle  from  above. 
(Original.) 


The  dead  larva  in  this  last  stage 
has  lost  by  evaporation  all  of  its 
moisture,  leaving  the  dry,  mummylike  remains  known  as  the  "scale." 

Anterior  third. — The  anterior  third  (fig.  22 ;  PI.  II,  Z)  through  dry- 
ing is  retracted  from  the  mouth  of  the  cell,  with  the  apex  drawn  still 
deeper  into  the  ceU  and  raised  toward  its  roof.  This  third  is  greatly 
wrinkled,  and,  being  of  a  very  dark-brown  color,  presents  often  an 
almost  black  appearance. 

Middle  third. — The  middle  third  (flg.  23;  PI.  II,  r),  is  deeply 
concave  from  side  to  side  and  may  show  renmants  of  the  segmental 
markings  of  the  larva.  The  surface  is  often  roughened  through 
drying.     Sometimes  both  longitudinal  and  transverse  trachese  are 


SACBTiOOD. 


21 


plainly  visible.  The  margin  frequently  presents  a  wavy  outline  cor- 
responding to  the  original  furrows  and  ridges  of  the  lateral  margin  of 
the  larva. 

Posterior  third.— The  posterior  third  (figs.  23  and  24)  extends  upon 
the  bottom  of  the  cell,  but  does  not  completely  cover  it.  A  lateral 
view  of  the  scale  (fig.  24)  shows  that  it  is  turned  upward  anteriorly 
and  drawn  somewhat  toward  the  bottom  of  the  cell.  The  ventral 
surface  is  concave,  often  roughenc-d,  and  directed  somewhat  forward. 
This  margin,  hke  that  of  the  middle  third,  has  a  tendency  toward 
being  irregular. 

The  scale.— The  scale  can  easily  be  removed  intact  from  the  cell. 
(PL  II,  aj.)  .  Indeed,  when  very  dry,  many  of  them  can  be  shaken 
from  the  brood  comb.  When  out  of  the  cell,  they  vary  markedly 
in  appearance.  The  anterior  third  is  of  a  deeper  brown  than  the 
the  other  two  thirds  as  a  rule.     The  dorsal  side  of  the  middle  and 


Fig,  24. — Scale,  or  larval  remains,  in  position  in  cell  out  lengthwise,  lateral  view.    (Original.) 

posterior  thirds  is  shaped  to  conform  to  the  floor  of  the  cell,  being  in 
general  convex,  with  a  surface  that  is  smooth  and  polished.  The 
margin  is  thin  and  wavy.  The  anterior  third  and  the  lateral  sides  of 
the  middle  and  posterior  thirds  being  turned  upward,  the  ventral  sur- 
face being  concave,  and  the  posterior  side  being  convex,  the  scale  in 
general  presents  a  boathke  appearance  and  could  be  styled  "gondola- 
shaped."  This  general  form  of  the  scale  has  been  referred  to  by 
beekeepers  as  being  that  of  a  Chinaman's  shoe.  When  completely 
dry,  the  scale  is  brittle  and  may  easily  be  ground  to  a  powder. 

Condition  of  the  virus  in  the  scale. — The  scales  in  sacbrood,  when  fed 
to  healthy  bees,  have  shown  no  evidence  of  being  infectious. 

The  length  of  time  that  dead  larvae  are  permitted  by  the  bees 
to  remain  in  the  cells  before  they  are  removed  varies.  They  may  be 
removed  soon  after  death,  they  may  remain  until  or  after  they  have 
become  a  dry  scale,  or  they  may  be  removed  at  any  intervening  stage 
in  their  decay.     Not  infrequently  they  are  permitted  to  remain  to  or 


22  BULLETIN  431,  TJ.  S.  DEPAETMENT   OF   AGKICULTUBE. 

through  the  stage  described  above  as  the  thu-d  stage  (figs.  7,  9, 
17,  and  18;  PI.  II  j,  p).  That  the  dead  larvffi  are  allowed  to  remain 
in  the  cells  often  for  weeks  is  in  part  the  cause  of  the  irregularity  ob- 
served ia  the  appearance  of  the  brood  combs  (p.  11).     (Pis.  I,  IV.) 

APPEARANCE  OF  THE  TISSIIES  OF  A  LAKVA  DEAD  OF  SACBROOD. 

The  gross  appearance  of  a  larva  during  its  decay  after  death  from 
sacbrood  has  just  been  described.-  The  sachke  appearance  o'f  the 
remains,  with  its  subcuticular  watery-like  fluid  and  its  granular 
content,  can  better  be  interpreted  by  knowing  something  of  the 
microscopic  structure  of  the  dead  larva. 

A  section  through  a  larva  (fig.  25,  A)  dead  of  sacbrood  shows  that 
the  fat  tissue  constitutes  the  greater  portion  of  the  bulk  of  the  body. 
The  fat  ceUs  (FC)  are  comparatively  large.  In  the  prepared  section 
when  considerably  magnified  (C)  they  are  seen  to  be  irregular 
in  outline,  with  an  irregular-shaped  nucleus  (Nu).  Bodies  stained 
black,  more  or  less  spherical  in  form  and  varying  in  size,  are  found 
in  them.  The  presence  of  these  cells  is  the  chief  cause  for  the 
granular  appearance  of  the  contents  of  larvae  dead  of  sacbrood.  This 
appearance  has  often  been  observed  by  beekeepers  and  is  a  weU- 
recognized  symptom  of  sacbrood. 

In  the  section  (A)  may  be  seen  a  molt  skin  (Cj),  which  is  at  a  con- 
siderable distance  from  the  hypodermis  (Hyp).  Another  cuticula 
(Cj)  is  already  quite  well  formed  and  lies  near  the  hypodermis.  Be- 
tween these  two  cuticulae  (Cj  and  C^)  during  the  earlier  stages  of 
decay  there  is  a  considerable  space  (" in tercuticular  space")  (IS). 
This  space  is  filled  with  a  watery-looking  fluid.  That  the  fluid  is  not 
water,  but  that  it  is  of  such  a  nature  that  a  coagulmn  is  formed  in  it 
during  the  preparation  of  the  tissues  for  study,  is  shown  by  the 
presence  of  a  coagulmn  in  the  sections. 

The  body  (B,  A)  wall  of  the  larva  is  composed  of  the  cuticula  (Cj), 
the  hypodermis  (Hyp)  and  the  basement  membrane  (BM).  The 
hypodermal  cells  may  be  present  in  the  mass  content  of  the  larval 
remains.  These  cells  are  comparatively  small.  Similar  ones  are  to 
be  found  in  the  tracheal  walls  (Tra).  These  cells,  however,  make 
up  only  a  small  portion  of  the  contents  of  the  sac. 

There  are  many  other  cellular  elements  to  be  found  in  the  decaying 
mass  of  larval  tissues,  some  of  which  contribute  to  this  granular  ap- 
pearance. Among  these  are  the  oenocytes  (Oe),  cells  (D)  larger  than 
the  fat  cells,  but  comparatively  few  in  number.  These  are  found 
among  the  fat  cells,  especially  in  the  ventral  half  of  the  body.  The 
oenocytes  in  the  prepared  tissues  are  irregular  in  outhne,  having  a 
nucleus  regular  in  outline.  The  cytoplasm  is  uniformly  granular  and 
does  not  contain  the  black  staining  bodies  found  in  the  fat  cells  (C). 


SACBROOD. 


23 


Fig.  25.— The  tissues  of  a  worker  larva  after  being  dead  of  sacbrood  about  one  week.  A,  cross  section, 
semidiagrammatic,  of  the  abdomen  in  the  region  of  the  ovaries,  showing  a  recently  cast  cuticula,  or 
molt  sWn  (Cj),  a  newly  formed  cuticula  (Ci),  the  hypodermis  (Hyp),  the  stomach  (St),  the  ovaries  (Ov), 
the  heart  (Ht),  the  ventral  nerve  cord  (VNC),  the  dorsal  diaphragm  (DDph),  tracheae  (Tra),  ceno- 
cytes  (Oe),  and  fat  cells  (FC).  Between  the  cuticula  C2  and  the  cuticula  Ci  is  a  considerable  intercu- 
tioular  space  (IS).  B  represents  the  body  wall  in  this  patholofjical  condition,  showing  the  cuticula  C2 
and  the  cuticula  Ci,  both  bearing  spines  (SCj  and  SCi),  and  theintercutioular  space  (IS)  in  which  is 
found  evidence  of  a  coagulum  formed  from  the  fluid  filling  the  space  by  the  action  of  the  fixing  fluids. 
The  remainder  of  the  body  wall,  the  hypodermis  (Hyp),  and  the  basement  membrane  (BM)  are  also 
shown.  C,  fat  cell  with  irregular  outline,  irregular  nucleus  (Nu),  and  deep  staining  bodies  (DSB). 
Dj  oenocyte  with  uniformly  staining  cytoplasm,  and  with  a  nucleus  (Nu)  havmg  a  uniform  outline. 
E,  a  portion  of  the  stomach  wall  showing  the  epithelium  (SEpth)  during  metamorphosis,  it  being  at 
this  time  quite  columnar  in  type,  and  the  musculature  (M).    (Original.) 


24  BULLETIN  431,  TJ.  S.  DEPARTMENT   OP   AGEICULTURE. 

The  molt  skin  (Cj)  is  probably  the  one  that  is  shed  nonnally.  about 
three  days  after  the  larva  is  capped.  The  cuticula  (Ci),  already  quite 
well  formed,  is  probably  the  one  which  normally  would  have  entered 
into  the  formation  of  the  molt  skin  that  is  cast  at  the  time  the  larva 
or  semipupa  changes  to  a  pupa.  The  molt  skin  (Cj)  constitutes  for 
the  most  part  the  sac  which  is  seen  to  inclose  the  decaying  larval 
mass  in  sacbrood,  the  cuticula  (Ci)  probably  assisting  somewhat 
at  times.  The  presence  of  the  subcuticular  fluid  is  made  more  intelli- 
gible by  these  facts.  Larvae  dying  of  sacbrood  at  an  earlier  or  later 
period  in  their  development  will  present  an  appearance  varying 
somewhat  from  that  just  described. 

Contrasted  with  the  stomach  (midintestine  or  midgut)  of  a  feeding 
larva,  the  stomach  (A,  St)  of  a  larva  at  the  age  at  which  it  dies  of  sac- 
brood is  small.  The  cells  lining  the  wall  of  the  organ  vary  con- 
siderably in  size  and  shape,  depending  upon  the  exact  time  at  which 
death  takes  place.  In  contrast  to  the  low  cells  of  the  stomach  wall  in 
younger  larvse,  the  cells  (E,  SEpth)  at  this  later  period  are  much  elon- 
gated. These  cells  would  also  at  times  be  found  in  the  decaying 
granular  mass  present  in  the  larval  remains. 

The  various  organs  of  the  body  contribute  to  the  cellular  content 
of  the  decaying  larval  mass.  At  the  period  at  which  the  larva  dies 
of  sacbrood,  the  cellular  changes  accompanying  metamorphosis  are 
particularly  marked.  This  condition  introduces  various  cellular  ele- 
ments into  the  decaying  larval  mass. 

The  granular  mass  from  the  larval  remains  in  sacbrood  is,  therefore, 
a  composite  affair.  Upon  examining  the  mass  microscopically,  it  wiU 
be  found  that  the  granular  appearance  is  due  for  the  most  part  to 
fat  cells  suspended  in  a  liquid.  The  Mquid  portion  seems  to  be 
chiefly  blood  of  the  larva,  or,  at  least,  derived  from  the  blood,  although 
augmented  most  probably  by  other  liquids  of  the  larva  and  possibly  by 
a  hquefaction  of  some  of  the  tissues  present.  The  granular  mass 
suspended  in  a  watery  fluid,  as  a  symptom  of  sacbrood,  is  by  these 
facts  rendered  more  easily  understood. 

CAUSE  OF  SACBROOD. 

DooUttle  (1881),  Jones  (1883),  Simmms  (1887),  Root  (1892  and 
1896),  Cook  (1902),  Dadant  (1906),  and  others  through  their  writ- 
ings have  pointed  out  the  fact  that  there  are  losses  sustained  from 
sacbrood.  There  has  been  no  consensus  of  opinion,  however,  as  to 
the  infectiousness  of  the  disease.  On  this  point  Dadant  (1906) 
writes: 

Wliatever  may  be  the  cause  of  this  disease  (so-called  Pickled  Brood),  and  although 
it  is  to  a  certain  extent  contagious,  it  often  passes  off  without  treatment.  But,  as 
colonies  may  be  entirely  ruined  by  it,  it  ought  not  to  be  neglected. 


SAGBKOOD.  25 

In  the  quotation  Dadant  expresses  the  belief  that  the  disease  is  an 
infectious  one.  This  view  has  been  proved  by  recent  studies  to  be  the 
correct  one.  Since  the  disease  is  one  of  a  somewhat  transient  nature, 
.often  subsiding  and  disappearing  quickly  without  treatment,  and  is 
quite  different  in  many  ways  from  the  f  oulbroods,  it  is  not  strange  that 
some  writers  should  have  held  that  it  is  not  infectious. 

PREDISPOSING  CAUSES. 

Beekeepers  have  known  for  many  years  certain  facts  concerning  the 
predisposing  causes  of  sacbrood.  Recent  studies  have  added  others 
relative  to  sex,  age,  race,  climatic  conditions,  season,  and  food  as 
possible  predisposing  factors  in  the  causation  of  the  disease. 

Age. — The  results  of  the  studies  suggest  that  adult  bees  are  not 
directly  susceptible  to  the  disease.  Pupse  are  rarely  affected  (PI. 
II,  zz).  If  one  succumbs  to  the  disease,  it  is  quite  soon  after  trans- 
formation from  the  larval  stage.  Primarily  it  is  the  larvae  that  are 
susceptible.  When  a  larva  dies  of  the  disease,  it  does  so  almost 
invariably  after  capping,  and  usually  during  the  2-day  period  immedi- 
ately preceding  the  time  for  the  change  to  a  pupa. 

Sex. — Worker  and  drone  larvse  may  become  infected.  Queen  larvte 
apparently  are  also  susceptible,  although  this  point  has  not  yet  been 
completely  demonstrated. 

Race. — No  complete  immunity  against  sacbrood  has  yet  been  found 
to  exist  in  any  race  of  bees  commonly  kept  in  America.  That  one 
race  is  less  susceptible  to  the  disease  than  another  may  be  said 
to  be  probable,  although  the  extent  of  such  immunity  has  not  been 
established. 

The  question:  "What  race  of  bees  is  there  in  the  diseased  colony? " 
was  asked  beekeepers  sending  samples  of  diseased  brood.  Out  of  140 
rephes  received  from  those  sending  sacbrood  samples,  53  reported 
hybrids,  49  reported  Itahans,  21  reported  blacks,  and  17  reported 
Itahan  hybrids.  These  replies  show  that  the  bees  commonly  kept  by 
American  beekeepers  are  susceptible,  although  their  relative  suscepti- 
bihty  is  not  shown. 

The  bees  which  have  been  inoculated  in  the  experimental  work 
on  sacbrood  have  been  largely  Italians  or  mixed  with  Itahan  blood. 
Blacks  have  also  been  used.  No  complete  immunity  was  observed 
in  any  colony  inoculated.  That  the  blacks  are  more  susceptible 
than  strains  having  Italian  blood  in  them  is  suggested  by  some  of  the 
results.  Facts  concerning  the  problem  of  immunity  as  relating  to 
bees  are  yet  altogether  too  meager  to  justify  more  definite  state- 
ments. 

Climate. — Historial  evidence  strongly  suggests  that  sacbrood  is 
found  in  Germany  (Langstroth,  1857),  England  (Simmins,  1887), 
58574°— Bull.  431—17 i 


26  BULLETIN  431,  TJ.  S.  DEPARTMENT   OF   AGEICTJLTUEE. 

and  Switzerland  (Bmri,  1906).  Beuhne  (1913)  reports  its  presence 
in  Australia,  and  Bahr  (1915)  has  encountered  a  brood  disorder 
among  bees  in  Denmark  which  he  finds  is  neither  of  the  foul  broods. 
He  had  examined  10  samples  of  it  but  had  not  studied  it  further. 
He  says  it  may  be  sacbrood. 

About  400  cases  of  sacbrood  have  been  diagnosed  by  Dr.  A.  H. 
McCray  and  the  writer  among  the  samples  of  brood  received  for 
examination  at  the  Bureau  of  Entomology.  A  few  of  these  were 
obtained  from  Canada.  Whether  the  disease  occurs  in  tropical 
chmates  or  the  coldest  chmates  in  which  bees  are  kept  has  not  yet 
been  completely  estabhshed. 

The  mountains  and  coast  plain  of  the  eastern  United  States,  the 
plains  of  the  Mississippi  VaUey  and  the  mountains,  plateaus,  and  coast 
plain  of  the  western  portion  of  the  country  have  contributed  to  the 
number  of  samples  examined.     It  occurs  in  the  South  and  the  North. 

Its  occurrence  in  such  widely  different  localities  is  proof  that  sac- 
brood is  of  such  a  natm-e  that  it  can  appear  under  widely  different 
climatic  conditions.  The  relative  frequency  of  the  disease,  further- 
more, is  not  materially  different  in  the  different  sections  of  the  country. 
It  must  be  said,  however,  that  the  extent,  if  any,  to  which  the  dis- 
ease is  affected  by  chmate  has  not  yet  been  determined. 

The  practical  import  of  these  observations  regarding  climate,  of 
particular  interest  here,  is  that  the  presence  of  sacbrood  in  any  region 
can  not  be  attributed  entirely  to  the  prevailing  chmatic  conditions. 

Season. — It  has  long  been  known  that  sacbrood  appears  most  often 
and  in  the  greatest  severity  during  the  spring  of  the  year.  As  is 
shown  by  the  results  obtained  in  the  diagnosis  of  it  in  the  laboratory, 
the  disease  may  appear  at  any  season  of  the  year  at  which  brood  is 
being  reared.  In  the  inoculation  experiments  sacbrood  has  been 
produced  with  ease  from  early  spring  to  October  21.  While  it  is  thus 
shown  that  the  brood  is  susceptible  to  sacbrood  at  all  seasons, 
various  factors  together  cause  the  disease  to  occur  with  greater 
frequency  during  the  spring. 

Food. — ^Before  it  was  known  that  sacbrood  is  an  infectious  disease 
the  quantity  or  quaUty  of  food  was  not  infrequently  mentioned  by 
beekeepers  as  being  the  cause  of  the  disease.  Since  a  filterable  virus 
has  been  shown  to  be  the  exciting  cause  of  the  disease,  it  is  left  to  be 
considered  whether  food  is  a  predisposing  cause.  The  distribution 
of  the  disease  mentioned  above,  under  the  heading  "Climate,"  here 
again  serves  a  useful  purpose.  Since  it  occurs  in  such  a  wide  range 
of  localities,  wherein  the  food  and  water  used  by  the  bees  vary  as 
greatly  almost  as  is  possible  in  the  United  States,  the  conclusion  may 
be  drawn  that  its  occurrence  is  not  dependent  upon  food  of  any 
restricted  character.  Furthermore,  sacbrood  is  found  in  colonies 
having  an  abundant  supply  of  food,  as  well  as  in  colonies  having  a 


SACBEOOD.  27 

scarcity.     It  has  been  produced  experimentally  in  colonies  under 
equally  varying  conditions  in  regard  to  the  quantity  of  food. 

While  it  is  possible  that  the  quantity  or  quality  of  food  may  influ- 
ence somewhat  the  course  of  the  disease  in  the  colony,  the  r61e  played 
by  food  in  the  causation  of  sacbrood  must  be  slight,  if  indeed  it  con- 
tributes at  all  appreciably  to  it.  Practically,  therefore,  for  the 
present  it  may  be  considered  that  neither  the  quality  nor  quantity 
of  food  predisposes  to  this  disease. 

EXCITING  CAUSE  OF  SACBHOOD. 

That  sacbrood  is  an  infectious  disease  was  demonstrated  by  the 
wi'iter  (1913)  through  experiments  performed  during  the  summer  of 
1912.  This  was  done  by  feeding  to  healthy  colonies  the  crushed 
tissues  of  larvse  dead  of  sacbrood,  suspended  in  sugar  sirup.  The 
experiments  were  performed  under  various  conditions,  and  it  was 
found  that  the  disease  could  be  produced  at  will,  demonstrating 
thereby  that  it  was  actually  an  infectious  one. 

In  the  crushed  larval  mass  no  microorganisms  were  found  either 
microscopically  or  culturally  to  which  the  infection  could  be  attrib- 
uted, although  the  experiments  had  proved  that  the  larva  dead  of 
the  disease  did  contain  the  infecting  agent.  This  led  to  the  next  step 
in  the  investigation,  which  was  to  determine  whether  the  virus  was 
so  small  that  it  had  not  been  observed,  and  whether  its  nature  would 
permit  its  passage  through  a  filter.  The  first  filter  used  for  this 
purpose  was  the  Berkefeld. 

The  process  by  which  the  filtration  is  done  is  briefly  this:  Larvae 
which  have  been  dead'  of  sacbrood  only  a  few  days  are  picked  from 
the  brood  comb  and  crushed.  The  crushed  mass  is  added  to  water  in 
the  proportion  of  1  part  larval  mass  to  10  parts  water.  A  higher 
dilution  may  be  used.  This  aqueous  suspension  is  allowed  to  stand  for 
some  hours,  preferably  overnight.  To  remove  the  fragments  of  the 
larval  tissues  stiU  remaining,  the  suspension  is  filtered,  using  filter 
paper.  The  filtrate  thus  obtained  is  then  filtered  by  the  use  of  the 
Berkefeld  filter  ^  (fig.  26)  properly  prepared.  The  filtering  in  the 
case  of  the  coarser  filters  especially  can  be  done  through  gravity 

alone. 

To  determine  whether  any  visible  microorganisms  are  present 
in  this  last  filtrate,  it  is  examined  microscopically  and  culturally. 
When  f oimd  to  be  apparently  free  from  such  microorganisms,  a  quan- 
tity of  it  may  be  added  to  sirup  and  the  mixture  fed  to  healthy  colo- 

1  The  Berkefeld  filter  consists  of  a  compact  material  (infusorial  earth)  in  the  form  of  a  cylinder.  A  glass 
mantel  (A)  in  which  is  fixed  the  filter  forms  a  cup  for  holding  the  fluid  to  be  filtered.  Havmg  filtered 
the  aqueous  suspension  of  crushed  sacbrood  larvae  through  paper,  the  filtrate  is  then  filtered  by  aUowmg 
it  to  pass  through  the  waUs  of  the  Berkefeld  cylinder  (B).  The  filtrate  from  this  filtration  is  collected 
into  a  sterile  flask  (F)  through  a  glass  tube  (D)  with  its  rubber  connection  (C).  In  flltermg  m  this  instance 
gravity  is  the  only  force  used. 


28  BULLETIN  431,  U.  S.  DEPAKTMENT   OF   AGEICULTUEE. 

nies.     When  all  this  is  properly  done,  sacbrood  will  appear  in  the 
inoculated  colonies.     This  shows  that  the  virus  '  of  this  disease,  to  a 


Fig.    26.— Berketeld   filter  (B)  -witli  the   glass  mantle  (A),  glass  tubing  (D),  a  connecting  rubber 
tubing  (C),  and  a  flask  (F)  with  a  cotton  plug  (E).    (Original.) 

certain  extent,  at  least,  passes  through  the  Berkefeld  filter.     With 
this  filter  the  virus  is  therefore  filterable. 

1  In  referring  to  the  infecting  agent  in  sacbrood,  the  term  "virus"  is  preferable  to  the  terms  "germ"  or 
"parasite."  In  relation  to  the  disease,  however,  its  meaning  is  the  same  as  that  conveyed  by  the  latter 
terms. 


SACBEOOD. 


29 


In  the  study  of  the  virus  of  sacbrood  use  has  been  made  also  of 
the  Pasteur-Chamberland  filter  '  (fig.  27).  This  is  a  clay  filter,  the 
pores  of  which  are  much  finer  than  those  of  the  Berkefeld  used.  In 
using  this  filter,  an  aqueous  suspension  of  larvse  dead  of  the  disease 
is  prepared  as  before.     This  is  filtered  by  the  aid  of  pressure  obtained 


Pig.  27. — A  convenient  apparatus  wmch  can  be  employed  in  using  the  Pasteur-CIiamberland, 
Berkefeld,  and  other  filters.  Pasteur-Chamberland  filter  (b)  with  a  glass  mantle  (a),  arubber  stopper  (c) 
through  which  passes  the  filter,  a  connecting  rubber  tubing  (d),  glass  tubing  (e),  a  perforated  rubber 
stopper  (f),  a  vacuum  jar  (g),  designed  by  the  writer,  in  which  is  placed  a  cotton-stoppered  and  steril- 
ized flask,  a  glass  stopcock  (h),  a  vacuum  gauge  (i),  a  reservoir  (m)  with  pressure-rubber  connections 
0),  and  a  vacuum  pump  (k).    (Original,) 

by  means  of  a  partial  vacuum  in  an  apparatus  devised  for  this  pur- 
pose. Filtrates  obtained  from  this  filter  when  fed  to  healthy  colonies 
produced  the  disease.     Since  the  virus  of  sacbrood  wiU  pass  through 

iThe  Pasteur-Chamberland  filter  consists  of  clay  molded  in  the  form  of  a  hollow  cylinder  and  baked. 
This  is  used  with  a  glass  cylinder  (a)  fitted  with  a  rubber  stopper  (c).  In  the  use  of  this  filter,  force  is 
employed.  This  was  obtained  for  these  experiments  through  the  use  of  a  jar  (g)  devised  by  the  writer  in 
which  a  partial  vacuum  can  be  produced.  In  this  jar,  is  placed  a  flask  plugged  with  cotton  and  sterilized. 
Connections  are  made  as  shown  in  the  illustration,  the  vacuum  being  produced  through  the  use  of  the 
pump  (k).    In  less  than  half  an  hour  usually  a  half-pint  of  filtrate  can  be  obtained  with  this  apparatus. 


30  BULLETIN  431,  U.  S.  0EPAETMENT  OF  AGKICULTUEE. 

the  pores  of  the  Pasteur-Chamberland  filter  also,  it  is  therefore  fil- 
terable and  is  very  properly  referred  to  as  a  "filterable"'  virus. 

In  considermg  the  virus  of  sacbrood  it  is  suggested  that  the  bee- 
keeper think  of  it  as  a  microorganism  ^  which  is  so  small  or  of  such 
a  nature  that  it  has  not  been  seen,  and  which  will  pass  through  the 
pores  of  fine  clay  filters.  This  conception  of  it  wiU  at  least  make  it 
more  easily  understood. 

WEAKENING  EFFECT  OF  SACBROOD  UPON  A  COLONY. 

The  first  inoculations  in  proving  that  sicbrood  is  an  infectious 
disease  were  made  on  June  25,  1912.  Two  colonies  were  used, 
each  being  fed  with  material  from  a  different  source.  The  inocu- 
lation feedings  were  made  on  successive  days.  Sacbrood  having 
been  produced  in  the  colonies,  the  inoculations  were  continued 
at  intervals  throughout  July  and  August.  During  this  period,  a 
large  amoimt  of  sacbrood  was  present  in  both  colonies.  By  the  end 
of  July  these  colonies  had  become  noticeably  weakened,  and  by  the 
end  of  August  they  had  become  very  much  weakened,  as  a  result  of 
the  sacbrood  present  in  them.  On  September  5  one  of  the  colonies 
swarmed  out. 

The  brood  (PI.  IV)  of  this  colony,  large  in  quantity,  was  practically 
all  dyiQg  of  sacbrood.  The  other  colony,  when  examined  on  Sep- 
tember 16,  was  found  to  be  very  weak.  At  this  time,  however,  most 
of  the  dead  brood  had  been  removed  and  healthy  brood  was  being 
reared.     This  colony  increased  in  strength  and  wintered  successfully. 

The  results  obtained  from  the  inoculation  of  these  two  colonies 
demonstrated  not  only  that  sacbrood  is  an  infectious  disease,  but 
also  that  the  disease  in  a  colony  tends  to  weaken  it.  The  results 
indicate  also  that  a  colony  may  be  destroyed  by 'the  disease,  or  it 
may  recover  from  it,  gain  in  strength,  and  winter  successfully. 

Each  year  since  1912  two  or  more  colonies  have  been  fed  sacbrood 
material  at  intervals  during  the  brood-rearing  season  for  the  purpose 
of  obtaining  disease  material  for  experimental  purposes.  The  inocu- 
lated colonies  in  aU  instances  have  shown  a  tendency  to  become 
weakened  as  a  result  of  the  inoculations. 

The  death  of  the  worker  larvae  is  the  primary  cause  for  the  weak- 
ness resulting  from  the  disease  in  a  colony.  Another  point  to  be 
thought  of  is  that  dead  sacbrood  larvae  remaining  in  the  cells  for 
weeks,  as  they  not  infrequently  do,  reduce  the  capacity  of  the  brood 
nest  for  brood  rearing,  which  has  a  tendency  also  to  weaken  the  colony. 

'  In  searching  the  tissues  of  larvse  dead  of  sacbrood  and  the  filtrates  obtained  from  them  nothing  has  been 
discovered  by  the  aid  of  the  microscope,  or  culturally,  which  has  yet  been  demonstrated  as  being  the  infect- 
ing agent.  This  being  true,  the  virus  could  be  spoken  of  tentatively  as  an  "ultramicroscopic  virus."  It 
is  preferable,  for  the  present,  howe^'e^,  to  refer  to  it  simply  as  a  filterable  virus. 

2  There  is  some  question  whether,  in  thn  case  of  diseases  having  a  virus  which  is  Alterable,  the  infecting 
agent  is  in  every  instance  a  microorganism.    The  evidence  is  strong,  however,  that  it  is. 


SAOBEOOD.  31 

AMOUNT  OF  VIRUS  REQUIRED  TO  PRODUCE  THE  DISEASE,  AND  THE 
RAPIDITY  OF  ITS  INCREASE. 

Assuming  the  virus  of  sacbrood  to  be  a  very  minute  microorgan- 
ism, the  number  of  germs  present  in  a  larva  dying  of  the  disease  must 
be  considered  as  exceedingly  large.  Whether  a  single  germ  taken 
up  by  a  larva  wiH  produce  the  disease  in  every  instance,  or  in  any 
instance,  is  not  known.  If  the  disease  does  result  at  any  time  from  the 
ingestion  of  a  single  germ,  aU  of  the  conditions,  it  may  be  assumed, 
must  be  especially  favorable  for  the  production  of  the  disease.  From 
what  is  known  of  diseases  of  other  animals  and  of  man,  and  from  the 
results  thus  far  obtained  in  the  study  of  sacbrood,  it  is  well,  at  present, 
to  assume  that  the  number  of  sacbrood  germs  taken  up  by  a  larva 
may  be  so  small  that  no  disease  results. 

It  is  certain,  however,  that  a  comparatively  small  nxmiber  of 
sacbrood  germs  ingested  by  a  lai-va  about  two  days  old  are  sufficient  to 
produce  the  disease.  That  the  few  germs  thus  taken  up  can  increase 
within  the  larva  during  an  incubation  period  of  five  or  six  days  to 
such  a  vast  number  as  is  assumed  to  be  present  in  a  larva  dying  of 
the  disease  indicates  the  extreme  rapidity  with  which  the  germs  are 
able  to  multiply. 

The  minimum  quantity  of  virus  necessary  to  produce  a  moderate 
infection  in  a  colony  has  not  been  definitely  determined.  It  was 
found  by  experiments,  however,  that  the  virus  contained  in  a  single 
larva  recently  dead  of  the  disease  was  sufficient  to  produce  a  large 
amount  of  sacbrood  in  a  colony. 

As  a  very  rough  estimate,  it  may  be  said  that  the  quantity  of  virus 
in  a  single  larva  dead  of  sacbrood  is  sufficient,  when  suspended  in 
half  a  pint  of  sirup  and  fed  to  a  healthy  colony,  to  produce  in- 
fection in  and  deaith  of  afc  least  3,000  larvse.  Startmg  then  with  the 
virus  contained  in  a  single  larva,  in  less  than  one  week  it  would 
easily  be  possible  to  have  3,000  larvse  dead  of  the  disease,  which 
means  that  the  virus  has  been  increased  3,000-fold  within  one  week. 
This  latter  amount  of  virus  would  be  sufiicient  to  produce  an  equal 
amount  of  infection  in  3,000  colonies,  increasmg  the  amount  of  virus 
again  3,000-fold.  In  less  than  two  weeks,  therefore,  theoretically 
it  would  be  possible  to  produce  a  sufficient  amount  of  virus  to  infect 
9,000,000  colonies,  more  colonies  probably  than  are  to  be  found  at 
present  in  the  United  States.  Carrymg  the  idea  somewhat  further, 
within  three  weeks,  theoretically  enough  virus  could  be  produced 
to  inoculate  every  colony  in  existence. 

These  facts  are  sufficient  to  indicate  somewhat  the  enormous 
rapidity  with  which  the  virus  of  sacbrood  is  capable  of  increasing. 


32  BULLETIN  431,  TJ.   S.  DEPAETMENT   OF    AGEICULTTJBE. 

METHODS  USED  IN  MAKING  EXPERIMENTAL  INOCULATIONS. 

The  laboratory  study  of  bee  diseases  being  new,  it  has  been  neces- 
sary in  many  instances  to  devise  new  methods.  In  the  experimental 
inoculations  of  bees  the  methods  used  have  undergone  revision  from 

time  to  time.  Those 
now  employed  have 
proved  quite  satis- 
factory. 

As  the  virus  of  sac- 
brood  has  not  been 
cultivated  in  the  lab- 
oratory artificially,  it 
has  been  necessary  in 
these  investigations 
to  inoculate  a  large 
number  of  colonies. 
A  nucleus  of  bees 
that  could  be  accom- 
modated on  from  3 
to  6  brood  frames 
was  found  to  serve 
very  satisfactorily 
the  purpose  of  an  ex- 
perimental colony. 
The  queen  should  al- 
ways be  clipped.  The 
frames  are  placed  in  one  side  of  a  10-frame  hive  body  (fig.  28).  Over 
the  entrance  to  the  hive  is  placed  wire  cloth,  leaving  a  small  space 
of  about  1  inch  in  length  on  the  side  occupied  by  the  brood  frames. 
Petri  dishes  *  (fig.  29)  serve  well  the  purpose  of  a  feeder.  Both 
halves  of  the  dish  are  used 
as  receptacles.  These  are 
placed,  preferably  about 
four  of  the  halves,  within 
the  hive  on  the  bottom 
board  on  the  side  not  occu- 
pied by  frames.  The  hives 
of  the  experimental  apiary 
(PI.  Ill)  are  arranged 
chiefly  in  pairs,  with  the  entrances  of  consecutive  rows  pointing  in 
opposite  directions.     The  space  occupied  by  the  apiary  should  be 

'  A  Petri  dish,  a  much-used  piece  of  apparatus  in  a  laboratory,  is  simply  a  shallow,  circular,  glass  dish 
with  a  flat  bottom  and  perpendicular  sides.  It  consists  of  two  halves,  a  bottom  and  a  top.  These  are 
very  similar.  The  top  half,  being  slightly  larger,  fits  over  the  bottom  one  when  the  two  halves  are  placed 
together. 


Fig.  28.— The  hive  as  it  is  employed  to  house  and  feed  a  colony  used 
for  experimental  inoculations.  Here  are  shown  four  Hoffman 
frames,  a  division  board,  four  open  Petri  dishes  as  feeders,  and  the  en- 
trance nearly  closed  with  wire  cloth,  the  opening  being  on  the  side 
of  the  hive  body  occupied  by  the  colony.  The  dimensions  indicated 
are  approximate.  The  angle  at  which  the  hive  was  photographed 
for  this  drawing  caused  its  length  to  appear  foreshortened.    ( Original. ) 


Fig.  29.— Petri  dish.    The  top  half  is  slightly  raised.    Tliosa 
used  here  are  4  inches  in  diameter.    (Original.) 


Plate 


Bui.  431 ,  U.  S.  Dept.  of  Agriculture. 


SACBTtOOD. 


33 


broken  up,  preferably  by  trees  or  shrubbery.  By  these  means,  it 
will  be  observed,  there  is  a  tendency  to  minimize  the  likelihood  of 
robbing,  swarming,  absconding,  and  accidental  straying  or  drifting 
of  bees  to  foreign  colonies. 

In  preparing  the  material  with  which  the  colony  is  inoculated, 
larvse  in  early  stages  of  the  disease  are  picked  from  the  brood 
frames,  crushed,  and  added  to  sugar  sirup.  The 
crushed  mass  from  10  or  more  sacbrood  larvse,  sus- 
pended in  somewhat  more  than  half  a  pint  of  sugar 
sirup,  has  been  found  to  be  a  suitable  quantity  of  the 
infective  material  to  use  in  making  an  inoculation. 
The  suspension  may  be  fed  to  the  bees  as  one  feeding 
or  more.  The  inoculation  feedings  should  be  made  as 
a  rule  toward  evening  to  avoid  the  tendency  to  rob, 
which  may  be  noticed  during  a  dearth  of  nectar.  Inocu- 
lations should  not  be  made  when  the  tendency  to  rob 
is  at  all  marked. 

Before  a  colony  is  inoculated  it  should  be  deter- 
mined that  its  activities  are  normal.  A  colony  should 
not  be  inoculated  for  several  days  after  it  has  been 
made  by  division,  or  immediately  after  its  removal 
from  a  foreign  location.  An  experimental  colony  when 
inoculated  should  have  larvse  of  all  ages,  and  a  queen 
doing  well. 

Between  five  and  six  days  after  a  colony  has  been 
inoculated  with  sacbrood  virus,  the  first  symptoms  of 
the  disease  are  to  be  expected.  The  finding  of  capped 
larvse  having  a  slightly  yellowish  hue  (fig.  12;  PI.  II, 
h,  h)  is  the  best  early  symptom  by  which  the  presence 
of  the  disease  may  be  known. 

Another  method  of  inoculation  may  be  used  and 
under  certain  circiimstances  is  desirable.  The  method 
is  more  direct  than  the  one  just  described.  The 
crushed  tissues  of  a  diseased  larva  are  suspended  in  a 
small  amount  of  water  or  thin  sugar  sirup.  With  a 
capillary  pipette  (fig.  30)  made  from  smaU  glass  tubing, 
a  very  small  amoimt  of  the  suspension  is  added  di- 
rectly to  the  food  which  surrounds  the  healthy  larva 
in  the  cell.  This  is  easily  done.  Having  drawn  some  of  the  suspen- 
sion into  the  pipette,  carefully  touch  the  food  iu  the  ceU  surround- 
ing the  larva  with  the  point  of  the  pipette.  A  small  amount  of  the 
suspension  will  flow  out  and  mix  with  the  food.  Larvse  approxi- 
mately two  days  of  age  should  be  selected  for  feeding.     A  dozen 


Fig.  30— Capillary 
pipette.  A  piece 
of  glass  tubing 
drawn  to  capil- 
lary size  at  one 
end.  Keduced  to 
three-fourths  of 
the  size  nsed. 
(Original.) 


34 


BULLETIN  431,  TJ.  S.  DEPARTMENT   OF   AGRICULTURE. 


or  more  should  be  fed  in  making  an  inoculation.  The  area  of 
brood  inoculated  may  be  designated  by  marking  on  the  brood  frame, 
or  by  removing  the  brood  from  around  the  area  inoculated,  thus 
marking  it  off. 

MEANS  FOR  THE  DESTRUCTION  OF  THE  VIRtS  OF  SACBROOD. 

Although  the  virus  of  sacbrood  may  increase  with  great  rapidity, 
fortunately  it  is  quite  as  readily  destroyed.  Nature  suppUes  many 
means  by  which  this  may  be  accomplished.  While  theoretically  a 
sufficient  amount  of  virus  may  be  produced  within  one  month  to 
inoculate  all  the  bees  in  existence,  within  another  month,  if  left  to 
natiu-al  means  alone,  practically  all  such  virus  would  be  destroyed. 
This  latter  fact  constitutes  one  of  the  chief  reasons  for  the  compara-. 
tively  rapid  self-recovery  of  colonies  from  this  disease. 

It  was  observed  in  the  experiments  that  larvse  dead  of  sacbrood 
when  left  in  the  brood  comb  ceased  to  be  infectious  in  less  than  one 
month  after  death. 

HEATING  REQUIRED  TO  DESTROY  SACBROOD  VIRUS  WHEN  SUSPENDED 

IN  WATER. 

Approximate  results  have  been  published  (White,  1914)  relative 
to  the  heating  that  is  necessary  to  destroy  the  virus  of  sacbrood 
when  it  is  suspended  in  water.  In  the  following  table  are  given 
some  results  which  have  been  obtained : 

Table  I. — Effect  of  heating  on  the  virus  of  sacbrood  suspended  in  waters 


Date  of  inoculation. 


Temperature. 


Time  of 

heating. 


Results  of  inoculation. 


Aug.  6, 1913.. 
Sept.  10, 1913 
Sept.  9, 1913., 
Sept.  18, 1913 
June  30, 1915. 
Sept.  10, 1913 
Aug.  28, 1915. 
Sept.  10, 1913 
Aug.  28, 1915, 
Aug.  26, 1913. 

Do 

Do 

Do 


°F. 
122 
131 
131 
135 
136 
136 
138 
140 
142 
149 
158 
167 
176 


MintUes. 
3D 
10 
20 
15 
10 
10 
10 
15 
10 
15 
15 
15 
15 


Sacbrood  produced. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


1  Fractions  will  be  omitted  in  this  paper,  the  nearest  whole  number  being  given. 

It  will  be  observed  from  Table  I  that  138°  F.  (59°  C.)  maintained 
for  10  minutes  was  sufficient  to  destroy  the  virus  of  sacbrood  in  the 
inoculation  experiments  recorded.  Technically,  in  view  of  the 
variable  factors  which  must  be  considered  in  experiments  of  this 
kind,  this  residt,  as  representing  the  thermal  death  point  of  the 
sacbrood  virus,  should  be  considered  as  being  only  approximate. 
For  practical  purposes,  however,  it  is  sufficient. 


SACBROOD. 

In  performing  these  experiments  a  crushed  mass, 
representing  from  10  to  20  larvse  recently  dead  of  the 
disease,  is  diluted  to  about  10  times  its  volume  with 
tap  water.  About  one-half  ounce  of  this  suspension  is 
placed  in  a  test  tube  (fig.  31),  almost  filling  it.  The 
tube  is  stoppered  with  a  perforated  cork,  bearing  a 
short  glass  tube  of  small  cahber  and  drawn  at  one  end 
to  capillary  size.  This  is  all  immersed  in  water  at  a 
temperature  to  which  it  is  desired  that  the  virus  shall 
be  heated.  It  requires  nearly  five  minutes  for  the  tem- 
perature of  the  suspension  in  the  tube  to  reach  that  of 
the  water  outside.  Aftei'  reaching  the  degree  desired 
the 'temperature  is  maintained  for  10  minutes,  after 
which  the  tube  is  removed  and  the  contents  added  to 
about  one-half  pint  of  sirup.  The  suspension  is  then 
fed  to  a  healthy  colony.  If  by  such  a  feeding  no  sac- 
brood  is  produced,  the  virus  is  considered  as  having 
been  destroyed  by  the  heating.  On  the  other  hand, 
if  the  disease  is  produced  it  follows  naturally  that  the 
virus  had  not  been  destroyed. 

HEATING  REQUIRED  TO  DESTROY  SACBROOD  VIRUS 
WHEN  SUSPENDED  IN  GLYCERINE. 


35 


■o  !i 


In  determining  the  amount  of  heating  that  is  necessary 
to  destroy  the  virus  of  a  disease  when  it  is  suspended 
in  a  liquid,  the  results  should  always  be  given  in  terms 
of  at  least  the  three  factors,  (1)  degree  of  temperature, 
(2)  time  of  heating,  and  (3)  the  medium  in  which  the 
virus  is  suspended. 

With  the  virus  of  sacbrood  the  results  vary  markedly; 
depending  upon  the  nature  of  the  liquid  in  which  the 
suspension  is  made.  To  illustrate  this  point  the  re- 
sults of  a  few  inoculation  experiments  are  given  here 
in  which  the  virus  was  heated  while  suspended  in 
glycerine. 


1 1 


a  » 

II 


Table  II. — Effect  produced  by  heating  the  virus  of  sacbrood  suspended 
in  glycerine. 


Date  of  inoculation. 

Temperature. 

Time  of 
heating. 

Results  of  inoculation. 

June  25, 1915 

'F. 
140 
149 
158 
160 
163 
167 

°C. 
60 
65 
70 
71 
73 
75 

Minutes. 
10 
10 
10 
10 
10 
10 

Sacbrood  produced. 
Do. 

June  24, 1915 . . . 

June  25, 1915 

Do. 

Aug.  28, 1915 

Do. 

Do 

Aug.  7, 1915 

Do. 

w 


3 
I 


36 


BULLETIN  431,  U.  S.  DEPAETMENT    OF    AGEICULTTJRE. 


In  these  inoculations  it  will  be  observed  that  a  temperature  some- 
what greater  than  158°  F.  (70°  C.)  maintained  for  10  minutes  was 
necessary  to  destroy  the  virus  of  sacbrood  when  it  was  suspended  in 
glycerine,  while  a  temperature  somewhat  less  than  140°  F.  (60°  C.) 
is  sufEcient  to  destroy  it  when  suspended  in  water  (p.  34).  The  same 
technique  was  employed  when  glycerine  was  used  as  the  suspending 
medium  as  was  employed  when  water  was  used  as  the  medium. 
The  same  strain  of  virus  was  used  in  both  instances.  The  point 
here  illustrated  is  of  special  interest  in  connection  with  the  heating 
of  honey  containing  the  virus  of  sacbrood. 

HEATING  REQUIRED  TO  DESTROY  SACBROOD  VIRUS  WHEN  SUSPENDED 

IN  HONEY. 

From  the  results  obtained  by  heating  the  virus  of  sacbrood  iu 
glycerine  as  given  above  it  might  be  expected  that  a  higher  tempera- 
ture would  be  necessary  to  destroy  the  virus  when  it  is  suspended  in 
honey  than  when  it  is  suspended  in  water. 

In  determining  the  heating  necessary  to  destroy  the  virus  when 
suspended  in  honey  the  technique  followed  was  similar  to  that 
employed  when  water  and  glycerine  suspensions  were  used.  The 
virus  used  in  the  inoculations  bearing  the  date  1915  was  of  the  same 
strain  in  all  instances. 

Table  III. — Results  obtained  vhen  the  virus  of  sacbrood  was  heated  in  honey. 


Date  of  inoculation. 


June  1, 1915. 

June  11, 1915, 

Do 

June  4, 1915. 
June  24, 1915. 

Do 

June  1, 1915. 
June  18, 1915, 
July  3, 1915.. 
Aug.  28, 1915 
Aug.  7, 1916. 
Aug.  28, 1915 
June  1,1915. 
Aug.  7, 1915. 
June  1, 1915. 


Temperature. 


°F. 
140 
145 
149 
154 
156 
158 
158 
158 
160 
160 
163 
163 
167 
167 
176 


'C. 
60 
63 
65 
68 
69 
70 
70 
70 
71 
71 
73 
73 
75 
75 
80 


Time  of 
heating. 


Minutes. 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 
10 


J 


Results  of  inoculation. 


Sacbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


As  shown  by  the  results  recorded  in  Table  III,  the  virus  of  sacbrood 
when  suspended  iu  honey  was  destroyed  in  10  minutes  at  a  tempera- 
ture very  near  158°  F.  (70°  C).  This  temperature  is  more  than  18°  F. 
(10°  C.)  greater  than  the  temperature  required  to  destroy  in  the  same 
time  the  virus  when  suspended  in  water  and  approximately  equal  to 
that  necessary  to  destroy  it  when  suspended  in  glycerine. 


SAOBROOD, 


37 


RESISTANCE  OF  SACBROOD  VIRUS  TO  DRYING  AT  ROOM  TEMPERATURE. 

In  the  experiments  made  for  the  purpose  of  determining  the  amount 
of  drying  which  the  virus  of  sacbrood  will  withstand,  larvse  recently- 
dead  of  the  disease  were  used.  These  are  crushed,  strained  through 
cheesecloth,  and  the  crushed  mass  poured  into  Petri  dishes  (fig.  32)  to 
the  extent  of  a  thin  layer  for  each  dish,  the  material  in  each  being  the 
crushed  remains  of  about  30  larvae.  These  are  placed  in  a  drawer, 
shielding  the  larval  material  from  the  light.  The  drying  then  pro- 
ceeds at  the  temperature  of  the  room.  This  temperature  varied 
greatly  from  day  to  day,  sometimes  being  as  high  as  93°  F.  (34°  C). 

At  intervals,  reckoned  in 
days,  after  the  preparation 
of  the  virus,  colonies  are 
inoculated.  An  aqueous 
suspension  is  made  of  the 
drying  larvaJ  content  con- 
tained in  a  Petri  dish. 
This  is  added  to  sirup,  and 

the  sirup  suspension  is  fed  to  a  healthy  colony 
gave  the  following  results : 


Fig.  32.— Open  Petri  dish.    One-half  of  Petri  dish,  either 
top  or  bottom.    (Original.) 


The  experiments 


Table  IV. — Resistance  of  sacbrood  virus  to  drying  at  room  temperature. 


Date  of  inoculation. 


Time  of  drying. 


Resxilts  of  inoculatidn. 


Aug.  8, 1914.. 
Aug.  14, 1914. 
Sept.  6, 1915. 
July  1, 1915. . 
Sept.  28, 1915 
Julys,  1915.. 
Sept.  3, 1915. 
Sept.  27, 1915. 
Oct.  9, 1914.. 
July  29, 1915. 
Sept.  3, 1915. 

Do 

May  22, 1915. 

Do 


3  days 

7days 

13  days 

16  days 

18  days 

20  days 

22  days 

26  days 

28  days 

28  days 

35  days 

45  days 

7  months  12  days . 
7  months  21  day^. 


Sacbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  sacbrood  produced. 

Do. 

No. 

Do. 

Do. 

Do. 

Do. 

Do. 


From  the  results  recorded  in  Table  IV  it  will  be  noted  that  the  virus 
of  sacbrood  in  the  experiment  referred  to  withstood  drying  at  room 
temperature  for  approximately  three  weeks. 

The  inoculations  made  during  the  third  week  indicated,  by  the  re- 
duced amount  of  sacbrood  produced,  that  much  of  the  virus  had 
already  been  destroyed.  Obtaining  negative  results  from  the  use  of 
larval  material  which  had  been  drying  more  than  seven  months  tends 
toward  eliminating  the  possibility  that  the  virus  possesses  a  resting 
stage. 


38 


BULLETIN  431,  XJ.   S.  DEPARTMENT    OF    AGEICULTTJEE. 


Similar  prelimiaary  experiments  made  to  determine  the  amount  of 
drying  which  the  virus  of  sacbrood  will  withstand  at  outdoor  tempera- 
ture and  at  incubator  temperature  (about  99°  F.  [37°  C.])  gave  results 
approximately  those  obtained  from  drying  at  room  temperature,  the 
time  being  somewhat  less  in  the  case  of  drying  at  incubator  tempera- 
ture. 

Prehminary  experiments  indicate  also  that  when  the  virus  is  mixed 
with  poUen  and  allowed  to  dry  the  period  for  which  it  remains  virulent 
is  iacreased  only  slightly. 

RESISTANCE  OF  SACBROOD  VIRUS  TO  DIRECT  SUNLIGHT  WHEN  DRY. 

In  the  experiments  made  to  determine  the  amount  of  sunlight 
which  the  virus  of  sacbrood  is  capable  of  resisting,  Petri-dish  prepara- 
tions similar  to  those  made  in  the  drying  experiment  were  prepared. 
After  drying  a  few  hours  in  the  room  the  uncovered  dish  is  exposed 
to  the  direct  rays  of  the  sun.  At  different  intervals,  measured  in 
hours,  inoculations  of  healthy  colonies  are  made  similar  to  those  in 
the  drying  experiments.     The  following  results  were  obtained: 

Table  V. — Resistance  of  the  virus  of  sacbrood,  when  dry,  to  direct  sunlight. 


Date  of  inoculation. 


Time  of 

exposure 

to  sun's 

rays. 


Results  of  inoculation. 


Sept.  17, 1915. 
July  29, 1915.. 
Sept.  17, 1915. 
Sept.  16, 1915. 

Do 

Do 

Aug.  25, 1915. 
Sept.  10, 1915. 

Do 

Sept.  9,1915.. 

Do 

Aug.  19,1915. 
July  16, 1915. 
Aug.  20, 1915. 
Sept.  11, 1915. 


Hours. 
2 
2J 


6 
4 
5 
7 
9 
12 
13 
18 
21 


Sacbrood  produced. 

Do.. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do, 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


The  results  recorded  in  Table  V  show  that  the  virus  of  sacbrood  in 
the  experiments  made  was  destroyed  in  from  four  to  seven  hours' 
exposure  to  the  direct  rays  of  the  sun.  The  results  obtained  also 
indicate  that  much  of  the  virus  was  destroyed  in  a  2-hour  exposure 
to  the  sun. 

It  vnR  be  readily  appreciated  that  the  time  that  the  virus  will 
resist  the  sun's  rays  will  depend  a  great  deal  upon  the  intensity  of 
the  rays  at  the  time  of  its  exposure  and  the  thickness  of  the  layer 
of  the  infective  larval  material  in  the  Petri  dish.    The  drying  that 


SACBKOOD. 


39 


would  naturally  take  place  during  the  exposure  to  the  sun  would 
tend  also  to  destroy  the  virus,  but  as  the  resistance  to  drying  is  better 
given  in  weeks  than  days,  this  factor  may  be  disregarded  here. 

RESISTANCE  OF  SACBROOD  VIRUS  TO  DIRECT  SUNLIGHT  WHEN  SUS- 
PENDED IN  WATER. 

In  the  experiments  made  for  the  purpose  of  determining  the  resist- 
ance of  the  virus  of  sacbrood  to  the  direct  rays  of  the  sun  when 
suspended  in  water,  Petri  dishes  were  again  used.  About  1^  ounces  of 
the  aqueous  suspension  containing  the  crushed  tissues  of  30  larvae  is 
poured  into  the  dish  and  exposed  to  the  direct  rays  of  the  sun.  After 
intervals  reckoned  in  hours  the  inoculations  of  healthy  colonies  are 
made.  The  contents  of  a  single  Petri  dish  are  added  to  about  one- 
half  pint  of  sirup  and  the  suspension  fed  to  a  healthy  colony.  The 
following  results  were  obtained  from  the  experiments: 

Table  VI. — Resistance  of  sacbrood  virus  to  the  direct  rays  of  the  sun  when  suspended  in 

water. 


Date  of  inoculation. 


Time  of 

exposure 

to  sun's 

rays. 


Results  of  inoculation. 


Sept.  10, 1915. 
Aug.  20, 191S. 
Sept.  14, 1915. 
Aag.24,igi5. 
Aug.  18, 1915. 
Sept.  9, 1915.. 
Sept.  10, 1915. 
Aug.  24, 1915. 

Do 

Aug.  16, 1915. 
Sept.  8, 1915.. 

Do 

Sept.  9, 1915.. 

Do 

Aug.  25, 1915. 
Aug.  20, 1915. 
Jtdyia,  1915. 
Aug.  26, 1919. 


Sacbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
"  Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


From  Table  VI  it  wiU  be  seen  that  when  suspended  in  water  the 
virus  of  sacbrood  was  killed  in  from  four  to  six  horn's. 

The  aqueous  suspensions  in  the  Petri  dishes  in  these  experiments 
did  not  reach  by  several  degrees  the  temperature  138°  F.  (59°  C.)  at 
which  the  virus  "is  destroyed  readily  by  heating  (p.  34).  Naturally 
experiments  of  the  natm-e  of  those  in  this  group  will  vary  in  all  cases 
with  the  intensity  of  the  sun's  rays  to  which  the  virus  is  exposed. 
The  exposures  were  made  in  these  experiments  between  9  and  4 
o'clock,  the  sun's  rays  toward  the  middle  of  the  day  being  most 
often  used. 


40 


BULLETIN  431,  U.  S.  DEPARTMENT   OP   AGBICULTtTEE. 


RESISTANCE  OF  SACBROOD  VIRUS  TO  DIRECT  SUNLIGHT  WHEN  SUS- 

PENDED  IN  HONEY. 

The  crushed  and  strained  tissue  mass  of  larvae  dead  of  sacbrood 
was  susj)ended  in  honey  and  exposed  to  the  direct  rays  of  the  sun. 
To  prevent  robbing  by  bees,  closed  Petri  dishes  were  used.  At 
intervals  reckoned  in  hours  healthy  colonies  were  inoculated,  each 
with  the  virus  from  a  single  Petri  dish.  The  exposures  were  made 
during  the  day  between  9  and  4  o'clock,  preference  being  given  to 
the  hours  near  midday.  The  group  of  experiments  conducted  on 
this  point  gave  the  following  results : 

Table  VII. — Resistance  of  the  sacbrood  virus  to  direct  sunlight  when  suspended  in  honey. 


Date  of  inoculation. 


Time  of 

exposure 

to  sun's 

rays. 


Besults  of  inoculation. 


Aug.  24, 1915. 

Do 

Aug.  18, 1915. 
Sept.  9, 1915.. 
Sept.  10, 1915. 
Aug.  24, 1915. 
Aug.  16,1915. 
Aug.  25, 1915. 
Sept.  8, 1915.. 

Do 

Sept.  9, 1915.. 

Do 

Aug.  25, 1915. 
Sept.  11, 1915. 
Aug.  26,  1915. 
Sept.  11, 1915, 


Hours. 

1 

2 

4 

4 

4 

5 

5 

5 

5 

6 

7 

8 

10 

12 

13 

18 


Sacbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


From  the  results  of  the  experiments  recorded  m  Table  VII  it  wUl 
be  observed  that  the  virus  of  sacbrood  when  suspended  in  honey 
was  destroyed  by  the  direct  rays  of  the  sun  in  from  five  to  six  hours. 
These  figures  represent  the  time  for  destruction  of  all  of  the  virus 
used  in  each  experiment.  The  results  obtained  from  the  experi- 
ments indicate,  however,  that  much  of  it  was  destroyed  earlier. 

LENGTH  OF  TIME  THAT  SACBROOD  VIRUS  REMAINS  VIRULENT  IN 

HONEY. 

In  devising  methods  for  the  treatment  of  sacbrood  it  is  of  particular 
interest  to  know  the  length  of  time  that  the  virus  will  remain  vindent 
when  it  is  ia  honey.  Experiments  have  been  made  to  gain  data  on 
this  point.  Larvae  recently  dead  of  sacbrood  are  crushed,  strained, 
and  suspended  ia  honey.  About  one-half  piat  of  the  suspension, 
representing  the  virus  from  about  30  dead  larvae,  is  placed  in  each  of 
a  number  of  glass  flasks.  These  are  allowed  to  stand  at  room  temper- 
ature, being  shielded  from  the  light  by  being  placed  in  a  closed  cabinet. 


SAOBEOOD. 


41 


After  periods  reckoned  in  days  inoculations  of  healthy  colonies  are 
made.     The  following  results  have  been  obtained : 

Table  VIII. — Length  of  time  the  virus  of  sacbrood  remains  virulent  in  honey. 


Date  of  Inoculation. 


June  17, 1915. 
June  4, 1915.. 
Oct.  2, 1915... 
Sept.  3, 1915.. 
July  29, 1915.. 
June  30, 1915. 

Do 

July  17, 1915.. 
Oct.  21, 1915.. 
Sept.  8,1915.. 
May  13, 1915.. 
May  6, 1915... 
May  4, 1915... 
May  18, 1915.. 
Sept.  3, 1915.. 


Time  virus 

was 

in 

Results  of  inoculation. 

honey. 

Mos.  Days. 

0 

20 

Sacbrood  produced. 

0 

23 

Do. 

0 

»() 

Do. 

0 

24 

No  disease  produced. 

0 

29 

Do. 

0 

33 

Do. 

0 

35 

Do. 

0 

3H 

Do. 

0 

49 

Do. 

0 

70 

Do. 

17 

10 

Do. 

7 

20 

Do. 

8 

2 

Do. 

8 

21 

Do. 

12 

1 

Do. 

I  Tlie  dead  brown  liuval  remains  were  not  cruslied  before  being  introduced  into  the  honey. 

The  experiments  recorded  in  Table  VIII  show  that  the  virus  of 
sacbrood  when  suspended  in  honey  at  room  temperature  remained 
virulent  for  three  weeks,  but  was  entirely  destroyed  before  the  end 
of  the  fifth  week.  It  is  most  likely  that  the  virus  in  most  instances 
is  destroyed  by  the  end  of  one  month  at  this  temperatiire. 

The  experiments  in  which  the  virus  had  been  allowed  to  remain 
in  the  honey  for  more  than  seven  months  suggest  that  there  is  prob- 
ably no  resting  stage  of  the  virus  to  be  considered  in  this  connection. 
The  facts  tend  to  indicate  that  the  vkus  does  not  receive  any  marked 
amount  of  protection  by  being  in  honey.  From  the  dates  of  the 
experiments  in  this  group  it  wiU  be  noted  that  the  virus  was  sub- 
jected to  summer  temperature.  The  evidence  at  hand  indicates  that 
it  remains  virulent  somewhat  longer  when  the  temperature  is  lower. 

RESISTANCE  OF  SACBROOD  VIRUS  TO  THE  PRESENCE  OF  FERMENTA- 

TIVE  PROCESSES. 

Fermentation  and  putrefaction  ^  are  other  means  by  which  the 
virus  of  sacbrood  may  be  destroyed  in  water.  A  crushed  and 
strained  mass  of  tissue  from  larvae  recently  dead  of  the  disease  is 
suspended  m  a  10  per  cent  sugar  (granulated  or  cane  sugar)  solution. 

1  "Fermentation"  has  reference  here  particularly  to  the  breaking  up  of  carbohydrate  substances  by 
the  growth  of  microorganisms,  the  sugars  in  honey  being  naturally  the  carbohydrates  especially  of  mterest 
in  these  discussions.  The  process  results  in  the  formation  of  a  large  number  of  suhstances-acids,  alcohols, 
etc.  The  odor  accompanying  such  a  process  could  not  be  called  offensive.  By  the  term  "putrefaction" 
is  meant  the  breaking  up  of  nitrogenous  organic  substances  by  microorganisms.  These  have  a  chemical 
composition  quite  different  from  the  carbohydrates.  When  broken  up  the  resulting  substances  are  more 
often  alkaline  in  nature.  The  odor  from  a  suspension  in  which  putrefactive  processes  are  gomg  on  is 
usually  distinctly  offensive. 


42 


BULLETIN  431,  U.  S.  DEPARTMENT  OP   AGRICTJLTTJRE. 


A  small  quantity  of  soil  is  added  to  inoculate  the  suspension  further. 
This  is  then  distributed  in  test  tubes  (fig.  33),  the  quantity  in  each  tube 
representing  the  virus  from  about  15  larvse.  These  suspensions  are 
allowed  to  remain  at  room  temperature,  shielded 
from  the  Ught.  Under  these  conditions  fermenta- 
tion goes  on  rather  rapidly. 

After  intervals  reckoned  in  days  colonies  free 
from  the  disease  are  inoculated,  each  with  the 
suspension  from  a  single  tube.  Results  from 
such  inoculations  are  given  in  the  following  table: 

Table  IX. — Resistance  of  sachrood  virus  to  fermentation  in  a  10 
per  cent  sugar  solution  at  room  temperature. 


v_y 


Date  of  inoculation. 

Period  of 
fermen- 
tation. 

Results  of  inoculation. 

Sept.  9, 1915. 

Days. 

1 

2 

3 

4 

3 

5 

5 

7 

9 

13 

34 

51 

85 

87 

90 

244 

Sacbrood  produced. 

Sept.  11, 1915..                      

Do. 

Do 

Do. 

Sept.  13, 1915  . 

Do. 

July  14, 1916 

No  disease  produced. 

July  22, 1916....                                

Do. 

Sept.  14, 1915  . 

Do. 

Sept.  22, 1916 

Do. 

July  10, 1916....                          

Do. 

June  10, 1915. . 

Do. 

July  7, 1914  ' 

Do. 

Aug.  27, 1914..              .           

Do. 

Do 

Do. 

Do 

Do. 

Do                                           

Do. 

Do 

Do. 

Fia.  33.— Test  tube 
bearing  a  cotton  plug, 
used  in  testing  the  ef- 
fect of  fermentation, 
putrefaction,  and  dis- 
infecting agents  on 
the  virus  of  sacbrood. 
(Original.) 


1  The  resultsrecorded  for  1914  were  obtained  with  a  suspension  of  crushed  larvse, 
in  various  stages  of  decay,  in  sirup  made  from  about  equal  parts  water  and  sugar. 

From  the  results  of  experiments  recorded  in  Table 
IX  it  win  be  noted  that  the  virus  of  sacbrood  was 
destroyed  in  from  three  to  five  days  in  the  presence 
of  fermentation  in  10  per  cent  cane  sugar  (saccharose) 
at  room  temperature. 

As  the  rapidity  of  fermentative  processes  varies 
with  the  temperature  present,  it  is  natural  to  sup- 
pose that  the  time  required  for  the  destruction  of 
the  virus  will  vary.  From  experiments  it  is  found 
that  at  incubator  temperature  the  time  is  slightly 
less,  and  at  outdoor  temperature  it  is  somewhat 
greater  than  at  room  temperature. 


RESISTANCE  OF  SACBROOD  VIRUS  TO  FERMENTATION   IN  DILUTED 
HONEY  AT  OUTDOOR  TEMPERATURE. 

Employing  the  egg  test  *  as  used  by  beekeepers  in  diluting  honey 
for  the  purpose  of  making  vinegar,  it  is  found  that  it  requires  about 

'  This  test  is  applied  in  the  following  manner:  Water  is  added  to  honey  until  an  egg  placed  in  the  mixture 
Is  nearly  submerged,  the  surface  remaining  above  the  liquid  being  only  about  as  large  as  a  10-cent  piece. 


SACBEOOD. 


43 


fom-  volumes  of  water  to  one  of  ripened  honey  to  obtain  the  strength 
recommended.  The  honey  solution  by  volmne,  therefore,  is  about 
20  per  cent  honey. 

A  suspension  of  the  virus  of  sacbrood  in  such  a  solution  is  dis- 
tributed in  test  tubes  placed  in  an  empty  hive  body  and  allowed  to 
ferment  at  outdoor  temperature.  After  periods  reckoned  in  days 
colonies  are  inoculated  as  was  done  in  case  of  the  sugar  solutions 
described  above.  The  following  results  were  obtained  from  the 
experiments  performed : 

Table  X. — Resistance  of  sacbrood  virus  to  fermentative  processes  in  a  20  per  cent  honey 
solution  at  outdoor  temperature. 


Date  of  inoculation. 


Results  of  inoculation. 


Sept.  11, 1915. 
Sept.  13, 1915. 
Sept.  14, 1915. 
Aug.  4, 1915.. 
Sept.  15, 1915. 
Sept.  14, 1915. 
Sept.  22, 1915. 
Sept.  17, 1915. 
Sept.  8, 1915.. 


Sacbrood  produced. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 


In  the  presence  of  fermentative  processes  taking  place  in  a  20  per 
cent  honey  solution  at  outdoor  temperature  it  wiU  be  observed  that 
the  virus  of  sacbrood  in  the  experiments  recorded  in  Table  X  was 
destroyed  in  six  days.  The  outdoor  temperature  during  these 
experiments  was  quite  warm.  Had  it  been  cooler,  the  time  for  the 
destruction  of  the  virus  would  have  been  somewhat  increased.  In 
the  making  of  vinegar  it  may  be  concluded  that  the  virus  of  sacbrood, 
should  it  be  present  in  the  honey  used,  would  be  destroyed  in  a  com- 
paratively short  time  as  a  result  of  fermentation. 

RESISTANCE  OF  SACBROOD  VIRUS  TO  THE  PRESENCE  OF  PUTREFACTIVE 

PROCESSES. 

Larvse  containing  the  virus  of  sacbrood  are  crushed  and  suspended 
m  water.  A  small  quantity  of  soil  is  added.  The  suspension  is 
stramed  and  distributed  in  test  tubes.  These  are  allowed  to  stand  at 
room  temperature  in  a  state  of  putrefaction.  After  periods  reckoned 
in  days  colonies  free  from  the  disease  are  moculated,  each  with  the 
contents  of  a  single  tube  added  to  sirup.  From  experiments  of  this 
kind  the  results  following  have  been  obtained. 


44  BULLETIN  431,  V.  S.  DEPARTMENT  OP  AGBICULTUEE. 

Table  XI.— Resistance  of  sacbrood  virus  to  putrefaction. 


Aug. 
Aug. 
Aug. 

July 
Sept. 
Sept. 
July 
July 
May 
Sept. 
Aug. 
Sept. 
Sept. 


iept 
uly 


Date  of  inoculation. 


6,1914.. 

7,1914.. 

10,1914. 
20, 1915. 

13, 1916 

14, 1915 
22,1915. 
8,1915.. 
22,1915. 
,  22, 1916. 

18, 1915. 
,  16, 1914. 
,  25, 1914. 
1,1915.. 


Results  of  inoculation. 


Sacbrood  produced. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 

Do. 


From  Table  XI  it  will  be  noted  that  the  virus  of  sacbrood  was 
destroyed  m.  the  experiments  recorded  in  from  7  to  10  days.  As  in 
the  case  of  fermentation,  so  in  the  case  of  putrefaction,  it  is  to  be 
expected  that  the  time  for  the  destruction  of  the  virus  will  vary 
appreciably  with  the  temperature  at  which  the  putrefactive  processes 
take  place. 

RESISTANCE  OF  SACBROOD  VIRUS  TO  CARBOLIC  ACID. 

Larvae  recently  dead  of  sacbrood  are  crushed  and  strained.  This 
larval  mass  is  diluted  with  carbolic  acid  in  aqueous  solution.  About 
10  parts  of  carboUc  acid  to  1  part  of  the  larval  mass  is  used.  This 
suspension  is  distributed  in  test  tubes  and  allowed  to  stand  at  room 
temperature.  Each  tube  contains  the  virus  from  about  15  larvae. 
After  periods,  reckoned  in  days,  colonies  free  from  disease  are  inocu- 
lated, each  with  the  contents  of  a  single  tube  added  to  sirup. 

Carbolic  acid  solutions  of  J,  1,  2,  and  4  per  cent  were  used  in  mak- 
ing the  suspensions.  The  following  results  were  obtained  from  the 
experiments : 

Table  XII. — Resistance  of  sacbrood  virus  to  carbolic  acid. 


Date  of  inoculation. 


Strength 
of  car- 

Time in 

bolic  acid 

used. 

Per  cent. 

Bays. 

,^ 

1 

1, 

10 

y 

24 

38 

4 

50 

i 

50 

i 

2.38 

1 

16 

25 

38 

50 

50 

261 

Results  of  inoculation. 


Sept.  3, 1914.. 
Sept.  18, 1914 . 
Sept.  3, 1914.. 
Sept.  17, 1914. 
Aug.  12, 1916. 
Aug.  20, 1916. 
May  14, 1915- . 
Sept.  3, 1914.. 
Sept.  18, 1914 . 
June  23, 1915.. 
Sept.  17, 1916. 
Aug.  12, 1916., 
Aug.  21, 1915.. 
June  4, 1915... 


Sacbrood  produced. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 
Sacbrood  produced. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 

Do. 


SACBROOD.  45 

Table  XII. — Resistance  of  sacbrood  viriis  to  carbolic  acid — Continued. 


Date  of  inoculation. 


Strength 

ot  car- 
bolic add 
used. 


Sept.  3, 1914.... 
Sept.  18,1914... 
June  23, 1915... 
Sept.  17, 1915... 
Aug.  12, 1915... 
Aug.  21, 1915... 


June  23, 1915.. 
July  1,1915... 


June  23, 1915. 
Aug.  12, 1915. 


Per  cent. 
2 
2 
2 
2 
2 
2 


Time  in 
suspen- 
sion. 


Days. 
1 

16 
25 
38 
42 
50 

Hmirg. 


Days. 
25 
50 


Besults  o(  inoculation. 


Saobrood  produced. 

Do. 

Do. 
No  disease  produced. 

Do. 

Do. 


Sacbrood  produced. 
Do. 


No  disease  produced. 
Do. 


From  the  preliminary  results  recorded  in  Table  XII  it  wiU  be 
observed  that  the  virus  of  sacbrood  shows  a  marked  resistance  to  the 
disinfecting  power  of  carbolic  acid.  Under  the  conditions  of  the 
experiments  the  virus  resisted  its  action  for  more  than  three  weeks 
in  ^,  1,  and  2  per  cent  aqueous  solutions. 

These  results  lead  naturally  to  a  consideration  of  the  effect  of 
drugs  on  the  virus  of  sacbrood  in  the  treatment  of  the  disease.  On 
this  point  complete  data  are  yet  wanting. 

While  the  disinfecting  power  of  a  compound,  as  shown  in  experi- 
ments such  as  those  described  above  for  carboHc  acid,  may  indicate 
something  as  to  the  value  of  the  compound  as  a  drug,  it  does  not 
necessarily  prove  its  value.  More  definite  proof  is  gained  through 
feeding  colonies  with  the  virus  suspended  in  honey  medicated  with 
the  drug,  and  then  continuing  to  feed  the  inoculated  colonies  with 
honey  similarly  medicated  daUy  thereafter  untU  the  time  for  the 
appearance  of  the  disease. 

To  illustrate  the  nature  of  experiments  which  are  being  conducted 
to  determine  the  value  of  drugs  in  the  treatment  of  sacbrood,  experi- 
ments with  quinine  and  carbolic  acid  are  here  referred  to.  A  colony 
was  fed  the  virus  of  sacbrood  suspended  in  honey  and  water,  equal 
parts,  to  which  was  added  5  grains  of  the  bisulphate  of  quinme  to 
one-half  pint  of  diluted  honey,  and  on  each  of  the  five  days  following 
the  inoculation  the  same  colony  was  fed  diluted  honey  containing  no 
virus,  but  medicated  with  quinine  in  the  same  way.  On  the  seventh 
day  following  the  inbciilation  with  the  virus  there  was  found  to  be  a 
large  quantity  of  sacbrood  produced  in  the  colony  so  inoculated  and 

treated. 

A  similar  experiment  in  which  carbohzed  honey  was  used  gave 
like  results.  These  experiments,  although  not  furnishmg  conclusive 
proof,  do  indicate  something  of  what  might  be  expected  from  the 
use  of  quinine  or  carbolic  acid  as  a  drug  in  the  treatment  of  sacbrood. 


46  BULLETIN  431,  U.  S.  DEPAETMENT   OF   AGBICULTUBE. 

Technically  the  foregoing  studies  should  be  thought  of  as  being 
prehminary.  Questions  relating  to  virulence  of  the  virus,  resistance 
of  the  bees,  technique,  and  many  other  factors  contribute  to  make 
results  such  as  these  vary.  For  practical  purposes,  however,  they 
are  sufficiently  complete.  In  estimating  the  time  necessaiy  for  the 
destruction  of  the  virus  in  practical  apiculture  by  any  of  the  fore- 
going tables  of  results  it  should  be  emphasized  that  the  time  element 
should  be  somewhat  increased,  inasmuch  as  the  conditions  present  in 
the  experiments  were  more  favorable  for  its  destruction  than  would 
ordinarily  be  the  case  in  practice. 

MODES  OF  TRANSMISSION  OF  SACBROOD. 

The  transmission  of  a  brood  disease  must  be  thought  of  as  taking 
place  (1)  from  diseased  to  healthy  brood  within  a  colony  and  (2)  from 
a  diseased  colony  to  a  healthy  one.  The  manner  in  which  sacbrood 
is  spread  naturally  depends  directly  upon  the  modes  by  which  the 
virus  of  the  disease  is  transmitted. 

As  is  shown  experimentally,  the  virus  of  sacbrood  produces  the 
disease  when  it  is  added  directly  to  the  food  of  young  larvae  or  when 
it  is  mixed  with  sirup  and  fed  to  a  colony.  From  this  fact  it  is  fair 
to  assume  that  sacbrood  may  result  whenever  the  food  or  water  used 
by  the  bees  contains  the  hving  virus  of  the  disease. 

Bees  have  a  tendency  to  remove  diseased  or  dead  larvae  from  the 
cells.  When  the  removal  is  attempted  about  the  time  of  death,  it 
is  done  piecemeal.  Each  fragment  removed  from  such  a  larva,  if 
fed  t(5  a  young  healthy  larva  within  a  week,  would  most  likely 
produce  sacbrood  in  the  larva.  Within  the  hive,  therefore,  the  dis- 
ease may  be  transmitted  to  healthy  larvae  more  or  less  directly  in 
this  way. 

Just  what  becomes  of  these  bits  of  tissue  removed  from  the  dis- 
eased larvae,  however,  is  not  known.  If  it  were  the  rule  that  the 
tissues  of  the  dead  larva  after  being  removed  in  fragments  were  fed 
unaltered  to  the  young  healthy  larva?  within  two  weeks  after  its 
removal,  it  would  seem  that  the  disease  would  increase  rapidly  in 
the  colony  as  a  result.  Such  an  increase,  however,  is  unusual,  the 
tendency  in  a  colony  being  in  most  cases  toward  a  recovery  from 
the  disease. 

This  fact  leads  one  to  think  of  other  possibilities  regarding  the 
destiny  of  the  infected  tissues  removed  as  fragments  from  the  dis- 
eased larvie.  If  the  infective  material  were  fed  to  the  older  larva;, 
death  probably  would  not  result.  Should  it  be  used  by  adult  bees  as 
food  for  themselves,  the  hkehhood  of  the  transmission  of  the  dis- 
ease under  such  chcumstances  would  apparently  be  very  materially 
reduced.     If  the  infective  material  were  stored  with  the  honey  and 


SACBEOOD.  47 

did  not  reach  the  brood  within  a  month  or  six  weeks,  it  is  not  prob- 
able that  the  disease  would  be  transmitted  mider  such  cu-cumstances 
(p.  41).  Should  the  dead  larva  or  any  fragments  of  them  be  car- 
ried out  of  the  hive,  the  virus  would  have  to  be  returned  to  the 
hive,  as  a  matter  of  course,  before  further  mfection  of  the  brood 
could  take  place  from  such  infective  material. 

It  is  left  to  be  considered  in  what  way  the  infective  material  if 
removed  from  the  hive  might  be  returned  to  the  brood  and  infect 
it.  Should  any  material  containing  the  virus  reach  the  water  sup- 
ply of  the  bees,  or  the  flowers  visited  by  the  bees,  it  is  within  the 
range  of  possibUity  that  some  of  the  hving  virus  might  be  returned 
to  the  hive  and  reach  healthy  young  larvae. 

While  out  of  the  hive,  however,  the  virus  must  withstand  certain 
destructive  agencies  in  nature.  Under  more  or  less  favorable  cir- 
cumstances it  would  withstand  drying  alone  for  a  few  weeks  (p.  37), 
but  if  exposed  to  the  sun  it  might  be  destroyed  in  a  few  hours,  (p  38). 
If  the  virus  were  subjected  to  fermentation  it  might  be  destroyed 
within  a  week  (p  43),  and  if  subjected  to  putrefaction,  within  two 
weeks  (p.  44). 

The  experimental  evidence  indicates  that  the  virus,  once  out  of 
the  hive  and  freed  from  the  adult  bees  removing  it,  during  the 
warmer  seasons  of  the  year,  at  least,  has  but  little  chance  of  being 
returned  to  the  hive  and  producing  any  noticeable  infection.  In  the 
experimental  apiary  (PI.  Ill)  a  large  number  of  colonies  have  been 
heavily  infected  with  sacbrood  through  experimental  inoculation, 
and  no  infection  was  observed  to  have  resulted  in  the  uninoculated 
colonies.  If  throughout  the  main  brood-rearing  season  the  usual 
source  of  infection  were  the  flowers  or  the  water  supply,  a  quite 
different  result  would  be  expected. 

Tentatively  it  may  be  concluded,  therefore,  that  the  probability  of 
the  transmission  of  the  virus  of  sacbrood  by  way  of  flowers  visited 
by  bees,  practically  considered,  is  quite  remote,  being,  however,  to 
a  limited  extent  theoretically  possible. 

It  would  seem  that  there  is  a  greater  likeUhood  of  the  water  supply 
being  a  source  of  infection  than  flowers.  The  chances  for  infection 
from  this  source,  should  it  occm-  at  all,  would  be  greater  in  the 
spring,  as  at  such  a  time  the  quantity  of  infective  material  in  dis- 
eased colonies  is  greater,  increasing  the  chances  that  some  of  it 
might  be  carried  to  the  water  supply  and  contaminate  it,  and  fur- 
thermore, the  destructive  agencies  in  nature  are  at  this  time  less 
efficient. 

Bees  drifting  or  straying  from  infected  colonies  to  healthy  ones 
must  be  thought  of  as  possible  transmitters  of  the  disease.  That 
the  disease  is  not  spread  to  any  great  extent  in  this  way  is  evidenced 


48  BULLETIN  431,  TJ.  S.  DEPARTMENT   OF   AGEICULTUBE. 

by  the  fact  that  colonies  in  the  apiary  that  were  not  inoculated 
experimentally  remained  free  from  disease,  although  many  colonies 
in  the  apiary  were  heavily  infected  at  the  time. 

Sacbrood  has  a  tendency  to  weaken  a  colony  in  which  it  is  present. 
Frequently  this  weakness  is  noticeable  and  often  marked.  Kob- 
bing,  which  occurs  not  infrequently  at  such  a  time,  results  in  the 
transmission  of  the  virus,  to  some  extent  at  least,  directly  to  healthy 
colonies.  Kobbing,  therefore,  must  always  be  considered  as  a  prob- 
able means  of  transmission. 

The  modes  of  transmission  of  sacbrood  within  the  colony  and  from 
colony  to  colony,  as  will  be  seen,  are  not  by  any  means  completely 
determined.  In  what  way  the  sacbrood  virus  is  carried  over  from 
one  brood-rearing  season  to  another  is  one  of  the  many  problems  con- 
cerning this  disease  that  are  yet  to  be  solved.  The  foregoing  facts, 
accompanied  by  the  brief  discussions,  it  is  hoped,  wiU  throw  some 
light  upon  this  important  phase  of  the  study — the  transmission  of 
this  disease — and  will  serve  as  an  aid  to  later  researches. 

DIAGNOSIS  OF  SACBROOD. 

The  diagnosis  of  sacbrood  can  be  made  from  the  symptoms  already 
described  (p.  10).  The  colony  may  or  may  not  be  noticeably  weak- 
ened. The  adult  bees  are  normal  in  appearance.  Scattered  here  and 
there  on  the  brood  frame  among  the  healthy  brood  are  found  dead 
larvae  in  the  late  larval  stage.  Usually  there  are  only  a  few  of  them, 
yet  sometimes  there  are  many.  These  larvae  may  be  in  capped  or 
uncapped  ceUs.  When  found  in  uncapped  cells,  however,  the  cap- 
pings  had  already  been  removed  by  the  bees  after  the  death  of  the 
larvae.  The  cap  over  a  dead  larva  in  a  cell  may  be  found  punctured 
or  not.     The  brood  possesses  no  abnormal  odor,  or  practically  none. 

The  post-mortem  appearances  of  larvae  dead  of  the  disease  are  espe- 
cially valuable  in  making  the  diagnosis.  The  larva  is  found  extended 
lengthwise  in  the  cell  and  on  its  dorsal  side.  Throughout  the  period 
of  decay  it  will  be  found  to  maintain  much  of  the  form  and  markings 
of  a  healthy  larva  of  the  age  at  which  it  died.  Soon  after  death  the 
larval  remains  are  slightly  yellow.  After  a  period  they  assume  a 
brownish  tint.  Since  the  brown  color  deepens  as  the  process  of  decay 
and  drying  takes  place,  the  remains  may  be  foimd  having  any  one  of 
a  number  of  shades  of  brown.  They  may  appear  at  times  almost 
black. 

After  death  the  cuticular  portion  of  the  body  wall  becomes  tough- 
ened, permitting  the  easy  removal  of  the  larva  intact  from  the  cell. 
When  removed,  the  saclike  appearance  of  the  remains  becomes  easily 
apparent.  Upon  rupturing  the  cuticular  sac  the  contents  are  found 
to  be  a  brownish,  granular-appearing  mass  suspended  in  a  compara- 


SACBROOD.  49 

livoly  small  quantity  of  more  or  less  clear  liquid.  The  scales  formed 
by  the  drying  of  the  decaying  remains  are  easily  removed  from  the 
cells.  After  becoming  quite  dry  many  of  them  indeed  can  be  shaken 
from  the  brood  comb. 

Upon  crushing  larvse  which  have  been  found  dead  for  some  time  but 
not  yet  dry,  a  marked  unpleasant  odor  will  be  noticed  if  the  crushed 
mass  is  held  near  the  nostrils. 

Microscopically  no  microorganisms  are  to  be  found  in  the  decay- 
ing remains  of  the  larviie.  Cultures  made  from  them  are  also  neg- 
ative. 

Differential  diagnosis. — Sacbrood  must  be  differentiated  from  the- 
other  brood  diseases. 

American  f  oulbrood  may  be  recognized  by  the  peculiar  odor  of  the 
brood  combs  when  the  odor  is  present.  The  body  wall  of  the  larval 
and  pupal  remains  is  easily  ruptured,  and  the  decaying  mass  becomes 
viscid,  giving  the  appearance  popularly  referred  to  as  "ropiness." 
The  scale  adheres  quite  firmly  to  the  floor  of  the  cell.  The  presence 
of  BaciUus  larvse  in  the  hrood  dead  of  the  disease  is  a  positive  means 
by  which  it  may  be  differentiated  from  sacbrood. 

European  foulbrood  may  be  recognized  by  the  fact  that  the  larvae 
as  a  rule  die  while  coiled  in  the  cell  and  before  an  endwise  position  is 
assumed.  In  the  majority  of  instances,  therefore,  death  takes  place 
before  the  cells  are  capped.  The  sachke  appearance  characterizing 
the  dead  larvse  in  sacbrood  is  absent.  The  granular  consistency  of 
the  decaying  mass  is  absent  also.  Microscopically,  a  large  number  of 
bacteria  are  found  in  larvae  dead  of  European  foulbrood,  but  are 
absent  in  larvse  dead  of  sacbrood.  The  presence  of  Bacillus  pluton 
is  a  positive  means  by  which  European  foulbrood  may  be  recognized. 
Bacillus  alvei  and  other  species  may  also  be  present. 

Sacbrood  must  also  be  differentiated  from  other  conditions  re- 
ferred to  as  chilled  brood,  overheated  brood,  and  starved  brood, 
which  occasionally  are  encountered.  This  can  be  done  by  a  compar- 
ison of  the  symptoms  presented  by  these  different  conditions  with  the 
symptoms  of  sacbrood,  and  the  history  of  the  cases.  Some  of  the 
larvse  dead  from  these  conditions  will  be  found  to  have  died  while 
yet  coiled  in  the  cell.  'This  fact  suggests  some  condition  other  ^;han 
sacbrood.  When  dying  later,  the  sachke  remains  characterizing  sac- 
brood are  not  present  in  conditions  other  than  sacbrood. 

PROGNOSIS. 

The  tendency  in  a  colony  affected  with  sacbrood  is  to  recover  from 
the  disease.  Colonies  which  during  the  spring  months  show  the  pres- 
ence of  more  or  less  disease,  by  midsummer  or  earlier  may,  and  very 


50  BULLETIN  431,  U.  S.  DEPARTMENT   OF   AGEICTJLTUEE. 

frequently  do,  contain  no  diseased  brood.  Experimentally  it  is  pos- 
sible to  destroy  a  colony  by  feeding  it  repeatedly  the  virus  of  sac- 
brood,  and  beekeepers  report  that  the  disease  sometimes  destroys 
colonies  in  their  apiaries.  The  percentage  of  colonies,  however,  that 
actually  die  out  as  a  direct  result  of  the  disease  is  small.  The  weak- 
ening of  the  colony  in  the  spring  of  the  year  not  only  reduces  or  entirely 
eliminates  the  profits  on  it  for  the  season,  but  may  also  cause  it  to 
be  in  a  weakened  condition  on  the  approach  of  winter. 

Whether  a  larva  once  infected  ever  recovers  from  the  disease  is  not 
known.  Reasoning  from  what  is  known  of  the  diseases  of  other  ani- 
mals and  man,  one  would  expect  that  a  larva  may  recover  from  sac- 
brood  infection.  It  is  known  that  many  larvae,  both  worker  and 
drone,  do  die.  From  the  information  thus  far  obtained  it  does  not 
appear  that  a  queenless  colony  would  be  likely  to  remain  so  as  a  con- 
sequence of  the  disease. 

As  to  the  prognosis  of  the  disease  in  a  colony  it  may  be  said,  there- 
fore, that  it  is  very  favorable  for  the  continued  existence  of  the  colony. 
As  to  the  economic  losses  to  be  expected  from  the  disease,  the  present 
studies  suggest  that  they  may  vary  from  losses  that  are  so  light  as  not 
to  be  detected  upon  examination  to  losses  that  may  equal  the  entire 
profits  of  the  colony  for  the  year.  Indeed,  at  times  the  death  of  the 
colony  takes  place  as  a  result  of  the  disease. 

RELATION  OF  THESE  STUDIES  TO  THE  TREATMENT  OF  SACBROOD. 

An  earher  paper  (White,  1908)  contains  a  brief  general  discussion 
of  the  relation  existmg  between  the  cause  of  bee  diseases  and  the 
treatment  of  them.  The  general  remarks  made  in  it  apply  also  to 
sacbrood.  No  doubt  the  beekeeper  in  studying  the  results  given 
here  has  already  observed  relations  existing  between  them  and  points 
which  should  be  incorporated  in  methods  for  treatment.  Mention- 
Lag  a  few  of  them  here  may  serve  to  suggest  still  others. 

That  the  weakness  resulting  in  a  sacbrood  colony  is  due  to  the 
death  of  worker  larvae;  that  adult  bees  are  not  susceptible  to  the 
disease;  that  queenlessness  is  rarely  to  be  expected  as  a  sequence 
of  the  disease;  that  the  disease  may  be  produced  with  ease  at  any 
time  of  the  year  that  brood  is  being  reared;  that  it  occurs  at  all 
seasons,  but  is  more  frequently  encoimtered  in  the  spring;  that  it 
is  ioxmd  in  localities  differing  widely  as  to  food  and  climatic  con- 
ditions; and  that  no  complete  racial  [immunity  to  the  disease  has 
yet  been  foimd  are  facts  concerning  the  predisposing  causes  of  sac- 
brood which  beekeepers  will  at  once  recognize  as  bearing  a  cIosq  rela- 
tion to  the  methods  by  which  the  disease  should  be  treated. 

As  sacbrood  can  not  occur  m  the  absence  of  its  exciting  cause 
(a  filterable  virus),  a  knowledge  of  this  cause  is  of  special  importance 
in  the  treatment  of  the  disease. 


SAOBEOOD.  51 

That  sacbrood  is  very  frequently  encomitered ;  that  it  is  infectious, 
but  that  it  is  more  benign  in  character  than  malignant;  that  it  does 
not  spread  rapidly  from  one  colony  to  another;  that  colonies  manifest 
a  strong  tendency  toward  self-recovery  from  the  disease;  that  this 
tendency  is  stronger  after  midsummer ;  that  the  disease  may  so  weaken 
a  colony  during  the  early  brood-rearing  season  that  the  profits  from 
it  may  be  much  reduced,  or  even  rendered  nil;  and  that  the  disease 
may  mdeed  destroy  the  colony  arc  facts  which  must  be  considered  in 
devising  logical  methods  for  its  treatment. 

That  the  virus  of  sacbrood  remains  virulent  in  larvae  dead  of  the 
disease  for  less  than  one  month;  that  it  remains  virulent  Iq  honey 
approximately  one  month ;  that  when  mixed  with  pollen  it  ceases  to  be 
virulent  after  about  one  month;  and  that  in  drying  no  virulence  is  to 
be  expected  after  one  month,  are  facts  that  accoimt  in  a  large  measure 
for  the  strong  tendency  to  recover  from  the  disease  manifested  by 
the  colony  and  that  furnish  information  concerning  the  use  of  combs 
from  sacbrood  colonies.  From  the  results  it  may  be  concluded  that 
it  is  better,  theoretically,  to  store  combs  from  sacbrood  colonies  for 
one  or  two  months  before  they  are  again  used,  provided  such  storing 
entails  no  particular  inconvenience  or  financial  loss  to  the  beekeeper. 
Further  experiments  show  that  brood  frames  from  badly-infected 
colonies  may  be  inserted  into  strong,  healthy  ones,  and  cause  thereby 
very  little  infection  and  consequently  only  a  shght  loss.  This  is 
especially  true  after  the  early  brood-rearing  season  of  the  year  is 
past.  Since  this  can  be  done,  it  is  qiute  probable  that  the  practical 
beekeeper  wiU  find  that  this  disposition  of  the  combs  will  be  the 
preferable  one  to  make.  At  any  event,  it  is  comforting  to  know  that 
it  is  never  necessary  to  destroy  the  combs  from  sacbrood  colonies  on 
account  of  the  disease. 

The  experimental  results  here  given  regarding  the  destruction  of 
the  virus  through  heating,  fermentation,  putrefaction,  drying,  and 
du-ect  sunlight  should  assist  materially  in  the  solution  of  the  problem 
of  the  transmission  of  sacbrood,  and  should  be  found  helpful  in  de- 
vising efficient  methods  for  the  treatment  of  the  disease. 

Toward  disinfecting  agents  it  is  shown  that  the  vims  of  sacbrood 
possesses,  in  some  instances  at  least,  marked  resistance.  These  and 
other  experimental  results  thus  far  obtained  indicate  that' the  use 
of  any  drug  m  the.  treatment  of  the  disease  should  not  be  depended 
upon  until  such  a  drug  has  been  proved  to  be  of  value. 

No  fear  need  be  entertamed  in  practical  apiculture  that  the  disease 
willbetransmittedbythehands  or  clothing  of  the  operator,  by  the  tools 
used  about  the  apiary,  through  the  medium  of  the  wind,  or  by  the 
queen .  It  would  seem  at  aU  tunes  superfluous  in  the  case  of  sacbrood 
to  flame  or  bum  the  inside  of  the  hive  or  to  treat  the  ground  about  a 
hive  containing  an  infected  colony. 


52  BULLETIN  431,  V.  S.  DEPARTMENT  OF   AGRICULTURE. 

There  is  but  little  danger  that  the  disease  will  be  Iratismitted  by 
way  of  flowers  visited  by  bees  from  sacbrood  colonies  and  later  from 
healthy  ones. 

Theoretically,  it  is  possible  that  the  disease  may  be  transmitted 
through  a  contamination  of  the  water  supply  by  bees  from  sacbrood 
colonies.  Whether  infection  ever  takes  place  in  this  way,  however, 
is  not  yet  known.  If  the  disease  is  ever  transmitted  in  this  way,  it 
would  seem  that  it  is  more  likely  to  take  place  in  the  spring  of  the 
year  than  at  any  other  season. 

While  there  is  yet  much  to  be  learned  about  sacbrood,  it  is  hoped 
that  by  carefully  considering  these  studies  the  be~ekeepers  will  be 
aided  in  devising  efficient  and  economical  methods  for  its  treatment. 

SUMMARY  AND  CONCLUSIONS. 

The  following  summary  and  statements  of  conclusions  seem  to  be 
justified  as  a  result  of  the  investigations  recorded  in  this  paper: 

(1)  Sacbrood  is  an  infectious  disease  of  the  brood  of  bees. 

(2)  Adult  bees  are  not  susceptible  to  the  disease. 

(3)  The  infecting  agent  causing  sacbrood  is  of  such  a  nature  that 
it  passes  through  the  pores  of  a  fine  clay  filter.  It  is  therefore  a 
filterable  virus. 

(4)  A  colony  may  be  inoculated  by  feeding  it  sirup  or  honey  con- 
taining the  virus. 

(5)  The  quantity  of  virus  contained  in  a  single  larva  recently  dead 
of  the  disease  is  sufficient  to  produce  quite  a  large  amount  of  sacbrood 
in  a  colony. 

(6)  The  period  from  time  of  inoculation  to  the  appearance  of  the 
first  sjonptoms  of  the  disease — the  incubation  period — is  approxi- 
mately six  days,  being  frequently  slightly  less. 

(7)  By  inoculation  the  disease  may  be  produced  at  any  season  of 
the  year  that  brood  is  being  reared. 

(8)  The  disease  is  more  often  encountered  during  the  first  half  of 
the  brood-rearing  season  than  during  the  second  half. 

(9)  It  occurs  among  bees  in  locaHties  having  as  wide  a  range  of 
climatic  conditions,  at  least,  as  are  found  in  the  United  States. 

(10)  The  course  of  the  disease  is  not  greatly  affected  by  the  char- 
acter or  quantity  of  the  food  obtained  and  used  by  the  bees. 

(11)  Larval  remains  recently  dead  of  the  disease  prove  to  be  very 
infectious  when  fed  to  bees.  Dead  larvae  which  have  been  in  the  brood 
comb  more  than  one  month  are  apparently  noninfectious. 

(12)  Colonies  possess  a  strong  tendency  to  recover  from  the  disease 
without  treatment. 

(13)  The  vu-us  of  sacbrood  suspended  in  water  and  heated  to 
138°  F.  (59°  C.)  was  destroyed  in  10  minutes.  Considering  the  vary- 
ing factors  which  enter  into  the  problem,  the  minimum  temperature 
necessary  to  destroy  this  virus  when  applied  for  10  minutes  should 


SAOBHOOD.  53 

be  found  at  all  times  to  lio  soinewh0ro  between  the  limits  of  131°  F 
(55°  C.)  and  l-i9°  F.  (65°  C). 

(14)  When  the  virus  of  sacbrood  is  suspended  in  honey  it  may  be 
destroyed  by  heating  the  suspension  for  10  minutes  at  approximately 
158°  F.  (70°  C). 

(15)  The  virus  resisted  drying  at  room  temperature  for  approxi- 
mately three  weeks. 

(16)  The  virus  when  diy  was  destroyed  by  the  direct  rays  of  the 
sun  in  from  four  to  seven  hours. 

(17)  The  virus  when  suspended  in  water  was  destroyed  by  the  direct 
rays  of  the  sun  in  from  four  to  six  hours. 

(18)  The  virus  when  suspended  in  honey  was  destroyed  by  the' 
dii-ect  rays  of  the  sun  in  from  five  to  six  hours. 

(19)  The  virus  when  suspended  in  honey  and  shielded  from  direct 
sunlight  remained  virulent,  for  slightly  less  than  one  month  at  room 
temperature  diu-ing  the  summer. 

(20)  The  virus  was  destroyed  in  approximately  five  days  in  the 
presence  of  fermentative  processes  taking  place  in  10  per  cent  sugar 
solution  at  room  temperature. 

(21)  In  the  presence  of  fermentative  processes  going  on  in  20  per 
cent  honey  solution  at  outdoor  temperature  the  virus  of  sacbrood  was 
destroyed  in  approximately  five  days. 

(22)  In  the  presence  of  putrefactive  processes  the  virus  remained 
virulent  for  approximately  10  days. 

(23)  The  virus  will  resist  |  per  cent,  1  per  cent,  and  2  per  cent 
aqueous  solutions  of  carbolic  acid,  respectively,  for  more  than  three 
weeks,  4  per  cent  being  more  efifeotive. 

(24)  Neither  carbolic  acid  nor  quinine  as  drugs  should  at  present 
be  relied  upon  in  the  treatment  of  sacbrood. 

(25)  Varying  factors  entering  into  many  of  the  problems  discussed 
in  this  paper  tend  to  vary  the  results  obtained.  In  such  problems 
the  results  here  given  must  be  considered  from  a  technical  point  of 
view  as  being  approximate  only.  They  are  sufficiently  exact  for 
application  by  the  beekeeper,  but  to  insure  the  destruction  of  the 
virus  in  practical  apiculture  the  time  element  indicated  from  these 
experiments  as  siifficient  should  be  increased  somewhat. 

LITERATURE  CITED. 

(1)  Bahe,  L. 

1915.  Sygdomme  hos  Honningbien  og  dens  Yngel.  Meddelelser  fra  den 
Kgl.  Veterinaer-og  Landboh^jskoles  Serumlaboratorium,  XXXVII, 
109  p.,  11  fig. 

(2)  Beuhne,  F.  K. 

1913.  Diseases  of  bees.  In  Jour.  Dept.  Agr.  Victoria,  v.  11,  pt.  8,  p.  487-t93, 
4  fig. 

(3)  BuERi,  R. 

1906.  Bakteriologische  Uatersuchungen  liber  die  Faulbrut  und  Sauerbrut 
der  Bienen.    40  p.,  1  pi.,  1  fig.    Aaran,  Switzerland. 


54  BULLETIN  431,  U.   S.  DEPAKTMENT   OF    AGEICULTURE. 

(4)  Cook,  A.  J. 

1904.  The  Bee-Keeper's  Guide  or  Manual  of  the  Apiary,  ed.  18,  543  p., 
295  fig.     Chicago. 

(5)  DOOLITTLE,  G.  M. 

1881.  Foul  brood.    In  Gleaniags  in  Bee  Culture,  v.  9,  no.  3,  p.  118-119. 

(6)  Dadant,  C.  p. 

1908.  Diseases  of  Bees.  Langstroth  on  the  Hive  and  Honey  Bee.  575  p. 
(p.  487),  229  fig.    Hamilton,  111. 

(7)  Howard,  Wm.  B. 

1896.  A  new  bee  disease — pickled  brood  or  white  fungus.  In  Amer.  Bee 
Jour.,  V.  36,  no.  37,  p.  577,  6  fig.;  also  in  ABO  of  Bee  Culture,  1903, 
p.  157-158. 

(8) . 

1898.  Pickled  brood  and  bee  paralysis.     In  Amer.  Bee  Jour.,  v.  38,  no.  34, 
p.  530-531. 
(9)  Jones,  A.  D. 

1883.  Symptoms  of  foul  brood.  In  The  American  Apiculturist,  v.  1,  no.  4, 
p.  79-80. 

(10)  KuflSTBINEE,  J. 

1910.  Zusammenstellung  der  Ergebnisse  des  vom  Mai  1903  bis  Dezember 
1909  unterauchten,  faulbrutverdachtigen  Wabenmaterials.  In 
Schweizerische  Bienen-Zeitung,  Yahrg.  33,  no.  4,  p.  187-189. 

(11)  Langstroth,  L.  L. 

1857.  A  practical  Treatise  on  the  Hive  and  Honey-Bee.  ed.  2,  534  p.  (p. 
275  ),  illus. 

(12)  [Editorial.] 

1892.  Is  it  a  new  bee  disease?  Something  that  resembles  foul  brood,  its 
causes  and  cure  not  definitely  known.  In  Gleanings  in  Bee  Culture, 
V.  20,  no.  15,  p.  594-595. 

(13) . 

1896.  Dead  brood — what  is  it?    How  distinguished  from  foul  brood.     In 
>  Gleanings  in  Bee  Culture,  v.  24,  no.  16,  p.  609-610. 

(14)  Root,  A.  I.  and  E.  R. 

1913.  ABC  and  XYZ  of  Bee  Culture.     717  p.,  illus.    Medina. 
Pickled  brood  and  its  cause,  p.  250. 

(15)  SiMMlNS,  S. 

1887.  Foul  brood,  dead  brood.  In,  British  Bee  Jour.,  v.  15,  no.  270,  p.  371- 
372;  also  in  Canad.  Bee  Jour.,  v.  3,  no.  28.  p.  576-577. 

(16)  White,  G.  F. 

1904.  The  further  investigation  of  the  diseases  affecting  the  apiaries  in  the 
State  of  New  York.  In  11th  Ann.  Rpt.  Comr.  Agr.  N.  Y.,  1903,  p 
103-114. 


(17) 
(18) 

(19) 


1908.  The  relation  of  the  etiology  (cause)  of  bee  diseases  t(5  the  treatment. 
U.  S.  Dept.  Agr.  Bur.  Ent.  Bui.  75,  pt.  4,  p.  33-42. 


1913.  Sacbrood,  a  disease  of  bees.  U.  S.  Dept.  Agr.  Bur.  Ent.  Circ.  169. 
5  p.;  Sackbrut.  Fine  Bienenkrankheit.  A  translation  by  Dr.  M. 
Kiistenmacher.     Berlin-StegUtz. 


1914.  Destruction  of  germs  of  infectious  bee  diseases  by  heating     U    S 
Dept.  Agr.  Bui.  92,  8  p.  (p.  4). 


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ADDITIONAL  COPIES 

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Circular  No.  79. 

United  States  Department  of  Agriculture, 

BUREAU    OF    ENTOMOLOGY, 
L.  O.  HOWARD,  Entomologist  and  Chief  of  Bureau. 


THE  BROOD  DISEASES  OF  BEES. 

By  E.  F.  Phillips,  Ph.  D., 
In  Charge  of  Bee  Culture. 

In  view  of  the  widespread  distribution  of  infectious  brood  diseases 
among  bees  in  the  United  States,  it  is  desirable  that  all  bee  keepers 
learn  to  distinguish  the  diseases  when  they  appear.  It  frequently 
happens  that  an  apiary  becomes  badly  infected  before  the  owner  real- 
izes that  any  disease  is  present,  or  it  may  be  that  any  dead  brood 
which  may  be  noticed  in  the  hives  is  attributed  to  chilling.  In  this 
way  disease  gets  a  start  which  makes  eradication  difficult. 

There  are  two  recognized  forms  of  disease  of  the  brood,  designated  as 
European  and  American  foul  brood,  which  are  particularly  virulent. 
In  some  ways  these  resemble  each  other,  but  there  are  certain  distin- 
guishing characters  which  make  it  possible  to  differentiate  the  two. 
Reports  are  sometimes  received  that  a  colony  is  infected  with  both 
diseases  at  the  same  time,  but  this  is  contrary  to  the  experience  of 
those  persons  most  conversant  with  these  conditions.  While  it  may 
be  possible  for  a  colony  to  have  the  infection  of  both  diseases  at  the 
^me  time,  it  is  not  by  any  means  the  rule,  and  such  cases  are  probably 
not  authentically  reported.  Since  both  diseases  are  caused  by  specific 
bacilli,  there  is  absolutely  no  ground  for  the  idea  held:  by  some  bee 
keepers  that  chilled  or  starved  brood  will  turn  to  one  or  the  other  of 
these  diseases.  Experience  of  the  best  practical  observers  is  also  in 
keeping  with  this.  For  a  discussion  of  the  causes  of  these  diseases  the 
reader  is  referred  to  Technical  Series,  No.  14,  of  the  Bureau  of  Ento- 
mology, "The  Bacteria  of  the  Apiary,  with  Special  Reference  to  Bee 
Diseases,"  by  Dr.  G.  F.  White. 

AMEEIOAN    FOUL   BEOOD. 

American  foul  brood  (often  called  simply  "foul  brood")  is  dis- 
tributed through  all  parts  of  the  United  States,  and  from  the  symptoms 
published  in  European  journals  and  texts  one  is  led  to  believe  that  it  is 
also  the  prevalent  brood  disease  in  Europe.  Although  it  is  found  in 
almost  all  sections  of  the  United  States,  there  are  many  localities 
entirely  free  from  disease  of  any  kind. 

The  adult  bees  of  an  infected  colony  are  usually  rather  inactive  and 

do  little  toward  cleaning  out  infected  material.     When  the  larv^  are 

first  affected  they  turn  to  a  light  chocolate  color,  and  in  the  advanced 

stages  of  decay  they  become  darker,  resembling  roasted  coffee  in  color.- 

5947—09 


Usually  the  larvae  are  attacked  at  about  the  time  of  capping,  and  most 
of  the  cells  containing  infected  larvae  are  capped.  As  decay  proceeds 
these  cappings  become  sunken  and  perforated,  and,  as  the  healthy 
brood  emerges,  the  comb  shows  the  scattered  cells  containing  larvae 
which  have  died  of  disease,  still  capped.  The  most  noticeable  charac- 
teristic of  this  infection  is  the  fact  that  when  a  small  stick  is  inserted 
in  a  larva  which  has  died  of  the  disease,  and  slowly  removed,  the 
broken-down  tissues  adhere  to  it  and  will  often  stretch  out  for  several 
inches  before  breaking.  When  the  larva  dries  it  forms  a  tightly 
adhering  scale  of  very  dark  brown  color,  which  can  best  be  observed 
when  the  comb  is  held  so  that  a  bright  light  strikes  the  lower  side 
wall.  Decaying  larvae  which  have  died  of  this  disease  have  a  very 
characteristic  odor  which  resembles  a  poor  quality  of  glue.  This 
disease  seldom  attacks  drone  or  queen  larvae.  It  appears  to  be  much 
more  virulent  in  the  western  part  of  the  United  States  than  in  the 
East. 

EUROPEAN    FOUL   BROOD. 

European  foul  brood  (often  called  "black  brood")  is  not  nearly  as 
widespread  in  the  United  States  as  is  American  foul  brood,  but  in  cer- 
tain parts  of  the  country  it  has  caused  enormous  losses.  It  is  steadily 
on  the  increase  and  is  constantly  being  reported  from  new  localities. 
It  is  therefore  desirable  that  bee  keepers  be  on  the  watch  for  it. 

Adult  bees  in  infected  colonies  are  not  very  active,  but  do  suc- 
ceed in  cleaning  out  some  of  the  dried  scales.  This  disease  attacks 
larvae  earlier  than  does  American  foul  brood,  and  a  comparatively 
small  percentage  of  the  diseased  brood  is  ever  capped.  The  diseased 
larvae  which  are  capped  over  have  sunken  and  perforated  cappings. 
The  larvae  when  first  attacked  show  a  small  yellow  spot  on  the  body 
near  the  head  and  move  uneasily  in  the  cell.  When  death  occurs  they 
turn  yellow,  then  brown,  and  finally  almost  black.  Decaying  larvae 
which  have  died  of  this  disease  do  not  usually  stretch  out  in  a  long 
thread  when  a  small  stick  is  inserted  and  slowly  removed.  Occasion- 
ally there  is  a  very  slight  "ropiness,"  but  this  is  never  very  marked. 
The  thoroughly  dried  larvae  form  irregular  scales  which  are  not  strongly 
adherent  to  the  lower  side  wall  of  the  cell.  There  is  very  little  odor 
from  decaying  larvae  which  have  died  from  this  disease,  and  when  an 
odor  is  noticeable  it  is  not  the  "glue-pot"  odor  of  the  American  foul 
brood,  but  more  nearly  resembles  that  of  soured  dead  brood.  This 
disease:  attacks  drone  and  queen  larvae  very  soon  after  the  colony  is 
infected.  It  is  as  a  rule  much  more  infectious  than  American  foul 
brood  and  spreads  more  rapidly.  On  the  other  hand,  it  sometimes 
happens  that  the  disease  will  disappear  of  its  own  accord,  a  thing  which 
the  author  never  knew  to  occur  in  a  genuine  case  of  American  foul 
brood.  European  foul  brood  is  most  destructive  during  the  spring  and 
early  summer,  often  almost  disappearing  in  late  summer  and  autumn. 

[Cir.  791 


TREATMENl'    OJ^    INFECTIOUS    DISEASES. 

The  treatment  for  both  American  foul  brood  and  European  foul 
brood  is  practically  the  same.  It  is  impossible  to  give  minute  direc- 
tions to  cover  every  case,  but  care  and  common  sense  will  enable  any 
bee  keeper  successfully  to  fight  diseases  of  brood. 

Drugs. — Drugs,  either  to  be  given  directly  in  food  or  to  be  used  for 
fumigating  combs,  can  not  be  recommended  for  either  of  these  dis- 
eases. 

Shaking  treatment. — To  cure  a  colony  of  either  form  of  foul  brood 
it  is  necessary  first  to  remove  from  the  hive  all  of  the  infected  material. 
This  is  done  by  shaking  the  bees  into  a  clean  hive  on  clean  frames  with 
small  strips  of  comb  foundation,  care  being  taken  that  infected  honey 
does  not  drop  from  the  infected  combs.  The  healthy  brood  in  the 
infected  combs  may  be  saved,  provided  there  is  enough  to  make  it 
profitable,  by  piling  up  combs  from  several  infected  hives  on  one  of 
the  weakest  of  the  diseased  colonies.  After  a  week  or  ten  days  all 
the  brood  which  is  worth  saving  will  have  hatched  out,  at  which  time 
all  these  combs  should  be  removed  and  the  colony  treated.  In  the 
case  of  box  hives  or  skeps  the  bees  may  be  drummed  out  into,  another 
box  or  preferably  into  a  hive  with  movable  frames.  Box  hives  are 
hard  to  inspect  for  disease  and  are  a  menace  to  all  other  bees  in  the 
neighborhood  in  a  region  where  disease  is  present. 

The  shaking  of  the  bees  from  combs  should  be  done  at  a  time  when 
the  other  bees  in  the  apiary  will  not  rob  and  thus  spread  disease,  or 
under  cover.  This  can  be  done  safely  in  the  evening  after  bees  have 
ceased  to  fly,  preferably  during  a  good  honey  flow.  Great  care  should 
be  exercised  to  keep  all  infected  material  away  from  other  bees  until 
it  can  be  completely  destroyed  or  the  combs  rendered  into  wax.  Wax 
from  diseased  colonies  should  be  rendered  by  some  means  in  which 
high  heating  is  used,  and  not  with  a  solar  wax  extractor.  The  honey 
from  a  diseased  colony  should  be  diluted  to  prevent  burning  and  then 
thoroughly  sterilized  by  hard  boiling  for  at  least  half  an  hour,  if  it  is  to 
be  fed  back  to  the  bees.  If  the  hive  is  again  used,  it  should  be  very 
thoroughly  cleaned,  and  special  care  should  be  taken  that  no  infected 
honey  or  comb  be  left  in  the  hive. 

It  is  frequently  necessary  to  repeat  the  treatment  by  shaking  the 
bees  onto  fresh  foundation  in  new  frames  after  four  or  five  days.  The 
bee  keeper  or  inspector  must  determine  whether  this  is  necessary,  but 
when  there  is  any  doubt  it  is  safer  to  repeat  the  operation  rather  than 
run  the  risk  of  reinfection.  If  repeated,  the  first  new  combs  should  be 
destroyed.  To  prevent  the  bees  from  deserting  the  strips  of  founda- 
tion the  queen  may  be  caged  in  the  hive  or  a  queen-excluding  zinc  put 
at  the  entrance. 

[Cir.  79] 


Treatment  with  bee  escape. — The  shaking  treatment  may  be  modified 
so  that  instead  of  shaking  the  bees  from  the  combs  the  hive  is  moved 
from  its  stand,  and  in  its  place  a  clean  hive  with  frames  and  founda- 
tion is  set.  The  queen  is  at  once  transferred  to  the  new  hive,  and  the 
field  bees  fly  there  when  they  next  return  from  the  field.  The  infected 
hive  is  then  placed  on  top  of  or  close  beside  the  clean  hive  and  a  bee 
esckpe  placed  over  the  entrance  of  the  hive  containing  disease,  so  that 
the  younger  bees  and  those  which  later  emerge  from  the  cells  may 
leave  the  hive  but  can  not  return.  They  therefore  join  the  colony  in 
the  new  hive. 

Fall  treatment. — If  it  is  desirable  to  treat  a  colony  so  late  in  the  fall 
that  it  would  be  impossible  for  the  bees  to  prepare  for  winter,  the 
treatnient  may  be  modified  by  shaking  the  bees  onto  combs  with 
plenty  of  honey  for  winter.  This  will  be  satisfactory  only  after  brood 
rearing  has  entirely  ceased.     In  such  cases  disease  rarely  reappears. 

In  the  Western  States,  where  American  foul  brood  is  particularly 
virulent,  it  is  desirable  thoroughly  to  disinfect  the  hive  by  burning  the 
inside  or  by  chemical  means  before  using  it  again.  This  is  not  always 
practiced  in  the  Eastern  States,  where  the  disease  is  much  milder. 
Some  persons  recommend  boiling  the  hives  or  disinfecting  them  with 
some  reliable  disinfectant  such  as  carbolic  acid  or  corrosive  sublimate. 
It  is  usually  not  profitable  to  save  frames  because  of  their  compara- 
tively small  value,  but  if  desired  they  may  be  disinfected.  Great  care 
should  be  exercised  in  cleaning  any  apparatus  It  does  not  pay  to 
treat  very  weak  colonies.  They  should  either  be  destroyed  at  once 
or  several  weak  ones  be  united  to  make  one  which  is  strong  enough  to 
build  up. 

Recently  some  new  "cures"  have  been  advocated  in  the  bee  jour- 
nals, particularly  for  European  foul  brood,  with  a  view  to  saving 
combs  from  infected  colonies.  The  cautious  bee  keeper  will  hardly 
experiment  with  such  methods,  especially  when  the  disease  is  just 
starting  in  his  locaKty  or  apiary,  but  will  eradicate  the  disease  at  once 
by  means  already  well  tried. 

In  kll  cases  great  care  should  be  exercised  that  the  bee  keeper  may 
not  himself  spread  the  infection  by  handling  healthy  colonies  before 
thoroughly  disinfecting  his  hands,  hive  tools,  and  even  smoker.  Since 
it  takes  but  a  very  small  amount  of  infected  material  to  start  disease  in 
a  previously  healthy  colony,  it  is  evident  that  too  much  care  can  not  be 
taken.  In  no  case  should  honey  from  unknown  sources  be  used  for 
feeding  bees.  Care  should  also  be  exercised  in  buying  queens,  since 
disease  is  often  transmitted  in  the  candy  used  in  shipping  cages, 
Combs  should  not  be  moved  from  hive  to  hive  in  infected  apiaries. 

[Cir.  79] 


PICKLE    BROOD. 

There  is  a  diseased  condition  of  the  brood  called  by  bee  keepers 
"pickle  brood,"  but  practically  nothing  is  known  of  its  cause.  It  is 
characterized  by  a  swollen,  watery  appearance  of  the  larva,  usually 
accompanied  by  black  color  of  the  head.  The  larvae  usually  lie  on 
their  backs  in  the  cell,  and  the  head  points  upward.  The  color  gradu- 
ally changes  from  light  yellow  to  brown  after  the  larva  dies.  There  is 
no  ropiness,  and  the  only  odor  is  that  of  sour  decaying  matter,  not  at 
all  like  that  of  American  foul  brood.  In  case  the  larvae  are  capped 
over,  the  cappings  do  not  become  dark,  as  in  the  case  of  the  contagious 
diseases,  but  they  may  be  punctured.  So  far  no  cause  can  be  given 
for  this  disease,  and  whether  or  not  it  is  contagious  is  a  disputed  point. 
Usually  no  treatment  is  necessary  beyond  feeding  during  a  dearth  of 
honey,  but  in  very  rare  cases  when  the  majority  of  larvae  in  a  comb  are 
dead  from  this  cause  the  frame  should  be  removed  and  a  clean  comb 
put  in  its  place  to  make  it  unnecessary  for  the  bees  to  clean  out  so 
much  dead  brood. 

CHILLED,  OVEEHEATED,  AND    STARVED    BROOD. 

Many  different  external  factors  may  cause  brood  to  die.  Such  dead 
brood  is  frequently  mistaken,  by  persons  unfamiliar  with  the  brood 
diseases,  for  one  or  the  other  of  them.  Careful  examination  will  soon 
determine  whether  dead  brood  is  the  result  of  disease  or  merely  some 
outside  change.  If  brood  dies  from  chilling  or  some  other  such  cause, 
it  is  usually  soon  carried  out  by  the  workers,  and  the  trouble  disap- 
pears. No  treatment  is  necessary.  Brood  which  dies  from  external 
causes  often  produces  a  strong  odor  in  the  colony,  but  wholly  unlike 
that  of  American  foul  brood,  merely  that  of  decaying  matter.  The 
color  of  such  brood  varies,  but  the  characteristic  colors  of  the  infec- 
tious diseases  are  usually  absent,  the  ordinary  color  of  dead  brood 
being  more  nearly  gray. 

Approved : 

James  Wilson, 

Secretary  of  Agriculture. 

Washington,  D.  C,  October  3,  1906. 

[Cir.  79]  „ 

o 


4- 


UNITED  STATES  DEPARTMENT  OF  AGRICULTURE 


BULLETIN  No.  804 

Contribution  from  the  Bureau  of  Entomology 
L.  O.  HOWARD,  Chief 


Washington,  D.  C. 


PROFESSIONAL  PAPER 


March  16, 1920 


A  STUDY  OF  THE  BEHAVIOR  OF  BEES  IN 
COLONIES  AFFECTED  BY  EUROPEAN  FOUL- 
BROOD  ^ 

By  Abnold  p.  Sturtevant 
Specialist  in  the  Bacteriology  of  Bee  Diseases 


CONTENTS 


Page 

Introduction 1 

Procedure 5 

Observations 8 

Summary  of  previous  experiments —  15 

Supplementary  observations 17 

Study    of    naturally    Infected 

colonies 17 

Behavior  of  bees  in  cleaning 

•            contaminated  cells 18 

Possible    infection    through 

queen 19 


Page 


Supplementary  observations — Contd. 
Distribution  of  introduced  in- 
fected material 20 

Age  at   which   larvje   are  in- 
fected         21 

Microscopical  bacteriological  observa- 
tions         24 

Summary  and  conclusions 28 

Literature  cited 28 


INTRODUCTION 

The  brood  diseases  of  bees  cause  annually  large  losses  of  bees  and 
consequently  of  the  honey  crop.  The  predominant  attitude  among 
beekeepers  has  long  been  how  best  to  eradicate  an  invading  bee  dis- 
ease after  the  attack  has  been  made.  They  depend  upon  this  pro- 
cedure, because  little  is  known  with  any  degree  of  certainty  concerning 
the  natural  conditions  which  might  prevent  or  control  the  onslaught 
of  the  disease.  As  a  result  of  this  attitude,  much  more  importance 
has  been  placed  on  the  significance  of  apiary  inspection  and  police- 
power  laws  and  of  purely  remedial  treatment,  the  reasons  for  which 
in  many  cases  are  imperfectly  understood.  But  the  old  adage  "  an 
ounce  of  prevention  is  worth  a  pound  of  cure  "  has  yet  to  be  refuted, 
particularly  with  regard  to  beekeeping.    In  the  reahn  of  human 

'A  series  of  investigations  was  started  in  the  spring  and  summer  of  1918  by  the 
Office  ^BLSiitoe  Investigations,  Bureau  of  Entomology,  for  the  purpose  of  faking 
m^hitensive  study  of  European  foulbrood  of  bees,  primarily  from  the  standpoint  of 
Z^^ZviTZ  relation  to  the  disease,  correlated  with  the  facts  and  practical 
obsfr^aUon"  alrA  known  to  the  beekeeper.  This  paper,  wMch  was  submitted  f^ 
pubUcation  January   13,   1919,   is  a  preliminary   report  on  the  beginning  of  the  In 

vestigation. 

134440°— BuU.  804—20^ 1 


2  BULLETIN   804,   V.   S.   DEPARTMENT   OF   AGKICULTURE 

medicine,  for  the  last  two  decades  at  least,  this  precept  has  been 
gaining  strength  so  that  to-day  preventive  medicine  stands  on  a  par 
with,  if  not  above,  most  of  the  other  branches  of  medicine.  Why  is 
it  not  logical  to  apply  this  principle  to  the  control  of  bee  diseases? 

Ever  since  European  foulbrood  of  bees  was  first  recognized  (in 
1894) ,  in  New  York  State,  as  a  distinct  brood  disease,  there  have  been 
much' controversy  and  speculation  concerning  the  etiology  of  the 
disease,  the  means  of  transmission,  the  method  of  spread,  and,  result- 
ing therefrom,  the  question  of  control.  From  the  laboratory  stand- 
point, the  etiology  of  the  disease  has  been  worked  out  quite  definitely 
bacteriologically  (12)  i.  But  as  yet  Bacillus  ■pluton,  the  accepted 
cause  of  European  foulbrood,  never  has  been  grown  in  pure  culture 
on  artificial  media,  although  it  has  been  definitely  identified  as  the 
cause  of  the  disease.  This  precludes  any  further  advance  along  this 
line  of  attack  for  the  time  being. 

From  the  side  of  practical  experience,  there  have  been  recorded 
large  numbers  of  observations,  many  of  them  of  a  similar  nature. 
These  observations  have  led  to  many  accepted  practices,  as,  for  in- 
stance, the  use  of  Italian  bees  and  strong  colonies  in  combating  the 
disease.  Although  the  weight  of  numbers  tends  to  give  substantia- 
tion to  observations,  the  scientific  explanation  of  how  these  things 
are  true  never  has  been  studied  carefully  and  coordinated  with  the 
practical  side  into  an  epidemiological  study  of  the  colony  under  dis- 
ease conditions  in  European  foulbrood.  ' 
The  history  of  bee  diseases  has  developed  mainly  along  two  lines. 
The  scientific  side  has  been  concerned  principally  with  determining 
the  causes  of  the  various  diseases  microbiologically,  the  method  of 
diagnosis,  and  conclusively  differentiating  them.  These  facts  have 
been  described  sufficiently  in  various  bulletins  of  the  Bureau  of  En- 
tomology and  will  not  be  discussed  here.  From  the  practical  side, 
countless  observations  have  been  recorded,  largely  in  the  bee  journals, 
in  which  various  manifestations  of  the  disease  and  experiences  with 
methods  of  treatment  have  been  discussed.  But  in  all  this  literature, 
particularly  with  regard  to  European  foulbrood,  there  are  few  ob- 
servations on  the  disease  and  on  the  behavior  of  the  bees  in  relation 
to  it  beyond  simple  description  of  symptoms. 

Early  in  the  experience  with  European  foulbrood  it  was  learned 
by  careful  observers  that  strong  colonies  are  essential  in  successfully 
combating  the  disease.  Later  the  value  of  Italian  bees  was  dis- 
covered. "West  (11),  a  New  York  State  apiary  inspector,  in  giving 
what  is  one  of  the  best  early  descriptions  of  European  foulbrood, 
makes  some  pertinent  observations  on  the  disease.  He  states  that 
when  diseased  brood  is  placed  above  a  strong,  healthy  colony,  with 
a  queen  excluder  between,  so  that  any  healthy  brood  may  emerge, 

1  Reference  is  made  by  number  in  parentliesls  to   "  Literature   cited,"   p.  28. 


BEES  IN   COLONIES  AFFECTED  BY  EUROPEAN  FOULBROOD  3 

the  diseased  larvse  are  cleaned  out  as  this  is  taking  place.  The  union 
with  a  healthy  colony  and  the  strength  gained  by  the  emergence  of 
so  many  young  bees  gives  the  colony  the  stimulus  to  eliminate  the 
disease.  He  notes,  as  have  many  other  beekeepers  since,  that  in 
August,  when  the  buckwheat  honey  flow  begins,  the  stronger  of  the 
diseased  colonies  are  stimulated  to  clean  up. 

Alexander  (1)  published  a  method  of  treatment  for  European  foul- 
brood,  the  principle  of  which,  after  many  varying  failures  and  suc- 
cesses, is  now  the  basis  for  the  present  method  of  treatment  most 
used;  that  is,  requeening  with  Italian  stock.  Alexander  mentions 
the  need  of  three  factors:  First,  the  necessity  of  requeening  with 
young  yellow  Italians,  as  hybrids  of  Italian  and  black  bees  are  prone 
to  contract  the  disease  in  the  first  place  and  also  are  more  likely  to 
succumb  to  it ;  second,  particularly  emphasized,  a  period  (at  least  27 
days,  according  to  Alexander)  of  queenlessness  in  which  to  allow  the 
bees  properly  to  clean  up  the  cells  and  polish  them,  preparatory  for 
eggs  of  a  new  queen ;  third,  a  factor  which  is  mentioned  only  casually 
but  which  is  equally  important  with  the  other  two,  the  direction  to 
unite  and  strengthen  diseased  colonies  before  treating.  So  little  em- 
phasis was  placed  on  this  that  the  majority  of  beekeepers  overlooked 
it  in  using  Alexander's  treatment  and  therefore  condemned  the  treat- 
ment as  unsuccessful  except  in  rare  cases. 

In  an  editorial  (8)  in  the  same  issue  of  the  journal  m  which  Mr. 
Alexander  was  writing,  the  question  was  raised  as  to  why  the  period 
of  broodlessness  caused  by  winter,  which  is  much  longer  than  27 
days,  does  not  always  prevent  a  recurrence  of  the  disease.  Mr.  Alex- 
ander  answered  this  question  by  explaining  that  when  the  queen 
stops  laying  in  the  fall,  the  bees  do  not  polish  up  the  cells  as  they 
do  earlier  in  the  season,  and  that  some  of  the  dried-down  material 
may  remain  until  the  next  spring.  The  opinion  also  is  given  in 
this  editorial  that  Italians  are  more  able  to  resist  the  disease  than 
hybrids  because  they  do  more  thorough  work  in  house  cleaning  and 
are  less  inclined  to  rob. 

Phillips  (6)  makes  the  statement  that  "European  foulbrood 
is  more  destructive  during  the  spring  and  early  summer  than 
at  other  times,  often  entirely  disappearing  during  the  late  summer 
and  early  autmrni,  or  during  a  heavy  honey  flow,"  but  gives  no  indi- 
cation as  to  how  this  takes  place.  The  same  year  Miller  (2)  pub- 
lished his  theory  of  the  relation  of  the  nurse  bees  to  the  spread  of 
European  foulbrood.  He  believes  that  the  nurse  bees  suck  up  the 
juices  of  a  freshly  diseased  larva  which  has  not  become  offensive,  and 
then  transmit  the  disease  when  feeding  the  healthy  larvae.  On  this 
supposition  he  believes  that  if  egg  laying  ceases  for  S  or  6  days  ("  the 
period  the  larvae  remain  unsealed  in  their  cells")  there  will  no 
longer  be  larvae  in  the  proper  condition  for  nurse  bees  to  feed  upon, 


4  BULIxETIN   804,   tr.   S.   DEPAETMENT   OF   AGEICTILTTJKB 

nor  healthy  unsealed  larvae  to  receive  the  infection,  and  the  disease 
will  thereby  come  to  an  end. 

Dr.  Miller  has  been  using  a  10-day  period  of  queenlessness  in  his 
treatment  of  European  foulbrood  sincfe  his  accidental  discovery  that 
10  days  were  sufficient,  but  in  a  later  article  (3)  in  enlarging  upon 
his  nurse-bee  theory  he  assumes  that  the  larva  is  fed  during  a  period 
of  5  days  but  is  not  effective  as  a  carrier  of  infection  during  the  whole 
time  as  probably  no  larvae  are  torn  open  until  they  are  2  or  3  days  old; 
thus  making  it  possible  to  shorten  the  queenless  period  even  more.  He 
admits  that  not  all  the  dead,  partially  dried  larvae  will  be  cleaned  out, 
but  believes  that  it  is  only  the  fresh  yellow  ones  which  are  infectious. 
He  also  states  that  nurse  bees  are  not  inclined  to  travel  far  on  the 
combs,  a  fact  which  may  explain  why  the  disease  may  be  found  con- 
fined to  one  comb  for  several  days  before  spreading  farther.  Dr. 
Miller  seems  to  have  overlooked  several  important  factors  which 
will  be  discussed  later. 

Quite  an  extensive  piece  of  investigation  was  carried  on  during  the 
summers  of  1915  and  1916  by  the  author  at  the  Massachusetts  Agri- 
cultural Experiment  Station  upon  the  effect  of  requeening  diseased 
colonies  with  various  strains  of  Italian  bees.  At  that  time  the  im- 
portance of  strong  colonies  with  the  requeening  had  not  been  em- 
phasized so  strongly  and  less  attention  was  paid  to  that  factor.  The 
records  show,  however,  that  in  a  total  of  50  colonies  observed,  cov- 
ering two  seasons,  of  10  strong  colonies  only  2  showed  recurrence, 
while  1  was  doubtful ;  of  20  medium-strength  colonies,  10  showed  re- 
currence with  2  doubtful ;  of  14  weak  colonies,  8  showed  recurrence. 
In  all  these  cases  the  new  queen  was  not  introduced  until  the  colony 
was  nearly  or  entirely  clean.  In  the  case  of  several  of  the  weaker 
colonies  it  was  necessary  to  strengthen  them  before  requeening  was 
possible,  in  order  to  save  the  colony.  One  or  two  of  these,  which  were 
united  and  requeened  with  Italian  stock,  were  the  best  colonies  the 
next  spring. 

Adding  some  strength  to  at  least  part  of  Miller's  theory  is  a  state- 
ment in  a  letter  by  G.  C.  Matthews,  formerly  of  this  bureau,  who 
wrote  in  February,  1918,  concerning  his  observations  in  California 
in  1914.  He  found  that  where  the  hives  stood  in  rows  of  pairs  the 
disease  continued  to  spread  down  each  row  to  corresponding  members 
of  each  pair.  This  ceased  when  he  rearranged  his  apiary  so  that 
the  rows  of  hives  were  at  least  10  feet  apart,  and  alternate  pairs 
of  hives  were  turned  at  right  angles.  No  pair  was  allowed  to  remain 
close  to  another  facing  the  same  way.  This  prevented  the  drifting 
of  nurse  bees,  which  he  believes  to  be  the  method  of  spreading  the 
disease.  Furthermore,  he  found  by  introducing  one  Italian  queen 
into  the  middle  colony  of  an  isolated  row  of  hybrid  bees  that  there 
was  considerable  drifting  of  nurse  bees.    Seven  days  after  the  brood 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBROOD  5 

from  the  Italian  queen  began  to  emerge,  yellow  bees  were  found  on 
either  side  in  several  of  the  hybrid  colonies.  Speaking  of  uniting 
weak  diseased  colonies  and  requeening,  Matthews  writes: 

Alter  two  or  three  were  put  together,  each  stack  of  brood  was  given  an 
Italian  cell.  When  young  queens  commenced  to  lay  there  was  still  disease  in 
many  of  those  hives,  but  as  the  queens  increased  in  laying  the  bees  cleaned 
out  an  ever-increasing  sphere  of  comb  •  for  a  brood  nest  until  they  had  the 
hives  free  of  disease.  But  in  no  case,  however  long  a  hive  might  be  queenless, 
did  I  see  the  disease  cleaned  out  before  a  virgin  appeared  In  the  hive.  In 
other  words,  a  virgin  had  to  be  present  before  the  bees  would  commence  their 
job  of  cleaning  up.  Therefore,  I  see  little  to  commend  the  practice  of  keeping 
diseased  colonies  queenless  21  days. 

A  new  bulletin  by  Phillips  (7)  has  been  issued  recently  by  the 
Department  of  Agriculture.  The  fundamental  idea  emphasized  is 
that  "in  keeping  European  foulbrood  under  control  it  is  far  more 
important  to  prevent  the  disease  from  getting  a  foothold  in  a  colony 
than  it  is  to  eradicate  the  disease  afterward."  This  bulletin,  aside 
from  discussing  symptoms  and  methods  of  treatment,  states  concisely 
for  the  first  time  the  facts  observed  in  apiary  practice  on  which 
successful  treatment  is  based,  and  without  an  imderstanding  of  which 
it  is  difficult  for  a  beekeeper  to  use  preventive  measures  with  any 
success. 

The  analysis  of  these  factors  of  response  in  behavior  to  treatment, 

as  stated  by  Phillips,  has  been  used  to  some  extent  as  a  foundation 

for  the  present  work  on  the  behavior  of  the  colony  in  relation  to 

disease,  in  an  endeavor  to  substantiate,  with  data  obtained  under 

controlled  conditions,  these  facts  that  are  constantly  observed  in 

apiary  practice  and,  if  possible,  to  eliminate  confusion  in  methods  of 

treatment. 

PROCEDURE 

Shortly  after  the  middle  of  May,  1918,  experiments  were  started 
in  Ithaca,  N.  Y.,  at  the  Cornell  Agricultural  College.  Through  the 
kindness  of  Prof.  J.  G.  Needham,  head  of  the  department  of  ento- 
mology, and  others  associated  with  him,  the  use  of  a  small,  isolated 
yard  of  bees  and  also  of  laboratory  facilities  was  offered  for  the 
purpose  of  carrying  on  these  investigations.  This  small  apiary  had 
been  used  previously  in  fruit-pollination  studies  and  had  no  record 
of  disease.  The  yard  was  admirably  located  in  a  naturally  well- 
protected  hollow  beyond  the  college  fruit  orchards,  about  a  mile  and 
a  half  from  the  main  college  apiary  or  other  apiaries,  with  high 
ground  and  woods  intervening.  The  author  and  the  Office  of  Bee- 
Culture  Investigations  are  under  deep  obligations  to  the  Cornell 
authorities  for  the  assistance  so  cordially  extended. 

Being  in  the  buckwheat  district,  the  general  locality  was  well 
adapted  to  the  work  because  of  the  desire  for  as  late  a  main  honey 


G  BULLETIN   804,   xr.   S.    DEPAETMENT   OF   AGEICTJLTURE 

flow  as  possible  in  order  not  to  have  ihe  influence  of  a  heavy  honey 
flow  until  other  factors  had  been  studied.  At  Ithaca  the  main  honey 
flow  is  generally  from  buckwheat,  coming  from  the  1st  to  the  mid- 
dle of  August.  Eather  unfortunately  for  the  best  results  from  the 
experiments,  however,  the  summer  of  1918  was  unusual  in  this  sec- 
tion, for  the  abnormally  heavy  honey  flow  from  clover  necessitated 
finishing  the  work  earlier  than  had  been  planned,  owing  to  the  great 
difficulty  of  artificially  infecting  colonies  during  the  heavy  honey 

flow. 

There  were  seven  colonies  in  the  original  experimental  apiary. 
At  first  it  was  intended  to  work  on  a  larger  scale,  but  the  trend  of 
the  observations  soon  led  to  the  plan  of  working  in  more  detail  and 
on  a  smaller  scale.  These  colonies  were  moved  some  distance  apart 
to  prevent  drifting  and  robbing.  Some  were  divided  and  some  were 
strengthened  in  an  effort  to  make  a  series  of  experiments  on  colonies 
of  different  strengths.  The  colonies  were  designated  by  letter  and 
the  combs  of  each  colony  by  number.  From  time  to  time  some  of 
these  colonies  were  artificially  infected  with  diseased  European  foul- 
brood  larvae  from  samples  sent  to  the  laboratory  for  diagnosis. 
Similar  colonies  were  held  intact  and  uninfected  for  controls.  The 
infection  was  made  by  feeding  diseased  larvae  macerated  in  sugar 
solution  (about  60  per  cent).  For  the  preliminary  experiments  10 
larvae  were  fed  in  about  250  c.  c.  of  sirup.  Later,  after  the  heavy 
honey  flow  had  begun,  it  was  necessary  greatly  to  increase  this  dose  in 
order  to  start  the  infection.  The  infected  sirup  was  fed  to  the  bees 
in  sterilized  glass  petri  dishes,  placed  on  top  of  the  frames  and  pro- 
tected by  an  empty  comb-honey  super  placed  on  the  regular  hive 
body  with  the  cover  on  top. 

At  the  time  of  inoculation,  the  condition  of  each  colony  was  noted 
as  to  age,  race,  condition  and  appearance  of  the  queen,  proportion 
of  nurse  bees  to  old  field  bees,  the  number  of  frames  of  brood  with 
ttie  amount  in  each,  its  age,  sealed  or  unsealed;  in  other  words,  the 
condition  of  the  colony  with  regard  to  factors  known  to  be  signifi- 
cant in  resisting  disease.  In  two  colonies  the  infected  sirup  was 
slightly  colored  with  harmless  eosin  dye  to  determine  where  the  fresh 
sirup  was  placed  and  its  ultimate  disposition.  At  first  daily  obser- 
vations were  made  to  determine  the  earliest  appearance  of  disease, 
the  period  of  incubation,  the  symptoms  exhibited,  and  the  rate  of 
increase. 

By  holding  up  each  comb  in  bright  sunlight  so  that  the  light  shone 
directly  on  the  larvae,  it  was  easy  to  detect  the  first  symptoms  of  the 
disease.  All  the  healthy  larvae  had  the  characteristic  firm,  well- 
rounded,  pearly-white,  glistening  appearance.  The  first  effect  of 
the  disease,  besides  an  abnormal  uneasy  movement,  was  a  loss  of  the 


BEES  IN   COLONIES  AFFECTED  BY  EUROPEAN  FOULBEOOD  7 

glistening  character  and  a  slight  tinge  of  grayish  or  creamy  dis- 
coloration which  would  not  be  noticed  except  in  direct  sunlight. 
These  larvae  showed  only  Bacillm  ^luton  present  when  examined 
microscopically,  as  will  be  mentioned  later.  Soon  after  these  first 
symptoms,  however,  the  more  noticeable  symptoms  appeared,  such 
as  a  larva  with  its  back  out,  the  increase  of  the  light  grayish  yellow 
color,  and,  later,  the  moist,  melting  appearance. 

A  statistical  record  was  kept  of  the  number  of  larvae  showing  new 
disease  at  each  observation,  the  number  previously  diseased  that  had 
been  cleaned  out  in  the  interval  since  the  previous  observation,  and 
those  remaining  over  in  the  cells  uncleaned  for  more  than  one  period 
between  observations.  At  various  times  observations  were  made  of 
the  behavior  and  types  of  bees  engaged  in  cleaning  up  and  the  fate 
of  the  material  removed.  Great  care  was  necessary  in  these  obser- 
vations to  disturb  the  colony  as  little  as  possible.  On  good  days 
it  was  sometimes  possible  to  remove  a  comb  carefully  from  the  hive 
and  to  watch  the  bees  continuing  at  their  work,  and  even  to  watch 
the  queen  laying  eggs.  An  'eight-frame  observation  hive  containing 
a  strong  healthy  colony  was  given  a  diseased  comb  from  time  to  time 
and  the  bees  were  observed  as  they  worked  on  it. 

One  of  the  difficulties  of  the  work  was  to  find  a  satisfactory  method 
of  recording  the  desired  data  for  each  comb.  At  first  the  diseased 
cells  were  marked  on  the  comb  by  a  circle  of  red  ceUoidin  around  the 
entrance  of  the  cell.  Although  this  dried  rapidly,  it  proved  unsatis- 
factory, as  the  bees,  in  their  attempt  to  remove  the  foreign  material, 
seemed  to, remove  both  diseased  and  healthy  larvae  indiscriminately. 
Next  small  pins  were  used,  inserted  in  the  cell  above  the  one  showing 
disease.  In  this  case  the  bees  tore  down  the  surrounding  cells  and 
completely  removed  the  pins,  many  of  which  were  found  on  the 
bottom  board.  Finally  a  method  of  plotting  the  diseased  cells  in  a 
comb  was  adopted.  An  empty  frame  was  laid  off  in  inch  squares  by 
means  of  heavy  black  thread.  This,  used  as  a  templet  superimposed 
on  a  comb,  aided  in  the  location  of  the  diseased  and  cleaned  out  cells, 
so  that  they  could  be  recorded  on  a  correspondingly  ruled  card  (fig.  1) . 
Placing  this  over  the  comb,  it  was  easy  to  locate  exactly  each  cell  and 
to  determine  how  long  the  diseased  material  remained,  thus  aiding 
in  following  the  course  of  the  disease  throughout  its  various  stages. 
The  only  difficulty  with  this  method  was  the  tediousness  of  the  obser-_ 
vations.  Therefore,  after  the  disease  had  become  definitely  estab- 
lished, daily  observations  of  each  colony  were  considered  unneces-^ 
sary.  Longer  periods  showed  just  as  well  what  was  happening  in  the 
colony.  Also,  after  the  disease  had  developed  enough  so  that  it  could 
be  definitely  predicted  whether  the  colony  would  recover  or  gradually^ 
be  exterminated,  observations  of  behavior  under  treatment  were 


8 


BULLETIN  804,  V.  S.   DEPAKTMENT  OF  AGBICULTUKE 


started,  the  method  and  degree  of  house  cleaning  being  watched  after 
the  colony  had  been  dequeened,  strengthened,  and  requeened  with  good 
Italian  stock.    Note  was  also  njade  of  any  recurrence  of  disease  and 


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Fio.  1. — Method  of  plotting  the  location  and  history  of  diseased  bee  larvae  in  the 
combs.  0  Freshly  diseased  larvas.  O-  Cells  that  have  been  cleaned  out.  (j)-Cells 
that  have  been  cleaned  out  and  filled  with  nectar.  (J)  Larvse  remaining  in  the  cells 
more  than  one  observation  period.  The  area  of  sealed  brood  was  the  amount 
present  at  the  time  of  infection  of  the  colony. 

apparent  reason  therefor.  In  other  words,  a  complete  study  was 
made  of  the  cycle  of  the  disease  and  of  the  activities  of  the  bees 
during  its  course. 

OBSERVATIONS 


COLONY  0 

Race. — Hybrid. 

Queen. — 1917,  dark  and  poor. 

Bees. — ^Workers  and  drones  very  dark,  almost  black,  very  excitable. 

Condition  of  colony  at  time  of  infection. — ^Brood  iu  four  frames,  a  little 
less  than  half  sealed,  besides  two  frames  of  eggs.  Bees  covering  about 
eight  frames,  medium  strength.  Slightly  more  field  bees  than  nurse 
bees,  because  of  having  divided  this  colony,  old  bees  returning  from  the 
division. 

Date  of  first  infection. — ^May  28,  1918. 

Material  used. — ^Ten  diseased  larv£e  from  sample  No.  5863,  macerated  in 
250  c.  c.  of  a  50  per  cent  sugar  sirup. 

First  appearance  of  disease  noted. — ^May  31,  1918,  three  day.3  after  inocu- 
lation. 

Age  of  larvw  first  attacked. — ^Three  to  four  days  after  hatching  from  the 
eggs. 

Colony  G  (fig.  2 ) ,  hybrids,  soon  succumbed  to  the  infection,  the  first 
diseased  larva  appearing  three  days  after  infection,  the  gross  diagnosis 
being  confirmed  by  the  finding  of  Bacillus  pluton  on  microscopic  ex- 
amination. The  spread  of  the  disease  was  rapid,  the  disease  being 
present  in  only  one  comb  on  the  third  day  and  in  seven  combs  on  the 
seventh  day.    All  of  this  early  spread  took  place  in  brood  unsealed 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBEOOD 


9 


at  the  time  of  infection.  The  first  high  peak  of  the  disease  coming 
on  the  nineteenth  day  was  followed  by  a  slight  improvement,  when 
for  a  time  the  house  cleaning  exceeded  the  occurrence  of  fresh  dis- 
ease.   This  was  probably  due  to  the  stimulus  of  the  increasing  honey 


M  M  M  M  §  M  M  M  ^  ^ 


c^iwdis'ss'o  ^t^d't// ya  ly-^ffA^^^ 


flow.  But  as  soon  as  the  next  series  of  eggs  hatched,  the  disease^ 
again  gained  the  upper  hand,  reaching  another  higher  peak  on  the 
thirty-first  day,  at  which  time  it  was  deemed  necessary  to  start  treat-^ 
ment.  It  had  become  evident  that  the  colony  was  being  overrun  by: 
the  disease.  More  and  more  dead  larvae  were  being  allowed  to  re-^ 
134440°— Bull.  804—20 i 


10  BTILLETI3Sr   804,   IT.   S.   DEPARTMENT   OF   AGRICULTUKE 


main  in  the  cells  for  several  days  without  being  cleaned  out.  Also' 
more  larvse  nearly  ready  for  pupation  were  being  affected.  Most  of 
these  instead  of  remaining  coiled  were  inclined  to  extend  on  the 
lower  side  wall  in  a  brownish  gray,  slimy  mass  and  exhibited  a  ten- 
dency to  be  viscid.  At  this  stage  of  decomposition,  when  a  stick  is 
inserted  the  mass  forms  a  coarse  granular  band  for  a  short  distance 
and  then  breaks  so  as  to  form  droplike  masses,  but  does  not  stretch 
out  in  a  fine  thread.  These  larval  masses  dried  down  to  rubbery 
dark  brown  scales  something  like  American  foulbrood  scales  in  ap- 
pearance, but  different  in  consistency.  These  scales  could  be  removed 
quite  easily  and  would  bend  like  a  piece  of  partially  granular  old 
rubber.  They  also  lay  irregularly  placed  in  the  cells,  often  spirally 
extended,  while  American  foulbrood  scales  are  uniformly  on  the 
lower  side  wall.  The  bacteriological  explanation  for  this  abnor- 
mal characteristic  will  be  discussed  later  under  bacteriological 
observations. 

The  predominance  of  these  rubbery  masses  and  scales  increased  as 
the  disease  progressed  and  the  bees  seemed  to  make  little  attempt  to 
clean  them  out,  even  after  the  queen  was  caged  on  the  thirty-first 
day,  thus  shutting  off  any  increase  of  fresh  larvse,  or  even  after  the 
queen  and  all  queen  cells  were  removed  on  the  thirty-seventh  day. 
On  the  thirty-ninth  and  also  on  the  forty-first  day,  five  and  four 
frames,  respectively,  of  emerging  brood  and  Italian  bees  were  united 
with  this  colony,  but  it  was  not  until  a  new  Italian  queen,  confined 
in  a  cage,  had  been  hung  in  on  the  forty-fifth  day  that  a  final  com- 
plete cleaning  up  was  made. 

This  new  queen  was  not  accepted,  however,  and  a  young  queen  was 
raised  from  the  brood  that  was  added  to  this  colony,  so  that  fur- 
ther observations  wer6  ended  here  although  the  virgin  queen  was 
killed  and  another  Italian  queen  introduced.  This  colony  was  re- 
ported healthy,  however,  about  the  middle  of  August. 

The  hybrid  bees  seemed  to  lack  ambition  to  fight  the  disease. 
When  combs  were  removed  from  the  colony,  the  bees  never  were  ob- 
served to  be  working  in  the  cells,  and  paid  little  attention  to  ma- 
terial partially  drawn  from  the  cells  and  crushed. 

COLONY  r 

Race. — Italian,  possibly  with  some  slight  hybrid  blood. 

Queen. — 1917,  fairly  good  condition. 

Bees. — Workers,  good  color,  fairly  quiet,  drones  inclined  to  be  darker. 

Condition  of  colony  at  time  of  infection. — Brood  in  three  frames,  a  little 
more  than  one-third  sealed.     Bees  covering  about  six  frames.     Build- 

_.  Ing  up  well.    Proportion  of  field  bees  to  nurse  bees  about  equal. 

Date  of  first  infection. — May  31,  1918. 

.Material  used. — Ten  diseased  larvse  from  sample  No.  5874,  macerated 
in  250  c.  c.  of  a  50  per  cent  sugar  sirup. 

First  appearance  of  disease  noted. — June  4,  1918,  four  days  after  in- 
fection. 

■^sejyf  larvw  first  attacked.— Vowr  days  after  hatching  from   the  egg. 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBBOOD         11 

Colony  F  (fig,  3),  which  was  the  next  one  to  be  infected,  although 
not  as  strong  as  colony  G,  was  of  Italian  stock  and  did  not  show  the 
appearance  of  disease  until  one  day  later.  On  the  fourth  day  one 
cell  appeared  in  each  of  two  combs.    It  was  not  until  the  twenty- 


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fifth  day  that  the  disease  ha4  spread  to  seven  combs,  the  total  number 
of  diseased  larvai  being,  as  a  whole,  less  than  in  the  hybrid  colony. 
There  was  not  brood  in  all  seven  combs  at  the  time  of  infection,  but 
the  brood  increased  faster  than  the  disease  spread.  After  the 
twenty-fourth  day  a  permanent  improvement  began  to  be  manifest. 


12  BULLETIN   804,   U.   S.   DEPABTMENT   OF   AGRICULTURE 

This  improvement  continued  after  the  queen  was  caged  and  became 
more  marked  after  she  was  removed  from  the  colony. 

These  bees  were  better  house  cleaners  as  well;  the  appearance  of 
larvae  remaining  over  more  than  one  observation  period  did  not  be- 
come evident  until  after  the  ninth  day,  compared  with  the  sixth  day 
in  colony  G.  At  no  time  were  there  as  many  of  the  larvae  nearly 
ready  to  pupate  that  were  gummy  or  rubbery.  Even  though  this 
colony  was  on  the  average  weaker  than  colony  G  all  the  time,  it 
handled  the  disease  much  better.  It  was  14  days  before  colony  G 
had  cleaned  up  to  such  an  extent  that  it  was  deemed  safe  to  intro- 
duce a  new  queen,  while  in  colony  F,  with  the  Italian  bees,  the 
combs  were  so  nearly  cleaned  of  everything  but  a  few  old  scales 
that  a  five-frame  nucleus  with  a  nfew  Italian  laying  queen  was  united 
with  this  colony  after  a  10-day  queenless  period  and  in  9  days 
more  everything  was  absolutely  clean  and  the  queen  was  laying  in 
the  combs  that  had  had  disease  in  them. 

When  an  observation  was  made  nine  days  after  the  new  queen's 
eggs  were  first  noted,  it  was  found  that  there  was  a  slight  recurrence 
of  disease  in  three  of  the  combs.  But,  unfortunately,  at  the  same 
time,  queen  cells  and  no  eggs  were  found,  denoting  that  for  some 
reason  this  queen  had  not  been  accepted.  Therefore  the  queen  cells 
were  all  removed  and  a  new  queen  was  introduced.  Although  the 
author's  observations  ended  of  necessity  soon  thereafter,  it  was 
reported  to  him  that  this  colony  was  doing  nicely  later  in  August 
and  was  perfectly  healthy.  If  the  first  new  queen  had  not  disap- 
peared, it  is  quite  probable  that  as  soon  as  a  sufficient  number  of 
her  bees  had  emerged  they  would  have  cleaned  up  the  recurring 
disease  in  the  same  manner  as  was  done  in  colony  J,  which  will  be 
mentioned  later. 

Several  times  in  this  colony,  during  the  cleaning-up  process,  bees 
were  watched  in  the  act  of  sucking  up  juices  of  diseased  larvae 
that  had  been  partially  removed'  from  the  cells  with  the  aid  of 
forceps. 

COLONY  H 

Race. — Hybrid,  a  division  of  Colony  G,  hybrid. 

Queen. — 1918.    Of  their  own  raising.    Poor. 

5ees.— Dark  hybrids,  almost  black,  excitable. 

Condition  of  colony  at  time  of  infection. — Brood  in  three  frames,  a  few 

eggs  in  one,  only  one  small  patch  sealed,  the  remainder  from  eggs  up  to 

4-day  larva.    Bees  covering  about  five  frames.    Fairly  good  proportion 

of  nurse  bees. 
Date  of  first  infection. — July  1,  1918;  second  infection,  July  8,  1918. 
Material  used.— 20  old,  dried,  rubbery,  diseased  scales  from  sample  No. 

5898,  macerated  in  250  e.  c.  of  a  50  per  cent  sugar  sirup,  colored  with 

eosin. 
First  appearance  of  disease  noted. — July  5,  doubtful.    Positive  July  8,  7 

days  after  infection. 
Age  of  larvm  first  attacked. — Four  days  after  hatching  from  the  egg. 


BEES  IN  COI^ONIES  AFFECTED  BY  EUROPEAN  FOULBROOD         13 

Colony  H  (fig.  4)  was  treated  as  a  double  experiment.  The  infec- 
tion of  this  colony  was  not  started  until  after  the  honey  flow  had  come 
on  quite  heavily.  Also,  instead  of  freshly  diseased  larvae,  old  brown 
rubbery  scales  were  used  that  showed  Baxdllus  pluton  present  micro- 
scopically, but  were  heavily  overgrown  by  Bacillus  alvei.  It  was  de- 
sired to  learn  whether  these  scales  were  still  infectious,  so  that  nurse 
bees  working  on  them,  cleaning  them  out,  might  get  infective  material 
on  their  feet  and  mouth  parts  which  could  be  carried  to  healthy  larvaj. 
This  was  noted  later  in  the  observation  hive,  where,  under  the  magni- 
fying glass,  bees  were  seen  trying  to  remove  some  of  these  rubbery 
scales,  first  moistening  them  with  their  tongues  and  then  pulling  at 
them  with  the  mandibles  and  front  feet. 

This  colony,  which  was  marked  hybrid  and  weak,  was  slow  in 
developing  the  disease,  partly  because  of  the  diluting  effect  of  the 


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Pig.  4. — The  course  of  lEuropean  foulbrood  in  colony  H. 

heavy  honey  flow  and  probably  partly  because  there  was  a  smaller 
number  of  infectious  organisms  present  in  the  scales  than  in  fresh 
larvaB.  This  is  explained  by  the  fact  that  the  secondary  putrefactive 
invading  organisms  would  tend  to  kill  off  the  primary  organism,  be- 
cause of  the  accumulation  of  the  products  of  the  putrefactive  action. 

On  the  seventh  day  before  the  disease  was  first  noted,  a  second 
infection  of  scales  macerated  in  sugar  sirup  was  given  this  colony 
to  counteract  the  effect  of  these  retarding  factors.  However,  later  on 
the  seventh  day,  diseased  larvae  were  found,  and  from  then  on  .the 
disease  started  to  spread  and  increase  irrespective  of  the  heavy  honey 
flow,  exhibiting  all  the  symptoms  and  tendencies  shown  in  colony  G, 
of  which  this  colony  was  a  division  before  infection. 

On  the  seventeenth  day  it  was  necessary  to  remove  the  queen  and 
start  treatment,  but  what  was  taking  place  was  evident.  This  removal 
of  the  queen  did  not  seem  to  have  a  very  marked  effect  on  the  house 


14 


BtJLLETIN  804,  XT.   S.  DEPAETMENT  OF  AGEICTJLTUKE 


cleaning  until  the  colony  was  united  with  colony  I,  a  slightly  dis- 
eased Italian  colony.  They  then  began  cleaning  the  H  combs,  and 
the  combined  colony  was  reported  clean  in  August. 

COLONY  A 

Race. — Italian  with  some  possible  slight  hybrid  blood. 

Queen. — 1918.     Of  their  own  raising. 

Bees. — ^Workers,  good  color;  fairly  quiet.  Drones,  some  slightly  darker 
than  pure  Italians. 

Condition  of  colony  at  time  of  infection. — Brood  in  seven  frames  about 
half  sealed.  Bees  covering  about  nine  frames  with  a  good  proportion 
of  young  nurse  bees.     Colony  strong  and  building  up. 

Date  of  first  infection. — July  2,  1918.    Second  infection,  July  6,  1918. 

Material  used. — ^First,  20  diseased  larvae  from  sample  No.  5937  macer- 
ated in  250  c.  c.  of  a  50  per  cent  sirup,  colored  with  eosin,  ab- 
normally heavy  infection;  second  infection,  20  diseased  larvse  from 
sample  No.  5953  in  250  c.  c.  of  uncolored  sirup. 

First  appearance  of  disease  noted. — July  8,  1918,  in  drone  brood,  six  days 
after  infection. 

Age  of  larvw  first  attacked. — Four  days  after  hatching  from  the  egg. 

Colony  A  (fig.  5)  was  a  fairly  strong  colony  of  Italians.     Like  col- 
ony H,  it  was  infected  after  the  heavy  honey  flow  had  started  and  was 


^^ss^a^.^ 


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Fig.  5. — The  course  of  European  foulbrood  In  colony  A. 

given  twice  the  amount  of  infective  material  colonies  F  and  G  received. 
Nothing  having  appeared  on  the  fourth  day,  a  second  infection  of 
the  same  amount  was  given.  On  the  sixth  day  6  diseased  larvae 
were  seen  in  three  combs.  This  colony,  however,  was  so  strong  that 
the  disease  obtained  very  little  foothold,  and  from  the  fourteenth 
day  began  to  decline,  or  at  least  failed  to  make  further  gains.  As 
a  side  experiment  in  this  colony  a  comb  of  eggs  laid  by  an  Italian 
queen  was  placed  in  between  two  combs  showing  disease.  If  there 
is  anything  in  the  belief  that  Italian  stock  is  more  resistant  to  disease, 
the  larvse  in  this  comb  should  not  have  developed  the  disease,  or  at 
least  not  so  soon.  However,  on  the  sixth  day  one  or  two  larvae  showed 
disease,  increasing  slightly  in  numbers  for  a  few  days  until  the  obser- 
vations were  of  necessity  stopped.  It  was  intended  to  perform  this 
experiment  with  several  variations,  such  as  placing  eggs  laid  by  an 
Italian  queen  in  a  diseased  hybrid  colony  and  placing  eggs  from 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBBOOD         15 

a  hybrid  queen  in  a  diseased  Italian  colony,  but  the  presence  of  the 
heavy  honey  flow  made  it  impracticable  to  carry  the  matter  further. 
This  colony  A  cleaned  up  readily  after  removal  of  the  queen  and 
was  reported  all  healthy  in  August.  Although  a  new  queen  was 
given  to  them  it  is  probable  that  a  period  of  queenlessness  and  the  re- 
turning of  the  same  queen  would  have  answered  just  as  well. 

COLONY  I 

i?oce.— Italian. 

Queen.~lQl7,  fairly  good  condition. 

Condition  of  colony  at  time  of  infection.— Se\en  frames  of  emerging 

brood  well  covered  with  young  bees.    A  strong  8-frame  colony. 
Date  of  first  infection. — July  10,  1918. 
Material  Msed.— Thirty  diseased  larvse  from  sample  No.  5959,  macerated 

In  250  c.  c.  of  a  50  per  cent  sugar  sirup.    This  was  abnormally  heavy 

Infection  of  diseased  material. 
First  appearance  of  disease  no«e(J.— July  15,  1918,  five  days  after  infection. 
Age  of  larvw  first  attacked.— Four  days  after  hatching  from  the  egg. 

This  colony  was  infected  during  the  heavy  honey  flow,  but 
although  given  a  heavy  infection  it  had  sufficient  strength,  aided  by 
the  heavy  honey  flow,  to  prevent  the  disease  from  spreading.  On 
July  15  there  were  a  few  diseased  larvae  in  two  combs.  On  July  24, 
14  days  after  inoculation,  there  were  only  a  few  diseased  larvae  in 
three  combs.  This  was  after  the  queen  had  been  removed  on  July 
18  and  the  colony  had  been  united  with  colony  H  on  the  20th. 

An  interesting  observation  was  that  under  the  magnifying  glass 
the  methods  of  the  nurse  bees  in  sucking  the  juices  from  dead  dis- 
eased larvae  and  the  pulling  of  the  skins  out  to  carry  them  away  could 
be  noted.  No  bee  worked  very  long  at  s.  time  on  one  larva.  One 
after  another  worked  until  all  was  completed. 

SUMMARY  OF  PREVIOUS  EXPERIMENTS 

Table  I  gives  a  partial  summary  of  the  data  thus  far  described. 

Table  I. — STiotoing  the  first  appearance  of  disease  noted  after  infection.  Also 
the  number  of  combs  showing  infection  and  the  spread  of  the  infection  from 
comb  to  comb  in  the  various  colonies  under  observation 


Col- 

Date 
infected. 

Days  after  iuleotion. 

ony. 

1 

2 

3 

4 

6 

6 

7 

8 

0 

10 

11 

12 

13 

14 

16 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 

26 

27 

28 

1918 
May  28 
May  31 
/July    1 
July    8 
July    2 
July     6 
July  10 

Number  of  combs. 

G>.... 

•• 

1 

3 
2 

3 

2 

4 
3 

5 
2 

4 

'4 
1 

7 

8 

Fi.... 

6 

S 

7 

... 

HI.... 

... 

2 

4 

( 

A>.... 

... 

... 

3 

Dtf 

5 

4 

P 

fir 

stt 

1  Experiments  started  before  the  beginning  of  the  heavy  honey  flow. 
>  Experiments  started  after  the  beguming  of  the  heavy  honey  flow. 


16  BULLETIN   804,   U.   S.   DEPARTMENT   OF   AGRICULTURE 

In  the  first  group,  colonies  G  and  F,  it  is  quite  apparent  that 
the  Italian  bees,  colony  F,  made  the  better  showing,  even  though  the 
hybrids  were  the  stronger  colony  in  the  beginning.  As  may  be 
seen  from  a  comparison  of  the  two  plots  in  figures  2  and  3,  in 
colony  G  wherever  there  was  a  lag  in  the  house  cleaning  there  was 
marked  increase  in  the  number  of  larvae  remaining  over  more  than 
one  observation  period,  and  these  increased  until  strengthening 
treatment  was  started.  On  the  other  hand,  in  colony  F  these  were 
removed  almost  entirely  by  the  time  the  strengthening  treatment 
was  started.  The  Italians  did  not  allow  the  disease  to  appear  as 
soon  or  to  spread  as  rapidly,  cleaned  house  better,  left  fewer  larvae 
to  dry  down  to  the  brownish  rubbery  scales,  and  responded  to  the 
increased  honey  flow  and  treatment  much  more  readily. 

In  the  second  group,  colonies  H,  A,  and  I,  the  Italian  colonies 
A  and  I  again  made  the  best  showing.  With  the  added  diluting 
effect  of  the  honey  flow,  they  allowed  the  disease  to  gain  no  foothold 
whatever,  while  the  hybrids,  though  aided  by  the  honey  flow,  soon 
succumbed  and  allowed  the  disease  to  gain  on  them.  It  is  evident 
that  the  Italian  bees  are  much  more  vigorous  house  cleaners.  In 
several  instances,  toward  the  end  of  the  egg  laying  of  the  old  queen, 
and  well  along  in  the  progress  of  the  disease,  cells  were  noted  on 
these  diagrams  which  had  previously  contained  diseased  larvae,  but 
which  had  been  cleaned  out,  and  then  in  which  disease  had  reap- 
peared after  other  eggs  had  been  laid  and  hatched  in  them.  They 
were  cells  in  which  fresh  nectar  had  not  been  placed  between  the  two 
series  of  larvae. 

It  was  also  noted  that  as  the  honey  flow  increased  and  as  the 
brood  became  more  scattered  from  the  effects  of  the  disease,  more 
and  more  fresh  nectar  was  placed  in  the  brood  nest  in  cells  from 
which  dead  larvae  had  been  removed.  Most  of  this  nectar,  however, 
was  moved  up  later,  particularly  after  the  bees  began  preparing  the 
brood  nest  for  a  new  queen  in  the  process  of  treatment.  That  the  ad- 
vent of  a  heavy  honey  flow  was  effective  in  controlling  the  disease  is 
evident,  particularly  in  the  length  of  time  between  the  infection  and 
the  first  appearance  of  disease.  The  data,  however,  show  little  dif- 
ference in  the  resistance  to  infection,  or  so-called  immunity,  being 
slightly  in  favor  of  the  Italians,  if  there  is  any  difference  at  all. 

Disregarding  the  effect  of  the  honey  flow,  the  period  of  incubation 
of  the  disease  is  apparently  between  3  and  4  days.  However,  it  was 
noted  that  after  Bacillus  pluton  was  first  observed  it  was  anywhere 
from  24  to  48  hours  before  many  characteristically  diseased  larvae 
were  observed.  Therefore,  the  actual  period  of  incubation  is  prob- 
ably from  24  to  48  hours. 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBKOOD         17 
SUPPLEMENTARY  OBSERVATIONS 

STUDY   OP  NATURALLY  INFECTED  COLONIES 

As  a  supplementary  study  to  the  preceding  artificial  infection  ex- 
periments, some  observations  were  made  upon  the  behavior  of  nat- 
urally infected  colonies  undergoing  treatment.  Through  the  kind- 
ness of  W.  L.  Bean,  of  McGraw,  N.  Y.,  it  was  possible  to  make  a 
series  of  such  observations.  In  his  apiary  of  about  30  colonies,  all 
hybrids,  the  majority  were  diseased  when  observed  June  8,  1918. 
Soon  thereafter  Mr.  Bean  kindly  loaned  two  of  these  diseased  colo- 
nies to  be  carried  to  Ithaca  for  closer  observation.  Mr.  Bean  at 
once  started  treating  his  bees,  requeening  with  Italian  stock  by  the 
method  of  introducing  a  queen  cell  almost  ready  to  emerge.  Appar- 
ently, this  method  was  successful,  for  in  the  latter  part  of  July  Mr. 
Bean  reported  all  treated  colonies  healthy  and  some  800  pounds  of 
surplus  honey. 

COLONY   J 

Race. — ^Hybrid. 

Queen. — Queenless  at  time  of  arrival  at  Ithaca.  Was  poor  hybrid  of  own 

raising,  probably  reared  while  disease  was  present  in  the  colony. 
Strength  in  spring. — Weak. 
Strength,  at  time  of  treatment.— Sc&ttered  brood  in  eight  frames.    Weak 

in  bees,  particularly  in  nurse  bees. 
Approximate  date  of  disease  first  noted. — ^May  31,  1918. 
Date  of  start  of  treatment  observations. — June  16,  1918. 

This  colony  made  no  effort  to  clean  up,  even  though  they  had  lost 
their  queen  shortly  before  being  brought  to  Ithaca.  On  the  18th  of 
June  six  frames  of  Italian  bees  and  emerging  brood  were  placed  on 
top  of  it.  At  once  house  cleaning  started,  a  reduction  of  50  per  cent 
being  noted  in  the  fresh,  moist,  melting  larvae  within  24  hours.  In 
this  colony  it  was  interesting  to  watch  the  bees  doing  the  house  clean- 
ing, particularly  when  diseased  larvae  in  various  stages  of  decomposi- 
tion were  partially  withdrawn  from  the  cell  with  a  pair  of  forceps. 
With  the  aid  of  a  powerful  hand  magnifying  glass  it  was  easy  to 
watch  them  suck  up  the  juices  of  the  dead  larvae,  even  those  which 
had  decomposed  to  the  extent  of  being  a  coffee  brown  in  color  and 
viscid  in  consistency.  No  bee  would  work  long  on  a  larva  but  would 
back  off  and  wipe  her  tongue  thoroughly  with  her  front  feet.  It  is 
conceivable  that  this  might  contaminate  her,  making  possible  car- 
riage of  the  infection  to  the  next  larva  fed,  even  though  the  juices  of 
the  diseased  larva  were  not  actually  fed  to  the  healthy  one.  The 
majority  of  bees  engaged  in  this  work  were  the  Italians.  From  these 
and  other  observations  of  a  similar  nature  there  is  no  doubt  that  the 
contamination  of  the  mouth  parts  is  the  primary  method  of  spread- 
ing the  disease  inside  the  oolony. 


18  BtTLLETIN  804,  U.   S.   DEPAKTMENT  OF  AGRICULTURE 

On  June  25  an  Italian  queen  was  introduced  in  a  cage  with  candy 
even  though'  a  few  scales  were  still  present.  This  was  fully  10  days 
after  the  colony  had  lost  its  queen,  if  not  a  little  longer.  On  June 
27  the  queen  was  out  and  laying  in  one  comb.  Eight  days  later,  on 
July  5,  a  recurrence  of  disease  was  noted,  one  larva  being  discolored 
and  sunken,  showing  Bacillus  pluton  on  microscopic  examination. 
From  that  time  on,  for  about  20  days  after  the  first  eggs  of  this 
queen  were  noted,  one  or  two  new  diseased  larvae  appeared  at  each 
observation,  the  number  decreasing,  however,  until  about  the  twenty- 
sixth  day  when  they  had  all  disappeared.  As  the  new  young  Italian 
bees  increased,  the  disease  decreased,  until  a  point  was  reached  where 
they  were  in  the  predominance  and  had  eliminated  the  disease  by 
their  activity.     This  was  also  observed  in  colony  F. 

COLONY  K 

Race. — ^Hybrid. 

Queen. — 1917.    Dark  hybrid  of  their  own  raising,  probably  from  diseased 

stock. 
Strength  in  spring. — ^Weak. 
Strength  at  time  of  treatment. — Eight  frames  of  scattered  brood  and 

hardly  enough  bees  to  cover  them. 
Approximate  date  of  disease  first  noted. — ^May  31,  1918. 
Date  of  start  of  treatment  observations. — June  20,  1918,  at  which  time 

the  queen  was  removed. 

Colony  K,  when  it  was  brought  to  Ithaca,  was  so  weak  that  it 
would  soon  have  died.  The  bees  made  no  attempt  to  clean  out  larvae 
that  had  been  partially  pulled  out  of  the  cells  with  forceps  and 
crushed.  On  June  26,  six  days  later,  they  were  still  showing  freshly 
diseased,  moist,  melting  larvae  from  eggs  laid  by  the  old  queen,  just 
before  removal.  At  this  time  five  frames  of  emerging  brood  and 
Italian  bees  were  given  this  colony.  On  the  27th  a  new  Italian  queen 
was  hung  in  with  the  cage  closed.  The  presence  of  the  new  queen, 
however,  seemed  to  give  added  impetus  to  the  house  cleaning  so  that 
by  July  1,  11  days  after  removal  of  the  queen,  they  were  prac- 
tically cleaned  up  and  the  cage  was  opened  with  candy  in  the  open- 
ing. V  Further  observations  on  this  colony  were  ended  because  they 
refused  to  accept  this  queen.  By  the  time  another  queen  finally  was 
accepted  and  was  laying  on  July  18,  it  was  too  late,  as  the  season's 
work  was  closed  by  the  23d. 

BEHAVIOR  OF  BEES  IN  CLEANING  CONTAMINATED  CELLS 

On  June  6,  1918,  a  sample  was  received  for  diagnosis  (No.  5898), 
consisting  of  an  entire  brood  comb,  containing  quite  an  area  of 
capped  honey.  About  one-half  of  each  side  of  the  comb  contained 
a  large  number  of  dead  and  diseased  European  foulbrood  larvse,  in 
stages  varying  from  the  yellowish,  moist,  melting  larvae  to  dried 
rubbery  scales  of  which  there  was  quite  a  large  proportion.    This  was 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBKOOD         19 

the  same  sample  from  which  infectious  dried  scales  were  used  to  in- 
fect colony  H.  After  this  comb  had  remained  in  the  laboratory, 
wrapped  in  paper  for  about  3  weeks,  it  was  placed  in  the  strong 
colony  in  the  observation  hive.  The  frame  was  first  placed  in  the 
middle  of  the  hive  for  about  an  hour  and  was  then  removed  to  the 
outside,  where  the  work  of  the  bees  on  it  could  be  watched.  A  large 
number  of  what  appeared  to  be  young  nurse  bees  were  already  hard 
at  work  on  the  dried  diseased  material.  The  bees,  working  on  the 
dried  gummy  masses,  would  wet  the  mass  with  their  tongues  for  a 
while  and  then  tear  at  them  with  their  mandibles,  at  times  removing 
pieces  large  enough  to  be  seen  from  the  outside.  Often  these  small 
pieces  were  apparently  dropped  to  the  bottom  board.  No  one  bee 
worked  long  at  one  place.  Those  bees  working  particularly  on  the 
fresh,  moist  material,  when  leaving,  would  carefully  wipe  their 
tongues  with  their  front  feet,  thereby  transferring  some  of  the  in- 
fection to  them.  Other  bees  were  at  work  carrying  away  the  larger, 
more  easily  removable  dead  masses.  The  entrance  also  was  watched 
to  see  if  any  of  this  material  was  carried  out.  Several  bees  were 
observed  carrying  out  portions  of  dead  larvae  or  pupae.  One  bee 
carried  a  piece  about  2  yards  before  dropping  it.  Others  dropped 
what  they  were  carrying  soon  after  leaving  the  entrance,  but  on  ex- 
amining the  surface  of  the  ground  about  the  entrance,  very  little 
material  could  be  distinguished,  so  that  apparently  most  of  the  ma- 
terial removed  must  have  been  carried  some  little  distance  before  being 
dropped.  After  about  an  hour's  work  it  was  apparent  that  consider- 
able progress  had  been  made.  This  comb  was  removed  before  it  was 
entirely  cleaned  and  later  placed  in  another  healthy  colony  for  obser- 
vation. It  was  quickly  cleaned  up  and  quite  a  bit  of  nectar  placed 
in  it  and,  eventually,  several  square  inches  of  brood.  Observations, 
however,  had  to  be  stopped  before  any  appearance  of  recurrence  was 
noted.  This  same  observation  hive  was  given  one  or  two  other  dis- 
eased combs  to  clean,  but  with  the  repeated  probable  infection  from 
these  sources  the  colony  was  so  strong  that  no  disease  was  noted  in 
it  during  the  entire  season  of  observations. 

POSSIBLE  INFECTION  THROUGH  QUEEN 

Colony  M  was  a  small  nucleus  made  to  receive  the  old  queen  from 
diseased  colony  K  from  McGraw,  N.  Y.  The  queen  was  introduced 
on  June  20,  1918.  For  a  while  she  laid  fairly  well,  it  being  neces- 
sary to  add  one  or  two  more  combs.  But  later  her  brood  became 
more  and  more  scattered.  Finally,  on  July  8,  there  was  observed  one 
dead  larva,  which  looked  suspicious,  but  which,  on  microscopic  ex- 
amination, proved  to  be  negative.  On  July  10,  however,  one  definite 
cell  appeared  and  several  other  slightly  yellowish,  abnormally  colored 
larvse.  This  dead  larva  contained  BaciUus  pluton.  From  then  on 
until  this  queen  was  killed  and  the  colony  united  with  another  dis- 


20 


BULLETIN   804,   TJ.   S.   DEPARTMENT   OE   AGKICULTTJRB 


eased  colony,  more  discolored  larva?  appeared,  showing  definitely  the 
•development  of  the  disease.  As  far  as  could  be  seen  the  only  source 
of  infection  was  the  queen  which  had  come  from  a  diseased  colony. 

This  occurrence  had  been  observed  previously  by  the  author  while 
employed  at  the  Massachusetts  Agricultural  Experiment  Station. 
During  the  summer  of  1916  eight  queens  taken  from  diseased  Euro- 
pean f  oulbrood  colonies  were  introduced  into  isolated,  healthy  nucleus 
colonies.  Of  these  eight  nuclei  three  developed  European  foulbrood, 
two  were  doubtful,  and  three  remained  healthy.  Several  such  in- 
stances have  been  mentioned  in  the  literature  of  beekeeping. 


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Fig.  6. — Distribution  of  cells  containing  infected  sugar  sirup  and  subsequent  spread 

of  tbe  disease  in  a  comb  taken  from   colony   H Area    covered 

by  brood  at  time  of  infection,  mostly  unsealed.  •  Location  of  cells  con- 
taining infected  colored  sugar  sirup  on  July  3,  1918.  ®  First  positive  diseased 
larvse  noted,  2  on  July  8.  Q  Number  of  new  diseased  larvaj  (4)  on  July  10. 
A  Number  of  diseased  larvae  (13)  on  July  12.  D  Number  of  diseased  larvae  (39) 
on  July  16.      O     Number  of  diseased  larvse  (52)  on  July  19. 

DISTRIBUTION  OF  INTRODUCED   INFECTED  MATERIAL 

An  interesting  experiment  was  carried  out  with  sugar  sirup,  colored 
by  a  small  amount  of  a  harmless  anilin  dye,  eosin,  used  as  an  indi- 
cator, which  gave  to  the  sirup  a  bright  red  color.  The  object  of  this 
experiment  was  to  determine  where  the  sirup,  or,  more  important, 
where  fresh  nectar  is  first  placed  in  the  hive  and  combs.  On  May  27 
two  colonies  were  fed  this  colored  sirup  from  above  some  time  before 
the  heavy  honey  flow  from  clover  started.  The  results  were  striking, 
for  in  nearly  every  case  the  colored  sirup  was  easily  discernible  in  the 
cells  and  the  greatest  part  of  the  sirup  was  located  in  quite  a  definite 
area.  These  colored  cells  were  either  scattered  among  the  cells  con- 
taining the  larvse  or  were  placed  in  a  ring  of  cells  adjacent  to  the 
brood  area  toward  the  top  of  the  comb,  little  being  placed  with  the 
solicJ  stores  (fig.  6).  Furthermore,  for  nearly  36  hours  after  the 
feeding  practically  all  the  young  nurse  bees  showed  a  marked  pinkish 
discoloration  of  the  anterior  end  of  their  abdomens,  denoting  the  dis- 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBEOOD         21 

tention  of  the  honey  stomachs  by  the  retention  of  the  colored  sirup 
therein.  About  half  the  bees  in  the  hives  were  discolored  in  this 
manner.  After  a  day  or  so,  however,  this  begfn  to  disappear.  Also 
the  number  of  cells  showing  the  pink  discoloration  began  to  disappear. 
Evidently  the  sirup  had  been  moved  up,  worked  over,  and  mixed  with 
other  nectar  or  consumed. 

Later,  some  time  after  the  heavy  honey  flow  had  started,  shortly 
after  July  1,  two  more  colonies  were  fed  colored  sirup,  this  time 
infected  with  diseased  larvae  macerated  therein. 

In  these  cases  the  discolored  abdomens  were  noted  about  as  before, 
but  the  colored  cells  were  less  numerous  and  the  color  less  striking. 
The  location  of  the  colored  cells  was  similar  to  that  in  the  former  ex- 
periment ;  that  is,  mainly  in  the  brood  area  or  just  contiguous  to  it 
and  mostly  above.  The  outside  combs,  containing  considerable  honey, 
showed  scarcely  any  of  the  colored  cells.  This  time  these  colored 
cells  disappeared  sooner,  showing  that  the  infected  material  must 
have  been  much  diluted  quite  soon  after  being  taken  up  from  the 
feeding  dishes. 

Figure  6  shows  the  method  of  plotting  the  location  of  diseased 
larvae  in  the  combs  and  also  the  location  of  the/cells  containing  the 
colored  sugar  sirup.  As  will  be  noted,  a  fairly  large  proportion  of 
these  cells  are  located  within  the  area  of  brood  at  the  time  of  feed- 
ing. It  is  interesting  to  note  the  tendency  of  diseased  brood  to  form 
concentric  circles,  showing  the  two  series  of  larvae  occurring  between 
the  dates  noted.  The  spreading  was  from  two  cells  at  first  to  quite  a 
large  number  at  the  last  observation  shown. 

AGE  AT  WHICH  LAEV^  ARE  INFECTED 

In  previous  observations  it  was  constantly  noted  that  the  larvae 
affected  by  European  foulbrood  were  regularly  at  least  4  days  old, 
the  age  at  which  the  coiled  larvae  completely  fill  the  bottom  of  the 
cells.  Occasionally  a  slightly  younger  and  smaller  larva  would 
become  diseased,  but  this  was  not  the  common  occurrence.  Further- 
more, in  the  cases  where  the  colored  sirup  was  fed  the  bees,  within 
24  to  36  hours  quite  a  number  of  larvae  averaging  4  days  old  could  be 
seen  discolored  from  having  been  fed  this  sirup,  while  it  was  notice- 
able that  the  younger  larvae  under  3  days  old  never  showed  the  dis- 
coloration. These  colored  larvae  were  examined  in  a  smear  under 
a  microscope,  but  the  infecting  organisms,  being  comparatively  few 
in  number,  had  not  increased  sufficiently  at  that  time  to  be  apparent. 

The  question  now  arises  as  to  the  age  at  which  the  larvae  first  are 
fed  nectar  or  infected  material.  There  has  been  much  controversy  over 
the  subject  of  composition  and  source  of  the  larval  food,  but  as  yet  no 
conclusive  scientific  evidence  has  been  presented.  Irrespective  of 
the  question  whether  the  food  at  various  stages  originates  from 
glands  or  is  regurgitated,  it  is  apparent  from  these  observations  that 


22 


BULLETIN  804,   V.   S.   DEPABTMENT  OF  AGRICULTURE 


there  must  be  a  difference  between  the  food  which  larvae  younger 
than  approximately  3  days  old  receive  and  that  fed  to  older  ones. 
Otherwise  the  younger  larvae  would  also  show  the  pink  coloration. 
Von  Planta  (9)  by  chemical  analyses,  of  questionable  exactness,  how- 
ever, makes  a  division  in  the  feeding  of  the  larvae  at  the  age  of  4 
days,  at  which  time  the  high  protein  and  low  sugar  content  change 
to  lower  protein  and  higher  sugar  content.  These  analyses  would 
tend  to  coincide  with  the  above  observations,  only  it  is  probable  that 
the  change  begins  earlier. 

Additional  data  upon  this  subject  are  recorded  in  Tables  II  and  III, 
although  the  observations  were  primarily  for  another  purpose.  In 
order  to  obtain  further  information  relating  to  a  possible  difference 
in  resistance  to  disease  between  Italian  and  hybrid  bees,  a  careful 
record  was  made  of  the  time  when  eggs  were  first  noted  in  empty 
combs  after  the  infection  of  the  colony  and  when  larvae  first  showed 
disease  thereafter.  In  the  case  of  comb  Special  No.  2,  the  eggs  were 
laid  by  an  Italian  queen  in  a  healthy  colony  and  then  placed  in  a 
diseased  colony.  Colonies  F,  A,  and  I  were  of  Italian  stock  while 
colonies  G,  H,  and  J  were  hybrid.  In  the  recurrence  of  disease  all 
were  given  new  Italian  queens.  As  has  been  mentioned  before,  as  soon 
as  the  bees  of  the  new  Italian  queens  emerged  in  sufficient  numbers 
the  disease  disappeared. 

Table  II 

THE  FIRST  APPEAEANCE  OF  DISEASE  IN  COMBS  IN  WHICH  EGGS  WERE  LAID  AFTER 
THE   COLONY  WAS  INFECTED 


Colony  and  comb  No. 

Number  of  dajB  after  eggs  were  first  noted  in  comb. 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

)2 

13 

14 

X 

0  6                        

X 

07                            

X 

G8              

X 
X 

F3                  

F4         

F7          

X 

H3                

X 
X 

H5     

A2                

X 
X 

A  4                 

A  6    

X 

A7           

A  8              

X 

X 

il.\. ::::;;:::::::::::::::::::::::::::::: 

X 

EECUREENCE  OF  DISEASE  AFTER  EGGS  OF  A  NEW  QUEEN  WERE  FIRST  NOTED  IN 

THE  COMBS 

J  2 

X 
X 

J  3 

J  4 

X 

J  5 

X 

J  6a 

X 

J  6b 

X 

J  7 

X 

J  8 

X 

F3 

X 
X 
X 

F4 

F? 

BEES  IN  COLONIES  AFlfECTED  BY  EUROPEAN  FOULBROOD 


23 


Table  III. — Average  time,  under  various  conditions,  in  which  disease  hecomes 
apparent  in  a  colony  after  infection  with  Europecm  foulbrood.  (Averages 
taken  from  Table  II) 


Before  the 

heavT 
honey  now. 


Colony  G,  hybrid.... 
Colony  F,  Italian.... 
Average  of  these  two, 


Days. 


7i 


During 
the  heavy 
honey  flow. 


Colony  H, hybrid 

Colony  A,  Italian 

Colony  I,  Italian 

Average  of  these  three 


Days. 


8i 
9 

8{i 


Becurrence 
of  disease, 
after  treat- 
ment, dur- 
ing honey 
flow. 


Colony  J,  hybrid  originally 
Colony  F,  Italian  originally. 
Average  of  these  two 


Days. 


9i 


The  data  shown,  particularly  in  Table  III,  tend  to  disprove  the 
theory  that  Italian  bees  have  a  natural  immunity  or  resistance.  If 
a  larger  number  of  observations  could  have  been  made,  the  variation 
would  have  appeared  less.  The  effect  of  the  honey  flow  is  evident, 
however. 

When  it  is  a  question  of  the  age  at  which  the  larvae  are  fed  material 
that  contains  infection,  these  figures  are  significant.  In  the  life 
history  of  the  bee,  3  days  are  spent  in  the  Qgg  and  from  5  to  6  days 
as  larva  before  capping,  making  a  period  of  9  days  in  all.  After 
3  days  in  the  egg  and  after  having  been  fed  predigested  food  for  3 
days,  with  the  additional  24  to  48  hour  period  of  incubation,  as 
was  observed  earlier  in  this  paper,  the  larva  ought  to  show  disease 
from  the  fourth  to  the  fifth  day  after  hatching,  or  the  seventh  to 
eighth  day  of  its  existence,  if  Von  Planta's  assumption  is  correct. 
From  actual  observation  this  was  found  to  be  true  and  from  observa- 
tion of  the  averages  in  Table  III  it  is  seen  that  the  first  appearance 
of  disease  occurs  between  the  seventh  and  ninth  days,  varying  with 
the  conditions  of  the  honey  flow. 

Eeferring  to  Dr.  Miller's  theories,  it  is  hard  to  believe  that  there 
is  not  plenty  of  highly  infectious  material  left  in  the  colony  after 
a  5  or  6  day  period  of  queenlessness.  Aside  from  actual  observations 
of  moist,  yellow,  melting  larvae  present  more  than  6  days  after  the 


24  BULLETIN  804,   V.   S.   DEPARTMENT  OF  AGEICITLTUEE 

queen  has  been  removed,  the  juices  of  which  the  workers  sucked  up 
with  avidity,  the  final  eggs  laid  will  be  just  at  the  stage  where  the  dis- 
ease first  appears;  that  is,  3  to  4  days  after  hatching,  at  the  end  of  a 
6-day  period.  Furthermore,  even  though  the  nurse  bees  do  not  feed 
to  healthy  larvae  the  material  that  is  taken  up  in  cleaning  out  the 
cells  in  varying  stages  of  decomposition,  infection,  even  from  scales, 
may  be  carried  on  the  feet,  mouth  parts,  and  tongue,  particularly, 
as  was  definitely  shown  with  colony  H,  since  these  scales  are  in- 
fectious. The  period  of  queenlessness  and  the  consequent  house 
cleaning  are  absolutely  dependent  on  the  strength  of  the  colony.  A 
strong  colony  cleans  up  rapidly,  particularly  after  the  introduction 
of  the  new  queen  in  a  cage  plugged  with  candy.  A  weak  colony,  on 
the  other  hand,  has  not  sufficient  bees  to  clean  even  after  complete 
introduction  of  a  queen,  and  the  disease  soon  appears  again.  Under 
average  conditions,  therefore,  it  would  appear  unsafe  to  allow  less 
than  a  10-day  period  of  queenlessness  in  treatment  of  European  £oul- 
brood. 

MICROSCOPICAL  BACTERIOLOGICAL  OBSERVATIONS 

A  large  number  of  microscopic  examinations  were  made  of  larvae 
under  various  conditions  for  the  positive  presence  of  the  characteris- 
tic groups  of  Bacillus  pluton.  These  examinations  were  made  mainly 
as  a  check  on  the  gross  observations  of  the  first  appearance  of  the 
disease.  Cover  glass  smears  were  made  of  crushed  larvae,  stained 
with  carbol  f  uchsin  and  mounted  in  Canada  balsam.  These  examina- 
tions were  made  at  regular  intervals  after  the  colonies  were  infected, 
larvae  of  all  ages  being  examined. 

It  was  found  in  the  smears  of  those  larvae  showing  the  first  slightly 
abnormal  symptoms  that  Bacillus  pluton  was  the  only  organism 
present.  This  substantiates  White's  (12)  observations  that  before  the 
disease  could  be  detected  by  gross  examination,  by  a  histological 
study  of  sections  of  larvae  during  the  period  of  incubation  it  was 
demonstrated  that  "  in  the  production  of  the  disease  Bacillus  pluton 
was  the  first  invader  of  the  healthy  larvae." 

As  the  disease  advanced  in  the  various  colonies,  observations  were 
made  of  larvae  in  various  stages  of  decomposition.  The  bacterial  con- 
tent was  found  to  vary  with  the  change  of  appearance  of  the  larvae 
during  decomposition.  The  presence  of  these  secondary  invaders 
easily  explains  the  atypical  appearance  of  certain  types  of  European 
foulbrood  that  heretofore  have  been  very  confusing  to  the  bee- 
keeper. 

For  a  short  time  after  the  death  of  the  larva,  the  color  remains  a 
moist,  creamy-grayish  yellow.  This  is  during  the  period  when  Bacil- 
lus pluton  and  such  occasional  secondary  invaders  as  Streptococcus 
apis  or  Bacterium  eurydice  and  other  organisms,  which  do  not  form 


BEES  IN  COLONIES  AFFECTED  BY  EUROPEAN  FOULBKOOD         25 

spores,  are  predominant  as  described  by  White  (12)  and  McGray 
(4).  Soon  the  putrefactive  spore- forming  organisms  increase  in 
number,  BaciUus  aZvei^  being  the  one  most  commonly  found.  This 
is  seen  particularly  in  the  case  of  the  more  mature  larv83,  which  when 
dying  extend  more  or  less  irregularly  in  the  cells,  becoming  the  gray- 
ish brown  slimy  masses  which  develop  into  the  dark  brown  granular 
rubbery  scales.  This  fact  has  been  observed  for  a  long  time  in  the 
many  samples  which  have  been  received  for  diagnosis.  A  partial 
description  of  these  scales  and  of  the  presence  of  Bacillus  alvei  in 
them  is  given  by  McCray  and  White  (5),  but  the  experimental 
observations  described  in  this  paper  added  to  diagnostic  observations 
show  that  this  condition  is  generally  much  more  pronounced  and 
common  than  described  by  these  writers  from  laboratory  observa- 
tions. The  rapid  increase  and  peculiar  process  of  decomposition  of 
BaciUus  alvei,  after  the  death  of  the  larva,  often  to  the  exclusion  of 
all  other  organisms,  accounts  for  this  abnormal  appearance.  In  the 
case  of  American  foulbrood,  almost  never  is  any  other  organism 
found  associated  with  the  disease  but  Bacillus  larvae,  the  cause  of 
the  disease.  This  accounts  for  the  constancy  of  the  symptoms  as 
compared  with  the  variation  of  symptoms  in  European  foulbrood 
where  there  may  be  several  secondary  invaders. 

Furthermore,  in  making  the  smears  of  the  diseased  larvae  upon 
cover  glasses,  the  peculiar  whitish  saclike  extrusion  of  the  larval  in- 
testines was  often  noticed  on  crushing  the  larvae  preparatory  to  smear- 
ing, which  White  (10)  describes  as  a  gross  diagnostic  character. 
When  this  sac  was  removed  and  smeared  separately,  it  was  always 
found  to  be  heavily  loaded  with  Bacillus  phiton.  Therefore  it  is  safe 
to  assume  that  the  intestinal  tract  is  the  primary  focus  of  infection, 
while  the  secondary  putrefaction  takes  place  mostly  in  the  body  tis- 
sues of  the  dead  larva. 

Coincident  with  the  microscopic  examination  of  larvae,  several  ex- 
aminations were  made  of  the  contents  of  the  ventriculus,  rectum,  and 
in  a  few  cases  of  the  honey  stomach  and  mouth  parts  of  bees.  These 
bees  were  presumably  nurse  bees  taken  from  diseased  combs,  some  in 
the  very  act  of  sucking  up  the  juices  of  dead  diseased  larvae.  Al- 
though insufficient  observations  were  made  to  give  conclusive  evidence, 
some  interesting  information  was  obtained. 

As  may  be  seen  from  Table  IV,  the  number  of  cases  where  Bacillus 
pluton  or  other  organisms  associated  with  infectious  material  were 
f  oimd  in  the  intestinal  contents  is  not  very  large.    However,  of  more 

^Badllus  alvei  originally  was  supposed  to  be  the  primary  cause  of  European  foulbrood, 
but  has  been  proved  by  White  and  others  to  be  only  a  common  secondary  invader.  Bacil- 
lus alvei  has  purely  putrefactive  functions.  From  its  cultural  and  biochemical  character- 
istics, Bacilhia  alvei  apparently  belongs  to  the  common  Baoillus  suitiUs  (hay  bacllIuB) 
group  of  Bpoie-toiming  organisms,  all  having  mainly  putrefactive  functions. 


26 


BULLETIN  804,  U.  S.   DEPAKTMENT  OF  AGRICULTURE 


importance  probably,  BacUhis  pluton  was  found  in  a  smear  made 
from  the  mouth  parts  of  a  nurse  bee  and  also  in  the  contents  of  the 
honey  stomach  of  another.  If  these  observations  had  been  carried  out 
systematically,  instead  of  only  casually,  it  is  expected  that  much  more 
positive  data  might  have  been  obtained  along  these  lines,  owing  to 
what  is  known  already  of  the  habits  of  house-cleaning  bees  working 
on  diseased  material. 

Table  IV. — The  results  of  the  microscopic  bacterial  examination  of  the  contents 
of  the  intestinal  tracts  of  nurse  tees  taken  from  diseased  colonies 


Microscopic  £iiidiiigs. 

G. 

F. 

H. 

A. 

I. 

J. 

K. 

Total. 

Positive inciZZtts  pZ«i07i,.. 

1 

11 

2 
24 

3 

2 

18 

1 

i 

9 

9 

17 

12 

6 
7 

97 

Baalim  alvei  or  doubtful  Bacillus 
pluton... 

n 

SUMMARY  AND  CONCLUSIONS 

In  arriving  at  the  following  conclusions  an  effort  has  been  made 
to  state  them  in  a  manner  which  will  indicate  the  substantiation  of 
previous  observations  made  both  in  the  laboratory  and  in  the  apiary. 
It  may  be  noted  that  many  of  these  conclusions  are  similar  to  some 
of  the  statements  made  in  Farmers'  Bulletin  975  in  the  summary  of 
facts  which  apiary  practice  has  brought  out. 

1.  European  foulbrood  is  an  infectious  disease.  BaciUvs  plioton 
was  found  to  be  the  primary  invader,  appearing  in  the  intestinal  tract 
of  larvae  before  death,  contemporary  with  the  first  slightly  apparent 
symptoms. 

2.  The  variation  in  the  appearance  of  the  diseased  larvae  after  death 
is  due  to  the  presence  or  absence  of  secondary  invaders. 

3.  The  period  of  incubation  for  European  foulbrood  was  found 
to  be  from  36  to  48  hours,  although  the  gross  symptoms  usually  do  not 
become  apparent  in  less  than  3  or  4  days,  varying  with  conditions 
of  honey  flow  and  strength  of  colony. 

4.  It  has  been  noted  in  apiary  practice  that  the  first  brood  of  the 
year  usually  escapes  with  little  loss.  During  the  first  5  to  7  days  the 
spread  of  the  disease  in  the  colony  after  infection  is  slow,  after  which 
the  increase  is  rapid  under  favorable  conditions.  The  critical  time, 
therefore,  to  detect  the  disease  and  start  treatment  is  early  in  its 
course,  thus  making  conditions  unfavorable. 

5.  The  evidence  tends  to  confirm  the  theory  that  one  of  the  ways 
the  disease  is  spread  in  the  colony  is  by  the  house-cleaning  bees,  and 
from  colony  to  colony  by  their  drifting.  It  is  quite  probable  that  the 
infective  organisms  are  carried  on  the  mouth  parts  and  pedal  appen- 
dages.   The  question  of  infection  from  intestinal  contents  or  from 


BEES  IN  COLONIES  AFFECTED  BY  EUBOPEAN  FOULBROOD    27 

the  source  of  larval  food  at  various  stages  needs  further  substantia- 
tion. 

6.  Irrespective  of  strength  of  colony,  the  Italian  bees  were  found 
to  resist  infection  much  better  than  hybrids  and  showed  more  ability 
to  overcome  the  disease. 

7.  This  apparent  resistance  of  the  Italian  bees  was  observed  to 
be  largely  due  to  the  more  vigorous  house-cleaning  characteristics 
rather  than  to  a  natural  resistance  or  immunity  to  the  disease. 
There  was  very  little  difference  in  the  apparent  period  of  incubation 
between  the  Italian  and  hybrid  colonies,  possibly  a  slight  difference 
in  favor  of  the  Italians.  Furthermore,  it  was  noted  that  often  there 
may  be  a  slight  recurrence  of  disease  in  the  brood  of  the  new  Italian 
queen  until  a  sufficient  number  of  her  bees  have  emerged  to  eliminate 
the  infection  by  house  cleaning.  Apparently,  infection  is  not  always 
entirely  removed  by  a  period  of  queenlessness. 

8.  As  a  rule,  requeening  is  necessary  in  the  treatment  of  European 
foulbrood,  except  possibly  in  the  strongest  Italian  colonies,  which 
show  only  slight  infection.  Where  a  considerable  quantity  of  dis- 
ease is  present,  sufficient  to  require  treatment,  it  was  found  unsafe 
to  use  a  period  of  less  than  10  days'  queenlessness,  due  to  the  infec- 
tious condition  of  the  diseased  material  remaining  and  the  accom- 
panying behavior  of  the  colony. 

9.  The  stronger  the  colony  in  Italian  bees,  the  more  rapid  was  the 
recovery. 

10.  A  heavy  honey  flow  tends  to  prevent  infection  from  gaining 
a  foothold.  It  also  tends  to  eliminate  the  disease  if  present  before 
the  start  of  the  heavy  honey  flow.  This  was  found  to  be  due  to  the 
effect  of  dilution  on  the  infection  because  of  the  influx  and  direct 
feeding  of  the  fresh  nectar  to  the  larvae. 

11.  European  foulbrood  is  a  disease  of  weak  colonies.  It  was 
found  to  be  difficult  effectually  to  infect  any  but  the  very  weak 
colonies  during  the  heavy  honey  flow.  Therefore,  colonies  kept 
strong  up  to  the  time  of  the  honey  flow  run  very  little  danger  of 
contracting  European  foulbrood.  This  and  others  of  the  facts  ob- 
served are  in  exact  harmony  with  facts  already  observed  in  apiary 
practice. 


LITERATURE  CITED 

(1)  Alexandbb,  E.  W. 

1905.  How  to  rid  your  apiary  of  black  brood/     In  Gleanings  in  Bee- 
Culture,  V.  33,  p.  1125. 

(2)  MnxEE,  C.  C. 

1911.  Fifty  years  among  tlie  bees. 

(3)  

1918.  European  foulbrood  and  its  treatment.     In  American  Bee  Journ., 
V.  58,  no.  7,  p.  232-234.    July. 

(4)  McCeat,  a.  H. 

1917.  Spore-forming  bacteria   of  tbe  apiary.     In  Jour.  Agr.   Research, 
V.  8,  no.  11,  p.  399-420,  pi.  93-94. 

(5)  and  White,  G.  F. 

1918.  The  diagnosis  of  bee  diseases  by  laboratory  methods.    U.  S.  Dept. 
Agr.  Bui.  671,  15  p.,  2  pi. 

(6)  Phillips,  E.  F. 

1911.  The  treatment  of  bee  diseases.     U.  S.  Dept.  Agr.  Farmers'  Bui.  442. 
May  6. 

(7)  

1918.  The  control  of  European  foulbrood.     V.  S.  Dept.  Agr.  Farmers'  Bui. 
975.     July. 

(8)  [Root,  E.  R.] 

1905.   (Editorial.)     In  Gleanings  in  Bee-Culture,  v.  33,  p.  1126.     Nov.  1. 

(9)  VoN  Planta,  a. 

1888.  TJeber  den  Futtersaft  der  Bienen.     In  Zeit.  f.  Phys.  Chemie  von 
Hoppe-Seyler,  v.  12,  p.  327-354. 

(10)  

1889.  Ueber  den  Futtersaft  der  Bienen.     In  Zeit.  f.  Phys.  Chemie  von 
Hoppe-Seyler,  v.  13,  p.  552-561. 

(11)  West,  N.  D. 

1899.  Foul  and  other  forms  of  diseased  brood  in  the  State  of  New  York. 
In  Gleanings  in  Bee-Culture,  v.  27,  p.  828.    Nov.  15. 

(12)  White,  G.  F. 

1912.  The  cause  of  European  foulbrood.    Clr.  1.57,  Bur.  Ent.,  U.  S.  Dept. 
Agr.    May  10. 

»  "  Black  brood  "  Is  an  old  name  for  European  foulbrood. 
28 


ADDITIONAL  COPIES 

OF  THIS  PUBLICATION  MAT  BE  PKOCUHED  FBOM 

THE  SUPERINTENDENT  OF  DOCUMENTS 

GOVERNMENT  PRINTING  OFFICE 

WASHmOTON,  D.  C. 

AT 

5  CENTS  PER  COPY 


laauerl  May  6, 1911. 

U.  S.  DEPARTMENT  OF  AGRICULTURE. 


FARMERS'   BULLETIN  442. 


THE  TREATMENT  OF  BEE  DISEASES. 


BY 


E.  F.  PHILLIPS,  Ph.  D., 
In  Charge  of  Bee  Culture,  Bureau  of  Entomology. 


WASHINGTON: 

GOVERNMENT    PRINTING    OFFICE. 

1911. 


442 


LETTER  OE  TRANSMITTAL. 


U.  »S.  Depaktment  of  Agriculture, 

Bureau  or  Entomology, 
Washington,  D.  G.,  February  2k-,  1911. 
Sir:  I  liav^e  the  honor  to  transmit  herewith  a  manuscript  entitled 
"  The  Treatment  of  Bee  Diseases,"  by  E.  F.  Phillips,  Ph.  D.,  in  charge 
of  bee  culture  in  this  bureau.     In  the  preparation  of  this  paper, 
which  is  intended  to  supersede  Circular  79,  of  this  bureau,  the  aim 
has  been  to  give  briefly  the  information  needed  by  the  beekeeper 
who  has  disease  in  his  apiary.    No  discussion  of  the  cause  or  distri- 
bution of  these  diseases  has  been  included.    I  recommend  the  publica- 
tion of  this  paper  as  a  Farmers'  Bulletin. 
Respectfully, 

L.  O.  Howard, 
Eniomologist  and  Chief  of  Bureau. 
Hon.  James  Wilson, 

Secretary  of  Agriculture. 

442 

2 


CONTENTS. 


Page. 

Introduction 5 

The  brood  diseases  of  bees 5 

Nature  of  the  diseases 7 

Names  of  the  diseases 7 

Symptoms 8 

American  foul  brood 8 

European  foul  brood 10 

The  so-called  "  pickle  brood  " 12 

Brood  dead  of  other  causes 12 

"  Bald-headed  brood  " 12 

Methods  of  spread 12 

Precautionary  measures 13 

Treatment  for  both  infectious  diseases 13 

Shaking  treatment 14 

Time  of  treatment 14 

Preparation 14 

Operation 14 

Saving  the  healthy'  brood 16 

Saving  the  wax 16 

Cleaning  the  hive 16 

Disposal  of  the  honey 17 

The  second  shake 17 

The  cost  of  shaking 17 

Treatment  with  bee  escape 17 

Fall  treatment 18 

Drugs 18 

Treatment  for  European  foul  brood 18 

Introduction  of  Italian  stock 19 

Dequeening 19 

Inspection  of  apiaries 19 

Examination  of  samples  of  diseased  brood ' 20 

The  diseases  of  adult  bees 20 

Dysentery 20 

The  so-called  paralysis 21 

Isle  of  Wight  disease 21 

Spring  dwindling 21 

Publications  of  the  Department  of  Agriculture  on  bee  diseases 22 

442 

3 


ILLUSTRATIONS. 


Page. , 

Fig .  1.  Work  of  tho  larger  wax  moth 6 

2.  American  foul  brood 8 

3.  The  ropiness  of  American  foul  brood 9 

4.  American  foul-brood  comb 9 

5.  European  foul  brood 11 

6.  Apparatus  for  the  shaking  treatment 15 

7.  Gasoline  torch 16 

442 

4 


THE  TREATMENT  OF  BEE  DISEASES. 


INTRODUCTION. 

The  diseases  which  attack  the  honey  bee  may  be  divided  into  two 
classes,  namelj',  tliosu  att'ecting  the  brood  and  those  to  which  the 
adult  bees  are  subject.  The  diseases  of  adult  bees  have  not  been  in- 
\estigated  sufficiently  to  make  it  possible  at  the  present  time  to  recom- 
mend methods  for  their  treatment.  In  the  present  bulletin,  tlierefore, 
only  a  brief  statement  concerning  these  diseases  will  be  made,  mainly 
for  the  purpose  of  indicating  the  present  state  of  knowledge  on  these 
subjects.  Concerning  the  diseases  of  the  brood  more  is  known,  and 
this  is  particularly  fortunate  since  they  are  far  more  destructive  in 
American  apiaries  than  are  the  diseases  of  the  adult  bees. 

The  causes  of  bee  diseases  will  not  be  discu.ssed  here.  For  informa- 
tion on  this  phase  of  the  subject  the  reader  is  referred  to  other  pub- 
lications of  the  Bureau  of  Entomology,  which  are  listed  at  the  end  of 
this  bulletin.  The  aim  of  this  bulletin  is  to  give  information  that 
can  be  used  by  the  practical  beekeeper  in  combating  bee  diseases. 

THE  BROOD  DISEASES  OF  BEES. 

The  brood  diseases  of  the  honey  bee  are  already  widely  distributed 
in  the  United  States  and  seem  to  be  spreading  rather  rapidly.  The 
loss  to  the  beekeepers  of  the  country,  owing  to  the  actual  death  of 
colonies  by  disease,  is  estimated  conservatively  at  $1,000,000  annually. 
This  does  not  include  the  loss  of  crops,  resulting  from  the  destruction 
of  colonies,  or  the  discouragement  to  the  beekeeper  which  often 
causes  him  to  give  up  the  business.  A  considerable  part  of  this  loss 
is  due  to  the  indifference  of  the  beekeepers  to  these  diseases  and  a  lack 
of  knowledge  concerning  them. 

It  frequently  happens  that  colonies  in  an  apiary  become  infected 
before  the  owner  realizes  that  disease  is  present.  He  may  errone- 
ously attribute  the  losses  observed  to'some  other  cause.  In  this  way 
the  disease  gets  a  start  which  makes  eradication  difficult  when  once 
the  cause  of  the  loss  has  been  discovered.  In  view  of  the  widespread 
distribution  of  these  diseases,  it  is  most  desirable  that  all  beekeepers 
learn  to  distinguish  the  diseases  when  they  appear  and  to  know  how 
to  keep  them  under  control. 

It  is  often  a  matter  of  surprise  to  beekeepers  to  learn  that  bees  are 
subject  to  disease.  The  most  frequent  source  of  confusion  is  the 
442  5 


e 


TREATMENT   OF   BEE   DISEASES. 


placing  of  the  blame  for  loss  of  colonies  on  some  cause  other  than 
disease.  The  poorer  class  of  beekeepers  attribute  their  losses  simply 
to  "  bad  luck,"  but  even  well-informed  beekeepers  err  in  this  matter. 


Fig.  1. — Work  of  the  larger  wax  moth  in  a  brood  comb.      (Original.) 

The  wax  moths  (see  %.  1)  are  most  frequently  blamed  for  the  death 
of  colonies,  whereas  they  do  no  damage  to  strong,  healthy  colonies, 
properly  cared  for,  but  enter  only  when  the  colony  is  weakened  by 
queenlessness,  lack  of  stores,  disease,  or  some  other  cause.     In  the 

442 


TREATMENT   OP   BEE  DISEASES.  7 

majority  of  the  reports  of  wax-moth  depredations  received  by  this 
department  which  can  be  investigated  it  is  found  that  the  trouble  is 
actually  an  outbreak  of  a  brood  disease. 

The  spraying  of  fruit  trees  while  in  bloom  is  possibly  injurious  to 
bees,  and  there  exists  among  beekeepers  a  strong  feeling  against  the 
jjractice.  Since  no  entomologist  now  recommends  that  fruit  trees  be 
sprayed  during  the  blooming  period,  this  is  probably  rarely  done  by 
progressive  fruit  growers.  However,  it  is  frequently  reported  by 
beekeepers  that  they  are  losing  bees  by  poisoning  due  to  spraying. 
A  number  of  cases  of  the  death  of  colonies,  reported  as  caused  by 
poisoning  due  to  spraying  while  trees  were  in  bloom,  have  been  found 
to  be  in  reality  outbreaks  of  European  foul  brood,  which  is  particu- 
larly prevalent  in  the  spring  and  early  summer. 

Other  circumstances  to  which  is  often  attributed  the  death  of  brood 
or  of  the  colony  are  chilling,  fumes  from  coke  ovens,  and  malicious 
poisoning.  The  wise  attitude  on  the  part  of  the  beekeeper  is  first  to 
suspect  diseases  as  being  the  cause  of  any  losses  which  he  may  sus- 
tain, and  to  be  sure  that  there  is  no  infectious  disease  present  before 
looking  elsewhere  for  a  cause. 

NATURE   OF   THE  DISEASES. 

There  are  two  recognized  infectious  diseases  of  the  brood  of  bees, 
now  known  as  American  foul  brood  and  European  foul  brood.  Both 
diseases  weaken  colonies  by  reducing  the  number  of  emerging  bees 
needed  to  replace  the  old  adult  bees  which  die  from  natural  or  other 
causes.  In  neither  case  are  adult  bees  affected,  so  far  as  known.  The 
means  used  by  the  beekeeper  in  deciding  which  disease  is  present  is  the 
difference  in  the  appearance  of  the  larva;  dead  of  the  two  diseases. 
That  the  diseases  are  entirely  distinct  can  not  now  be  doubted,  since 
they  show  certain  differences  in  the  age  of  the  larvae  affected,  in  their 
response  to  treatment,  and  in  the  appearance  of  the  dead  larvae. 
This  is  made  still  more  certain  by  a  study  of  the  bacteria  present 
in  the  dead  larvae.  Reports  are  sometimes  received  that  a  colony 
is  infected  with  both  diseases  at  the  same  time.  While  this  is  pos- 
sible, it  is  not  by  any  means  the  rule,  and  such  cases  are  usually 
not  authentically  reported.  There  is  no  evidence  that  chilled  or 
starved  brood  develops  into  an  infectious  disease  or  that  dead  brood 
favors  the  development  of  a  disease. 

NAMES  OF   THE  DISEASES. 

The  names  American  foul  brood  and  European  foul  brood  were 
applied  to  these  diseases  by  the  Bureau  of  Entomology,  of  this  de- 
partrnent,  to  clear  Up  the  confusion  in  names  which  formerly  existed. 
By  retaining  the  words  "  foul  brood  "  in  each  name  the  disease- 
inspection  laws  then  in  force  could  be  interpreted  as  applying  to 

442 


8 


TEEATMEXT   OF   BEE   DISEASES. 


both  diseases.  These  names  were  in  no  way  intended  to  designate 
geographical  distribution,  since  both  diseases  did  exist  and  do  now 
exist  in  both  Europe  and  America,  but  were  chosen  primarily  because 
they  were  convenient  and  easily  remembered  names.  Their  only 
significance  is  in  indicating  where  the  diseases  were  first  seriously 
investigated.  It  was  particularly  desirable  to  change  the  name  of 
the  disease  now  known  as  European  foul  brood,  since  "  black  brood  "" 
entirely  fails  to  be  descriptive  and  is  misleading. 

SYMPTOMS. 

The  presence  of  a  particular  disease  in  a  colony  of  bees  can  be 
ascertained  most  reliably  by  a  bacteriological  examination,  since  the 
symptoms  are  somewhat  variable.  It  is  possible,  however,  to  describe 
the  usual  manifestations  of  the  diseases,  and  the  usual  differences,  so 
that  the  beekeeper  can  in  most  cases  tell  which  disease  is  present. 

American   Foul  Brood. 

American  foul  brood  is  frequently  called  simply  "  foul  brood." 
It  usually  shows  itself  in  the  larva  just  about  the  time  that  the  larva 
fills  the  cell  and  after  it  has  ceased  feeding  and  has  begun  pupation. 


Fig.  2. — American  foul  brood  :  o,  5,  J,  normal  sealed  cells ; 
c.  ;.  sunken  cappings,  showing  perforations ;  g^  sunken 
oappin:::  not  perforated;  7i.  I,  m,  n,  g,  r,  larvae  affected  by 
disease ;  e,  i,  p,  /<,  scales  formed  from  dried-down  larvse ; 
(?,  o,  pupfe  affected  by  disease.  Three  times  natural  size. 
(Orisrinal.) 


At  this  time  it  is  sealed  over  in  the  comb  (fig.  2.  a,  h,  /).  The  first 
indication  of  the  infection  is  a  slight  brownish  discoloration  and 
the  loss  of  the  well-rounded  appearance  of  the  normal  larva  (fig. 
2.  I).    At  this  stage  the  disease  is  not  usuallj'  recognized  by  the  bee- 

442 


TREATMENT   OF   BEE   DISEASES. 


9 


keeper.  The  larva  gradually  sinks  down  in  the  cell  and  becomes 
darker  in  color  (fig.  2,  /i,  m),  and  the  posterior  end  lies  against  the 
bottom  of  the  cell.  Frequently  the  segmentation  of  the  larva  is 
clearly  marked.  By  the  time  it  has  partially  dried  down  and  has 
became  quite  dark 
brown  (coffee  col- 
ored) the  most 
typical  character- 
istic of  this  disease 
manifests  itself. 
If  a  match  stick 
or  tooth-pick  is  in- 
serted into  the  de- 
caying mass  and 
^vithdrawn  the  larval  remains  adhere  to  it  and  are  drawn  out  in  a 
thread  (fig.  3),  which  sometimes  extends  for  several  inches  before 
breaking.  This  ropiness  is  the  chief  characteristic  used  by  the  bee- 
keejjer  in  diagnosing  this  disease.  The  larva  continues  to  dry  down 
and  gradually  loses  its  ropiness  until  it  finally  becomes  merely  a 


Fig.  3. — The  ropiness  of  American  foul  brood.      (Original.) 


Pig.  4. — American  foui-brood  comb,  showing  irregular  patches  of  sunken  cappings  and  scales. 
The  position  of  the  comb  indicates  the  best  way  to  view  the  scales.      (Original.) 

scale  on  the  lower  side  wall  and  base  of  the  cell  (fig.  2,  e,  p,  s).  The 
scale  formed  by  the  dried-down  larva  adheres  tightly  to  the  cell  and 
can  be  removed  with  difficulty  from  the  cell  wall.  The  scales  can 
best  be  observed  when  the  comb  is  held  with  the  top  inclined  toward 
the  observer  so  that  a  bright  light  strikes  the  lower  si'de  wall  (fig.  4). 
83568°— Bull.  442—11 2 


10  TEEATMEXT    OP    BEE    DISEASES. 

A  very  characteristic  and  usually  penetrating  odor  is  often  iiotice- 
able  in  the  decaying  larvae.  This  can  perhaps  best  be  likened  to  the 
odor  of  heated  glue. 

The  majority  of  the  larva?  which  die  of  this  disease  are  attacked 
after  being  sealed  in  the  cells.  The  cappings  are  often  entirely  re- 
moved by  the  bees,  but  when  they  are  left  they  usually  become 
sunken  (fig.  2.  g,  c,  j)  and  frequently  perforated  (fig.  2,  e,  j).  As  the 
healthy  brood  emerges  the  comb  shows  the  scattered  sunken  cappings 
covering  dead  larvae  (fig.  4) ,  giving  it  a  characteristic  appearance. 

Pupse  also  may  die  of  this  disease,  in  which  case  they,  too,  dry  down 
(fig.  2,  0,  d).  become  ropy,  and  have  the  characteristic  odor  and  color. 
The  tongue  frequently  adheres  to  the  upper  side  wall  and  often 
remains  there  even  after  the  pupa  has  dried  down  to  a  scale.  Younger 
unsealed  larvae  are  sometimes  affected.  Usually  the  disease  attacks 
onh"  worker  brood,  but  occasional  cases  are  found  in  which  queen 
and  drone  brood  are  diseased.  It  is  not  certain  that  race  of  bees, 
season,  or  climate  have  any  effect  on  the  virulence  of  this  disease, 
except  that  in  warmer  climates,  where  the  breeding  season  is  pro- 
longed, the  rapidity  of  devastation  is  more  marked. 

European  Foul  Brood. 

European  foul  brood  was  formerly  called  "  black  brood  "  or  "  New 
York  bee  disease."  The  name  "  black  brood  "  was  a  poor  one,  for  the 
color  of  the  dead  brood  is  rarely  black  or  even  very  dark  brown. 
European  foul  brood  usually  attacks  the  larva  at  an  earlier  stage  of 
its  development  than  American  foul  brood  and  while  it  is  still  curled 
up  at  the  base  of  the  cell  (fig.  5,  ;•).  A  small  percentage  of  larv'se 
dies  after  capping,  but  sometimes  quite  young  larvae  are  attacked  (fig. 
5,  e.  m).  Sunken  and  perforated  cappings  are  sometimes  observed 
just  as  in  American  foul  brood  (fig.  2,  c,  g,  j).  The  earliest  indication 
of  the  disease  is  a  slight  yellow  or  gray  discoloration  and  uneasy 
movement  of  the  larva  in  the  cell.  The  larva  loses  its  well-rounded, 
opaque  appearance  and  becomes  slightly  translucent,  so  that  the 
tracheae  may  become  prominent  (fig.  5,  &),  giving  the  larvae  a  clearly 
segmented  appearance.  The  larva  is  usually  flattened  against  the 
base  of  the  cell,  but  may  turn  so  that  the  ends  of  the  larva  are  to  the 
rear  of  the  cell  (fig.  5,  p),  or  may  fall  away  from  the  base  (fig.  5, 
e,  g,  1) .  Later  the  color  changes  to  a  decided  yellow  or  gray  and  the 
translucency  is  lost  (fig.  5,  q,  h).  The  yellow  color  may  be  taken  as 
the  chief  characteristic  of  this  disease.  The  dead  larva  appears  as  a 
moist,  somewhat  collapsed  mass,  giving  the  appearance  of  being 
melted.  "Wlien  the  remains  have  become  almost  dry  (fig.  5,  c)  the 
tracheae  sometimes  become  conspicuous  again,  this  time  by  retaining 
their  shape,  while  the  rest  of  the  body  content  dries  around  them. 
Finally  all  that  is  left  of  the  larva  is  a  grayish-brown  scale  against 

442 


TREATMENT   OF   BEE   DISEASES. 


11 


the  base  of  the  cell  (fig.  5,  /,  h),  or  a  shapeless  mass  on  the  lower  side 
wall  if  the  larva  did  not  retain  its  normal  position  (fig.  5,  n,  o). 
Very  few  scales  ai-e  black.  The  scales  are  not  adhesive,  but  are  easily 
removed,  and  the  bees  carry  out  a  great  many  in  their  efforts  to  clean 
house. 

Decaying  larvae  which  have  died  of  this  disease  are  usually  not 
ropy  as  in  American  foul  brood,  but  a  slight  ropiness  is  sometimes 
observed.  There  is  usually  little  odor  in  European  foul  brood,  but 
sometimes  a  sour  odor  is  present,  which  reminds  one  of  yeast  fer- 
mentation. This  disease  attacks  drone  and  queen  larvae  ^  almost  as 
quickly  as  those  of  the  workers. 


Fig.  5. — European  foul  brood:  a,  j,  h,  normal  sealed  cells; 
l>t  Cj  dj  ej  g,  i,  X,  nij  p,  q,  larvae  affected  by  disease  ;  r,  nar- 
mal  larva  at  age  attacked  by  disease ;  f,  h,  n,  o,  dried-down 
larvie  or  scales.     Three  times  natural  size.      (Original.) 


European  foul  brood  is  more  destructive  during  the  spring  and 
early  summer  than  at  other  times,  often  entirely  disappearing  during 
late  summer  and  autumn,  or  during  a  heavy  honey  flow.  Italian  bees 
seem  to  be  better  able  to  resist  the  ravages  of  this  disease  than  any 
other  race.  The  disease  at  times  spreads  with  startling  rapidity  and 
is  most  destructive.  Where  it  is  prevalent  a  considerably  larger  per- 
centage of  colonies  is  affected  than  is  usual  for  American  foul  brood. 
This  disease  is  very  variable  in  its  symptoms  and  other  manifesta- 
tions and  is  often  a  puzzle  to  the  beekeeper. 

1  The  tendency  of  this  disease  to  attack  queen  larvae  is  a  serious  drawback  in  treat- 
ment.    Frequently  the  bees  of  a  diseased  colony  attempt  to  supersede  their  queen,  but 
the  larvte   in   the   queen   cells   often   die,   leaving  the  colony   hopelessly   queenless.     The 
colony  is  thus  depleted  very  rapidly. 
442 


12  TEEATMENT    OF    BEE    DISEASES. 

Tlie  So-Called  "  Pickle  Brood." 

Id  addition  to  the  two  infectious  diseases  just  described,  brood 
dead  from  other  causes  is  often  observed.  The  most  common  disease 
of  this  kind  is  what  is  known  among  beekeepers  as  "  pickle  brood."' 
This  name  is  seemingly  applied  to  a  great  many  different  appear- 
ances and  nothing  is  known  of  the  cause  or  methods  of  spread.  The 
most  typical  form  kills  the  larva  when  it  has  extended  itself  in 
the  cell.  The  larva  usually  lies  on  its  back  with  the  head  turned 
upward.  The  color  varies,  but  is  frequently  light  yellow  or  brown, 
and  the  head  is  often  almost  black.  The  body  is  swollen  and  the 
contents  watery,  and  the  head  may  be  quite  hard.  There  is  no 
ropiness.  In  case  the  larva  are  sealed  before  djdng  the  cappings 
are  usually  normal.  The  name  usually  applied  to  this  condition  was 
unwisely  chosen,  and  for  the  present  and  until  more  is  known  con- 
cerning the  disease  it  is  sjDoken  of  as  the  "  so-called  pickle  brood." 

This  trouble  does  not  appear  to  be  infectious  and  is  usually  not 
serious,  except  that  in  the  aggregate  it  may  cause  loss  by  weakening 
colonies.  Xo  treatment  is  necessar}',  as  the  trouble  usually  soon  dis- 
appears. The  most  serious  aspect  of  this  disease  is  that  it  is  often 
mistaken  for  one  of  the  infectious  diseases,  and  the  colony  is  need- 
lessly treated. 

Brood  dead  of  other  causes. 

Many  different  external  factors  may  cause  brood  to  die.  If  brood 
is  killed  by  chilling  in  the  spring  or  fall,  or  by  overheating  in  ex- 
tremely hot  weather,  or  in  shipping  colonies  of  bees,  or  by  starvation, 
the  loss  is  often  mistaken^  attributed  to  an  infectious  disease.  Such 
dead  brood  is  soon  removed  by  the  bees.  When  the  cause  is  removed 
the  trouble  then  soon  disappears.  Allien  a  considerable  quantity  of 
brood  is  killed  a  disagreeable  odor  is  usually  present. 

"  Bald-headed  brood.'' 

It  sometimes  happens  that  unsealed  or  only  partially  sealed  pupae, 
known  as  "bald-headed  brood,''  are  observed  in  the  hive,  and  fre- 
quently beginners  mistake  such  a  condition  for  disease.  The  par- 
tially built  capping  is  often  mistaken  for  the  punctured  capping  of 
American  foul  brood.  If,  on  examination,  the  pupae  are  normal  no 
fear  need  be  entertained. 

METHODS  OF  SPREAD. 

Both  American  foul  brood  and  European  foul  brood  spread  from 
colony  to  colony  and  from  apiary  to  apiary  in  much  the  same  way. 
The  common  means  of  carrying  the  virus  is  in  honey  which  has  be- 
come contaminated.  The  disease  may  be  carried  when  bees  rob  a 
hive  in  which  a  colony  has  died  of  disease  or  may  be  transmitted  by 

442 


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TREATMENT   OF   BEE   DISEASES.  13 

the  use  of  honey  from  diseased  colonies  for  feeding  bees.  It  is  not 
ahvays  necessary  that  bees  be  intentionally  fed  for  them  to  get  dis- 
ease from  contaminated  honey.  Discarded  honey  receptacles  which 
have  contained  honey  from  a  contaminated  colony,  if  not  thoroughly 
cleaned,  may  contain  enough  honey  to  carry  disease  to  a  healthy 
I'.piary.  This  may  occur  in  the  vicinity  of  bakeries  or  confectionery 
shops,  or  may  even  occur  when  empty  honey  bottles  are  thrown  out 
from  private  houses.  It  is  also  possible  to  introduce  disease  into  a 
colony  in  introducing  queen  bees  purchased  from  a  distance,  probably 
due  to  the  use  of  contaminated  honey  in  making  the  candy  to  supply 
tlie  queen  cages. 

Precautionary   Measures. 

In  combating  diseases  it  is  much  better  to  prevent  disease  from 
getting  a  foothold  than  it  is  to  eradicate  it  after  it  has  begun  its 
work.  All  beekeepers,  wherever  located,  should  practice  the  fol- 
lowing precautionary  measures : 

(1)  If  a  colony  becomes  weak  from  any  cause,  or  if  disease  is 
suspected,  contract  the  entrance  to  prevent  robbing,  and  if  robbing 
is  imminent  close  the  entrance  entirely. 

(2)  Never  feed  honey  purchased  on  the  open  market.  In  case  of 
doubt  as  to  the  source  of  honey  feed  sugar  sirup. 

(o)  If  within  the  range  of  possibility,  see  that  no  honey  that  comes 
from  diseased  apiaries  is  sold  in  the  neighborhood.  This  may  some- 
times be  accomplished  by  cultivating  the  home  market  so  that  there 
will  be  no  incentive  for  bringing  in  other  honey. 

(4)  In  introducing  purchased  queens,  transfer  them  to  clean  cages 
provided  with  candy  known  to  be  free  from  contamination,  and 
destroy  the  old  cage,  candy,  and  accompanying  Avorkers.  Of  course, 
if  it  is  certain  that  the  queen  comes  from  a  healthy  apiary  this  is  not 
necessary. 

(5)  Colonies  of  bees  should  never  be  purchased  unless  it  is  cer- 
tain that  they  are  free  from  disease. 

(6)  The  purchase  of  old  combs  or  second-hand  supplies  is  dan- 
gerous, unless  it  is  certain  that  they  came  from  healthy  apiaries. 

TREATMENT   FOR  BOTH   INFECTIOUS  DISEASES. 

The  treatment  of  an  infectious  bee  disease  consists  primarily  in  the 
elimination  or  removal  of  the  cause  of  the  disease.  It  is  definitely 
known  that  American  foul  brood  is  caused  by  a  bacillus  named 
Bacillus  larvce.  In  treating  this  disease,  therefore,  the  aim  of  the 
manipulation  is  to  remove  or  destroy  all  of  the  bacteria  of  this 
species.  It  should  be  remembered  that  the  effort  is  not  to  save  the 
larvse  that  are  already  dead  or  dying,  but  to  stop  the  further  de- 

442 


14  TREATMENT    OP   BEE   DISEASES. 

vastatioii  of  the  disease  by  removing  all  material  capable  of  trans- 
mitting the  cause  of  the  trouble. 

The  cause  of  European  foul  brood  is  not  definitely  known,  but  the 
same  principles  of  treatment  doubtless  apply  in  this  disease  also. 
In  all  of  the  operations  great  pains  should  be  taken  not  to  spread  the 
disease  through  carelessness.  After  handling  a  diseased  colony  the 
hands  of  the  operator  should  be  washed  with  water  to  remove  any 
honey  that  may  be  on  them.  It  does  not  pay  to  treat  colonies  that 
are  considerably  weakened  by  disease.  In  case  there  are  several 
such  colonies  they  should  be  united  to  form  strong,  vigorous  colonies 
before  or  during  treatment. 

In  discussing  treatment  it  is  assumed  that  hives  with  movable 

frames  are  in  use.     Box  hives  are  a  menace  in  regions  where  disease 

is  present.     These  may  be  treated  for  disease  by  drumming  the  colony 

into  another  box  and  then  hiving  it  like  a  SAvarm  in  a  hive,  but  box 

hives  are  not  profitable  and  are  especially  to  be  condemned  where 

disease  is   present  on  account  of  the  difficulty   in  inspecting  and 

treating. 

Shaking  Treatment. 

The  shaking  treatment  consists  essentially  in  the  removal  of  all 
infected  material  from  the  colony,  and  in  compelling  the  colony  to 
take  a  fresh  start  by  building  new  combs  and  gathering  fresh  stores. 
This  is  done  by  shaking  the  bees  from  the  old  combs  into  a  clean  hive 
on  clean  frames. 

Time  of  treatment. — The  shaking  treatment  should  be  given  during 
a  flow  of  honey,  so  that  other  bees  in  the  apiary  will  not  be  inclined 
to  rob.  If  this  is  not  possible  the  operation  may  be  performed  under 
a  tent  made  of  mosquito  netting.  The  best  time  is  during  the  middle 
of  a  clear  day  when  a  large  number  of  bees  are  in  the  field.  It  is 
sometimes  recommended  that  shaking  be  done  in  the  evening,  but 
this  is  impossible  if  many  colonies  are  to  be  treated.  The  colony 
can  be  handled  more  quickly  when  the  field  force  is  out  of  the  hive. 

Preparation. — All  implements  that  will  be  needed,  such  as  queen 
and  drone  trap,  hive  tool,  and  lighted  smoker,  should  be  in  readiness 
before  the  operation  is  begun.  A  complete  clean  hive  with  frames 
is  provided,  as  well  as  a  tightly  closed  hive  body  in  which  to  put  the 
contaminated  combs  after  shaking.  An  extra  hive  cover  or  some 
similar  apparatus  should  be  provided  to  serve  as  a  runway  for  the 
bees  as  they  enter  the  new  hive.  The  new  frames  should  contain 
strips  of  comb  foundation  from  one-fourth  to  1  inch  wide.  Full 
sheets  are  not  desirable,  and  if  combs  built  on  full  sheets  of  founda- 
tion are  desired  they  may  be  built  later. 

Operation.— The  old  hive  containing  the  diseased  colony  (fig.  6,  .4) 
is  now  lifted  to  one  side  out  of  the  flight  of  returning  field  bees 
and  the  clean  hive  (B)  set  exactly  in  its  place.     The  cover  ((?)  is 

442 


TREATMENT   OP   BEE   DISEASES.  15 

now  taken  off  and  a  few  frames  {E)  removed  from  the  center  of  the 
hive.  If  unspaced  frames  are  used,  those  remaining  in  the  hive 
should  be  pushed  tightly  to  either  side  of  the  hive,  thus  making  a 
barrier  beyond  which  the  bees  can  not  crawl  as  they  move  to  the  top 
of  the  hive  after  shaking.  This  largely  prevents  them  from  getting 
on  the  outside  of  the  hive.  If  self-spacing  frames  are  used,  a  couple 
of  thin  boards  laid  on  the  top  bars  on  either  side  will  accomplish  the 
same  result.  The  runway  {D)  is  put  in  place  in  front  of  the  entrance. 
The  old  hive  is  now  opened  for  the  first  time.  The  frames  are 
removed  one  at  a  time,  lowered  part  way  into  the  new  hive,  and  with 
a  quick  downward  shake  the  bees  are  dislodged.  The  frames  are 
then  put  into  the  extra  hive  body  (C)  and  immediately  covered  to 
prevent  robbing.  After  all  the  frames  are  shaken  the  bees  remaining 
on  the  sides  of  the  old  hive  {A)  are  shaken  out. 


Fig.  6. — ^Apparatus  for  the  shaking  treatment :  A,  Hive  containing  diseased  colony  (for- 
merly in  position  of  B)  ;  B,  clean  hive  ;  G,  empty  hive  to  receive  combs  after  shaking ; 
D,  hive  cover  used  as  runway ;  E,  frames  removed  from  B  to  give  room  for  shaking ; 
Ft  queen  and  drone  trap;  0-,  cover  tor  clean  hive,  B.     (Original.) 

If  honey  is  coming  in  freely,  so  that  thin  honey  is  shaken  out  of 
the  combs,  cover  the  runway  (Z>)  with  newspapers  and  shake  the  bees 
in  front  of  the  new  hive  {B),  leaving  all  frames  in  place  and  the 
cover  on.  After  the  operation  the  soiled  newspapers  should  be  de- 
stroyed. In  shaking  in  front  of  the  entrance  the  first  one  or  two 
frames  should  be  so  shaken  that  the  bees  are  thrown  again,  t  the 
entrance,  where  they  can  locate  the  hive  quickly.  They  thei  fan 
their  wings  and.  the  others  follow  them  into  the  hive.  If  this  is 
not  done  the  bees  may  wander  about  and  get  under  the  hive  or  in 
some  other  undesirable  place. 

After  the  bees  are  mostly  in  the  new  hive  a  queen  and  drone  trap 
{F)  or  a  strip  of  perforated  zinc  is  placed  over  the  entrance  to 
prevent  the  colony  from  deserting  the  hive.  The  queen  can  not 
pass  through  the  openings  in  the  perforated  zinc  and  the  workers 
will  not  leave  without  her.  By  the  time  that  new  combs  are  built 
and  new  brood  is  ready  to  be  fed,  any  contaminated  honey  carried 
by  the  bees  into  their  new  hive  will  have  been  consumed  and  the 

442 


16 


TREATMENT    OF    BEE    DISEASES. 


disease  will  rarely  reappear.    If  it  should,  a  repetition  of  the  treat- 
ment will  be  necessary. 

Saving  the  healthy  brood. — The  old  combs  are  now  quickly  removed. 
If  several  colonies  are  being  treated  at  one  time  it  may  pay  to  stack 
several  hive  bodies  containing  contaminated  combs  over  a  weak 
diseased  colony  to  allow  most  of  the  healthy  brood  to  emerge,  thereby 
strengthening  the  weak  colony.  After  10  or  12  days  this  colony  is 
treated  in  turn  and  all  the  combs  rendered  into  wax.  If  only  one 
or  two  colonies  in  a  large  ajjiary  are  being  treated  it  will  not  pay  fo 
do  this. 

Saving  the  wax. — Any  but  a  very  small  apiary  should  have  in- 
cluded in  its  equipment  a  wax  press  for  removing  wax  from  old 
combs.     After  the  contaminated  frames  are  taken  to  the  honey  house 

the  combs  should  be  kept  carefullj' 
covered,  so  that  no  bees  can  reach 
them  until  the  Avax  can  be  ren- 
dered. This  should  not  be  de- 
layed Aery  long  or  the  comb^  may 
be  ruined  by  wax  moths.  The 
slumgum  or  refuse  remaining 
after  the  wax  is  removed  should 
be  burned.  Contaminated  combs 
should  not  be  put  into  a  solar  wax 
extractor  for  fear  of  spreading  the 
dis^^ase.  The  wax  from  contami- 
nated combs  may  safely  be  used 
for  the  manufacture  of  comb 
foundation. 

Cleaning    the    hive. — The     hive 

which  has  contained  the  diseased 

(Origiaai.,        ^^^j^^^^.      ^j^^^j^j      ^^      thoroughly 

cleaned  of  all  wax  and  honey,  and  it  is  desirable  that  it  be  care- 
fully disinfected  by  burning  out  the  inside  with  a  gasoline  blue- 
flame  torch  (fig.  T).  If  this  piece  of  apparatus  is  not  available 
several  hive  bodies  ma}'  be  piled  together  on  a  hive  bottom  and 
some  gasoline  or  kerosene  poured  on  the  sides  and  on  some  straw 
or  excelsior  at  the  bottom.  This  is  then  ignited  and  after  burn- 
ing for  a  few  seconds  a  close-fitting  hive  cover  is  placed  on  top 
of  the  pile  to  extinguish  the  flames.  The  inside  of  the  hive  bodies 
should  be  charred  to  a  light  brown.  The  careful  cleaning  and  dis- 
infection of  frames  always  costs  considerably  more  in  labor  than 
new  frames  would  cost,  but  these  also  may  be  carefully  cleaned  and 
used  again.  Frames  may  be  cleaned  by  boiling  in  water  for  about 
half  an  hour,  but  this  frequently  causes  them  to  warp  badly.  The 
disinfection  of  hives  and  frames  with  chemicals  is  not  recommended, 
as  the  ordinary  strengths  used  are  valueless  for  the  purpose. 

442 


Fig. 


-Gasoline   torch. 


TREATMENT  OP  BEE  DISEASES.  l7 

Disposal  of  the  honey. — If  there  is  a  considerable  quantity  of  honey 
m  the  contaminated  combs  it  may  be  extracted.  This  honey  is  not 
safe  to  feed  to  bees  without  boiliii"-,  but  it  is  absolutely  safe  for  human 
consumption.  If  there  is  a  comparatively  small  quantity  it  may  be 
consimied  in  the  beekeeper's  family,  care  being  taken  that  none  of  it 
is  placed  so  that  the  bees  can  ever  get  it. 

To  put  such  honey  on  the  market  is  contrary  to  law  in  some  States. 
There  is  always  danger  that  an  emptied  receptacle  will  be  thrown 
out  where  bees  can  have  access  to  it,  thus  causing  a  new  outbreak  of 
disease.  It  can  be  safely  used  for  feeding  to  bees,  provided  it  is 
diluted  with  at  least  an  equal  volume  of  water  to  prevent  burning, 
and  boiled  in  a  closed  vessel  for  not  less  than  one-half  hour,  count- 
ing from  the  time  that  the  diluted  honey  first  boils  vigorously.  The 
honey  will  not  be  sterilized  if  it  is  heated  in  a  vessel  set  inside  of 
another  containing  boiling  water.  Boiled  honey  can  not  be  sold  as 
honey.  It  is  good  only  as  a  food  for  bees,  and  even  then  should 
never  be  used  for  winter  stores,  as  it  will  probably  cause  dysentery. 

The  second  shake. — Some  beekeepers  prefer  to  shake  the  bees  first 
onto  frames  containing  strips  of  foundation  as  above  described,  and 
in  four  dajs  to  shake  the  colony  a  second  time  onto  full  sheets  of 
foundation,  destroying  all  comb  built  after  the  first  treatment. 
This  insures  better  combs  than  the  use  of  strips  of  foundation,  but  is 
a  severe  drOjin  on  the  strength  of  the  colony.  Since  it  is  desirable  to 
have  combs  built  on  full  sheets,  the  best  policy  is  to  replace  any  ir- 
regular combs  with  full  sheets  of  foundation  or  good  combs  later  in 
the  season. 

The  cost  of  shaking. — If  the  treatment  just  described  is  given  at  the 
beginning  of  a  good  honey  flow,  it  is  practically  equivalent  to  arti- 
ficial swarming  and  results  in  an  actual  increase  in  the  surplus  honey, 
especially  in  the  case  of  comb-honey  production.  The  wax  rendered 
from  the  combs  will  sell  for  enough  to  pay  for  the  foundation  used 
if  full  sheets  of  foundation  are  employed.  Since  a  colony  so  treated 
actually  appears  to  work  with  greater  vigor  than  a  colony  not  so 
manipulated,  the  cost  of  treatment  is  small.  If  treatment  must  be 
given  at  some  other  time,  so  that  the  colony  must  be  fed,  the  cost  is 
materially  increased.  In  feeding,  it  is  best  to  use  sugar  sirup,  or 
honey  that  is  known  to  have  come  from  healthy  colonies. 

Treatment  with.  Bee  Escape. 

As  a  substitute  for  the  shaking  treatment  just  described,  the  bees 
may  be  removed  from  their  old  combs  by  means  of  a  bee  escape.  The 
old  hive  is  moved  to  one  side  and  in  its  place  is  set  a  clean  hive  with 
clean  frames  and  foundation.  The  queen  is  at  once  transferred  to 
the  new  hive  and  the  field  bees  fly  there  on  their  return  from  the 

442 


18  TREATMENT  OF  BEE  DISEASES. 

field.  The  infected  hive  is  now  placed  on  top  of  or  close  beside  the 
clean  hive  and  a  bee  escape  placed  over  the  entrance,  so  that  the 
younger  bees  and  those  which  later  emerge  from  the  cells  may  leave 
the  contaminated  hive  but  can  not  return.  They  therefore  join  the 
colony  in  the  new  hive.  If  desired,  the  infected  hive  may  be  placed 
above  the  clean  hive  and  a  tin  tube  about  1  inch  in  diameter  placed 
from  the  old  entrance  so  that  the  lower  end  is  just  above  the  open 
entrance  of  the  new  hive.  The  bees  follow  down  this  tube  and  on 
their  return  enter  the  new  hive.  When  all  of  the  healthy  brood  has 
emerged  from  the  infected  combs  the  old  hive  is  removed.  This 
treatment  induces  less  excitement  in  the  apiary  and  is  preferred  by 
many  experienced  beekeeperb.  Care  should  be  taken  that  the  old 
hive  is  absolutelj-  tight  to  prevent  robbing.  The  old  hive  and  its 
contents  of  honej'  and  wax  are  treated  as  indicated  under  the  shak- 
ing treatment. 

Fall  Treatment. 

If  it  is  necessary  to  treat  a  colony  so  late  in  the  fall  that  it  would 
be  impossible  for  the  bees  to  prepare  for  winter,  the  treatment  may 
be  modified  by  shaking  the  bees  onto  combs  entirely  full  of  honey 
so  that  there  is  no  place  for  any  brood  to  be  reared.  This  will  usually 
be  satisfactory  only  after  brood  rearing  has  entirely  ceased.  Unless 
a  colon}'  is  (juite  strong  it  does  not  paj'  to  treat  in  the  fall,  but  it 
should  be  destroyed  or  united  to  another  colony.  In  case  a  diseased 
colony  dies  outdoors  in  the  winter  there  is  danger  that  other  bees 
may  have  opportunity  to  rob  the  hive  before  the  beekeepers  can  close 
the  entrance.  In  case  bees  are  wintered  in  the  cellar  it  is  more  ad- 
visable to  risk  wintering  before  treatment,  for  if  the  colony  does  die 
the  hive  will  not  ^3e  robbed. 

Drugs. 

Many  European  writers  have  in  the  past  advocated  the  use  of 
various  drugs  for  feeding,  in  sugar  sirup,  to  diseased  colonies,  or  the 
fumigation  of  contaminated  combs.  In  the  case  of  American  foul 
brood,  of  which  the  cause  is  known,  it  has  been  found  that  the  drugs 
recommended  are  not  of  the  slightest  value  and  no  time  should  be 
wasted  in  their  use. 

TREATMENT  FOR  EUROPEAN  FOUL  BROOD. 

European  foul  brood  is  a  very  peculiar  disease  and  its  cause  has 
not  yet  been  satisfactorily  determined.  It  is,  therefore,  impossible  to 
discuss  the  treatment  of  this  disease  as  definitely  as  that  of  American 
foul  brood.  From  the  experience  of  many  careful  beekeepers  it  is, 
however,  possible  to  suggest  some  additional  manipulations  which 
may  be  tried  by  experienced  beekeepers.  The  treatments  given  pre- 
viously are  strongly  recommended  for  this  disease. 

442 


TREATMENT  OF   BEE  DISEASES.  19 

Introduction  of  Italian  Stock. 

Since,  as  stated  previously  (p.  11),  Italian  bees  seem  to  be  better 
able  to  withstand  European  foul  brood  than  are  other  races,  it  is 
recommended  that  apiaries  in  rejiions  where  this  diseas^e  is  prevalent 
be  requeened  with  young,  vigorous  Italian  queens  of  good  stock. 
This  should  be  done  whether  or  not  the  shaking  treatment  is  given. 

Dequeening. 

It  has  been  found  that  tlie  removal  of  the  queen  and  the  keeping 
of  the  colony  queenless  for  a  period  often  results  in  the  disappearance 
of  European  foul  brood.  The  length  of  time  that  this  should  be  done 
is  in  dispute.  ^Ir.  E.  "W  Alexander,  who  advocated  this  method,^ 
recommended  that  the  colony  be  kept  queenless  (by  cutting  out  all 
queen  cells  at  the  end  of  9  days)  for  a  period  of  20  days,  at  which 
time  a  cell  containing  a  queen  of  Italian  stock  ready  to  emerge  is  to 
be  given  the  colony.  The  young  queen  will  thus  begin  to  laj'  in  about 
27  daj's  after  the  old  queen  has  been  removed,  or  in  at  least  3  days 
after  the  last  of  the  drone  brood  has  emerged.  Other  writers  have 
advocated  a  shorter  time. 

The  dequeening  treatment  is  not  alwaj's  successful,  and  it  is  there- 
fore recommended  that  care  be  exercised  in  trying  it.  Since  there  is 
a  considerable  percentage  of  successful  results,  this  would  indicate 
that  there  is  an  important  princijDle  involved.  It  should  not  be  for- 
gotten, however,  that  European  foul  brood  often  disappears  in  the 
late  summer  of  its  own  accord  if  the  case  is  not  severe  (p.  11),  and  it 
is  probable  that  in  many  of  the  cases  of  dequeening  reported  as  suc- 
cessful the  disease  would  have  disappeared  without  the  treatment. 
This  treatment  is  suggested  only  for  the  experienced  beekeeper. 

IWSPECTION   OF   APIAKIES. 

Several  States  have  passed  laws  providing  for  the  inspection  of 
apiaries  for  contagious  disease  and  creating  the  office  of  apiary 
inspector.  The  men  holding  these  offices  are  usually  practical  bee- 
keepers, capable  of  giving  excellent  advice  regarding  disease,  and 
it  is  desirable,  when  disease  exists  in  a  community,  that  the  owners 
of  apiaries  take  steps  to  learn  who  the  inspector  is  and  to  notify 
him  of  the  existence  of  disease.  The  Bureau  of  Entomology  of  this 
department  can  usually  give  information  concerning  the  inspector 
and  is  always  glad  to  be  of  service  in  bringing  the  beekeepers  and 
inspectors  in  touch  with  one  another. 

Apiary  inspection  has  proved  beneficial  to  the  beekeeping  industry 
in  spreading  information   concerning  the   nature,   symptoms,   and 

^  Alexander,  E.  W. — How  to  rid  your  apiary  of  black  brood.     Gleanings  in  Bee  Culture, 
vol.  33,  pp.  1123-1127.  1905. 
442 


20  TKEATMEXT   OF   BEE   DISEASES. 

treatment  of  the  contagious  diseases  and  particularly  in  compelling 
negligent  and  careless  beekeepers  to  treat  their  diseased  colonies.  It 
is  quite  possible  for  the  individual  beekeeper  to  clean  up  his  own 
apiary  by  following  the  directions  given  in  this  bulletin,  but  unless 
all  of  the  beekeepers  in  the  neighborhood  do  the  same  thing  there 
will  probably  be  a  recurrence  of  the  trouble  due  to  infection  from 
outside  apiaries.  It  is  therefore  manifestly  to  the  advantage  of  the 
beekeepers  that  they  cooperate  with  the  inspectors  in  the  fight  against 
diseases.  1 

EXAMINATION  OP   SAMPLES   OP  DISEASED   BROOD. 

The  Bureau  of  Entomology  of  this  department  is  prepared  to 
assist  in  the  diagnosis  of  disease  in  cases  where  the  beekeeper  is 
unable  to  tell  whether  or  not  disease  is  present,  or  to  determine  which 
disease  is  in  his  apiary.  Samples  of  brood  comb  about  5  inches 
square  containing  diseased  or  dead  larvae  should  be  sent  by  mail 
in  a  strong  wooden  or  tin  box.  The  comb  should  not  be  wrapped  in 
paper  or  cotton,  but  should  be  cut  to  fit  the  box  closelj'.  It  is  not 
possible  to  diagnose  from  empty  combs,  and  no  honey  should  be 
included  in  the  sample,  as  it  is  valueless  in  diagnosis  and  will  prob- 
ably spoil  the  sample  as  well  as  other  mail  matter.  The  name  of  the 
sender  must  always  appear  on  the  package,  and  any  available  data 
should  be  sent  in  a  separate  letter.  Xever  inclose  a  letter  in  the  box 
with  the  sample. 

THE  DISEASES  OF  ADULT  BEES. 

The  diseases  affecting  adult  bees  are  but  imperfectly  known.  At 
present  four  are  known  to  beekeepers  by  name.  "\A'liether  these  are 
entirely  distinct  or  whether  mider  each  name  one  or  more  diseases  are 
included  is  not  known.  As  stated  in  the  introduction,  these  diseases 
have  not  been  sufficiently  investigated  to  give  much  help  to  the 
practical  beekeeper. 

DYSENTERY. 

Dysentery  affects  bees  only  in  the  winter  and  is  manifested  by  a 
distension  of  the  abdomen,  due  to  an  accumulation  of  fecal  matter 
in  the  intestine.  When  a  day  warm  enough  for  flight  occurs  the 
bees  fly  from  the  hive  to  cleanse  themselves,  and  the  hive  and  sur- 
roundings are  spotted  with  yellow  excreta.  After  a  good  cleansing 
flight  the  trouble  usually  disappears,  but  if  the  bees  are  unable  to  fly 
they  often  die  in  great  numbers.  It  is  generally  believed  that  dysen- 
tery is  due  to  improper  winter  stores,  the  honey  containing-  too  high 
a  percentage  of  indigestible  matter.  Honeydew  honey  almost  always 
produces  dysentery,  while  bees  wintered  on  high-class  honey  or  sugar 
sirup   are  not  affected.     From  the  wide  experience  of  many  bee- 

442 


TREATMENT   OF   BEE   DISEASES.  21 

keepers  in  this  matter  it  is  safe  to  assume  that  this  explanation  of 
the  disease  is  the  correct  one,  and  consequently  great  care  should 
be  exercised  that  the  colonies  are  provided  with  good  stores  for 
winter. 

Recently  it  has  been  claimed  that  there  are  two  types  of  dysentery, 
one  form  as  above  described  and  another  form  which  is  infectious. 
American  beekeepers  are  not  familiar  with  an  infectious  dysentery, 
and  in  practical  manipulations  it  is  necessary  to  consider  only  the 
type  above  described. 

THE   SO-CALLED  PARALYSIS. 

It  is  quite  possible  that  under  the  name  "paralysis"  are  included 
several  distinct  diseases.  This  is  indicated  by  the  variety  of  symp- 
toms reported  by  beekeepers  and  the  number  of  different  seasons  and 
conditions  under  which  the  disease  is  supposed  to  occur.  The  usual 
manifestation  described  is  that  the  worker  bees  are  seen  crawling  in 
front  of  the  hive  with  their  abdomens  trembling.  The  abdomens 
are  also  frequently  distended.  The  bees  often  climb  grass  blades 
and  on  attempting  to  fly  from  the  top  fall  again  to  the  ground. 
Frequently  the  bees  so  affected  are  almost  hairless.  The  same  trem- 
bling motion  may  often  be  observed  on  opening  the  hive.  The  colony 
is  often  depleted  very  rapidly.  There  is  no  evidence  that  the  disease 
is  infectious. 

The  cause  of  this  peculiar  trouble  is  unknown,  and  no  remedy  can 
be  recommended.  It  is  claimed  by  some  writers  that  a  salt-water 
spray  applied  to  the  combs  or  salt  or  sulphur  sprinkled  on  the  top 
bars  or  entrance  is  sometimes  an  effective  remedy. 

ISLE  OF  WIGHT  DISEASE. 

Recently  a  supposedly  infectious  disease  of  adult  bees  has  deci- 
mated the  bees  on  the  Isle  of  Wight  and  is  said  to  be  spreading  in 
England.  It  resembles  somewhat  the  so-called  paralysis.  No  treat- 
ment other  than  destruction  to  prevent  the  spread  of  the  disease  has 
been  recommended.  So  far  as  is  known  no  trouble  of  this  kind  has 
been  experienced  in  America. 

SPRIN,G  DWINDLING. 

It  sometimes  happens  that  the  adult  bees  in  a  colony  die  off  in  the 
spring  more  rapidly  than  they  are  replaced  by  emerging  brood. 
This  dwindling  may  be  diminished  somewhat  by  keeping  the  colony 
warm  and  by  stimulative  feeding,  so  that  all  of  the  energy  of  the 
old  bees  may  be  used  to  the  best  advantage.  This  condition  is  prob- 
ably due  to  the  fact  that  the  colony  goes  into  winter  with  too  large 
a  percentage  of  old  worn-out  bees.  To  prevent  this,  brood  rearing 
should  be  continued  as  late  as  possible  in  the  fall;  if  necessary,  by 
stimulative  feeding. 

442 


22  TREATMENT   OF   BEE   DISEASES. 

PUBLICATIONS  OF  THE  DEPARTMENT  OF  AGRICULTURE  ON  BEE 

DISEASES. 

There  are  several  other  publications  of  the  Bureau  of  Entomology 
of  this  department  which  deal  with  bee  diseases.  They  may  be 
obtained  on  request  to  the  Editor  and  Chief  of  the  Division  of  Pub- 
lications, Department  of  Agriculture,  and  are  the  following: 

Circular  Xo.  94,  "  The  Ciiuse  of  American  Foul  Brood."    By  G.  F.  White,  Ph.  D. 
1907.     1  pp. 

This  publication  contains  a  brief  account  of  the  investigations  which  demonstrated 
for  the  first  time  the  cause  of  one  of  the  brood  diseases  of  bees,  American  foul  brood. 

Bulletin  No.  70.  "  Report  of  the  Meeting  of  Inspectors  of  Apiaries,  San  Antonio, 
Tex.,  Xovember  12,  190Li."     19U7.     79  pp.,  1  pi. 

Contains  an  account  of  tlie  history  of  bee-disease  investigations,  the  relationship  of 
bacteria  to  bee  diseases,  and  a  discussion  of  treatment  by  various  inspectors  of  apiaries 
and  other  practical  beelieepers  who  are  familiar  with  diseases  of  bees. 

Bulletin  Xo.  75.  Part  II.  "Wax  ^Motbs  and  American  Foul  Brood."     By  E.  F. 
Phillips,  Ph.  D.     1907.     Pp.  19-212,  3  pis. 

An  account  of  the  behavior  of  tbo  two  species  of  wax  moths  on  combs  containing 
American  foul  brood,  showing  that  moths  do  not  clean  up  the  disease-carrying  scales. 

Bulletin  Xo.   7o,  Part  III,   "  Bee  Diseases  in   Massachusetts."     By  Burton  X. 
Gates.     1908.     Pp.  23-32,  map. 

.\n  account  of  the  distribution  of  the  brood  diseases  of  bees  in  the  State,  with  brief 
directions  for  controlling  them. 

Bulletin  Xd.  7."j.  I'art  1\'.  "The  Kelation  of  the  Etiology   (Cause)  of  Bee  Dis- 
eases to  the  Treatment."     By  G.  F.  White,  Ph.  D.     190S.     Pp.  33-42. 

The  necessity  for  a  linowledge  of  the  causes  of  bee  diseases  before  rational  treatment 
is  possible  is  pointed  out.  The  present  state  of  linowlodge  of  tlie  causes  of  disease  is 
summarized. 

Technical  Scries.  Xo.  14,  "  The  Bacteria  of  the  Apiary,  with  Spoc-ial  Reference 
to  Bee  Diseases."     By  G.  F.  White.  Ph.  D.     19(ir,.     ."O  pp. 

A  study  of  the  bacteria  present  in  both  the  healthy  and  tlie   diseased  colony,  with 
special  reference  to  the  diseases  of  bees. 
442 


FARMERS'  BULLETINS. 

Bulletins  in  this  list  will  be  sent  free,  so  long  as  the  supply  lasts,  to  any  resident  of  the  United  States, 
on  application  to  his  Senator,  Representative,  or  Dologato  In  Congress,  or  to  the  Secretary  of  Agri- 
culture, Washington,  D.  C.  Because  of  the  limitnl  supplif,  applicants  are  urged  to  select  onltj  a  few  num- 
(lers,  choosing  those  which  are  of  special  interest  to  them.  Residents  of  foreign  countries  shoulil  apply  to 
the  Superintendent  of  Documents,  Government  Printing  Office,  Washington,  D.  C,  who  has  these 
bulletins  for  sale.  Price  5  cents  each  to  Canada,  Cuba,  and  Mexico;  (i  cents  to  other  foreign  countries. 
The  bulletins  entitlect "  Experiment  Station  Work"  give  briefly  the  results  of  experiments  performed 
by  tbe  State  experiment  stations. 
22.  The  Feeding  of  Farm  Animals. 

27.  Flax  for  Seed  and  Fiber. 

28.  Weeds:  And  How  to  Kill  Them, 

80.  Grape  Diseases  on  the  Pacific  Coast. 
32.  Silos  and  Silage. 

34.  Meats:  Composition  and  Cooicing. 

35.  Potato  Culture. 

36.  Cotton  Seed  and  Its  Products. 
44.  Commercial  Fertilizers. 

48.  The  Manuring  of  Cotton. 

49.  Sheep  Feeding. 
61.  Standard  Varieties  of  Chickens. 
52.  Tbe  Sugar  Beet. 
54.  Some  Common  Birds. 

65.  The  Dairy  Herd. 

66.  Experiment  Station  Work— I. 

60.  Methods  of  Curing  Tobacco. 

61.  Asparagus  Culture. 

62.  Marketing  Farm  Produce. 

64.  Ducks  and  Geese. 

65.  E.xperiment  Station  Work— II. 
69.  Experiment  Station  Work- III. 
73.  Experiment  Station  Work— IV. 

77.  The  Liming  of  Soils. 

78.  Experiment  Station  Work— V. 

79.  Experiment  Station  Work— VI. 

81.  Corn  Culture  in  the  South. 

82.  The  Culture  of  Tobacco. 

83.  Tobacco  Soils. 

84.  Experiment  Station  Work— VII. 

85.  Fisn  as  Food. 

86.  Thirty  Poisonous  Plants. 

87.  Experiment  Station  Work— VIII. 

88.  Alkali  Lands. 

91.  Potato  Diseases  and  Treatment. 

92.  Experiment  Station  Work— IX. 

93.  Sugar  as  Food. 

96.  Raising  Sheep  for  Mutton. 

97.  Experiment  Station  Work — X. 
99.  Insect  Enemies  of  Shade  Trees. 

101.  Millets. 

103.  Experiment  Station  Work- XI. 

104.  Notes  on  Frost. 

105.  Experiment  Station  Work- XII. 

106.  Breeds  of  Dairy  Cattle. 

113.  The  Apple  and  How  to  Grow  It. 

114.  Experiment  Station  Work— XIV. 

118.  Grape  Growing  in  the  South. 

119.  Experiment  Station  Work— XV. 

120.  Insects  ASecting  Tobacco. 

121.  Beans,  Peas,  and  Other  Legumes  as  Food. 

122.  Experiment  Station  Work— XVI. 

126.  Practical  Suggestions  for  Farm  Buildings. 

127.  Important  Insecticides. 

128.  Eggs  and  Their  Uses  as  Food. 
131.  Household  Tests  for  Detection  of  Oleomar- 
garine and  Renovated  Butter. 

133.  Experiment  Station  Work- XVIII. 

134.  Tree  Planting  on  Rnral  School  Grounds. 

135.  Sorghum  Sirup  Manufacture. 

137.  The  Angora  Goat. 

138.  Irrigation  In  Field  and  Garden. 

139.  Emmer:  A  Grain  for  theSemiarid  Regions. 

140.  Pineapple  Growing. 
142.  Nutrition  and  Nutritive  Value  of  Food. 

144.  Experiment  Station  Work— XIX. 

145.  Carbon  Bisulphid  as  an  Insecticide. 

149.  Experiment  Station  Work — XX. 

150.  Clearing  New  Land. 
152.  Scabies  of  Cattle. 

'154.  Home  Fruit  Garden:  Preparation  and  Care. 

155.  How  Insects  Affect  Health  in  Rural  Districts. 

156.  The  Home  Vineyard. 

157.  The  Propagation  of  Plants. 

158.  How  to  Biuld  Small  Irrigation  Ditches. 
162.  Experiment  Station  Work— XXI. 
164.  Rape  as  a  Forage  Crop. 

166.  Cheese  Making  on  the  Farm. 

167.  Cassava. 

169.  Experiment  Station  Work— XXII. 

170.  Principles  of  Horse  Feeding. 
172.  Scale  Insects  and  Uites  on  Citrus  T^ees. 


(I) 


173.  Primer  of  Forestry.    Part  I:  The  Forest. 

174.  Broom  Corn. 

176.  Home  Manufacture  and  Use  of  Unfermented 
Grape  Juice. 

176.  Cranberry  Culture. 

177.  Squab  Raising. 

178.  Insects  Injurious  in  Cranberry  Culture. 

179.  Horseshoeing. 

181.  Pruning. 

182.  Poultry  as  Food. 

183.  Meat  on  the  Farm:  Butchering,  Curing,  etc. 

185.  Beautifying  the  Home  Grounds. 

186.  Experiment  Station  Work— XXIII. 

187.  Drainage  of  Farm  Lands. 

188.  Weeds  Used  in  Medicine. 

190.  Experiment  Station  Work— XXIV. 

192.  Barnyard  Manure. 

193.  Experiment  Station  Work— XXV. 

194.  Alfalfa  Seed. 
Annual  Flowering  Plants. 
Usefulness  of  the  American  Toad. 
Importation  of  Game  Birds  and  Eggs  for 

Propagation. 
Strawberries. 
Turkeys. 

Cream  Separator  on  Western  Farms. 
Experiment  Station  Work— XXVI. 
Canned  Fruits,  Preserves,  and  Jellies. 

204.  The  Cultivation  of  Mushrooms. 

205.  Pig  Management. 

206.  Milk  Fever  and  Its  Treatment. 

209.  Controlling  the  Boll  Weevil  in  Cotton  Seed 

and  at  Ginneries. 

210.  Experiment  Station  Work— XXVII. 
213.  Raspberries. 

218.  The  School  Garden. 

219.  Lessons  from  the  Grain  Rust  Epidemic  of  1904. 

220.  Tomatoes. 

221.  Fungous  Diseases  of  tbe  Cranberry. 

222.  Experiment  Station  Work— XXVIII. 

223.  Miscellaneous  Cotton  Insects  in  Texas. 

224.  Canadian  Field  Peas. 

225.  Experiment  Station  Work— XXIX. 

227.  Experiment  Station  Work— XXX. 

228.  Forest  Planting  and  Farm  Management. 

229.  The  Production  of  Good  Seed  Com. 

231.  Spraying  for  Cucumber  and  Melon  Diseases. 

232.  Okra:  Its  Culture  and  Uses. 

233.  Experiment  Station  Work— XXXI. 

234.  The  Guinea  Fowl. 

235.  Preparation  of  Cement  Concrete. 

236.  Incubation  and  Incubators. 

237.  Experiment  Station  Work— XXXII. 

238.  Citrus  Fruit  Growing  in  the  Gulf  States. 

239.  The  Corrosion  of  Fence  Wire. 

241.  Butter  Making  on  the  Farm. 

242.  An  Example  of  Model  Farming. 

243.  Fungicides  and  Their  Use  in  Preventing  Dis- 

eases of  Fruits. 

244.  Experiment  Station  Work— XXXIII. 

245.  Renovation  of  Worn-out  Soils. 

246.  Saccharine  Sorghums  for  Forage. 

248.  The  Lawn. 

249.  Cereal  Breakfast  Foods. 

250.  The  Prevention  of  Stinking  Smut  of  Wheat 

and  Loose  Smut  of  Oats. 

251.  Experiment  Station  Work— XXXIV 

252.  Maple  Sugar  and  Sirup. 

253.  The  Germination  of  Seed  Corn. 

254.  Cucumbers. 

255.  The  Home  Vegetable  Garden. 

256.  Preparation  of  Vegetables  for  the  Table. 

257.  Soil  Fertility. 

258.  Texas  or  Tick  Fever  and  Its  Prevention. 

259.  Experiment  Station  Work— XXXV. 

260.  Seed  of  Red  Clover  and  Its  Impurities. 

262.  Experiment  Station  Work— XXXVI. 

263.  Practical  Information  for  Beginners  in  Irri- 

gation. 

264.  The  Brown-tail  Moth  and  How  to  Control  It. 

266.  Management  of  Soils  to  Conserve  Moisture. 

267.  Experiment  Station  Work— XXXVII. 


II 


269, 
270, 
271, 

272, 
273. 
274, 
276, 
276. 
277. 
278. 
279. 
280. 
281. 
282. 
283. 

284. 


287, 
288, 
289, 
290, 
291. 
292. 
293. 
294. 
295. 
296. 
298. 
299. 

301. 
302. 


304. 
305. 
306. 
307. 
309. 
310. 
311. 
312. 
313. 
314. 

316. 
317. 
318. 
320. 
321. 
322. 
323. 

324. 
325. 
326. 
328. 
329. 


332. 
333. 
334. 
336. 


341. 
342. 
343. 


346. 
346. 

347. 
348. 
349. 
360. 
851. 
352. 
353. 


Industrial  Alcohol;  Uses  and  Statistics. 

Modern  Conveniences  for  the  Farm  Home. 

Forage  Crop  Practices  in  Western  Oregon 
and  Western  Washington. 

A  Successful  Hog  and  Seed-corn  Farm. 

Experiment  Station  Work— XXXVIII. 

Flax  Culture. 

The  Gipsy  Moth  and  How  to  Control  It. 

Experiment  Station  Work— XXXIX. 

Alcohol  and  Gasoline  in  Farm  Engines. 

Leguminous  Crops  for  Green  Manuring. 

A  Method  of  Eradicating  Johnson  Grass. 

A  Profitable  Tenant  Dairy  Farm. 

Experiment  Station  Work — XL. 

Celery. 

Spraying  for  Apple  Diseases  and  the  Codling 
Moth  in  the  Ozarks. 

Insect  and  Fungous  Enemies  of  the  Grape 
East  of  the  Rocky  Mountains. 

Comparative  Value  of  Whole  Cotton  Seed 
and  Cotton-seed  Meal  in  Fertilizing  Cotton. 

Poultry  Management. 

Nonsaccharine  Sorghums. 

Beans. 

The  Cotton  BoUworm. 

Evaporation  of  Apples. 

Cost  of  Filling  Silos. 

Use  of  Fruit  as  Food. 

Farm  Practice  in  Columbia  Basin  Uplands. 

Potatoes  and  Other  Root  Crops  as  Food. 

Experiment  Station  Work — XLI. 

Food  Value  of  Corn  and  Corn  Products. 

Diversified  Farming  Under  the  Plantation 
System. 

Home-grown  Tea. 

Sea  Island  -Cotton:  Its  Culture,  Improve- 
ment, aUd  Diseases. 

Corn  Harvesting  Machinery. 

Growing  and  Curing  Hops. 

Experiment  Station  Work — XLII. 

Dodder  in  Relation  to  Farm  Seeds. 

Roselle:  Its  Culture  and  Uses. 

Experiment  Station  Work — XLIII. 

A  Successful  Alabama  Diversification  Farm. 

Sand-clay  and  Burnt-clay  Roads. 

A  Successful  Southern  Hay  Farm. 

Harvesting  and  Storing  Corn. 

A  Method  of  Breeding  Early  Cotton  to  Es- 
cape Boll-weevil  Damage. 

Experiment  Station  Work— XLIV. 

Experiment  Station  Work— XLV. 

Cowpeas. 

Experiment  Station  Work — XLVI. 

The  Use  of  the  Split-log  Dragon  Earth  Roads. 

Milo  as  a  Dry-land  Grain  Crop. 

Clover  Farming  on  the  Sandy  Jack-pine 
Lands  of  the  North. 

Sweet  Potatoes. 

Small  Farms  in  the  Com  Belt, 

Building  Up  a  Run-down  Cotton  Plantation. 

Silver  Fox  Farming. 

Experiment  Station  Work— XLVII. 

Deer  Farming  in  the  United  States. 

Forage  Crops  for  Hogs  in  Kansas  and  Okla- 
homa. 

Nuts  and  Their  Uses  as  Food. 

Cotton  Wilt. 

Experiment  Station  Work— XLVIII. 

Harmful  and  Beneficial  Mammals  of  the 
Arid  Interior.' 

Cropping  Systems  lor  New  England  Dairy 
Farms. 

Macadam  Roads. 

Alfalfa 

The  Basket  Willow. 

Experiment  Station  Work- XLIX. 

The  Cultivation  of  Tobacco  in  Kentucky 
and  Tennessee. 

The  Boll  Weevil  Problem,  with  Special  Refer- 
ence to  Means  of  Reducing  Damage. 

Some  Common  Disinfectants. 

The  Computation  of  Rations  for  Farm  Ani- 
mals by  the  Use  of  Energy  Values. 

The  Repair  of  Farm  Equipment. 

Bacteria  in  Milk. 

The  Dairy  Industry  in  the  South. 

The  Dehorning  of  Cattle. 

TheTubereulinTestofCattleforTuberculosis 

The  Nevada  Mouse  Plague  of  1907-8. 

Experiment  Station  Work— L. 


364.  Onion  Culture. 

355.  A  Successful  Poultry  and  Dairy  Farm. 

357.  Methodsof  Poultry  Management  at  theMaine 
Agricultural  Experiment  Station. 

368.  A  Primer  of  Forestry.  Partll:  Practical  For- 
estry. 

359.  Canning  Vegetables  in  the  Home. 

360.  Experiment  Station  Work— LI. 

361.  Meadow  Fescue:  Its  Culture  and  Uses. 

362.  Conditions  AflectingtheValueofMarketHay. 

363.  The  Use  of  Milk  as  Pood. 

364.  A  Profitable  Cotton  Farm. 

365.  Farm    Management   in    Northern    Potato- 

growing  Sections. 

366.  Experiment  Station  Work— LII. 

367.  Lightning  and  Lightning  Conductors. 

368.  The  Eradication  of  Bindweed,  or  Wild  Morn- 

ing-glory. 

369.  How  to  Destroy  Rata. 

370.  Replanning  a  Farm  for  Profit. 

371.  Drainage  of  Irrigated  Lands. 

372.  Soy  Beans. 

373.  Irrigation  of  Alfalfa. 

374.  Experiment  Station  Work — LIII. 

376.  Care  of  Food  in  the  Home. 

377.  Harmfulness  of  Headache  Mixtures. 

378.  Methods  of  Exterminating  Texas-fever  Tick. 

379.  Hog  Cholera. 

380.  The  Loco-weed  Disease. 

381.  Experiment  Station  Work— LIV. 

382.  The  Adulteration  of  Forage-plant  Seeds. 

383.  How  to  Destroy  English  Sparrows, 

384.  Experiment  Station  Work— LV. 

385.  Boys'  and  Girls' Agricultural  Clubs. 

386.  PotatoCultureon  Irrigated  Farmsof  the  West. 

387.  ThePreservativeTreatmentof  Farm  Timbers. 

388.  Experiment  Station  Work— LVI. 

389.  Bread  and  Bread  Making. 

390.  Pheasant  Raising  in  the  United  States. 

391.  Economical  Use  of  Meat  in  the  Home. 

392.  Irrigation  of  Sugar  Beets. 

393.  Habit-forming  Agents. 

394.  Windmills  in  Irrigation  in  Semiarid  West. 

395.  Sixty-day  and  Kherson  Oats. 

396.  The  Muskrat. 

397.  Bees. 

398.  Farm  Practice  in  the  Use  of  Commercial  Fer- 

tilizers in  the  South  Atlantic  States. 

399.  Irrigation  of  Grain . 

400.  A  More  Profitable  Corn-planting  Method. 

401.  Protection  of  Orchards  in  Northwest  from 

Spring  Fro.stH  by  Fires  and  Smudges. 

402.  Canada  Bluegrass;  Its  Culture  and  Uses. 

403.  The  Construction  of  Concrete  Pence  Posts. 

404.  Irrigation  of  Orchards. 

405.  Experiment  Station  Work— L VII. 

406.  Soil  Conservation. 

407.  The  Potato  as  a  Truck  Crop. 

408.  School  Exercises  in  Plant  Production. 

409.  School  Lessons  on  Corn. 

4)  0.  Potato  Culls  as  a  Sourceof  Industrial  AlcohoL 

411.  Feeding  Hogs  in  the  South. 

412.  Expeiiment  Station  Work— LVIII. 

413.  The  Care  of  Milk  and  Its  Use  in  the  Home. 

414.  Corn  Cultivation. 

415.  Seed  Corn. 

416.  Cigar-leaf  Tobacco  in  Pennsylvania. 

417.  Rice  Culture. 

418.  Game  Laws  for  1910. 

419.  Experiment  Station  Work— LIX. 

420.  Oats:  Distribution  and  Uses. 

421.  Control  of  Blowing  Soils. 

422.  Demonstration  Work  on  Southern  Farms. 

423.  Forest  Nurseries  for  Schools. 

424.  Oats:  Growing  the  Crop. 

125.  Experiment  Station  Work— LX. 

426.  Caiining  Peaches  on  the  Farm. 

427.  Barley  Culture  in  the  Southern  States. 

428.  Testing  Farm  Seeds  in  the  Home  and  in  the 

Rural  School. 

429.  IndustrialAlcohol:  SourcesandManufacture. 

430.  Experiment  Station  Work— LXI. 

431.  The  Peanut. 

432.  How  a  City  Family  Managed  a  Farm. 

433.  Cabbage. 

434.  The  Home  Production  of  Onion  Seed  and  Sets. 

435.  Experiment  Station  Work— LXII. 

436.  Winter  Oats  for  the  South. 

437.  A  Si-stem  of  Tenant  Farming  and  Its  Re- 

sults. 


o 


/ 


FARMERS'  BULLETIN  975 


THE  CONTROL  OF  EUROPEAN 
FOULBROOD 


E.  F.  PHILLIPS 

Apieulturist 


UNITED  STATES 
DEPARTMENT  OF  AGRICULTURE 


WASHINarON  :  GOVERNMENT  PRINTINQ  OEFICE  :  IflZl 


EUROPEAN  FOULBROOD  is  a  disease  of  the 
brood  of  bees  which  has  caused  great  losses  to 
American  beekeepers.  It  was  first  recognized  as  a 
distinct  disease  in  the  United  States  by  New  York 
beekeepers  in  1894,  but  it  has  probably  been  present 
in  the  United  States  for  a  long  ti'me. 

It  is  important  that  the  beekeeper  know  whether 
European  or  American  foulbrood  is  in  his  apiary,  for 
the  two  do  not  respond  to  the  same  treatment.  In 
European  foulbrood  control  the  most  important  step 
is  to  prevent  the  entrance  of  the  disease  by  keeping 
all  colonies  strong  and  by  having  all  stock  resistant 
to  the  disease.  This  can  be  done  successfully  even 
though  the  disease  is  in  the  neighborhood. 

In  case,  through  failure  to  take  all  precautions, 
the  disease  does  enter,  there  are  certain  practices  by 
which  the  disease  can  be  readily  eliminated,  but  all 
of  these  must  be  used  with  care. 

The  facts  about  the  disease  on  which  the  pre- 
ventive and  remedial  measures  are  based  are  dis- 
cussed in  this  bulletin. 


Contribution  from  the  Bureau  of  Entomology 

L.  O.  HOWAHD,  Chief 

Issued  July,  1918 
Washington,  D.  C.  Reprint  December,  1921 


THE  CONTROL  OF  EUROPEAN  FOULBROOD. 


CONTENTS. 


Page. 

Diffloulties  of  control 3 

Name  of  the  disease 3 

Symptoms 4 

Basis  of  treatment 7 


Pag». 

Preventive  measures 10 

Bemedial  measures 13 

Oood  beekeeping  will  eradicate  the  disease. . .       IS 


DIFFICULTIES  OF  CONTROL. 

EUEOPEAN  FOULBROOD  has  caused  much  trouble  in  treat- 
ment and  causes  more  anxiety  among  beekeepers  than  does 
American  f oulbrood.  It  is  recognized  generally  that  European  foul- 
brood  requires  less  drastic  methods  than  does  American  foulbrood, 
but  seemingly  one  cannot  always  be  so  sure  of  the  efficacy  of  the 
treatment,  and  it  is  often  said  by  beekeepers  that  European  foul- 
brood  "  does  not  fight  fair."  The  difficulty  seems  to  lie  in  the  fact 
that  the  course  of  the  disease  in  the  colony  has  not  been  sufficiently 
studied  and  the  features  of  treatment  have  not  been  adequately 
analyzed.  It  is  not  enough  simply  to  know  the  name  of  the  organ- 
ism which  causes  the  disease,  but  it  is  essential  to  know  the  habits  of 
the  germ  in  the  colony. 

European  foulbrood  was  first  recognized  in  New  York  State  in 
1894,  and  previous  to  that  time  no  adequate  diflferentiation  had  been 
made  between  this  disease  and  American  foulbrood.  Various  writers, 
especially  those  in  Europe,  had  recorded  two  types  of  brood  diseases 
and  had  differentiated  them  sufficiently  to  call  one  mild  and  the  other 
virulent.  Careful  observations  of  beekeepers,  as  well  as  bacteriologi- 
cal investigations,  have  shown  that  the  two  diseases  are  entirely  dis- 
tinct, that  one  does  not  change  to  the  other,  and  that  in  treatment 
they  behave  differently. 

Now  that  the  symptoms  of  the  two  diseases  have  been  carefully 
studied,  one  can  examine  the  earlier  literature  and  find  indications 
that  European  foulbrood  was  rather  widespread  in  the  United  States 
before  it  was  recognized  as  a  distinct  disease.  At  any  rate  it  appears 
certain  that  all  the  European  foulbrood  in  the  country  did  not 
spread  from  the  first  recognized  outbreak  in  New  York  State.  New 
York  beekeepers  with  justice  objected  to  the  name  "  New  York  bee 
disease  "  which  was  at  one  time  applied  to  the  disease. 

NAME  OF  THE  DISEASE. 

When  American  beekeepers  first  differentiated  this  disease  the 
name  "  black  brood  "  was  generally  applied  to  it.    When  the  investi- 

79121°— 21— Bull.  976  3 


4  FARMEES     BXJLLETIN   975. 

gation  of  bee-disease  control  was  inaugurated  by  the  Bureau  of  Ento- 
mology it  was  recognized  that  this  name  was  not  well  chosen,  for 
black  is  not  the  predominating  color  of  the  dead  larvae.  If  any  color 
designation  were  to  be  used,  yellow  would  be  best,  but  color  is  not  a 
safe  guide,  as  this  is  a  variable  symptom.  Any  descriptive  name 
seemed  unsafe  for  a  disease  with  such  variable  manifestations,  and 
the  author  therefore  proposed  that  the  name  be  changed.  After  con- 
sultation with  beekeepers  and  apiary  inspectors  it  was  decided  to 
adopt  the  name  European  foulbrood.  This  was  first  used  in  a  cir- 
cular ^  of  the  Bureau  of  Entomology  and  the  name  has  been  gener- 
ally accepted  by  beekeepers  throughout  the  country.  The  adjective 
"European"  was  chosen  because  it  appeared  that  this  disease  had 
first  been  subjected  to  bacteriological  investigation  by  European  in- 
vestigators, while  the  other  disease,  American  foulbrood,  had  not 
been  investigated  carefully  until  such  work  was  undertaken  in  Amer- 
ica. The  names  obviously  are  not  intended  to  convey  the  idea  that 
the  diseases  originated  one  in  America  and  the  other  in  Europe,  for 
the  honeybee  is  not  native  to  America.  The  names  were  chosen 
simply  that  beekeepers  might  have  names  which  could  be  used  with 
safety,  and  which  would  not  lead  to  confusion  by  being  descriptive. 

SYMPTOMS. 

The  beekeeper  should  know  whether  he  has  to  deal  with  American 
or  European  foulbrood,  for  they  do  not  respond  to  the  same  treat- 
ment. The  symptoms  of  European  foulbrood  are  simply  the  out- 
ward manifestations  of  the  disease,  being  chiefly  the  appearance  of 
the  larvae  after  death.  The  symptoms  are  therefore  variable.  The 
most  accurate  method  of  diagnosis  is  by  bacteriological  examination, 
but  this  is,  of  course,  not  possible  in  apiary  practice.  In  cases  of 
doubt  samples  should  be  sent  to  the  Bureau  of  Entomology  for 
diagnosis.^ 

In  regions  where  both  diseases  occur,  beekeepers  at  times  experi- 
ence difficulty  in  differentiating  them,  due  chiefly  to  insufficient  ob- 
servation of  the  symptoms.  If  European  foulbrood  appears  in  an 
apiary  in  the  spring,  and  if  American  foulbrood  is  then  observed 
later,  the  beekeeper  may  erroneously  conclude  that  both  types  are 

1  Phillips,  E.  F.  Tlie  brood  diseases  of  bees.  tJ.  S.  Dept.  Agr.  Bur.  Ent.  Clrc.  79. 
5    p.      1906. 

=  If  dead  brooiJ  is  observed  and  the  beekeeper  is  not  able  to  diagnose  it  with  accuracy, 
samples  may  be  sent  the  Bureau  of  Entomology  for  examination.  A  piece  of  comb  con- 
taining dead  larvse  about  4  by  5  inches  should  be  cut  out  and  mailed  In  a  heavy  paste- 
board or  wooden  box.  Tin  boxes  should  never  be  used,  as  the  brood  usually  molds  in 
transit,  making  examination  impossible.  The  sample  should  not  be  wrapped  before 
being  placed  in  the  box.     A  suitable  box  for  sending  samples  will  be  mailed  on  request. 

It  is  not  possible  to  diagnose  from  empty  combs,  and  no  honey  should  be  included  in 
the  sample,  as  it  is  valueless  in  diagnosis  and  will  probably  spoil  the  sample  as  well  as 
other  mail  matter,  'xne  name  of  the  sender  must  always  appear  on  the  package,  and 
any  available  data  should  be  sent  in  a.  letter.  Never  inclose  a  letter  In  the  box  with 
the  sample. 


CONTROL  OF  ETTROPEAN   FOTJLBROOD.  5 

I  manifestations  of  one  disease,  or  that  European  foulbrood  changes 
to  American  foulbrood.  Such  is  not  the  case.  It  is  therefore  essen- 
tial that  the  symptoms  be  studied  with  great  care,  since  to  treat 
American  foulbrood  by  methods  applicable  only  to  European  foul- 
brood will  result  in  the  spread  rather  than  in  the  eradication  of  the 
disease. 

(1)  Age  of  larvce  affectea. — European  foulbrood  usually  attacks 
the  larva  at  an  early  stage  of  its  development,  while  it  is  still  curled 
up  at  the  base  of  the  cell  (fig.  1,  E).  At  the  time  of  the  first  mani- 
festation of  disease  the  larva  is  about  three  days  old,  from  the 
hatching  of  the  egg.    A  very  small  percentage  of  larvae  die  after 


Fig.  1. — Portion  of  comb  showing  the  effect  of  European  foulbiood 
upon  the  larvae:  a,j,k,  Normal  sealed  cells;  b,c,d,  e,  g,  i,  I,  m,  p,  g, 
larvse  affected  by  disease;  r,  normal  larva  at  age  attacked  by  disease- 
/,  liinfOf  dried^down  larvae  or  scales.    Three  times  natural  size. 


capping,  but  sometimes  quite  young  larvae  are  attacked  (fig.  1,  E,  M) . 
Sunken  and  perforated  cappings,  which  are  such  common  symptoms 
of  American  foulbrood,  are  sometimes  seen  in  colonies  suffering  with 
European  foulbrood. 

(2)  Early  sym/ptoms. — The  earliest  indications  of  the  disease  are 
a  slight  yellow  or  gray  discoloration  and  the  uneasy  movement  of 
the  larva  in  the  cell.  The  larva  loses  its  well-rounded,  opaque  ap- 
pearance and  becomes  slightly  translucent,  so  that  the  tracheae  may 
become  prominent  (fig.  1,  B),  giving  the  larva  a  clearly  segmented 
appearance. 

(3)  Position  of  larnw. — The  larva  may  be  flattened  against  the 
base  of  the  cell,  may  turn  so  that  the  two  ends  are  to  the  rear  of  the 
cell  (fig.  1,  P) ,  or  may  fall  away  from  the  base  (fig.  1,  E,  G,  L) .    The 


6  farmers'  bulletin  975. 

position  of  the  larva  is  one  of  the  best  means  of  differentiating 
American  foulbrood  and  European  foulbrood.  In  American  foul- 
brood  the  larvae  almost  without  exception  are  found  on  the  lower 
side  wall,  while  in  European  foulbrood  they  may  be  there,  or  at  the 
base  of  the  cell,  or  on  any  of  the  side  walls,  even  the  upper  one. 

(4)  Color. — As  the  decay  proceeds  the  color  changes  to  a  decided 
yellW  or  gray  and  the  translucency  is  lost  (fig.  1,  Q,  H) .  When  the 
disease  first  appears  in  a  region  the  yellow  color  of  the  decaying 
larvae  seems  more  constant  than  later,  due  probably  to  the  fact  that 
as  the  disease  spreads  the  germ  causing  the  disease  is  accompanied 
by  other  organisms.  The  yellow  color  may  be  taken  as  the  chief 
characteristic  of  the  disease.  The  dead  larva  appears  as  a  moist, 
somewhat  collapsed  mass,  giving  the  appearance  of  being  melted. 

(5)  Scale. — When  the  remains  have  become  almost  dry  (fig.  1,  C), 
the  tracheae  sometimes  become  conspicuous  again,  this  time  by  re- 
taining their  shape,  while  the  rest  of  the  body  content  dries  around 
them.  Finally  all  that  is  left  of  the  larva  is  a  yellow  or  grayish- 
brown  scale  against  the  base  of  the  cell  (fig.  1,  F,  H),  or  a  shapeless 
mass  on  one  of  the  side  walls  if  the  larva  did  not  retain  its  normal 
position  before  death  (fig.  1,  N,  O).     Very  few  scales  are  black. 

(6)  Adhesion  to  cell. — At  no  time  during  the  decay  does  the  larva 
adhere  to  the  wax  closely,  but  is  easily  removed,  and  the  bees  carry 
out  a  great  many  of  them  in  their  efforts  to  clean  house. 

(7)  Usual  lack  of  ropiness. — A  slight  ropiness  is  sometimes  ob- 
served in  the  decaying  larvae.  This  is  not,  however,  at  all  like  the 
fine  ropiness  observed  in  larvae  dead  of  American  foulbrood,  but  the 
decaying  mass  behaves  more  like  an  old  rubber  band  which  has  lost 
its  elasticity  and  which  breaks  when  stretched. 

(8)  Odor. — ^There  is  usually  little  odor  in  European  foulbrood,  but 
sometimes  a  sour  odor  is  present  which  reminds  one  of  yeast  fermen- 
tation. This  odor  is  quite  constant  in  some  regions  and  seems  to 
come  from  the  decay  due  to  organisms  other  than  the  one  which 
causes  European  foulbrood. 

(9)  Sex. — A  symptom  of  the  greatest  importance  is  the  fact  that 
the  disease  attacks  drone  and  queen  larvae  ^  nearly  as  quickly  as  those 
of  the  workers. 

(10)  Epidemic  cMracter. — In  regions  where  the  disease  occurs  a 
considerably  larger  percentage  of  colonies  is  affected  than  is  usual 
for  American  foulbrood.  However,  not  many  colonies  die  of  Euro- 
pean foulbrood,  but  the  chief  trouble  is  that  weakened  colonies  suc- 
cumb during  winter  unless  well  cared  for.     The  disease  spreads  at 

1  The  tendency  of  this  disease  to  attack  queen  larvse  is  a  serious  drawback  in  treat- 
ment. Frequently  the  bees  of  a  diseased  colony  attempt  to  supersede  their  queen,  but 
the  larvoi  In  the  queen  cells  often  die,  leaving  the  colony  hopelessly  queenless.  '  The 
colony  Is  thus  depleted  rapidly. 


CONTROL  OF  EUROPEAN  FOULBROOD.  7 

times  with  startling  rapidity,  much  more  rapidly  than  American 
foulbrood. 

(11)  Variability. — In  all  its  symptoms  European  foulbrood  is 
more  variable  than  is  American  foulbrood.  Color  is  perhaps  the 
most  constant  symptom. 


BASIS  OF  TREATMENT. 

The  confusion  in  the  treatment  of  the  disease  is  due  to  a  failure  to 
analyze  the  factors  forming  the  basis  of  treatment.  Various  treat- 
ments have  been  described  in  the  beekeeping  journals  as  distinct 
when  they  were  simply  modifications  of  the  same  treatment. 

(1)  European  foulbrood  is  a  disease  of  weak  colonies.  While  at 
times  one  may  observe  larvae  dead  of  this  disease  in  strong  colonies, 
usually  .they  are  removed  before  the  disease  can  do  much  harm.  It 
should  be  pointed  out,  further,  that  it  is  the  colony  which  is  failing 
to  increase  in  strength  in  the  spring  which  is  most  seriously  affected, 
for  a  small  colony  which  is  rich  in  young  and  vigorous  bees  and 
which  is  increasing  in  strength  is  often  able  to  overcome  the  disease. 
It  is  therefore  a  disease  of  weak  rather  than  small  colonies. 

(2)  The  disease  is  prevalent  in  the  spring  and  early  summer. 
While  at  times  it  is  observed  at  other  periods  of  the  year,  this  is  not 
usual.  Samples  of  European  foulbrood  have  been  received  by  the 
Bureau  of  Entomology  in  every  month  of  the  year,  but,  as  will  be 
seen  from  Table  I,  they  are  far  more  commonly  received  in  the  early 
part  of  the  active  season.  These  samples  are  listed  according  to  the 
date  of  receipt  at  the  bureau  laboratory.  The  highest  number  is  re- 
ceived in  June  and  the  average  date  for  the  removal  of  these  samples 
from  the  hives  is  probably  a  few  days  previous  to  June  15,  perhaps 
June  10.  The  earliest  samples  received  are  regularly  those  from 
California,  where  the  season  opens  early.  There  is  a  sudden  increase 
in  May  and  June  and  almost  as  rapid-  a  decline  later.  The  few  sam- 
ples received  from  October  to  April  may  be  largely  disregarded,  as 
they  are  almost  without  exception  dried  material  of  unknown  age. 

Table  I. — Distril)Ution  of  European  foulbrood  by  months,  including  all  posi- 
tively diagnosed  samples  received  by  the  Bureau  of  Entomology  from  1908 
to  December,  1917. 


Month. 


Total 

Califor- 

number. 

nia. 

3 

0 

4 

3 

17- 

10 

33 

17 

ISO' 

24 

334 

30 

240 

20 

164 

9 

9S 

8 

17 

1 

7 

3 

2 

1 

New 
York. 


January... 
February. . 

March 

April 

May 

June 

July 

August 

September 
October... 
November. 
December. 


0 

0 

0 

2 

23 

50 

41 

20 

8 

3 

1 

0 


8  FAEMEES'   BtTLLETIH"  9^5. 

(3)  The  disease  disappears  later  in  the  summer  unless  the  colony 
has  become  so  badly  weakened  that  it  can  not  remove  the  dead  larvae. 
Such  weakened  colonies  usually  die  in  winter  or  in  a  time  of  dearth. 
Colonies  do  not  as  a  rule  die  as  a  direct  result  of  European  foul- 
brood.  There  may  still  remain  some  dead  larvae  in  the  combs,  show- 
ing that  the  bees  have  not  been  able  to  remove  all  of  them,  but  in  any 
but  the  worst  cases  even  these  disappear.  If  conditions  which  com- 
monly prevail  in  early  summer  again  appear  there  may  be  a  recur- 
rence of  the  disease  the  same  season. 

(4)  This  disappearance  of  the  disease  usually  accompanies  the 
beginning  of  the  honey  flow.  At  this  time,  unless  the  colony  has 
already  reached  maximum  strength,  there  is  a  rapid  increase  in 
brood  rearing  and  the  colony  increases  in  strength,  bringing  about 
conditions  unfavorable  for  the  development  of  the  disease.  •  If  the 
honey  flow  fails,  the  disease  may  continue  and  under  such  condi- 
tions is  at  its  worst.  It  should  be  noted  that  in  regions  where  the 
early  honey  flows  are  uncertain  or  usually  lacking  European  foul- 
brood  has  done  the  most  damage,  for  in  years  of  failure  the  disease 
spreads  with  such  rapidity  that  the  entire  region  becomes  badly 
infected.  European  foulbrood  is  rarely  observed  in  regions  where 
an  early  honey  flow  is  certain. 

(5)  The  .earliest  brood  of  the  year  usually  escapes  with  little  loss. 
This  important  fact  has  been  overlooked  in  previous  discussions  of 
this  disease,  but  it  is  evident  from  Table  I.  The  scarcity  of  Euro- 
pean foulbrood  in  the  early  spring  was  mentioned  in  the  earliest 
accounts  of  its  prevalence  in  New  York.  This  in  all  probability  is 
due  to  the  fact  that  the  colonies  have  been  able  to  remove  most  of 
the  disease  dui'ing  the  previous  summer  and  there  has  been  left  only 
a  little  of  the  infecting  material. 

(6)  Some  bees  resist  the  disease  more  successfully  than  others.  It 
has  been  found  through  the  experience  of  beekeepers  generally  that 
the  three-banded  Italian  bees  are  best  for  this  purpose.  These  bees 
have  a  further  advantage  in  that  they  give  excellent  results  in  all 
lines  of  beekeeping  activity,  and  it  is  therefore  safe  to  recommend 
them  as  the  best.  This  does  not  at  all  indicate  that  other  races  of 
bees  would  not  give  as  good  results,  as  far  as  European  foulbrood 
control  is  concerned,  but  that  it  is  easier  to  get  good  three-banded 
Italian  than  good  bees  of  any  other  race.  The  resistance  appears  to 
be  either  a  form  of  immunity  or  a  greater  ability  to  remove  the  dead 
larvse  completely. 

(7)  European  foulbrood  is  an  infectious  disease.  This  was  clearly 
shown  by  the  experience  of  beekeepers  before  the  disease  was  investi- 
gated from  a  bacteriological  standpoint,  and  these  investigations  have 
supported  the  observations  of  the  beekeeper.     The  bacteriological 


CONTROL  OF  EUBOPEAN  FOULBEOOD.  9 

work  has  shown,  further,  that  the  disease  is  caused  by  an  organism  * 
which  has  never  been  found  in  any  other  brood  disease  of  bees.  The 
cause  of  the  disease  is,  therefore,  a  specific  organism,  and  the  disease 
is  entirely  distinct  from  American  foulbrood.  This  is  an  important 
point,  for  there  has  in  the  past  been  considerable  confusion  in  that 
a  few  beekeepers  have  claimed  that  one  disease  changes  to  the  other. 
It  should  be  made  clear  that  this  supposition  is  not  supported  by 
any  careful  observation  in  the  apiary,  and  that  it  was  recognized 
generally  by  beekeepers  before  the  bacteriological  investigations 
were  made  that  the  diseases  were  distinct. 

(8)  The  organism  causing  European  foulbrood  does  not  seem  from 
observations  in  the  apiary  to  be  so  difficult  to  eradicate  as  does  the 
one  causing  American  foulbrood.  This  is  partially  confirmed  by  the 
bacteriological  observations  also. 

(9)  When  a  bee  larva  dies  of  European  foulbrood  the  decaying 
mass  does  not  adhere  closely  to  the  cell  wall  at  any  time  in  the  decay 
or  when  it  has  dried  down  to  a  scale  in  the  back  or  on  the  side  walls  of 
the  cell.  Dead  larvae  may  therefore  be  removed  easily  by  the  bees 
if  conditions  are  favorable  for  this  cleaning. 

(10)  The  bees  are  able  under  suitable  conditions  of  colony  strength 
and  resistance  to  clean  the  cells  so  thoroughly  that  when  future  larvae 
are  reared  in  these  cells  the  disease  is  not  contracted. 

(11)  The  method  of  spread  of  the  disease  is  not  well  known,  al- 
though there  is  some  evidence  that  the  infection  is  carried  chiefly  by 
nurse  bees.  It  has  been  observed  that  under  some  circurstances  it  may 
be  transmitted  through  feeding,  but  the  experience  of  beekeepers  indi- 
cates that  contaminated  honey  is  not  the  common  means  of  carrying 
the  disease.  It  is  well  known  that  honey  from  infected  colonies  may 
be  given  to  healthy  colonies  with  entire  safety  provided  the  healthy 
colonies  are  in  such  condition  that  they  are  able  to  resist  the  disease. 
It  is  therefore  not  necessary  to  disinfect  the  honey  from  colonies 
having  European  foulbrood,  as  is  the  case  with  that  from  colonies 
suffering  from  American  foulbrood. 

(12)  It  has  not  been  found  necessary  to  disinfect  hives,  combs,  or 
frames  from  diseased  colonies.  This  does  not  indicate  that  the  germ 
causing  the  disease  is  absent  from  such  material,  but  that  if  present 
it  does  not  do  any  damage. 

(13)  While  the  disease  spreads  with  great  rapidity  at  times,  it 
does  not  seem  to  be  so  malignant  as  is  American  foulbrood,  since 
many  colonies  exposed  to  infection  fail  to  contract  the  disease. 

These  facts  concerning  the  disease  have  been  discovered  in  the 
apiary  rather  than  in  the  laboratory.  The  facts  are  supported  by 
repeated  observations,  and  while  the  records  of  observation  are  not  as 
accurately  made  as  are  those  of  the  laboratory  the  correctness  of  most 

^  Bacillus  pluton. 


1Q  PABMEES'  BULLETIN"   9^5. 

of  the  facts  is  attested  by  the  experience  of  hundreds  of  beekeepers. 
In  certain  cases  the  findings  have  been  corroborated  by  bacterio- 
logical investigation.^  The  methods  of  treatment  have  also  all  been 
devised  in  the  apiary. 

The  difficulty  in  drawing  conclusions  from  practical  observations 
is  that  too  often  beekeepers  fail  to  show  the  ways  in  which  their  ex- 
perience differs  from  that  of  others  or  in  what  manner  the  same 
principles  have  been  applied  in  a  slightly  different  manner. 

PREVENTIVE  MEASURES. 

In  keeping  European  foulbrood  under  control  it  is  far  more  im- 
portant to  prevent  the  disease  from  getting  a  foothold  in  a  colony 
than  it  is  to  eradicate  the  disease  afterward. 

This  is  not  true  of  American  foulbrood,  for  reliable  and  practicable 
preventive  measures  have  not  been  found  for  that  disease. 

(1)  The  use  of  resistant  stock  is  of  the  greatest  importance,  other- 
wise there  is  no  hope  of  warding  off  the  disease  when  it  enters  a  re- 
gion or  of  eradicating  it  froir  the  apiary  after  it  is  once  introduced. 
The  use  of  strong,  vigorous  Italian  stock  is  best  from  the  standpoint 
of  honey-production,  and  every  beekeeper  should  therefore  see  that 
his  apiary  is  provided  with  such  queens  even  before  European  foul- 
brood appears  in  the  unmediate  neighborhood  or  in  the  apiary.  When 
the  disease  is  absent  it  is  quite  permissible  for  the  beekeeper  to  save 
any  mismated  queens  which  show  themselves  to  be  good,  but  when 
European  foulbrood  is  near  by  this  course  is  unsafe,  and  in  no  case 
should  a  mismated  queen  be  used  as  breeding  stock.  The  purity  of 
mating  of  queens  then  becomes  a  matter  of  first  importance  and  this 
entails  more  work  than  is  necessary  in  the  ordinary  practices  of  the 
apiary. 

It  is  not  enough  simply  that  queens  be  pure  bred  and  purely  mated, 
however,  for  it  often  occurs  that  a  queen  will  be  poor  from  other 
causes.  Whenever  a  queen  shows  signs  of  failing  it  is  good  bee- 
keeping to  replace  her  with  a  good  queen.  When  European  foul- 
brood is  present  this  becomes  far  more  important. 

Not  all  Italian  stock  is  equally  resistant  to  European  foulbrood,  and 
when  the  disease  is  nearby  it  becomes  important  that  the  beekeeper 
find  out  which  stock  is  best.  Not  all  queens  sold  as  Italians  are  pure 
bred.  By  far  the  best  plan  is  to  buy  a  few  untested  Italian  queens 
from  each  of  several  queen  breeders  and  after  these  have  been  under 
observation  for  a  short  time  the  beekeeper  will  be  able  to  choose 
from  the  lot  those  best  suited  for  breeding  purposes.  It  is  not  so 
good  a  practice  to  buy  a  breeding  queen,  for  such  queens  do  not  ship 

1  Bacteriological  studies  of  bee  diseases  have  been  useful  to  practical  beekeepers  in 
explaining  the  reasons  for  success  or  failure  with  various  treatments  attempted.  These 
studies  have  been  especially  important,  however,  because  through  them  methods  of 
laboratory  diagnosis  of  the  different  diseases  have  been  wmrked  out. 


CONTROL  OF  EUROPEAN  EOULBEOOD.  11 

SO  well  in  the  mails,  and  even  a  breeding  queen  of  the  most  resistant 
stock  might  allow  her  colony  to  become  infected  simply  because  she 
had  been  so  injured  in  the  mails  that  she  could  not  keep  up  egg- 
laying  properly.  The  buying  of  untested  queens  is  to  be  advised  at 
all  times,  for  until  more  accurate  work  in  breeding  is  done  the  indi- 
vidual beekeeper  can  choose  breeding  stock  as  well  as  most  breeders. 

It  would  be  possible  to  recommend  certain  stock  as  the  best  were 
it  not  for  the  fact  that  the  stock  of  the  various  queen  breeders  is  not 
constant.  The  stock  which  in  one  year  makes  the  best  showing  possi- 
bly can  not  be  duplicated  by  the  queen  breeders  the  next  year.  The 
best  course  therefore  is  for  each  beekeeper,  or  possibly  a  group  of 
beekeepers,  to  try  out  several  strains  of  Italian  bees  to  find  which  is 
best.  Having  done  this,  they  can  continue  to  breed  from  the  best 
stock  obtained,  and  they  can  do  as  well  by  that  means  as  they  can  if 
they  continue  to  buy  queens  from  the  queen  breeders. 

(2)  Strength  of  colony  is  fully  as  important  as  resistant  stock. 
Unfortunately  too  many  beekeepers  fail  to  provide  conditions  neces- 
sary to  the  bees  in  order  that  the  colonies  may  be  at  the  proper 
strength  in  time  to  combat  European  foulbrood  successfully.  It  is 
good  beekeeping  to  have  all  colonies  strong,  and  nothing  leads  to 
large  honey  crops  as  does  this  factor,  yet  throughout  the  country 
there  are  thousands  of  beekeepers  who  annually  fail  to  get  half  the 
crop  through  failure  to  have  strong  colonies  at  the  right  time.  When 
the  honey-flow  comes  early  in  the  season,  as  is  the  case  throughout 
most  of  the  United  States,  it  is  important  that  every  colony  be  at 
maximum  strength  early  in  the  spring.  Since  European  foulbrood 
appears  in  the  spring  and  early  summer,  good  beekeeping  practice 
again  coincides  with  the  requirements  for  preventing  the  ravages 
of  this  disease. 

One  difficulty  arises  from  the  fact  that  there  is  no  standard  for 
strength  of  colony  and  what  one  beekeeper  considers  a  strong  colony 
may  be  considered  weak  by  another  and  better  beekeeper.  At  the 
opening  of  the  honey-flow  every  colony  from  which  a  full  crop  is  to 
be  expected  should  be  strong  enough  to  have  10  full  combs  of 
Langstroth  size  filled  with  brood.  Of  course  this  brood  may  be  in  a 
larger  number  of  combs,  since  the  bees  usually  store  some  honey  at 
the  top  of  each  comb,  but  it  is  easy  to  estimate  the  brood  in  terms 
of  full  combs.  If  now  we  accept  the  same  standard  for  the  desired 
strength  of  colony  for  the  purpose  of  resisting  European  foulbrood, 
we  will  have  a  condition  under  which  (assuming  resistant  stock)  this 
disease  will  never  get  a  start  in  any  colony  in  the  apiary.  It  is  of 
course  recognized  that  such  a  standard  is  seldom  realized  before  or 
at  the  beginning  of  the  honey-flow,  and  this  fact  is  the  reason  for 
the  loss  of  so  much  honey  as  well  as  the  fullexplanation  of  the  rav- 
ages of  European  foulbrood  in  so  many  places.  It  is  suggested  that 
each  beekeeper  in  a  region  where  European  foulbrood  exists  ask 


12  FAEMEES'   BULLETIN   975. 

himself  whether  his  colonies  are  actually  in  as  good  condition  at  the 
opening  of  the  year  as  he  has  supposed  and  that  he  find  out  how 
strong  the  colonies  may  be  made  by  providing  the  best  of  conditions 
for  the  development  of  the  colony  population.  A  beekeeper  whose 
colonies  do  not  measure  up  to  this  standard  should  not  condemn  the 
standard  until  he  assures  himself  that  it  is  entirely  impossible,  under 
his  conditions,  to  reach  it. 

Obviously  the  proper  wintering  of  bees  becomes  a  matter  of  the 
highest  importance  in  regions  where  European  foulbrood  is  found. 
Those  who  fail  to  practice  good  wintering  are  the  ones  who  first 
lose  so  many  colonies  that  they  become  discouraged  and  give  up  bee- 
keeping, while  those  whose  wintering  has  been  better  are  able  to 
treat  the  disease  although  their  standard  of  colony  strength  may  not 
be  high  enough  entirely  to  ward  it  off. 

As  was  pointed  out  earlier,  the  first  brood  of  the  year  usually 
escapes  with  little  loss.  If  proper  conditions  are  provided  for  winter, 
either  in  the  cellar  or  outdoors,  brood-rearing  is  delayed,  whereas  in 
poor  wintering  brood-rearing  may  begin  during  the  coldest  period 
of  the  winter.^  If  then  brood-rearing  is  delayed  by  protection,  it 
will  begin  as  a  reaction  to  incoming  nectar  and  pollen.  The  vitality 
of  the  bees  has  not  been  destroyed  by  unseasonable  brood-rearing 
and  the  colony  can  rear  large  quantities  of  brood  from  the  very 
beginning.  This  can,  of  course,  occur  only  when  the  colony  has 
proper  spring  protection.  The  earliest  brood  will  emerge  without 
appreciable  loss  from  disease,  the  colony  is  increased  in  strength  at 
once,  and  its  capacity  for  brood-rearing  is  great.  Provided  the  stock 
is  resistant,  the  colony  is  then  able  to  ward  off  the  disease.  To 
bring  about  all  the  proper  conditions  with  the  least  labor  on  the  part 
of  the  beekeeper  and  the  least  waste  of  effort  on  the  part  of  the  bees, 
it  is  desirable  to  winter  outdoor  colonies  in  two  hive-bodies,  which 
has  been  recommended  by  this  department  for  other  reasons  also. 
Good  beekeeping,  in  so  far  as  handling  the  bees  is  concerned,  con- 
sists of  providing  conditions  in  the  fall  so  that  the  colony  is  full  of 
young,  vigorous  bees  for  winter;  of  providing  conditions  of  protec- 
tion and  good  stores  such  that  the  bees  are  not  depleted  in  numbers 
and  vitality  during  the  winter  by  excessive  heat-production ;  of  pro- 
viding plenty  of  stores,  adequate  room  for  breeding,  and  abundant 
protection  during  the  period  of  heavy  brood-rearing  in  spring;  and 
of  preventing  reduction  in  the  strength  of  the  colony  by  swarming. 
All  of  these  things,  and  there  are  no  others  of  importance,  pertain 
to  keeping  colonies  strong.  The  beekeeper  who  provides  conditions 
such  that  the  bees  can  keep  up  their  own  strength  will  not  only  reap 
the  honey-crop  but  he  will  escape  the  ravages  of  European  foulbrood. 
To  a  large  degree  the  failure  of  American  beekeepers  to  get  their 
colonies  strong  enough  is  due  to  the  use  of  small  hives  that  are  in- 

>  The  explanation  is  given  in  the  publications  of  the  Bureau  of  Entomology  on  wintering. 


CONTROL  OF  EUROPEAN  POULBROOD.  13 

suiEciently  protected  during  the  winter  and  spring.  The  single- 
walled  hive  was  first  made  as  a  means  of  reducing  the  cost.  Such 
a  hive  is  a  good  tool  for  the  beekeeper  but  it  is  a  poor  home  for  the 
bees.  When  the  10-frame  hive  was  found  too  large  to  be  filled  with 
bees  in  time  for  them  to  go  into  the  supers  as  soon  as  the  honey-flow 
opened,  instead  of  protecting  the  hive  the  use  of  the  8-frame  hive 
was  commonly  adopted.  This  hive  is  in  rather  general  use  through- 
out the  United  States,  although  fortunately  it  is  now  being  replaced 
by  the  10-frame  hive  in  many  localities.  In  order  that  the  beekeeper 
may  reduce  his  labor,  it  would  be  well  to  raise  the  standard  of  colony 
strength  by  providing  better  protection  and  more  room  for  the  bees. 
This  will  to  a  large  degree  eliminate  the  spring  manipulations  so 
often  practiced,  will  get  better  crops,  and  will  make  European  foul- 
brood  a  minor  trouble  of  the  apiary. 

REMEDIAL  MEASURES. 

When  strong  colonies  headed  by  vigorous  queens  of  resistant  stock 
are  present,  European  f oulbrood  will  usually  make  little  if  any  head- 
way, yet  from  time  to  time  there  may  appear  cases  which  require 
treatment.  The  shaking  treatment  used  for  American  f oulbrood^ 
is  often  advocated  for  European  foulbrood  and  is  recommended  by 
many  inspectors  of  apiaries.  It  was  recommended  in  previous  pub- 
lications of  this  department,  but  later  observations  show  that  other 
methods  are  more  reliable.  If  colonies  are  given  young  Italian 
queens  at  the  time  of  shaking,  results  will  usually  be  good,  but  unless 
this  is  done  shaking  is  of  little  or  no  value.  Some  beekeepers  prac- 
tice heavy  feeding  of  either  honey  or  sugar  sirup  when  European 
foulbrood  appears.  This  often  gives  good  results,  for  it  brings  about 
the  conditions  which  are  advocated  as  preventive  measures,  although 
as  applied  it  constitutes  a  remedial  measure.  The  same  amount  of 
stores  left  with  the  colony  the  previous  fall  will  usually  do  more 
good  than  heavy  spring  feeding  as  a  means  of  disease  control. 

The  remedial  measures  here  described  should  be  used  only  to  re- 
move the  disease  if  it  enters  the  apiary.  Preventive  measures  should 
then  be  employed  to  avoid  a  recurrence  of  the  disease. 

(1)  The  dead  larvae  are  easily  removed  from  the  cells,  and  the  re- 
medial treatment  serves  to  provide  conditions  such  that  these  may 
be  removed  by  the  bees  during  a  period  when  no  new  diseased  ma- 
terial is  appearing  in  the  combs.  Usually  the  queen  is  removed  from 
the  colony,  and,  since  a  queen  whose  colony  becomes  badly  infected  is 
rarely  of  any  value,  she  is  killed.  In  five  or  six  days  all  queen  cells 
are  removed,  so  that  the  colony  is  hopelessly  queenless.  The  workers 
do  not  clean  out  the  diseased  cells  so  rapidly  unless  they  have  a  queen 

1  For  a  description  of  this  treatment  the  reader  is  referred  to  Farmers'  Bulletin  442, 
"  The  Treatment  of  Bee  Diseases." 


]^4  farmers'  bulletin  975. 

or  a  queen  cell.  As  soon  as  the  dead  larvae  are  removed,  which  may 
be  easily  determined  by  examinations,  the  colony  is  given  a  young 
vigorous  Italian  queen  of  resistant  stock.  If  only  a  few  diseased 
cells  are  observed  and  if  the  colony  is  fairly  populous  the  queen  may 
simply  be  caged  and  released  later  when  the  dead  brood  is  removed. 

The  length  of  time  necessary  for  the  cleaning  out  of  the  dead  larvae 
varies  with  the  strength  of  the  colony,  and  for  weak  colonies  it  may  be 
necessary  to  wait  until  all  brood  has  emerged  before  giving  a  young 
queen.^  This  method  should  not  be  employed  unless  each  colony  has 
enough  bees  to  sustain  at  least  five  combs  full  of  brood.  Some  col- 
onies seem  to  clean  out  dead  brood  more  rapidly  than  others  of  the 
same  strength.  If  the  honey-flow  comes  early  it  will  usually  be  pos- 
sible to  reduce  the  period  of  queenlessness  to  a  few  days.  A  bee- 
keeper may  use  the  time  necessary  for  cleaning  up  as  an  indication 
of  the  strength  of  his  colonies,  for  if  he  finds  a  long  time  needed  he 
may  be  sure  that  his  colonies,  for  some  reason,  are  not  as  prosperous 
as  they  should  be.  If  it  is  certain  that  there  will  be  no  honey-flow 
until  midsummer  or  later  it  is  not  so  necessary,  from  the  standpoint 
of  good  beekeeping,  to  have  all  colonies  strong  so  early  in  the  year, 
but  it  is  surely  an  exceptional  locality  where  there  is  nothing  for  the 
bees  to  get  in  early  summer. 

Where  the  beekeeper  is  dependent  on  a  late  honey-flow  it  is  often 
desirable  to  move  the  bees  during  the  early  part  of  the  season  to  some 
place  where  nectar  may  be  obtained.  This  will  often  be  easier  and 
less  expensive  than  treating  the  colonies.  For  example,  the  author 
was  shown  a  location  in  the  west  where  European  foulbrood  caused 
great  annoyance  during  the  spring,  while  apiaries  not  many  miles 
away  were  able  to  get  enough  nectar  to  ward  off  the  disease  and  at 
the  same  time  to  give  the  beekeeper  enough  profit  to  justify  the  ex- 
pense and  time  of  moving.  In  such  a  case  preventive  measures  are 
cheaper  and  better  than  the  remedial  measures  here  described.  Apiary 
inspectors  should  exercise  judgment  in  such  cases  and  permit  the 
moving  of  colonies  to  such  places,  provided  they  are  sure  that  due 
precautions  will  be  taken.  No  precautions  need  be  demanded  if  the 
new  location  is  already  infected. 

^  This  method  of  treatment  was  described  in  Its  essentials  in  1905,  In  an  article 
published  in  a  periodical  devoted  to  beekeeping.  The  writer  of  that  article  advised 
that  the  colony  be  left  queenless  for  three  days  after  all  drone-brood  has  emerged,  thus 
making  a  queenless  period  of  27  days.  Later  other  beekeepers  tried  shorter  periods 
with  success.  It  should  be  remembered  that  the  apiaries  belonging  to  the  writer  of  the 
article  referred  to  were  located  in  the  buckwheat  region  of  New  York,  and  that  he  used 
a  small  hive,  and  on  account  of  these  conditions  It  may  be  safely  assumed  that  at  the  time 
when  European  foulbrood  attacks  colonies  his  colonies  were  unusually  weak.  Those 
who  have  found  a  shorter  time  sufficient  have  been  located  in  regions  where  the  colony 
strength  may  be  developed  earlier  because  of  earlier  honey-flows,  or  perhaps  in  some  cases 
these  beekeepers  wintered  better,  so  that  in  the  spring  their  colonies  were  in  better 
condition  to  resist  the  ravages  of  the  disease.  It  would  be  quite  possible  to  refer  to 
apiaries  where  the  wintering  is  good  and  where  the  spring  care  Is  sufficient  to  elimi- 
nate entirely  the  period  of  queenlessness. 


CONTROL  OF  ETJBOPBAN  POTTLBEOOP.  15 

The  methods  of  requeening  and  rearing  the  queens  are  matters 
aside  from  the  treatment  of  European  foulbrood,  but  in  many  cases 
the  directions  have  been  obscured  by  including  all  such  details. 
Usually  it  is  easier  to  introduce  a  queencell  of  the  proper  age  for  the 
queen  to  emerge  and  mate  by  the  time  egg  laying  may  again  proceed 
safely  in  the  colony. 

(2)  A  substitute  for  the  treatment  just  described  introduces  no 
new  principle.  The  colonies  found  to  have  European  foulbrood  are 
graded  according  to  strength,  and  half  or  more  of  the  stronger  ones 
are  shaken  to  dry  extracting  combs  (not  comb  foundation)  at  the 
same  time  that  the  old  queens  are  killed  and  replaced  by  young,  vig- 
orous stock.  No  colony  too  weak  to  have  five  frames  of  brood  should 
be  so  treated.  If  there  is  no  honey  coming  in,  the  combs  may  contain 
some  honey,  and  it  is  immaterial  whether  or  not  it  comes  from  a  col- 
ony having  European  foulbrood.  The  removed  brood  is  now  stacked 
on  the  weaker  diseased  colonies  so  that  they  may  be  increased  in 
strength.  Just  as  soon  as  these  have  reached  the  degree  of  strength 
possessed  by  the  first  colonies  shaken,  they,  too,  may  be  shaken  to 
drawn  combs  containing  no  brood,  and  the  diseased  brood  is  given  to 
the  remaining  few  diseased  colonies.  Usually  by  the  time  that  the 
last  colonies  are  ready  for  treatment  it  will  be  found  that  treatment 
is  not  necessary,  for  in  many  cases  the  dead  brood  will  have  been 
removed.  If  necessary,  of  course,  every  diseased  colony  may  be 
treated. 

This  substitution  for  the  more  usual  method  of  treatment  has  cer- 
tain advantages.  No  colony  is  left  queenless  and,  as  a  result,  the  total 
brood  reared  in  the  apiary  is  increased.  No  brood  is  wasted,  and  the 
colonies  which  receive  the  most  of  the  combs  containing  diseased 
brood  are  usually  made  sufficiently  strong  to  gather  a  good  crop. 

(3)  Another  method  which  is  much  used  is  to  place  all  the  brood 
combs  of  the  infected  colony  except  one  in  the  second  hive  body  over 
a  queen-excluder  and  to  place  the  queen  below  with  the  one  frame  of 
brood  and  frames  containing  foundation  or  even  drawn  combs.  Others 
prefer  to  put  the  queen  and  one  frame  of  brood  above.  Of  course 
only  good  Italian  queens  should  be  used.     , 

It  is  interesting  to  note  that  the  methods  used  in  the  control  of 
European  foulbrood  are  exactly  the  same  as  are  used  in  remedial 
methods  for  swarm  control.^  Either  the  queen  or  the  brood  is  re- 
moved or  the  queen  and  brood  are  separated  within  the  hive.  Such  a 
similarity  is  probably  of  significance,  but  this  at  present  is  merely 
a  matter  of  speculation. 

GOOD  BEEKEEPING  WILL  ERADICATE  THE  DISEASE. 

It  can  not  be  emphasized  too  strongly  that  the  practices  of  good 
beekeeping  are  those  which  result  in  the  eradication  of  European 

^  See  Farmers'  Bulletin  503,  "  Comb  Honey." 


16  FABMERS'  BULLETIN  975. 

foulbrood.  It  does  not  follow  that  because  a  beekeeper  is  troubled 
with  European  foulbrood  he  is  a  poor  beekeeper,  for  he  may  have 
had  good  results  before  the  disease  appeared.  With  the  entrance  of 
the  disease,  however,  he  can  change  his  system  so  as  to  overcome 
the  trouble  and  he  may  do  this  with  assurance  that  the  changes  are 
such  as  to  result  in  good  beekeeping.  Unlike  American  foulbrood, 
the  disease  does  not  make  it  necessary  that  anything  of  value  be  de- 
stroyed by  the  beekeeper,  and  if  the  proper  system  of  management 
for  the  particular  locality  can  be  found  it  will  result,  in  most  circum- 
stances, in  larger  crops  than  are  usually  obtained. 

O 


Reprinted  from  Journal  of  Economic  Entomology 
Vol.  27,  No.  3,  June,  1934. 


STUDIES  ON  THE  BACTERIA  ASSOCIATED  WITH 
EUROPEAN  FOULBROOD 

By    C.    E.    BuRNSiDE,    Assistant    Apiculturist,    Bureau    of    Entomology,    United 
States  Department  of  Agriculture 

The  etiology  of  European  foulbrood  of  bees  is  an  unsettled  problem, 
several  theories  having  been  advanced  regarding  the  cause  of  this  dis- 
ease. In  1885  Cheshire  and  Cheyne  (2)  described  Bacillus  alvei,  which 
they  claimed  was  the  cause  of  the  brood  disease  now  known  as  European 
foulbrood.  In  1907  Maassen  (9)  stated  his  belief  that  the  etiology  of  the 
mild  form  of  foulbrood  (European  foulbrood)  is  not  uniform  but  that 
the  disease  is  caused  principally  by  Streptococcus  apis  and  B.  alvei. 
White  (11,  12,  13)  was  unsuccessful  in  attempts  to  produce  typical 
European  foulbrood  with  cultures  of  B.  alvei,  S.  apis,  or  Bacterium 
eurydice  and  concluded  that  this  disease  is  caused  by  a  new  species. 
Bacillus  pluton  White,  which  failed  to  grow  on  artificial  media.  Bor- 
chert  {1  p.  12)  and  Lehmann  and  Newman  {4  p.  236),  of  Germany,  have 
pointed  out  that  uncertainty  still  exists  concerning  the  etiology  of 
European  foulbrood.  Wharton  (14)  reported  having  cultured  B.  pluton 
and  producing  infection  in  a  colony  of  black  bees  by  inoculation  with 
cultures  derived  from  primary  colonies.  Lochhead  (5)  says  that  this 
organism  cultured  by  Wharton  "appears  to  be  closely  related  if  not  iden- 
tical with  Streptococcus  apis  described  by  Maassen."  Wharton  (14) 
also  says  that  "cultures  of  B.  pluton  have  been  observed  to  change  to  B. 
alvei  form  resembling  biologically  the  B.  alvei  isolated  from  infected 
larvae.''  Lochhead  (5,  6)  reported  the  origin  of  a  coccoid  bacillus  in 
cultures  of  B.  alvei.  The  coccoid  was  isolated  and  stabilized  and  is  said 
to  have  "all  the  appearance  of  what  White  calls  Bacillus  pluton.''  Both 
Lochhead  and  Wharton  question  the  secondary-organism  theory  of 
White  as  regards  European  foulbrood. 

At  the  time  White  conducted  his  studies  on  European  foulbrood  it 
was  generally  believed  that  bacterial  species  remain  constant  in  mor- 
phological and  cultural  characteristics.  In  recent  years  evidence  has 
been  constantly  increasing  that  bacteria  are  capable  of  morphological. 


June,  '34]     burnside  :  bacteria  associated  with  European  foulbroou  657 

cultural,  and  biological  transformation,  and  the  old  doctrine  of  fixity  of 
bacterial  species  is  gradually  giving  way  before  this  evidence.  Chief 
among  the  investigators  in  this  field  is  Mellon,  whose  extensive  works 
have  demonstrated  that  many  species  of  bacteria,  when  cultured  under 
different  environments,  produce  mutants  and  variants  with  greater  fre- 
quency than  is  commonly  supposed.  Works  of  Mellon,  Hadly  {3), 
Lohnis  and  Smith  {7,  8),  and  others  strongly  indicate  the  existence  of 
life  cycles  among  bacteria  similar  to  life  cycles  among  the  fungi.  Mellon 
{10)  has  aptly  stated  what  seems  to  be  the  situation  in  the  following  quo- 
tation: "Thus  the  analogy  is  complete,  constituting  rather  formidable 
evidence  for  our  contention  that  biologically  bacteria  may  be  properly 
regarded  as  fungi  which  have  been  telescoped  down  into  a  state  of  exist- 
ence where  their  life  cycles,  although  much  compressed  and  often  abbre- 
viated, are  still  not  obliterated." 

In  1928  the  writer  started  observations  and  experiments  on  European 
foulbrood  to  obtain  evidence  in  support  of  one  or  another  of  the  theories 
regarding  the  cause  of  this  disease.  He  repeated  experiments  of  others 
but  sometimes  interpreted  them  differently,  and  he  also  performed  new 
experiments.  Experimental  results  were  not  always  so  conclusive  as 
might  be  desired  and  the  significance  of  observations  was  not  always  ap- 
parent. Observations  and  experimental  results  which  may  aid,  directly 
or  indirectly,  in  arriving  at  a  true  conception  of  the  etiology  of  European 
foulbrood  are  reported  in  this  paper. 

Morphology  of  Bacteria  in  Affected  Brood. — Wide  variation  was 
observed  in  the  morphology  of  bacteria  present  in  sick  or  dead  brood. 
(Plate  6,  A,  B,  and  C.)  In  recently  infected  larvae  the  bacteria  were 
mostly  very  short  rods  occurring  singly,  in  pairs,  or  m  short  chains. 
Medium-long  rods  were  sometimes  present,  but  no  distinctly  pointed 
cells  were  found  during  early  infection.  As  the  disease  progressed  and 
bacteria  increased  in  number,  variability  in  their  morphology  increased. 
Most  frequently  coccoid  cells  predominated,  but  at  times  moderately  long 
rods  were  equally  numerous.  Cells  of  the  B.  pluton  type  (Plate  6,  C) 
originated  from  the  coccoid  cells  at  about  the  time  multiplication  of  bac- 
teria was  checked  by  overcrowding.  The  pointed  condition  appeared  to 
be  an  expression  of  dormancy,  since  these  cells  usually  occurred  singly 
in  coherent  masses  with  rarely  any  indication  of  active  division.  In 
very  late  infection  pointed  cells  usually  predominated,  but  among  differ- 
ent larvae  and  different  colonies  the  proportion  ranged  from  10  per  cent 
or  even  less  to  nearly  100  per  cent.  In  some  larvae  coccoid  cells  pre- 
dominated (Plate  6,  A),  in  others  moderately  long  rods  were  most 
numerous  (Plate  6,  B),  and  occasionally  long,  slender,  faintly  staining 


•J  K  L 

Bacterial  Forms  from  Larvae  Infected  with  European  Foulbrood 

(x  1,500) 
A,  B,  and  C,  Smears  from  the  stomach  of  different  larvae  in  an  advanced  stage 
of  infection,  showing  difference  in  morphology  of  the  bacteria.     In  A  only  a  few 


June,  '34]     burnside  :  bacteria  associated  with  European  foulbrood  659 

rods  were  present  in  small  numbers  (Plate  6,  C).  Thus  it  is  apparent 
that  the  morphological  forms  encountered  in  sick  larvae  present  a  com- 
plex and  variable  picture. 

When  bacterial  growth  occurred  after  death  of  larvae,  it  usually  con- 
sisted almost  entirely  of  moderate-sized  rods,  of  which  a  variable  per- 
centage formed  spores  of  B.  alvei.  (Plate  6,  D.)  Occasionally  coccoid 
bacilli  indistinguishable  morphologically  from  bacilli  that  grow  in  the  di- 
gestive tract  of  sick  larvae  caused  decay  of  the  body  tissues  after  death. 
In  still  other  larvae  decay  was  caused  by  both  the  rod  and  the  coccoid 
form. 

Cultures  from  Sick  or  Dead  Brood  Yielded  Different  Morph- 
ological Forms. — Rough  inoculation  of  bouillon  agar  slants  from  sick 
or  dead  brood  most  frequently  yielded  cultures  of  B.  alvei  which  sporu- 
lated  promptly.  Many  cultures,  particularly  those  prepared  from  the 
digestive  tract  of  larvae  in  an  early  stage  of  infection,  yielded  a  coccoid 
organism  in  apparently  pure  culture  which  morphologically  and  cul- 
turally closely  resembled  5".  apis,  and  there  seems  to  be  little  doubt  that 
it  is  identical  with  the  form  described  by  Maassen  (P)  in  1908  and  later 
studied  by  White  (^11, 12, 13),  Wharton  {14),  and  Lochhead  (5).  On 
egg-yolk  agar  many  cells  of  this  form  become  lancet-shaped  and  were 

of  the  cells  are  still  dividing;  the  majority  are  coccoid  with  rounded  ends,  while 
some  are  more  or  less  pointed.  In  B  the  coccoid  cells,  short  rods,  and  medium 
long  rods  are  about  equally  numerous.  In  C  most  of  the  cells  have  pointed  ends 
and  are  typical  of  the  type  known  as  Bacillus  pluton;  two  long,  slender  rods,  such 
as  occur  in  small  numbers  in  infected  larvae,  are  also  seen. 

D,  Spores  of  Bacillus  alvei  from  the  decayed  remains  of  a  larva  dead  of  European 
foulbrood. 

E,  Pure  culture  of  Streptococcus  apis  from  agar  culture  containing  unheated  egg 
yolk.  In  some  cultures  50  per  cent  or  more  of  the  cells  become  more  or  less 
pointed  and  are  indistinguishable  morphologically  from  Bacillus  pluton. 

F,  Bacillus  alvei  and  Streptococcus  apis  from  an  agar  culture  prepared  directly 
from  a  sick  larva.     (2  days  at  36°  C.) 

G,  Streptococcus  apis  from  a  culture  prepared  directly  from  a  sick  larva  in  brood 
filtrate.     (2  days  at  36°  C.) 

H,  Asporogenic  agar  culture  of  Bacillus  alvei,  which  morphologically  closely 
resembles  Bacterium  eurydice.    (5  days  at  20°  C.) 

/,  Threadlike  rods  from  an  asporogenic  agar  culture  of  Bacillus  alvei.  (17  days 
at  20°  C.) 

/,  Rods  from  an  asporogenic  bouiUon-agar  culture  of  Bacillus  alvei  with  beaded 
and  granular  protoplasm.     (S  days  at  36°  C.) 

K,  Culture  of  Streptococcus  apis  in  bouillon  broth,  showing  rods  of  B.  alvei 
which  appeared  after  6  days  at  36°  C. 

L,  Pure  culture  of  Streptococcus  apis  from  bouillon  agar  to  which  10  per  cent 
honey  was  added.     (,36°  C.) 


660  JOURNAL  OF  ECONOMIC  ENTOMOLOGY  [Vol.  27 

indistinguishable  morphologically  from  B.  plitton.  (Plate  6,  E.)  Many 
cultures  yielded  both  B.  alvei  and  6".  apis.  (Plate  6,  F)  In  some  cul- 
tures the  cells  of  the  coccoid  form  were  observed  to  be  dissociated.  Oc- 
casional cultures  of  B.  alvei  prepared  from  sick  or  dead  brood — the  rela- 
tive number  varied  in  different  samples  of  infected  brood  comb — grew 
slowly  and  sporulation  was  delayed  and  incomplete.  In  a  few  instances 
rough  inoculation  from  affected  brood  yielded  cultures  of  rods  which 
did  not  form  spores  at  all  when  cultivated  at  room  temperature.  Cul- 
tures of  asporogenic  rods  were  also  obtained,  some  of  which  closely  re- 
sembled B.  enrydice,  by  plating  directly  from  sick  larvae  at  room 
temperature. 

In  a  few  cultures  on  bouillon  agar  or  egg-yolk  agar  prepared  with 
bacteria  from  the  digestive  tract  of  sick  larvae  no  growth  was  detected. 
From  the  same  larvae,  however,  prompt  and  abundant  growth  was  ob- 
tained in  dilute  sterile  filtrate  prepared  from  macerated  honeybee  larvae. 
In  filtrate  medium  a  coccoid  organism  resembling  .S".  apis  (Plate  6,  G) 
was  usually  obtained,  but  some  cultures  yielded  also  small  or  moderate- 
sized  rods.  It  is  evident  that  failure  to  obtain  growth  on  ordinary  nu- 
trient agar  does  not  prove  the  absence  of  culturable  bacteria. 

Cultures  from  healthy-appearing  larvae  from  different  infected  colo- 
nies yielded  in  a  variable  percentage  of  the  tubes  apparently  one  or  an- 
other of  the  same  forms  obtained  in  cultures  from  sick  or  dead  larvae 
{B.  alvei  or  S.  apis).  When  combs  of  brood  were  removed  from  colonies 
shortly  after  infection  had  subsided  and  were  kept  either  at  room 
temperature  or  at  36°  C,  none  of  the  larvae  dying  of  starvation  or 
chilling  were  noticeably  decayed  by  B.  alvei,  even  though  this  organism 
was  found  by  cultural  tests  to  be  present  in  the  digestive  tract  of  more 
than  90  per  cent  of  them. 

Bacteria  Present  in  Honey  from  Infected  Colonies. — Bacillus 
alvei  was  found  to  be  abundant  in  honey  and  pollen  from  the  brood 
chamber  of  infected  colonies.  In  advanced  cases  inoculations  of  nutrient 
agar  with  a  single  loopful  of  honey  (about  0.001  cc)  practically  always 
yielded  B.  alvei,  while  a  few  also  yielded  5".  apis.  In  early  or  mild  cases 
part  of  the  cultures  prepared  with  honey  or  pollen  yielded  B.  alvei. 

Bacteria  Found  in  Colonies  with  European  Foulbrood  Not 
Present  in  Healthy  Colonies. — In  striking  contracts  to  the  preva- 
lence of  bacteria  in  larvae  from  infected  colonies  is  the  complete  absence 
of  these  forms  in  healthy  colonies.  The  writer  has  made  microscopical 
examinations  of  and  prepared  cultures  numbering  well  into  the  thousands 
from  larvae  dead  of  American  foulbrood,  sacbrood,  fungus  diseases,  plant 
poisoning,  and  other  brood  disorders,  as  well  as  from  healthy  larvae. 


June,  '34]     burnside  :  bacteria  associated  with  European  foulbrood  661 

without  having  found  or  obtained  B.  alvei  or  6".  apis  in  culture,  except 
on  rare  occasions  when  mixed  infection  was  suspected.  Lilcewise  cul- 
tures prepared  with  honey  and  pollen  from  healthy  colonies  in  which 
European  foulbrood  never  existed  have  never  yielded  B.  alvei. 

The  writer's  observations  on  this  point  differ  from  those  of  Maassen 
(P),  who  claims  to  have  found  B.  alvei  present  in  some  cases  in  larvae 
dead  of  "virulent  foulbrood"  (American  foulbrood).  In  a  few  cases 
the  writer  obtained  B.  alvei  in  cultures  from  combs  infected  with  Amer- 
ican foulbrood,  but  a  thorough  inspection  of  the  brood  xomb  and  of  the 
scales  used  in  preparing  the  cultures  generally  revealed  mixed  infection 
and  occasionally  a  scale  of  European  foulbrood  which  resembled  that  of 
American  foulbrood.  It  seems  possible  that  Maassen  may  likewise  have 
been  dealing  with  cases  of  mixed  infection. 

Transmission  of  European  Foulbrood  with  Cultures. — When 
conditions  are  favorable,  typical  European  foulbrood  is  readily  trans- 
mitted by  inoculation  with  bacteria  taken  from  the  digestive  tract  of  sick 
or  dead  brood.  On  the  other  hand,  typical  European  foulbrood  has 
only  rarely  been  produced  by  inoculation  with  cultures,  although  several 
investigators,  in  inoculation  experiments  with  cultures  of  B.  alvei 
(rods  and  spores),  have  obtained  an  atypical  infection.  In  the  writer's 
experiments  an  occasional  larva  or  pupa  was  attacked  by  B.  alvei  when  a 
water  suspension  of  sporulating  cultures  recently  isolated  from  infected 
brood  was  sprayed  over  developing  brood.  It  appears  that  B.  alvei  in  the 
usual  sporogenic  state  may,  under  favorable  circumstances,  produce 
disease  in  larvae  or  pupae,  but  this  disease  is  not  typical  European  foul- 
brood. 

Likewise,  attempts  to  produce  European  foulbrood  by  inoculation  with 
pure  cultures  of  5".  apis  have  usually  been  unsuccessful.  Maassen  (9) 
failed  to  demonstrate  pathogenesis  for  6".  apis  by  feeding  pure  cultures, 
and  White  {12)  states  that  "No  disease  results  when  the  brood  of  bees 
is  fed  cultures  of  Streptococcus  apis  either  by  the  direct  or  indirect 
method."  In  speaking  of  the  coccoid  form  of  B.  alvei,  Lochhead  (d) 
states,  "Our  attempts  to  produce  the  disease  in  a  colony  of  black  bees 
through  feeding  cultures  of  the  coccus  have  so  far  been  inconclusive." 
On  the  other  hand,  Wharton  {14),  in  inoculation  experiments  with  cul- 
tures of  a  coccoid  bacillus  which  Lochhead  (5)  says  "appeared  to  be 
closely  related  to,  if  not  identical  with.  Streptococcus  apis,"  claims  to 
have  produced  typical  European  foulbrood.  Concerning  this  experiment 
Wharton  says,  "The  writer  has  obtained  infection  in  a  healthy  colony  of 
black  bees  in  four  days,  using  as  inoculum  cultures  of  the  organism  de- 
rived from  isolated  colonies.     The  symptoms   of  the   diseased  larvae 


662  JOURNAL  OF  ECONOMIC  ENTOMOLOGY  [Vol.  27 

accorded  with  those  observed  in  naturally  infected  larvae  and  the  micro- 
scopical picture  was  typical — B.  alvei  forms  being  also  present,  though 
only  in  small  numbers."  If  Wharton's  cultures  were  pure,  as  he  as- 
sumes, to  him  belongs  the  credit  of  first  producing  typical  European 
foulbrood  by  inoculation  with  pure  cultures. 

The  writer's  inoculation  experiments  with  5".  apis  and  with  non-spore- 
forming  rod  cultures  (resembling  B.  eurydice)  isolated  from  sick  or  dead 
brood  gave  results  that  were  largely  negative  or  inconclusive.  On  one 
occasion  typical  European  foulbrood  was  produced  by  inoculation  with 
cultures  of  6".  apis  freshly  isolated  from  sick  larvae.  In  isolating  pure 
cultures  plating  was  ordinarily  done  two  or  more  times.  Occasionally 
larvae  inoculated  with  such  cultures  appeared  to  become  infected  and 
were  removed  by  the  bees,  but  the  symptoms  were  not  typical  of 
European  foulbrood  and  the  infection  disappeared  promptly. 

In  an  experiment  performed  in  1933,  bacteria  from  the  digestive  tract 
of  a  naturally  infected  larva  were  streaked  on  egg-yolk-agar  plates. 
After  24  hours  at  34°  C.  isolated  colonies  of  S.  apis  were  touched  with  a 
platinum  loop  and  cultures  were  prepared  on  egg-yolk-agar  slants.  With 
the  abundant  growth  obtained  on  these  slants  after  44  hours  at  36°  C, 
a  colony  of  black  bees  was  inoculated  by  spraying  the  bacteria,  in  water 
suspension,  over  two  combs  of  young  and  hatching  larvae.  Two  days 
later  numerous  coccoid  bacteria  were  found  within  the  digestive  tract 
of  some  of  the  larvae.  On  the  following  day  coccoid  bacteria  had  greatly 
increased  in  number  in  many  of  the  inoculated  larvae  and  larvae  were 
being  removed  rapidly  by  the  bees.  On  the  fourth  day  more  than  90 
per  cent  of  the  inoculated  larvae  had  been  removed.  None  of  those 
remaining  showed  outward  symptoms,  but  upon  microscopical  exami- 
nation coccoid  bacteria  morphologically  identical  with  the  bacteria  in 
the  inoculum  were  so  abundant  within  the  digestive  tract  that  infection 
could  be  definitely  ascertained.  All  the  unsealed  brood  in  the  inoculated 
combs  was  finally  removed  by  the  bees  and  no  dead  larvae  were  found 
in  the  cells. 

A  water  suspension  of  bacteria  from  the  artificially  infected  larvae  was 
next  sprayed  over  another  comb  of  young  brood  in  the  same  colony. 
After  3  days  fully  25  per  cent  of  the  inoculated  larvae  in  this  comb  were 
dead  or  dying  from  infection,  of  which  the  gross  symptoms  and  the 
bacteriological  picture  were  typical  of  European  foulbrood.  Pointed  or 
lancet-shaped  cells  {B.  pluton)  were  at  first  absent  or  present  only  in 
small  numbers,  but  later  they  became  numerous.  The  coccoid  bacillus 
was  reisolated,  but  out  of  about  100  cultures  B.  alvei  was  obtained  in 
only  one.     With  cultures  prepared  by  rough  transfer  from  those  with 


June,  '34]     burnside  :  bacteria  associated  with  European  foulbrood  663 

which  infection  was  obtained  three  succeeding  experiments  gave  negative 
results.  The  results  of  this  experiment  and  the  comparable  experiment 
performed  by  Wharton  (14)  seem  to  point  to  retention  of  virulence  by 
5".  apis  during  only  about  two  generations  on  artificial  culture  media.  It 
is  recognized,  however,  that  the  purity  of  such  recently  isolated  cultures 
may  be  questioned. 

Pleomorphism  and  Variability  in  Bacillus  alvei.  Several  in- 
vestigators have  observed  variation  in  size  and  shape  of  individual  cells 
in  cultures  of  B.  alvei.  Maassen  (P)  says  that  cultures  of  B.  alvei 
degenerate  on  the  usual  artificial  medium  and  that  nuclei  or  granules 
develop  in  the  plasma  while  the  ability  to  form  spores  disappears.  Loch- 
head  (5,  6),  using  a  special  nutrient  agar,  observed  the  origin  of  coccoid 
cells  from  rods  of  B.  alvei,  which  he  reported  (6)  to  be  indistinguishable 
morphologically  from  B.  pluton. 

In  the  writer's  experiments  B.  alvei,  in  the  form  in  which  it  is  usually 
isolated  from  dead  brood,  grew  luxuriantly,  spread  rapidly  over  the 
agar,  and  formed  spores  promptly  and  abundantly  on  bouillon  agar  and 
on  egg-yolk  agar  at  36°  C.  (Plate  7,  A.)  In  repeated  transfers  at  36° 
C.  on  these  agars  no  morphological  or  cultural  changes  were  observed. 
In  bouillon  broth,  potato  broth,  and  milk,  and  in  media  containing  sterile 
filtrate  prepared  from  honeybee  larvae,  the  luxuriance  of  growth  and  the 
tendency  to  form  spores  gradually  decreased  in  repeated  transfers.  After 
about  10  generations  in  potato  broth,  cultures  prepared  by  rough  trans- 
fers to  bouillon  agar  and  egg-yolk  agar  grew  slowly  while  "sporulation 
was  incomplete  and  delayed  or  lacking.  Growth  either  spread  slowly  or 
was  confined  to  small  colonies.  (Plate?,  S.)  By  planting  and  culturing 
from  isolated  colonies,  strictly  asporogenic  cultures  were  obtained  which 
in  repeated  transfers  remained  asporogenic.  When  cultured  at  room 
temperature  the  transformation  in  potato  broth  from  a  sporogenic  to  an 
asporogenic  condition  was  more  rapid.  Bouillon  broth  seemed  less 
effective  in  producing  the  change,  and  results  with  filtrates  from  honey- 
bee larvae  were  irregular. 

These  asporogenic  cultures  of  B.  alvei  varied  in  morphology  and  cul- 
tural characteristics  (Plate  6,  H,  I,  J),  but  in  some  cases  the  resemblance 
to  cultures  of  asporogenic  rods  isolated  by  plating  from  sick  larvae  was 
marked.  It  seems  probable,  therefore,  that  B.  alvei  may  exist  in  infected 
larvae  in  either  sporogenic  or  asporogenic  condition. 

Morphologically  and  culturally  the  characteristics  of  some  of  the  cul- 
tures were  indistinguishable  from  the  characteristics  given  by  White 
(11)  for  B.  eurydice.  (Plate  7,  B;  plate  6,  H.)  Concerning  this  form 
White  (13)  says:  "In  studying  this  species  cultures  were  isolated  which 


Plate  7 


Types  of  Growth  of  Bacillus  alvei 

A,  Two  spreading  colonies  of  Bacillus  alvei  on  a  bouillon- 
agar  plate,  showing  difiference  in  type  of  growth. 

B,  Bacillus  alvei  on  glucose-agar  plate  growing  in  small 
colonies  after  transformation  from  a  sporogenic  to  an 
asporogenic   condition. 


June,  '34]     burnside  :  bacteria  associateu  with  European  foulbrood  665 

in  some  respects  differed  from  it.  Whether  these  are  different  species 
or  belong  to  a  group  of  which  B.  eurydice  is  a  representative  has  not  been 
definitely  determined."  Concerning  methods  of  culture  White  further 
says :  "Incubation  must  be  carried  out  at  room  temperature.  Growth  of 
the  species  is  always  slow  and  never  luxuriant."  In  view  of  the  writer's 
observations  it  seems  probable  that  the  culture  described  by  White  as 
B.  eurydice  and  cultures  which  "in  some  respects  differed  from  it"  may 
have  been  asporogenic  variants  of  B.  alvei. 

The  variability  of  B.  alvei  in  morphology  and  cultural  characteristics 
appeared  to  depend  upon  the  physiological  condition  of  the  organism  as 
well  as  upon  the  culture  medium.  To  retain  viability  of  cultures  frequent 
transfers  were  necessary.  The  description  of  the  organism  given  below 
is  of  cultures  produced  as  follows :  Agar  slant  cultures  of  sporogenic 
B.  alvei  were  prepared  from  isolated  colonies.  A  water  suspension  of 
spores  was  boiled  for  3  to  5  minutes,  after  which  the  organism  was,  cul- 
tured by  transferring  for  10  generations  in  potato  broth.  Cultures  pre- 
pared from  isolated  colonies  on  agar  plate  by  transfer  to  nutrient  agar  on 
which  sporulation  is  ordinarily  prompt  were  then  asporogenic  at  room 
temperature. 

Glucose-agar  plate. — Colonies  slightly  convex  and  rounded  with  uni- 
form outline,  1  to  2  mm  in  diameter,  grayish  by  reflected  light,  bluish 
gray  by  transmitted  light ;  under  a  binocular  appearing  very  light  brown 
and  finely  granular. 

Morphology. — Variable;  rods  nonmotile  and  asporogenic,  occurring 
singly,  in  pairs,  or  in  chains,  ends  rounded ;  protoplasm  homogeneous  or 
granular  or  broken ;  smaller  and  more  slender  than  sporogenic  B.  alvei 
in  some  cultures,  of  equal  dimensions  in  others. 

Staining  properties. — Stained  readily  with  the  usual  dyes  and  Gram- 
negative  ;  gra:nules  sometimes  darkly  staining  and  Gram-positive. 

Oxygen  requirements. — Growth  Occurring  under  anaerobic  conditions 
but  more  luxuriant  in  the  presence  of  air. 

Bouillon. — Medium  slightly  clouded  after  48  hours,  a  slightly  viscid 
sediment  forming  slowly  at  bottom  of  tubes. 

Sugars. — With  the  usual  sugars  acid  but  no  gas  produced ;  both  arms 
of  tube  clouded,  but  growth  most  luxuriant  in  open  arm;  litmus  dis- 
charged. 

Brood  filtrate. — In  some  cultures  brood  filtrate  added  to  the  medium 
increased  growth,  but  in  other  cultures  no  effect  observed ;  growth  also 
variable  in  water  solution  of  filtrate. 

Milk.- — Slight  growth  with  little  or  no  change  apparent  in  either  litmus 
milk  or  plain  milk. 


666  JOURNAL  OF  ECONOMIC  ENTOMOLOGY  [Vol.  27 

Potato  broth. — Growth  slow,  with  sHght  uniform  clouding  and  slight 
sediment. 

Potato. — Feeble,  grayish  growth. 

Gelatine  stab. — No  liquefaction. 

In  asporogenic  cultures  of  B.  alvei  coccoid  bodies  were  observed  which 
morphologically  resembled  the  coccoid  bodies  observed  by  Lochhead  ((5), 
but  attempts  to  isolate  this  form  have  thus  far  been  unsuccessful. 

In  recently  formed  asporogenic  cultures  the  protoplasm  (from  bouillon 
or  glucose  agar)  was  usually  homogeneous.  After  several  transfers, 
especially  on  egg-yolk  agar,  the  protoplasm  often  became  granular  or 
broken.  At  times  the  rods  assumed  a  beaded  appearance  resembling 
chains  of  coccoid  cells.  One  culture  in  brood-filtrate  medium  assumed 
a  decided  coccoid  appearance  with  many  forms  indistinguishable  mor- 
phologically from  chains  of  coccoids  observed  in  cultures  of  5".  apis.  Rods 
were  frequently  observed  in  a  state  of  dissociation,  and  in  some  cultures 
few  rods  remained  undissociated  after  4  or  5  days'  incubation. 

Pleomorphism  in  Streptococcus  apis.  —  On  ordinary  bouillon  agar 
.y.  apis,  when  freshly  isolated,  appears  in  diplococcoid  form  with  occa- 
sional single  cells  and  short  chains.  The  cells  are  only  rarely  spherical, 
their  length  being  usually  aproximately  lj4  times  their  thickness.  In 
bouillon  broth  the  tendency  to  grow  in  chains  is  accentuated,  while  on 
nutrient  agar  containing  egg  yolk  the  cells  are  smaller  than  in  bouillon 
agar  and  appear  singly  or  in  pairs.  The  ends  are  sharply  rounded  and 
frequently  pointed,  many  forms  being  morphologically  indistinguishable 
from  B.  pluton.  In  some  of  the  cultures  on  egg-yolk  agar  approximate- 
ly 50  per  cent  of  the  cells  became  more  or  less  pointed  after  multiplication 
ceased.  (Plate  6,  E.)  After  prolonged  cultivation  further  changes  in 
morphology  have  been  observed  from  time  to  time. 

Wharton  {14)  reported  that  his  morphological  studies  suggest  the 
identity  of  B.  pluton  and  B.  alvei  and  stated  that  "Cultures  of  B.  pluton 
have  been  observed  to  change  to  B.  alvei  form,  resembling  biologically 
the  B.  alvei  isolated  from  infected  larvae."  In  a  few  instances  the 
writer's  cultures  of  6".  apis  derived  originally  from  isolated  colonies  have 
yielded  rods  (Plate  6,  K)  and  eventually  spores  of  B.  alvei.  This  has 
been  observed  only  in  broth  cultures  prepared  by  transfer  from  old 
cultures  on  nutrient  agar.  After  incubation  for  7  to  12  days  at  27°  C, 
rods  of  B.  alvei  appeared  in  small  numbers,  but  nothing  was  determined 
concerning  their  origin.  Spores  were  produced  in  the  original  broth 
cultures  and  in  transfers  on  nutrient  agar.  On  other  occasions  rods  that 
failed  either  to  grow  or  to  produce  spores  in  transfers  originated  in 
broth  cultures.    Occasionally  rods  with  length  equal  to  about  five  times 


June, '34]     burn  side:  bacteria  associated  with  European  toulbrood  667 

their  thickness,  shorter  rods,  coccoid  cells,  and  lancet-shaped  cells  were 
observed  in  the  same  chains  in  broth  cultures  of  5".  apis.  In  cultures  on 
bouillon  agar  to  which  10  per  cent  honey  was  added,  some  of  the  cells 
were  increased  in  size,  many  were  distinctly  rod-shaped,  while  others 
assumed  lancet  shapes  indistinguishable  from  B '  alvei  ( Plate  6,  L ) . 
In  broth  cultures  of  5'.  apis  containing  both  honey  and  unheated  egg 
yolk,  several  variants  were  observed  after  2  days,  including  large,  irregu- 
lar, barrel-shaped,  and  spherical  cells,  occurring  usually  in  pairs  or 
in  chains. 

Conclusions. — Several  morphologically  different  bacteria  forms  are 
more  or  less  constantly  present  in  honeybee  larvae  sick  or  dead  of 
European  foulbrood.  These  forms  are  absent  in  larvae  sick  or  dead  of 
other  causes. 

No  evidence  has  yet  been  obtained  which  satisfactorily  explains  the 
etiology  of  European  foulbrood  or  why  these  different  bacterial  forms 
are  constantly  associated  with  this  disease. 

It  has  been  found  that  Bacillus  alvei  is  capable  of  morphological,  cul- 
tural, and  biological  transformation  and  is  also  capable  of  stabilization, 
at  least  temporarily,  as  a  sporogenic  rod,  an  asporogenic  rod  resembling 
Bacterium  eurydice,  or  a  coccoid  resembling  Bacillus  pluton. 

There  seems  to  be  insufficient  reason  for  assuming  that  the  lancet- 
shaped  bacterial  cell,  B.  pluton,  found  in  late  stages  of  infection  in 
European  foulbrood,  is  of  different  genus  and  species  from  the  similar 
form  Streptococcus  apis,  which  is  readily  obtained  in  culture  from  sick 
larvae. 

The  identity  of  Streptococcus  apis  and  Bacillus  pluton  is  suggested  by 
morphological  similarity,  by  the  fact  that  the  pointed  or  lancet  shape  is 
a  variable  character  in  both  forms  and  appears  to  be  only  an  expression 
of  restricted  growth  or  dormancy  accentuated  in  infected  larvae,  and 
also  by  the  usual,  if  not  invariable,  occurrence  of  Streptococcus  apis  in 
recently  infected  larvae,  and  by  the  fact  that  typical  European  foul- 
brood was  produced  in  Wharton's  and  in  the  writer's  experiments  when 
young  brood  was  inoculated  with  cultures  of  6".  apis  prepared  with 
isolated  colonies. 

That  Bacillus  pluton  and  Streptococcus  apis  are  variants,  or  stages  in 
the  life  history,  of  Bacillus  alvei  is  suggested  by  the  occurrence  of  vari- 
ants resembling  B.  pluton  in  pure  cultures  of  B.  alvei  and  by  the  apparent 
origin  on  rare  occasions  of  sporogenic  B.  alvei  in  cultures  5.  apis. 

The  transformation  at  room  temperature  of  sporogenic  B.  alvei  into 
an  asporogenic  nonmotile  rod  which  morphologically,  culturally,  and  bio- 


668  JOURNAL  OF  ECONOMIC  ENTOMOLOGY  [Vol.  27 

logicall)'  is  closely  allied  to  Bacterium  eurvdice  likewise  suggests  the 
identity  of  these  forms. 

Regarding  the  etiology  of  European  foulbrood  and  the  variety  of  bac- 
terial forms  present  in  sick  and  dead  larvae  much  remains  to  be  deter- 
mined. The  writer  is  of  the  opinion  that  the  evidence  now  available 
points  more  strongly  to  a  pleomorphic  organism  as  the  etiological  factor 
in  this  disease  than  to  the  secondary  organism  theory  advanced  by 
White. 

Literature  Cited 

1.  BoRCHERT,  A.     1926.     Die  seuchenhaften  Krankheiten  der  Honigbiene.     98  p., 

illus.    Berlin. 

2.  Cheshire,  F.  R.,  and  Cheyne,  W.  W.  1885.  The  pathogenic  history  and  his- 
tory under  cultivation  of  a  new  bacillus  (S.  alve'i),  the  cause  of  a  disease  of  the 
hive  bee  hitherto  known  as  foul  brood.  Jour.  Roy.  Micros.  Soc.  (2)  5  pt.  4)  : 
581-601. 

3.  Hadley,  p.  1927.  Microbic  dissociation.  The  instability  of  bacterial  species 
with  special  reference  to  active  dissociation  and  transmissible  autolysis.  Jour. 
Infect.  Diseases  40:  1-312,  illus. 

4.  Lehmann,  K.  B.,  and  Newman,  R.  O.  1927.  Bakteriologie,  inbesondere 
bakteriologische  Diagnostik.  II  Band.  AUgememeine  und  spezielle  Bakteriologie. 
876  p.      Munich. 

5.  LocHHEAD,  A.  G.  1928.  The  etiology  of  European  foul-brood  of  bees.  Science 
67:159-160. 

6.  .    1928.    Studies  on  the  etiology  of  European  foulbrood  of  bees.  4th 

Intern.  Cong.  Ent.  Trans.,  v.  2,  p.  1005-1009,  illus. 

7.  Lohnis,  F.,  and  Smith,  N.  R.  1916.  Life  cycles  of  the  bacteria.  Jour.  Agr. 
Research  6  :  675-702,  illus. 

8.  .  1923.     Studies  upon  the  life  cycles  of  the  bacteria — Part  II :  Life 

history  of  Azotobacter.    Jour.  Agr.  Research  23 :  401-432,  illus. 

9.  Maassen,  a.  1908.  Zur  atiologie  der  sogenannten  Faulbrut  der  Honigbienen. 
Arbeiten  K.  Biol.  Anst.  Land  u.  Forstw.  6:  53-70,  illus. 

10.  Mellon,  R.  P.     1926.  Studies  in  microbic  heredity.     VI.     The  infective  and 

toxonomic  significance  of  a  newly  described  ascospore  stage  for  the  fungi  of 
blastomycosis.    Jour.  Bact.  11:229-252,  illus. 

11.  White,  G.  F.     1912.     The  cause  of  European  foulbrood.     U.   S.  Dept.  Agr. 
Bur.  Ent.  Circ.  157,  IS  p.,  illus. 

12.  .     1920.     European  foulbrood.     U.   S.  Dept.  Agr.  Bui.  810,  39  p., 

illus. 

13-  .     1920.     Some  observations  on  European  foulbrood.     Amer    Bee 

Jour.  60 :  225-227,  266-268,  illus. 
14.  Wharton,  D,  R.  A.   1928.    Etiology  of  European  foul-brood  of  bees.     Science 

66:451-452. 


Reprinted  from  the  Journal  of  Economic  Entomology,  Vol.  14,  February,  1921,  No.  i 


MIXED  INFECTION  IN  THE  BROOD  DISEASES  OF  BEES 

By  Arnold  P.  Sturtevant,  Specialist  in  the  Bacteriology  of  Bee  Diseases,  Bureau  oj 
Entomology,  United  States  Department  of  Agriculture 

The  two  principal  brood  diseases  of  bees,  European  foulbrood  and 
American  foulbrood,  heretofore  have  not  been  found  associated  together 
commonly  in  the  same  colony.  The  generally  accepted  belief  has  been 
that  it  is  indeed  a  rare  occurrence  to  find  both  diseases  under  these 
conditions.  Sacbrood,  on  the  other  hand,  is  much  more  Often  found  in 
greater  or  less  quantity  associated  with  either  European  foulbrood  or 
American  foulbrood,  but  seldom  assuming  dangerous  proportions, 
either  alone  or  in  conjunction  with  the  others.  Statistics  for  the  past 
few  years,  however,  show  that  these  cases  of  what  may  be  called  mixed 
infection  are  probably  more  common  than  was  previously  supposed  and 
may  account  for  some  of  the  puzzling  instances  where  colonies  have  not 
responded  to  treatment  in  the  customary  manner,  thereby  causing 
beekeepers  to  believe  they  have  some  new  form  of  brood  disease,  or  that 
the  disease  is  showing  some  new  unheard  of  characteristics. 

Cases  of  so-called  mixed  infections  are  not  at  all  tmcommon  among 
human  diseases.  Where  this  condition  occurs,  such  as  when  a  person 
affected  with  typhoid  fever  develops  pneumonia  at  the  same  time,  it  is 
always  the  individual  to  whom  the  term  mixed  infection  is  applied. 
It  is  a  somewhat  different  matter  in  the  case  of  the  brood  diseases  of 
bees.  In  the  first  place,  so  far  as  is  known,  the  organisms  causing  these 
two  diseases,  Bacillus  larvae  of  American  foulbrood  and  Bacillus  pluton 
of  European  foulbrood,  have  never  been  found  together  in  the  same 
individual  larva.     It   is,  therefore,  the   colony   as  whole  which  is  to 


128  JOURNAL    OF    ECONOMIC    ENTOMOLOGY  [Vol.   14 

be  considered  as  the  individual  unit,  as  is  the  case  in  the  majority  of 
the  manipulations  of  beekeeping  practice.  This  fact  makes  the  problem 
slightly  different  from  a  case  of  mixed  infection  as  considered  from  the 
point  of  view  of  hiunan  medicine.  However,  since  different  individuals 
are  involved  in  the  mixed  infections  there  is  no  "a  priori"  reason  for 
considering  such  cases  as  impossible. 

The  first  published  report  of  an  authentic  instance  where  both  Ameri- 
can and  European  foulbrood  were  found  together  in  the  same  comb  from 
a  diseased  colony  was  reported  by  McCray.i  This  report  was  concern- 
ing a  sample  (4982)  received  at  the  laborator)-  for  diagnosis  May  4,  1916, 
from  Stanislaus  County,  California.  Previous  to  this  case  only  one  other 
such  sample  (2598  from  Brown  County,  Wisconsin  in  1911)  had  been 
received  for  diagnosis,  showing  the  presence  of  both  diseases,  but  no 
report  concerning  it  was  pubHshed.  These  two  samples  were  the  only 
known  authentic  cases  on  record  either  in  the  Bee-Culture  Laboratory 
among  practically  5000  samples  received  up  to  1916,  or  in  the  beekeeping 
literature.  These  two  cases  were  considered  to  be  interesting  in  that 
they  demonstrated  that  the  presence  of  both  diseases  at  the  same  time 
in  a  colony  was  possible,  but  not  much  importance  was  given  the  matter 
because  of  their  rare  occurrence.  White^  states  that  "such  a  double 
infection  has  been  encountered  in  the  writer's  experience  very  rarely. 
In  such  diagnoses,  therefore,  after  European  foulbrood  had  been  found 
in  the  sample,  American  foulbrood  is  seldom  looked  for."  This  practice 
has  been  the  custom  generally  as  well  when  American  foulbrood  was 
found  present  in  a  sample,  no  further  search  for  European  foulbrood 
being  made  unless  there  were  present  strikingly  prominent  symptoms 
abnormal  for  American  foulbrood.  As  a  result  the  diagnostic  records 
of  the  Office  of  Bee-Culture  show  but  six  cases  of  mixed  infection  up  to 
December  31,  1918,  among  the  approximately  6000  sample  records. 

Developments  during  the  year  1919,  however,  showed  that  mixed  or 
double  infection  is  more  probable  than  had  been  previously  supposed. 
These  facts  were  particularly  impressed  upon  the  writer  during  the 
spring  of  1919  while  on  a  trip  investigating  the  bee  disease  conditions  in 
the  State  of  CaUfomia.  While  in  the  field  during  a  period  of  less  than 
one  month,  and  in  three  different  counties  of  the  State  of  Cahfomia,  six 
cases  were  found  showing  both  American  foulbrood  and  European  foul- 
brood in  the  same  colonies.  Each  case  was  diagnosed  posivitely  at 
once  in  the  field  by  means  of  microscopic  examination  of  dead  larvae 
showing  characteristic  symptoms  of  the  two  diseases  and  found  to  con- 
tain the  specific  causative  organisms.     It  is  interesting  to  note  that  three 

'McCray,  A.  H.  1916.  Report  of  the  finding  of  American  Foulbrood  and  European 
foulbrood  in  the  same  comb.     Jour,  of  Eco.  Ext.  Vol.  IX,  p.  379. 

2 White,  G.  P.,  1920.     European  foulbrood .     U.  S.  Dept.  of  Agric.  Bui.  810. 


February,  '21] 


sturtevant:  mixed  infections 


129 


of  the  six  samples  were  found  in  Stanislaus  County  in  the  same  locality 
as  the  sample  reported  by  McCray  in  1916.  These  cases  were  all  found 
in  regions  where  both  diseases  are  exceedingly  prevalent  and  of  long 
standing.  A  few  of  the  samples  were  fairly  self  evident  from  gross 
appearances,  but  the  majority  required  a  more  minute  examination. 

From  that  time  on,  particularly  after  returning  to  the  laboratory  in 
Washington,  more  careful  examination  was  made,  both  gross  and 
microscopic  of  all  samples  received  because  of  suspicions  aroused  by 
the  unusual  prevalence  of  the  obvious  cases  found  in  California.  This 
was  done  in  order  to  eliminate  the  danger  of  overlooking  cases  where  one 
disease  might  be  predominant  over  the  other,  whether  both  diseases 
were  suspected  or  not,  causing  the  less  prominent  to  be  overlooked. 

As  a  result,  during  the  remainder  of  the  year  1919  from  June  until 
December,  twelve  more  such  samples  were  received  in  the  laboratory 
from  various  parts  of  the  country,  (18  in  all  for  that  year,  total  24)  all  of 
which  proved  upon  careful  diagnosis  to  contain  both  American  foul- 
brood  and  European  foulbrood  in  the  same  sample  of  comb.  Further- 
more, during  the  year  1920,  up  until  November  15th,  fourteen  more 
such  samples  were  received,  making  a  total  in  all  of  38.  Tables  1  and  2 
give  the  data  from  sample  records. 


Table  I. — Cases  of  Mixed  Infection  from  Laboratory  Records 


Apparent  primary 

Date 

Lab.  No 

..     State 

County 

invader  from  gross                   Remarks 

appearance 

9-20-11 

2598 

Wisconsin 

Brown 

? 

Diagnosed  by  G.  F.  White 

5-  4-16 

4982 

California 

Stanislaus 

American  fb. 

Diagnosed  by  A.  H.  McCray 

6-  3-16 

5061 

California 

Stanislaus 

American  fb. 

Diagnosed  by  A.  H.  McCray 

5-16-17 

5392 

Missouri 

Jasper 

Probably  Afb. 

5-  9-18 

5836 

Mississippi 

Washington 

? 

Apparently  about  equa 

10-  9-18 

6122 

Wisconsin 

Barron 

? 

More  Efb  than  Afb 

4-19-19 

6437 

California 

Santa  Barbara 

Probably  Efb. 

One  cell  Afb. 

4-26-19 

6441 

California 

Sacramento 

American  fb. 

From  history  of  case 

4-26-19 

6442 

California 

Sacramento 

American  fb. 

4-28-19 

6445 

California 

Stanislaus 

European  fb. 

Few  cells  Afb. 

4-30-19 

6449 

California 

Stanislaus 

American  fb. 

Pew  cells  Efb. 

5-  1-19 

6452 

California 

Stanislaus 

European  fb. 

From  history  of  case 

5-20-19 

6304 

Missouri 

Lewis 

? 

6-11-19 

6401 

Ohio 

Ashtabula 

? 

6-27-19 

6498 

Iowa 

Johnson 

American  fb. 

Efb  early  stages,  also  Sacbrood 

8-  1-19 

6629 

Ohio 

Trumbull 

? 

8-15-19 

6672 

Connecticut 

Tolland 

Probably  Efb. 

Afb  slight  amount 

8-25-19 

6698 

Kansas 

Cherokee 

? 

8-29-19 

6716 

New  York 

Cayuga 

American  fb. 

Efb  active  Afb  scales 

9-  2-19 

6721 

Washington 

Pacific 

7 

9-  2-19 

6722 

Washington 

Pacific 

? 

Efb  more  prominent 

9-19-19 

6768 

California 

Santa  Barbara 

7 

Afb  1st  disease  reported  for  county 

9-26-19 

6778 

California 

Santa  Barbara 

7 

10-  5-19 

6834 

California 

Santa  Cruz 

? 

5-12-20 

6985 

California 

Butte 

European  fb. 

Afb  one  or  two  cells 

5-29-20 

7023 

Michigan 

Calhoun 

? 

5-29-20 

7025 

Michigan 

Calhoun 

? 

5-29-20 

7026 

Wisconsin 

Fond  du  Lac 

European  fb. 

Few  cells  Afb 

6-17-20 

7119 

Washington 

Lewis 

5 

6-17-20 

7120 

Washington 

Lewis 

? 

Also  Sacbrood 

6-22-20 

7143 

New  York 

Allegany 

European  fb. 

Few  cells  Afb. 

6-24-20 

7158 

Pennsylvania 

,  Crawford 

? 

6-26-20 

7172 

New  York 

Cayuga 

? 

6-26-20 

7174 

New  York 

Cayuga 

? 

6-26-20 

7177 

Pennsylvania 

Crawford 

? 

7-21-20 

7335 

New  York 

Seneca 

Probably  Afb. 

8-  S-20 

7386 

Indiana 

Blackford 

? 

8-  5-20 

7387 

Indiana 

Blackford 

? 

130                                                JOURNAL    OF    ECONOMIC   ENTOMOLOGY  [Vol.  14 

Table  II. — Samples  of  Mixed  Infection  by  Years 

Samples  of  Total  Samples 

Year                                                                                                       mixed  infection  received 

1911 1  1042 

1916 2  374 

1917 1  449 

1918 2  429 

1919 18  693 

1920 14  698 


1905-1920  38  7568 

This  marked  apparent  increase  in  cases  of  mixed  infection  carries 
the  subject  over  from  one  of  scientific  interest  to  one  of  practical  im- 
portance. As  is  shown  in  Table  III,  the  38  samples  of  mixed  infection 
have  come  from  24  counties  in  thirteen  states,  most  of  these  located  in 
prominent  beekeepiag  regions.  In  eleven  of  these  thirteen  states  both- 
European  foulbrood  and  American  foulbrood  as  shown  by  samples  of 
disease  received  in  the  laboratory  for  diagnosis  are  prevalent  and  of 
long  standing.  There  are  only  about  three  or  four  other  states  where 
both  diseases  have  been  found  in  quantity  from  which  samples  of  mixed 
infection  have  not  been  received,  while  only  from  two  states  of  the 
many  where  the  diseases  are  only  occasionally  bad  have  such  samples 
been  received. 

Table  III. — Samples  of  Mixed  Infection  by  States  and  Counties 

State  Counties        Samples 

California 5  12 

Connecticut   

Indiana  

Iowa    

Kansas    

Michigan    

Mississippi   

Missouri   2  2 

New  York 3  5 

Ohio 2  2 

Pennsylvania 1  2 

Wisconsin  2  3 

Washington    2  4 

Statistics  obtained  from  the  sample  records,  however,  are  not  entirely 
conclusive  since  a  majority  of  the  samples  come  to  the  laboratory 
unsolicited.  If  a  careful  survey  could  be  made  of  the  regions  where  the 
brood  diseases  are  bad  and  widespread,  probably  many  more  such  cases 
would  come  to  light. 


February,  '21]  sturtevant;  mixed  infections  131 

Table  IV. — Distribution  of  Samples  of  Mixed  Infection  by  Months 

April    5 

May 9 

June 10 

July  1 

August 6 

September 5 

October 1 

November 1 

These  samples  of  mixed  infection  have  been  examined  in  eight  out  of 
the  twelve  months  of  the  year,  April  to  November  inclusive,  as  shown  in 
Table  IV.  Twenty -four  of  the  total  38  samples,  nearly  65  per  cent., 
were  examined  during  the  months  of  April,  May  and  June,  the  months 
during  which  European  foulbrood  is  most  prevalent.^  In  contrast  to 
the  spring  months,  eleven  samples  of  mixed  infection  were  examined 
during  August  and  September,  and  only  one  each  in  July,  October  and 
November,  a  total  of  fourteen. 

The  question,  however,  of  which  diesase  is  most  often  the  primary 
invader  in  a  colony  is  difficult  to  answer,  particularly  without  a  history 
of  the  colony  and  locality.  (Table  I) .  If  only  dried  adhesive  American 
foulbrood  scales  are  found,  accompanied  by  numerous  coiled  fresh  moist 
melting  larvae  of  European  foulbrood,  it  is  not  difficult  to  say  that 
American  foulbrood  was  the  primary  invader,  perhaps  during  the  pre- 
vious season,  as  was  the  case  of  the  sample  reported  by  McCray.  But 
often  there  is  no  such  demarkation.  Because  the  presence  of  American 
foulbrood  depletes  the  strength  of  the  colony  this  increases  the  probabil- 
ity of  European  foulbrood  infection. 

Since  the  requirements  of  the  treatment  of  the  two  diseases  are  so 
entirely  different,  the  necessity  for  correct  diagnosis  becomes  of  im- 
portance, particularly  in  regions  where  both  diseases  have  been  prevalent 
for  some  time.  The  presence  of  both  diseases  in  the  same  colonies  or 
even  in  the  same  apiary  is  a  complicating  factor  in  the  diagnosis  and 
treatment.  Furthermore  there  is  danger  from  the  possibility  of  con- 
tinued and  confusing  losses  due  to  the  ignorance  of  the  presence  of  mixed 
infection  in  colonies  under  such  circumstances  and  resulting  therefrom, 
improper  treatment  which  would  only  continue  the  losses. 

Several  samples  have  been  received  for  diagnosis  which  beekeepers 
have  thought  contained  both  diseases  and  which  indeed  seemed  to  have 
some  of  the  characteristics  of  each.  Upon  careful  examination,  however, 
both  gross  and  microscopic,  these  have  mostly  proven  to  be  definitely 
not  mixed  infections.     The  recognition  of  cases  of  mixed  infection  in 

'Phillips,  E.  p.,  1918.  The  control  of  European  foulbrood.  U.  S.  Dept.  of  Agnc. 
Farmers'  Bulletin  975,  16  pp. 


132  JOURNAL    OF   ECONOMIC   ENTOMOLOGY  [Vol.  14 

colonies  is  often  difficult  because  of  the  fact,  as  is  particularly  the  case 
with  European  foulbrood,  there  are  many  irregularities  and  variations 
in  symptoms  that  often  add  to  the  confusion  of  the  beekeeper  in  making 
gross  diagnosis  hurriedly  in  the  field.  In  order  to  more  easily  differen- 
tiate some  of  these  confusing  sjonptoms  to  assist  in  gross  diagnosis,  they 
may  be  divided  into  three  classes.  Occasionally  in  an  unusually  virulent 
case  of  American  foulbrood  or  in  one  where  the  bees  have  deserted  the 
brood  because  of  its  foul  condition  allowing  what  healthy  brood  there 
is  to  starve,  larvae  will  be  found  which  have  died  while  still  coiled  in  the 
cell,  among  the  typical  American  foulbrood  larvae.^  These  coiled 
larvae  often  have  much  the  same  appearance  as  typical  European  foul- 
brood coiled  larvae.  However,  the  consistency  is  generally  quite  dif- 
ferent from  European  foulbrood,  more  like  the  typical  slimy  glue-like 
consistency  of  American  foulbrood  material.  As  a  rule,  however,  the 
symptoms  of  American  foulbrood  are  uniformly  constant  because  of  the 
fact  that  Bacillus  larvae  is  almost  always  the  only  invader  of  the  larvae 
causing  death  and  a  type  of  decomposition  which  prevents  growth  of 
other  organisms.     Several  such  cases  were  found  in  California. 

A  second  class  of  confusing  sjanptoms  are  found  in  samples  which 
come  particularly  from  regions  where  European  foulbrood  has  been 
allowed  to  run  unchecked  for  a  long  time.  Such  samples  were  found 
in  certain  sections  of  California  and  have  been  received  from  various 
other  sections  of  the  country.  These  samples  show  along  with  more 
or  less  of  the  typically  coiled  European  foulbrood  larvae,  large  numbers 
of  larvae  which  have  died  after  extending  and  even  being  sealed  in  the 
cell,  showing  a  consistency  somewhat  like  that  of  American  foulbrood 
but  more  lumpy  or  like  an  old  partly  rotten  rubber  band.^  Sometimes 
scales  are  found  extended  in  the  cells  in  such  large  n-umbers  as  to  appear 
on  casual  examination  like  an  old  comb  of  American  foulbrood.  Close 
examination,  however,  shows  the  consistency,  irregular  shape  and  posi- 
tion with  lack  of  adherence  to  the  cell  wall  to  be  different  from  that  in 
American  foulbrood.  This  type  was  found  to  be  quite  prevalent  in 
California. 

The  third  class  is  composed  of  cases  of  actual  mixed  infection  where 
typical  American  foulbrood,  ropy  larvae  or  scales,  are  associated  in  the 
same  comb  with  typical  European  foulbrood,  coiled  moist  melting  larvae, 
or  possibly  occasionally  the  abnormal  rubbery  irregular  larvae  mentioned 
above.  The  active  stage  of  the  two  diseases  often  seems  to  be  locaKzed 
more  or  less  in  different  parts  of  the  comb.     This  is  probably  due  to 

*White,  G.  F.  1920.    American  foulbrood.     U.  S.  Dept.  of  Agric.  Bui.  No.  809. 
^Sturtevant,  A.  P. ,  1920.     A  study  of  the  behavior  of  colonies  affected  by  European 
foulbrood  of  bees.     U.  S.  Dept.  of  Agric.  Bui.  No.  804. 


February,  '21]  sturtevant:  mixed  infections  133 

the  fact  that  the  queen  would  tend  to  desert  that  section  of  the  comb 
containing  the  American  foulbrood,  particularly  where  this  disease  was 
the  primary  invader.  In  many  cases  one  or  the  other  of  the  diseases 
will  be  more  prominent,  at  least  in  the  active  stages.  This  fact  may 
be  one  of  the  causes  for  cases  of  mixed  infection  having  been  overlooked, 
the  beekeeper  seeing  only  the  prominent  outstanding  symptoms.  There- 
fore in  cases  where  there  is  doubt  or  suspicion  that  both  diseases  may  be 
present  in  the  same  colony,  a  positive  laboratory  diagnosis  often  appears 
to  be  desirable. 

As  is  well  known,  the  shaking  method  of  treatment  in  its  essentials 
is  so  far  the  only  successful  way  of  treating  American  foulbrood.'  The 
nature  of  Bacillus  larvae  has  prevented  success  along  any  other  line, 
because  of  its  ability  to  form  exceedingly  resistant  spores  and  especially 
to  decompose  the  dead  larva  in  such  a  way  as  to  cause  the  mass  contain- 
ing large  numbers  of  these  spores  to  adhere  to  the  cell  wall  as  if  glued. 
It  has  been  learned  furthermore,  often  by  sad  experience,  that  the 
shaking  treatment  is  practically  never  successful  in  the  treatment  of 
European  foulbrood;  in  fact,  often  when  used  causes  the  disease  to  be 
spread  all  the  more  because  of  the  weakening  effect  the  shaking  has  on  the 
colonies.'  The  requirements  for  the  successful  treatment  of  European 
foulbrood  have  been  found  to  be  fundamentally  dependent  upon  ade- 
quately strengthening  the  colonies  with  young  bees  sufficiently  to  throw 
off  the  disease,'  at  the  same  time  combined  with  the  requeening  of  the 
diseased  colonies  with  vigorous  young  Italian  queens,  permitting  the 
bees  themselves  to  remove  the  infected  material. 

The  apparent  logical  solution  of  the  problem  of  the  treatment  for  a 
known  case  of  mixed  infection,  therefore,  is  to  combine  the  treatments  for 
both  American  foulbrood  and  European  foulbrood  as  a  single  treatment. 
In  other  words,  the  one  or  more  colonies  known  or  strongly  suspected 
to  have  mixed  infection  should  be  shaken  as  for  American  foulbrood, 
requeening  them  with  vigorous  young  Italian  queens  and  later  strength- 
ening them  by  the  addition  of  young  bees  or  hatching  brood  from  a 
healthy  colony,  or  by  uniting  later.  Strength  of  colony  is  the  important- 
factor  combined  with  the  shaking  and  requeening  with  vigorous  Italian 
stock. 

The  problem  of  the  control  of  mixed  infections  of  American  foulbrood 
and  European  foulbrood  is  primarily  associated  with  the  control  of 
European  foulbrood.     In  localities  where  both  diseases  are  prevalent 


'Phillips,  E.  P.  1920.  The  control  of  American  foulbrood.  U.  S.  Dept.  of  Agric, 
Farmers'  Bulletin  No.  1084. 

'Phillips,  E.  F.  1918.  The  control  of  European  foulbrood.  U.  S.  Dept.  of  Agric, 
Farmers'  Bulletin  No.  975. 


134  JOURNAL   OF   ECONOMIC   ENTOMOLOGY  [Vol.  14 

and  there  is  suspicion  of  both  being  present  in  the  same  apiary,  and  pos- 
sibly even  some  as  mixed  infection  in  the  same  colony,  control  of  the 
two  diseases  will  depend  upon  the  elimination  of  European  foulbrood 
first.  This  should  be  done  by  treating  the  entire  apiary  for  European 
foulbrood,  by  strengthening  and  requeening  all  the  colonies  with  young 
and  vigorous  Italian  queens,  which  is  after  all  only  good  beekeeping. 
After  the  elimination  of  European  foulbrood  it  will  be  a  simple  matter 
to  determine  those  colonies  that  have  not  responded  to  this  treatment, 
as  being  American  foulbrood.  This  method  is  possible  because  of  the 
fact  that  American  foulbrood  seldom  spreads  with  the  rapidity  of  Euro- 
pean foulbrood,  particularly  if  care  is  taken  to  prevent  robbing  and  mixing 
up  of  combs.  Those  colonies  which  continue  to  show  American  foul- 
brood remaining  may  now  be  given  the  usual  shaking  treatment. 


K-228 


f 


RELATION  OF  COMMERCIAL  HONEY  TO  THE  SPREAD 
OF  AMERICAN  FOULBROOD 


BY 
A.  p.  STURTEVANT 

(Contribution  from  Bureau  of  Entomology) 


Reprinted  from  JOURNAL  OF  AGRICULTURAL  RESEARCH 

Vol.  45,  No.  S    :    :    :    Washington,  D.  C,  September  1,  1932 

(Pages  257-285) 


ISSUED  BY  AUTHORITY  OF  THE  SECRETARY  OF  AGRICULTURE 

WITH  THE  COOPERATION  OF  THE  ASSOCIATION  OF 

LAND-GRANT  COLLEGES  AND  UNIVERSITIES 


U.  S.  GOVERNMENT  PRINTING  OFFICE  :  1932 


JOINT  COMMITTEE  ON  POLICY  AND  MANUSCRIPTS 


FOE  THE   UNITED   STATES   DEPAETMENT       FOE  THE  ASSOCIATION  OF  lAND-GEANT 
OF  AGEICUITUEE  COIIEGES  AND   UNIVEESITIES 

H.  G.  KNIGHT,  Chairman  S.  W.  FLETCHER 

aief,  Bureau  „/  CKerai^ry  a^  Soil,  ^S^af^f/ZS^i.fS'^"'''  ^"^ 

F.  L.  CAMPBELL  S.  B.  DOTEN 

ETUomolosjist,  Bureau  of  Eittomology  Director,  Nevada  Agricultural  Experiment 

Station 

JOHN  W.  ROBERTS  C.  G.  WILLIAMS 

Senior  Patkologiat,  Bureau  of  Plard  Director,  Ohio  Agricultural  Experiment 

Iniuetry  Station 

EDITOEIAL  SUFEEVISION 

M.  C.  MERRILL 

Chief  of  Publicatiom,  United  States  Departmem.  of  Agriculture 


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RELATION   OF   COMMERCIAL  HONEY  TO  THE  SPREAD 
OF  AMERICAN  FOULBROOD ' 

By  A.  P.  Sttjrtevant  ' 

Associate  ApicuUurist,  Division  of  Bee  Culture,  Bureau  of  Entomology,  United  States- 
Department  of  Agriculture 

INTRODUCTION 

The  relation  and  importance  of  commercial  honey  to  the  spread  of 
American  foulbrood  of  bees  has  occupied  the  attention  of  the  bee- 
keeping industry  more  or  less  prominently  for  many  years.  The- 
theoiy  has  been  promulgated  that  honey  which  has  not  come  from, 
disease-free  apiaries  is  dangerous  because  of  the  possibility  of  its  dis- 
seminating American  foulbrood.  A  few  States  and  at  least  one  for- 
eign country  require  that  honey  intended  for  interstate  shipment  be 
accompanied  by  a  certificate  from  the  bee  inspector  of  the  State  in' 
which  the  honey  originated  to  the  effect  that  such  honey  was  produced^ 
in  apiaries  free  from  American  foulbrood. 

It  is  a  well-established  fact  that  honey  taken  directly  from  the 
combs  of  the  brood  chambers  of  colonies  affected  by  American  foul- 
brood is  capable  of  producing  the  disease  if  fed  to  healthy  colonies. 
Since  commercial  beekeeping  practice  bans  the  extracting  of  honey 
from  the  brood  nest,  it  is  difficult  to  understand  how  heavily  infected 
honey,  in  large  quantities,  could  get  on  the  market.  Whether  honey' 
from  supers  that  have  been  on  colonies  affected  with  American  foul- 
brood is  of  serious  importance  in  transmitting  the  disease  is  still  open 
to  question.  White  {SO,  p.  S5y  says:  "The  likelihood  that  the  disease" 
will  be  transmitted  by  combs  from  diseased  colonies,  which  contain 
honey  but  no  brood,  probably  is  frequently  overestimated."  On  the" 
other  hand,  Millen  {23)  found  that  combs  built  from  foundation  and 
completely  filled  above  an  excluder  with  honey  from  colonies  that 
had  been  destroyed  by  American  foulbrood  produced  disease  in  all  of 
10  colonies  made  from  package  bees  to  which  one  comb  each  of  the 
honey  had  been  given.  Corkins  {8)  expressed  the  belief,  as  a  result-' 
of  preliminary  studies,  that  "Extracted  honey  produced  above  an 
excluder  in  a  colony  in  the  early  stages  of  American  foulbrood  is" 
insignificant  in  the  spread  of  this  disease  through  commercial  honey."' 
The  conflicting  nature  of  these  observations  emphasizes  the  need  for 
further  research  before  the  certification  of  honey  is  required  as  a; 
means  of  alleviating  the  foulbrood  situation. 

In  both  animal  and  plant  disease  bacteriology  it  is  known  that 
pathogenic  microorganisms  may  vary  considerably,  even  within  indi- 

'  Eeceived  for  publication  Feb.  1, 1932;  issued  September,  1932. 

'  For  advice  and  assistance  the  writer  Ls  indebted  to  Profs.  C.  L.  Corliins  and  O.  H.  Gilbert,  of  the  Uni- 
versity of  Wyoming;  Prof.  K.  Q.  Hiohmond,  deputy  State  entomologist,  apiary  investigations,  Colorado' 
Agricultural  College;  H.  Bauohfuss,  of  Englewood,  Colo.;  N.  L.  Henthorne,  of  Greeley,  Colo.;  and  C.  H„ 
Banney,  of  Lander,  Wyo.  Appreciation  is  also  expressed  for  the  many  courtesies  extended  by  H.  C. 
Hilton,  supervisor  of  the  Medicine  Bow  National  Forest. 

'  Beference  is  made  by  number  (italics)  to  Literature  Cited,  p.  284. 

Journal  of  Agricultural  Eesearch,  Vol.  45,  No.  6  ■ 

Washington,  D.  C.  Sept.  1, 1932 

Key  No.  K-228 

(257) 


258  Journal  oj  Agricultural  Research  voi.  45,  No.  5 


vidual  species,  in  virulence  and  in  ability  to  produce  disease.     Fur- 
thermore, as  stated  by  Zinsser  {31,  p.  188-189) — 

Whether  or  not  infection  occurs  depends  also  upon  the  number  of  bacteria 
which  gain  entrance  to  the  animal  tissues.  A  small  number  of  bacteria,  even 
though  of  proper  species  and  of  sufficient  virulence,  may  easily  be  overcome  by 
the  first  onslaught  of  the  defensive  forces  of  the  body.  Bacteria,  therefore,  must 
be  in  sufficient  number  to  overcome  local  defenses  and  to  gain  a  definite  foothold 
and  carry  on  their  life  processes,  before  they  can  give  rise  to  an  infection.  The 
more  virulent  the  germ,  other  conditions  being  equal,  the  smaller  the  number 
necessary  for  the  production  of  disease.  The  introduction  of  a  single  individual 
of  the  anthrax  species,  it  is  claimed,  is  often  sufficient  to  cause  fatal  infection; 
while  forms  less  well  adapted  to  the  parasitic  mode  of  life  will  gain  a  foothold  in 
the  animal  body  only  after  the  introduction  of  large  numbers. 

In  the  case  of  American  foulbrood  the  quantity  of  infectious  mate- 
rial that  honey  must  carry  in  order  to  produce  disease  in  a  colony  has 
never  been  determined.  White  (SO,  p.  ^0,  footnote  1)  states,  in  con- 
nection with  inoculating  healthy  colonies  experimentally  with  Bacillus 
larvae: 

It  was  found  that  less  than  one  scale  is  sufficient  disease  material  to  produce  a 
considerable  amount  of  disease  in  the  colony.  In  some  experiments  one  scale, 
therefore,  might  supply  all  the  spores  needed  although  the  use  of  a  somewhat 
greater  quantity  of  material  is  advisable  in  most  instances. 

While  infected  honey  no  doubt  does  become  mixed  with  disease-free 
honey,  it  is  probable  in  many  cases  that,  because  of  the  practice  of 
using  large  settling  and  storage  tanks,  infected  honey  would  be  so 
diluted  with  spore-free  honey  as  to  make  the  spore  content  insufficient 
to  produce  infection  even  if  fed  to  healthy  bee  larvae.  Therefore,  one 
object  of  these  investigations  was  to  determine  the  minimum  number 
of  spores  of  Bacillus  larvae  in  honey  necessary  to  produce  American 
foulbrood  in  healthy  colonies  of  bees  as  correlated  with  the  infectivity 
or  spore  content  of  the  average  commercial  honey. 

In  order  to  obtain  information  relative  to  this  subject,  experiments 
were  conducted  in  the  apiary  over  a  period  of  five  years.  In  these 
experiments  honey  or  sugar  sirup  with  a  known  content  of  spores  of 
Bacillus  larvae  was  fed  to  healthy  colonies  and  the  minimum  number 
of  spores  that  would  produce  infection  was  determined.  At  the  same 
time  laboratory  studies  were  carried  on  with  cultures  of  spores  of  B. 
larvae,  concerning  certain  growth  phases  of  the  organism,  particularly 
the  minimum  number  of  spores  that  would  produce  vegetative  growth 
on  artificial  culture  media.  Methods  for  demonstrating  the  presence 
or  absence  of  spores  of  B.  larvae  in  samples  of  commercial  honeys  were 
also  investigated,  and  these  honeys  were  studied  in  relation  to  their 
infectiousness  as  correlated  with  the  spore-feeding  experiments. 
These  three  phases  of  the  investigation  will  be  discussed  in  the  order 
mentioned. 

MINIMUM  NUMBER  OF   SPORES    OF   BACILLUS    LARVAE    NECES- 
SARY TO  PRODUCE  DISEASE  IN  HEALTHY  COLONIES  OF  BEES 

methods  of  proceduee 

Location  of  Experiments 

These  investigations  were  started  during  the  summer  of  1926  in  a 
small  experimental  apiary  located  about  half  a  mile  from  the  bee 
culture  laboratory  of  the  Bm-eau  of  Entomology  at  Somerset,  Md. 
The  location  at  Somerset  was  undesirable,  however,  because  of  its 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foutbrood       253 

close  proximity  to  the  apiary  connected  with  the  laboratory  and  tO' 
other  privately  owned  colonies  of  bees,  necessitating  extreme  pre- 
cautions to  prevent  spread  of  the  disease.  In  1927  the  experimental 
work  was  transferred  to  the  Intermountain  States  bee  culture  field 
laboratory  at  Laramie,  Wyo.*  In  Wyoming  an  ideal  isolated  location 
was  foimd  about  14  miles  east  of  Laramie  in  the  Medicine  Bow  Na- 
tional Forest,  the  nearest  colonies  of  bees  being  at  least  14  miles  away 
and  probably  farther.  Since  this  location  is  more  than  8,000  feet 
above  sea  level,  there  is  only  a  slight  nectar  flow  from  wild  flowers, 
which  assures  the  immediate  use  of  any  inoculated  simp  fed  to  colonies 
of  bees.  In  fact,  after  the  middle  of  the  summer  it  was  found  neces- 
sary in  most  cases  to  feed  the  experimental  colonies  with  uninocu- 
lated  sugar  sirup  in  order  to  prevent  starvation. 

In  1927  and  1928  the  colonies  used  for  experimentation  were  located 
in  two  yards  between  a  quarter  and  a  half  mile  apart.  The  arrange- 
ment of  the  colonies  in  the  two  yards  was  such  as  to  prevent  drifting' 
as  rnuch  as  possible.  In  1929  and  1930,  in  order  to  limit  still  further 
the  danger  of  transmission  of  disease  because  of  drifting  or  robbing,. 
20  colonies  were  stationed  in  pairs,  so  arranged  as  to  minimize  the 
danger  from  drifting,  in  10  isolated  locations  at  least  a  quarter  of  a. 
mile  apart. 

Make-up  of  Colonies 

Five-frame  nucleus  hives  were  used  for  the  spore-feeding  experi- 
ments. The  colonies  were  prepared  either  with  two  or  three  frames, 
of  brood,  honey,  and  adhering  bees  taken  from  healthy  colonies, 
together  with  a  young  laying  queen,  or,  as  in  1927,  1928,  and  1929, 
by  placing  a  2-pound  package  of  bees  containing  a  laying  queen  on 
foundation  or  on  combs  containing  honey  from  healthy  colonies  and 
feeding  them  sugar  sirup.  During  a  good  honey  flow  these  small 
colonies  were  allowed  to  build  up  in  the  apiary  connected  with  the 
laboratory  until  they  consisted  of  three  or  four  frames  of  brood  before 
they  were  moved  to  the  isolated  locations.  The  bees  making  up  the 
colonies  used  for  the  feeding  experiments  from  1927  to  193Q  at  Lara- 
mie, Wyo.,  were  all  from  the  same  general  strain. 

Material  Used  for  Inoculation 

Spores  of  Bacillus  larvae  were  obtained  from  American  foulbrood 
scales  in  combs  taken  from  diseased  colonies  located  in  the  States  of 
Maryland,  Iowa,  and  Wyoming.  The  strain  used  at  Somerset,  Md., 
was  obtained  from  a  sample  sent  to  that  laboratory  for  diagnosis. 
Two  different  strains  were  used  at  Laramie  during  1927,  1928,  and 
1929,  one  obtained  from  a  diseased  colony  in  the  experimental  apiary 
belonging  to  the  University  of  W^yoming  and  one  obtained  from  a  bee- 
keeper at  Lander,  Wyo.  In  1930  three  other  strains  were  used  in 
the  feeding  experiments,  one  from  Iowa  and  two  from  apiaries  in 

Wyoming. 

Preparation  of  Spore  Suspensions 

In  preparing  the  spores  for  feeding  to  the  healthy  colonies,  scales 
were  removed  from  the  combs  by  means  of  sterile  forceps  (the  neces- 
sary precautions  being  taken  against  contamination)  and  placed  in 

<  This  laboratory  is  maintained  cooperatively  by  tbe  University  of  Wyoming  and  the  U.  S.  Department 
of  Agriculture. 


260  Journal  oj  Agricultural  Research  voi.  45,  No.  5 

a  flask  containing  50  c  c  of  sterile  water  and  glass  beads.  After  the 
scales  had  softened  in  the  water,  the  flask  was  shaken  for  one-halt 
hour  to  insure  complete  maceration  of  the  scales.  The  suspension 
was  then  filtered  through  two  thin  layers  of  sterile  absorbent  cotton 
into  another  sterile  flask  in  order  to  remove  any  lumps  or  debris. 

In  preparing  the  stock  suspensions  of  spores,  at  first  75  to  100 
scales  were  taken  by  counting.  Later  it  was  found  that  the  average 
American  foulbrood  scale  weighs  0.0223  g.  Therefore,  the  100  scales 
for  the  stock  suspensions  were  obtained  by  weight,  the  scales  bemg 
•weighed  in  a  sterile  covered  glass  dish  before  they  were  deposited 
in  the  flask  of  sterile  water. 

After  the  suspension  had  been  filtered  and  tested  for  contamma- 
tion  and  was  ready  for  use,  the  number  of  spores  per  cubic  centimeter 
was  determined  by  the  following  method:  By  means  of  a  blood- 
diluting  pipette  giving  a  dilution  of  1  to  20,  the  spore  suspension  was 
diluted  with  a  weak  solution  of  carbol  fuchsin  and  a  drop  placed  in 
the  counting  chamber  of  a  Helber  bacteria-counting  cell  0.02  mm 
deep  and  ruled  in  squares  of  0.0025  mm^  each.*  With  the  use  of 
two  15  X  eyepieces  in  a  binocular  microscope  and  a  1.8-mm  oil- 
immersion  objective,  the  spores  in  25  squares  of  the  Helber  chamber 
were  counted.     Then  by  means  of  the  formula 

Total  spores  counted  X  dilution  X  20,000  X  1,000 
Number  of  squares  counted 

the  approximate  number  of  spores  per  cubic  centimeter  in  the  sus- 
pension was  determined. 

Later  this  method  was  checked  by  the  method  of  Breed  and  Brew 
(2)  for  counting  bacteria  in  milk.  With  the  aid  of  a  binocular  micro- 
scope having  two  15  X  eyepieces  and  a  1.8  mm  oil-immersion  ob- 
jective, the  area  of  a  circle  etched  on  an  ocular  micrometer  disk  was 
determined  by  means  of  a  stage  micrometer.  One  one-hundredth 
cubic  centimeter  of  a  1  to  100  dilution  of  the  stock  suspension  of 
spores  was  placed  on  a  glass  slide  on  which  1  cm^  had  been  ruled 
with  a  diamond  pencil.  This  was  mixed  with  a  small  loopful  of 
carbol  fuchsin  stain  and  the  whole  spread  over  the  1  cm  ^  of  surface  * 
and  allowed  to  dry  uniformly.  The  number  of  spores  per  cubic 
centimeter  of  the  stock  suspension  was  determined  according  to  the 
formula 

Area  1  cm  ^  total  number  of  spores  counted  X  dflution  X  100. 

Area  of  circular  field  number  of  circular  fields  counted 

These  two  methods  were  found  to  check  fairly  closely  within  the 
limits  of  the  precision  of  the  methods  used  in  counting.  Further- 
more, by  both  methods  it  was  found  that  in  the  majority  of  cases  100 
scales  in  50  c  c  of  water  give  approximately  5,000,000,000  spores  per 
cubic  centimeter  for  each  suspension  made  up  in  this  way.  Therefore, 
this  number  was  used  as  a  standard  for  making  all  dilutions. 

»  Mm'  and  cm'  are  the  abbreviations  tor  square  millimeter  and  square  centimeter,  respectively,  recently 
adopted  by  the  Style  Manual  for  United  States  Government  printing. 


Sept,  1,1932     Commercial  Honey  and  Spread  qf  American  Foulbrood       261 

After  a  considerable  number  of  counts  had  been  taken  in  making 
up  several  stock  suspensions  of  spores,  counting  was  eliminated  and 
the  spore  content  of  the  stock  suspensions  was  standardized  according 
to  the  method  described  by  Gates  {11,  p.  114),  as  follows:  "The 
opacity  of  a  bacterial  suspension  is  measured  by  the  length  of  a  col- 
umn of  the  suspension  required  to  cause  the  disappearance  of  a  wire 
loop."  An  instrument  known  as  a  suspensiometer  was  used  for  this 
purpose.  The  use  of  this  method  saved  considerable  time  and  labor 
without  appreciably  affecting  the  precision  of  the  counts.  One  liter 
of  a  50  per  cent  solution  of  sugar  in  water  was  used  as  the  standard 
quantity  of  inoculated  sirup  fed  to  each  experimental  colony.  A 
series  of  dilutions  of  the  original  stock  suspension  containing  5,000,- 
000,000  spores  was  made  by  adding  different  quantities  of  the  spore 
suspension  to  1  liter  of  sugar  sirup.  In  this  way  the  approximate 
total  number  of  spores  in  each  liter  of  sugar  sirup  to  be  fed  to  colonies 
of  bees  was  known. 

Method  op  Inoculating  Colonies 

In  1926  at  Somerset,  Md.,  the  sugar  sirup  containing  the  various 
dilutions  of  spores  was  fed  to  the  colonies  by  means  of  galvanized-iron 
troughs  that  were  hung  inside  the  hives  after  two  combs  had  been 
removed.  In  these  troughs  sterile  excelsior  was  placed  for  the  bees 
to  walk  on  in  order  to  prevent  them  from  drowning.  This  method 
was  found  unsatisfactory,  however.  At  Laramie,  Wyo.,  the  sugar 
sirup  containing  the  spores  was  first  placed  in  Boardman  feeders,  but 
owing  to  the  danger  of  robbing  at  the  entrance  of  the  hives,  the 
method  finally  used  was  to  invert  the  jars  in  holes  bored  in  the  hive 
covers.  In  this  way  any  leakage  into  the  hives  was  cleaned  up  by  the 
bees  without  danger  of  causing  robbing.  To  prevent  the  jars  from 
being  broken  or  knocked  over,  box  covers  were  placed  over  them  and 
fastened  to  the  hive  covers.  Each  colony  was  usually  inoculated  only 
once  with  an  individual  dilution  of  spores.  Duplicate  colonies  were 
inoculated  with  each  dilution  of  spores.  Uninoculated  check  colonies 
were  placed  among  those  that  were  inoculated. 

PRIMARY  OBSERVATIONS 

Observations  of  the  condition  of  the  brood  were  made  at  least  once 
a  week,  and  sometimes  oftener,  after  the  colony  was  given  the  liter  of 
inoculated  sirup.  In  1926  at  Somerset,  Md.,  as  soon  as  diseased 
larvae  appeared  in  a  colony,  the  colony  was  killed  and  at  once  re- 
moved from  the  apiary.  Because  of  the  isolated  location  near  Lara- 
mie, Wyo.,  the  colonies  were  left  until  the  end  of  the  brood-rearing 
season,  when  fiaal  observations  were  made. 

The  results  of  the  spore-feeding  experiments  are  shown  in  Table  1. 


262 


Journal  oj  Agricultural  Research 


Vol.  45,  No.  5 


Table  1. — Results  of  spore-feeding  experiments  " 

[Duplicate  colonies  of  bees  (A  and  B)  were  used  in  the  first  i  years,  and  triplicate  colonies  (A,  B,  and  C> 

in  1930] 


Extent  of  foulbrood  in — 

1926 

1927 

1928, 
repeat 

192« 

1929 

1930, 

final 

Total  number 
of  spores  fed 

During 
season 

Pinal 

During 
season 

Final 

During 
season 

Final 

A 

B 

A       B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

c 

5,  000,  000,  000 
2,  600,  000,  000 
1,  000,  000,  000 
750,  000,  000 
600,  000,  000 
3,50,000,000 
200,  000,  000 
17.5,  000,  000 
150,000,000 
126,  000,  000 
100,  000,  000 
76  000  000 

+ 
+ 

?+ 
+ 
+ 
+ 
+ 
0 

^+ 

+ 
+ 

+ 
+ 

+ 

+ 
+ 

+ 

?+ 
0 
0 

+ 

0 

+ 
?+ 

+ 
■>+ 
?+ 
?+ 
0 

+ 

+ 

0 
0 

* 

0 

+ 
+ 

+ 

* 

+ 

+ 

+ 

+ 

* 

* 

* 

0 

+ 

0 
0 

_i_ 
+ 

0 

0 

+ 
0 
0 

+ 
+ 
+ 

0 
0 

+ 
+ 

* 

+ 
0 
0 
0 
0 
0 
0 
0 

n 

0 

+ 
0 
0 
0 
0 
0 
0 

n 

+ 
0 
0 
0 
0 
0 
0 
0 

n 

0 

* 

0 
0 
0 
0 
0 
0 
0 

60,  000,  000 

0 

0 

0 
0 

0 
0 

0 

25,  000,  000 

n 

10  000  000 

0 

0 

5, 000, 000 

2,  600, 000 

1,  600, 000 

600,  000 

100,  000 

Controls 

l+,12-0 

l+,2-0 

l+,2-0 

1-0 

l+,3-0 

H-,3-0 

8- 

-0 

8 

-0 

2-0 

'  +,  Positive  American  foulbrood;  ?+,  probable  American  foulbrood,  very  slight  and  unconfirmed  and 
disappearing  by  end  of  brood-rearing  season;  0,  no  disease  found  during  season;  — *,  disease  cleaned  out  by 
end  of  brood  rearing;  — ,  no  recurrence  in  second  season. 

In  1926  a  total  of  200,000,000  spores  fed  to  a  colony  was  the 
smallest  number  that  produced  disease;  in  1927,  on  the  other  hand, 
75,000,000  was  the  smallest  number.  However,  in  the  latter  year 
the  spores  were  obtained  from  another  locality  in  which  environ- 
mental conditions  were  quite  different.  In  an  effort  to  obtain  check 
results,  the  feeding  experiments  were  repeated  in  1928.  Through  an 
error  in  maldng  up  the  spore  dilutions,  which  was  not  discovered  until 
too  late  for  rectification,  no  colony  received  less  than  50,000,000 
spores.  This  season  one  colony  of  the  pair  receiving  an  inoculation  of 
50,000,000  spores  became  infected.  The  feeding  experiments  were 
repeated  again  in  1929,  with  dilutions  of  spores  from  75,000,000 
down  to  100,000 — ^considerably  less  than  the  minimum  number  in 
1928.  Again  only  one  colony  of  the  pair  receiving  a  total  of  50,000,000 
became  infected.  As  a  result  of  two  years'  experiments  this  was 
foimd  to  be  the  apparent  minimum  number  of  spores  of  Bacillus  larvae 
capable  of  producing  infection  when  fed  in  1  liter  of  sugar  sirup.  In 
1930  spores  from  three  different  locaUties  were  fed  in  duplicate  to  six 
healthy  colonies  in  dilutions  of  50,000,000  and  25,000,000  without 
prod\icing  disease. 

It  is  therefore  apparent  that  a  certain  minimum  number  or  mass 
of  spores  is  required  to  start  the  initial  action  capable  of  producing 
American  foulbrood  in  healthy  larvae.  Under  the  conditions  of  these 
expenments  this  minimum  number  was  approximately  50,000,000 
spores  of  inoculum  per  liter  of  sirup. 

SECONDARY  OBSERVATIONS 

During  the  first  tliree  years  of  the  experiments,  or  previous  to  1929, 
at  which  time  the  experimental  colonies  were  isolated  in  pairs,  certain 
of  the  unmoculated  control  colonies  developed  disease,  1  out  of  13  in 


Bept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood       263 

1926,  1  out  of  3  in  1927,  and  1  out  of  4  in  1928.  It  was  assumed  that 
the  disease  was  probably  not  spread  by  robbing,  since  no  active  rob- 
bing was  observed  at  any  time.  In  practically  every  case  where  a 
control  colony  became  infected,  it  was  so  located  in  relation  to  the 
inoculated  colonies  that  drifting  of  young  nurse  bees  during  play 
flights  could  account  for  the  spread  of  the  disease,  in  one  or  two  cases 
quite  definitely  so.  In  1929  all  eight  uninoculated  colonies,  although 
they  were  not  located  with  the  inoculated  colonies  but  were  within 
robbing  range  of  all,  remained  free  from  disease.  The  prevention  of 
drifting  apparently  eliminated  the  casual  spread  of  disease. 

Occasionally  a  colony  of  bees  affected  with  American  foulbrood  will 
try  to  clean  out  the  diseased  remains,  often  removing  parts  of  the 
scales  and  sometimes  actually  tearing  a  comb  down  to  the  midrib  in 
order  to  do  this.     White  (SO,  p.  34-35)  states: 

There  is  considerable  evidence  to  support  the  belief  that  occasionally  in  cases 
of  light  infection  the  disease  may  disappear  unaided  by  treatment.  *  *  *  j^ 
should  be  emphasized  that  such  a  course  for  the  disease,  if  it  occurs  at  all,  is 
unusual.  Although  American  foulbrood  spreads  more  or  less  rapidly  within  an 
infected  colony,  the  fact  remains  that  it  frequently  does  not. 

Lineburg  (16)  in  1925  reported  that  in  two  colonies  which  were 
diseased  in  the  spring  the  disease  apparently  disappeared  later  in  the 
season.  Three  colonies  were  divided  and  used  for  maldng  increase 
in  June  and  July,  but  all  remained  free  from  disease,  at  least  until  the 
end  of  that  season.  Further  observations  were  not  reported.  Cor- 
kins  (8)  in  1928  reported  five  colonies  which  were  given  combs  con- 
taining scales  of  American  foulbrood  at  the  beginning  of  the  honey 
flow  of  1927  and  developed  no  disease  up  to  July  10,  1928.  Two 
other  colonies  were  observed  to  have  cleaned  out  the  disease  and 
remained  healthy  for  an  entire  season.  However,  during  the  several 
years  of  his  experimental  work  on  American  foulbrood,  the  writer 
never  observed  a  colony  in  which  the  disease  was  permanently 
cleaned  out  until  1927.  In  that  year,  of  16  colonies  inoculated  with 
various  dilutions  of  spores,  4  colonies,  2  of  which  received  more  than 
the  probable  minimum  dose  causing  infection,  showed  no  disease 
during  the  season.  The  disease  completely  disappeared  by  the  end 
of  brood  rearing  ia  10  of  the  12  other  colonies  that  had  showed  either 
positive  or  probable  disease  some  time  during  the  summer.  In  1928 
package  bees  were  placed  on  the  combs  of  seven  qf  these  colonies 
that  had  apparently  cleaned  out  the  disease  during  the  previous 
summer  and  on  two  that  had  been  inoculated  with  presumably  a 
sufiicient  number  of  spores  but  which  had  remained  healthy.  Three 
of  the  seven  developed  disease  again  the  second  season,  while  four 
remained  healthy  during  the  entire  season.  Neither  of  the  two 
inoculated  colonies  that  had  remained  free  from  disease  in  1927 
developed  it  in  1928.  Of  the  11  colonies  inoculated  in  1928  that 
developed  disease,  4  cleaned  up  the  disease  by  the  end  of  the  brood- 
rearing  season  and  2  inoculated  colonies  showed  no  disease.  In 
1929,  1  of  the  2  colonies  developing  disease  cleaned  up  by  the  end  of 
the  brood-rearing  season,  making  a  total  of  15  cases  in  which  the 
disease  was  cleaned  up  by  the  end  of  brood  rearing.  Two  of  the 
colonies  inoculated  with  the  minimum  infectious  dose  or  more  showed 
no  disease  during  that  summer. 

It  is  possible  that,  in  the  high  altitude  of  Laramie,  and  in  similar 
places  where  the  air  is  very  dry,  the  scales  of  American  foulbrood 
131772—32 2 


264  Journal  oj  Agricultural  Research  vu.  45,  No.  5 

become  dried  without  adhering  so  tenaciously  to  the  cell  walls  as 
they  do  in  more  humid  climates  at  lower  altitudes.  These  observa- 
tions iadicate  the  necessity  of  further  work  on  the  resistance  of  bees  to 
the  disease  and  variation  in  virulence  of  different  strains  of  the 
organism. 

INOCULATION    OF    INDIVIDUAL    BEE    LARVAE    WITH    DEFINITE 
NUMBERS  OF  SPORES  OF  BACILLUS  LARVAE 

In  the  light  of  the  results  of  the  foregoing  experiments,  in  which 
colonies  were  inoculated  with  presumably  a  quantity  of  spores 
sufficient  to  produce  infection  but  in  which  no  disease  developed,  the 
question  arises  as  to  what  became  of  the  spores  in  the  sugar  sirup, 
some  of  which  presumably  were  fed  to  healthy  larvae.  In  those 
colonies  developing  disease  that  received  a  minimum  number  of 
spores,  how  many  spores  did  each  larva  developing  the  disease  receive? 
In  order  to  obtain  information  on  these  points,  a  preliminary  series  of 
experiments  was  planned  in  which  individual  larvae  were  inoculated 
with  known  numbers  of  spores. 

Touraanoff  (29)  reports  that  he  was  unable  to  cause  infection  by 
giving  individual  larvae  a  drop  of  a  rich  emulsion  of  a  culture  of 
Bacillus  larvae  in  salt  solution.  He  found  that  many  of  the  larvae  so 
treated  were  removed  from  the  cells  by  the  bees,  and  those  remaining 
failed  to  develop  disease.  He  further  found  that  larvae  given  only 
uninoculated  salt  solution  were  also  removed  in  the  same  way. 
Therefore,  in  the  present  experiments  sugar  sirup  was  used  instead 
of  salt  solution.  In  a  comb  from  a  healthy  colony  containing  numer- 
ous coiled  larvae,  a  drop  of  an  uninoculated  50  per  cent  solution  of 
sugar  in  water  was  placed  in  each  cell  containing  a  larva,  as  near  the 
mouth  parts  of  the  larva  as  possible.  The  rim  of  each  cell  so  treated 
was  marked  with  a  paint  consisting  of  1  part  of  liquid  white  shellac, 
1  part  of  a  paint  pigment,  and  4  parts  of  ethyl  alcohol.  The  sugar 
sirup  was  slightly  colored  with  water-soluble  eosin  in  order  to  aid 
m  determining  the  effect.  Frequent  observations  showed  that 
practically  all  larvae  that  were  fed  this  colored  sugar  sirup  developed 
normally  and  were  sealed  over,  the  pigment  markings  still  being 
present  on  the  edges  of  the  cappings.  In  most  of  the  cells  a  residue  of 
colored  sirup  could  be  observed  for  several  hours  after  the  larvae 
had  fed. 

A  series  of  5-frame  nuclei  was  prepared,  each  containing  one  or  two 
combs  havmg  a  large  number  of  unsealed  larvae.  A  set  of  dilutions  of 
spores  was  made  from  a  stock  suspension  with  a  steriUzed  50  per  cent 
sugar  su-up  m  such  a  way  that  each  0.01  c  c  of  the  dilution  would 
£?^*f  1^  a^  approximate  Imown  number  of  spores,  as  indicated  in 
iable  2.  btenhzed  2  c  c  Luer  tuberculin  hypodermic  syringes  Grad- 
uated m  0.01  c  c  the  needles  of  which  had  been  blunted,  were  "used 
in  inoculating  the  cells  containing  coiled  larvae.  Fifty  or  more 
coiled  larvae  at  least  4  days  old  were  each  given  0.01  c  c  of  a  dilution 
ot  spores,  each  dilution  being  given  to  larvae  in  one  comb  in  a  separate 
colony,  and  the  cells  so  inoculated  were  distinctively  marked  A  few 
larvae  that  had  just  been  sealed  also  were  inoculated  by  puncturing 
the  cappmg  with  the  inoculating  needle  and  depositing  the  0.01  c  c  in 
the  ceU  Observations  were  taken  at  the  end  of  24  hours  and  at 
frequent  intervals   thereafter   until   the   end    of   the   brood-rearing 


Sept.  1,1032    Commercial  Honey  and  Spread  fff  American  Foulbrood      265 


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j3§oOiO        O        OiON^ 

<o-^"o"i>r,  ^-    ^ 

266  Journal  oj  Agricultural  Research  ^'ol.  45,  No.  5 

In  the  first  series  of  inoculations  the  number  of  spores  fed  each 
larva  ranged  from  approximately  5,000  down  to  1.  None  of  the 
larvae  inoculated  developed  disease.  (Table  2.)  Later  a  second 
series  of  inoculations  was  made.  The  same  colonies  were  used  be- 
cause of  the  limited  number  available^  but  the  larvae  inoculated  were 
in  a  different  comb  in  each  colony  and  a  different  color  was  used  to 
mark  the  cells.  In  these  inoculations  the  number  of  spores  fed 
ranged  from  5,000,000  down  to  1,000  per  larva.  No  disease  devel- 
oped from  this  set  of  inoculations. 

It  vv-as  thought  possible  that  the  nurse  bees  might  be  removing 
most,  if  not  all,  of  the  inoculated  sugar  sirup  before  the  larvae  had 
had  time  to  ingest  a  sufficient  number  of  spores  to  bring  about  infection. 
Therefore,  in  a  third  series  of  experiments  each  inoculated  comb  was 
placed  in  a  screen-wire  queen-nucleus  introducing  cage,  and  this  cage 
was  put  back  in  the  colony  for  periods  ranging  from  one-half  to  one 
hour  before  the  unprotected  comb  was  replaced  in  the  colony,  thus 
theoretically  giving  the  larvae  time  to  ingest  some  of  the  sugar  sirup 
before  the  nurse  bees  had  access  to  the  inoculated  cells.  In  these 
tests  the  larvae  were  kept  from  the  bees  so  long  that  many  of  them, 
becoming  hungry,  were  starting  to  crawl  from  the  cells.  The  number 
of  spores  fed  ranged  from  50,000,000  down  to  500,000  per  larva. 
Twenty-four  hours  after  the  larvae  were  fed  it  was  found  that  all  re- 
ceiving 50,000,000  and  25,000,000  spores  had  been  removed  from  the 
cells,  while  those  receiving  a  smaller  number  of  spores  were  either 
partly  removed  or  remained  in  the  cells,  according  to  the  strength  of 
the  dilution  and  the  length  of  time  that  the  larvae  were  kept  away 
from  the  nurse  bees.     (Table  2.) 

Two  days  later  another  set  of  larvae  was  inoculated  with  the  same 
dilutions  as  were  previously  used  for  these  colonies  but  on  the  other 
■side  of  the  same  combs.  In  this  series  the  combs  were  kept  away 
from_  the  bees  for  periods  ranging  from  5  minutes  for  the  heaviest 
dilution  to  30  minutes  for  the  weakest.  Again  all  the  larvae  receiving 
the  50,000,000  and  25,000,000  spores  were  removed,  while  those 
receiving  the  5,000,000,  which  were  kept  from  the  bees  for  half  an 
hour,  were  partly  removed,  and  those  receiving  7,500,000  or  10,000,000 
were  not  removed.  Apparently  there  are  two  factors  concerned  in 
the  removal  of  the  larvae — the  length  of  time  they  are  kept  away 
from  the  bees  and  the  amount  of  foreign  matter  in  the  sirup,  as  indi- 
cated by  the  spore  content,  that  is  given  to  the  larvae. 

The  results  of  the  last  two  series  of  inoculations  showed  that  in  the 
colonies  in  wliicli  the  larvae  were  not  removed,  or  were  not  entirely 
removed,  several  larvae  in  the  colony  receiving  10,000,000  spores  per 
larva  developed  disease,  while  those  in  the  colonies  receiving  a  smaller 
number  remained  healthy.  (Table  2.)  This  work  should  be  repeated 
with  a  different  colony  for  each  set  of  inoculations,  although  appar- 
ently the  disease  did  not  spread  in  the  colonies  used.  Only  one  colony 
of  the  entire  number  developed  disease,  xilthough  a  certain  degree 
of  success  was  obtained,  these  results  seem  to  bear  out  Toumanoff's 
{29)  conclusion  that  the  artificial  infection  of  individual  larvae  is  not 
brought  about  so  easily  as  one  had  been  in  the  habit  of  believing. 
Apparently,  also,  a  considerable  number  of  spores  are  necessary  to 
establish  an  infection  under  these  conditions. 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulhrood       267 

MINIMUM  NUMBER  OF  SPORES  OF  BACILLUS  LARVAE  PRODUCING. 
VEGETATIVE  GROWTH  ON  ARTIFICIAL  CULTURE  MEDIA 

Bacteria  are  known  to  pass  tlirough  a  definite  cycle  of  growth,  par- 
ticularly when  cells  from  an  old  culture  are  transferred  to  fresh 
culture  media.  The  growth  stages  have  been  described  by  Buchanan 
(3;  IS,  Ch.  Tl,  Henrici  (12),  and  Winslow  (IS,  Ch.  VI)  somewhat  as. 
follows:  The  initial  stationary  phase  during  which  no  growth  takes 
place;  the  logarithmic  phase  when  the  organisms  begin  to  divide, 
slowly  at  first  but  gradually  accelerating;  and  so  on  through  the  com- 
plete cycle  of  growth.     Henrici  (IS,  p.  21,  24)  has  observed  that — 

Various  factors,  as  temperature;  the  size,  the  age,  and  previous  history  of  the? 
inoculum;  and  the  composition  and  nutrient  value  of  the  medium,  influence  the- 
growth  curves  of  bacteria.  *  *  *  Qf  the  various  factors  which  influence  the 
rate  of  growth  and  form  of  the  growth  curve,  the  initial  number  of  cells  introduced! 
into  a  unit  volume  of  medium  seems  to  be  one  of  the  most  important. 

Robertson  (25),  in  studies  of  cultures  of  certain  protozoa,  has 
shown  that  growth  seems  to  be  stimulated  by  the  presence  of  other 
cells  of  the  same  type.  This  characteristic  has  been  described  at 
various  times  as  mass  action  or  communal  activity. 

Early  in  1929,  in  conjunction  with  the  spore-feeding  experiments.-. 
in  the  apiary,  an  investigation  was  started  to  determine  whether 
there  is  a  similar  manifestation  of  mass  action  in  the  vegetative 
growth  of  spores  of  Bacillus  larvae  on  artificial  culture  media.  In  a^ 
preliminary  paper  on  this  subject  the  writer  (27,  p.  4-56)  made  the 
following  observations :  Starting  with  a  seeding  of  5,000,000,000  spores^ 
of  B.  larvae  on  a  suitable  slanted  solid  culture  medium,  it  was  found 
at  the  end  of  48  hours'  incubation  at  37°  C.  that  growth  had  occurred, 
in  the  original  and  in  a  diluted  seeding  containing  60,000,000  spores, 
but  not  in  one  containing  50,000,000  spores.  Growth  occurred  in  a 
diluted  seeding  containing  only  5,000,000  spores  after  six  days' 
incubation,  and  in  one  containing  700,000  spores  after  10  days'  incu- 
bation. (Table  4,  Group  1.)  These  observations  indicated  that  a. 
certain  initial  mass  of  spores  is  necessary  to  start  vegetative  growth.. 
Furthermore,  although  the  growth  results  were  rather  irregular  owin^ 
to  the  comparatively  small  number  of  cultures  made,  they  seemed  to 
show  that,  within  certain  limits,  the  smaller  the  seeding  the  longer 
the  incubation  period  necessary  to  obtain  germination  of  the  spores 
and  vegetative  growth.  From  this  preliminary  work  it  was  assumed 
that  the  lower  limits  of  dilution  of  the  stock  suspension  that  would 
give  growth  on  longer  incubation  had  not  been  reached. 

Ahrens  (1)  has  observed,  in  cultural  studies  of  scales  treated  with- 
formalin  solution  for  different  lengths  of  time,  that  growth  may: 
occur  in  cultures  from  such  scales  after  varying  periods  of  incubation; 
up  to  30  days,  depending  on  the  length  of  treatment  and  the  per- 
centage of  formalin  in  the  solution.  Burnside  (7)  states,  in  connec- 
tion with  studies  of  disinfection  of  American  foulbrood  combs  by- 
fumigation  with  formaldehyde  gas,  that  "it  is  probable  that  if  scales; 
had  been  washed  and  the  incubation  period  increased,  growth  of 
Bacillus  larvae  would  have  been  obtained  in  some  instances  whem 
negative  results  were  recorded." 

Therefore,  a  single  trial  series  of  cultures  was  run  (No.  7,  Table  4)^ 
the  total  incubation  period  being  30  days.  Results  from  this  set  of 
cultures  showed  that  in  some  cases  growth  was  obtained  aftier  30i 


268  Journal  oj  Agricultural  Research  voi.  45,  No.  s 

days'  incubation  where  no  growth  was  observed  after  10  days'  incu- 
bation. Work  on  this  phase  of  the  problem  was  continued  during 
the  summer  and  fall  of  1930.  Several  sets  of  cultures  were  made  in 
which  Bacillus  larvae  from  eight  different  localities  were  used  in  a 
series  of  seedings  with  a  decreasing  number  of  spores  for  each  lot  of 
the  organism  and  all  incubated  for  30  days.     (Table  4,  Group  2.) 

methods  op  procedure 

Culture  Media 

A  culture  medium  was  used  similar  to  that  employed  by  the  writer 
in  the  preliminary  experiments  (^7)  and  also  in  earlier  cultural  work 
with  Bacillus  larvae  (26} — -that  is,  a  combination  of  the  medium  made 
of  yeast-extract  and  egg-yolk  suspension  and  the  carrot-extract 
medium  of  Lochhead  (18).  The  yeast-carrot  extract  medium  was 
prepared  as  follows: 

(A)  Dried  yeast grams__     10 

Peptone do 10 

Buffer  (sodium  glycerophosphate) do 2.  5 

Water  (distilled) cubic  centimeters  ._  500 

This  solution  was  heated  in  flowing  steam  for  one-half  hour  and,  after  a  table- 
spoonful  of  siliceous  earth  had  been  added  to  assist  in  the  filtration  and  clarifica- 
tion, it  was  filtered  through  filter  paper  on  a  perforated  porcelain  funnel  with 
suction. 

(B)  Two  hundred  grams  of  cleaned  carrots  was  macerated  in  a  meat  grindei, 
added  to  500  c  c  of  distilled  water,  and  allowed  to  stand  for  at  least  30  minutes, 
preferably  longer.  The  macerated  carrot  was  removed  by  filtration  through 
fine  muslin,  as  much  liquid  as  possible  being  squeezed  from  the  mass.  The 
filtrate  was  then  clarified  by  the  addition  of  siliceous  earth  and  filtration  in  the 
same  manner  as  the  yeast-extract  medium. 

(C)  The  final  base  medium  was  prepared  by  mixing  500  c  c  of  A  with  200  c  c 
of  B  and  adding  700  c  c  of  a  3  per  cent  solution  of  washed  agar. 

The  reactidn  of  the  medium  was  so  adjusted  that  when  2  c  c  of 
sterile  egg-yolk  suspension,  prepared  as  described  in  a  previous  paper 
(26),  was  added  to  10  c  c  of  the  yeast-carrot  extract  base  medium  by 
means  of  the  apparatus  shown  in  Figure  1,  and  described  previously 
(26),  the  pH  value  was  6.8.  The  medium  was  then  sterilized  in  the 
Autoclave  at  15  pounds'  pressure  (sea  level)  for  15  minutes.  After  it 
iad  cooled  to  45°  C,  20  drops,  or  about  2  c  c,  of  the  sterile  egg-yolk 
suspension  was  added  to  each  tube  of  medium,  mixed  by  shaking, 
and  the  medium  was  then  allowed  to  solidify  in  a  slanting  position. 

The  Lochhead  yeast-extract  medium  was  tried  without  the  addition 
of  egg-yolk  suspension,  but  although  it  gave  good  growth  with  the 
heavier  seedings  of  spores,  the  combination  medium  was  found  to  give 
more  uniform  germination  and  heavier  vegetative  growth  with  the 
more  dilute  seedings.  The  addition  of  the  carrot  extract,  while  pos- 
sibly adding  somewhat  to  the  growth-producing  qualities  of  the  med- 
ium, served  m  these  experiments  as  an  indicator  for  vegetative  growth 
because  of  the  abiUty  of  Bacillus  larvae  to  produce  nitrite  in  the  carrot- 
extract  medmm  without  the  addition  of  potassium  nitrate  (18). 

Pkeparation  op  Dilutions  of  Spores 

The  stock  suspensions  of  spores  of  Bacillus  larvae  were  made  up  as 
described  earlier  m  this  paper.  A  series  of  primary  dilutions,  each 
one-tenth  of  the  preceding  dilution,  was  then  made  up  in  sterile  125 


Sept.  1, 1932    Commercial  Honey  and  Spread  oj  American  Foulbrood      269 


c  c  flasks  bv  adding  4  c  c  of  a  dilution  to  36  c  c  of  sterUe  water.  The 
series  of  dilutions  containing  gradually  decreasing  numbers  of  spores 
per  cubic  centimeter  to  be  used  in  inoculating  the  culture  medium 
were  then  prepared  as  indicated  in  Table  4.  Sterile  burettes  were 
used  in  adding  the  proper  proportions  of  spore  suspension  or  spore- 
suspension  dilutions  to  the  proper  quantities  of  sterile  water  in  sterile 
test  tubes,  in  order  to  make  up  the  desired  series  of  dilutions  contain- 
ing approximately  known  numbers  of  spores. 

Inoculation  op  Culture  Medium 

Swann  has  observed  that  in  old  cultures  of  anthrax  a  considerable 
percentage  of  spores  are  dead  and  therefore  never  germinate.  Be- 
cause of  the  possibility  that  some  of  the 
spores  in  the  stock  suspensions  of  Bacillus 
larvae  might  not  be  viable,  an  effort  was  made 
to  determine  the  approximate  proportions  of 
viable  and  dead  spores  in  the  stock  suspen- 
sions. Since  the  determination  of  viable 
spores  of  B.  larvae  by  means  of  plate  cultures 
is  difficult  because  of  the  opaqueness  of  the 
special  culture  medium  that  is  required,  an 
attempt  was  made  to  determine  the  percent- 
age of  viable  spores  by  the  differential  stain- 
ing method  of  Burke  (4)  as  modified  by  Koser 
and  MUls  (IS).  The  procedure  is  as  follows : 
A  small  quantity  of  the  spore  suspension  is 
spread  in  a  thin  film  on  a  slide  and  allowed 
to  dry  without  heating.  The  slide,  after 
immersion  in  a  solution  of  carbol  fuchsin  at 
room  temperature  for  two  minutes,  is  washed 
in  water  and  decolorized  with  absolute  ace- 
tone for  a  few  seconds,  washed  again,  and 
immersed  in  Loefiler's  alkaline  methylene 
blue  for  two  minutes,  washed,  dried,  and 
examined.  Very  few  solid-staining  forms 
were  observed  in  any  of  the  suspensions  ex-  ^'<'™t^tinrotKii  sXensS  ^'' 
amined,  possibly  one  or  two  spores  in  several 

fields.  It  was  therefore  assumed  that  the  number  of  nonviable  spores 
could  be  considered  as  negligible  and  probably  within  the  limits  of 
the  precision  of  the  measurements  as  indicated  by  this  procedure. 

One  cubic  centimeter  of  each  dilution  was  added  to  duplicate  tubes 
of  the  slanted  solid  medium  by  means  of  sterile  Ice  pipettes,  each 
cubic  centimeter  of  inoculum  containing  an  approximately  known 
number  of  spores  of  Bacillus  larvae.  After  inoculation  the  cultures 
were  incubated  at  37°  C.  In  order  to  prevent  the  liquid  in  the  tubes 
from  drying  out  on  long  incubation,  from  time  to  time,  as  the  water 
of  condensation  evaporated,  2  or  3  c  c  of  sterile  broth  similar  in  com- 
position to  that  of  the  base  medium,  without  the  egg,  was  added  to 
each  tube  by  means  of  the  apparatus  shown  in  Figure  1.  A  total  of 
556  cultures  was  made  during  this  series  of  experiments. 


270  Journal  of  Agricultural  Research  voi.  45,  No.  5 

Method  of  Making  Observations 

The  culture  tubes  were  incubated  for  30  days  at  37°  C.  Each  tube 
was  examined  usually  every  24  hours  during  this  period.  The  pres- 
ence or  absence  of  vegetative  growth  was  noted  at  each  observation, 
and  ia  cases  of  slight  or  doubtful  growth  the  vegetative  growth  was 
checked  both  by  microscopic  examination  of  a  stained  smear  and  by 
testing  for  nitrite  production  in  the  culture  medium  by  the  sulphanUic 
acid  and  alpha-naphthylamine  acetate  test.  After  a  large  number  of 
such  observations  had  been  made,  it  was  found  that  vegetative  ger- 
mination of  spores  of  Bacillus  larvae,  almost  too  slight  to  be  seen, 
would  give  a  definite  pink  color  on  the  addition  of  the  reagents. 

Lochhead  {17,  p.  14)  sta,tes: 

It  was  found,  however,  that  ordinary  nitrate-reducing  species,  such  as  B. 
cereus  or  Es.  coli,  which  are  able  to  form  nitrites  readily  in  nitrate  media,  were 
unable  to  produce  nitrites  in  recognizable  amount  in  the  peptone-carrot  media, 
though  capable  of  doing  so  upon  the  addition  of  nitrates.  Bacillus  larvse  under 
the  same  condition  readily  forms  nitrites  without  the  addition  of  nitrate  to  the 
medium. 

Despite  this  statement,  a  series  of  miscellaneous  organisms  was 
tested  in  standard  nitrate  broth,  in  carrot-extract  broth,  and  on 
carrot-extract  agar.  Several  organisms  that  commonly  reduce 
nitrates  and  a  few  that  do  not  were  used .  (Table  3.)  Observations 
were  made  at  short  intervals  during  the  first  24  hours.  Most  of 
these  organisms  gave  positive  nitrite  tests  within  a  few  hours  after 
inoculation  in  all  the  media  used,  but  in  the  carrot-extract  medium 
the  nitrate  had  apparently  disappeared  in  most  cases  after  24  hours' 
incubation,  and  in  all  cases  after  48  hours.  The  same  organisms 
on  standard  nitrate  medium  still  gave  positive  tests  after  48  hours' 
incubation.  A  positive  nitrite  test  was  obtained  in  cultures  of 
Bacillus  larvae  that  were  incubated  for  5  days  and  in  one  culture 
that  was  incubated  for  4  days  and  then  allowed  to  stand  at  room 
temperature  for  16  days  more  before  testing.  Therefore,  it  appears 
probable — at  least  the  results  in  Table  3  indicate — that  in  the  case 
of  many  contaminating  organisms  having  the  power  to  reduce  nitrite 
that  might  get  into  the  culture  tubes  inoculated  with  spores  of  B. 
larvae  the  nitrite,  if  produced  by  the  contaminating  organism,  would 
have  disappeared  after  48  hours'  incubation,  leaving  contamination 
to  be  determined  by  gross  appearence  of  the  culture  and  microscopic 
examination.  Nevertheless,  in  order  to  be  sure  that  contaminating 
growth  of  any  kind  was  not  giving  erroneous  results  with  the  nitrite 
test  when  this  was  used  alone,  any  suspicious-looking  growth  in  the 
culture  tubes  was  examined  under  the  microscope  before  it  was  tested 
with  the  reagents  for  nitrite  production.  Even  though  a  positive 
mtrite  test  might  be  observed  in  some  cases,  the  contaminations  were 
recorded  only  as  such. 

OBSERVATIONS  AND  EESULTS 

In  no  instance  was  positive  growth  obtained  in  cultures  moculated 
with  less  than  50,000  spores,  even  after  30  days'  incubation,  and 
growth  with  50,000  spores  was  obtained  from  only  two  of  the  eight 
lots  of  spores  used,  namely,  Nos.  19  and  23.  (Table  4.)  In  the 
other  six  strains  the  mimmum  number  of  spores  that  produced  positive 
growth  ranged  from  5,000,000  to  70,000. 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood       271 


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Sept.  1, 1932     Commercial  Honey  and  Spread  oj  American  Foulbrood      273 


COMCOWCi-     _._ 

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274  Journal  oj  Agricultural  Research  voi.  45,  No.  5 

The  length  of  the  incubation  period  in  relation  to  the  decreasing 
number  of  spores  used  varied  greatly  with  the  different  lots  of  spores, 
even  with  the  duplicate  inoculations  of  each  lot.  Table  5  gives  the 
results  of  positive  cultures  obtained  in  relation  to  the  period  of  incu- 
bation and  the  dilution  of  the  spores.  The  coefficient  of  correlation 
{14,  -p.  179)  for  the  positive  cultiu-es  only,  in  relation  to  length  of 
incubation  and  dilution  of  spores,  was  found  to  be  0.3558 ±0.0440. 
While  this  does  not  show  a  strong  correlation,  it  indicates  that  with 
the  smaller  numbers  of  spores  there  is  a  tendency  for  growth  to  take 
place  with  longer  periods  of  incubation.  However,  when  the  cases 
of  positive  growth  were  correlated  with  the  dilution  and  incubation 
time  on  the  basis  of  the  percentage  of  positive  cultures  to  negative 
cultures  for  each  observation  period  of  incubation  time,  an  insignifi- 
cant negative  correlation  was  obtained.  Apparently  there  is  a 
variable  uncontrollable  factor  present,  more  obvious  when  spores  are 
used  from  different  lots  of  the  organism,  which  makes  it  impossible 
to  correlate  the  other  factors  closely.  However,  the  data  summarized 
in  Table  6  indicate  that,  of  the  120  cultvu-es  made  with  seedings  of 
between  5,000,000,000  and  9,000,000  spores  per  seeding,  98.33  per 
cent  showed  growth  at  the  end  of  10  days'  incubation,  while  100 
per  cent  (120  cultures)  showed  growth  after  30  days'  incubation. 
This  is  56.87  per  cent  of  the  211  total  cultures  showing  growth 
after  30  days. 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood      275 


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o"o"o"o'  o"o'o"o"o'o'o'o"o  o'o  o"o"o'c 
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O  O  O"  O  O'o"  ClT  Oi"  CO  t-^  CC  "ri" -*"  CO  M  rH 

000000  01001^(010^ 

Total  posi 

cultures 

Total    nega 

cultures.- 
Total  cultui 

276 


Journal  oj  Agricultural  Research 


Vol.  45,  No.  s 


1 

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cultures 

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total  of  all 
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6, 000, 000, 000- 

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600, 000 

400, 000-50, 000 

40,  000-0 

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o 

Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood       277 

Of  the  171  cultures  made  with  seedings  between  8,000,000  and 
500,000  spores  per  seeding,  48,  or  28.07  per  cent,  showed  growth  at 
the  end  of  10  da>s'  incubation,  while  79,  or  46.20  per  cent,  showed 
growth  after  30  days'  incubation.  The  latter  number  is  37.44  per 
cent  of  the  211  total  cultures  showing  growth  after  30  days'  incubation. 

Of  the  142  cultures  made  with  seedings  between  400,000  and  50,000' 
spores  per  seeding,  only  4,  or  2.82  per  cent,  showed  growth  at  the  end 
of  10  days'  incubation,  while  12,  or  8.45  per  cent,  showed  growth 
after  30  days'  incubation.  The  latter  figure  is  5.69  per  cent  of  the 
211  cultures  showing  growth  after  30  days'  incubation. 

Of  the  123  cultures  made  with  seedings  of  40,000  or  fewer  spores. 
per  seeding,  no  growth  was  obtained  after  30  days'  incubation. 

Of  the  556  cultures  made  with  all  seedings,  30.58  per  cent  showed 
growth  at  the  end  of  10  days'  incubation  and  69.42  per  cent  showed 
no  growth.  The  170  positive  cultures  after  10  days'  incubation  is- 
80.57  per  cent  (not  shown  in  Table  6)  of  the  211  total  positive 
cultures  obtained.  In  the  interval  between  the  10  and  30  day  incuba- 
tion periods,  19.43  per  cent  (not  shown  in  Table  6)  of  the  211  total 
positive  cultures,  or  another  7.37  per  cent  of  all  cultures  made,  showed 
growth,  making  a  total  of  only  37.95  per  cent  of  all  cultures  which 
showed  growth  at  the  end  of  30  days'  incubation,  with  62.05  per  cent 
still  showing  no  growth. 

The  initial  growth  phases  as  described  by  Buchanan  (5;  IS,  Ch.  V) 
are  clearly  more  marked  with  spores  than  with  simple  vegetative 
organisms,  since  there  is  a  varying  length  of  time  necessary  for  spores 
to  germinate  and  start  growing  after  implantation  in  a  suitable 
medium.  In  the  light  of  observations  on  other  spore-forming 
organisms,  it  is  probable  that  this  factor,  which  seems  to  cause  varia- 
tions in  the  germination  time  of  Bacillus  larvae  even  within  a  lot 
from  a  single  source,  is  what  has  been  termed  "dormancy."  Burke 
(5,  p.  283) ,  working  with  Clostridium  botulinum,  foimd : 

The  individual  (unheated)  spores  in  a  given  culture  of  CI.  botuUnum  vary  greatlj' 
in  the  time  required  for  germination  under  optimum  growth  conditions.  The 
majority  germinate  relatively  quickly,  but  a  few  lie  dormant  for  a  longer  time. 
One  hundred  and  forty-four  days  is  the  maximum  period  of  dormancy  recorded 
here     *     *     *. 

Burke  states : 

The  primary  factors  which  cause  the  spore  to  lie  dormant  for  long  periods  of 
time  under  optimum  growth  conditions  are  believed  to  be  inherent  in  the  spore 
itself.  It  is  thought  that  relative  permeability  of  the  spore  wall  is  one  of  the 
factors.  Environmental  conditions  may  secondarily  modify  the  period  of 
dormancy. 

Burke,  Sprague,  and  Barnes  (6,  p.  560)  observed  the  same  phe- 
nomenon with  such  non  spore-bearing  bacteria  as  Bacillus  coli 
(  =  Escherichia  coli).  They  found  that  spores  of  B.  subtilis  remained 
dormant  39  days  and  those  of  B.  megatherium  90  days,  although  a 
large  majority  developed  in  4  or  5  days.     They  believe: 

Dormancy  must  be  considered  a  factor  in  infection.  It  reduces  the  chances  of 
infection  by  reducing  the  number  of  organisms  that  would  otherwise  start  to 
grow  at  one  time.  Since  the  cells  begin  to  multiply  at  different  times,  the  body 
has  an  opportunity  to  initiate  defensive  reactions  before  all  the  cells  develop. 
If  dormant  for  a  sufficient  period,  the  organisms  will  be  excluded  from  the  body 
before  development  takes  place. 


278  Journal  of  Agricultural  Research  voi.  45,  No.  6 

Swann  {28)  has  observed  that  there  is  a  variation  in  the  germination 
time  of  anthrax  spores,  depending  on  the  age  and  condition  of  the 
spores. 

Morrison  and  Rettger  {24,  j).  339)  recently  stated — 

Because  of  the  marked  variability  of  germination,  depending  upon  the  stimuli 
supplied  in  the  environment,  the  deduction  is  made  that  bacterial  spores  in  the 
process  of  germination  are  vitally  active  bodies  having  requirements  for  meta- 
bolic function  which  are  the  same  as  or  more  exacting  and  specific  than  those  of 
the  vegetative  cells. 

Experimental  evidence  is  presented  to  show  that  the  dormancy  of  aerobic 
bacterial  spores  is  largely,  if  not  entirely,  determined  by  conditions  in  the  environ- 
ment of  the  spores,  and  that  these  factors  must  be  taken  into  consideration, 
perhaps  specifically  for  each  species,  before  so-called  "inherent "  or  "  normal "  dor- 
mancy of  bacterial  spores  can  be  established. 

This  phase  of  the  work  with  Bacillus  larvae  is  being  repeated  with 
the  organism  obtained  from  a  single  source  in  an  effort  to  determine 
the  importance  of  this  variable  factor  of  dormancy. 

SPORES  OF  BACILLUS  LARVAE  IN  COMMERCIAL  HONEY 

A  few  instances  have  been  reported  in  the  bee  journals,  such  as  that 
by  Merrill  {22),  in  which  American  foulbrood  has  developed  as  a 
result  of  bees  having  access  to  cans  of  infected  honey  that  have  been 
carelessly  thrown  out.  Without  doubt  in  some  cases  honey  has  been 
allowed  to  get  on  the  market  from  infected  colonies  through  negligence 
of  the  beekeepers  and  without  being  diluted  by  mixing  or  blending 
with  honey  from  disease-free  apiaries.  On  the  other  hand,  Fracker 
{10,  p.  379-380)  has  shown,  by  a  study  of  disease-inspection  statistics 
for  Wisconsin: 

1.  In  Wisconsin  the  introduction  of  this  disease  into  the  State  and  into  many 
individual  localities  is  definitely  known  to  have  been  in  specific  importations  of 
bees  and  equipment. 

2.  Cases  of  infection  in  which  the  source  appears  to  be  infected  honey  in  the 
•channels  of  trade  are  comparatively  rare. 

3.  Even  near  such  a  large  center  as  Milwaukee  the  infection  percentage  is 
greatest  m  locahties  of  active  movement,  such  as  greenhouse  areas,  and  is  relatively 
low  within  the  city  itself. 

4.  Towns  and  cities  of  from  3,000  to  40,000  which  have  been  natural  markets 
for  infected  honey  from  near-by  counties,  have  remained  for  years  free  from 
disease  either  until  the  present  or  until  infected  bees  and  equipment  were 
introduced. 

5.  No  new  centers  of  infection  are  known  to  have  been  started  since  the  policv 
ot  limiting  movement  of  bees  and  equipment  was  begun  in  1919 

6    These  observations  appear  to  be  confirmed  by  conditions  in  the  South,  in 

T^12l  f^!  t  f""^  *^®  "S-T-i  °S  ^'=*'^^  *^'slit  of  the  bee  tends  to  continue 
through  the  peak  of  honey  distribution. 

Furthermore,  F.  L.  Thomas,  State  entomologist  of  Texas,  in  an 
unpublished  manuscript  states: 

K  J^^i;!^-""^*" ^V^  *^?  estimates  with  reference  to  the  quantity  of  honey  that  is 

brought  into  Texas  ma  year  is  19  carloads.     Most  of  this  honey  is  produced  in 

California,  Colorado,  New  Mexico,  Utah,  and  Wyoming      *     *     *    P™aucea  in 

If   19   carloads  of  foulbrood-infected  honey  are  distributed   annually  in  this 

time  tnipp^'?h-'!?'°°^^^"'-I°  '"t^^"'^  ^^^*  °"^  inspectors  would  have  1  hard 
time  to  keep  this  disease  withm  bounds.     In  fact,  I  would  exDect  to  find  thni  thP 

Srat^ATrl'e  sLfe^tthJj°"°^  TT""  '"^  thefrttemp'tfto^er^adl^^^^^^^^ 
wS  ■?-!,"  t^    share  of  the  honey  which  is  imported  is  sold  in  west  and  north- 

west Texas  where  practically  no  bees  are  kept.  The  amount  which  is  dbtributed 
in  the  beekeeping  territory  of  the  State  is  evidently  less  dangerous  than  is  com- 
mon^^y  supposed.     The  following  facts,  I  think,  will  prove  tStatement 

hafS  ca^FeT?ntf'?or^''"  I'-  ^^^°f -t?  ^"^^^^  ^1,  1926,  the  insTect  on  work 
has  been  carried  into  100  counties.     Fifty-six  counties  were  found  to  be  free 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood      279 

from  contagious  or  infectious  diseases  of  bees,  but  in  the  other  44  counties  Ameri- 
can foulbrood  lias  been  present. 

An  average  of  668  beekeepers  have  been  visited  each  year  and  38,661  colonies 
examined  with  the  result  that  an  average  of  430  colonies,  or  1.11  per  cent,  have 
been  found  to  be  diseased. 

American  foulbrood  is  found  now  in  only  23  counties,  21  of  the  44  counties 
having  been  cleaned  up.  In  12  of  the  counties  where  disease  occurs,  only  30 
colonies  were  found  to  be  infected  out  of  7,642  examined — less  than  0.4  of  1  per 
cent.     Six  counties  had  one  diseased  colony  each. 

About  40  per  cent  of  the  beekeepers  and  60  per  cent  of  the  colonies  are  rein- 
spected  from  year  to  year;  the  remainder,  being  free  of  disease  and  considered 
out  of  danger,  are  dropped  and  "new  territory"  is  taken  over  and  examined  for 
presence  of  foulbrood.  By  "new  territory"  is  meant  beekeepers  and  their 
colonies  visited  and  inspected  for  the  first  time.  An  average  of  228  diseased 
colonies  are  discovered  each  year  in  "new  territory."  This  is  1.6  per  cent  of  the 
total  number  of  colonies  examined  in  this  territory. 

The  reinspection  which  has  been  made  in  the  counties  where  disease  has  been 
present  shows  that  there  have  been  both  gains  and  losses.  But  a  net  gain  has 
resulted  which  has  averaged  21  beekeepers  and  368  colonies  freed  from  American 
foulbrood  and  quarantine  annually. 

From  these  facts  it  is  easily  seen  that  definite  and  really  rapid  progress  in 
eradicating  the  disease  is  being  made.  Rarely  do  our  inspectors  find  new  out- 
breaks of  disease  that  can  not  be  traced  to  careless  beekeeping  methods,  bees 
robbing  infected  and  weakened  colonies,  or  to  the  use  of  old  and  infected  equip- 
ment. 

It  is  not  my  intention  to  imply  that  honey  is  not  a  carrier  of  American  foul- 
brood. The  above  evidence  simply  indicates  that  the  honey  which  has  been 
imported  into  Texas  has  not  been  as  dangerous  a  source  of  disease  to  bees  as  is 
sometimes  thought. 

Practically  no  work  has  been  reported  on  the  microbiology  of 
honey  other  than  that  in  connection  with  the  spoilage  of  honey 
through  fermentation  by  yeasts  {19,  21),  and  no  work  appears  to 
have  been  done  on  the  Bacillus  larvae  spore  content  of  commercial 
honey.  In  1925  the  writer  undertook  to  devise  a  method  for  demon- 
strating, at  least  qualitatively,  the  presence  or  absence  of  spores  of 
B.  larvae  in  honey  and  their  significance  in  relation  to  the  results  of 
the  spore-feeding  experiments.  Difficulties  were  encountered  in 
obtaiaing  cultures  of  B.  larvae  from  honey.  It  was  impossible  to 
obtain  vegetative  growth  of  this  organism,  even  when  a  considerable 
number  of  spores  had  previously  been  added  to  honey,  because  of  the 
difficulty  of  eliminating  contaminating  organisms  that  developed 
rapidly  in  the  honey,  completely  overgrowing  any  possible  vegetative 
growth  of  B.  larvae  before  it  could  get  well  started.  Therefore, 
methods  of  concentratiag  the  spores  from  the  honey  and  of  identifying 
them  by  means  of  microscopic  examination  were  attempted.  Because 
spores  of  B.  larvae  have  a  characteristic  appearance  in  stained  smears 
{20,  -p.  9),  it  was  assumed  that  this  method  might  give  at  least  tentative 

6V1(1gI1C6 

METHODS  OF,PE0CEDURE 

The  first  method  attempted  was  the  filtration  of  honey  diluted 
with  water  through  a  membrane  of  ether-alcohol  collodion  or  through 
filter  paper  impregnated  with  an  acetic  acid  solution  of  collodion 
{9).  Apparatus  was  devised  in  which  both  suction  and  pressure 
were  tried  in  this  filtering  process.  Stained  smears  were  made  of  the 
sediment'  retained  on  the  surface  of  the  filter.  In  several  cases 
spores  of  Bacillus  larvae  were  observed  in  stained  smears  of  the  sedi- 
ment filtered  out  of  honey  known  to  have  a  large  spore  content. 
However,  with  honey  containing  fewer  spores  it  was  found  impossible 
to  concentrate  them  on  a  small  enough  area  of  filter  in  sufficient 


280  Journal  of  Agricultural  Research  voi. «,  No,  s 

numbers  to  recover  and  identify  them  under  the  microscope.  Even 
with  a  comparatively  large  filtering  surface,  the  process  was  so  slow 
that  the  diluted  honey  would  frequently  start  to  ferment  before  it 
had  all  passed  through  the  fUter.  A  filter  of  smaller  area  would 
become  clogged,  preventing  the  passage  of  a  sufficient  quantity  of 
honey. 

Several  unsuccessful  attempts  were  made  to  recover  spores  of  Bacil- 
lus larvae  from  honey  by  centrifuging  samples  diluted  with  an  equal 
quantity  of  water.  After  considerable  experimentation  with  honey 
of  known  spore  content,  it  was  found  that  it  was  necessary  to  dilute 
the  honey  to  a  much  greater  extent — 1  part  to  at  least  9  of  water — ia 
order  to  throw  the  spores  down  with  the  sediment.  Apparently  the 
specific  gravity  of  these  spores  is  so  low  that  on  centrifuging  they 
remain  in  suspension  in  only  slightly  diluted  honey. 

The  procedure  finally  used  for  demonstrating  the  presence  of  spores 
of  Bacillus  larvae  in  honey  is  as  follows:  Five  c  c  of  warmed  honey 
is  thoroughly  mixed  with  45  c  c  of  distilled  water  in  a  50  c  c  cone- 
shaped  centrifuge  tube  made  of  heat-resistant  glass.  Duplicate 
quantities  of  each  sample  of  honey  are  made  up  for  examination. 
The  diluted  honey  is  then  centrifuged  at  2,000  revolutions  per  minute 
for  one-half  hour.  Because  of  the  difficulty  of  obtaining  a  satisfac- 
tory stained  smear  from  the  sediment  thrown  down  in  the  presence 
of  the  sugars  of  the  honey  solution,  all  but  2  c  c  of  the  solution  in  each 
centrifuge  tube  is  drawn  off  by  means  of  a  50  c  c  pipette.  Another 
45  c  c  of  distilled  water  is  added ,  the  sediment  is  thoroughly  shaken 
up  in  the  water,  and  the  tabes  are  centrifuged  again  for  20  minutes. 
After  all  but  2  c  c  or  less  of  the  wash  water  has  been  removed, 
0.01  c  c  of  the  sediment  is  removed  by  means  of  a  capillary  pipette 
and  smeared  on  a  cover  glass  over  a  surface  of  1  cm^,  a  small  loopful 
of  carbol  fuchsin  being  mixed  with  the  material  before  it  is  allowed 
to  dry.  After  drying  by  gentle  heat,  the  cover  glass  is  mounted 
on  a  slide  by  means  of  a  drop  of  distilled  water  and  the  smear  is 
examined  with  an  oil-immersion  objective.  Spores  of  B.  larvae  are 
identified  by  their  size  and  shape  in  conjunction  with  their  distinctive 
habit  of  breaking  loose  from  the  stained  mass  of  the  smear  and  of 
showing  a  delicate  Brownian  movement  in  the  thin  film  of  water 
between  the  two  pieces  of  glass.  In  a  few  samples  only  one  or  two 
spores  were  seen  in  numerous  fields  examined  or  the  spores  did  not 
have  the  typical  appearance  of  spores  of  B.  larvae.  In  such  cases 
another  test,  in  which  twice  as  much  honey  was  used,  was  made 
from  the  sample. 

OBSERVATIONS 

One  hundred  and  ninety-one  samples  of  honey  were  examined  by 
this  method.  (Table  7.)  Of  tlfese,  187  were  regular  commercial 
samples  purchased  in  the  open  market  and  2  were  from  the  experi- 
mental apiary  at  Laramie.  The  other  two  were  miscellaneous 
samples,  one  of  which  was  obtained  from  a  brood  comb  from  a  dis- 
eased colony  and  the  bther  from  a  cappings  melter  which  had  been 
used  with  combs  from  an  infected  apiary. 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood      281 


Table  7. — Results  of  the  examination  of  samples  of  honey  for  the  presence  of  spores 

of  Bacillus  larvae 


Source 

Samples 
tested 

Samples 
showing 
positive 
presence 
of  spores 
resem- 
bling 
Bacillus 
larvae 

Samples 
showing 

no 
evidence 

of 
spores  ° 

Commercial  samples  from  30  States 

187 
2 
2 

15 

172 

2 

2 

Total 

191 

17 

174 

"  29  of  these  samples  were  doubtful  on  the  first  examination,  but  repeated  examinations  gave  negative 
Tesults  in  each  case. 

Of  the  187  samples  of  commercial  honey  obtained  from  30  different 
States  or  Territories,  15,  or  8  per  cent,  showed  the  presence  of  a  suf- 
ficient number  of  spores  resembling  spores  of  Bacillus  larvae  to  be 
designated  as  positive.  In  29  of  the  commercial  samples,  or  15.5  per 
cent,  one  or  two  doubtful  spores  were  seen  in  each  case,  but  on 
repeated  examinations  none  of  these  samples  could  be  considered 
positive.  Two  of  the  four  miscellaneous  samples  from  infected 
sources  were  also  found  to  contain  spores  of  B.  larvae. 

Five  of  the  samples  showing  the  presence  of  spores  of  Bacillus 
larvae  were  fed  to  healthy  5-frame  colonies  during  the  summer  of  1930. 
These  samples  consisted  of  from  a  pint  to  a  quart  of  honey.  No  evi- 
dence of  American  foulbrood  appeared  in  any  of  the  five  colonies 
during  the  entire  brood-rearing  season. 

In  order  to  determine  the  approximate  number  of  spores  in  the 
samples  of  honey  in  which  the  presence  of  Bacillus  larvae  was  demon- 
strated, a  series  of  dilutions  of  spores  was  prepared  as  described  for 
the  work  with  cultures.  A  stained  smear  was  made  of  0.01  c  c  of 
each  dilution  spread  over  a  1-cm^  surface  of  cover  glass  mounted  with 
water  and  examined  with  the  oil-immersion  objective.  By  this 
means  a  definitely  recognizable  number  of  spores  could  be  found 
down  to  the  dilution  of  2,000,000  spores  per  cubic  centimeter,  with  a 
few  single  spores  seen  in  occasional  fields  down  to  the  dilution  of 
500,000  spores  per  cubic  centimeter.  (Table  8.)  Then  1  c  c  of  each 
dilution  was  added  to  5  c  c  of  distilled  water  in  15  c  c  centrifuge  tubes 
and  centrifuged  at  2,000  revolutions  per  minute  for  20  minutes.  A 
stained  smear  made  from  0.01  c  c  of  each  sediment  showed  a  definitely 
recognizable  number  of  spores  down  to  the  5,000-spore  dilution,  with 
one  or  two  doubtful  spores  in  several  fields  from  the  500-spore  dilu- 
tion. The  sample  containing  the  50,000-spore  dilution,  which  would 
be  comparable  to  the  sugar  sirup  containing  the  minimum  number  of 
spores  per  cubic  centimeter  fed  to  colonies  in  the  spore-feeding  experi- 
ments that  produced  infection,  showed  a  great  many  more  spores  in 
each  field  examined  by  this  method  than  did  the  sample  of  commer- 
cial honey  that  showed  the  greatest  number  of  spores.  Therefore, 
until  a  better  quantitative  method  is  devised,  it  seems  reasonable  to 
believe,  from  the  indications  of  the  preliminary  work  on  this  problem, 
that,  even  though  the  presence  of  a  few  spores  of  B.  larvae  may  be 


282 


Journal  of  Agricultural  Research 


Vol.  45,  No.  5 


demonstrated  in  5  c  c  quantities  from  a  comparatively  small  per- 
centage of  samples  of  commercial  honey,  the  numbers  are  far  below 
the  minimum  necessary  to  produce  infection  when  such  honey  is 
used  in  healthy  colonies  of  bees.  Before  definite  conclusions  can 
be  drawn,  it  will  be  desirable  to  examine  many  more  samples  of  coxn- 
mercial  honey  and  to  feed  to  healthy  colonies  samples  of  honey  in 
which  the  presence  of  spores  has  been  demonstrated. 

Table  8. — Microscopic  examination  of  dilutions  for  spores  of  Bacillus  larvae  " 


Number  of 
spores  per 
cubic  centi- 
meter in  each 
dUution 

Direct 
exami- 
nation 
of  0.01 
cubic 
centi- 
meter 

Exami- 
nation 
of  sedi- 
ment 
after 
centri- 
fuging 
1  cubic 
centi- 
meter 

Number  of 
spores  per 
cubic  centi- 
meter in  each 
dilution 

Direct 
exami- 
nation 
of  0.01 
cubic 
centi- 
meter 

Exami- 
nation 
of  sedi- 
ment 
after 
centrl- 
fuging 
1  cubic 
centi- 
meter 

Number  of 
spores  per 
cubic  centi- 
meter in  each 
dilution 

Direct 
exami- 
nation 
of  0.01 
cubic 
centi- 
meter 

Exami- 
nation 
of  sedi- 
ment 
after 
centri- 
fuging 
1  cubic 
centi- 
meter 

5,  000,  000,  000 

4,  000,  000,  000 

3,  000,  000,  000 

2,  000,  000,  000 

1,  000,  000,  000 

500,  000,  000 

400,  000,  000 

300,  000,  000 

200,  000,  000 

100.  000,  000 

90,  000.  000 

80,  000,  000 

70,  000,  000 

-j- 
-r 
+ 
-t- 
-1- 
-1- 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 

H- 
-1- 
-t- 
+ 
-t- 
-1- 
-t- 
-t- 
+ 
-1- 
+ 
+ 
+ 
+ 
+ 
+ 
+ 
+ 

10, 000, 000 

9, 000, 000 

8,  000,  000 

7,  000,  000 

6,  000,  000 

5,  000,  000 

4,000,000 

3,  000,  000 

2,  000,  000 

1,  000,  000 

900,  000 

800,  000 

700,  000 

eoo,  000 

SOO,  000 
400,  000 
300,  000 
200, 000 

-1- 
-+- 
-f 
-1- 
+ 
+ 
-f- 

+ 
±? 

— ^y- 

+ 
+ 
+ 
+ 
+ 
+ 
+ 
-1- 
+ 
-1- 
-f 
+ 
+ 
-i- 
+ 
-t- 
-1- 
+ 

100, 000 

90, 000 

80, 000 

70,  000 

60, 000 

60,000 

40, 000 

30,  ( 00 

20,  000 

10,  000 

5,000 

4,000 

3,  f  00 

2,000 

1,000 

500 

50 

6 

"'"-'"" 

+ 
+ 
-1- 
+ 
-t- 
+ 
+ 

4- 
-i- 
-1- 
+ 

60,  000,  000 

50,  000,  000 

40, 000,  000 
30,  000,  000 
20,  000,  000 

± 

<•  -j-  indicates  that  spores  were  found;  —  indicates  that  spores  were  not  found,  by  microscopic  examina- 
tion; ±  indicates  that  the  result  was  doubtful;  ±?  indicates  that  the  positive  was  more  doubtful  than 
the  negative;  — ?  indicates  that  the  absence  of  spores  was  not  definite. 

SUMMARY  AND  CONCLUSIONS 

As  a  result  of  five  years'  study  it  has  been  found  that,  in  order  to 
produce  American  foulbrood  infection  in  a  healthy  colony  of  bees, 
the  sugar  sirup  used  for  inoculation  must  contain  a  certain  initial 
number  of  spores  of  Bacillus  larvae.  Seventy-three  colonies  were 
inoculated  during  this  time  with  numbers  of  spores  ranging  from 
approximately  5,000,000,000  to  100,000  per  colony;  30  of  these 
colonies  receiving  50,000,000  spores  or  less.  Of  these  30  colonies,  2 
out  of  11  receiving  50,000,000  spores  showed  infection,  but  no  colony 
receiving  less  than  that  number  of  spores  developed  disease.  There- 
fore, the  minimum  infectious  dose  of  B.  larvae  for  a  colony  of  bees 
seems  to  be  approximately  50,000,000  spores  in  1  liter  of  sugar  sirup. 

PreUminary  experiments  in  which  individual  bee  larvae  were  given 
known  numbers  of  spores  of  Bacillus  larvae  in  0.01  c  c  quantities  of 
sugar  sirup  show  that  infection  can  be  produced  bv  this  method, 
but  with  considerable  difficulty.  From  50  to  100  larvae  were 
inoculated  with  each  dilution  of  spores,  ranging  in  number  from 
approxinaately  50,000,000  spores  to,  theoretically,  1  spore  per  larva. 
The  minimum  infectious  dose  was  found  to  be  10,000,000  spores  per 
larva  fed  in  0.01  c  c  of  sugar  sirup.     These  results  indicate  that  the 


Sept.  1, 1932     Commercial  Honey  and  Spread  of  American  Foulbrood       283 

minimum  dose  of  spores  of  B.  larvae  that  will  produce  American  foul- 
brood  infection  must  be  large. 

The  germination  of  spores  of  Bacillus  larvae  and  vegetative  growth 
on  a  suitable  artificial  culture  medium  resulting  from  the  inoculation 
of  556  culture  tubes  with  seedings  varying  from  approximately 
50,000,000,000  to  500  spores  per  culture  also  shows  that  a  certain 
minimum  initial  number  of  spores  in  the  inoculum  is  necessary  in 
order  to  produce  growth.  This  minimum  number  of  spores  produc- 
ing vegetative  growth  on  a  medium  consisting  of  yeast-carrot  extract, 
egg-yolk  suspension,  and  agar  was  found  to  be  approximately  50,000 
in  1  c  c  of  suspension  inoculated. 

The  production  of  nitrite  in  this  medium  by  the  vegetative  growth 
of  Bacillus  larvae  serves  as  a  fairly  delicate  and  reliable  indicator  of 
such  growth. 

There  was  a  tendency  for  the  seedings  containing  the  smaller  num- 
bers of  spores  of  Bacillus  larvae  to  require  a  longer  period  of  incubation 
than  the  larger  seedings  in  order  to  produce  vegetative  growth. 
However,  there  was  a  considerable  variation  in  the  germination  time 
of  many  of  the  seedings  of  spores,  in  one  case  a  seeding  of  9,000,000 
spores  requiring  27  days'  incubation  to  produce  growth  and  another 
of  70,000  spores  requiring  only  6  days.  This  variation,  thought  to 
be  due  to  the  variable  character  known  as  dormancy  in  bacterial 
spores,  prevented  more  than  a  slight  correlation. 

In  the  group  of  cultures  comprising  seedings  between  5,000,000,000 
and  9,000,000  spores,  only  1.67  per  cent  required  more  than  10  days' 
incubation  to  produce  vegetative  growth,  100  per  cent  having  shown 
growth  after  30  days.  In  the  group  of  cultures  comprising  seedings 
between  8,000,000  and  500,000  spores,  71.93  per  cent  required  more 
than  10  days'  incubation,  while  53.81  per  cent  showed  no  growth  at 
the  end  of  30  days'  incubation.  In  the  group  of  cultures  comprising 
seedings  between  400,000  and  50,000  spores,  97.18  per  cent  required 
more  than  10  days'  incubation,  while  91.55  per  cent  of  the  group 
showed  no  growth  at  the  end  of  30  days.  Below  50,000  spores  no 
growth  was  obtained.  In  other  words,  below  a  seeding  of  9,000,000 
spores  an  increasing  number  of  the  smaller  spore  seedings  required  a 
longer  period  of  incubation.  About  80  per  cent  of  all  the  positive 
cultures  were  obtained  during  the  first  10  days  of  incubation,  although 
this  was  approximately  only  30  per  cent  of  all  the  cultures  made;  at 
the  end  of  30  days'  incubation  only  about  38  per  cent  of  all  the  cul- 
tures had  shown  any  growth. 

It  was  found  possible  to  demonstrate  the  presence  of  spores  of 
Bacillus  larvae  in  15  out  of  187,  or  in  8  per  cent,  of  the  samples  of 
commercial  honey  examined  by  means  of  the  centrifuge  and  the 
microscope.  "The  preliminary  results  indicate  that,  even  though 
spores  of  B.  larvae  may  be  demonstrated  in  a  certain  percentage  of 
samples  of  commercial  honey,  in  most  instances  they  are  probably 
present  in  such  small  numbers  as  to  be  less  than  the  minimum 
number,  50,000,000  per  liter,  found  to  be  capable  of  producing  dis- 
ease, and  therefore  are  ineffective  in  the  spread  of  American  foul- 
brood. 


284  Journal  of  Agricultural  Research  voi.  45,  No.  s- 

LITERATURE  CITED 

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1930.  NEW  FACTS  ABOTTT  FORMALIN  TREATMENT.     Amer.  Bee  Joup.  70: 

61-62. 

(2)  Breed,  R.  S.,  and  Brew,  J.  D. 

1916.    COtTNTING  BACTERIA  BY  MEANS   OF  THE  MICROSCOPE.       N.   Y.   State 

Agr.  Expt.  Sta.  Tech.  Bui.  49,  31  p.,  illus. 

(3)  Buchanan,  R.  E. 

1918.  LIFE  PHASES  IN  A  BACTERIAL  CULTURE.     Joui.    Infect.    Diseases 
23:109-125,  illus. 

(4)  Burke,  G.  S. 

1923.  studies  on  the  thermal  death  time  of  spores  op  clostridium 
botulinum.     2.  the    differential   staining   of   living   and 
DEAD  SPORES.     Jour.  Infect.  Diseases  32 :  [433]-438,  illus. 
(5) 

1923.  STUDIES  ON  THE  THERMAL  DEATH  TIME  OF  SPORES  OP  CLOSTRIDIUM 
BOTULINUM.      3.    DORMANCY    OR     SLOW    GERMINATION    OF    SPORES 

UNDER  OPTIMUM  GROWTH  CONDITIONS.     Jour.  Infect.   Diseases 
33:  [2741-284. 

(6)  Burke,  V.,  Spkague,  A.,  and  Barnes,  La  V. 

1925.  DORMANCY  IN  BACTERIA.     Jour.  Infect.  Diseases  36:  [565]-560. 

(7)  BURNSIDE,  C.  E. 

1931.  DISINFECTION    OF   AMERICAN   POULBKOOD    COMBS   BY   FUMIGATION  BY 

FORMALDEHYDE.     Bee  World  12:3-7,  16-19. 

(8)  CORKINS,  C.  L. 

1928.  QUARTERLY  REPORT.     Wyo.  Beeline  5 :  25-26. 

(9)  Elford,  W.  J. 

1928.  ULTRAFILTRATION.  (AN  HISTORICAL  SURVEY,  WITH  SOME  REMARKS 
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See.  (3)  48:36-45,  illus. 

(10)  Fracker,  S.  B. 

1925.  are  commercial  honey  shipments  largely  responsible  foe  the 

DISSEMINATION    OP    AMERICAN    FOULBROOD?      Jour.     EcOn.     Ent. 

18:372-380. 

(11)  Gates,  F.  L. 

1920.  a  method  op  standardizing  bacterial  suspensions.  jour. 
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(12)  Henrici,  a.  T. 

1928.  morphologic  variation  and  the  rate  op  growth  of  bacteria. 
194  p.,  illus.,  Springfield,  111.,  and  Baltimore,  Md.  (Mono- 
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(13)  Jordan,  E.  O.,  AND  Falk,  I.  S.,  editors. 

1928.    THE     NEWER     KNOWLEDGE     OF    BACTERIOLOGY     AND     IMMUNOLOGY. 

1196  p.,  iUus.     Chicago. 

(14)  Kelley,  T.  L. 

1923.  STATISTICAL  METHOD.     390  p.,  illus.  New  York. 

(15)  KosER,  S.  A.,  AND  Mills,  J.  H. 

1925.    DIFFERENTIAL   STAINING   OP   LIVING   AND   DEAD   BACTERIAL   SPORES 

Jour.  Bact.  10:25-36. 

(16)  LiNEBURG,  B. 

1925.  STRAIN  OF  IMMUNE  BEES.     Gleanings  Bee  Cult.  53 :  709-710. 

(17)  LOCHHEAD,  A.  G. 

[1927.]    FURTHER  STUDIES  OF   BACILLUS   LARV^,    THE    CAUSE    OF   AMERICAN 

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1926:13-16. 


1928. 


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(19)  AND  Heron,  D.  A. 

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1928.  SPREADING  FOULBROOD.     Beekeeper  36: 134. 

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(27)  

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o 


K-269 


QUANTITATIVE  DEMONSTRATION  OF  THE  PRESENCE 

OF  SPORES  OF  BACILLUS  LARVAE  IN  HONEY 

CONTAMINATED  BY  CONTACT  WITH 

AMERICAN  FOULBROOD 


BY 


A.  P.  STURTEVANT 


(Contribution  from  Bureau  of  Entomology  and  Plant  Quarantine) 


Reprinted  from  JOURNAL  OF  AGRICULTURAL  RESEARCH 

Vol.  52,  No.  9     :     :     :     :    Washington,  D.  C,  May  1,  1936 

(Pages  597-704) 


ISSUED  BY  AUTHORITY  OF  THE  SECRETARY  OF  AGRICULTURE 

WITH  THE   COOPERATION   OF   THE   ASSOCIATION   OF 

LAND-GRANT  COLLEGES  AND  UNIVERSITIES 


U.  S.  GOVERNMENT  PRINTING  OFFICE  :  1936 


JOINT  COMMITTEE  ON  POLICY  AND  MANUSCRIPTS 


TOR  THE    UiriTED    STATES    DEPARTMENT  FOE  THE  ASSOCIATIOIT   OF  LASfD-GEANT 

OF  AGRICTJITURE  COIIEGES  AND  UNIVERSITIES 

H.  G.  KNIGHT,  Chairman  S.  W.  FLETCHER 

Chief,  Bureau  of  Chemistry  and  Soils  Director  of  Research,  Pennsylvania  Agri- 
cultural Experiment  Station 

F.L.CAMPBELL  j    y   p.TT 

^lT?^,lf  b^„";S^{''""'™''"""'  '  director,  Kansas  AgricuUural  Experiment 


and  Plant  Quarantine 


Station 


JOHN  W.  ROBERTS  C.  E.  LADD 

Principal  Pathologist,  Bureau  of  Plant  Director,  New  York  {Cornell)  Agricultural 

Industry  Experiment  Station 


EDITORIAI  STTPEEVISION 

M.  C.  MERRILL 

Chief  of  Publications,  United  States  Department  of  Agriculture 


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QUANTITATIVE  DEMONSTRATION  OF  THE  PRESENCE 
OF  SPORES  OF  BACILLUS  LARVAE  IN  HONEY  CON- 

S^5!?i^^'^^°   ^Y  CONTACT  WITH  AMERICAN  FOUL- 
BROOD  ^ 

By  A.  P.  Sttjbtevant  ' 
Associate  apiculturist,  Division  of  Bee  Culture,  Bureau  of  Entomology  and  Plant 
Quarantine,  United  States  Department  of  Agriculture 

INTRODUCTION 

In  a  previous  paper  ^  the  writer  showed  that  it  is  possible  to 
demonstrate  the  presence  of  spores  of  Bacillus  larvae,  the  cause  of 
American  foulbrood,  in  samples  of  commercial  honey  that  have  had 
contact  with  American  foulbrood  in  the  course  of  their  production  or 
prepa,ration  for  the  market.  Siace  this  work  was  reported,  25 
additional  samples,  making  a  total  of  212  samples  of  commercial 
honey,  obtained  on  the  open  market  from  28  States  and  2  Territories 
have  been  examined  by  the  same  method,  and  spores  of  B.  larvae 
have  been  found  in  17,  or  8  percent,  of  these  samples.^  In  most 
cases  the  spores  were  present  in  relatively  small  numbers. 

The  method  of  examiaation  used  in  the  work  thus  far  reported 
gave  only  a  qualitative  indication  of  the  number  of  spores  present, 
the  observations  being  recorded  as  showing  "the  presence  of  a  suflfi- 
cient  number  of  spores  resembling  spores  of  B.  larvae  to  be  designated 
as  positive."*  This  araounted  to  from  one  or  two  definite  spores  to 
a  very  few  spores  seen  in  numerous  microscopic  fields  of  each  stained 
sediment  examined.  The  primary  object  was  to  demonstrate  only 
their  presence  or  absence.  It  was  assumed  that  iu  most  cases  the 
number  of  spores  found  was  considerably  smaller  than  would  be 
foimd  in  honey  containing  numbers  comparable  with  the  observed 
minimum  infective  dose  of  50,000,000  per  Hter. 

The  only  way  of  demonstrating  the  accuracy  of  this  assumption 
has  been  to  feed  such  "positive"  samples  of  commercial  honey  to 
healthy  colonies  of  bees.  This  was  done  with  15  of  the  16  samples 
in  which  spores  were  demonstrated,  and  only  1  sample,  or  6.7  per- 
cent, was  found  to  contain  sufficient  infection  to  produce  the  disease 
in  a  healthy  colony.  These  investigations  indicate  that  the  require- 
ment of  certification  of  honey,  as  has  been  proposed  and  even  placed 
in  operation  in  certain  States,  is  not  a  justifiable  measure  in  the 
control  of  American  foulbrood  under  the  present  conditions  of 
inspection  and  control  of  disease  in  this  country. 

To  permit  a  more  accurate,  quantitative  study  of  the  infectivity 
of  honey  that  has  been  in  contact  with  American  foulbrood,  on  the 

'  Beeeived  for  publication  Jan.  27,  1936;  issued  June  1936.  Tliis  investigation  was  carried  on  at 
the  Intermountain  States  laboratory  of  the  Division  of  Bee  Culture,  which  is  maintained  cooperatively 
by  the  University  of  Wyoming  and  the  Bureau  of  Entomology  and  Plant  Quarantine,  XJ.  S.  Department 
of  Agriculture. 

'  Acknowledgments  are  due  to  P.  E.  Hall,  associate  professor  of  commerce,  University  of  Wyoming,  for 
advice  and  assistance  in  the  statistical  analysis  of  the  data. 

'  STOETEVANT,   a.    p.     EELATION   of  COMMEBCIAL  honey   to   the   SPKEAD   or  AMEEICAN  rOULBEOOD. 

Jour.  Agr.  Research  45: 257-285,  illiu.    1932. 

*  Stdetevant,  a.  p.  honey  or  the  inteemodntain  eegion.  Gleanings  Bee  Cult.  63: 463-468,  illus. 
1935. 

»  Sttjetevant,  a.  p.    See  footnote  3. 

Journal  of  Agricultural  Research,  Vol.  62,  no.  9 

Washington,  D.  O.  May  1,  1936 

Key  no.  K:-269 
57176—36  (697) 


698  Journal  oj  Agricvltural  Research  voi.  62,  no.  9 

basis  of  its  spore  content^that  is,  a  detailed  study  of  the  distribution 
of  spores  of  B.  larvae  m.  the  honey  from  infected  hives  or  apiaries, 
or  in  commercial  honey  obtained  on  the  open  market,  or  of  the  effect 
of  mixing  infected  honey  with  disease-free  honey  in  the  course  of 
production  or  blending  and  preparation  for  the  market — a  more 
detailed  iavestigation  has  been  made  of  the  spore  content  of  honey 
containing  approximately  known  numbers  of  spores.  This  has  been 
accomphshed  by  an  improved  and  more  accurate  method  of  deter- 
mining the  number  of  spores  in  such  honey,  and  the  accuracy  of  the 
results  and  method  has  been  demonstrated  by  means  of  a  statistical 
analysis  of  the  data  obtained. 

METHOD  OF  OBTAINING  THE  DATA 

PREPARATION  OF  SAMPLES  OF  HONEY 

A  series  of  samples  of  honey  containing  approximately  known 
numbers  of  spores  per  cubic  centimeter  were  prepared  in  the  manner 
described  previously,'  by  adding  to  100-cc  quantities  of  spore-free 
honey  the  necessary  quantities  of  various  dilutions  of  a  stock  suspen- 
sion of  spores  of  Bacillus  larvae  containing  approximately  5,000,000,000 
spores  per  cubic  centimeter.  Five  samples  of  honey  were  prepared 
in  this  way  containing  approximately  1,000,000,  800,000,  500,000, 
300,000,  and  50,000  spores  per  cubic  centimeter,  respectively.  These 
samples,  each  considered  as  a  unit  and  not  as  a  dilution  of  the 
1,000,000-spore  sample,  were  heated  in  a  water  bath  to  120°-130° 
F.,  and  then  thoroughly  mixed  with  a  mechanical  stirrer  for  5  minutes. 
Duplicate  5-cc  quantities  of  each  sample  were  then  placed  in  50-cc 
conical  centrifuge  tubes,  and  45  cc  of  distilled  water  of  approximately 
the  same  temperature  was  added.  When  the  honey  and  water  were 
completely  mixed,  the  samples  were  centrifuged  at  2,000  revolutions 
per  minute  for  45  minutes.  All  but  about  1  cc  of  the  supernatant 
honey-water  solution  of  each  sample  was  then  removed  by  means  of 
a  pipette  and  suction.  Again  approximately  45  cc  of  distilled  water 
was  added,  and  after  thorough  mixing  the  suspensions  were  centri- 
fuged for  30  minutes  longer.  The  removal  of  the  supernatant  solu- 
tion was  repeated  until  all  but  approximately  0.1  cc'  of  the  water 
had  been  removed  from  each  centrifuge  tube,  and  each  sample  of 
sediment  was  completely  suspended  in  this  remaining  quantity  of 
water  by  blowing  gently  through  a  capUlary  pipette  dipped  into  the 
water.  Duphcate  0.01-cc  quantities  of  each  suspension  were  then 
transferred  with  the  capillary  pipette  (calibrated  to  deliver  0.01  cc) 
to  microscope  cover  glasses.  Circular  cover  glasses,  size  12,  no.  1 
thickness,  having  an  area  of  1.13  cm  2,  proved  satisfactory  for  this 
P^Pj®^"  u  ^^^'^  ^^  *°  ^  "^™)  loopful  of  carbolfuchsm  stam  was 
added  to  the  drop  of  suspension  on  the  cover  glass  and  thoroughly 
mixed  with  it.  This  stained  liquid  was  then  spread  uniformly  over 
a  1-cm  area  of  the  cover  glass,  a  narrow  ring  at  the  outside  edge 
being  left  uncovered.  The  smears  were  allowed  to  dry  in  the  air 
and  were  then  mounted  on  microscope  slides  either  with  water  or  pref- 
erably, with  Canada  balsam,  for  examination  under  the  microscope, 
ihese  stamed  smears  were  not  washed  in  water,  as  this  might  have 
caused  some  spores  to  be  lost. 

•  Stuetevant,  a.  p.    See  footnote  3. 

'  A  mark  was  placed  on  the  outside  of  the  conical  centrifuge  tubes  to  indicate  the  0.1-co  volume. 


Uay  1, 1036 


Spores  <yf  Bacillus  Larval  in  Honey 


699 


The  foregoing  process  gives  a  concentration  of  spores  in  the  sedi- 
ment from  the  5-cc  samples  of  honey  suspended  in  0.1  cc  of  water, 
or  one-fiftieth  the  original  volume. 

METHOD  OF  COUNTING  SPORES 

A  method  simUar  to  that  of  Breed  and  Brew  *  for  counting  bacteri'a 
in  milk  was  used  for  counting  the  spores  of  Bacillus  larvae  in  these 
staiued  smears.  This  method  is  similar  to  that  described  in  a  previous 
paper  ®  and  is  represented  by  the  formula 


Number  of  spores  per  cubic  centimeter= 


KNXXIOOXD 


N 

where  K  is  the  factor  for  the  number  of  circular  fields  per  1-cm^  area, 
N  is  the  number  of  circular  fields  counted,  X  is  the  actual  mean 
number  of  spores  per  field,  100  is  the  factor  that  gives  the  number  of 
spores  per  cubic  centimeter  from  0.01  cc  of  the  suspension,  and  D 
is  the  dilution. 

Table  1. — Spore  counts  in  stained  smears  of  the  sediments  resulting  from  the  cen- 
irifuging  of  duplicate  5-cc  portions  of  five  samples  of  honey  containing  known 
numbers  of  spores  of  Bacillus  larvae 


Spore  counts  in  samples '  containing  the  indicated  number  of 
spores  per  cubic  centimeter 

Field  no. 

60,000 

300,000 

600,000 

800,000 

1,000,000 

A 

B 

A 

B 

A 

B 

A 

B 

A 

B 

1 

2 
2 
1 
0 
2 
1 
0 
2 
1 
0 
1 
1 
1 
2 
1 
2 
1 
2 
0 
0 
2 
2 
1 
3 
1 
0 
1 
2 
2 
2 

1 
2 
1 
2 
0 
3 
1 

I 
2 

1 
3 
3 
1 
0 
1 
0 
0 
2 
1 
1 
3 
1 
1 
1 
1 
1 
1 
2 
2 

7 
8 
8 
7 
9 
9 
8 
7 
8 
9 
7 

ID 
9 
7 
8 
8 
6 
6 
7 
9 

10 

11 
7 
6 
7 
6 
S 
7 
8 

10 

8 
9 

10 
6 
6 
7 
7 
8 
6 

12 

11 
6 
6 
9 

10 
5 
9 
6 

10 
6 

16 

10 
8 
6 
9 
7 

10 
8 

10 
8 

14 
12 
12 
10 
10 
12 
15 
12 
16 

\l 
16 
13 
18 
12 
10 
11 
13 
17 
13 
13 
8 
18 
16 
12 
16 
10 
12 
11 
12 

16 
12 
13 
16 
11 
13 
14 
13 
16 
13 
12 
12 
10 
14 
13 
11 
16 
14 
16 
18 
15 
16 
10 
12 
15 
10 
14 
16 
11 
11 

19 
18 
24 
22 
20 
18 
21 
27 
19 
21 
16 
21 
22 
25 
21 
20 
18 
26 
24 
26 
16 
19 
20 
18 
21 
21 
26 
22 
22 
25 

21 
20 
18 
18 
20 
17 
18 
17 
21 
20 
19 
20 
22 
24 
26 
26 
28 
23 
23 
28 
23 
18 
16 
22 
21 
20 
18 
26 
28 
22 

24 
24 
26 
23 
3D 
23 
26 
29 

Jg 
25 
28 
27 
25 
29 
24 

25 
27 
25 
26 
27 
•     28 
29 
27 
34 
22 
29 
21 

21 

2 

29 

3 

38 

i .._ 

24 

S 

34 

6: 

29 

7. 

fl 

8 

21 

9 

26 

10 

31 

11 

36 

12 

26 

13 

33 

14 

22 

16 

26 

16 .                 ..      . 

26 

17 

18 

28 

19 ....:. 

34 

20   . 

30 

21 

32 

22 

2S 

23..  . 

25 

24 

34 

26 

28 

26 .      . 

22 

27 ;..^ 

23 

28. .i ^ ; 

27 

29 

28 

30 

3rt 

Total :: .;..... 

38 

39 

233 

244 

393 

400 

638 

641 

791 

836 

.        Total  for  60  fields.-i,-, 

Mean  niiniber  M  sporfeS  pet  field 

7 
1.21 

7  , 
333 

4' 
7.9 

7 
500 

,75 
13.2 

h 

1,2 
21.3 

1^7 

1.' 
27.1 

36 
ODD 

'  A  and  B  represent  duplicate  portions  of  the  samples. 


'  Bebed,  B.  S.;  aitd  Sbkw',  J.  li.   fcocNTJNa  baoieeia  bt  means  of  ihe  micSoscope.   N.  Y.  StSte 
Agr,,  Expt.  eta.  TSoH,  Bull.  4fl,  31  pp..  Ulus.    1916. 
'  STTETEtANT,  A.  F.    SeB  foOtndt^  3. 


700 


Journal  of  Agricultural  Research 


Vol.  62,  no.  9 


An  ocular  micrometer  disk,  such  as  is  used  for  counting  bacteria  in 
milk,  was  used  in  counting  spores  in  the  fields  of  the  stamed  smears 
The  area  of  the  circle  etched  on  this  disk  was  found  to  be  O.OOUUbUb^ 
cm^  when  used  in  a  biaocular  microscope  with  15  X  paired  eyepieces 
anda  1.8-mm  oil-immersion  objective.    Therefore,  the  factor  it  became 

16,441.96.  ^    ,       ,      ,.     . 

The  spores  in  30  fields  from  each  of  the  duphcate  smears  were 
counted,  making  a  total  of  60  fields  (AT)  for  each  honey-spore  sample. 
The  fields  were  counted  at  random  from  various  parts  of  the  smear. 
From  these  counts  the  actual  mean  number  of  spores  per  field  re- 
covered in  60  fields  for  each  honey-spore  sample  was  determined 

Substituting  the  values  for  K  and  N  and  0.02  (1/50)  for  D,  the 
spore  dilution  in  the  foregoing  formula  gives 

Number  of  spores  per  cubic  centimeter 

_16,442X60XX100X0.02_g^  ^^^^ 

COMPUTATION  OF  THEORETICAL  MEAN  NUMBER  OP  SPORES  PER  FIELD 

The  theoretical  mean  numbers  of  spores  per  field  that  should  be 
recovered  from  each  of  five  honey-spore  samples  used,  under  ideal 
conditions  where  there  is  no  loss  of  spores  during  the  process,  were 
calculated  by  the  foregoing  formula,  which  for  this  purpose  may  be 
stated  as  follows: 


X-- 


Number  of  spores  per  cubic  centimeter 
32,884 


X  now  designates  the  theoretical  mean  number  of  spores  per  field. 
In  table  2  these  values  are  given  in  comparison  with  the  corresponding 
actual  mean  number  of  spores  per  field  for  each  honey-spore  sample. 

Table  2. — Relation  between  the  actual  and  the  theoretical  mean  numbers  of  spores  of 
Bacillus  larvae  per  field  recovered  from  five  samples  of  honey  containing  known 
numbers  of  spores  per  cubic  centimeter 


Spores  per  cubic  centimeter  in  sample  (number) 


Mean  spores  per  field 


Theoretical 


Actual 


Standard 
deviation 


Ratio  of 
actual  mean 
to  theoret- 
ical mean 


1,000,000. 
800,000.. 
500,000-. 
300,000.. 
50,000... 


Number 

30. 4100 

24. 3279 

16. 2050 

9.1230 

1.6205 


Number 

27.  lOOOiO.  3554 

21. 3167±  .  2751 

13.2167±  .2011 

7. 9500it  .  1708 

1. 2833±  .  0747 


Number 
4. 0812 
3. 1596 
2.3100 
1.9615 
.8582 


Percent 
89.12 
87.62 
86.92 
87.14 
84.40 


RESULTS  OBTAINED  BY  USE  OF  THE  METHOD 

By  the  method  used,  the  actual  mean  number  of  spores  per  field 
obtained  by  counting  60  fields  from  each  honey-spore  sample  differed 
from  the  calculated  theoretical  mean  number  of  spores  per  field  by 
10.88  percent  for  the  honey  containing  1,000,000  spores  per  cubic 
centimeter  to  15.60  percent  for  the  honey  containing  50,000  spores  per 


May  1, 1930 


Spores  of  Bacillus  Larvae  in  Honey 


701 


cubic  centimeter  (table  2).  This  difference,  which  is  relatively 
constant  for  each  sample,  may  be  due  to  the  fact  that  some  spores 
are  lost  during  the  centrifuging,  but  more  probably  to  the  fact  that 
a  certain  proportion  of  the  spores  in  each  smear  are  covered  up  and 
not  seen  in  the  masses  of  stained  debris  always  present  even  in  honey 
of  the  highest  quality. 

DETERMINATION  OF  ACCURACY  OF  THE  METHOD 

STATISTICAL  ANALYSIS  OP  THE  DATA 

Since  the  data  obtained  for  the  actual  mean  number  of  spores  per 
field  (table  1)  for  each  honey-spore  sample,  if  plotted  against  the  data 
calculated  for  the  theoretical  mean  number  of  spores  per  field  (table 
2),  give  practically  a  straight  line  having  a  trend  similar  to  that  of 
a  line  plotted  for  the  theoretical  data  alone,  the  relation  between  the 
theoretical  means  and  the  actual  means,  for  the  five  honey-spore 
samples  used,  was  determined  by  the  customary  statistical  methods. 

The  standard  deviation  and  the  probable  error  for  the  actual  mean 
number  of  spores  per  field  were  determined  from  frequency  tables 
prepared  from  the  original  data  (table  1)  for  each  honey-spore 
sample  used  '°  (table  2).  The  actual  means  were  derived  from  large 
samples  (60  fields  each),  and  the  calculated  probable  errors  and 
standard  deviations  were  shown  statistically  to  be  small. 

The  coefficient  of  correlation  "  between  the  values  for  the  actual 
mean  number  and  those  for  the  theoretical  mean  number  of  spores 
per  field  for  each  sample  as  given  in  table  2  was  found  to  be  0.9999 ± 
0.0001. 

The  relation  between  the  actual  mean  number  of  spores  per  field 
recovered  from  each  honey-spore  sample  and  the  corresponding  most 
probable  values  estimated  from  the  theoretical  mean  number  of  spores 
per  field  for  each  sample  was  determined  by  use  of  the  regression 
equation  for  the  actual  mean  number  of  spores.  This  was  found  to 
be  r=0.8905-X'— 0.1791.  Substituting  the  various  values  of  the 
theoretical  mean  number  of  spores  per  field  (table  2)  for  X  in  this 
equation  gave  the  most  probable  estimated  values  for  the  actual  mean 
number  of  spores  per  field  (Y)  that  should  have  been  recovered  from 
each  sample  (table  3).  These  most  probable  estimated  values  were 
found  to  be  in  excellent  agreement  with  the  actual  values  obtained. 

Table  3. — Theoretical  and  actual  mean  numbers  of  spores  per  field  and  the  most 
probable  estimated  theoretical  and  actual  mean  numbers  of  spores  per  field 


Number  of  spores  per  cubic  centimeter  in 
sample 

Mean  number  of  spores  per  field 

Theoretical 

Estimated 
theoretical 

Actual 

Estimated 
actual 

1,000,000 

30.4100 
24.3280 
16.2050 
9.1230 
1.6205 

30.8313 
24. 1378 
16.0431 
9. 1297 
1.  6443 

27.1000 
21. 3167 
13.  2167 
7.9500 
1.2833 

26. 9010 

800,000 

21. 4860 

600,000 

13. 3610 

300,000 

7.9449 

60,000 

1. 1749 

"  Chaddoce,  B.  E.    PEINCIPLE3  AND  METHODS  OF  STATisTica.    pp.  160-164,  240-241.     Boston,  New 
York  [etc.].    1926. 
"  Oboxton,  F.  E.,  and  Cowden,  D.  J.    practical  bdsiness  statistics,    p.  416.    New  York.    1934. 


702  Journal  oj  Agricultural  Research  voi.  62,  no. » 

The  purpose  of  this  investigatioa,  however,  was  to  develop  an 
equation  with  which,  if  the  actual  mean  number  of  spores  per  field 
is  obtained  with  sufficient  accuracy,  the  theoretical  number  of  spores 
per  field  may  be  estimated,  thereby  giving  the  data  necessary  for 
estimating  the  number  of  spores  per  cubic  centimeter  in  an  unknown 
sample  of  honey.  The  regression  equation  or  the  theoretical  mean 
number  of  spores  per  field  can  be  used  for  this  piirpose,  and  was  found 
to  be  Z=1.1228F+0.2034.  Substituting  for  Y  in  this  equation,  the 
various  values  of  the  actual  mean  number  of  spores  per  field,  as 
obtained  in  table  1,  gave  the  most  probable  estimated  values  for  the 
theoretical  mean  number  of  spores  per  field  that  should  be  obtained 
from  the  actual  counts  for  each  honey-spore  sample  (table  3).  By 
this  method  of  estimation  these-  values  were  found  to  agree  closely 
with  the  original  calculated  values  for  the  theoretical  mean  number 
of  spores  per  field  for  each  honey-spore  sample  (table  2). 

DETERMINATION  OF  PERMISSIBLE  LIMITS  OF  ERROR 

The  analysis  of  the  data  so  far  indicates  the  accuracy  of  the  method 
outUned  above  for  determining  the  most  probable  actual  mean  spore 
count  per  field  from  the  mean  of  60  fields  counted.  Variations  in  the 
counts  may  occur  in  individual  samples,  however,  owing  to  the  failure 
to  recover  all  the  spores,  as  stated  previously. 

The  permissible  limits  of  error  in  the  statistical  analysis  of  such 
cases  are  customarily  determined  by  use  of  the  standard  error  of 
estimate.  This,  for  the  most  probable  estimated  actual  means 
derived  from  the  theoretical  means,  was  found  to  be  small,  ±0.1298 
spore,  and  indicates  the  closeness  with  which  new  estimated  values 
may  be  expected  to  approximate  the  true  but  unknown  values.  Since 
two  of  the  five  actual  means  fall  within  ±0.1298  spore  of  the  esti- 
mated actual  means  while  the  other  three  are  only  from  0.11  to  0.26 
percent  outside  this  zone,  within  which  approximately  two- thirds 
of  the  observations  may  be  expected  to  fall  in  relation  to  the  most 
probable  values,  a  sufficient  accuracy  for  the  method  is  indicated. 

The  standard  error  of  estimate  for  the  most  probable  theoretical 
means  derived  from  the  actual  means  (which  were  found  to  agree 
closely  with  the  estimated  actual  means)  was  found  to  be  ±0.1458 
spore.  As  is  to  be  expected  in  this  case,  again  two  of  the  original 
theoretical  means  fall  within  the  zone  of  ±0.1458  spore  while  the 
other  three  are  only  from  0.11  to  0.25  percent  outside  this  zone. 
However,  since  ±  3  times  the  standard  error  of  estimate,  which  should 
include  99.7  percent  of  all  observations,  is  used  customarily  in  delin- 
eating the  largest  error  to  which  statistical  analyses  of  this  type  are 
subject,  it  is  found  that  all  the  theoretical  means  fall  well  within  this 
zone,  or  within  ±  0.4374  spore.  This  indicates  the  probable  accuracy 
of  estimating  the  number  of  spores  per  cubic  centimeter  in  an  unknown 
sample  by  calculating  the  most  probable  theoretical  number  of  spores 
per  field  from  the  actual  mean  number  counted. 

PRACTICAL  APPLICATION  OF  THE  METHOD 

In  a  previous  paper  ^^  it  was  shown  that  during  observations  cover- 
ing 5  years  no  cases  of  American  foulbrood  developed  in  19  colonies 
of  bees  fed  less  than  approximately  50,000,000  spores  of  BaciUus 

"  Stuktbtant,  a.  p.    See  table  1  of  reterence  In  footnote  3. 


May  1, 1936  Spores  of  BocUlus  Larvae  in  Honey  703 

larvae  in  1  liter  of  sugar  sirup,  or  less  than  50,000  spores  per  cubic 
centimeter.  Of  11  colonies  fed  50,000  spores  per  cubic  centimeter,  2 
developed  disease  and  9  remained  healthy;  of  6  colonies  fed  75,000 
per  cubic  centimeter,  3  developed  positive  disease  and  I  probable 
disease,  and  2  remained  healthy;  of  6  colonies  fed  100,000  per  cubic 
centimeter,  2  were  positive,  1  probable,  and  3  remained  healthy;  of 
4  colonies  fed  200,000  spores  per  cubic  centimeter,  3  were  positive 
and  1  probable.  Thus  it  was  assumed  that  50,000  spores  per  cubic 
centimeter  of  sirup  could  be .  considered  the  critical  number  or 
minimum  infectious  dose  of  spores  that  will  produce  disease,  when 
1  liter  is  used  as  the  unit  volume  to  be  fed. 

Since  the  foregoing  analysis  of  the  data  indicates,  by  the  method  of 
estimating  used,  that  the  actual  mean  number  of  spores  per  field  falls 
well  withm  the  limits  of  permissible  error  for  the  estimated  actual 
means  (±3  times  the  standard  error  of  estimate),  the  most  probable 
value  for  such  a  mean  for  use  in  determining  the  number  of  spores  per 
cubic  centimeter  of  an  unknown  sample  is  the  actual  mean  number  of 
spores  per  field  determined  by  counting  30  fields  each  from  stained 
smears  from  two  centrifuged  sediments  of  this^ample.  If  the  formula 
X=1.1228F+0.2034  is  used  to  estimate  X,  the  most  probable 
theoretical  number  of  spores  that  should  have  been  recovered,  when 
Y  represents  the  actual  mean  number  of  spores  per  field,  and  if  this 
value  is  then  multiplied  by  32,884,  the  most  probable  number  of 
spores  per  cubic  centimeter  in  the  unknown  sample  can  be  calculated. 
Applying  the  limits  of  error  for  X,±3  times  the  standard  error  of  esti- 
mate, or  ±0.4374  spore,  and  carrying  it  through  into  the  second  for- 
mula will  give  the  possible  range  in  which  the  number  of  spores  per 
cubic  centimeter  might  fall  within  the  precision  of  the  method. 

Further  work  is  in  progress  to  determine  whether  the  same  accuracy 
will  be  obtained  by  counting  a  smaller  number  of  fields  to  obtain  the 
mean  number  of  spores  per  field  from  a  larger  number  of  smears  from 
sediments. 

Since  in  the  experimental  work  the  samples  of  known  spore  content 
contained  approximately  round  numbers  of  spores — multiples  of 
50,000 — ^it  probably  would  be  sufficiently  accurate  to  designate  the 
number  of  spores  as  the  nearest  multiple  of  50,000  to  the  actual  figures 
derived  from  the  formulas.  When  using  the  limits  of  error  0± 0.4374 
spore  per  field,  for  the  estimated  mean  number  of  spores  per  field,  it 
wiU  be  found  that  for  numbers  below  100,000  there  will  be  some 
overlapping  between  10,000-spore  increments,  and  the  value  will 
have  to  be  expressed  approximately  (for  example,  the  honey  contains 
between  40,000  and  60,000  spores  per  cubic  centimeter);  neverthless 
the  honey  can  still  be  designated  either  as  dangerous  or  as  not 
dangerous. 

SUMMARY 

Previous  work  on  the  qualitative  demonstration  of  the  presence  or 
absence  of  spores  of  Bacillus  larvae  in  honey  that  has  been  in  contact 
with  American  foulbrood  has  been  followed  by  the  development  of  a 
quantitative  method  for  determining  the  approximate  number  of 
spores  per  cubic  centimeter  in  such  honey.  The  method  is  represented 
by  the  formula 

Number  of  spores  per  cubic  centimeter= ^^ 


704  Journal  oj  Agricultural  Research    voi.  52,  no.  9,  May  1,  i936 

where  Kis  the  factor  for  the  number  of  circular  fields  per  1-cm^  area, 
N  is  the  number  of  circular  fields  counted,  X  is  the  actual  mean  num- 
ber of  spores  per  field,  100  is  the  factor  that  gives  the  number  of  spores 
per  cubic  centimeter  from  0.01  cc  of  the  suspension,  and  D  is  the  dilu- 
tion. The  mean  number  of  spores  of  Bacillus  larvae  per  field  counted 
in  60  fields  of  stained  smears  made  from  the  sediments  obtained 
by  centrifuging  5-cc  quantities  of  honey  contaiuing  approximately 
known  numbers  of  spores  have  been  determined  by  this  method. 

The  mean  actual  spore  count  per  field  was  determined  for  a  series 
of  samples  of  honey  prepared  to  contaia  approximately  1,000,000, 
800,000,  500,000,  300,000,  and  50,000  spores  per  cubic  centimeter. 
The  mean  theoretical  spore  count  per  field  that  should  have  been 
recovered  was  determined  by  use  of  the  formula 

y_  Number  of  spores  per  cubic  centimeter 
32,884 

The  actual  mean  numbers  of  spores  per  field  were  similar  in  trend 
to  the  calculated  theoretical  means  but  were  from  10.88  to  15.60  per- 
cent smaller.  A  statistical  analysis  of  the  data  to  determine  the 
accuracy  of  the  method  showed  that  the  calculated  probable  errors 
and  standard  deviations  were  small.  The  coefficient  of  correlation 
between  the  actual  and  the  theoretical  mean  number  of  spores  per 
field  for  each  sample  was  found  to  be  0.9999±0.0001. 

_The  relation  between  the  actual  mean  number  of  spores  per  field 
(F)  and  the  corresponding  most  probable  values  that  should  have  been 
recovered,  estimated  from  the  theoretical  mean  number  of  spores  per 
field  (X),  was  determiaed  by  means  of  the  regression  equation 
F=0.8905Z'— 0.1791.  These  most  probable  estimated  values  were 
found  to  be  in  excellent  agreement  with  the  actual  values  obtained, 
well  within  the  customary  limits  of  ±3  times  the  standard  error  of 
estimate,  which  was  found  to  be  ±0.1298  spore. 

_The  most  probable  theoretical  mean  number  of  spores  per  field 
(X)  was  estimated  by  means  of  the  regression  equation  X=1.1228F+ 
0.2034.  These  values  were  found  to  be  in  excellent  agreement  with 
the  original  calculated  values  for  the  theoretical  mean,  weU  within  ±3 
times  the  standard  error  of  estimate,  ±0.1458  spore. 

The  statistical  analysis  of  the  data  therefore  indicates  that  the 
method  used  is  sufficiently  accurate  for  determining  the  spore  content 
of  unknown  samples  of  honey.  For  this  purpose  the  following  formulas 
are  used: 

X=1.1228F+0.2034±0.4374 

where  F=the  actual  mean  number  of  spores  per  field  counted  from 
60  fields,  and 

Number  of  spores  per  cubic  centimeter=32,884X 

O 


K-12!i 


THE   DEVELOPMENT  OF  AMERICAN   FOULBROOI)   IN 

RELATION  TO  THE  METABOLISM  OF  ITS 

CAUSATIVE  ORGANISM 


BY 

A.  P.  STUR  1  EVANT 

(Contribution  from  Bureau  of  Entomology) 


Reprinted  from  JOURNAL  OF  AGRICULTURAL  RESEARCH 

Vol.  XXVIII,  No.  2      .-      :      :     Washington,  D.  C,  April  12,  1924 


PUBLISHED  BY  AUTHORITY  OF  THE  SECRETARY  OF  AGRICULTURE,  WITH 
THE  COOPERATION  OF  THE  ASSOCIATION  OF  LAND-GRANT  COLLEGES 


•WASHINGTON-ipOVERNMEHT  PRINT|N<3  OFFICK:192l 


THE  DEVELOPMENT  OF  AMERICAN  FOULBROOD  IN 
RELATION  TO  THE  METABOLISM  OF  ITS  CAUSATIVE 
ORGANISM 

By  A.  P.  Sturtevant' 

Apricnltural  Assistant,  Bee  Culture  Investigations,  Bureau  of  Entomology,  United 

States  Department  of  Agriculture 

INTRODUCTION 

American  foulbrood  is  one  of  the  two  serious  diseases  affecting  the  brood  of 
the  honeybee.  The  specific  cause  of  this  disease  is  a  pathogenic,  spore-forming 
microorganism,  known  as  Bacillus  larvae.  The  occurrence  of  this  organism  in 
uniformly  pure  culture,  accompanied  by  the  gross  effects  of  its  activity,  as  mani- 
fested by  the  characteristic  appearance  and  age  of  the  diseased  and  dead  larvsB, 
differentiates  American  foulbrood  from  the  other  serious  brood  disease  of  bees, 
European  foulbrood.  The  latter  disease  is  caused  by  an  entirely  different  non- 
spore-forming  organism.  Bacillus  pluton,  which  causes  a  different  manifestation 
of  gross  symptoms,  complicated  by  the  action  of  various  secondary  invaders. 

Certain  limited  facts  concerning  the  characteristics  of  the  various  types  of 
bacteria  concerned  in  causing  or  associated  with  these  brood  diseases  have  been 
studied,  from  which  various  practical  applications  have  been  derived.  As  has 
been  stated  by  Phillips  {S9y,  "Bacteriological  studies  of  bee  diseases  have  been 
useful  to  practical  beekeepers  in  explaining  the  reasons  for  success  or  failure 
with  various  treatments  attempted.  These  studies  have  been  especially  impor- 
tant, however,  because  through  them'  methods  of  laboratory  diagnosis  of  the 
different  diseases  have  been  worked  out." 

Advancement  in  knowledge  concerning  the  etiological  and  biochemical  rela- 
tionships of  the  brood  diseases,  particularly  concerning  differences  in  charac- 
teristics as  related  to  gross  symptoms,  has  been  limited,  however,  because  of  the 
peculiar  growth  requirements  of  the  causative  organisms.  There  are  funda- 
mental differences  between  American  foulbrood  and  European  foulbrood,  par- 
ticularly as  to  characteristics  of  development,  which,  although  recognized,  have 
not  been  adequately  explained  by  the  incomplete  data  so  far  obtained  on  the 
metabolism  of  the  causative  organisms. 

The  present  investigation  was  undertaken  to  obtain  further  data  concerning 
the  growth  requirements  of  Bacillus  larvae,  the  cause  of  American  foulbrood,  by 
which  to  explain  these  differences  in  the  symptoms  and  development  of  the  two 
diseases.  Through  improved  methods  of  cultivation,  a  study  has  been  made  of 
factors  concerned  in  the  metabolism  of  Bacillus  larvae  correlated  with  certain 
hitherto  unrecognized  biochemical  factors  associated  with  the  metabolism  of  the 
normal  honeybee  larva.  The  results  obtained  add  materially  to  the  knowledge 
of  the  biology  of  the  brood  diseases. 

'  Acknowledgments  are  due  to  Dr.  B.  E.  Whitmore,  professor  of  bacteriology  and  preventive  medicine 
of  the  Oeorge  Washington  University,  for  much  valuable  advice  and  many  suggestions,  and  to  Dr.  E.  F. 
Phillips,  apiculturist.  Bureau  of  Entomology,  United  States  Department  of  Agriculture,  under  whose 
direct  supervision  this  work  was  done.  Presented  in  part  satisfaction  of  the  requirements  for  the  degree 
of  doctor  of  philosophy  at  the  George  Washington  University,  April  21, 1923.  This  work  was  completed 
April  10, 1923. 

2  Reference  is  made  by  number  (italic)  to  "  Literature  cited,"  p.  165-168. 

Journal  of  Agricultural  Research,  Vol.  XXVIII,  No.  2 

Washington.  D.  C.  Apr.  12, 1924 

Key  No.  K;-128 
5095-24t 1  (129) 


130  Journal  of  Agricultural  Research        voi.  xxvm,  no.  2 

THE  RELATION  OF  CONTRIBUTING  CAUSES  TO  THE  COMPARATIVE 
DEVELOPMENT  OF  THE  TWO  SERIOUS  BROOD  DISEASES  OF 
BEES 

In  order  to  understand  the  basis  upon  which  the  consideration  of  this  problem 
has  been  developed,  it  is  necessary  to  make  a  comparative  study  of  certain  of 
the  characteristics  of  the  two  brood  diseases,  American  foulbrood  and  European 
foulbrood,  aside  from  their  etiology.  It  will  be  apparent  from  this  study  that 
certain  contributing  causes,  although  recognized  and  described,  have  not  been 
further  analyzed  to  any  extent,  particularly  in  relation  to  specific  etiology. 
The  experimental  work  of  the  present  investigation  is  concerned  primarily 
with.  American  foulbrood,  however,  since  the  causative  organism,  Bacillus 
larvae,  can  be  isolated  and  grown  in  pure  culture,  while  as  yet  no  artificial  medium 
suitable  for  the  growth  of  Bacillus  pluton,  the  cause  of  European  foulbrood, 
has  been  devised. 

RACE 

It  is  an  accepted  fact  that  in  American  foulbrood  the  race  or  strain  of  bees 
has  little  or  no  relation  to  the  development  of  or  the  resistance  to  the  disease. 
This,  aside  from  apparent  lack  of  immunity  or  resistance  of  any  of  the  races, 
may  be  explained  partially  by  the  fact  that  the  decomposed  material  resulting 
from  the  death  of  the  larvae  is  of  such  a  nature  that  the  bees  can  not  to  any 
extent  remove  it  from  the  combs  after  the  disease  has  once  become  established. 
The  dried-down  masses  (scales)  are  practically  glued  to  the  cell  walls.  Bacillus 
larvae  forms  resistant  spores  which  allow  the  disease  to  be  carried  and  spread 
almost  indefinitely  by  means  of  the  honey  and  old  scales. 

In  European  foulbrood,  on  the  contrary,  Italian  bees  seem  to  have  some  character- 
istic which  makes  them  more  resistant  or  vigorous  in  combating  infection  under 
the  proper  conditions.  The  results  of  bacterial  decomposition  of  the  diseased 
remains,  even  at  their  worst,  are  such  that,  if  the  colony  is  able  to  build  up  or 
is  made  sufficiently  strong  in  worker  bees,  they  are  able  to  remove  these  remains, 
thereby  removing  the  infection  sufficiently  to  prevent  its  further  development. 
Bacillus  pluton  does  not  form  spores  and  lives  only  a  comparatively  short  time 
under  unfavorable  conditions  for  growth,  as  in  honey  or  on  long  drying.  Fur- 
thermore, as  has  been  demonstrated  by  the  writer  in  a  previous  paper  (45), 
this  apparent  resistance  of  the  Italian  bees  was  observed  to  be  due  largely  to 
the  racial  characteristic  of  removing  all  foreign  materials  more  promptly  from 
the  hive  than  do  common  black  bees  or  hybrids,  rather  than  to  any  natural 
resistance  or  immunity  to  the  disease. 

STRENGTH  OF  COLONY 

If  a  colony  of  bees  has  been  exposed  to  infection  from  American  foulbrood, 
the  strength  of  the  colony  apparently  has  no  direct  relation  to  the  development 
of  the  disease,  except  that  strong  colonies  are  usually  the  ones  which  rob  the 
weaker  infected  colonies,  thereby  spreading  the  infection  through  the  apiary. 
As  suggested  above,  European  foulbrood  attacks  primarily  the  weak  colonies 
which  have  an  insufficient  force  of  bees  to  remove  the  infected  material.  Dis- 
eased combs  from  such  a  colony  can  be  placed  in  a  strong  healthy  colony  of 
Italian  bees  with  no  resulting  disease.  This  would  be  fatal  in  the  case  of  Ameri- 
can foulbrood. 

SEX 

There  has  been  slight  mention  in  the  literature  of  the  relation  of  the  sex  of  the 
bee  larvsB  to  the  development  of  disease.  Phillips  (38)  states  with  regard  to 
European  foulbrood:  "A  symptom  of  greatest  importance  is  the  fact  that  the 


Apr.  12, 1924  Development  of  AmeHcan  Foulbrood  131 

disease  attacks  drone  and  queen  larvse  nearly  as  quickly  as  those  of  the  workers . 
The  tendency  of  this  disease  to  attack  queen  larvse  is  a  serious  drawback  in  treat- 
ment. Frequently  bees  of  a  diseased  colony  attempt  to  supersede  their  queen 
but  the  larvffl  in  the  queen  cells  often  die,  leaving  the  colony  hopelessly  queenless. 
The  colony  is  thus  depleted  rapidly." 

In  American  foulbrood,  according  to  PhiUips  (39),  "Usually  the  disease  attacks 
only  worker  brood,  but  rare  cases  are  found  in  which  queen  and  drone  brood  are 
diseased."  White  (BS)  states,  however:  "That  worker,  drone,  and  queen  larvae 
are  all  susceptible  to  the  disease  has  been  demonstrated  during  these  [White's] 
studies.  Affected  drone  brood  is  encountered  less  often  in  the  diagnosis  of  this 
disease  than  in  that  of  European  foulbrood.  The  writer  has  encountered  queen 
larvsB  affected  by  American  foulbrood  in  experimental  colonies  only,  although 
very  probably  diseased  queen  larvse  do  occur  in  nature  also."  A  few  samples  of 
diseased  brood  containing  American  foulbrood  sent  to  the  Bee  Culture  Labora- 
tory for  diagnosis  have  been  found  to  contain  affected  drone  larvse  as  well  as  one 
or  two  cases  of  diseased  queen  larvse.  Although  beekeepers  believe  that  in 
American  foulbrood  drone  brood  is  so  seldom  affected  that  the  absence  of  dis- 
eased drone  brood  is  a  diagnostic  character,  the  fact  that  occasionally  drone  larvse 
do  die  of  the  disease  makes  it  possible  that  some  other  factor  than  nonsuscepti- 
bility  of  sex  is  concerned.  No  accurate  data  are  available  on  this  subject.  The 
work  of  this  paper  is  concerned  only  with  worker  brood,  because  the  great  pre- 
ponderance of  worker  brood  affected  gives  slight  importance  to  the  comparatively 
few  drone  larvae  in  the  average  colony. 

AGE 

The  general  characteristic  difference  in  age  between  larvae  dying  of  American 
foulbrood  and  those  dying  of  European  foulbrood,  mentioned  at  the  beginning 
of  this  paper,  has  been  one  of  the  chief  factors  in  the  differentiation  between  the 
two  diseases.  Originally  there  was  considered  to  be  only  one  disease,  "foul- 
brood." Although  beekeepers  have  long  known  that  brood  of  various  ages  is 
attacked  by  brood  disease,  it  seems  not  to  have  been  until  about  1880  that  the 
difference  in  age  at  the  time  of  attack  was  used  to  separate  foulbrood  into  two 
distinct  forms,  one  "easily  curable"  and  the  other  "virulent."  Dzierzon  (SI) 
was  the  first  thus  to  differentiate  definitely  into  two  types  of  disease,  according  to 
the  difference  in  symptoms  and  age  a|t  time  of  attack.  He  stated  that  in  the 
curable  disease,  "  More  of  the  larvse  die  still  unsealed,  while  they  are  still  coiled 
in  the  bottom  of  the  cell  *  *  *.  xhe  brood  which  does  not  die  before  sealing 
mostly  attains  to  perfection  *  *  *.  This  is  exactly  the  reverse  in  the  malig- 
nant kind  of  foulbrood.  In  this  the  Jarvae  do  not  generallydie  before  they  have 
raised  themselves  from  the  bottom  of  the  cell,  have  been  sealed  and  begun  to 
change  into  nymphs." 

Cheshire  {13)  who  probably  was  the  first  to  investigate  the  bacteria  associated 
with  what,  in  the  light  of  present  knowledge,  is  known  as  European  foulbrood, 
was  inclined  to  agree  at  first  with  the  distinctions  made  by  Dzierzon.  He  soon 
stated  (,14),  however,  that  Dzierzon  was  in  error  and  that  there  is  only  the  one 
disease,  foulbrood,  which  he  supposed  was  caused  by  an  organism  to  which  he 
gave  the  name  Bacillus  alvei.  Cheshire  and  Cheyne  (15)  described  Bacillus  alvei 
as  a  spore-forming  bacillus  which  they  constantly  found  associated  with  a  dis- 
eased condition  of  the  brood  and  recognized  only  as  "foulbrood."  The  results 
of  this  work  caused  considerable  confusion  to  beekeepers  and  investigators,  both 
in  this  country  and  abroad,  for  more  than  a  decade. 

In  this  country  some  time  after  1890  it  became  evident  to  certain  beekeepers , 
particularly  in  New  York  State,  that  they  were  dealing  with  two  distinct  dis- 
eases.    The  newly  recognized  form,  which  was  found  to  attack  the  coiled  larvse. 


132 


JouttmI  of  Agricultural  Research         voi.  xxviii,  No.  2 


was  at  first  erroneously  called  "black  brood,"  to  distinguish  it  from  the  "foul- 
brood"  of  sealed  larvae.  "Black  brood"  assumed  epidemic  proportions  in  New 
York  State  by  1897.  This  gave  rise  in  American  beekeeping  literature  to  descrip- 
tions of  two  distinct  diseases,  as  far  as  the  age  of  the  larvse  attacked  and  the 
appearance  from  the  resulting  decomposition  were  concerned. 

RESULTING   DETERMINATION   OF   ETIOLOGY 

As  a  result  of  the  increasing  devastation  by  this  new  disease,  work  was  started 
in  New  York  State  in  1902  (53),  which  was  later  carried  on  by  White  {49,  50), 
on  the  bacteriology  of  these  brood  diseases,  by  which  doubt  was  cast  upon 
Bacillus  alvei  being  the  cause  of  any  disease,  although  it  was  found  to  be  asso- 
ciated only  with  European  foulbrood.  Furthermore,  a  new  spore-forming 
bacillus  distinct  from  Bacillus  alvei  was  observed  and  cultivated  on  special 
culture  media  from  the  disease  attacking  the  sealed  larvae.  This  organism  was 
at  first  designated  Bacillus  X  but  was  later  named  Bacillus  larvae  (figs.  1  and  2) . 
Subsequently  this  was  found  to  be  the  cause  of  American  foulbrood  by  experi- 
mental inoculation  of  healthy  colonies  with  pure  cultures  (51).     The  symptoms 


Fig.  1.— Spores  of  Bacillus  larvae. 
(McCray  (SI)) 


Fig.  2.— Vegetative  rod  form  of  Bacillus  larvae. 
(White  (SS)) 


were  accurately  described  and  differentiated  by  Phillips  (37),  definite  new  names 
being  used  for  the  first  time  in  order  to  eliminate  confusion,  as  follows:  Ameri- 
can foulbrood,  formerly  known  as  "foulbrood"  ("Usually  the  larvae  are  attacked 
at  about  the  time  of  capping,  and  most  of  the  cells  containing  infected  larvae 
are  capped");  and  European  foulbrood,  originally  called  "black  brood"  ("This 
disease  attacks  the  larvae  earlier  than  does  American  foulbrood,  and  a  com- 
paratively small  percentage  of  diseased  brood  is  ever  capped  ") . 

Maassen  (27)  in  Germany  described  at  about  the  same  time  what  is  now  ac- 
cepted as  the  same  organism  as  Bacillus  larvae,  a  spore-forming  organism  con- 
stantly found  to  be  present  in  the  diseased  brood  dying  after  sealing,  "  Nymphen- 
seuche."  He  gave  the  name  Bacillus  brandenburgiensis  to  this  organism.  Burri 
(IS)  in  Switzerland  also  recognized  the  fact  that  the  spores  present  in  large 
numbers  in  scales  in  the  "nymph"  disease  were  a  new  species  that  was  difficult 
of  cultivation. 

White  (6S)  later  showed  conclusively  that  Bacillus  alvei  is  not  the  cause  of 
European  foulbrood  but  is  only  one  of  several  secondary  invaders.  He  demon- 
strated that  the  probable  cause  of  European  foulbrood  Is  a  nonspore-forming 
organism  which  he  called  Bacillus  pluton.     This  organism  develops  before  the 


Apr.  12, 1924  Development  of  American  Foulirood  133 

death  of  the  larva  in  the  intestinal  tract  and  usually  kills  before  sealing  takes 
place,  as  differentiated  from  American  foulbrood  as  described  above.  Unfor- 
tunately, as  yet  it  has  been  impossible  to  grow  this  organism  in  pure  culture  on 
artificial  culture  media. 

Further  work  has  been  done  by  various  investigators  on  certain  laboratory 
phases  of  the  bacteriology  and  diagnosis  of  the  two  diseases,  but  no  additional 
information  has  been  obtained  concerning  the  etiological  and  biochemical  rela- 
tionships of  the  causative  organisms  which  would  aid  fti  the  solution  of  the 
present  problem. 

BASIS  FOR  INVESTIGATIONS 

Throughout  all  the  discussion  of  symptoms  of  the  brood  diseases  in  the  litera- 
ture, particularly  in  relation  to  the  different  ages  at  which  the  diseases  attack 
during  the  life  history  of  the  larvse,  there  has  been  no  adequate  explanation  of 
the  reason  for  this  apparent  fundamental  difference. 

Maassen  (2S)  in  the  case  of  American  foulbrood  made  the  observation  that, 
"according  to  the  microscopic  findings  from  section  preparations.  Bacillus 
brandenburgiensis  [Bacillus  larvae]  does  not  come  to  luxuriant  development  in 
the  intestine  of  the  larva,  though  this  is  the  case  with  Bacillus  alvei  and  with 
Streptococcus  apis  [in  'sourbrood'].  It  finds  much  more  promising  nourishment 
in  the  fat  bodies  of  the  larva.  Apparently  the  bacillus  finds  opportunity  to  press 
its  way  into  the  fat  bodies  shortly  before  the  pupation  of  the  bee,  at  the  begin- 
ning of  the  natural  changes  in  the  intestinal  tube.  From  this  it  seems  clear 
why  the  larvae  containing  Bacillus  brandenburgiensis  die  after  sealing."  In  part 
this  is  probably  correct,  since  it  may  easily  be  observed  that  soon  after  capping  the 
tissues  of  the  healthy  larva  become  more  or  less  granular  and  watery  in  consistency , 
at  which  time  it  is  almost  impossible  to  distinguish  the  intestinal  tract.  It  is  also 
difficult  to  remove  the  larva  in  this  condition  from  the  cell  without  rupturing  the 
skin  envelope.  This  process  is  described  more  in  detail  later.  It  does  not 
explain,  however,  why  the  spores  of  Bacillus  larvae  do  not  germinate  and  increase 
in  numbers  suflBciently  to  kill  the  larva  much  earlier  during  the  feeding  period, 
as  in  the  case  of  European  foulbrood.  A  vague  and  only  partially  correct  sug- 
gestion was  given  in  an  earlier  paper  by  the  writer  {4S),  in  which  the  following 
theory  was  stated:  "Bacillus  larvae  gains  entrance  to  the  larva  generally  in  the 
spore  stage,  in  the  larval  food.  This  occurs  at  about  the  same  stage  as  in  Euro- 
pean foulbrood,  while  the  larva  is  still  coiled  in  the  cell.  Only  rarely,  however, 
do  coiled  larvae  die.  This  is  apparently  because  it  takes  some  time  for  the  rest- 
ing stage  spores  to  germinate  into  the  active  vegetative  rods.  This  causes  death, 
as  a  rule,  to  occur  later  in  the  life  history  of  the  larva." 

KEIATION    OF    THE    BROOD    DISEASES    TO    THE    LIFE    HISTORY    OF    THE 

HONEYBEE  LARVA 

The  development  of  the  honeybee  may  be  divided  in  general  as  follows:  After 
the  egg  is  laid  there  is  a  period  of  three  day's  incubation  before  it  hatches  into 
the  larva.  The  larval  stage,  during  which  active  feeding  and  growth  occur, 
comprises  four  and  a  half  to  five  and  a  half  or  six  days.  At  the  end  of  the  feeding 
period  the  larva  is  sealed  in  the  cell,  where  it  spins  its  cocoon.  Metamorphosis 
then  occurs,  and  the  fully  formed  adult  bee  emerges  in  about  12  days,  making  a 
complete  developmental  period  of  approximately  21  days.  According  to  White 
(63) ,  there  is  a  prepupal  period  in  healthy  brood  of  four  days  after  sealing  occurs 
before  the  actual  change  in  the  external  form  to  that  of  the  adult  bee  takes  place. 
During  the  first  t^o  days  after  capping,  the  larva  is  active  in  the  cell,  consuming 
any  remaining  food  and  spinning  a  cocoon.  Some  time  during  this  period  ac- 
cording to  Straus  (^S),  or  just  previous  to  capping  according  to  Zander  (57) 


134 


Journal  of  Agricultural  Research         voi.  xxvni,  No.  2 


Fig.  3.— Healthy  prepupa 
approximately  8  days  old, 
having  reached  the  quies- 
cent stage.  This  is  the 
age  at  which  the  majority 
of  larvSD  die  from  Ameri- 
can foulbrood.  End  view. 
(White  (55)) 


the  larval  intestine,  which  up  to  this  time  has  been  a  blind  sac,  is  connected  with 
the  end  gut,  allowing  defecation  to  take  place.  There  is  then  two  days  of  quies- 
cence, during  which  the  larva  extends  in  the  cell  and  lies  motionless,  while  internal 
change.s  preparatory  to  metamorphosis  occur  (figs.  3  and  4) .  These  changes  (7) 
consist  of  the  almost  complete  histolysis  of  the  fat  body  of  the  larva  in  order  to 
furnish  nutriment  for  the  formation  of  imaginal  tissues.  This  is  made  possible 
by  the  physiological  and  morphological  changes  occurring 
Tn  this  stage  of  the  development  of  the  larva.  Extended 
investigations  have  been  made  of  these  physiological  and 
morphological  changes,  but  they  need  not  be  summarized 
further  here,  since  the  present  work  has  been  solely  of  a 
biochemical  character.  It  is  noticeable,  however,  that  the 
intestines  of  mature  larvse  even  for  a  short  time  after  cap- 
ping are  full  of  material  colored  by  the  pollen  content, 
while  the  intestines  of  the  prepupae,  after  they  have 
extended  in  the  cell,  are  colorless. 

It  is  during  the  latter  two-day  prepupal  period  that 
according  to  Maassen  {28)  the  invasion  of  the  fat  body  by 
Bacillus  larvae  occurs  and  that  according  to  White  (SS) 
the  majority  of  the  brood  dies  in  American  foulbrood. 

In  European  foulbrood,  on  the  contrary,  the  majority 
of  the  larvae  in  typical  cases  of  this  disease  die  before 
sealing  and  after  reaching  an  age  of  Si  to  4  days  from 
the  time  of  hatching  of  the  egg  (56)  (fig.  5) .  In  certain 
abnormal  cases  in  European  foulbrood  death  may  occur  after  capping  (46), 
but  this  almost  always  occurs  during  the  first  two  days  of  the  prepupal  stage, 
when  the  larva  in  most  cases  is  still  moving  about  in  the  cell,  usually  causing 
a  gross  appearance  quite  different  from  that  of 
dead  of  American  foulbrood. 


PEELISnNARY  EXPERIMENTS 

While  studying  the  bacterial  flora  associated  with  the 
early  stages  of  European  foulbrood  in  the  larval  intestine 
certain  results  were  obtained  which  suggested  a  possible 
explanation  of  the  delayed  development  in  American 
foulbrood.  Until  death  takes  place  in  European  foulbrood 
the  growth  of  the  organism  causing  the  disease  and  certain 
secondary  associated  forms  occur  only  within  the  intes- 
tine (52);  that  is,  within  the  peritrophic  membrane,  but 
not  in  actual  contact  with  living  tissues  of  the  larva.  It 
is  only  after  death  that  the  secondary  invaders,  particu- 
larly Bacillus  alvei,  invade  the  body  tissues  (^5) . 

Another  important  distinction  which  must  be  consid- 
ered is  that  the  feeding  of  the  larva  is  not  the  same  ' 
throughout  larval  life.  Von  Planta  (40)  has  shown  ^'o- ■'•"Healthy  prepupa. 
that  for  the  first  part  of  the  feeding  period  one  type  of  ^'^^^'^-  <^'''"=(*«' 
food  is  used  by  the  larva  and  that  at  a  later  stage  a  food  different  in  chemical 
and  physical  composition  is  provided.  Young  larv*  receive  a  food  for  a  time 
after  hatchmg  that  is  much  richer  in  fat  and  albuminous  material  but  lower 
m  sugar  content  than  that  fed  to  older  larv«.  The  food  of  the  older  larv«, 
which  IS  known  to  consist  mainly  of  honey  or  nectar  and  pollen,  is  much 
ingher  m  sugar  content,  while  there  is  a  considerable  decrease  in  fat  and 
albuminous  material.     The  sugar  in  the  food  of  the   older  larv^,  particulariy 


Apr.  12,  1924 


Development  of  American  Foulbrood 


135 


that  of  larvse  at  the  age  when  European  foulbrood  makes  its  attack,  comprises 
nearly  45  per  cent  of  the  dried  substance,  or  nearly  14  per  cent  of  the  fresh 
substance.  From  these  facts  it  may  be  assumed  that,  because  of  the  great 
amount  of  food  given  the  larva  at  this  age,  there  must  be  present  in  the  larval 
intestine,  at  all  times  during  the  active  feeding  period,  considerable  amounts 
of  this  food  rich  in  sugar  unassimilated,  up  to  and  even  after  active  feeding 
ceases.  A  number  of  larval  and  prepupal  intestines  were  dissected  from 
healthy  larvse  and  tested  roughly  with  Benedict's  qualitative  solution  {34) 
for  the  presence  of  reducing  sugars.  The  results  indicated  the  presence  of 
relatively  large  amounts  of  reducing  sugar  in  the  intestines  of  larvse  just  prior 
to  sealing.  Little  or  no  reducing  sugar  could  be  demonstrated  in  the  intestines 
of  sealed  larvse  or  prepupoe. 

It  may  therefore  be  assumed  that  certain  of  the  organisms  associated  with  the 
early  stages  of  European  foulbrood  are  able  to  grow  in  the  presence  of  a  high 
sugar  concentration.  Experiments  were  devised  in 
which  a  medium  containing  10  per  cent  dextrose  was 
used.  It  was  found  that  while  a  few  types  of  organ- 
isms, such  as  one  resembling  Streptococcus  apis  {S8), 
could  be  grown  in  varying  numbers,  an  organism  sim- 
ilar to  that  described  by  Maassen  (S9),  resembling  the 
larger'forms  of  Bacillus  pluton,  called  Bacillus  lanceolatus, 
could  be  isolated  and  grown  from  over  50  per  cent 
of  the  samples  cultured.  As  described  by  the  writer 
(.4'^),  "This  organism  was  found  to  grow  best  on  a 
10  per  cent  dextrose  yeast  extract  agar  with  a  reac- 
tion slightly  acid.  It  is  differentiated  from  Bacillus 
pluton  and  Streptococcus  apis  in  being  gram-negative, 
and  does  not  grow  readily  if  at  all  in  media  with- 
out sugar."  From  these  studies  it  was  suggested 
that  possibly  this  comparatively  high  sugar  content  of 
the  unassimilated  food  in  the  larval  intestine  may 
have  an  influence  on  the  germination  of  the  spores  and  growth  of  Bacillus 
larvae  and  that  a  change  may  occur  when  the  sugar  content  is  suflBciently  re- 
duced by  assimilation  in  the  larval  intestine  after  it  has  been  capped  and  when 
the  intestines  have  been  emptied  by  the  opening  of  the  ventriculus  into  the 
end  gut.  Therefore,  with  these  preliminary  observations  as  a  basis,  experi- 
nental  work  on  this  subject  was  begun  during  the  spring  of  1922. 

GROWTH  OF  BACILLUS  LARVAE  IN  CULTURES  IN  RELATION  TO 
VARIATION  IN  SUGAR  CONCENTRATION 

The  first  step  in  the  substantiation  of  this  theoretical  assumption  is  to  de- 
termine whether  there  is  a  correlation  between  germination  of  the  spores  of 
Bacillus  larvae  and  vigor  of  vegetative  growth  and  variations  in  concentration  of 
reducing  sugars  in  culture  media.  Ordinary  culture  media  are  unsuitable  for 
the  growth  and  isolation  of  Bacillus  larvae;  in  fact,  one  of  the  confirmatory  tests 
for  this  organism  in  laboratory  diagnosis  of  American  foulbrood  (31)  is  the 
absence  of  growth  on  plain  beef  infusion  agar  plates,  since  the  spores  wiU  not 
germinate  thereon.  There  are  rarely  any  secondary  invaders  associated  with 
Bacillus  larvae  in  the  decayed  material,  and  these  plates  practically  never  show 
growth. 

GROWTH  EBQUIEEMENTS  OF  BACILLUS  LARVAE 

Various  special  culture  media  have  been  devised  which  answer  more  or  less 
satisfactorily  the  requirements  for  the  ordinary  growth  of  the  organism.     The 


Fig.  6.— Healthy  coilea  larva 
at  age  of  maximum  intestinal 
sugar  content  and  approxi- 
mately the  age  when  the  ma- 
jority die  from  European  foul- 
brood.   (White  (iff)) 


136  Journal  of  Agricultural  Research      -  voi.  xxvin.  No.  2 

spores  of  Bacillus  larvae  will  germinate  and  grow  feebly  on  an  agar  medium  in  the 
preparation  of  which  healthy  bee  larvse  are  used  as  is  meat  in  ordinary  culture 
media,  sterilizing  as  usual  by  heat  in  an  autoclave  (49).  However  {51),  if  a 
broth  made  by  macerating  healthy  bee  larvae  in  several  times  their  volume  of 
water  is  sterilized  without  heating  by  filtering  through  sterile  bacteria-proof 
filters  and  then  is  pipetted  aseptically  into  tubes  of  previously  sterilized  liquefied 
agar  cooled  to  50°  C,  the  resulting  medium  gives  much  better  growth.  This 
medium  is  nevertheless  unsatisfactory,  owing  to  difficulties  of  prepara,tion,  and 
particularly  because  of  lack  of  material  for  its  preparation  except  during  the 
brood-rearing  season.  White  {5f)  therefore  devised  a  medium  which  consists  of 
a  suspension  of  the  yolk  of  an  egg  aseptically  in  70  cc.  of  sterile  water,  1  cc.  of 
which  suspension  is  added  by  sterile  pipette  to  each  5  cc.  of  ordinary  sterilized 
tubed  agar  medium  which  has  been  melted  and  cooled  to  60°  C.  Growth  occurs 
on  this  medium  quite  abundantly,  although  with  the  technic  described  great 
care  must  be  taken  to  prevent  contamination. 

Maassen  {2S)  has  also  devised  a  medium  made  from  a  mixture  of  equal  parts 
of  a  broth  from  calf  or  pig  brain  and  a  solution  of  egg  albumin  in  water,  to  which 
1.8  per  cent  agar  and  1  per  cent  each  of  Witte's  and  Chapoteaut's  peptone  are 
added,  after  which  it  is  filtered,  tubed,  and  sterilized.  This  medium  gives  an 
almost  neutral  or  weakly  acid  reaction  to  blue  litmus  paper.  Maassen  also 
found  that  the  vegetative  forms  develop  abundantly  if  grown  on  a  meat  and 
water  medium  if  it  is  acid  in  reaction  and  if  0.25  per  cent  of  pollen  and  1.5  per  cent 
of  Aschmann's  or  Chapoteaut's  peptone  are  added,  but  that  the  former  medium  is 
more  favorable.  Both  media  are  found  to  deteriorate  on  too  much  heating.  It 
is  also  stated  that  in  acid  peptone  bouillon,  in  bouillon  of  bee  larvae,  and  in  the 
brain  bouillon,  the  bacillus  may  be  cultivated,  although  growth  is  slow,  the  bouillon 
becoming  weakly   turbid   and  a  thick  slimy  deposit  gradually  being  formed. 

For  the  purpose  of  the  present  experiments,  after  consideration  of  the  advan- 
tages or  disadvantages  of  the  various  media  so  far  described,  a  modification  of  the 
egg-yolk  suspension  medium  of  White  was  adopted  as  the  most  satisfactory 
general  medium.  During  the  course  of  the  experiments  some  modifications  were 
made  both  in  the  medium  and  in  the  technic  of  preparation. 

PREPAEATION  OF  YEAST-EXTEACT  AGAR  BASE 

Because  of  most  satisfactory  results  in  other  work  with  various  brood  disease 
cultures,  a  yeast-extract  agar  described  by  Ayers  and  Rupp  (2)  was  used  instead  of 
beef  infusion  agar  as  a  base,  because  of  the  ease  of  preparation  and  the  uniformity 
of  the  medium.  Spores  of  Bacillus  larvae  on  the  surface  of  a  slant  of  this  agar 
germinate  to  some  extent  on  this  medium  alone,  and  vegetative  cultures  from  egg- 
yolk  suspension  agar  transferred  to  the  yeast  medium  grow  fairly  vigorously. 
The  addition  of  egg-yolk  suspension  to  the  yeast-extract  agar  increased  the  vigor 
of  growth  and  longevity  of  cultures. 

One  liter  of  the  yeast  extract  agar  is  prepared  as  follows: 

Dried  yeast '. grama..     10 

Peptone do 10 

Buffer  (sodium  glycero-phosphate) do 5 

Water cc  "^   gOo 

This  is  heated  in  fiowing  steam  for  one-half  hour,  then  adjusted  to  a  hydrogen- 
ion  concentration  of  Pb=7.6  to  7.8  by  the  colorimetric  method  of  Clark  and 
Lubs  {16,  17).  The  broth  is  then  boiled  for  one  minute  over  an  open  flame  and 
filtered  through  filter  paper  on  a  perforated  porcelain  funnel,  using  siliceous  earth 
to  clarify.     To  this  broth  is  added  an  equal  amount  (500  cc.)  of  double  strength 


Apr.  12, 1924 


Development  of  American  Foulbrood 


137 


(3  per  cent)  solution  of  agar,  washed  and  filtered  by  the  method  described  by 
Ayers,  Mudge,  and  Rupp  (S).  The  final  hydrogen-ion  concentration  reaction  is 
adjusted  so  that  upon  addition  of  1  cc.  of  the  egg-yolk  suspension  to  10  cc.  of 
the  yeast-extract  agar  the  reaction  is  about  Pn=6.8.'  The  normal  hydrogen-ion 
concentration  value  of  the  contents  of  the  larval  intestine  at  various  ages  during 
the  active  feeding  period  with  honey  and  pollen  and  just  after  sealing  averages 
Ph  =  6.8,  varying  to  slightly  more  acid  with  the  amount  and  type  of  pollen  in 
the  food  material.  Intestines  were  dissected  out  from  the  larvis  and  macerated 
in  10  cc.  of  neutral  distilled  water  and  compared  colorimetrically  with  known 
buffer  solutions,  using  brom  thymol  blue  as  an  indicator.  Fabian  and  Parks  (SS) 
found  this  value  to  be  Pa  =6.6  by  macerating  the  entire  larva  in  water.  From 
earlier  unpublished  work  by  the  writer,  as  well  as  by  the  above-mentioned  inves- 
tigators, the  optimum  hydrogen-ion  concentration  for  the  growth  of  Bacillus 
larvae  was  found  to  be  approximately  Ph=6.8.  The  yeast  extract  medium  is 
tubed,  sterilized  in  the  autoclave  at  15  pounds  pressure  for  15  minutes,  and 
stored  until  needed. 


^ 


PKEPARATION  OF  EGG-YOLK  SUSPENSION 

The  egg  yolk  can  be  diluted  much  more  than  was  directed  in  the  original 
formula  with  even  better  results,  the  more  dilute  suspension  giving  a  more 
transparent  medium  with  fully  as  profuse  growth. 
A  wide-mouthed  flask  containing  200  cc.  water, 
sterilized  with  a  cotton  plug  protected  by  a  paper 
cap,  is  used  for  each  egg  yolk.  At  times,  from  0.5 
per  cent  to  1  per  cent  of  a  neutral  buffer  salt  is 
added  to  the  water  previous  to  sterilization.  This 
holds  in  check  the  slow  increase  in  acidity  observed 
on  long  standing.  A  small  amount  of  normal  so- 
dium hydroxid  (2  to  3  cc.)  is  also  added  to  the  flasks 
before  sterilization  to  bring  the  resulting  reaction 
of  the  egg  suspension  nearer  to  the  desired  reaction 
for  the  final  medium. 

APPARATUS  TO  REPLACE  PIPETTING 

An  apparatus  was  devised  (fig.  6)  which  to  a 
great  extent  eliminates  the  danger  of  contamination 
of  agar  tubes  when  adding  egg-yolk  suspension, 
and  also  makes  possible  the  preparation  of  a  large 
quantity  of  medium  in  a  short  time.  As  a  rule 
egg-yolk  suspension  can  be  stored  or  withdrawn  at 
any  time  after  the  apparatus  has  been  set  up,  until 
aU  used  up,  unless  the  egg  yolk  itself  is  not  sterile. 
A  two-holed  rubber  stopper,  of  correct  size  to  fit 
the  flask  containing  the  egg-yolk  suspension,  is 
fltted  with  two  tubes,  one  of  small  bore  to  reach  nearly  to  the  surface  of  the 
liquid  when  placed  in  the  egg-yolk  flask,  and  a  second  larger  tube  fitted  flush  to 
the  inner  surface  of  the  stopper,  protruding  outward  about  IJ  inches.  A  piece  of 
rubber  tubing  5  inches  long  is  fitted  to  this  tube,  closed  with  a  pinchcock.  To 
this  rubber  tube  is  attached  a  delivery  tube  which  passes  through  another 
rubber  stopper  placed  in  one  end  of  a  glass  cylinder  1\  inches  in  diameter  and 
4  inches  long,  to  about  half  its  length.  This  forms  a  protective  beU  for  tha 
delivery  tube  similar  to  that  used  In  filling  vaccine  or  antitoxin  ampules.     The 

5095— 24t 2 


Fig  6. — Apparatus  to  replace  pipet- 
ting of  egg-yolk  suspension 


138 


Journal  of  Agricultural  Research         voi.  xxviii,  No.  2 


entire  apparatus  is  sterilized  in  the  autoclave,  using  a  temporary  empty  flask 
into  which  the  stopper  for  the  culture  flask  is  placed,  and  all  is  wrapped  in  paper 
with  a  paper  protective  cap  over  the  open  end  of  the  delivery  bell.  Before  use, 
the  apparatus  is  removed  from  the  paper  and  the  stopper  is  carefully  removed 
from  the  empty  flask  so  as  to  prevent  contamination  and  is  fastened  firmly  in 
the  fliask  containing  the  egg-yolk  suspension.  After  placing  the  pinch  cock  in 
position,  the  apparatus  is  carefuUy  inverted  and  hung 
on  a  ring  stand.  The  small-bore  glass  tube  in  the  flask 
now  reaches  a  little  above  the  surface  of  the  hquid  and 
serves  for  an  air  inlet.  By  means  of  this  apparatus, 
sterile  egg-yolk  suspension  can  be  added  to  tubes  of 
sterile  base  medium,  with  little  danger  of  external  con- 
tamination, by  inserting  the  tube  under  the  protective 
beU. 

METHOD   OF   ISOLATION   OF   PURE    CULTURES 

OF    BACILLUS    LARVAE 

Fig.  7.— American  foiUbrood  j.         .     j 

scale.   End  view.    (White        When  medium  IS  desired  for  the  isolation  or  cultiva- 

(««))  tion  of  Bacillus  larvae,  tubes  of  the  yeast-extract  agar  are 

melted  in  a  water  bath  and  cooled  to  55°  C,  after 

which  from  1  to  2  cc.  of  egg-yolk  suspension  is  added  for  each  10  cc.  of  base,  by 

means  of  the  apparatus  described  above.     The  contents  of  the  tubes  are  well 

mixed  and  then  slanted. 

From  a  comb  containing  decaying  material  dead  of  the  disease,  a  dried  scale 
(figs.  7  and  8)  is  removed  with  a  sterilized  needle  scalpel  (also  used  for  removing 
cappings)  and  dropped  into  the  water  of  condensation  in  the  culture  tube  to 
soften.  It  is  then  smeared  over  the  surface  of  the  agar  with  an  inoculating 
needle.  If  ropy  gluelike  material  is  available  it  is  more  satisfactory  (fig.  9).  A 
large  loopful  of  this  is  removed  from  the  cell,  from  which 
the  capping  has  been  aseptically  removed  by  means  of 
an  inoculating  needle,  and  is  streaked  over  the  surface 
of  the  agar.  A  heavy  initial  inoculum  gives  best  re- 
sults, as  it  is  often  difficult  to  obtain  growth  with  a  small 
amount.  It  is  quite  easy  to  obtain  pure  cultures  by  this 
procedure,  since  almost  never  are  secondary  contam- 
inations found  associated  with  Bacillus  larvae.  Plating 
may  be  carried  out  from  these  initial  cultures  if  abso- 
lute surety  is  desired,  but  initial  growth  is  obtained 
much  more  easily  by  the  tube  culture  method.  Germi- 
nation of  spores  and  some  growth  take  place  during  the 
first  24  hours'  incubation  at  37°  C,  but  maximum  growth 
is  not  obtained  much  before  48  hours. 

EXPERIMENTAL  PROCEDURE,  USING  AGAR   SLANTS 


Fio.  8.— American  foulbrood 
scale.    Side  view.    (White 


To  determine  whether  there  is  a  correlation  between 
germination  of  spores  and  vegetative  growth  of  Bacillus 
larvae  SkuA  the  concentration  of  sugar  in  the  culture 
medium,  a  series  of  tubes  is  prepared  with  varying 
percentages  of  dextrose,  from  0.5  per  cent  to  10  per  cent  (Table  I).  These  are 
prepared  by  adding  the  required  amounts  of  dextrose  to  50  cc.  portions  of  the 
yeast-extract  agar  base,  which  is  then  tubed  and  sterilized  at  10  pounds  pres- 
sure for  15  minutes.     On  cooling  to  55°  C,  1  cc.  of  sterile  egg-yolk  suspension  is 


Apr.  12, 1924 


Development  of  American  Foulbrood 


139 


added  to  each  tube  and  it  is  then  slanted.  Series  of  agar  slants  varying  in  sugar 
concentration  are  inoculated  with  either  vegetative  cultures  or  diseased  material 
containing  only  spores.  To  determine  spore  germination  an  approximately 
uniform  amount,  about  one  2-mm.  loopful  of  ropy  material,  when  available,  is 
used  for  inoculation  of  slants,  otherwise  a  scale  softened 
as  described  above.  If  no  visible  growth  takes  place 
after  48  hours'  incubation,  stained  smears  are  made,  to 
determine  whether  any  germination  has  occurred.  In 
the  case  of  the  determination  of  growth  from  vegetative 
culture,  a  single  uniform  streak  is  made  on  the  agar  slant, 
using  one  2-mm.  loopful  of  growth  from  a  48-hour  cul- 
ture of  Bacillus  larvae  prevously  isolated  and  cultivated. 
After  48  hours'  incubation,  as  well  as  after  about  one 
week,  comparative  observations  are  made  of  the  relative 
amount  and  character  of  the  growth.  Where  little  or  no 
growth  has  occurred,  stained  smears  are  made  from  the 
streak  to  see  what  has  happened  to  the  organisms.  These 
experiments  were  carried  out  with  a  number  of  different 
strains  of  vegetative  cultures  and  from  a  number  of  different  samples  of  American 
foulbrood. 


Fig.  9.— Partially  decom- 
posed American  foulbrood 
larva  at  the  stage  of  ropy 
consistency.    (White  C55)) 


Table  I. — The  effect  of  varying  the  sugar  concentration  in  egg-yolk  suspension 
medium  (1)  on  germination  and  vegetative  growth  from  spores;  and  {2)  on  vege- 
tative growth  from  vigorous  vegetative  cultures  of  Bacillus  larvae.  <» 


Test  material 


Per  cent  dextrose  in  medium 


Control         0.6         0.7         1.0 


1.3 


Spores _ 

Vegetative  cultures - 


++++ 


++ 
+++ 


+++ 
+++ 


++++ 
++++ 


++++ 
++++ 


+++ 
++++ 


+++   ++ 
+++  +++ 


Per  cent  dextrose  in  medium 


Test  material 


2.2i       2.6     2.75       3.0       3.S      4.0       4.6       5.0       7.6       10.0 


Spores ._ 

Vegetative  cultures - 


+ 
++ 


+ 
++ 


+ 
++ 


+ 
++ 


o  Tbe  following  symbols  are  used: 
+  Slight  growth. 
++  Fair  growth. 
+++  Good  growth. 
++++  Heavy  growth.     - 


±  Doubtful. 

—  No  evidence  of  growth 

O  Slight  germination  of 


EXPERIMENTAL  PROCEDURE,  USING  PLATE  CULTURES 


The  egg-yolk  suspension  agar  is  not  entirely  satisfactory  for  counting  colonies 
in  plate  cultures,  since  the  egg  yolk  gives  the  medium  a  cloudy,  semiopaque  ap- 
pearance. However,  by  using  the  supernatant  fluid  from  the  egg-yolk  suspen- 
sion or  a  somiewhat  smaller  amount  of  the  suspension  for  each  tube  of  yeast  extract 
agar  (10  to  15  drops),  a  fairly  satisfactory  plate  culture  is  obtained  if  the  proper 
amount  of  inoculum  is  used.  The  following  procedure  is  used:  To  a  series  of 
melted  tubes  of  yeast-extract  agar  containing  varying  amounts  of  dextrose  as 
described  above  (Table  II)  the  egg-yolk  suspension  is  added  and  the  desired  in- 
oculation of  the  tube  made  while  the  medium  is  still  liquid.  The  tubes  are  agi- 
tated to  mix  the  contents  thoroughly  and  then  poured  into  sterile  Petri  dishes. 


140 


Journal  of  Agricultural  Research  voi.  xxviii,  No.  i 


These  on  cooling  are  inverted  and  incubated  for  48  hours  at  37°  C,  after  which 
counts  are  made.  If  the  plates  are  flooded  with  a  dilute  solution  of  fuchsin  or 
eosin  before  counting,  the  colonies  are  more  easily  differentiated  for  counting  in 
the  semiopaque  medium.  Vegetative  cultures  only  were  used  for  plating.  A 
suspension  of  one  loopful  of  culture  in  3  cc.  of  sterile  broth  is  made  and  one 
loopful  of  that  is  used  to  inoculate  each  plate.  Dilution  in  sterile  water  was 
also  tried,  using  1  cc.  of  the  dilution  for  each  plate,  but  without  success,  since 
there  seems  to  be  a  minimum  amount  of  initial  inoculum  required,  below  which 
it  is  difficult  to]^obtain  growth. 

Table  II. — Average  number  of  colonies  per  ^-mpi.  loopful  of  vegetative  culture  sus- 
pension in  broth  on  plates  of  varying  sugar  concentration 


Per  cent  dextrose 

Average 

number 

of  colonies 

Per  cent  dextrose 

Average 
number 
of  colonies 

1,600 

1,690 

1,660 

914 

2.0 

0.5 

2.  6 

150 

1.0 

3.0 

1.6 

3.6 

0 

OBSERVATIONS 

SPORE    GERMINATION    AND    GROWTH    IN    RELATION    TO    SUGAR    CONCENTRATION 

At  different  times  during  the  investigation  seven  different  series  of  culture 
tubes  were  made,  using  as  material  for  inoculation  either  scales  or,  in  most 
cases,  ropy  remains  heavily  laden  with  spores  of  Bacillus  larvae,  but  no  vegetative 
rods.  This  material  was  taken  from  six  different  samples  of  diseased  brood 
from  different  localities.  From  these  series  of  cultures,  varying  in  sugar  con- 
centration from  0.5  per  cent  to  10  per  cent  dextrose,  it  was  found  that  active 
growth  occurs  up  to  and  including  2.5  per  cent  dextrose,  although  some  growth 
occurs  occasionally  up  to  3  per  cent  (Table  I).  The  exact  limits  varied  slightly 
with  different  strains  as  well  as  with  variation  in  the  amount  of  inoculum.  Even 
up  to  10  per  cent  dextrose  concentration,  a  varying  small  number  of  spores 
germinate,  as  is  demonstrated  by  stained  smears,  but  they  give  no  further  evi- 
dence of  vegetative  growth  upon  the  culture  medium. 

GROWTH  FROM  ACTIVE  VEGETATIVE  CULTURES 

In  a  similar  manner  five  different  series  of  tubes  with  varying  sugar  con- 
centrations were  made,  using  24-hour  cultures  of  three  different  characteristic 
vegetative  cultures  of  Bacillus  larvae,  previously  isolated  and  accustomed  to 
growth  on  artificial  culture  media  for  different  lengths  of  'time.  Good  growth 
occurs  on  the  average  up  to  2.5  per  cent  to  3  per  cent  dextrose  concentration, 
with  evidence  of  varying  slight  growth  up  to  4  per  cent  and  in  one  case  up  to 
4.5  per  cent  (Table  I) .  In  the  latter  case  much  of  the  variation  is  due  to  varia- 
tion in  the  amount  of  initial  inoculum.  If  a  heavy  inoculation  is  made  on  the 
surface  of  the  agar  tubes,  the  upper  sugar  concentration  limits  for  inhibition  of 
growth  are  increased,  although  in  these  cases  the  growth  was  meager  at  best. 
Stained  smears,  however,  made  after  a  few  days,  from  the  higher  sugar  con- 
centrations particularly,  soon  showed  the  pecuHar  disintegration  of  the  rods 
noted  by  White  {65)  as  taking  place  in  old  cultures  and  where  spore  formation 
is  inhibited,  such  as  in  the  presence  of  sugar.  This,  according  to  observations 
of  Sturges  and  Rettger  (,44)  on  other  organisms,  suggests  that  this  disintegration 
of  the  rods  is  the  result  of  autolysis. 


Apr.  12, 1024 


Development  of  American  Foulbrood 


141 


CUANTITATIVE    GROWTH    IN    PLATE    CULTUBE8 

Great  difficulty  is  found  in  obtaining  satisfactory  plate  cultures.  Only  two 
series  of  plate  cultures  were  obtained  which  could  be  counted  successfully.  The 
average  number  of  colonies  showed  a  definite  decrease  with  increased  sugar 
concentration,  with  no  growth  at  3  per  cent  or  higher.  (Table  II,  fig.  10). 
As  stated  above,  the  plate  method,  with  the  small  amount  of  initial  inoculum 
necessary  for  accurate  counts,  is  not  a  satisfactory  method  for  obtaining  growth 
of  Bacillus  larvae  under  these  conditions,  although  the  method  may  be  used  for 
obtaining  pure  cultures. 

From  these  observations  (Table  I)  it  is,  therefore,  safe  to  conclude  that  a  con- 
centration of  reducing  sugar  of  approximately  3  to  4  per  cent  or  more  inhibits 
the  growth  of  Bacillus  larvae,  although  slight  germination  of  spores  may  take 
place  at  higher  sugar  concentrations. 


O.S 


/.O  AS  2.0  2.S 

PER  CSA/r  DEXTROSE 


3.0 


Fig.  10. — Average  number  of  colonies  per  4-min.  loopful  of  vegetative  culture  suspension  witli  varying  suga  r 

concentration  (Table  II) 


QUANTJTATIVE  DETERMINATION  OF  UNASSIMILATED  SUGAR  IN 
THE  LARVAL  INTESTINE  AT  VARIOUS  AGE  PERIODS 

In  the  preliminary  experiments  it  was  shown  that  unassimilated  sugar  is 
present  in  the  intestinal  contents  of  the  actively  feeding  larva,  which  apparently 
i  s  assimilated  completely  by  the  time  the  prepupa  has  extended  in  the  cell  and  has 
become  quiescent.  Since  it  is  demonstrated  that  a  direct  relation  exists  between 
the  growth  of  Bacillus  larvae  in  suitable  culture  media  and  its  reducing  sugar  con- 
centration, it  is  now  necessary  to  determine  quantitatively  the  amount  of  unas- 
similated sugar  in  the  intestine  of  the  feeding  larva  and  in  the  intestine  of  the 
prepupa,  in  order  to  determine  whether  reducing  sugar  concentration  has  any 
bearing  on  the  time  of  attack  by  American  foulbrood. 

COMPOSITION   OF  BROOD  FOOD 

The  older  bee  larvae  {40)  receive  a  food  consisting  of  a  mixture  of  honey  or 
nectar  and  poUen,  rich  in  sugar,  chiefly  reducing  sugar.  This  sugar  constitutes 
about  45  per  cent  of  the  dried  substance,  or  over  13  per  cent  of  the  fresh  substance. 
The  food  of  younger  larvae  contains  only  about  5  per  cent  of  sugar  in  the  fresh 


142 


Journal  of  Agricultural  Research         voi.  xxviii,  no.  2 


material  (Table  III,  fig.  11).  Nelson  and  Sturtevant  {SB)  and  Lineburg  {M) 
have  shown  that  the  change  in  the  composition  of  this  food  comes  definitely  soon 
after  the  second  day,  instead  of  the  fourth  day,  as  stated  by  Von  Planta,  after 
which  increasingly  large  amounts  of  honey  and  pollen  are  fed  up  until  the  time  of 
sealing.  The  larva  is  fed  during  this  period  about  as  fast  as  it  can  ingest  the  food. 
From  this  it  is  reasonable  to  suppose  that  there  must  be  a  constant  surplus  of 
unassimilated  food  in  the  larval  intestine  until  after  feeding  has  ceased. 

Table  III. — Percentage  corn-position  0/  worker  brood  food,  calculated  from  Von 
Planta  (40),  and  on  the  basis  of  his  assumption  of  70  per  cent  water  content 


■Under  four  days 

Over  four  days 

Substance 

Dried 
substance 

Fresh 
substance 

Dried 
substance 

Fresh 
substance 

Nitrogenous                            -              

Per  cent 
53.38 
8.38 
18.09 

Per  cent 
16.01 
2.51 
6.43 

Per  cent 

27.87 

3.69 

44.93 

Per  cent 
8.36 

Fat           - 

1.11 

13.48 

COMPOSITION  OF  HONEY 

The  average  chemical  analysis  of  American  honeys  has  been  shown  by  Browne 
(9)  to  be  as  follows:  Moisture  17.59  per  cent,  invert  sugar  74.41  per  cent,  sucrose 
1.98  per  cent,  ash  0.23  per  cent,  dextrin  2.09  per  cent,  undetermined  3.70  per 
cent.  Approximately  the  same  percentages  have  been  found  by  all  other  workers 
in  this  field.  The  maximum  sucrose  content  of  honey  is  given  in  American 
standards  for  food  analysis  as  8  per  cent,  although  a  few  samples  have  been  found 
with  a  slightly  higher  sucrose  content.  In  the  utilization  of  honey  as  food  by 
either  the  adult  bee  or  the  larva,  it  may  be  assumed  that  sucrose  is  rapidly 
hydrolized.  In  any  analysis  of  the  stomach  content  of  the  bee  larva  for  sugar 
content,  therefore,  after  the  change  in  larval  food  has  occurred  and  when  honey 
enters  directly  into  its  composition,  it  may  safely  be  assumed  that  a  determination 
of  the  amount  of  reducing  sugar  will  indicate  the  amount  of  unassimilated  sugar 
in  the  intestine,  since  there  will  be  but  a  small  additional  sugar  content  from 
sucrose,  if  any  of  the  latter  sugar  still  remains.  In  determining  the  sugar  content 
of  the  whole  larva,  as  was  done  in  most  of  the  present  work,  it  may  be  assumed 
that  there  is  a  comparatively  small  amount  of  reducing  sugar  in  the  blood  stream, 
because  of  the  exceedingly  rapid  transformation  of  these  sugars  into  fat  and 
glycogen  which  are  known  to  occur  in  the  bee  larva.  It  is  therefore  concluded 
that  the  sugar  found  in  the  whole  larva  is  virtually  that  which  occurs  in  the  intes- 
tine alone,  and  this  greatly  simplifies  the  work  of  analysis. 

COMPOSITION     OP     THE     LARVA     AT     DIFFERENT     AGE     PERIODS 

The  work  of  Straus  (43)  on  the  chemical  composition  of  the  worker  and  drone 
brood  during  their  different  developmental  stages  gives  the  results  of  the  metabo- 
lism of  this  food,  as  indicated  by  the  presence  of  fat  and  glycogen  stored  in  the 
so-caUed  fat  body  of  the  larva  (Table  IV,  fig.  12).  He  was  unable  to  demon- 
strate more  than  a  trace  of  what  he  terms  reducing  substances,  except  in  one 
case  in  which  only  a  sHght  amount  was  found.  He  behoves  that  this  is  because 
the  sugar  of  the  larval  food  is  assimilated  so  rapidly,  as  is  indicated  in  the  larval 
composition  by  the  exceedingly  rapid  increase  in  the  amount  of  glycogen  and 
fat  until  after  feeding  has  ceased. 


Apr.  12, 1924 


Development  of  American  Foulbrood 


143 


/e 

\ 

\ 

I 

/4 

\ 

1 

\ 

r- 

SOG/tA 

> 

/2 

\ 

\ 

\\ 

/O 

A 

n 

1 

1 

L. 

— _ 

wrRoc 

£Noas 

/ 

1 

e 

4 

4 





— -\ 
\ 

z 

N 

\ 

\ 

fi^T 

a^rs  /         z        3        4        s        6        7 

FiQ.  11.— Per  cent  composition  of  worker  brood  food  (Table  III) 


144 


Journal  of  Agricultural  Research         voi.  xxvni,  No.  a 


'f        £        6         7        a        s        /o       /J 

Fio.  12.— Average  chemical  composition  ol  worker  larvae  at  different  ages  (Table  IV) 


Apr.  12,  1924 


Development  of  American  Foulbrood 


145 


Table  IV. — Average  chemical  composition  of  worker  larvce  at  (liferent  ages,  compiled 

from  Straus  (43) 


Weight 
of larvft 

Glycogen 

Fat 

Nitrogen 

Age 

Grams 
per  larva 

Per  cent 

of  fresh 

substance 

Grams 
per  larva 

Per  cent 

of  fresh 

substance 

Grams 
per  larva 

Per  cent 

of  fresh 

substance 

Reducing 
sugar 

Days 
X 

Qrams 
0.00030 
.00340 
.03000 
.10010 
.  12775 
.14290 
.  16140 
.14300 
.14200 
.14500 
.13000 

2 

0.00008 
.0012 
.0055 
.0072 
.0088 
.0092 
.0089 
.0075 
.0076 
.0066 

2.60 
2.76 
5.68 
6.67 
6.95 
6.43 
6.35 
6.21 
6.24 
4.21 

0.00004 
.00005 
.0031 
.0047 

=  .0067 
.0060 
.0051 
.0062 
.0049 
.0047 

i.63 
1.64 
3.60 
3.64 
"3.98 
3.71 
3.53 
3.66 
3.60 
3.26 

0. 00009 
.0006 
.0016 
.  0010 
.0019 
.0018 
.0027 
.0022 
.0022 
.0023 

2.86 
2.04 
1.44 
1.47 
1.45 
1.22 
1.51 
1.60 
1.68 
1.68 

3 

4            

5 

0 

6         

7 

Trace. 

8 

Trace. 

0                      

10 

0.0002 

11            

o  Calculated  by  interpolation  and  averaging. 

CHOICE  OF  REAGENT 

It  was  necessary  to  devise  a  special  technic  for  the  determination  of  the  unassimi- 
lated  reducing  sugar  in  the  larva  by  the  application  of  procedures  used  in  other 
analyses  where  small  amounts  of  reducing  sugars  must  be  determined,  such  as  in 
urine  analysis.  After  studying  the  various  methods  of  sugar  analysis,  a  volumetric 
titration  method  seemed  the  most  promising. 

For  the  purpose  of  determining  quantitatively  the  unassimilated  sugar  in  the 
bee  larva  at  different  ages,  the  modified  copper  sulphate  solution  of  Benedict  (5) 
was  chosen,  mainly  because,  as  in  urine  analysis,  it  has  proved  more  satisfactory 
than  any  other  titration  method  for  determining  small  amounts  of  reducing 
sugars  quantitatively,  and  because  this  solution  keeps  indefinitely  .without 
deteriorating.  The  potassium  sulphocyanate  in  the  solution  produces,  upon 
reductioil  of  the  sugar,  a  white  precipitate  of  cuprous  sulphocyanate,  which  per- 
mits the  end  point  of  the  reaction  to  be  more  accurately  determined  than  with 
Fehling's  solution.  A  trace  of  ferrocyanid  is  added  to  prevent  precipitation  of  red 
cuprous  oxid  which,  may  be  caused  by  certain  impurities,  which  would  interfere 
with  the  determination  of  the  end  point.  The  test  solution  is  standardized  to  a 
known  solution  of  dextrose  so  that  5   cc.  equals  0.0102  grams  of  dextrose. 

CHOICE  OF  LAKVAE 

Since  there  is  little  likelihood  of  there  being  any  appreciable  amount  of  sugar 
elsewhere  than  in  the  intestine,  analyses  were  made  of  entire  larvse,  because  of 
the  great  difficulty  attending  the  dissection  of  the  intestines.  Larvse  for  analysis 
were  chosen  from  combs  having  large  areas  of  brood  of  uniform  size  and  age.  In 
most  cases  35  larvae  as  nearly  of  the  same  size  as  possible  were  carefully  removed 
from  the  cells  by  means  of  a  pair  of  fine  forceps,  care  being  taken  to  remove  as  little 
uningested  food  as  possible.  Any  visible  amount  of  adhering  food  was  removed 
with  filter  paper  and  the  25  larvse  were  weighed.  Several  series  were  weighed  for 
each  age  above  the  two-day  age  period  through  to  about  the  fourth  day  after 

capping. 

DETERMINATION  OF  AGE  OF  LARVAE 

When  choosing  larva  for  the  analysis,  the  approximate  age  was  determined  by 
comparison  with  drawings  to  scale  by  Nelson  and  Sturtevant  (35)  of  larvse  of 
known  age  at  various  age  periods,  24  hours  apart.     Nelson  and  Sturtevant,  as 
5095— 24t 3 


146  Journal  of  Agricultural  ResearcJi         voi.  xxvm,  No.  2 

well  as  Straus  (Table  IV),  also  give  weights  for  larvse  of  known  age,  but 
in  order  to  eliminate  the  danger  of  variations  due  to  the  eflfect  of  different 
seasonal  and  environmental  conditions,  the  average  age  of  the  larvse  analyzed 
from  various  groups  of  25  was  determined  by  comparison  with  a  series  of  weigh- 
ings of  larvse  of  known  age  that  "were  made  during  this  same  period  (35) .  The 
various  series  of  weights,  with  the  corresponding  determinations  of  reducing 
sugar,  were  arranged  in  age  groups,  24  hours  apart,  as  shown  ip  Table  V.  In 
some  cases,  such  as  the  small  two-day  larvae,  or  the  quiescent  prepupffi,  where 
the  amount  of  unassimilated  sugar  is  small,  50  larvae  were  taken  for  analysis, 
but  usually  25  proved  satisfactory. 

PREPAEATION  OF  MATERIAL  FOE  ANALYSIS 

Several  difficulties  were  encountered  in  the  preparation  of  material  for  sugar 
determination.  At  first,  attempts  to  extract  the  sugar  were  made  by  macerating 
the  larvae  with  distilled  water  and  filtering  through  filter  paper.  This  produced 
a  cloudy  opalescent  liquid,  indicating  the  presence  of  colloidal  material,  and  this 
solution  did  not  give  the  characteristic  reaction  with  the  Benedict  reagent. 
Various  clarification  methods  were  tried.  Precipitation  with  both  neutral  and 
basic  lead  acetate  (10,  p.  276)  solutions  proved  unsatisfactory,  something  stiU 
remaining  to  interfere  with  the  reaction.  Mercuric  nitrate  solution,  which  is 
sometimes  used  to  clarify  liquids  of  animal  origin  such  as  blood,  urine,  and  milk, 
was  tried  {10,  p.  447).  This  method  occasionally  gave  good  results,  mainly 
with  the  younger  larvae,  but  often  with  older  larvae  and  prepupae  the  colloidlike 
material  still  remained  in  the  filtrate,  interfering  with  the  reaction.  Furthermore, 
because  of  the  numerous  filtrations  necessary  to  remove  successive  precipitates, 
it  was  feared  that  more  or  less  sugar  is  lost  by  adsorption  to  those  precipitates, 
even  with  careful  washing.  An  attempt  was  made  to  clarify  by  filtration  with 
suction  through  a  celloidin  membrane,  and  this  gave  a  clear  solution  which  reacted 
well  with  the  test  solution,  but  the  method  required  too  great  time.  The  method 
finally  adopted  was  by  extraction  with  50  per  cent  alcohol,  similar  to  the  method 
used  in  the  extraction  of  sugars  from  grains  and  similar  products  (It).  This 
method  proved  successful,  since  the  alcohol  causes  precipitation  of  all  solid 
matter,  giving  a  clear  filtrate  which  reacted  properly  with  the  Benedict's  reagent. 
Since  glycogen  in  water  solution  is  colloidal  in  nature,  and  thereby  difficult  to 
remove  by  filtration  from  such  a  solution,  it  is  doubtless  the  glycogen  present  in 
the  larva  which  prevented  clarification  and  interfered  with  the  reaction.  It  is 
possible  for  this  reason  that  Straus  (43)  failed  to  demonstrate  reducing  sugars. 
To  determine  this  point,  a  small  amount  of  glycogen  was  added  to  a  known  solu- 
tion of  dextrose  and  tested  with  the  copper  sulphate  solution,  and  the  known 
reducing  sugars  could  not  now  be  demonstrated  quantitatively.  Since  glycogen 
is  insoluble  in  alcohol  {10,  p.  44S)  the  50  per  cent  alcohol  precipitates  the  glycogen 
and  thereby  removes  materials  interfering  with  the  reaction  in  the  filtrate. 
Even  though  there  may  stiU  be  a  small  loss  of  reducing  sugar  by  adsorption  or  by 
some  other  means,  the  results  obtained  are  of  value  for  purposes  of  comparison. 
If  any  reducing  sugar  is  lost  by  the  method  adopted,  the  amount  is  exceedingly 
small  and  may  therefore  be  disregarded,  since  repeated  washings  failed  to  demon- 
strate its  presence. 

TECHNIC  ADOPTED 

After  weighing,  the  larvae  are  renioved  to  a  small  porcelain  mortar  and  mace- 
rated in  30  CO.  of  50  per  cent  alcohol.  This  material  is  then  washed  carefully 
into  a  small  flask  and  allowed  to  stand  from  two  to  three  hours  before  filtering. 
The  precipitate  is  washed  with  60  per  cent  alcohol.  The  filtrate  is  then  made 
up  to  50  cc.  with  distilled  water,  and  run  into  a  burette.     Five  cc.  of  the  stand. 


Apr.  12,  1S24 


Development  of  American  Foulhrood 


147 


ardized  Benedict's  solution  are  placed  in  a  white  porcelain  casserole  and  di- 
luted with  an  equal  amount  of  distilled  water.  To  this  are  added  about  5  grams 
of  anhydrous  sodium  carbonate  and  a  small  amount  of  ground  pumice.  This 
solution  is  brought  to  a  boil  and  the  larval  extract  is  run  in  slowly,  drop  by 
drop  at  the  end,  until  the  blue  color  disappears  and  a  white  precipitate  forms. 
From  the  number  of  cc.  of  larval  extract  used,  the  milligrams  of  sugar  per  larva 
and  the  per  cent  of  sugar  per  larva  are  calculated  (Table  V) . 

Table  V. —  Unassimilated  sugar  in  intestinal  content  of  larvx  at  different  ages 


Larvse  of  known  age, 
Sturtevant  (SS) 

Larvffl  analyzed  for  presence  of  unassimilated  sugar  {weights  in  grams) 

Ago 

Aver- 

Limits by 
weiglit 
tor  age 
groups 

Weight 

Num- 
ber 

Aver- 
age 

Ex- 

Equiv- 
alent 

CuSO) 

Equiv- 
alent 
dex- 
trose 

Dex- 
trose 

Sugar 

in 

age 

Date 

of 

of 

weight 

tract 

number 

solu- 

per 

days 

weight 

sample 

lar- 

VEB 

otl 
larva 

used" 

of 
larva 

tion 

per 
larva 

larva 

Oram 

Qrarfi 

Wit 

Oram 

Gram 

Cc. 

Cc. 

Oram 

Oram 

P.d. 

2 

0.004745 
.024626 

Up  to 
0. 014685. 

0. 014685  to 

7-18 

0.6233 

50 

0.01247 

60 

60 

6 

No  re- 
action. 

0 

0 

3 

7-25 

.4967 

25 

.  01987 

60 

25 

5 

No  re- 

0 

0 

0.059308. 

action. 

7-25 

1. 1072 

25 

> .04429 

44 

22 

5 

0.01020 

0.  000463 

1.13 

8-2 

'  1.  0979 

25 

'.04392 

23.6 

23.5 

6 

.  01020 

.000434 

.98 

5-9 

•  2.  2906 

50 

.04581 

90 

46 

10 

.  02040 

.  000463 

.94 

8-11 

1. 1706 

25 

.  04682 

42 

21 

6 

.  01030 

.  000490 

1.04 

Average 
0. 093990 

7-27 

1.4009 

25 

.06604 

20.25 

10. 126 

6 

.  01020 

.001007 

1.79 

.043222 

.000476 

.98 

0. 059308  to 

4 

8-2 

1. 6749 

25 

.06700 

21 

10.5 

5 

.01020 

.00097 

1.44 

0. 120369. 

8-11 

■>  1.  6817 

25 

.  06727 

11 

9.16 

6 

.  01030 

.  00112 

1.66 

8-11 

1.  9372 

25 

.  07749 

12.76 

.6.375 

5 

.  01030 

.00161 

2.07 

8-11 

1.  9916 

25 

.07966 

18.1 

9.05 

5 

.  01030 

.00113 

1.41 

7-31 

2.3044 

26 

». 09218 

6.8 

3.4 

6 

.01020 

.00300 

3.25 

8-2 

2.  3566 

26 

' .09426 

8.6 

4.3 

5 

.  01020 

.00237 

2.51 

8-30 

2. 3733 

26 

.  09493 

6.68 

3.29 

6 

.  01030 

.  00313 

3.29 

7-25 

2.  4901 

26 

.  09960 

5.8 

2.9 

6 

.  01020 

.  .  00351 

3.52 

8-2 

2.  6748 

26 

.  10699 

8.0 

4.0 

5 

.01020 

.00255 

2.37 

.8-17 

2.6843 

25 

.  10737 

4.76 

2.38 

5 

.01030 

.00431 

4.01 

8-18 

2.  7781 

26 

.  11112 

4.35 

2.175 

6 

.01030 

.00473 

4.25 

8-2 

2.  7919 

25 

.11168 

7.0 

3.5 

6 

.01020 

.00291 

2.61 

8-18 

2.8205 

25 

.11282 

4.35 

2.175 

5 

.  01030 

.  00473 

4.19 

6-1 

2.8332 

25 

.  11333 

12.7 

6.35 

8.9 

.  018166 

.00286 

2.52 

8-11 

2.8972 

25 

.11689 

6.8 

3.4 

5 

.  01030 

.  00303 

2.61 

8-4 

2.9274 

25 

.  11710 

7.1 

3.65 

5 

.01020 

.00287 

2.45 

8-2 

2.9505 

25 

.11802 

7.1 

3.55 

5 

.  01020 

.00287 

2.43 

8-31 

2. 9749 

26 

.11900 

5.2 

2.6 

6 

.01030 

.00396 

3.32 

8-2 

2.9908 

25 

.  11963 

6.0 

3.0 

5 

.01020 

. 00340 

2.84 

8-17 

3.0038 

25 

.12015 

4.5 

2.25 

5 

.  01030 

.00457 

3.80 

Average 
0. 146748 

8-4 

3.0105 

25 

.12042 

6.5 

3.25 

6 

.  01020 

.00314 

2.61 

. 10314 

.00299 

2.82 

0. 120369  to 

6 

6-1 

c  3.  0961 

26 

.12384 

27.0 

6.76 

10 

.02040 

.  00317 

2.67 

0. 160876. 

8-18 

3. 1148 

25 

.12459 

4.2 

2.1 

6 

.  01030 

.  00490 

3.93 

8-18 

3.1953 

25 

.  12781 

4.2 

2.1 

6 

.  01030 

.00490 

3.83 

8-11 

3.  2141 

25 

.12856 

6.4 

3.2 

5 

.  01030 

.00322 

2.61 

7-26 

3.3153 

25 

.  13261 

5.6 

2.8 

5 

.01020 

.00364 

2.75 

7-31 

'  3.  3278 

25   .13311 

5.0 

2.5 

5 

.01020 

.00408 

3.06 

8-11 

3.3479 

25   .13392 

6.5 

3.25 

5 

.01030 

. 00317 

2.36 

8-10 

3.  3602 

25   .13441 

6.15 

3.075 

6 

.  01030 

.  00334 

2.45 

7-31 

3.3689 

25   .13476 

tl 

2.05 

5 

.  01020 

.  00497 

3.69 

8-11 

3.  3706 

25   .13482 

6.1 

3.05 

6 

.01030 

.00337 

2.49 

7-27 

3.  3721 

25   .13488 

4.6 

2.25 

5 

.01020 

.  00463 

3.35 

7-25 

3. 4029 

25   .13612 

3.4 

1.7 

5 

.01020 

.  00600 

4.41 

7-25 

3.4620 

26   .13848 

5.25 

2.626 

5 

,01020 

.00388 

2.80 

8-17 

3.4644 

25   .13868 

3.5 

1.75 

5 

.01030 

.00688 

4.23 

8-31 

3.  4776 

25   .13910 

4.6 

2.3 

5 

.01030 

.00448 

3.22 

7-25 

/  3.  6156 

25   .14462 

7.92 

3.96 

6.07 

.01238 

.  00312 

2.15 

8-17 

3.  6394 

25   .14558 

3.6 

1.8 

6 

.01030 

.00572 

3.92 

8-17 

/  3.  6971 

25   .14788 

4.2 

2.1 

5 

.01030 

.  00490 

3.31 

Average 

8-18 

/  3.  7164 

25   .14866 

5.2 

2.6 

5 

.01030 

.00396 

2.66 

. 13691 

.00428 

3.14 



«  Unless  otherwise  stated,  total  cc.  of  extract  equals  50 
'  Total  extract,  25  cc.  only. 
'  Total  extract,  100  cc. 


<*  Total  extract,  30  cc.  only. 

'  Just  sealed,  early. 

f  Just  sealed,  still  coiled. 


148 


Journal'  of  Agricultural  Research         voi.  xxviii.  No.  2 


Table  V. — Unassimilated  sugar  in  intestinal  content  of  larvx  at  different  ages — 

Continued 


Larvae  of  knownage, 
Sturtevant  (35^ 

Larvas  analyzed  for  presence  of  unassimilated  sugar  (weights  in  grams) 

Age 

in 

days 

Aver- 
age 
weight 

Limits  by 
weight 
for  age 
groups 

Date 

Weight 
sample 

Num- 
ber 
of 
lar- 
vae 

Aver- 
age 
weight 
of  1 

larva 

Ex- 
tract 
used 

Equiv- 
alent 

□umber 

of 

larva 

CuSO( 
solu- 
tion 

Equiv- 
alent 
dex- 
trose 

Dex- 
trose 
per 
larva 

Sugar 
per 
larva 

6 

Gram 
0. 165005 

Average 
0. 141648 

Average 
0. 137165 

Average 
0. 133152 

Gram. 
0. 160876  to 
maximum 
and  down 
to  0.148326. 

19a 
7-18 
8-31 
8-11 
8-31 
8-31 
7-27 

Grams 
'  3. 8012 
3.8038 
3.  8925 
«  3.  9783 
4.1249 
3.  7706 

26 
25 
25 
25 
26 
25 

Gram 
0. 16205 
.  15215 
.  15570 
.  16913 
. 16600 
.  15082 

cc. 
18.05 
6.4 
4.35 
6.0 
7.0 
6.65 

4.51 

2.7 

2.175 

3.0 

3.5 

3.33 

«. 
9.6 
6 
5 
6 
5 
5 

Gram 
0. 19584 
.  01030 
.01030 
.  01030 
.  01030 
.01020 

Gram 
0. 00434 
.00381 
.00473 
.00343 
.00294 
.00306 

P.ct. 
2.85 
2.50 
3.03 
2.15 
1.78 
2.03 

.15581 

.  00372 

2.39 

0. 148326  to 
0. 139406. 

7 

8-18 
8-10 
8-10 
8-4 

8-18 

3.  6980 
3.6600 

*  3.  5835 
3.  5572 

•  3.  4871 

25 
25 
26 
26 
25 

.  14792 
.  14280 
.  14334 
.  14229 
. 13948 

7.3 
8.9 

11.5 
7.4 

45.0 

3.66 
4.45 
5.75 
3.7 
2Z5 

6 
6 
5 
5 
5 

.01030 
.01030 
.  01030 
.01030 
.  01030 

.00282 
.00231 
.00179 
.00275 
.00050 

1.91 
1.66 
1.25 
1.93 
.36 

.  14397 

.  00203 

1  40 

0. 139406  to 
0. 135158. 

S 

7-26 
7-18 

3.4358 
1  3. 4453 

25 
26 

. 13743 
^ .  13781 

50.0 
50.0 

25 
25 

5 
6 

No  re- 
action. 
No  re- 
action. 

0 
0 

0 
0 

.  13762 

1 

u 

fl 

8-4 

'  3.  3232 

25 

.13293 

50.0 

26 

6 

No  re- 
action. 

0 

<:  Total  extract,  100  cc. 

«  All  sealed,  coiled  or  with  backs  out.    Feeding  ended  and  spinning  of  cocoons  started. 

*  Cocoon  partially  spun,  still  some  color  in  the  intestine. 

i  Cocoon  not  quite  finished,  still  moving  somewhat,  no  color  in  intestine. 
i  Quiescent  prepupae,  intestines  colorless,  empty,  histolysis  started.  • 

*  First  indication  of  change  in  external  form. 


OBSERVATIONS 

Over  60  samples  of  25  larvae  each  of  various  ages,  containing  over  1,600  indi- 
vidual larvae,  were  analyzed  for  the  presence  of  reducing  sugars.  The  largest 
number  of  analyses  were  made  on  larvae  from  3i  to  6J  days  of  age  during  the 
active  honey  and  poUen  feeding  period.  At  least  five  analyses  were  made  of 
each  of  the  other  age  periods  which  might  show  the  presence  of  sugar.  To  obtain 
averages  with  a  small  probable  error,  the  analyses  are  grouped  by  age  periods  of 
24  hours  each,  as  described  earlier  (Table  V,  fig.  13).  All  larva  in  the  two- 
day  group,  as  well  as  one  sample  of  larvae  nearly  as  heavy  as  the  three-day  aver- 
age larva,  showed  no  reducing  sugar.  Larvae  in  the  three-day  group,  averaging 
0.043222  gm.  in  weight,  gave  0.000475  gm.  of  reducing  sugar  per  larva,  or 
0.98  per  cent  concentration.  Larvae  in  the  four-day  group,  averaging  0.10314 
gm.  in  weight,  gave  0.00299  gm.  of  reducing  sugar  per  larva,  or  2.82  per  cent 
concentration.  Larva;  in  the  five-day  group,  comprising  those  just  prior  to 
seahng,  with  a  few  just  sealed,  averaging  0.13591  gm.  in  weight,  gave  0.00428 
gm.  of  reducing  sugar  per  larva,  or  3.14  per  cent  concentration.  In  the  five-day 
group  there  were  two  samples  which  gave  a  concentration  of  over  4  per  cent,  the 
maximum  being  4.41  per  cent.  The  six-day  group,  comprised  entirely  of  larva 
that  had  been  sealed,  had  finished  feeding  and  had  started  spinning,  averaging 
0.15581  gm.  in  weight,  gave  0.00372  gm.  of  reducing  sugar  per  larva,  or  2.39  per 
cent  concentration.     This  group  contains  larvae  of  maximum  size  (fig.  14).     From 


Apr.  12,  1924 


Development  of  American  Foulhrood 


149 


this  point  on  the  gross  weight  decreases  as  preparation  for  metamorphosis  begins. 
The  seven-day  group,  comprising  larvse  which  are  still  moving  about  in  spinning, 
and  most  of  which  show  only  a  slight  remaining  color  in  the  intestines,  indicating 


SO 


/■40 


/20 


\ 

N 

I 


/oo 


eo 


eo 


40 


20 


\4 

\ 

1 

// 

\ 

/ 

/ 

N 

^ 

/ 

i 

^ 

*v^ 

// 

/ 



^ 

—— 

V 

1 

// 

1 

/C/VOW/V  /tG£. 

: W£/0/fr  OF  i^/fM£  //V 

i 

sao^p. 

If 

i 

1 

i 
// 

^i 

1 

1 

/ 

t 

I 

1- 

i 

1 

1  / 

^ 

'I 

^ 
\ 

5 

: 

i 

3  4  £  6  7  e  3 

y^G£  /A/  O^I<S 

Fig.  13.— Unassimilated  sugar  in  larvae  at  different  ages  (Table  V) 


/O 


that  the  connection  between  ventriculus  and  end  gut  is  made,  averaging  0.14397 
gm.  in  weight,  gave  0.00203  gm.  of  reducing  sugar  per  larva,  or  1.40  per  cent 
concentration.     One  sample  in  this  group  gave  as  low  as  0.36  per  cent.     Larvae 


150 


Journal  of  Agricultural  Research  voi.  xxvm,  No  2 


Fig.  14.— Healthy  larva  at 
age  of  maximum  size,  just 
after  sealing  and  before  the 
start  of  the  cocoon-spin- 
ning period  (White  (56)) 


Of  the  eight-day  group,  averaging  0.13762  gm.  in  weight,  showed  a  total  absence 

of  reducing  sugar.  These  larvs  represent  the  two-day  quiescent  prepupal  stage 
(fig.  3  and  4).  They  have  stretched  out  motionless  in 
the  cell,  the  intestines  are  entirely  empty  and  colorless, 
and  the  histolysis  of  the  tissues  preliminary  to  metamor- 
phosis has  begun. 

From  these  observations  it  is  seen  that  there  is  an 
amount  of  reducing  sugar  in  the  entire  actively  feeding 
larva  which  would  seriously  interfere  with  the  germina- 
tion and  growth  of  Bacillus  larvae,  provided  the  entire  bee 
larva  were  to  serve  as  the  medium  for  its  growth.  Since 
this  reducing  sugar  does  not  exist  equally  distributed 
throughout  the  bee  larva,  and  since  at  this  stage  the 
organisms  are  found  almost  solely  in  the  intestinal  tract, 
it  is  certain  that  the  reducing  sugar  concentration  of  the 
intestine  is  sufficient  to  prevent  the  germination  of 
Bacillus  larvae,  so  that  death  from  American  foulbrood  is 

delayed  until  after  the  larva  has  been  sealed  in  the  cell  and  has  become  quiescent. 

This  will  be  discussed  more  in  detail  later. 

SUPPLEMENTARY   STUDIES    ON    THE   BIOCHEMICAL   REACTIONS 
OF  BACILLUS  LARVAE 

Up  to  the  present  time  few  facts  have  been  determined  concerning  the 
biochemical  reactions  of  Bacillus  larvae,  mainly  because  of  a  lack  of  suitable 
culture  media.  White  states  {65),  "Carbohydrate  liquid  media  as  ordinarily 
prepared  are  not  suitable  for  the  growth  of  Bacillus  larvae.  In  some  of  these 
after  a  considerable  period  a  slight  growth  may  appear  at  the  bottom  of  the 
tubes.  A  little  brood-filtrate  or  egg-suspension  added  to  the  media  improves  it. 
No  visible  gas  is  formed,  but  in  some  instances  slight  acidity  Is  produced.  No 
growth  takes  place  in  plain  or  in  brood-filtrate  gelatin  at  temperatures  at  which 
it  remains  congealed."  Maassen  states  {28) ,  "  The  bacillus  also  grows  on  nutrient 
gelatin.  Upon  a  nutrient  gelatin  medium  which  had  been  made  from  the  pre- 
viously mentioned  nutrient  liquids,  and  an  almost  completely  neutralized  gelatin 
(a  so-called  emulsion  of  gelatin),  there  resulted  growth  although  very  slowly, 
from  which  a  quite  gradual  liquefaction  of  the  gelatin  resulted.  Liquefaction 
did  not  occur  in  the  presence  of  grape  sugar  (dextrose) .  Through  the  addition  of 
1  per  cent  grape  sugar  the  growth-producing  ability  of  the  gelatin  as  well  as  of  other 
nutrient  media  was  noticeably  improved.  On  the  most  favorable  media  no 
special  chemical  properties  were  shown,  with  the  exception  of  the  ability  to 
peptonize.  The  destruction  of  the  albuminous  bodies  occurred  very  slowly 
and  with  little  characteristic  appearance.  Only  in  worn-out  cultures  could 
any  odor  resembling  foul  glue  be  detected  after  a  tipie.''  There  are,  however, 
certain  characteristic  manifestations  in  American  foulbrood  resulting  from  the 
growth  and  metabolism  of  Bacillus  larvae,  aside  from  the  gross  symptoms  and 
appearances,  which  only  a  more  complete  knowledge  of  the  biocheniical  activity 
of  the  organism  can  explain. 

From  the  previous  cultural  experiments  (Table  I)  it  may  be  seen  that  apparently 
Bacillus  larvae  can  utiUze  in  its  metabolism  a  certain  amount  of  reducing  sugar 
(dextrose),  although  this  sugar  is  not  necessary  to  the  development  of  the 
organism.  In  the  larva  which  is  attacked  by  American  foulbrood  there  may  be 
two  sources  of  sugar,  that  present  unassimilated  in  the  intestine  and  that  hydro- 
lyzed  from  the  stored  glycogen.  Hydrolysis  of  glycogen  may  occur  in  connection 
with  histolysis  of  the  tissues  preparatory  to  metamorphosis  through  enzym 


Apr.  11,  1924 


Development  of  American  Foulbrood 


151 


action,  or  Bacillus  larvae  itself  may  have  the  ability  to  produce  enzymes  which 
hydrolyze  the  glycogen,  or  it  may  be  a  combination  of  both.  Through  the 
utilization  of  this  reducing  sugar  one  would  expect  that  there  at  least  would  be  a 
considerable  production  of  acid,  but,  as  stated  earlier,  the  hydrogen-ion  concen- 
tration of  dead  ropy  material  is  nei^er  found  to  vary  much  from  Ph=6.6  to  6.8. 
Since  the  data  available  concerning  the  biochemical  reactions  of  Bacillus  larvae 
offer  no  explanation  of  this  hydrogen-ion  concentration,  a  series  of  experiments 
was  devised,  the  results  of  which  add  materially  to  the  knowledge  concerning 
the  biochemical  reactions  and  relationships  of  Bacillus  larvae.  In  certain 
cases  where,  because  of  the  limitations  on  growth,  cultural  growth  has  failed, 
it  was  found  possible  to  obtain  the  desired  information  by  examination  of  the 
diseased  larval  remains. 


/ 
/ 
/ 

/ 
y 

/ 

\ 

/ 

/ 
/ 

\ 

\ 

\ 

V 

\ 

/ 

\ 

\ 
> 

/    X   / 



--- 





'^--^ 

f/ 

^ 

/     "v. 

/v/Ty? 

OGe/^ 

^- 





, 

/ 

^ 

s         e         7 


/o         // 


Fig.  15. — Per  cent  composition  of  worker  larvas  at  different  ages  (Tables  IV  and  V) 


UTILIZATION  OF  GLYCOGEN 


According  to  Straus  (Table  IV,  fig.  15)  the  greatest  percentage  of  stored 
glycogen  occurs  just  after  sealing,  when  feeding  has  ceased.  If  an  emulsion  of 
the  tissues  of  a  larva  of  this  age,  or  slightly  older,  at  the  age  when  prepupae  usu- 
ally die  of  American  foulbrood,  is  tested  for  the  presence  of  glycogen  with  iodin 
solution,'  the  resulting  deep  reddish  brown  color  shows  that  there  are  large 
amounts  of  glycogen  present.  If  a  prepupa  which  has  ]'ust  died  from  disease, 
sUmy  in  consistency,  light  brown  in  color,  and  which  in  the  microscopic  picture 
still  shows  the  presence  of  vegetative  rods,  is  tested  with  iodin  solution,  it  will 

*  Glycogen  treated  with  iodin  solution  gives  a  color  varying  from  brown  to  wine  red,  which  disappears 
upon  heating  to  60°  C,  but  returns  again  upon  cooling.  Soluble  plant  starch  with  iodin  solution' gives 
the  following  reactions:  Amylodeitrin,  first  dextrin  of  conversion,  dark  blue;  erythrodextrin,  second  dex- 
trin of  conversion,  red;  intermediate  steps  give  various  shades  of  purple  or  lavender. 


152  Journal  of  Agricultural  Besearch  voi.  xxviii,  No.  2 

be  found  that  most  of  the  glycogen  has  disappeared,  although  the  iodin  solu- 
tion gives  a  light  yellowish  brown  color.  The  presence  of  a  trace  of  reducing 
sugar  also  occasionally  can  be  demonstrated  with  Benedict's  solution  in  dis- 
eased material  of  this  type  where  vegetative  organisms  are  stiU  actively  present. 
In  material  which  has  decomposed  completely,  has  reached  the  dark  brown 
ropy  stage  (fig.  9),  and  contains  only  spores  of  Bacillus  larvae,  glycogen  is  found 
to  be  completelj'  absent,  nor  can  any  reducing  sugar  be  demonstrated,  the 
sugars  having  been  completely  destroyed. 

This  type  of  material  stained  with  Sudan  III  or  osmic  acid  {S^,  p.  78)  shows 
fat  globules  in  practically  the  same  condition  and  amount  as  in  healthy  larvae, 
so  that  fat  is  apparently  not  acted  upon  by  Bacillus  larvae  even  after  drying  down 
to  the  scale  stage. 

Glycogen  of  the  fat  body  of  the  healthy  larva  is  hydrolyzed  to  dextrose  to  be 
used  in  metamorphosis,  by  the  action  of  enzyms  during  the  histolytic  processes 
subsequent  to  sealing  and  prior  to  metamorphosis.  This  enzym  action  is  demon- 
strated by  the  following  e.xperiments: 

EXPERIMENTAL    PBOCEDURE 

Several  series  of  50  healthy  prepupae  each  that  had  reached  the  period  of 
quiescence  were  macerated  in  25  cubic  centimeters  of  50  per  cent  alcohol  and 
incubated  at  37°  C.  for  from  3  to  24  hours.  The  extract  was  then  filtered  and 
diluted  with  an  equal  amount  of  water.  A  series  .of  test  tubes  were  prepared, 
using  for  each  tube  5  cubic  centimeters  of  this  extract  and  5  cubic  centimeters  of 
0.4  per  cent  glycogen  in  water,  and  also  another  series  using  5  cubic  centimeters 
each  of  a  0.1  per  cent  soluble  starch.  Both  glycogen  and  starch  were  used,  since 
it  has  been  shown  by  Bradley  and  KeUersberger  (S),  as  well  as  bj*  experiments 
by  the  writer  using  commercial  Taka-diastase,  that  diastase  acts  similarly  on 
both  glycogen  and  starch.  These  tubes  were  incubated  for  various  periods  and 
then  tested  with  iodin  solution  for  the  presence  of  glycogen  and  starch  (Table 
VI) .  Hydrolysis  of  both  glycogen  and  starch  seems  to  be  complete  after  incuba- 
tion for  about  five  hours,  and  positively  complete  after  incubation  overnight, 
demonstrating  the  presence  of  diastase  in  the  prepupae. 

In  another  experiment  50  prepupae  were  macerated  in  50  cc.  of  water  and  in- 
cubated at  37°  C.  for  24  hours.  Then  sufiicient  95  per  cent  alcohol  was  added  to 
precipitate  any  glycogen  present,  and  the  solution  was  filtered  and  tested  with 
both  the  qualitative  and  the  quantitative  Benedict's  solutions.  In  both  cases 
definite  traces  of  reducing  sugar  could  be  demonstrated,  none  having  been  present 
in  the  original  solution  before  incubation,  again  demonstrating  enzym  activity 
of  the  larval  tissues.  This  may  have  been  due  to  action  by  bacterial  contamina- 
tion, but  if  such  had  been  the  case  the  sugar  would  probably  have  been  fermented 
and  could  not  have  been  demonstrated. 

In  a  similar  manner  extracts  with  50  per  cent  alcohol  were  made  of  ropy  dis- 
eased material,  enzym  activity  being  demonstrated  in  the  same  manner  as  above. 
This,  however,  does  not  indicate  whether  the  organism  causing  the  disease  has 
any  diastatic  power  or  whether  the  reaction  was  due  to  enzyms  remaining  in 
the  decomposed  tissues.  Further  extracts  were  made  with  25  per  cent  and  50 
per  cent  alcohol  of  several  48-hour  vegetative  cultures  of  Bacillus  larvae  grown  on 
egg-yolk  suspension  medium.  These  extracts  showed  definite  enzym  activity 
with  glycogen  after  a  few  hours'  incubation,  and  more  positive  activity  after 
incubation  overnight  (Table  VI),  while  with  starch  marked  hydrolysis  was  shown 
'after  only  a  few  hours'  incubation. 


Apr.  12, 1924 


Development  of  American  Foulbrood 


153 


Table  VI. — Test  for  diasiaiic  action  with  alcoholic  extracf* 


Color  with  lodin  after  incubation  of— 

Test  material 

Ohour 

ihour 

2i  hours 

6i  hours 

18  hours 

5 

CQ 

1 

3 

CQ 

1 
5 

M 

3 

CO 

3 

OQ 

3 

1 

Extract  of  healthy  pre- 

++++ 

(brown) 

++++ 
++++ 

(blue) 

+++ 

++ 

++ 

++ 

+ 

+ 
++ 

+ 

+ 
++ 

± 

± 
+ 

Ertract  of  decomposed 

ropy  remains. 

Extract  of  vegetative 

++++ 

<*  The  following  symbols  are  used: 
++++  Deep  color,  brown  or  blue. 
+++  Slightly  lighter  brown  than  check  or  wine 
color. 
++  Light  coffee  brown  or  lavender. 


+  Trace  faint  brown  or  trace  taint  lavender. 
—  No  color  or  only  iodln  color,  showing  com. 
plete  diastatic  action. 


To  further  determine  the  production  of  diastase  by  Bacillus  larvae,  a  series  of 
Petri  dishes  were  poured,  using  yeast-extract  egg-yolk  suspension  agar,  to  which 
had  been  added  respectively  0.25  per  cent  and  1  per  cent  of  glycogen  and  0.25  per 
cent  and  1  per  cent  of  starch,  this  being  an  adaptation  from  methods  described 
by  Vedder  (45)  and  by  AUen  (i).  After  solidification  of  the  media  in  the  Petri 
dishes,  smears  were  made  upon  the  surface  of  the  agar  from  48-hour  cultures  of 
various  previously  isolated  strains  of  Bacillus  larvae.  After  several  days  the  plates 
were  examined,  first  by  holding  up  to  the  light  and  then  later  by  flooding  with 
iodin  solution,  and  comparing  with  control  plates  containing  no  starch  or  glycogen. 
In  nearly  all  the  plates  good  growth  had  occurred,  causing  clear  areas  to  be  pro- 
duced in  the  cloudy  culture  medium  extending  slightly  beyond  the  edge  of  the 
area  of  growth.  When  flooded  with  iodin  the  halo  around  the  culture  growth, 
although  not  wide,  was  more  prominently  differentiated  from  the  surrounding 
medium,  showing  in  both  glycogen  and  starch  plates.  These  results,  in  con- 
junction with  those  of  the  extraction  experiments,  demonstrate  that  weak  dia- 
static action  is  produced  by  Bacillus  larvae. 

ACID  PRODUCTION 

It  has  been  shown  that  there  is  still  an  appreciable  amount  of  sugar  (reducing 
sugars  in  the  food  remaining  in  the  intestines  and  dextrose  available  from  gly- 
cogen) present  in  the  larva  after  sealing  and  in  the  prepupa  at  the  age  when 
American  foulbrood  attacks,  available  for  fermentation  (Tables  IV  and  V).  In 
the  various  cultural  investigations  both  by  others  and  by  the  present  writer,  there 
is  no  evidence  of  carbon  dioxid  production.  It  would  be  expected,  however,  that 
at  least  some  acid  would  be  produced  from  the  bacterial  fermentation  of  these 
sugars,  which  is  known  to  be  present.  To  determine  this  more  definitely  than 
heretofore,  a  culture  medium  was  devised  for  the  qualitative  determination  of 
acid  production,  which  gave  good  vigorous  growth  of  Bacillus  larvae. 

The  method  used  Is  an  adaptation  of  the  method  of  using  agar  slants  for  detect- 
ing acid  formation,  instead  of  liquid  medium,  described  by  Conn  and  Hucker  {18) , 
in  which  the  change  in  reaction  can  readily  be  seen.  The  regulation  yeast- 
extract  egg-yolk  suspension  agar  was  prepared  for  this  purpose  by  adding  to  the 
yeast  extract  base  before  sterilization  an  indicator  in  the  proper  amount  both  to 
the  plain  medium  and  also  to  a  portion  to  which  was  added  1  per  cent  of  dextrose. 


154 


Journal  of  Agricultural  Research         voi.  xxvin.  No.  2 


Brom  thymol  blue  was  first  used,  as  it  covers  the  range  of  the  supposed  optimum 
reaction  for  Bacillus  larvx  as  described  earlier.  Baker  (4)  also  has  shown  that 
brom  thymol  blue,  used  in  about  a  0.0024  per  cent  concentration  in  culture  media, 
gives  the  most  desirable  color  for  comparison,  without  inhibiting  acid  fermentation. 
This  concentration  was  obtained  by  using  12  cc.  of  a  0.2  per  cent  alcoholic  solu- 
tion of  the  indicator  per  Uter.  After  marked  acid  production  in  the  dextrose 
tubes  was  demonstrated  with  brom  thymol  blue,  brom  cresol  purple  was  used  as 
suggested  by  Conn  and  Hucker  {18)  in  a  0.001  per  cent  concentration  as  a  check 
on  the  end  point.  This  concentration  was  obtained  by  using  8  cc.  of  a  0.2  per 
cent  alcoholic  solution  of  the  indicator  per  liter.  The  yeast-extract  base,  both 
with  and  without  dextrose,  was  adjusted  so  that  after  the  addition  of  the  egg- 
yolk  suspension  the  final  medium  would  have  a  primary  reaction  of  approximately 
Ph=7.2,  a  definite  blue  grass  green  in  the  case  of  brom  thymol  blue  and  a  marked 
purplish  tinge  with  brom  cresol  purple,  except  in  one  series,  where  the  primary 
reaction  of  the  plain  medium  was  Ph=7.6.  These  tubes  after  being  slanted  were 
inoculated  as  usual,  both  with  vegetative  cultures  and  with  diseased  material 
containing  spores.  The  change  in  reaction  was  noted  after  different  lengths  of 
incubation,  and  the  final  reaction  was  determined  by  comparison  with  standard 
buffer  tubes  used  in  combination  with  tubes  of  plain  egg-yolk  suspension  media 
slanted  in  the  same  manner.  The  approximate  increase  in  hydrogen-ion  con- 
centration was  determined  by  this  comparison  (Table  VII). 

Table  VII. — Acid  production  by  Bacillus  larvae 


Brom  thymol  blue  indicator 

Brom  cresol  purple  indicator 

Culture  No. 

Plain  medium 

1  per  cent  dextrose 

Plain  medium 

1  per  cent  dextrose 

Control 

Inocu- 
lated 

Control 

Inocu- 
lated 

Control 

Inocu- 
lated 

Control 

Inocu- 
lated 

9693-1 

Ph 

7.6 
7.2 
7.6 
6.8-7.0 
7.2 
7.6 
7.6 
7.6 

Ph 

6.8 

6.6 

•7.4 

±6.6 

6.6-6.8 

"7.4 

"7.4 

7.0-7.2 

Ph 
7.2 
7.2 
7.2 
6.6 
7.2 
7.2 
7.2 
7.2 

Ph 
6.0 
6.0 
6.2 
6.0 
6.0-6.2 
6.4 
6.0 
6.0 

Ph 

Ph 
W 

Ph 

Ph 

S.8 

9834-1 

9834-2 

(') 

(•) 

W 

6.0 

9863 

9857 

9867 

i 

i 

6.2 
6.8 
6.8 

9869 

9874 

'  Doubtful  growth. 

*  Beyond  end  point,  no  growth. 

'  No  change  in  color,  good  growth. 


''  Beyond  end  point,  good  growth. 
«  No  change  in  color,  no  growth. 


OBSERVATIONS 

Several  interesting  facts  were  observed  from  these  experiments.  Addition  of 
buffer  salts  to  the  media  delayed  the  approach  to  the  final  hydrogen-ion  con- 
centration reaction  somewhat,  but  eventuaUy  practicaUy  the  same  end  point 
was  reached.  Also,  in  one  series  of  media  in  which  the  plain  medium  was 
adjusted  to  about  Ph=7.6,  little  if  any  growth  occurred  in  these  tubes  except 
with  two  strains  of  Bacillus  larvae,  indicating  that  the  alkaline  limit  for  growth 
is  about  at  this  point.  In  cases  where  the  initial  reaction  of  the  plain  medium 
was  Ph=7.2,  the  final  reaction  averaged  Ph=6.6  to  Ph=6.8  (Table  VII).  In 
the  case  of  the  medium  to  which  1  per  cent  dextrose  had  been  added,  the  final 
reaction  averaged  about  Ph=6.0  for  brom  thymol  blue  and  from  Ph=5.8  to 
Ph=6.0  for  brom  cresol  purple  (Table  VII).  WhUe,  therefore,  only  a  sUght 
change  in  reaction  occurred  in  media  without  sugar,  a  marked  production  of 


Apr.  12, 1024  Development  of  American  Foulbrood  155 

acid  was  indicated  in  the  tubes  to  which  1  per  cent  dextrose  had  been  added. 
The  maximum  production  of  acid,  however,  required  approximately  48  hours 
or  more,  the  fermentation  of  the  sugar  apparently  being  relatively  slow.  As 
has  been  stated,  however,"  the  reaction  of  diseased  material  in  various  stages  of 
decomposition  and  drying  down  is  never  found  to  reach  a  hydrogen-ion  concen- 
tration of  more  than  Ph  =6.6,  and  usually  averages  Ph  =6.8. 

PROTEIN  DECOMPOSITION 

It  is  known  that  certain  organisms  have  the  ability  to  break  down  protein 
material  under  proper  conditions,  with  the  production  of  amino  acids  and  alka- 
line decomposition  substances,  which  latter  tend  to  neutralize  any  acid  produced 
from  fermentation  of  sugar.  If  it  can  be  shown  that  Bacillus  larvae  has  this 
ability,  it  will  explain  the  fact  that  the  remains  of  larvje  dead  from  American  foul- 
brood  do  not  show  a  greater  acid  reaction  resulting  from  the  fermentation  of  the 
sugar  of  the  intestinal  contents.  A  series  of  experiments  was  devised  to  demon- 
strate whether  such  is  the  case  with  Bacillus  larvae. 

The  prepupa  at  the  age  attacked  by  Americafi  foulbrood  contains  nitrogenous 
substances  as  shown  by  the  Kjeldahl  nitrogen  determination  equivalent  to  1.45 
per  cent  nitrogen  (4S) .  The  source  of  this  nitrogen  is  mainly  albuminous  mate- 
rial, one  of  the  constituents  of  the  larval  fat  body.  Its  exact  composition  has 
not  been  determined,  but  without  doubt  it  is  complex  in  nature.  There  are 
certain  color  reaction  tests  by  means  of  which  the  constitution  of  this  nitrogenous 
material  may  be  indicated. 

A  delicate  test  for  the  presence  of  coagulable  protein  is  that  of  Heller  (3S,  p. 
1067).  A  suspension  of  healthy  prepupse  in  water,  treated  by  pouring  about 
4  cc.  of  concentrated  nitric  acid  down  the  side  of  the  inclined  test  tube,  causes  a 
white  ring  to  form  at  the  junction  of  the  two  liquids.  Decomposed  ropy  material 
tested  in  this  way  gives  no  indication  of  such  a  ring,  indicating  that  the  complex 
protein  has  disappeared. 

One  of  the  most  characteristic  reactions  for  complex  protein  is  the  biuret  test 
(SS,  p.  915).  If  some  healthy  prepupse  are  suspended  in  a  few  cubic  centimeters 
of  10  per  cent  sodium  hydroxid  and  are  treated  with  a  few  drops  of  a  0.5  per 
cent  copper  sulphate  solution,  a  distinct  pinkish-violet  color  is  formed,  again 
indicating  the  presence  of  complex  protein  material.  Decomposed  ropy  material 
tested  in  this  way  gives  no  indication  of  this  color,  again  indicating  the  complete 
disappearance  of  the  complex  protein. 

There  is  also  the  xantho-proteic  reaction  (SS  p.  916),  which  is  given  both  by 
solid  and  by  dissolved  protein,  and  indicates  the  presence  of  the  amino-acids, 
tryptophan,  tyrosin,  or  phenylalanin  in  the  protein  molecule,  or  in  solution. 
Tryptophan  gives  the  reaction  most  intensely.  Both  healthy  prepupse  and 
ropy  material,  boiled  with  concentrated  nitric  acid,  produce  a  lemon-yellow 
color  which  on  cooling  and  neutralizing  with  sodium  hydroxid  changes  to  an 
orange,  denoting  a  positive  reaction. 

An  even  more  delicate  reaction  for  protein  is  that  with  Millon's  solution  (S2, 
p.  916).  A  few  cubic  centimeters  of  a  suspension  of  healthy  prepupae,  treated 
with  a  few  drops  of  Millon's  reagent  and  boiled,  cause  a  brick-red  precipitate  to 
form,  leaving  the  liquid  practically  clear.  A  solution  of  decomposed  ropy 
material,  treated  in  the  same  way  with  Millon's  reagent  and  boiled,  causes  a 
somewhat  similar  reddish  precipitate,  but  the  solution  is  also  distinctly  colored 
simDarly,  indicating  that  the  protein  has  been  changed  in  some  way,  part  at 
least  being  soluble  in  water.  Tyrosin  is  the  only  amino  acid  in  protein  that 
gives  this  reaction. 


]^  5  6  Journal  of  Agricultural  Research  voi.  xxviii,  no.  2 

Since  tryptophan  is  probably  one  of  the  principal  constituents  of  the  protein 

molecule  in  the  healthy  prepupa  as  well  as  in  solution  in  diseased  material, 

certain  tests  were  made  to  determine  its  presence,  because  this  amino-acid  is 

'  easily  utilizable  by  bacteria  and  gives  decomposition  products  indicating  the 

nature  of  bacterial  action.     The  following  tests  are  specific  tryptophan  reactions: 

AdamUewicz  reaction  {SS,  p.  917).— A  suspension  of  healthy  prepups  or  of 
diseased  material  in  glacial  acetic  acid,  treated  by  pouring  concentrated  sul- 
phuric acid  down  the  side  of  the  inclined  tube,  causes  a  violet  ring  to  form  at 
the  junction  of  the  two  liquids,  indicating  the  presence  of  tryptophan,  either 
as  part  of  the  complex  molecule  or  in  solution. 

Rhodes  reaction  {41).—To  a  suspension  of  healthy  prepup*  or  of  diseased 
material  in  water,  a  few  drops  of  a  wea