Destruction of germs of infectious bee diseases by heating

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USDEPARTNENT OP AGRICULTURE

No. 92

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Contribution from the Bureau of Entomology, L. O. Howard, Chief.
May 15, 1914.

DESTRUCTION OF GERMS OF INFECTIOUS BEE
DISEASES BY HEATING.

By G. F. Waite, M. D., Ph. D., Expert, Engaged in the Investigation of Bee Diseases.

INTRODUCTION.

To reduce the losses due to bee diseases beekeepers have often
employed heat in one form or another. The direct flame has been
used in scorching or burning the inside of hives that have housed
infected colonies. Before being fed back to bees honey is often
heated for the purpose of destroying the germs of bee diseases, should
any be present. Heat is used in the rendering of wax and in the mak-
ing of comb foundation. It is natural and very appropriate, there-
fore, that beekeepers should inquire about the amount of heating
that is necessary to destroy the germs that produce diseases among
bees.

As no work had been done to determine the facts relative to this
question with any degree of accuracy, the writer has performed during
the last two years a number of experiments for the purpose of ascer-
taining them. Of these experiments 55 are summarized in the three
tables included in this paper. It may be of interest to beekeepers to
know in a general way how these experiments were made. A brief
description of the methods used will serve also to make the tables
more readily understood. An aqueous suspension of larvee sick or
dead of the disease is made and placed in a smail glass tube. This
tube is immersed in water of the temperature desired in the heating.
After the germ-containing material is heated in this way 1t must be
tested to determine whether or not the germs have been destroyed.
In the case of American foul brood this can be done by inoculating a
suitable artificial medium with the heated material and observing the

presence or absence of growth of Bacillus larve, the germ of this dis-

ease. As there is no artificial medium now known suitable for culti-

vating the infecting agent of either European foul brood, sacbrood,

NotEe.—This paper is of interest to beekeepers in all parts of the United States; it was read before the

New York State Beekeepers’ Association, February 10, 1914, at Ithaca, N. Y.
35960°—14 :

2 BULLETIN 92, U. S. DEPARTMENT OF AGRICULTURE.

or Nosema disease, healthy colonies of bees must be inoculated in
making the test in case of these diseases. This is done by feeding the
bees the heated germ-containing material in sirup. If the disease is
produced by this feeding, naturally the infecting agent has not been
destroyed by the heating; but if the disease is not produced, it vir-
tually has been destroyed by it. By repeated experiments of this kind
in which the temperature used in the heating is varied, the minimum >
temperature at which any virus is killed can be determined. As will
be seen from the tables, 13 experiments for European foul brood,
22 for sacbrood, and 20 for Nosema disease were made in which healthy
colonies were inoculated with heated germ-containing material
from these three diseases, respectively. In the last disease the
stomachs from diseased bees furnished the germ-containing material
for heating and feeding. In these experiments the temperature was
maintained for 10 minutes as a rule.

DISEASES OF THE BROOD OF BEES.

Nearly a century and a half ago the name “foul brood” was used
for a destructive brood disorder of bees, and for almost a century
later it was apparently the custom to diagnose as foul brood any
destructive disease of the brood. About half a century ago bee-
keepers began to note that all of the brood diseases are not the same.
They began, therefore, to write of different forms of foul brood.
At the present time it is known that there are at least three infectious
diseases of the brood of bees. All of these diseases are more or less
destructive, and it is quite likely that each of them has now and then
been diagnosed as foul brood. In America these brood diseases are
now known, as European foul brood, American foul brood, and sac-
brood.

EUROPEAN FOUL BROOD.

In European foul brood death occurs early, the larve dying usually
before the time for cell capping. There is no viscidity (ropiness) to
the decaying larve as a rule, and no pronounced odor present.

Numerous samples of this disease have been examined from the
United States, andsome from Canada. Its presence also in England,
Germany, Switzerland, and Denmark is strongly suggested by written
reports from these countries. It is very probable that the disease
has a much wider geographical distribution than these facts indicate.

Two years ago the fact was demonstrated that the germ causing
European foul brood is the microorganism to which the name Bacillus
pluton is given. In a paper! announcing the fact it was stated that
the studies then made indicated that the germ is easily killed by heat.
This belief has been confirmed by further experiments.

1 White, G. F., 1912. The Cause of European Foul Brood. U.S. Dept. Agriculture, Bureau of Ento-
mology, Cir. No. 157.

DESTRUCTION OF GERMS OF BEE DISEASES BY HEATING. 3

Table I gives a brief summary of 13 inoculation experiments per-
formed for the purpose of determining approximately the amount of
heating necessary to destroy the germ of European foul brood.

TABLE I.—A summary of the experiments made to determine approximately the minimum
amount of heating necessary to destroy the germ causing European foul brood.

Teen veneer v Stine! Results of inoculation.
iG: Min.

Pept. au 1912 75 to 80 ‘10 | No disease produced.
cee tee 65 to 70 10 | Do.

Sao. 23, 1912) 64 to 66 10 | Do.

Oct. 12) 1912 64 to 65 10 | Do.

Oct. 1,1912 62 to 63 10 Do.

Oct. 8, 1912 G2ACONGSW ee eee Do.

Oct. 10,1912 62 to 63 10 | Disease produced.

Oct. 4,1912 61 to 62 10 Do.

Aug. 8, 1913 60 20 | Do.

Sept. 3,1912 60 10 Do.

Sept. 20, 1912 58 to 60 LON Do.

Sept. 28, 1912 57 to 60 20 | Do.

Sept. 20, 1912 55 to 56 10 | Do.

Tt will be observed by an inspection of Table I that European foul
brood was produced in every instance where healthy colonies were
fed disease material which had been heated for 10 minutes at tempera-
tures below 63°C. (145.4° F.), but that no disease was produced when
temperatures higher than 63° C. (145.4° F.) were used for the same
length of time. The minimum temperature that can be used, there-
fore, in destroying the germ of European foul brood, if it is applied
for 10 minutes, lies somewhere between 60° C. (140° F.) and 65° C.
(149° F.), being near 63° C. (145.4° F.).

AMERICAN FOUL BROOD.

American foul brood is the disease of the brood of bees that is best
known to beekeepers and is the one the presence of which they have
been able to recognize most easily. In this disease the larvee usually
die after the cells containing them are capped. ‘The disease is charac-
terized especially by the marked viscidity (ropiness) manifested by the
decaying larve that are dead of the disease. The pronounced odor
noticeable within hives housing colonies affected by this disease, espe-
cially in its later stages, 1s another well-known characteristic.

This disease is very widely distributed geographically. Samples
of it have been received from many localities in the United States,
from Switzerland, New Zealand, Germany, England, and France, and
it is very probable that it has a much wider geographical distribution
even than is indicated by these facts.

Until seven years ago the cause! of American foul brood was not
known. At that time the fact was demonstrated positively that the

1 White, G. F., 1907. The Cause of American Foul Brood. U.S. Dept.of Agriculture, Bureau of Ento-
mology, Cir. No.94.

4 BULLETIN 92, U. S. DEPARTMENT OF AGRICULTURE.

germ causing the disease is the one to which the name Bacillus larve
is given.

The facts obtained to date are too meager to justify anything ©
more than a general statement regarding the minimum amount of
heating that can be employed in rendering material containing the
germ of American foul brood noninfectious. Taking rather wide
limits, it may safely be said that the minimum temperature at which
this can be done, if the temperature is applied for 10 minutes, lies
somewhere between 90° C. (194° F.) and 100° C. (212° F.). Itseems
quite probable, indeed, that a temperature less than 98° C. (208.4° F.)
will suffice if applied for 10 minutes. When 100° C. was used the
spores of Bacillus larve were killed in less than five minutes.

SACBROOD.

Observant beekeepers have for many years noted the presence of
dead brood which seemed to them to be different from that dead of
foul brood. Some were inclined to believe that the disease was
an infectious one; a larger number apparently were disposed to
ascribe the trouble to such causes as an unsatisfactory queen, starva-
tion, and thelike. This brood disease has been recently demonstrated
to be an infectious one, and the name “‘sacbrood”’ has been given to it.
Larve that die of this disease do so almost invariably after the time
of cell capping. The most characteristic symptom of the disease is
the saclike appearance of the dead larve when they are removed from
the cell. This fact suggested the name “sacbrood”’ for the disease.

Sacbrood is frequently met with. Its presence has been diagnosed
by Dr. A. H. McCray and the writer in 367 samples received from 44
States of the Union and in 13 samples received from Canada. Reports
from England, Switzerland, and Australia indicate strongly that this
disease exists in these countries also. It is very probable that it has
a much wider geographical distribution than is shown by these facts.

More than a year ago it was again the writer’s fortune to determine
the cause of another brood disease. Unlike the cause of either
Kuropean foul brood or American foul brood, the. infecting agent
causing sacbrood has not yet been seen. It was demonstrated,
however, that the infecting agent in this disease passes through the
pores of earthenware filters. For this reason the cause of sacbrood
is spoken of as a filterable virus.

In a paper! announcing the cause of sacbrood the statement is
made that the germ causing the disease is destroyed by a com-
paratively small amount of heat. This belief is confirmed by the
results of the experiments summarized in Table II.

1 White, G. F., 1913. Sacbrood,a Disease of Bees. U.S. Dept. of Agriculture, Bureau of Entomology,
Cir. No. 169.

DESTRUCTION OF GERMS OF BEE DISEASES BY HEATING. 5

TABLE II.—A summary of the experiments made to determine approximately the minimum
amount of heating necessary to render sacbrood material noninfectious.

Dates of inocu- Time of

lation. Temperature. poating. | Results of inoculation.

: “GE Minutes.
July 27,1912 95 to 100 2 | No disease produced.
Aug. 8,1912 95 to 100 2 Do.
Aug. 29,1912 75 to 80 10 Do.
Sept. 5, 1912 65 to 70 20 Do.
Sept. 3,1912 55 to 60 20 Do.
Aug. 26,1913 80 15 Do
(0) 75 15 Do
Do 70 15 Do
Do 65 15 Do
) 65 15 Do
Sept. 2,1913 65 15 Do
Sept. 3,1913 60 20 Do
Sept. 9, 1913 60 15 Do
Sept. 10, 1913 60 15 Do
Sept. 17, 1913 60 10 Do
Sept. 10, 1913 58 10 Do.
Sept. 17,1913 58 10 Do.
Sept. 18, 1913 57 10 | Sacbrood produced.
Sept. 9,1913 55 20 Do.
Sept. 10, 1913 55 10 Do
Sept. 17,1913 55 10 Do.
Aug 6,1913 50 30 Do

From Table II it will be observed that when larve dead of sacbrood
were heated 10 minutes at a temperature of 57° C. (134.6° F.) or less
and then fed to a healthy colony, sacbrood was produced; if, on the
other hand, the dead larve used in making the feeding were heated
to 58° C. (136.4° F.) or higher, the disease was not produced. The
conclusion to be drawn from these experiments is that the minimum
temperature, when maintained for 10 minutes, at which the infecting
agent causing sacbrood is destroyed lies somewhere between 55° C.
(131° F.) and 60° C. (140° F.), being near 58° C. (136.4° F.).

DISEASES OF ADULT BEES.

Very little is known about the diseases of adult bees. Many names
have been used for the purpose of designating them, but the number
of such diseases is probably small. There is only one adult disease
that can be diagnosed at present by laboratory methods. This one is

the Nosema disease.
NOSEMA DISEASE.

Fifty-seven years ago Dr. Donhoff made a more or less brief study
of a disease of adult bees in Germany. He observed that the stomach
was the organ that was primarily affected. By feeding to healthy
colonies in sirup the crushed stomachs from affected bees Déonhoff
demonstrated that the disease could be transmitted to healthy colo-
nies. It was therefore infectious.

The work by Dénhoff had been practically forgotten, apparently,
when Zander,’ of Erlangen, Germany, five years ago observed the

1 Zander, E., Aug., 1909. Tierische Parasiten als Krankheitserreger bei der Biene. Miinchener Bienen-
zeitung.

6 BULLETIN 92, U. S. DEPARTMENT OF AGRICULTURE.

presence of a disease among adult bees. From the evidence at hand
it seems most probable that the disorder encountered by Dénhoff and
the one encountered by Zander are one and the same disease.

Aside from rediscovering the disease Zander has identified the germ
causing it as a protozoan (a one-celled animal parasite) and has given
to it the name Nosema apis. For the disease he has used the name
‘“Nosema Seuche.” This is an appropriate one, as it suggests some-
what the nature of the disease. The name ‘‘ Nosema disease,’ which
the writer suggests as the common name for this disease, is, it will be
observed, only a translation of the German name used by Zander.

The germ Nosema apis gains entrance to the body of the bee by way
of the alimentary canal. In the walls of the stomach the growth and
multiplication of the parasite take place to an enormous extent,
causing the abnormal appearance manifested by the organ. When the
disease reaches an advanced stage the stomach is white and fragile
and reveals upon a microscopic examination the presence of the para-
site in very large numbers. In the spring of the year, especially,
many weak colonies show upon examination a high percentage of
Nosema-infected bees.- Quite often, indeed, in the examinations that
have been made o7 such colonies, 50 to 90 per cent of the bees in sam-
ples taken from them were found to be infected with the parasite.
It is an interesting and important fact that a very large number of
colonies which are strong and apparently doing well are found upon
examination to contain at least a small percentage of Nosema-infected
bees.

Nosema apis has a very wide geographic distribution. It has al-
ready been encountered in Germany by a number of investigators;
it has been found in Australia, Switzerland, and England. The
writer has found it in samples of bees received from 27 different States
in the United States and in two samples of adult bees from Canada.

From the facts gathered it would seem that many of the cases called
‘‘spring dwindling’ by the beekeepers are caused, in part at least, by
Nosema apis. This statement is not by any means to be interpreted
as saying that Nosema disease and spring dwindling are always the
same.

It has been demonstrated experimentally that colonies can be
weakened and killed by feeding to them material containing Nosema
apis. For this and other reasons it seems certain that the disease
causes a loss to apiaries, but, for want of sufficient data, the extent
of such loss can not now be estimated at all definitely. From the
facts at hand one is justified in at least drawing the conclusion that
Nosema infection in a colony tends to weaken the colony. Nosema
apis is therefore a germ in which the beekeeper is economically
interested.

DESTRUCTION OF GERMS OF BEE DISEASES BY HEATING. 7

For thepurpose of determining approximately the minimum amount
of heating that is sufficient to destroy the germ Nosema apis the mnoc-
ulation experiments summarized in Table Ii] were made.

TasBLE III.—Summary of experiments in which the germ, Nosema apis, was heated and
fed to healthy colonies.

. Tempera- -
Tae ts eg ture nod : ees Results of inoculation.
tion in heating. 8.
°¢. Minutes. 3
Oct. 29,1912 95 to 100 5 | No Nosema infection produced.
Nov. 12,1912 95 to 100 5 Do.
Oct. 29,1912 80 20 Do.
Nov. 9, 1912 80 10 Do.
Nov. 11, 1912 68 to 70 10 Do
OP au 68 to 70 10 Do
Nov. 12, 1912 65 20 Do
Jan. 8,1913 65 10 Do
Nov. 11, 1912 60 10 Do
(NEES eee 60 10 Do
Nov. 20, 1912 60 10 Do
Feb. 8, 1913 58 10 Do.
Oct. 4,1913 58 10 Do.
Feb. 8,1913 57 to 58 15 Do.
Oct. 15,1913 57 10 Do.
Dore 3 57 10 Do.
Oct. 4,1918 56 10 | Nosema infection produced.
Oct. 15,1913 56 10 Do.
Jan. 8,1913 55 20 Do.
Jan. 31,1913 55 10 Do.

It will be observed from Table III that when Nosema apis was
heated to 57° C. (134.6° F.) or higher for 10 minutes and fed to
healthy bees no infection took place, but when held at tempera-
tures below 57° C. (134.6° F.) for the same period of time the bees
became Nosema infected. It is shown, therefore, that the minimum
temperature that will destroy the germ Nosema apis in 10 minutes
lies somewhere between 55° C. (131° F.) and 60° C, (140° F.), being
quite near 57° C, (134.6° F.).

By way of parenthesis it might be well to say a word or two further
regarding Nosema disease. The studies of this disease disclose the
interesting fact that it is not a new one in American apiaries. There
is no cause, therefore, for anticipating any additional losses to our
apiaries. Indeed, since the presence of the disease is known, hopes
may be entertained that methods will be determined for reducing the
losses due to it. Considerable work must yet be done, however,
before methods for its control can be recommended.

Nosema disease is being studied in England, Germany, Switzer-
land, and Australia. During the last two years the writer has de-
voted considerable time to its study in America. The plan is to
continue the studies during the present year, after which it is hoped
a further discussion of this disease will be justified.

8 BULLETIN 92, U. S. DEPARTMENT OF AGRICULTURE.

SUMMARY AND GENERAL REMARKS.

The results of these experiments show that when they are main-
tained for 10 mimutes the minimum temperatures that can be used ©
for destroying the germs of the four bee diseases now known to be
infectious are as follows:

(1) The minimum temperature for European foul brood lies some-
where between 60° C. (140° F.) and 65° C. (149° F.), being approxi-
mately 63° C. (145.4° F.).

(2) The mmimum temperature for American foul brood lies some-
where between 90° C. (194° F.) and 100° C. (212°) F., being probably
less than 98° C. (208.4° F.).

(3) The minimum temperature for sacbrood lies somewhere between
55° C. (131° F.) and 60° C. (140° F.), being approximately 58° C.
(136.4° F.).

(4) The minimum temperature for Nosema disease lies between
55° C. (131° F.) and 60° C. (140° F.), bemg approximately 57° C.
(134.6° F.).

It will be noted, therefore, that 63° C. (145.4° F.) for European
foul brood, 98° C. (208.4° F.) for American foul brood, 58° C. (136.4° F.)
for sacbrood, and 57° C. (134.6° F.) for Nosema disease are the ap-
proximate minimum temperatures at which the germs of these dis-
eases, respectively, are destroyed. Since there are varying factors
in experiments of this nature that tend to produce slight variations
in results, these temperatures are referred to as being approxi-
mate. It is probable that future experiments may cause slight
changes to be made im these conclusions. Notbing more than a com-
paratively shght variation is to be expected, however. In practice
the beekeeper, in destroying these germs by heating, will naturally
use a quantity of heat somewhat in excess of the mimimum amount
that is absolutely necessary.

Some generalizations may now be made which will be of interest to
the beekeeper. The melting point of beeswax is between 62° C.
(143.6° F.) and 64° C. (147.2° F.), inclusive. It will be observed that
this same temperature in 10 minutes will destroy the germ causing
European foul brood, and that it is about 10° F. above that which will
destroy the germs of sacbrood and Nosema disease. A further inter-
esting generalization may be made concerning the heating of honey. —
Honey when heated to 160° F. reaches a temperature 15° F. above the ~
temperature necessary to destroy the germ of European foul brood ~
and about 25° F. above the temperature that will destroy the infect-
ing agents of sacbrood and Nosemadisease. The infecting agents of
these three diseases of the bee, therefore, will be destroyed when the
temperature of 160° F. is used in the commercial handling of honey.
Finally, it is believed that the results of this work on the thermal
death point of the viruses of the bee diseases will be directly applica-
ble to the control of these diseases.

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