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Dr. Roger a. Morse
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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
THE BACTEEIA OF THE APIABY.
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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
Grejsdalen den 11. Septbr. 1904. (Efter Foredragsholderens
manuskript.) RoskUde [1904] 17 p. ( Saertryk af " Tldsskrif t
for Biavl," Nr. 16 og 17, 1904.
(2)
1915. Sygdomme hos Honningbien og dens Yngel. Meddelelser fra den
Kgl. Veterinser-og Landboh^jskoles Serumlaboratorinm
XXXVII. 109 p., 11 fig.
EUROPEAN FOULBROOD. 35
(3) Btjkri, R.
1906. Bacteriologlsche Untersuchungen fiber die Faulbrut und Saw-r-
brut der Blenen. 39 p., 1 pi. Vorwort von U. Kramer, January.
(4) Cheshihe, F. R., and Cheyne, W. W.
1885. The pathogenic history and history under cultivation of a new
bacillus (B. alvel), the cause of a disease of the hive bee
hitherto known as foul brood. In Jour. Roy. Micros. Soe. Ser.
II, vol. V, pt. 2, p. 581-601, pi. X-XI, August.
(5) Ford, W. W., Laubach,.C. A., Lawrence, J. S., and Rice, J. L.
1916. Studies on aerobic spore-bearing non-pathogenic bacteria. Pt. II.
In Jour, of Bact.. vol. I, No. .5, p. 493-533, 15 pis. September.
(6) Howard, W. R.
1900. New York bee disease, or black brood. In Gleanings in Bee Culture,
V. 28, no. 4, p. 121-127, February 15.
(7) Maassen, Aisert.
1907. t'ber die sogenannte Faulbrut der Honigbienen. Mitteilungen
aus der Kaiserlichen blologischen Anstalt fiir Land- und Forst-
wirtschaft, Hft. 4, p. 51-53, 6 fig. February.
(8)
1908. Vber die unter der Namen " Faulbrut " bekannten seuchenhaften
Bruterkrankungen der Honigbiene. IMittellungen aus der
Kaiserlichen konlgliehen Anstalt fiir Land- und Forstwirtschaft,
Hft. 7, 24 p., 4 pi. September.
(9) McCrat, a. H.
1917. Spore-forming bacteria of the apiary. In V. S. Dept. Agr. Jour.
of Agr. Research, v. 8, no. 11, p. 399-^20, 6 fig., pi. 93-94,
March 12.
(10) McCrat, A. H., and White, G. F.
1918. The diagnosis of bee diseases by laboratory methods. U. S. Dept.
Agr. Bui. 671. 15 p., 2 pi. June 21.
(11) Moore, Veranus A., and White, G. F.
1903. A preliminary investigation into the cause of the infectious bee
diseases prevailing in the State of New York. In N. Y. [State]
Dept. Agr. 10th Ann. Rept. Com. Agr. for 1902, p. 255-260, 2 pi.
January 15.
(12) Muck, Oswald.
1915. Seuchen der Bienenbrut. In Wiener tierarztlichen Monatsschrift,
Jahrg. 11, Hft. 3, p. 124-139, 6 fig., 2 pi.
(13) White, G. F.
1906. The bacteria of the apiary, with special reference to bee dis-
eases. XJ. S. Dept. Agr. Bur. Ent. Tech. Ser. no. 14. 50 p.
(14)
1908. Miscellaneous papers on apiculture. The relation of the etiology
(cause) of bee diseases to the treatment. XJ. S. Dept. Agr. Bur.
Ent. Bui. 75, pt. 4, p. 33-42. December 26.
(15)
1912. The cause of European foulbrood. U. S. Dept. Agr. Bur. Ent.
Giro. 157. 15 p., 10 fig. May 10.
(16)
1914. Destruction of germs of infectious bee diseases by heating. U. S.
Dept. Agr. Bui. 92. 8 p. May 15.
36 EVLLETIN 810, U. S. DEPARTMENT OF AGRICULTURE.
(17) White, G. F. — Continued.
1917. Sacbrood. U. S. Dept. Agr. Bui. 431. 55 p., 33 fig., 4 pi. Feb-
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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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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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 ^ ^
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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
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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.
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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
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conclusions must be approved from the statistical viewpoint by someone (named)
competent to judge. All computations should be verified.
Station manuscripts and correspondence concerning them should be addressed
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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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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
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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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Total nega
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Total cultui
276
Journal oj Agricultural Research
Vol. 45, No. s
1
nj
g
s
1
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Percent-
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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
ON MEMBRANE PREPARATION TECHNIQUE). Jour. Rov. MicrOS.
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.
Expt. Med. 31:105-114, illus.
(12) Henrici, a. T.
1928. morphologic variation and the rate op growth of bacteria.
194 p., illus., Springfield, 111., and Baltimore, Md. (Mono-
graphs on Agricultural and Industrial Microbiology, v. 1.)
(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
FOULBROOD OP BEES. Canada Expt. Farms, Div. Bact. Rpt.
(18)
1926:13-16.
1928.
CULTURAL STUDIES OP BACILLUS LARV« (WHITE). Sci. Agr. 9r
80—89, illus.
(19) AND Heron, D. A.
1929. MICROBIOLOGICAL STUDIES OF HONEY. I. HONEY FERMENTATION
AND ITS CAUSE. II. INFECTION OF HONEY BY SUGAR-TOLER-
(9m MnPo-, a'^'J?' ^^"^^w Canada Dept. Agr. Bui. (n. s.) 116, 47 p., illus.
(20) McCray, a. H., AND White, G. F. ' > f <
1918. THE DIAGNOSIS OP BEE DISEASES BY LABORATORY METHODS. U. S.
Dept. Agr. Bui. 671, 15 p., iUus.
Sept. 1, 1932 Commercial Honey and Spread of American Foulbrood 285
(21) Marvin, G. E.
1928. the occurence and characteristics op certain yeasts found
IN FERMENTED HONEY. Jour. Econ. Ent. 21:363-370, illus.
(22) Merrill, J. H.
1927. AN INITIAL OUTBREAK OF FOULBROOD. Amer. Bee Jour. 67:414-
415.
(23) MiLLEN, F. E.
1928. SPREADING FOULBROOD. Beekeeper 36: 134.
(24) Morrison, E. W., and Rettger, L. F.
1930. bacterial spores. ii. a study of bacterial spore germination
IN RELATION TO ENVIRONMENT. Jour. Bact. 20:313-342.
(26) Robertson, T. B.
1923. THE CHEMICAL BASIS OP GROWTH AND SENESCENCE. 389 p., illus.
Philadelphia and London. [Original not seen.]
(26) Sturtevant, A. P.
1924. the development op American foulbrood in relation to the
METABOLISM OF ITS CAUSATIVE ORGANISM. Jour. Agr. Research
28:129-168, illus.
(27)
1930. PRELIMINARY REPORT CONCERNING FACTORS RELATED TO CERTAIN
OF THE GROWTH PHASES OP BACILLUS LARVAE. Jour. EoOn. Ent.
23:453-459.
(28) SwANN, M. B. R.
1924. ON THE GERMINATION PERIOD AND MORTALITY OP THE SPORES OP
BACILLUS ANTHHACis. Jour. Path, and Bact. 27:130-134.
(29) TOUMANOPF, K.
1929. NOTE SUR l'iNFECTION DBS LARVES d'aBEILLES PAR BACILLUS.
LARV.E. Bul. Acad. V(St. France 2:45-49.
(30) White, G. F.
1920. AMERICAN FOULBROOD. U. S. Dspt. Agr. Bul. 809, 46 p., illus.
(31) Zinsser, H.
1927. a textbook of bacteriology; a treatise on the application
op bacteriology and immunology to the etiology, diag-
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eases, for students and practitioners op medicine and
PUBLIC HEALTH . . . Rewritten, rev. and reset . . . Ed. 6, 1053.
p., illus. New York and London.
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
Articles for publication in the Journal must bear the formal approval of the
■chief of the department bureau, or of the director of the e.xperiment station from
which the paper emanates. Each manuscript must be accompanied by a state-
ment that it has been read and approved by one or more persons (named) famiUar
with the subject. The data as represented by tables, graphs, summaries, and
conclusions must be approved from the statistical viewpoint by someone (named)
competent to judge. All computations should be verified.
Station rnanuscripts and correspondence concerning them should be addressed
to S. W. Fletcher, Director of Research, Pennsylvania Agricultural Experiment
Station, State College, Pa.
Published on the 1st and 15th of each month. This volume will consist of 12
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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
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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
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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 
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