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ORIGINAL ARTICLES
STUDIES ON THE DIPHTHERITIC FORM OF FOWL-POX
IN INDIA
BY
R. L. KAURA, B.V.Sc., M.R.C.V.S.,
Assistant Serologist,
AND
S. GANAPATHY IYER, G.M.V.C.,
Veterinary Inspector,
Imperial Veterinary Research Institute, Muktesar
[Received for publication on 14th May, 1936]
(With Plates XX & XXI)
INTRODUCTION
Fowl-pox is a contagious disease of birds due to a filtrable virus
and is characterised by the appearance of wart-like nodules on the head and
cheesy diphtheritic membranes in the buccal cavity with or without oculo-nasal
discharge. One or all of the above lesions may be present in the same bird.
At the instigation of this Institute in 1934, specimens in the form of dried
crusts from suspected cases of fowl-pox, have been received from various parts
of India for confirmation, and this has enabled the writers to study the disease.
As detailed in Table I, so far 22 specimens have been received, out of which
15 were found positive to fowl-pox, and of these, three received from the Madras
Presidency gave rise to the development of diphtheritic lesions in the buccal
cavity in addition to the cutaneous lesions, on subinoculation over the feather
follicles of healthy fowls. Advantage was taken of this opportunity to study
this diphtheritic condition in some detail.
A review of the past literature shows that the nomenclature of this disease
has been rather confusing. Each lesion appears to have been named separately
and amongst other names we have chicken-pox, contagious epithelioma, canker,
avian diphtheria, and roup, although it is now known that these conditions are
only different manifestations of the same disease determined by one and the
same virus.
(313)
314 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, IV
While studying the diphtheritic form Moore [1885], Marshall [1900] and
Jackley [1918] were of opinion that bacteria might be the cause but could not
confirm their identity. Jowett [1909] stated that comb and mouth lesions were
etiologically distinct. Ward [1904] was the first to relate roup etiologically to
chicken-pox. Haring and Kofoid [1911] demonstrated cell inclusions of chicken-
pox in the cheese-like lesions of the mouth and throat of fowls. Hadley and
Beach [1913] believed that the head and mouth forms were different manifesta-
tions of the same disease. Mack and Records [1915] showed evidence that chicken-
pox and avian diphtheria were etiologically identical but did not draw positive
conclusions. Arloing [1911-12] differentiated false and true fowl-pox diphtheria
by histological methods. Jackley [loc. cit.] while studying roup, incriminated
an organism of the Pasteurella group. Beach, Lothe and Halpin [1915] showed
in an outbreak of chicken-pox and avian diphtheria that the high mortality was
due to secondary invading bacteria although the primary cause was a filtrable
virus. From the study of Brumley and Snook [1916] it seems that although there
is a virus and complicating conditions induced by secondary bacterial infection,
neither factor alone would cause a typical disease. Doyle and Minett [1927]
studied the identity of wart-like growths on the head and diphtheritic lesions
in the mouth and confirmed that comb and mouth lesions in the case of fowl-pox
were due to the same virus and immunity to one form could be set up with
material from another form. There was no evidence that bacteria alone could
produce typical mouth lesions, the primary action of the specific virus being
essential.
PURPOSE OF EXPERIMENTS
Experiments have been conducted to study the transmissibility of the virus
of the diphtheritic lesions, its tendency for generalization, its etiological identity
with the ordinary cutaneous form of fowl-pox as commonly found in India, filtra¬
bility of the causative virus and its immunological identity with our stock fowl-
pox virus, which was obtained through the courtesy of the Veterinary Laboratory
of the Ministry of Agriculture, Weybridge, England.
METHOD OF EXPERIMENTAL TRANSMISSION
A specimen of crusts was powdered and a 1 per cent emulsion was made
after the method of Doyle and Minett [loc. cit.] with a slight modification that
instead of ordinary saline, 80 per cent glycerinated saline was used in its prepara-
tion. The inoculum thus prepared was applied by means of a swab over a scari-
fied portion of the comb in the case of cocks, and in other birds over the feather
follicles of the leg after pulling out a few feathers. Two healthy fowls and a
fowl immune to our stock fowl-pox virus (Weybridge strain) were used for each
specimen and the results were reported after a fortnight's observation from the
DIPHTHERITIC FORM OF FOWL-POX IN INDIA
315
date of inoculation, except in the case of negative results, when the fowls were
subjected to an immunity test with the stock virus to exclude the possibility of
their having a natural immunity to fowl-pox.
In no case, except for the special purposes recorded in this paper, was direct
inoculation ever done over the broken or unbroken mucous membrane of the
mouth.
All the specimens which were received from outside were subjected to biolo-
gical test, for evidence of fowl-pox infection, in the manner described above,
with the following results :—
TABLE I
Results of biological test on specimens, for evidence of fowl-pox infection
Serial
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Speci-
men
No.
56
69
70
85
117
121
122
123
130
139
150
151
197
220
246
Received from
Principal, Madras Veteri-
nary College.
Superintendent,
Civil
Veterinary Department,
Bangalore.
Veterinary
Assistant
Surgeon,
Jamtara,
(B. & O.).
Special Officer, C. V. D.,
Muzaffarpur, (B. & O.).
V. A. S., Conjeevaram,
(Madras).
D. V. S., Nagpur, (C. P.) .
V. A. S.,
Cuddalore,
(Madras).
Principal, Madras Veteri-
nary College.
V. A. S., Kumbakonam,
(Madras).
D. V. S., Lucknow, (U. P.).
V. A. S., Rajapalyam,
(Madras).
V. A. S.,
Kothapeta,
(Madras).
V. A. S.,
Anakapalle,
(Madras).
V. A. S., Deoria, (U. P.). .
V. A. S.,
Kumbakonam,
(Madras).
Date of
receipt
9th Feb., 1935 .
14th Feb., 1935
15th Feb., 1935
22nd Feb., 1935
14th Mar., 1935
15th Mar., 1935
Do. .
Do. .
18th Mar., 1935
22nd Mar., 1935
25th Mar., 1935
Do. .
20th April, 1935
4th May, 1935 .
16th May, 1935
Nature of
material
Dried
crusts.
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Result of biologica
test
Healthy
fowls
Positive
Do. .
Do. .
Do. .
Negative
Positive
Negative
Do. .
Do. .
Immune
fowls
(Wey¬
bridge
strain)
Negative
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Unfit for examination
Positive
Do. .
Do. .
Negative
Do. .
Negative
Do. .
Do. .
Do. .
Do. .
Remarks.
Cutaneous le-
sions.
Do.
Do.
Do.
. . .
Cutaneous le-
sions.
. . .
. . .
. . .
?
Cutaneous le-
sions.
Do.
Do.
. . .
. . .
316 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, IV
TABLE I—(continued)
Serial
No.
16
17
18
19
20
21
22
Speci-
men
No.
285
397
429
39
84
129
193
Received from
V. A. S., Hassanpur,
(U. P.).
V. A. S., Cannanore,
(Madras).
Ditto
.
Ditto
.
V. I. O., Lucknow,
(U. P.).
V. A. S., Chingleput,
(Madras).
Ditto .
Date of
receipt
12th June, 1935
29th Aug., 1935
13th Sept., 1935
20th Jan., 1936
15th Feb., 1936
11th Mar., 1936
6th April, 1936
Nature
of
material
Dried
crusts.
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Result of biological
test
Healthy
fowls
Positive
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Immune
fowls
(Wey¬
bridge
strain)
Negative
Do. .
Do. .
Do. .
Do. .
Do. .
Do. .
Remarks.
Cutaneous le-
sions.
Cutaneous and
buccal
le-
sions.
Do.
Cutaneous le-
sions.
Do.
Do.
Cutaneous and
buccal
le-
sions.
Conclusion :—From the above table, it is evident that the fowls immunized
with the Weybridge strain of fowl-pox virus can resist the Indian strain of this
virus proving their immunological identity.
TRANSMISSION EXPERIMENTS
On subinoculation over the feather follicles of the leg, emulsion prepared
from specimen No. 397/1935, received from Cannanore, Madras, gave rise to the
development of fowl-pox lesions over the leg and diphtheritic membranes in the
buccal cavity in the case of healthy fowls which eventually died, whereas the
immune bird showed no lesions of the disease and remained quite healthy. Post
mortem examination on the dead fowls revealed extensive buccal lesions extend-
ing into the throat (Plate XX, Fig. 1). On histo-pathological examination, the
usual features of a diphtheritic lesion were observed and on cultural examina-
tion Escherichia coli communior and Streptococcus faecalis were isolated.
Death was not very common in fowls exhibiting cutaneous lesions only,
and in the above case the fowls evidently died on account of the acute buccal
form of the disease.
Ind. Jour. Vety. Sci. & Anim. Husb., Vol. VI, Pt. IV.
PLATE XX
FIG. 1. Buccal diphtheritic lesions of fowl No. 229.
FIG. 2. Buccal diphtheritic lesions reproduced in fowl
No. 256, from the buccal material of fowl No. 229.
DIPHTHERITIC FORM OF FOWL-POX IN INDIA
317
Two more specimens (Nos. 429/1935 and 193/1936) were received which on
biological test also gave rise to the development of the buccal form of the disease
in addition to localisation over the feather follicles of the inoculated leg in healthy
fowls, whereas the immune fowls did not react.
In neither of the above cases was the virus emulsion applied in the mouth
but on the leg only. From the above tests it is confirmed that the etiology of the
mouth form and the cutaneous form is one and the same, and the virus in the
above cases has produced the mouth form without any direct inoculation into
the buccal mucous membrane. In other words it seems that the above samples
of crusts on subinoculation into healthy fowls induced a reaction of a greater
severity and probably caused generalisation of the disease.
TABLE II
Transmission experiments
Specimen No. 397/1935
318
THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, IV
From Table II it will be observed that inoculation of the leg alone in the
case of fowl No. 255 gave rise to the lesions in the mouth in addition to the
localisation over the feather follicles of the leg, giving a suggestion of generalisa-
tion of the virus in the body of the fowl (Plate XXI). Fowl No. 256 which
was inoculated over the scarified buccal mucous membrane showed the develop-
ment of lesions only in the mouth and not in any other part of the body where
all the feathers were intact (Plate XX, Fig. 2). This is in accord with the
findings of Doyle and Minett [loc. cit.] who failed to infect a fowl's leg from
which feathers had not been plucked.
Two fowls Nos. 303 and 304 were subinoculated with the virus in the mouth
on 15th November 1935 (Table II), and at the same time feathers were plucked
from the leg but no application of the virus was done on that site in either case.
Both died after a week showing severe diphtheritic lesions in the buccal cavity,
but without showing any lesion over the plucked area. The severe mouth lesions
apparently caused the death of the birds too quickly to allow the cutaneous lesions
to develop through generalisation.
Two fowls were scarified in the mouth with a sterile scalpel but no virus
was inoculated. No lesions were observed in the mouth in either case, indicating
that the micro-organisms normally inhabiting the buccal cavity of fowls were
not able to produce the diphtheritic lesions without the presence of fowl-pox
virus.
VIABILITY OF THE VIRUS IN FILTRATES
Material for filtration was obtained from the buccal lesions of two fowls
and cutaneous lesions of fowl No. 255, in which the lesions were produced from
the same original diphtheritic buccal material of fowl No. 228. The material
was gently removed with sterile forceps, and, after a mild wash in normal saline
solution, was pulped and emulsified with the same solution to make a 1 per cent
emulsion. The emulsion was left at room temperature before filtration for twenty
minutes for autolysis to take place. Filtrates were obtained through Cham¬
berland L. 3, Berkefeld ' V ' and Berkefeld ' N '. Every precaution was taken
to avoid leakage and contamination during the process of filtration. Healthy
fowls were inoculated with the filtrates, as detailed below in Table III and were
subsequently put to immunity test with the stock virus, with the result that
Berkefeld ' V ' filtrate was found to be viable whereas Chamberland L. 3 and
Berkefeld ' N' filtrates were found to be sterile.
Ind. Jour. Vety. Sci. & Anim. Husb., Vol. VI, Pt. IV.
PLATE XXI
Leg lesions produced in fowl No 255, from the
buccal material of fowl No. 229. (This fowl also
showed diphtheritic lesions in the buccal cavity.)
DIPHTHERITlC FORM OF FOWL-POX IN INDIA
319
TABLE III
Filtration Experiments
Serial
No.
1
2
3
4
5
Fowl
No.
282
305
306
218
247
Date of
inoculation
22nd Oct., 1935
19th Oct., 1935
Do. .
2nd Dec, 1935.
Do. .
Site of
inoculation
Feather follicles
and
buccal
mucous mem-
brane.
Feather follicles
Do. .
Do. .
DO.
.
Inoculum
Chamberland
L. 3
filtrate
(Buccal material
from fowl No.
256).
Berkefeld ' V ' fil-
trate (Cutaneous
material
from
fowl No. 255).
Do. .
Berkefeld 'N' fil-
trate
(Buccal
material
from
fowl No. 297).
Do. .
Reaction
Negative .
Died
on
28th Nov.,
1935 due
to other
causes.
P o s i t i ve
leg and
mouth
lesions.
Negative .
Do. .
Result
of immunity
test with
fowl-pox
virus (Wey¬
bridge
strain)
Reacted .
. . .
No
re-
action.
Destroyed
on 17th
Dec., 1935
for experi¬
mental
purposes.
Reacted .
Remarks.
Aviable.
?
Viable.
Aviable.
Do.
* The material in all the five cases was derived from a single strain (Buccal lesions of Fowl No. 228 which
reacted to specimen No. 397/1935).
DISCUSSION
From the results of the transmission experiments it is evident that the diph-
theritic form of fowl-pox is also observed in India, besides the cutaneous form,
and that the two forms are due to the same virus, which is immunologically
indistinguishable from our stock Weybridge strain of this virus.
Positive results with the Berkefeld ' V' filtrate of the material originally
obtained from the buccal cavity of fowl No. 228, exhibiting the diphtheritic
form of fowl-pox and inability of the micro-organisms normally inhabiting the
buccal cavity of fowls to produce such lesions without the help of the virus, in
spite of scarification, show that the above described lesions are due to a specific
virus.
With this particular strain of virus, buccal lesions developed almost invari-
ably, in addition to the cutaneous lesions, even when the material was inoculated
only on the feather follicles. But when the buccal cavity was inoculated with
the material it killed the bird rather quickly and no distant cutaneous lesions
were able to develop through generalisation, in spite of pulling out the feathers
and thereby injuring the feather follicles. It was only in one case (fowl No. 256)
320 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, IV
that the bird lived for about four weeks after the inoculation of the material
in the buccal cavity, possibly due to varying individual susceptibility, but in
that case the feathers had not been plucked and lesions were noticed only in the
mouth. The explanation appears to be that unbroken skin does not take the
virus from the body. Lesions of the oculo-nasal form of the disease were also
observed in subsequent passages of the same virus.
Doyle and Minett [loc. cit.] described the possibility of generalised symptoms
being set up by inoculation of the virus intravenously, subcutaneously and intra-
muscularly, although the lesions developed in a varying degree of severity.
In comparison with those cases where only localised cutaneous lesions deve-
loped, there was a greater tendency for fatal termination when generalisation
occurred, with the additional production of mouth lesions as was observed with
the strain under study. In view of its highly virulent nature it is being main-
tained at this Institute.
SUMMARY
(1) A survey of the past work on fowl-pox and several other allied condi-
tions, now known to be etiologically one and the same, has been made.
(2) Experimental transmission of diphtheritic lesions from the mouth of
fowls has confirmed that the causal agent, a filtrable virus, is the same as that
which produces fowl-pox lesions on the skin in India.
(3) The Indian strain of fowl-pox virus is immunologically indistinguish-
able from the Weybridge strain.
(4) There seems to be a tendency for generalisation with this particular
strain of the virus (obtained from Cannanore, Madras) although this has not
been seen in the case of specimens received from other provinces in India, so far.
(5) Filtration experiments have been conducted with this strain of the virus
and Berkefeld ' V' filtrate has given positive results unlike Chamberland L. 3
and Berkefeld ' N ' filtrates.
ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Mr. F. Ware, F.R.C.V.S.,
I.V.S., Director of this Institute, for the facilities so kindly afforded by him, and
to Mr. J. R. Haddow, B.Sc., M.R.C.V.S. D.V.S.M., I.V.S., Scrologist, for
the valuable criticism and helpful advice kindly given by him, during the pro-
secution of this work. Our thanks are also due to Mr. J. S. Rao, Artist of this
Institute, for the illustrations.
DIPHTHERITIC FORM OF FOWL-POX IN INDIA
321
REFERENCES
Arloing (1911-12). Amer. Vet. Rev., 15, 717.
Beach, A. B., Lothe, H., and Halpin, G. R. (1915). J. Inf. Dis., 17, No. 3, 554.
Brumley, O. V., and Snook, T. H. (1916). Ohio Sta. Uni. Bull., 20, No. 17.
Doyle, T. M., and Minett, F. C. (1927). J. Comp. Path. & Ther., 40, 247.
Hadley, P. R., and Beach, B. A. (1913). Proc. Amer. Vet. Med. Assoc., 704.
Haring, C. M., and Kofoid, C. A. (1911). Amer. Vet. Rev., 40, 717.
Jackley, J. G. (1918). J. Amer. Vet. Med. Assoc., 52, 853.
Jowett, W. (1909). J. Comp. Path. & Ther., 22, 22.
Mack and Records, (1915). Nev. Sta. Bull., 82.
Marshall, H. (1900). Rhode Island Station Report, 223.
Moore, V. A. (1885). Uni. Sta. Dep. Agri. Bull., No. 8, B. A. 1.
Ward, A. R. (1904). Quoted in Trans. 1st World's Poult. Cong., 1, 214.
OBSERVATIONS ON DOYLE'S DISEASE OF FOWLS
BY
R. N. NAIK, G.B.V.C.,
Veterinary Investigation Officer, Bombay Presidency
[Received for publication on 27th August, 1936]
Doyle's disease, which is commonly known in India as Ranikhet disease,
is a disease of the greatest economic importance as it ranks first amongst the
diseases of fowls in causing mortality. It was first recognised in the United
Kingdom by Doyle in 1926 and in India by Dr. Edwards in 1927. Since then
it has been found prevalent in certain other parts of the world ; Piecard found
it in Java in 1928, Rodier in the Philippine in 1929 and Ochi and Hashimoto in
Korea in the same year. In the Bombay Presidency the disease was first recog-
nised by me in the year 1932 and materials from the artificially infected fowls
were forwarded to the Imperial Institute of Veterinary Research, Muktesar, for
confirmation. It is also understood that the disease is very extensively pre-
valent in various parts of India. It is a fatal disease and whenever it breaks out
in a village it wipes out the major portion of the fowl population if not the whole
and as a result poultry-breeding industry is sustaining very heavy losses every
year.
Unfortunately no curative treatment or prophylaxis against the disease
has been found out as yet. All efforts made by different workers in this respect
have, so far, proved abortive ; Doyle [1927] tried heat-killed virus without suc-
cess ; he, however, experimented with an anti-serum prepared from ducks
and found that birds treated with it failed to contract the disease by contact
but died when artificially infected. Cooper [1931], on the other hand, succeeded
in protecting birds against artificial infection by injecting serum prepared from
immune fowls. But as the quantity of serum obtained from a fowl or a duck is
extremely small this contribution to the knowledge of immunology has served
no practical purposes in dealing with the disease. Doyle [1927] further found,
in Great Britain, that formalinised virus when applied by the subcutaneous route
afforded considerable protection against natural infection but not against arti-
ficial one, whereas all attempts made in India at the Imperial Institute of Veteri-
nary Research, Muktesar, to prepare such a product either by treating the vital
organs with chloroform or formalin or by desiccating them in vacuo have proved
of no avail.
In the lay press in India one frequently comes across reports, evidently
written by fowl-breeders having little knowledge of the disease, and medicines
claiming good results obtained by the oral administration of kerosine oil or some
other stuff. But it is needless to say here that they have proved utterly useless
in saving the life of affected fowls.
( 322 )