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148 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, II
It would thus appear from the foregoing extracts that the observations recorded
by various workers, in regard to the nature and extent of morbid disturbances evoked
by the occurrence of filarial embryos in the circulating blood of the different species
of animals, afford one reasonable ground for thinking that the clinical symptoms
that have been attributed to a microfilarial invasion in these animals may (at any
rate, in a large proportion of the recorded cases) have been, in reality, due to causes
other than microfilariasis 1 .
TREATMENT OF EQUINE MICROFILARIASIS.
As one would expect, in the absence of precise knowledge as to the pathogenic
rôle played by filarial embryos, the various forms of therapeutic treatment designed
to combat these parasites have been of a very empirical character. So far as the
equine species is concerned, Wirth [1917] found that no special treatment was called
for, as the disease generally ran a favourable course, although recovery did not take
place before two to four months from the onset of the disease. Wirth, however,
tested the efficacy of atoxyl, without any beneficial results. Yakimoff and his col-
laborators [1915] found the intravenous injection of salvarsan valueless. Aldigé
[1920] would appear to have given a fairly extensive trial to the various products
utilized in the treatment of trypanosomiasis, namely, orpiment, atoxyl, trypanblue,
trypanred, and tartar emetic, but except for the last, all the drugs tested by him
yielded negative results. With tartar emetic, the injection of the drug in from 1.2`
to 1.4 gramme doses was followed by the disappearance of the parasites and an
amelioration of the symptoms, provided the injections were repeated from four to
twelve times (one or two injections were found by him to be valueless). In the case
of the affected horses in the Remount Depot mentioned above, treatment was
attempted by the intravenous administration of single doses (50 c. c. of a 10 per
cent. solution) of " Bayer 205 ", but the results were negative.
As a matter of fact, only a limited number of drugs would appear to have been
tested as to their efficacy for animal microfilariasis. Of these, mention may be
made of galyl, which was employed by Curasson [1920], in doses of 0.5 c. c. of a 1
per cent. solution per kilo., intravenously, in the treatment of microfilariasis of the
carrion crow, but the application of this remedy resulted only in a partial recovery.
For the treatment of the microfilariasis of the dog (due to Mf. immitis), Itagaki
and his collaborators [1927], in Japan, employed sodium antimonyl tartrate, admi-
nistered intravenously in doses ranging from 0.003 to 0.004 gramme per kilo. body
weight, at intervals of one or more days. The treatment is claimed by them to
have resulted in a disappearance of the embryos from the circulation of the affected
animals.
1 A view which would seem warranted even allowing for the possibility that the parasites con-
cerned in the cases described by the serveral authors were not specifically identical.
REVIEW OF EQUINE MICROFILARIASIS
149
REFERENCES.
Aldigé (1920). Rec. Med. Vet. 96, 46-54.
Beal (1920). Report on Live-stock Industries of the Northern Territories of Gold Coast. [Abstr. in Trop.
Vet. Bull. 9, 48; 1921.]
Blanck (1927). Arch. f. Wissenschaft. U. Prakt. Tierheilk. 55, 557-579. [Abstr. in Trop. Vet. Bull., 15,
106; 1927.]
Bodkin and Cleare (1916). Bull. Entom. Res. 7, 179-190.
Curasson (1920). Bull. Soc. Path. Exot. 13, 654-655.
Davis (1928). Ann. Trop. Med. and Parasit. 22, 59-61.
Dios and Zuccarini (1926). Compt. Rend. Soc. Biol. 95, 827.
Foley (1921). Ann. Inst. Past. 35, 212-217.
Foley, Catanei and Vialatte (1926). Arch. Inst. Pasteur d'Algerie, 4, 485-518. [Abstr. in Trop. Vet.
Bull. 16, 17-18; 1928.]
Ford (1919). Vet. Journ. 75, 45-54.
Iliesco (1923). Arch. Veterinaria, 17, 116-119. [Abstr. in Trop. Vet. Bull. 14, 13; 1925.] Deals
with hæmorrhages caused by P. multipapillosa.
Itagaki and Makino (1927). Journ. Jap. Soc. Vet. Sci. 6, 15-23.
Littlewood (1915). Minist. Agric. Annual Report of the Vet. Service for the year 1914 (Egypt.)
Littlewood (1921). Ibid. for the year 1919-20.
Macalister (1917). Mem. Dept. Agric. India, Vet. Ser. 2, 207-261.
Martin (1917). Berl. Tierärzt. Wochenschr. 33, 520-524.
Oguni (1927). Journ. Jap. Soc. Trop. Vet. Sci. 6, 188-190.
Richardson (1921). Annual Report Dept. Agric. Uganda Protect. for the year ended 31st March 1920.
[Abstr. in Trop. Vet. Bull. 9, 134; 1921.]
Smit (1925). Nederl.-Indië Blad. V. Diergeneesk, 37, 529-534. [Abstr. in Trop. Vet. Bull. 14, 61;
1926.]
Smit and Ihle (1925). Centralbl. f. Bakt. I. Abt. Orig. 96, 30-32.
Teague and Clark (1918). Journ. Infect. Dis. 22, 154-158.
Velu (1921). Trop. Vet. Bull. 9, 261-262.
Vleming (1917). Tidjs. v. Vergelijk. Geneesk. 2, 109-116; 180-193. [Abstr. in Trop. Vet. Bull 7,
207-208; 1919.]
Wirth (1917). Zeitschr. f. Infektionskr. parasit. Krankheit. u. Hyg. d. Haustiere, 18, 380-413. [Abstr.
in Trop. Vet. Bull. 5, 237-248 ; 1917.]
Yakimoff, Schochos, Koselkin, Winogradow, and Demidow (1915). Ibid. 16, 275-286. [Abstr. in
Trop. Vet. Bull. 6, 96-97; 1918.]
Yakimoff and his collaborators (1916). Bull. Soc. Path. Exot. 9, 219-226.
Yakimoff and his collaborators (1917). Bull. Soc. Path. Exot. 10, 102-105.
Zeiss (1927). Deut. Tierärzt. Wochenschr. 35, 364-367.
SELECTED ARTICLE
INTERIM REPORT ON THE IMMUNISATION OF DRAFT
ANIMALS IN BURMA AGAINST ANTHRAX.
BY
D. T. MITCHELL, M.R.C.V.S.,
Director of Veterinary Services, Burma.
(Reprinted from Bulletin No. 1 of 1930 of the Veterinary Department, Burma.)
DISTRIBUTION.
The distribution of anthrax in this Province is widespread. In the last two
years, during which time an effort has been made to increase the number of blood
smears submitted for microscopic examination from dead animals in the districts,
the disease has been recorded from practically every district.
It usually occurs in sporadic form and results in the death of only a few ani-
mals in each outbreak, so there is every reason to presume that many of these out-
breaks in cattle and buffaloes pass unnoticed. Epidemics have been recorded in
wild animals in the neighbourhood of an outbreak in domestic stock, and as practi-
cally all species, with the exception of carnivora, are highly susceptible, such an
epidemic seriously complicates the ordinary preventive measures of segregation or
isolation.
Where animals are concentrated in any area, the disease is liable to assume
epidemic form. As examples of this, we have the recent mortality (1928-29) in the
horses of the Rangoon Paper Chase Club and the annual epidemics which have
occurred up to the present in the elephants of the timber firms, working in infected
forest areas.
MORTALITY FROM ANTHRAX.
It is very difficult to arrive at any estimate of the losses which are occurring in
cattle and buffaloes from this disease as it can only be definitely diagnosed by mi-
croscopic examination of blood smears. The difficulties at present existing in get-
ting smears from animals which die in the village-tracts are almost insuperable and
( 150 )
IMMUNIZATION OF DRAFT ANIMALS AGAINST ANTHRAX
151
many years will require to elapse before the villager can be taught to realise the
importance of a correct and early diagnosis in the prevention of epidemic disease
in stock. The grouping of black quarter, hæmorrhagic septicæmia and anthrax
under one heading in the annual report of the Department indicates the present
position regarding diagnosis. So long as the returns submitted by the Thugyi
remain the principal source of information in compiling mortality statistics, little
reliance can be placed on these as an index of distribution of disease or mortality in
animals.
In the case of elephants the methods of recording mortality and submitting
material for a laboratory diagnosis are well organised by the firms concerned, and
the records can be relied upon. The mortality from anthrax in these returns over
a period of years is a staggering figure when converted into terms of cash. One
reads of losses from anthrax varying from 20 to 90 animals per annum in the herds
of one of the firms concerned.
ECONOMIC VALUE.
The average total annual mortality from anthrax in working elephants in
Burma has not yet been worked out, but it may be taken for the purpose of illustra-
tion at the low estimate of 60 animals. The average value of these animals is about
Rs. 5,000 each or Rs. 3,00,000. Averaging the season's work of one elephant at 80
tons of timber, the royalties to the Government in respect to 60 elephants works out
at about Rs. 1,16,160 which are indefinitely or permanently deferred.
To this must also be added the loss in holding up the work in a forest where an
outbreak occurs and the loss of profit on the timber which might have been extract-
ed, etc., which cannot be estimated.
These figures in relation to one class of animal and one section of Agriculture
only gave one some idea of the economic importance of the problem and indicated
that research into methods of prevention should be considered first among the very
urgent problems which confronted the Veterinary Department in Burma.
RESEARCH WORK BEGUN.
A Special Research Officer was appointed in December 1927, but it was not
found possible to commence serious work on anthrax until nearly a year later, Octo-
ber 1928. This was due to (a) the necessity for training a staff in general laboratory
duties, (b) the provision of internal fittings for the laboratory, (c) the absence of
suitable apparatus and equipment which had to be purchased locally, borrowed or
ordered from England, (d) repairs to Incinerator, (e) arrangements for the supply of
small laboratory animals, etc., etc.
152 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, II
Considerable difficulty was experienced in procuring a local strain of anthrax
from each species. Specimen bottles for despatch of a piece of dried ear from sus-
pected cases were distributed, but the fear of spreading infection prevented speci-
mens being freely submitted. Only one specimen from an elephant was found to be
infective, although smears from animals, dead of the disease, were frequently being
received.
STRAINS USED.
This specimen came from an elephant of Messrs. T. D. Findlay of Nyaunglebin
on 8th March 1928 and the strain was used in these tests as the virulent anthrax
emulsion. Strains from cattle and horses were secured from local outbreaks in the
Insein area.
For the production of the vaccine a strain was used which was brought from
South Africa in 1927 and was of " Bovine " origin. This was obtained with other
anthrax strains, through the courtesy of the Director of Veterinary Services, Union
of South Africa.
NATURE OF VACCINE.
The preparation of the vaccine was carried out on lines similar to those employ-
ed in South Africa and recorded in the Annual Report of the Department Union of
South Africa. The finished vaccine consists of a suspension of living anthrax spores,
freed from the bacilli, from a strain attenuated to a fixed degree by incubation at a
temperature higher than the optimium, in a solution of water and glycerine. It is
therefore a very stable product and one which, even in jungle conditions in Burma,
may be expected to remain unchanged for a considerable period if given a reason-
able amount of protection from light and heat.
NATURE OF THE VIRULENT ANTHRAX MATERIAL.
The virulent anthrax suspension was prepared by isolating the strain from the
case in the elephant at Nyaunglebin and growing it under ordinary incubator condi-
tions in the laboratory. The virulent suspension was prepared by emulsifying the
spores in old cultures in water and glycerine. For use, this emulsion was standar-
dised on sheep. The minimum amount required to kill a sheep being named in this
report " The M. L. D." which was found to be 0.00005 c.c.
The virulency of this strain was found to be about double that of a virulent
cattle strain which has been brought from South Africa.
The dose of the vaccine was calculated on the amount required to protect a
sheep one month later against 1,000 M.L.D. of virulent spore emulsion by an experi-
ment on a series of these animals.
IMMUNIZATION OF DRAFT ANIMALS AGAINST ANTHRAX
153
Having thus produced a supply of standardised attenuated anthrax spore emul-
sion for use as a vaccine and a standardised virulent anthrax spore emulsion of
elephant strain for use as a test of the immunity produced, it was possible to con-
sider arrangements for estimating the value of the vaccine prepared from the South
Africa strain against the local Burmese strains of anthrax. The strains in the various
species of animals in Burma do not show very much variation and the history of
outbreaks indicate that in all probability the strains present in elephants, cattle and
buffaloes are the same or may be considered so at present from the point of view of
immunisation.
TESTS ON SHEEP.
Preliminary laboratory test on sheep were commenced on 5th January 1929.
Batch (I) anthrax vaccine was used and varying doses were given from 0.1 c.c. to
20.0 c.c.
The results from this batch of sheep were unsatisfactory. The animals were
allowed to graze in the Research Laboratory compound and a very heavy mortality
occurred a few days after inoculation from a condition resembling plant poisoning.
None of the animals showed anthrax on blood examination. It was later established
by animal experiment that this condition was due to eating the thorny branches of
the " sensitive plant " (Mimosapudica) which was very plentiful and at an attractive
stage. The sheep had come from the dry zone and were not accustomed to the local
Insein vegetation.
The test was repeated on 18th February 1929 on a further batch of sheep which,
on this occasion, were stable throughout the tests.
Vaccine in doses varying from 0.002 c.c. to 10.0 c.c. were injected into two
animals each. The inoculation of the vaccine was followed by a distinct thermal
reaction about the third day. A month was allowed for establishment of immunity,
and on 28th March 500 and 1,000 M.L.D. of virulent cultures were injected into each
of the pairs of vaccinated sheep.
The sheep which received a dose of 0.002 c.c. vaccine died of acute anthrax in
48 hours, of those which received 0.005 c.c., one died after being injected with 1,000
M.L.D. of virulent emulsion and the second sheep which received 500 M.L.D. of
virulent culture survived. All sheep receiving a dose of 0.01 c.c. or higher survived.
These tests were considered to be very satisfactory as they indicated—
(1) That the vaccine was safe in administration up 10 c.c. to sheep.
(2) That a good immunity against the local elephant strain was produced by the
bovine vaccine strain.
(3) The dose of vaccine required to protect against a standard dose of culture
of 500 M.L.D. or 1,000 M.L.D., viz., 0.01 c.c.
154 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, II
TESTS ON CATTLE.
The vaccine was then tested out on cattle at the dose indicated in the previous
experiments and multiples :—
3 cattle receiving 0.01 c.c. each.
3 cattle receiving 0.03 c.c. each.
3 cattle receiving 0.05 c.c. each.
The cattle were two years old, locally purchased animals which had been through
rinderpest tests a short time previously.
All the animals exhibited a very marked thermal reaction of 2—4 degrees
commencing on the day after the inoculation and persisting for about 48 hours.
The animals were not visibly ill and did not go off feed. No swellings occurred at
the site of the inoculation and after four days the animals were apparently normal.
It was not considered necessary to test the immunity produced in cattle as ex-
perience in South Africa showed that, given a good reaction, a good immunity
lasting 12—16 months was obtained.
The test indicated—
(1) That a standard dose of vaccine produced a good temperature reaction with
rapid recovery.
(2) That multiples of the standard dose up to five times constituted a dose
which was still safe for cattle.
(3) That the temperature reaction did not depend on the size of the dose given.
PRELIMINARY TESTS ON ELEPHANTS.
It was now considered that the Laboratory tests were sufficiently conclusive to
warrant the vaccine being tested out on elephants, and in consultation with the
Manager of the Bombay Burmah Trading Corporation, a test was arranged in a small
lot of three elephants at Pyinmana on 25th April 1929. The dose given was 0.01
c.c. of stock vaccine diluted to 1 c.c. for ease of inoculation.
Two of these elephants gave a definite thermal reaction and all recovered in a
few days. A small local swelling appeared in two animals at the site of inoculation
but this disappeared later. The site of inoculation was on the chest wall about
midway between fore and hind leg at the level of the elbow. U Tun Hlaing,
Research Veterinary Inspector, carried out the inoculations and superintended the
experiment throughout.
RETURN OF SPECIAL RESEACH OFFICER TO AFRICA.
While this test was in progress, the period of secondment of the Special Research
Officer was approaching termination and it was necessary for him to return to South
IMMUNIZATION OF DRAFT ANIMALS AGAINST ANTHRAX
155
Africa. The vaccine and stock emulsions, cultures, etc., were placed on ice until
work could be resumed.
In the following month an outbreak occurred among elephants belonging to
Messrs. MacGregor & Co., Limited, Toungoo, at Gwe Chaung. Vaccine was asked
for and issued for the inoculation of three remaining elephants.
The inoculation was performed on 24th May by the firm's Veterinary Assistant,
Mr. Birch, and inoculations were done in the neck. From the details submitted
it is difficult to arrive at any satisfactory conclusion regarding this test. The test
was carried in the presence of active anthrax infection and there was a possibility of
one or other of the animals being already infected. A dose of carbolic acid had been
administered 12 days previously and the vaccination was inadvertently done in the
neck instead of on the side. One animal developed no thermal reaction but the
remaining animals showed a marked rise in temperature 4—5 days after inoculation.
In one of the animals the reaction was very severe (3.5° F.). The temperature of
this animal returned to normal in 6 days but he was still definitely ill and getting
weaker. The animal eventually died on the 13th day following the
inoculation. The death was not attributed to anthrax and a post-mortem was
held which supported this. Smears taken after death from the spleen and blood
were returned negative from the laboratory. This case is difficult to explain
except by presuming that there was some complicating factor which under the
influence of the reaction due to the vaccination was able to produce a fatal ending.
It certainly was not directly due to the vaccine or anthrax bacilli would have been
found on examination of the spleen smears. The case may be regarded as an
accident of vaccination such as is occasionally met with in the other species.
During July Messrs. Steel Brothers decided to have an experimental batch
of elephants vaccinated at the Swa and arrangements were made for U Tun Hla-
ing to do the inoculations and superintend the tests.
Nine elephants were injected with 1 c. c. anthrax vaccine (0.01 c. c. stock
emulsion) under the skin on the chest wall on 2nd July and a further batch of 4
animals on 10th July, making 13 animals in all.
Most of the animals showed a temperature reaction between the 4th and 7th
day after inoculation. Swellings of various sizes appeared from 2" to 4" in diameter
but two animals developed dangerous swellings at the site of inoculation.
The clinical symptoms and thermal reactions had in all but one case subsided
14 days after the inoculation. Recovery from the local swelling in this case took
about three weeks.
Results. In all tests up to this point 19 animals had been vaccinated and in
the majority of cases a thermal reaction had occurred four to five days after ino-
culation.
156 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ I, II
Two animals showed large swellings at the site of the inoculation and one
animal had died from some complicating factor.
These results were as good as could be excepted when it is considered that
the inoculations were carried out under jungle conditions by men who had no
previous experience in inoculation of elephants. The swellings were probably due
to some contamination. The skin of an elephant is difficult to render sterile and
the insertion of the needle requires a considerable muscular effort until one gets
accustomed to it.
From these results it was concluded that the vaccine was safe for use in
elephants in doses of 1 c. c. (0.01 c. c. stock vaccine.)
RETURN OF RESEARCH OFFICER TO BURMA.
No further inoculation work was carried out in elephants between this date
and the return of the Research Officer to Burma in October 1929.
The next step contemplated was the estimation of the degree of immunity,
if any, produced by this vaccine on elephants. This phase of the work had already
been discussed with the representatives of the timber firms at an informal meeting
before departure from Burma and it was then accepted that an experiment involv-
ing a number of vaccinated elephants, inoculated subsequently with virulent cul-
tures, would be necessary before the issue of the vaccine for general immunisation
work could be considered.
ARRANGEMENT FOR THE CRUCIAL TEST.
At a combined meeting on October 31st, 1929, representatives from four firms
agreed to share the cost of the animals for the experiment and the incidental ex-
penditure in connection with transport and supervision.
A site was suggested on the Toungoo-Thandaung road at Shwenyaungbin as
suitable for the test as it provided the conditions laid down of (1) good water, (2)
good fodder for the animals, (3) no cattle or buffaloes in the vicinity, (4) plenty of
timber for cremation of carcases and (5) convenient to the main line. This area
was inspected and approved. The decision to use virulent cultures of elephant
strain anthrax on the animals in this test and the anticipated deaths in control
animals from anthrax necessitated the strictest care and supervision at every stage
of the experiment in order to prevent permanent infection of the site by the
disease.
A retest of the vaccine which had been in storage for eight months was made
on six 2 years old cattle at the Research Laboratory in November 1929 when it
was found that the vaccine was active, although it did not give rise to so acute