oS

th nena

3 1924

ALBERT R. MANN
LIBRARY

New YorK STATE COLLEGES

OF
AGRICULTURE AND HoME ECONOMICS

CORNELL UNIVERSITY

EVERETT FRANKLIN PHILLIPS

BEEKEEPING LIBRARY

BULLETIN No. 431

, Conéribution from the Bureau of Timer
L. O. HOWARD, Chief

“

\ UNITED STATES DEPARTMENT OF AGRICULTURE

Washington, D. C. PROFESSIONAL PAPER February 9, 1917

=e

SACBROOD

By

G. F. WHITE
Expert, Engaged in the Investigation of Bee Diseases

é gt
ae Page
“Gntroduction . . 2 6 «0 ee ee © ~~ 1 | Resistance of Sachrood Virus to Direct
, anes) Seceane ees est eS es 2 Sunlight When Dry
‘Name of the Diseas se « « © « 4 | Resistance of Sacbrood Virus to Direct
Appearance of Healthy Brood at the Age Sunlight When Suspended in Water .
“"@t Which it DiesofSachbraod . .. . 6 | Resistance of Sach Virus to Direct
ik Spiestuns of Sac rood 2. 2 2 ew 10 Besitant When Suspended in Honey .
Cause ofSachbrood. . . . . 2. = = 24 Length of Time omg Sacbrood Virus. Re-
Vee Lhe of Sacbrood Upon a mains Virulent in Honey
oak cad ju, had 30 |. Resistance of Sacbrood Virusto thePres-
amount of ‘Virus Required to Prodace ' ence of Fermentative Processes ._.
_the Disease, and the Rapidity of its Resistance of Sacbrood_ Virus to Fer-
31 mentation in Diluted Honey at Out-
1 ods Used in Making Experimental door Temperatare .
» Tnoculations 32 | Resistance of S: Sacbrood Virusto thePres-
Mgans for for the Destruction of the Virus of ence of Putretactive Processes
le eo s 34 | Resistance of Sacbrood Virus to Carbolic
Hent serena to Destroy Sacbrood Acid a a
‘When ended In Water’ . . 34 | Modes: of Transmission of Sacbrood +;
to Destroy Sacbrood Diagnosis of Sacbrood . . « « « = +
ay Suspended in Glycerine . 865 | Prognosia
ecuired™ to Destroy Sacbrood Relation of These Studies io the Treat-
hay Sespended tn Honey - « 36 | ment of Sacbrood . . . + 2 e's «
6 of Sachrood Virua to Drying poorest rb gai oe eae

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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. F, Waite,

Experi, Engaged in the Investigation of Bee Dimer

CONTENTS.
Introduction 2.6. seein. 2 sescneses aoeesee es 1 | Resistance of sacbrood virus to direct sunlight
Historical account ....... 2 WHEN: GEY eee -oioitcie rinse en ssueieseiisSeieeis 38
Name of the disease 4 | Resistance of sacbrood virus to direct sunlight
Appearance of healthy brood at theageat which when suspended in water..............-.... 39
it dies of s#ebr00d wx. :1o4 ce susseeeeeeweawe 6 | Resistance of sacbrood virus to direct sunlight
Symptoms of sacbrood .. . 10 when suspended in honey...........-..--+-. 40
Cause of sacbrood.........--..-.----02-e-- eee 24 ) Length of time that sacbrood virus remains
Weakening effect of sacbrood upon a colony... 30 virulentin honey. ........---...2.--....eee-e 40
Amount of virus required to produce the dis- Resistance of sacbrood virus to the presence of
ease, and the rapidity of its increase......... 31 fermentative processes. ...-......---..-.000-- 41
Methods used in making experimental inocula- —_—(| ‘Resistance of sacbrood virus to fermentation in
HONS scseswera neste pe eeeeeeeieneeeeenes 32 diluted honey at outdoor temperature....... 42
Means for the destruction of the virus of sac- Resistance of sacbrood virus to the presence of
F EGO ans cisic aha Sicfsiccdsindags Ses citissociaveas apaieglbe cine, 34 putrefactive processes. ......-..2-.-6--2 2.0 ee 43
Heating required to destroy sacbrood virus | Resistance of sacbrood virus to carbolic acid... 44
when suspended in water......-..-...-..... 34 | Modes of transmission of sacbrood...........-. 46
Heating required to destroy sacbrood virus Diagnosis of sacbrood.......-......---...ee005 48
when suspended in glycerine................ BO | PPORMOSIS soccsskis comands 2: ence eres meamaee 49
Heating required to destroy sacbrood virus Relation of these studies to the treatment of
when suspended in honey.......-........--. 36 SACDEOOG sais isi53 <icwioeieibee slasisisisjereaieisiseniincieie ence 50
Resistance of sacbrood virus to drying at room Summary and conclusions... 52
temperature ......02.- een e cece eens Lisiseters 37 | Literature cited. .......cccceec cence en cnnecence 53
INTRODUCTION.
Sacbrood is an infectious 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 malignant.
nature and somewhat transient in its character.

It is insidious in its
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 in which
the disease is present, a fact which makes the disease one of great
economic importance.

58574°—Bull, 481—17-—1

Missing Page

SACBROOD. . 3

It will be noted that Jones also recognized that there was a disease
that resembled somewhat the genuine foul brodd, 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.

Simmins (1887), writing from Rottingdean, England, points out
the difference between ‘‘deadbrood’’ and foulbrood:

That foul brood is often confused with simple dead brood I am well aware. co.

But that every bee 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.

Tn addition to emphasizing the difference between ‘‘deadbrood”
and ‘‘foulbrood,” Simmins says that these two diseases are in turn to
be differentiated from chilled brood. He adds the additional fact
also that Cheshire had examined 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 in 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 diseaséd condition in their apiaries
which they are diagnosing as “pickled brood.’’ The larve 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 secede 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 occurred in the older larva, and showed a gray to blackish colora-

4 BULLETIN 431, U. S, DEPARTMENT OF AGRICULTURE,

tion, partially drying the larve until mummified, These larve 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
examined by Burri and himself 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, Kursteiner 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 unlike 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 little 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 larves
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. Itis only the knowledge concerning the disease and its name
that is of recent origin. It is far better, and in all probability 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 unawares 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 knowing of it hope may be entertained 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 William R. Howard of Texas. The
condition which he described under this term he declared was caused

SACBROOD. 5

by a fungus to which he gave the name Aspergillus pollini. In a
second article (1898) he writes that pupe and adult bees, as well as
the larve, are attacked by the disease, stating his belief that the
disease in adult bees had been diagnosed as paralysis. Technically,
therefore, the term “pickled brood’’ refers to an infectious disorder _
of bees affecting 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 beekeepers 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
likely included under the term “pickled brood’’ as it is popularly
used. Brood dead of starvation and that found 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?’ In the replies
received more than one disease was sometimes suggested as being
suspected. Out of 189 replies received from beekeepers sending
samples of sacbrood, European foulbrood was suggested in 55 replies,
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 natural to suppose that sacbrood would have been one of the
conditions occasionally referred to under the term “pickled brood.”’

6 BULLETIN 431, U. 8, DEPARTMENT OF AGRICULTURE.

As sacbrood has been proved, however, to be a distinct disease and
different from all other disorders, naturally it is incorrect to use the
terms ‘‘sacbrood”’ and ‘pickled brood” synonymously, either in 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 willbegiven. In thisdescription
the same method will be used and simi-
lar terms employed as will be found in
the description of the symptoms of the
disease.

Tt 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 all cells in the un-
oe ea oe angi capped areas will be without caps while
wall (L),and the wrinkling of theinnersur- practically all in the capped areas will
face of the cell near the opening, indicating bec apped.

the presence of a mass of cocoons (C), are ‘ :
shown. Enlarged about 8 diameters. Since the brood that dies of sac-

Coneinah) 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. (Thecaphas been removed by the beesfrom
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
(A,) 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
“tpickled 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.” , (

SAOBROOD. 7

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 cell 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 will be referred to as the lateral
walls (fig. 1, L). The bottom is concave on the inside. The cap

SN
WN

DAASEES

,
a
7
7
a:
4
>
2
2
7
7

Fie. 3.—End view of cell capped. The 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
Fic. 2.—Empty arimee cell cut in half along it is found to be less convex and

the long axis of the cell, showing cocoons (C) somewhat irregular . The cap should

ee areca Sepae remain normally for the most part

two walls which paseo floor (F) of entire (fig. 8). While this is the rule,

the cell. Enlarged about 8 diameters. there are exceptions toit. The bee-

Qian keeper is familiar with the appear-
ance which suggests that it had not been entirely completed (fig. 11;
Pl. II, 6).

The long axis of the cell is nearly horizontal, the bottom of the cell
being normally only slightly 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 cies of sacbrood is found.

8 BULLETIN 431, U. 8, DEPARTMENT OF AGRICULTURE.
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 larve dead of the disease. As an aid in interpreting the
description of these appearances a description of the healthy larve
is first made. i

Larve' that die of sacbrood do so almost invariably after capping
and at some time during the four days just preceding the change in
form of the maturing bee to that of a true pupa.

During the first 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-

AT MT PT

Fic. 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.) 7

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 larve die of sacbrood during the first two days
cr 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 larve 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,

1 As beekeepers usually refer to the brood at this age as “larve,” the term is used here to designate the
developing bee at this stage of its growth.

SACBROOD. 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.
Tts 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 larve at this
age might be thought of as white, they
are in fact more or less bluish white in
color. Itis 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 thirds (PT).

Anterior third.—On removing the cap
from a ‘cell the anterior cone-shaped See :
portion of the larva is seen (fig. 5; Pl. ae aoe ee Can Ie ae ee

1 wes torn and turned aside with forceps. En-
ft @) ay pee oe paler ce _larged about 8 diameters. Original.)
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; Pl. IT, 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 margins. In some of the segments a trans-
verse trachea may be seen appearing as a very fine, scarcely per-

58574°—Bull. 431—17-—2

10 BULLETIN 431, U. 8, DEPARTMENT OF AGRICULTURE.

ceptible, white line. Sometimes there may be seen a narrow area
along the median line of the ventral surface 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 similar
in appearance to the one on the dorsal side, but less distinct. A cross
section of this third is slightly elliptical in outline. The middle third
passes more or less gradually into the posterior third. The juncture
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 surface 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.

TISSUES OF A HEALTHY LARVA AT THE AGE
AT WHICH IT DIES OF SACBROOD.

—

We
i
\

SSSSSss

Upon removing a larva in the late
larval stage and puncturing its body
wall lightly, a clear fluid almost
water-like in appearance flows out.
This fluid consists chiefly of larval
blood. By heating it, or by treat-
ing it with any one of a number
__ Fig. pears Bae pees from 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 primarily in the

SACBROOD. 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.
A symptom of disease manifested by an individual bee, broadly con-
sidered, is, in fact, alsoacolonysymptom. 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 larvz dead of the disease.
With sacbrood thus produced in ex-
perimental colonies the symptoms of
the disease have been studied, and the
description 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 OBSERVED FROM A CASUAL
EXAMINATION, Fic. 7.—Larva dead of sacbrood lying in the

7 cell as viewed from above and at an angle.
The presence of dead brood is usually — tt may have been dead a month. Cap of

the first symptom observed. Anirreg- — Guiremoved Oy Tees. Tnlarged sbout &
ularity in the appearance of the brood , ;

nest (PI. I, figs. 1 and 2; Pl. 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
naturally 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. 8, DEPARTMENT OF AGRICULTURE.

stage is reached. It is rare to find a pupa dead of sacbrood (Pl. I,
zz). The larve: 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 in cells which
have been uncapped (fig. 9), as bees
often remove the caps from cells
containing dead larve. Caps that
are not removed are more often en-
tire, yet not infrequently they are
found to have been punctured by
the bees. Usually only one puncture
is found in a cap (Pl. II, d), but
there may be two (fig. 10) or even
more (Pl. II,f). The punctures vary
in size, sometimes approximating
that of a pinhead, although usually
smaller, and are often irregular in

Pu, 8nd view of npotcll ic ouéline, Sometimes a cap (fig. 11,
lar to the one shown in figure 9. The cap Pl. II, b) has a hole through it which
pene sani ters, (Oniba om suggests by its position and uniform
circumference that it has never been

completed. Through such an opening (fig. 11; Pl. IT, e) or through
one of the larger punctures the dead larva may be seen within the cell.

A larva recently dead of sacbrood is slightly yellow. Thecolorina
few days changes to brown. Theshade
deepens as the process of decay con-
tinues, until 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, dufing 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 saclike remains, dur-
ing @ certain period of its decay, is ,. Bae rene ee
watery and granular in appearance. larva dead of sacbrood. The sage th
Much of the time the form of the oe the same as in figure 8.
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

PLATE I.

@' ae “elas |

Bul. 431, U.S, Dept. of Agriculture.

7 See <So..0%e

Te ee ia
& Meg’ Feted<8a%9's

ae

(ORIGINAL.)

Fic. 1.—MARKED SACBROOD INFECTION. SIZE SLIGHTLY LESS THAN NATURAL.

OF DECAY OF LARVA. Eaas, YOUNG LARVA IN DIFFERENT STAGES OF DEVELOP-

MENT, AND DISEASED LARVA: IN SAME AREA. NATURAL SIZE. (ORIGINAL.)
. SACBROOD PRODUCED BY EXPERIMENTAL INOCULATION.

Fie. 2.—HeAvy SACBROOD INFECTION, SHOWING A NUMBER OF DIFFERENT STAGES

Bul. 431, U. S, Dept. of Agriculture. PLaTE Il.

ComPARISON OF A HEALTHY LARVA AND THE REMAINS OF LARVZ DEAD OF
SACBROOD.

a, A cap of a healthy larva; 6,¢,d,e,and f, caps over larvex in first, second, third, fourth, and
fifth stages of decay, respectively; g, a healthy larva, end view; h, 7, j, k, and/,an end view
of the five stages of decay; m, a healthy larva viewed from above; N, 0, p, gq, and 7, cor-
responding view of the five stages of decay; sand y, healthy larva removed from the
cell; ¢, u, v, w, and z, larval remains in different stages of decay removed from the cell;
ww, @ larva recently dead of sacbrood with the anterior third removed by the bees; x, a
scale removed from the cell; 2a, larval remains from which a small portion has been
removed by bees: yy, almost a pupa; 2z, a pupa dead of sacbrood which had only recently
transformed. (Original.) :

SACBROOD., 18

“scale” (figs. 22; 23; Pl. II, 1,7, and zx). This scale is not adherent
to the cell wall. :

In sacbrood the brood éombs may be said to have no odor. Larve
undergoing 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 larve dead
of the disease, however, will make it
possible to avoid any such confusion.
‘A more -.careful study of the dead
larve is therefore justified. e4

iy,

APPEARANCE OF LARVA! DEAD OF SACBROOD. [Fic. 10.—Cap of cell containing the remains
. ; P of a larva dead of sacbrood. The cap is
No signs in a larva dying of sac- slightly sunkenand bears two perforations

brood have yet been discovered by ™2d°Py the Pees. COriginal.)

which the exact time of death may be determined. As the larve 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. In 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
i N 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-
ss I acareese cenetanenn. mie scription that would be correct for

pecans "a never huviee been com- 4 dead larva on one day, there-
pleted. (Original.) fore, may and probably would be
incorrect for the same larva on the following day. Moreover, all
larve 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 continual change in appear-
ance is here considered in five more or less arbitrary stages. As the

Wp, iN ;
as

= SS ~
ISS

N WY lige =

14 BULLETIN 431, U. S. DEPARTMENT OF AGRICULTURE,

same plan will be followed and similar terms will be used in describing
these stages as were employed in the description of a healthy larva
of the same age, the interpretation of the description will be aided
if the appearance of a healthy larva as described above is borne in
mind.
First Stace.

Uncapping a larva showing the first symptoms of the disease, it

will be observed that it has assumed a slightly yellowish appearance.

Fig. 12.—First stage: Larva showing first
symptoms of sacbrood and presenting the
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 i
and extreme cephalic end of the an-
terior third (fig. 12; Pl. II, 6, h) may f ;
have assumed, and frequently do as-

SHS, SOE Or less transparent oP yaa, 13.—First stage: Ventral view of larva
pearance (represented in the figure by dead of sacbrood as seen from above and at
shading). The position and the sur- fi0;2>,sivite a ventral view of all three
face markings of the anterior third are SSE re ne ee eee
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-like 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-

SACBROOD. 15

verse trachese 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 still 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; Pl. II, ww) dead
of sacbrood is found in the cell. Such remnants vary in size. The

Fie. 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.) :

Fia. 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 in
evidence. By microscopic examination the granular appearance is
found to be due chiefly to fat cells.

Condition of the virus in the first stage.—When larve of this stage
are crushed, suspended in sirup, and fed to healthy bees, a large

16 BULLETIN 431, U. §. DEPARTMENT OF AGRICULTURE.

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.

SEconpD 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

Fic. 17.—Third stage: Dorsal view of an-
terior third 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; PL. II, 7) ag arule 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
little 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 slightly from that observed in the preceding
one. The apex is farther from the roof of the cell (Pl. II, 2). The
deflection is more marked and is seen in a greater number of larva.
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. (Original.)

SACBROOD. 17

The ventral surface of these two thirds (fig. 16, Pl. II, 0) is less con-
vex from side to side. The ridges and furrows, representing the seg-
ments, are less pronounced. The lateral margins are still deeply
notched. The prominent angle seen on the ventral side of a healthy
larva, at the juncture 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-
vee 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 larve at this stage of decay are
still in some instances infectious. The
amount of infection produced when
such larve are used in making in-
oculations is very much less, how-
ever, than when larve in the first
stage are used.

Tarrp Sraae.

The color of the dead larva of this
stage is quite brown, that of the an- Fie. 18.—Third stage: Larva dead of sacbrood,
terior third being a deeper shade than en ee
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 brown.
This third still preserves its conelike form (figs. 17 and 9; Pl. II, 4),
the distance of the apex from the roof of the cell 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.

§8574°—Bull. 431—17—8

18 BULLETIN 431, U. S, DEPARTMENT OF AGRICULTURE.

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 lighter. The ventral surface of this third (figs. 18
and 7) is less convex from side to side than in the preceding stage,
and the segmental markings, while still 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;
Pl. 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 still 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-

Kg

te

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
still maintains, in a general way, the form and markings of the normal
larva (fig. 4). The turgidity is gone, although the position in the
cell 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 little 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 larve at this stage

SACBROOD. 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.

Fourtra Srace.

The brown color of the larval remains has further deepened, the
anterior third 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 conelike third

Fig. 20.—Fourth stage: Remains of larva
dead of sacbrood. (Original.)

is often nearer the roof of the cell in
‘this stage than in the preceding one.
As aresult 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, &) of decay almost obliter-
ated through the wrinkling of the
surface, due to drying.

Middle third.—This third ig de- Fig. 21.—Fourth stage: Remains of Jarva dead
éide dly brown, but lighter fa sbnde of sacbrood, ventral view. (Original. )
than the anterior third. The ventral surface (fig. 21; Pl. 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 (Pl. II, g) may or may not be
darker than the middle third, but it-is not darker than the anterior

20 BULLETIN 431, U. S. DEPARTMENT OF AGRICULTURE.

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 remains, but are much less pronounced.
The subcuticular fluid is no longer in evidence.

Consistency of the contents of the sac.—Upon tearing the sac, the
contents are found to be less fluid than in preceding stages. The
decaying tissue mass is still granular in appearance. As the drying

Fig. 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 fourth stage do
not seem to be infectious.

Firrn Stace.

Fig. 23.—Fifth stage: Scale, or larval remains,
The dead larva in this last sta: ge in sacbrood viewed at an angle from above.

: (Original.)
has lost by evaporation all of its coi

moisture, leaving the dry, mummylike remains known as the ‘‘scale.”

Anterior third.—The anterior third (fig. 22; Pl. II, 1) through dry-
ing is retracted from the mouth of the cell, with the apex drawn still
deeper into the cell 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 (fig. 23; Pl. II, 7), is deeply
concave from side to side and may show remnants of the segmental
markings of the larva. The surface is often roughened through
drying. Sometimes both longitudinal and transverse trachew are

SACBROOD. 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 roughened, and directed somewhat forward.
‘This margin, like that of the middle third, has a tendency toward
being irregular. :

The scale.—The scale can easily be removed intact from the cell.
(Pl. TI, x.) 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

| iF

UittitionionuiaHiMesunaosvoutll!Z

Yi fj YY WWW
1 Dp | { TIA .
ssi il gee
| ee, | | i Zz
| A RO ZB
\ a RT ANAM MN aa) ZG

4

Fic. 24.—Scale, or larval remains, in position in cell cut 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 boatlike 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 larve 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, U. 8, DEPARTMENT OF AGRICULTURE.

through the stage described above as the third stage (figs. 7, 9,
17, and 18; Pl. Ij, p). That the dead larve are allowed to remain
in the cells often for weeks is in part the cause of the irregularity ob-
served in the appearance of the brood combs (p. 11). (Pls. I, IV.)

APPEARANCE OF THE TISSUES OF A LARVA DEAD OF SACBROOD.

The gross appearance of a larva during its decay after death from
sacbrood has just been described. The saclike appearance of 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 cells (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 larve dead of sacbrood. This
appearance has often been observed by beekeepers and is a well-
recognized symptom of sacbrood.

In the section (A) may be seen a molt skin (C,), which is at a con-
siderable distance from the hypodermis (Hyp). Another cuticula
(C,) is already quite well formed and lies near the hypodermis. Be-
tween these two cuticule (C, and C,) during the earlier stages of
decay there is a considerable space (‘intercuticular 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 coagulum is formed in it
during the preparation of the tissues for study, is shown by the
presence of a coagulum in the sections.

The body (B, A) wall of the larva is composed of the cuticula (C,),
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 cenocytes (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
cenocytes in the prepared tissues are irregular in outline, 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. 93

Fi. 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 skin (C2), a newly formed cuticula (C;), the hypodermis (Hyp), the stomach (St), the ovaries (Ov),
the heart (Ht), the ventral nerve cord (VNC), the dorsal diaphragm (DDph), tracheze (Tra), ceno-
cytes (Oe), and fat cells (FC). Between the cuticula C2 and the cuticula C; is a considerable intercu-
ticular space (IS). B repfesents the body wall in this pathological condition, showing the cuticula Cs
and the cuticula C1, both bearing spines (SC, and SC), and the intercuticular 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).
D, cenocyte with uniformly staining cytoplasm, and with a nucleus (Nu) having a uniform outline.
E, 2 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, U. 8. DEPARTMENT OF AGRICULTURE.

The molt skin (C,) is probably the one that is shed normally about
three days after the larva is capped. The cuticula (C,), 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 (C,) constitutes for
the most part the sac which is seen to inclose the decaying larval
mass in sacbrood, the cuticula (C,) probably assisting somewhat
at times. The presence of the subcuticular fluid is made more intelli-
gible by these facts. Larve 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 larvee, 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 will
be found that the granular appearance is due for the most part to
fat cells suspended in a liquid. The liquid 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 liquefaction 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.

Doolittle (1881), Jones (1883), Simmins (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:

Whatever 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.

SACBROOD, 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 foulbroods, 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. Pupe are rarely affected (Pl.
II, zz). If one succumbs to the disease, it is quite soon after trans-
formation from the larval stage. Primarily it is the larve 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 larve may become infected. Queen larve
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
replies received from those sending sacbrood samples,’ 53 reported
hybrids, 49 reported Italians, 21 reported blacks, and 17 reported
Italian hybrids. These replies show that the bees commonly kept by
American beekeepers are susceptible, although their relative suscepti-
bility is not shown.

The bees which have been inoculated in the experimental work
on sacbrood have been largely Italians or mixed with Italian 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. is :

Climate.—Historial evidence strongly suggests that sacbrood is
found in Germany (Langstroth, 1857), England (Simmins, 1887),

58574°—Bull. 431—17-—4

26 BULLETIN 431, U. 8. DEPARTMENT OF AGRICULTURE.

and Switzerland (Burri, 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
climates or the coldest climates in which bees are kept has not yet
been completely established.

The mountains and coast plain of the eastern United States, the
plains of the Mississippi Valley 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 nature 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 climate 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 climatic 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 quality 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 vaty 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

SACBROOD. 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 réle 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 SACBROOD.

That sacbrood is an infectious disease was demonstrated by the
writer (1913) through experiments performed during the summer of
1912. This was done by feeding to healthy colonies the crushed
tissues of larvee 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.

Tn 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: Larve
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 still 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 found to be apparently free from such microorganisms, a quan-
tity of it may be added to sirup and the mixture fed to healthy colo-

1The Berkefeld filter consists of a compact material (infusorial earth) in the form of acylinder. A glass
mantel (A) in which is fixed the filter forms a cup for holding the fiuid to be filtered. Having filtered
the aqueous suspension of crushed sacbrood Jarvee through paper, the filtrate is then filtered by allowing
it to pass through the walls of the Berkefeld cylinder (B). The filtrate from this filtration is collected
into asterile flask (F) through a glass mre (D) with itsrubber connection (C). In filtering in this instance
gravity is the only force used.

28 BULLETIN 481, U. 8, DEPARTMENT OF AGRICULTURE.

nies. When all this is properly done, sachrood will appear in the
inoculated colonies. This shows that the virus ' of this disease, to a

Fig. 26,—Berkefeld filter (B) with 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” of

“parasite.” In relation to the disease, however, its meaning is the same as that conveyed by the latter
terms,

SACBROOD. 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 larve dead of the disease
is prepared as before. This is filtered by the aid of pressure obtained

CNA

\

Fic. 27.—A convenient apparatus which can be employed in using the Pasteur-Chamberland,
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 sterile
ized flask, a glass stopcock (h), a vacuum gauge (i), @ reservoir (m) with pressure-rubber connections
(j), 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 will pass through

1The 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. 8, DEPARTMENT OF AGRICULTURE.

the pores of the Pasteur-Chamberland filter also, it is therefore fil-
terable and is very properly referred to as a ‘‘filterable’”* virus.

In considering 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 whieh will pass through the
pores of fine clay filters. This conception of it will at least make it
more easily understood.

WEAKENING EFFECT OF SACBROOD UPON A COLONY.

The first inoculations in proving that sacbrood 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 amount 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 dying 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 all instances have shown a tendency to become
weakened as a result of the inoculations.

The death of the worker larve 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 larve 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.

1In searching the tissues of larvee 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 “Caltramicroscopie virus.” It
is preferable, for the present, however, to refer to it simply as a filterable virus. :
2 There is some question whether, in the case of diseases having a virus which is filterable,

: ; e : the infecting
agent is in every instance a microorganism. The evidence is strong, however, that it is.

SACBROOD. 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 will produce the disease in every instance, or in any
instance, isnot known. If the disease does result at any time from the
ingestion of a single germ, all 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 number of
sacbrood germs ingested by a larva 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 death of at least 3,000 larve. Starting then with the
virus contained in.a single larva, in less than one week it would
easily be possible to have 3,000 larve 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 sufficient to produce an equal
amount of infection in 3,000 colonies, increasing 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. Carrying 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, U. 8S. DEPARTMENT OF AGRICULTURE.
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
‘noculations of bees the methods used have undergone revision from

time to time. Those

2 ee now employed have

a 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

Spiral was found to serve
i et er ee OT Ss rety staatiolonly
frames, a division board, four open Petri dishes as feeders, and the en- the purpose of an ex-
trance nearly closed with wire cloth, the opening being on the side :
of the hive body.occupied by the colony. The dimensions indicated perimental colon y-
are approximate. The angle at which the hive was photographed The queen should al-
for this drawing caused its length toappear foreshortened. (Original.) ways be clippe d. 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 oceupied 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 byframes. The hives
of the experimental apiary Fie. 29—Potridish. The top half is slightly raised. Those
(Pl. IIT) are arrange d used here are 4 inches in diameter. (Original.)
chiefly in pairs, with the entrances of consecutive rows pointing in
opposite directions. The space occupied by the apiary should be

un

ai
|
&
NM
A i &
a je

Li
dees

Pw eee

te

uamenreansey

v

La

1A 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 halvesare placed
together.

(IVNIDINO)

“GALONGNOD 3YSM GIG] 4O YAWWNS 3H Q S0VW SLNAWIYSdXy NOILVINOON| SHL HOIHM NI SSINOIOD $G JO AYVIdY IWLNAWIYSdXq SHL JO MIA V

PLATE III.

£
3
a=
3
2
=
i)
=<
3s
Bb
o
[=]
7)
3
—
«9
vt
3
a

Bul. 431, U. S. Dept. of Agriculture, PLATE IV.

UCED BY FEEDING THE VIRUS OF THE

DISEASE, (ORIGINAL.)

BrRooD FRAME GONTAINING SACBROOD, TAKEN FROM AN EXPERIMENTAL COLONY IN WHICH THE DISEASE was Prop

SACBROOD. 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,
larvee 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 larve, 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 adearthof 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 aforeign location. An experimental colony when
inoculated should have larve 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
larve having a slightly yellowish hue (fig. 12; Pl. II,
b, 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 circumstances is desirable. The method
is more direct than the one just described. The

crushed tissues of a diseased larva are suspended in a re gy Gard

small amount of water or thin sugar sirup. With a drawn to “capie

lary size at one

capillary pipette (fig. 30) made from small glass tubing, — end. “Reduced to
a very small amount of the suspension is added di- three oe at
rectly to the food which surrounds the healthy larva "8"

in the cell. Thisis easily done. Having drawn some of the suspen-
sion into the pipette, carefully touch the food in the cell surround-
ing the larva with the point of the pipette. A small amount of the
suspension will flow out and mix with the food. Larve approxi-

mately two days of age should be selected for feeding. A dozen

mS

34 BULLETIN 431, U. 8, 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 VIRUS OF SACBROOD.

Although the virus of sacbrood may increase with great rapidity,
fortunately it is quite as readily destroyed. Nature supplies 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
natural 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 larve 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.—Efect of heating on the virus of sacbrood suspended in water.)

: * Time of ‘ .
Date of inoculation. Temperature. heating. Results of inoculation.
oF. °C. Minutes. :
122 50 30 | Sacbrood produced.
131 55 10 Do.
131 55 20 Do.
135 57 15 Do.
136 58 10 Do.
136 58 | 10 | No disease produced.
138 59 10 Do,
1 60 15 Do.
142 61 10 Do
149 65 15 Do.
158 70 15 Do
167 75 15 Do.
176 80 15 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 result, 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 larve 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 caliber 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. After 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.

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 afew inoculation experiments are given here
in which the virus was heated while suspended in
glycerin.

Tae I].—Hfect produced by heating the virus of sacbrood suspended

in glycerine.
i ‘ Time of eae
Date of inoculation. Temperature. heating. Results of inoculation.
°F. °C. | Minutes.
140 60 10 | Sacbrood produced.
149 65 10 Do.
158 70 10 Do.
160 71 10 Do.
163 73 10 | No disease produced.
167 75 10 Do. 2

Fig. 31,—Tube in which a suspension of sacbrood larve is placed for heating. It consists of a test tube one-half inch in diameter supplied with a perforated stopper

oo
On

through which passes a short piece of glass tubing drawn at one end to capillary size. (Original.)

86 BULLETIN 431, U. 8. DEPARTMENT OF AGRICULTURE.

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 sufficient 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 in
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 II].—Results obtained when the virus of sacbrood was heated in honey.

3 : Time of . ;
Date of inoculation. Temperature. heating. Results of inoculation.
oF, °C. | Minutes.
PUNE, LOUD Sc coerce tesnsmaees saunas axe eeeted 140 60 10 | Sacbrood produced.
June 11, 1915... ate one 145 43 10 Do.
D 149 65 10 Do.
154) 68 10 Do.
156 69 10 Do,
158 70 10 Do.
158 70 10 | No disease produced.
158 70 10 Do.
160 71 10 Do.
Ss 160 7 10 Do.
5 1 73 10 Do.
. 28, 163 2B 10 Do
June 1, 1915... ‘ 167 75 10 Do
‘Aug. 7, 1915... CAS the 167 15 10 Do.
DONG LOLS 6 orca ecee dethrone aiasie deat tenia | 176 80 10 Do.

As shown by the results recorded in Table III, the virus of sacbrood
when suspended in 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.

SACBROOD, 87
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, larve 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 larve. 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 larval content con-
tained in a Petri dish. ia. 32.—Open Petri dish. One-half of Petri dish, either
This is added to sirup, and ns ee deers
the sirup suspension is fed to a healthy colony. The experiments
gave the following results:

i}
Tasie IV.—Resistance of sacbrood virus to drying at room .temperature.

Date of inoculation. Time of drying. Results of inoculation.

...-| Sacbrood produced.
, 0.

Oct.9, 1914.0... 000s sees eee sere esses ees.) 28 days. clile. Ll} No.
July 29, 1915... sag : nate i
Sept, 3, 1915... ----| 85 days....... meee Do.
:11] 7 months 12 days.....211! :
[5 anek een ewarpsr ater Oconee eraet ow eur te 7 months 21 days......... 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.

388 BULLETIN 431, U. S. DEPARTMENT OF AGRICULTURE.

Similar preliminary 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.

Preliminary experiments indicate also that when the virus is mixed
with pollen and allowed to dry the period for which it remains virulent
is increased 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:

TasLe V.—Resistance of the virus of sacbrood, when dry, to direct sunlight.

Time of
Date of inoculation. Se. Results of inoculation.
jays.
Hours.
Sue DENIS ic tcc di ak durian wegen tans DESAI SEAR AE 2 | Sacbrood produced.
July 20, 1018. occ ccec at vicc caceauncnensecsaceacaad+onbe tates gee 3 23 Do.
OD GET, MOND ecserc Avec eatare ybremrtias aecsaisine arclasaraistaswiele sieitleticice 3 | Do.
Sept. 16, 1915 4 Do.
IS pesos asa. aati hig RN Sitsip ein ai nei Scbetes 5 Do.
DO er srcate scepeiere 2eaig eicraratane aya ain jwecre eeaneeraee! atadectoen nines clersicea 6 Do.
Aug, 25,1915... 6 Do.
Bent 10) OS eee seman uo eye nga ares eee Senremae 4 | No disease produced.
DO! ge siccuiswiana uence at sete eawenen annie bums wreaey 5 Do.
Sept. 9, 1915. 73 Do.
DO vajascsane sie 9 Do.
Aug. 19, 1915........-..- = 12 Do.
July 16, 1915............ = 13 Do.
PUIG 20) LOL Gs 5. cre ocarorctacarateiss Sere nevencstary wat thearasie se see dee meee cane 18 Do.
OPt ed Ly DOG weiccreeisiscggtsatierescem a sit ete mae aisinladaeisene ewer ae 21 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 will 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

SACBROOD. 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 14 ounces of
the aqueous suspension containing the crushed tissues of 30 larve 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.

Time of
Date of inoculation. la Results of inoculation.
rays.

. Hours. ;
Sept: 10) 1015 pic's )sctreasireenaenemeeuvesmasennaiee gemeuenio: sees 1 | Sacbrood produced.
Aug. 20, 1915... 2 Do.

Sept. 14, 1915 2 Do.
Aug. 24, 1915 2 Do.
Aug. 18, 1915 3 Do.
Sept. 9, 1915. 4 Do.
Sept. b, 1915... 4 Do.
es 4, 1915... 5 Do.
neues siaiocin 4 | No disease produced
pees 16, 1915... 5 Do.
Sept. 8, 1915. 5 Do.
DO sects 6 Do.
Bert e 1915... 7 Do.
is mintentnats 8 Do.
ae 25, 1915 10 Do.
Aug. 20; 1915 12. Do.
July 13, 1915. 1B Do.
Aug. 26, DONS scriatera seeien sient cians Slee och ei edielt aecesiste cree sa tunics 13 Do.

From Table VI it will be seen that when suspended in water the
virus of sacbrood was killed in from four to six hours.

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 nature 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 eed:

40 BULLETIN 431, U. S. DEPARTMENT OF AGRICULTURE.

RESISTANCE OF SACBROOD VIRUS TO DIRECT SUNLIGHT WHEN SUS-
PENDED IN HONEY.

The crushed and strained tissue mass of larvee dead of sacbrood
was suspended 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.

Time of |
Date of inoculation. ee Results of inoculation.
jays.
Hours. .
Aug. oe 1915 Bankes produced.
Sete re 0.
Aug. 1S. 1915 4 Do.
Sept. 9, NDIB estos eee 4 Do.
Sept. 10, 1915 4 Do.
Aug. 24, 1915 5 Do.
Aug. 16, 1915 5 | No disease produced.
ug. 25, 1915 5 Do.
Sept. 8, "1915.0... 5 Do.
‘DO.....-- 6 Do.
Rept 4 1915... ve Do.
Ss eeisesia8 8 Do.
Aue 25, 1915... 10 Do.
Sept. it, 1915... 12 Do.
Aug. 26, 71915... 13 Do.
Sept. i, 1915..... 18 Do.

From the results of the experiments recorded in Table VII it will
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.

Tn devising methods for the treatment of sacbrood it is of particular
interest to know the length of time that the virus will remain virulent
when it isin honey. Experiments have been made to gain data on
this point. Larve recently dead of sacbrood are crushed, strained,
and suspended in honey. About one-half pint of the suspension,
representing the virus from about 30 dead larve, 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.

Es

SACBROOD. 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.

Time virus :
Date of inoculation. was in Results of inoculation.
honey.
Mos. ae
POPO TT scree c.u ees awreeuner wea anuars ox teuaares teak es - 0 10 eee pene
Tune 4, MOTB rece e oat tate sie tulsa te een beonn 0 28
Oct. 2, 1915. sone 0 630 De
BOP be 8p 1915 sc cnwwinns vediecimeane weit cae 0 24] No disease produced.
GUY 20 LOY acc asctiseotrasc cage loaierreiey tere 0 Do.
June 80, 1915......2 202s eee eee eee 0 33 Do.
DO eats cciemenarns ayseetinnssais canes 0 35 Do.
Tuly 17, 1915... 0 36 Do.
Oct. 21; 1915. 0 49 Do.
Sept. 3) 1915 0 70 Do.
May 13, 1915, 17 10 Do.
May 6, 1915. 7 20 Do.
May 4; MOTB Go ooefs:ctcterent re hice BCA eet ak eo Sa armies ab Atee. 8 Do.
May 18 MOUG oe ccesccesccsnivs cueeeedc weet wens asimeee se mceciea: 8 al Do.
Sept. 3, 1915...............0.. i atrtueguum anne bases ean atundoe 122 «1 Do.

1 The dead brown larval remains were not crushed 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 temperature.

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 virus does not receive any marked
amount of protection by being in honey. From the dates of the
experiments in this group it will 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 larve recently dead of the disease is
suspended in 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 microorgarisms, the sugars in honey being naturally the carbohydrates especially of interest
in these discussions. The process results in the formation of a large number of substances—acids, alcohols,
ete. 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 going on is
usually distinctly offensive.

¢

42 BULLETIN 431, U. 8, DEPARTMENT OF AGRICULTURE.

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 larve, These suspensions are

: allowed to remain at room temperature, shielded
from the light. 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:

Taste TX.—Resistance of sacbrood virus to fermentation in a 10
per cent sugar solution at room temperature.

(ae => Period of
Date of inoculation. fermen- Results of inoculation.
' tation.
Days.
PO Os TD sya ices atte vip sscw waomininiel? 1 | Sachrood produced.

Sept. 11, 1915. a 2 Do.
D 3 Do.
4 Do.

3 | No disease produced.
5 Do.
5 Do.
Hf Do.
9 Do.
13 Do.
34 Do.
51 Do.
85 Do.
87 Do.
90 Do.
244 Do.

1Theresultsrecorded for 1914 were obtained with a suspension of crushed larve,
in various stages of decay, in sirup made from about equal parts Water and sugar.
From the results of experiments recorded in Table
TX it will be noted that the virus of sacbrood was
destroyed in from three to five days in the presence
of fermentation in 10 per cent canesugar (saccharose)
| at room temperature.

J As the rapidity of fermentative processes varies
with the temperature present, it is natural to sup-
Fig, 33.—Test tube hi hi : ‘red f a ° f
bearing acotton plug, Pose that the time required for the destruction o
used in testing the ef? ¢he virus will vary. From experiments it is found

fect of fermentation, . : 2 .
putrefaction, and dis) that at incubator temperature the time is slightly
infecting agents on less, and at outdoor temperature it is somewhat

the virus of sacbrood.
(Original.) 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

1 This test is applied in the following manner: Water is added to honey until an egg placed in the mixture
nearly submerged, the surface remaining above the liquid being only about as large as a 10-cent piece.

SACBROOD. 43

four volumes of water to one of ripened honey to obtain the strength
recommended. The honey solution by volume, 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 ina 20 per cent honey
solution at outdoor temperature.

Time of
Date of inoculation. fermen- Results of inoculation.
tation.

Days.
E Bane ocd produced.

Do.
No disease produced.
0.

Sansone
is}
5

>

In the presence of fermentative processes taking place in a 20 per
cent honey solution at outdoor temperature it will 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.

Larve containing the virus of sacbrood are crushed and suspended
in water. A small quantity of soil is added. The suspension is
strained 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 inoculated, 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, U. S, DEPARTMENT OF AGRICULTURE.

TaBLy XI.—Resistance of sacbrood virus to putrefaction.

Time of 7 7
Date of inoculation. putrefac-| Results of inoculation.
tion. |
Days.
1 | Sacbrood produced.
2 Do.
3 Do.
3 Do.
4 Do. ‘
5 Do,
5 Do. .
7 Do.
9 Do.
7 | No disease produced.
10 Do. |
14 Do.
14 Do.
16 Do.

From Table XI it will be noted that the virus of sacbrood was
destroyed in 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.

Larve recently dead of sacbrood are crushed and strained. This
larval mass is diluted with carbolic acid in aqueous solution. About
10 parts of carbolic 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 larve.
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 4, 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.

Strength Time in
Date of inoculation. Pr a| Suspen- | Results of inoculation.
used. sion.
Per cent.| Days. ;
4 1 | Sachbrood produced.
4 16 Do.
4 24 Do.
4 38 | No disease produced.
pa 50 Do.
i] gs] be
0.
Sept. 3, 1914..... 1 1 | Sacbrood produced.
Sept. 18, 1914... be 16 Do.
June 23, 1915. 1 25 | Do.
Sept. 17, 1915 1 38 | No disease produced.
Aug. 12, 1915 1 50 Do.
‘Aug. 21, 1915 1 50 Do.
June 4, 1915... . 1 251 Do.

SACBROOD. 45

TasLe XII.—Resistance of sacbrood virus to car bolic acid—Continued.

Strength | time in

Date of inoculation.. of car. | suspen- | Results of inoculation.
bolic ad sion,

used. | :

Per cent. | ' Days.

2 1 | Sacbrood produced.
2 16 Do.
2 25 Do.
2 38 | No disease produced.
2 42 Do.
2 50 | Do.
a Hours
DUNG 23} VOU BD sscsccteer coe osetia ons Sein el Lol ae 4 3 | Sacbrood produced.
THUY hy SOM ec eae secon ord vd un ciReIRW Hoa abe hore eaee 4 7 Do.
Days.
FUNC: 28 OLE oon iwinaisae none eeceem eth eoneeceeeeees 4 25 | No disease produced.
AUG. 12, LOG ss casevcie cis cae scanners sue eeceieled ate ewomelgete 4 50 Do.

From the preliminary results recorded in Table XII it will 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 4, 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 carbolic 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 daily thereafter until 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 quinine 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 inoculation 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 carbolized honey was used gave
like results. These experiments, although not furnishing 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, DEPARTMENT OF AGRICULTURE.

Technically the foregoing studies should be thought of as being
preliminary. 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 necessary for the
destruction of the virus in practical apiculture by any of the fore-
going tables of results it should be emphasizéed 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 larve 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 living virus of the disease.

Bees have a tendency to remove diseased or dead larve 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 to 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 larve more or less directly in
this way.

Just what becomes of these bits of tissue removed from the dis-
eased larve, 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 larve 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 larve. If the infective material were fed to the older larve,
death probably would not result. Should it be used by adult bees as
food for themselves, the likelihood of the transmission of the dis-
ease under such circumstances would apparently be very materially
reduced. If the infective material were stored with the honey and

SACBROOD. 47

did not reach the brood within a month or six weeks, it is not prob-
able that the disease would be transmitted under such circumstances
(p. 41). Should the dead larve 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 infection 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 possibility that some of the living virus might be returned
to the hive and reach healthy young larve.

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 48), and if subjected to putrefaction, within two
weeks (p. 44). t

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 (Pl. III) 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 likelihood of the water supply
being a source of infection than flowers. The chances for infection

‘from this source, should it occur 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, U. 8, DEPARTMENT OF AGRICULTURE.

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. Rob-
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. Robbing, 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, will 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
larve in the late larval stage. Usually there are only a few of them,

‘yet sometimes there are many. These larve may be in capped or
uncapped cells. When found in uncapped cells, however, the cap-
pings had already been removed by the bees after the death of the
larvee. 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 larve 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 found 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

tively 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 larvee 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 larve. Cultures made from them are also neg-
ative.

Differential diagnosis.—Sacbrood must be differentiated from the
other brood diseases.

American foulbrood 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 Bacillus larve in the brood 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 larvee
as a rule die while coiled in the cell and before an endwise position is
assumed. Inthe majority of instances, therefore, death takes place
before the cells are capped. The saclike appearance characterizing
the dead larve in sacbrood is absent. The granular consistency of
the decaying mass is absent also. Microscopically, a large number of
bacteria are found in larve dead of European foulbrood, but are
absent in larve 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 ebe 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
larve dead from these conditions will be found to have died while
yet coiled in the cell. This fact suggests some condition other than
sacbrood. When dying later, the saclike remains characterizing sac-
brood are not present in conditions other than sacbrood.

PROGNOSIS.

The tendeney 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 AGRICULTURE.

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 larve, 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 earlier paper (White, 1908) contains a brief general discussion
of the relation existing 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-
ing 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 larve; 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 encountered in the spring; that it
- is found in localities differing widely as to food and climatic con-
ditions; and that no complete racial fimmunity to the disease has
yet been found are facts concerning the predisposing causes of sac-
brood which beekeepers will at once recognize as bearing a close rela-
tion to the methods by which the disease should be treated.

As sacbrood can not occur in the absence of its exciting cause
(a filterable virus), a knowledge of this cause is of special importance
in the treatment of the disease.

SACBROOD. 51

That sacbrood is very frequently encountered; 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 indeed destroy the colony are facts which must be considered in
devising logical methods for its treatment.

That the virus of sacbrood remains virulent in larve dead of the
disease for less than one month; that it remains virulent in 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 account 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 slight loss. This is
especially true after the early brood-rearing season of the year is
past. Since this can be done, it is quite probable that the practical
beekeeper will 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
direct 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 virus 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 in 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 entertained in practical apiculture that the disease
willbe transmitted by the hands 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 all times superfluous in the case of sacbrood
to flame or burn the inside of the hive or to treat the ground about a
hive containing an infected colony.

52 BULLETIN 431, U. S, DEPARTMENT: OF AGRICULTURE.

There is but little danger that the disease will be transmitted 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 beekeepers 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 récently 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 symptoms 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 localities 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 larvee 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 virus 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

SACBROOD. 53

be found at all times to lie somewhere between the limits of 131° F.
(55° C.) and 149° 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 dry 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
direct 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 during 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 4 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 effective.

(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 sufficient should be increased somewhat.

LITERATURE CITED.
(1) Bane, L.
1915. Sygdomme hos Honningbien og dens Yngel. Meddelelser fra den
Kgl. Veterinzer-og Landboh¢jskoles Serumlaboratorium, XXXVIT,
109 p., 11 fig.
(2) Beugye, F. R.
1913. Diseases of bees. In Jour. Dept. Agr. Victoria, v.11, pt. 8, p. 487-493,
4 fig.
(3) Burrr, R. .

1906. Bakteriologische Untersuchungen itber die Faulbrut und Sauerbrut
der Bienen. 40 p., 1 pl.,1 fig. Aaran, Switzerland.

54 BULLETIN 431, U. S. DEPARTMENT OF AGRICULTURE.

(4) Coox, A. J.
1904. The Bee-Keeper’s Guide or Manual of the Apiary. ed. 18, 543 p.,
295 fig. Chicago.
(5) DoourttLe, G. M.
1881. Foul brood. Jn Gleanings in Bee Culture, v. 9, no. 3, p. 118-119.
(6) Dapant, C. P.
1908, Diseases of Bees. Langstroth on the Hive and Honey Bee. 575 p.
(p. 487), 229 fig. Hamilton, Ill.
(7) Howarp, Wm. R.
1896. A new bee disease—pickled brood or white fungus. Jn Amer. Bee
Jour., v. 36, no. 37, p. 577, 6 fig.; also in ABC of Bee Culture, 1903,
p. 157-158.
1898. Pickled brood and bee paralysis. In Amer. Bee Jour., v. 38, no. 34,
p. 530-531.
(9) Jongs, A. D.
1883. Symptoms of foul brood. Jn The American Apiculturist, v. 1, no. 4,
p. 79-80.
(10) Ktiastemer, J.
1910. Zusammenstellung der Ergebnisse des vom Mai 1903 bis Dezember
1909 untersuchten, faulbrutverdaéchtigen. Wabenmaterials. In
Schweizerische Bienen-Zeitung, Yahrg. 33, no. 4, p. 187-189.
(11) Lanestrrota, 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. Jn Gleanings in Bee Culture,
v. 20, no. 15, p. 594-595.

(8)

(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) Smumins, S. ‘
1887. Foul brood, dead brood. Jn British Bee Jour., v. 15, no. 270, p. 371-
372; also in Canad. Bee Jour., v. 3, no. 28. p. 576-577.
(16) Warts, G. F. ;
1904. The further investigation of the diseases affecting the apiaries in the
State of New York. Jn 11th Ann. Rpt. Comr. Agr. N. Y., 1903, p.

103-114.
(17) :
1908. The relation of the etiology (cause) of bee diseases to the treatment.
U. 8. Dept. Agr. Bur. Ent. Bul. 75, pt. 4, p. 38-42.
(18) :
1913. Sacbrood, a disease of bees. U.S. Dept. Agr. Bur. Ent. Circ. 169.
5 p.; Sackbrut. Eine Bienenkrankheit. A translation by Dr. M.
Kiigtenmacher. Berlin-Steglitz.
(19)

1914. Destruction of germs of infectious bee diseases by heating. U. S.
Dept. Agr. Bul. 92, 8 p. (p. 4).

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