NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
Indian J. Vet. Sci. & Anim. Husb., Vol. XIII, Part I ]
PLATE
I
FIG. 1. Experimental Newcastle disease. Note ruffled feathers and crouched appearance.
FIG. 2. Experimental Newcastle disease. Note breathing through the mouth.
PLATE II
[ Indian J. Vet, Sci. & Anim. Husb., Vol. XIII, Part I
FIG. 1. Experimental Newcastle disease. Note paralysis of leg and wing,
closed eyes and oral breathing.
FIG. 2. Experimental Newcastle disease in a pigeon. Note paralysis of leg and wing.
FIG. 3. Experimental Newcastle disease in chicks.
Note the chick in extremis on the 2nd day after infection.
Indian J. Vet. Sci. & Anim. Husb., Vol. XIII, Part I ]
PLATE
III
FIG. 1. Two specimens of proventriculus of fowls
died of experimental Newcastle disease.
Note petechiae on the mucous membrane.
FIG. 2. Rectum, ileum and caeca of a fowl which died of
experimental Newcastle disease. Note the
extensive ulceration and the characteristic bran-
like deposit at the ileo-caecal valve.
FIG. 3. Pieces of intestine of a fowl which died of experimental Newcastle disease. Note the
catarrhal enteritis and haemorrhages.
PLATE IV
[ Indian J. Vet Sci. & Anim: Husb., Vol. XIII, Part I
1. 14 days old chicken embryo showing
minute sharply outlined haemorrhages
on the wings and legs
FIG. 2.
14 days old chorio-allan¬
toic membrane of a
developing chicken embryo
showing lesions of New-
castle disease (natural size)
FIG. 3. The lesions in Fig. 2 magnified.
Note the greyish white necrotic
areas on the chorio-allantoic
membrane representing areas of
ectodermal proliferation, vacuola-
tion & necrosis ×25 (See also
Fig. 5)
FIG. 4. Normal section of 14 days old chorio-
allantoic membrane of the developing
chicken embryo × 60
FIG. 5. Microphotograph of 14 days old Newcastle
disease infected chorio-allantoic membrane
showing ectodermal proliferation, vacuola-
tion and necrosis, and cellular infiltration
of mesoderm ×100
FIG. 6. Same section as in Fig. 5 but magnified showing vacuolation of the
ectodermal cells with inclusion-like bodies ×300
10
Studies on Newcastle (Ranikhet) Disease Virus
II. EXPERIMENTAL WORK
Purpose of work
From the foregoing it will be seen that there is no aspect of the problem of
greater importance than the control of the disease. The author had been
engaged on this investigation whilst on study-leave in Great Britain on deputa¬
tion from the Government of India. During a large amount of experimental
work with fowl-passage virus, in an attempt to produce a satisfactory immu-
nizing agent, no useful results were achieved.
General experimental procedure
The fowls used for the different experimental inoculations were males and
females of Rhode Island Red, White Leghorn and Light Sussex breeds, from
six months to two years of age. The birds were mostly bred at the Ministry's
Veterinary Laboratory, Weybridge, and it is remarkable that their susceptibil-
ity to experimental infection with passaged Newcastle disease virus was very
high. The birds, before coming in under experiment, were housed in fold units.
During periods when there was a shortage of healthy fowls, a few third-year
birds were included for certain inoculations without affecting the results.
After inoculation, the birds were kept in individual coops measuring about
3 × 3 ft. made of corrugated sheet iron unless otherwise stated. The usual
grower's mash and grain were fed. Water was given individually to birds in
separate galvanized trays. The dead birds were removed each day, post-
mortem examination made and the carcases incinerated. The coops in which
the birds had died were sterilized. In certain experiments, where infection
was intended to be conveyed through contact with soiled litter, food and water,
contaminated coops were retained and the experimental birds were left in
them. Many of the experiments were conducted during the winter months.
The virus contained in organs, such as liver and spleen and occasionally brain
from the experimentally infected fowls, collected after death, was preserved in
50 per cent glycerol saline and stored in the refrigerator 0.5°C. when not
required for use.
The standard virus, which has been referred to in the succeeding pages,
unless otherwise stated, was represented by a 1 per cent saline suspension of
equal parts by weight of liver and spleen from one or more fowls which had died
of experimental Newcastle disease virus infection. The emulsion was rendered
cell-free by spinning at 500 revolutions per minute for about three minutes and
the supernatant fluid was then pipetted off.
In experiments involving the use of egg cultivated virus, a 10 per cent
emulsion in saline or broth of infected chorio-allantois, or whole embryo
infected experimentally, was used as the material containing the virus.
Search for a vaccine
(a) Formalized vaccine. (i) Formalized liver and spleen vaccine. To liver
and spleen suspension of fowl-passage virus, formalin was added to a concen-
tration of 0.2 per cent and the mixture was attenuated at 37°C. for 72 hours,
during which period it was agitated by gentle shaking twice a day to distribute
the sediment uniformly. The vaccine was tested for bacterial contamination
S.
GANAPATHY
IYER
11
and, if found free, was inoculated into fowls. Two doses of 5 c.c. each were
injected into each fowl at a weekly interval; two fowls were injected by the
subcutaneous route ; two by the intramuscular route ; and two intravenously.
Results. As will be seen from Table I, the two fowls which received two
doses of the vaccine into the muscle both died, one seven days and the other 11
days after the last dose of the vaccine. The cause of death was Newcastle
disease. Since the birds died sometime after vaccination with the first dose,
it was presumed that the vaccine was free from any active virus. Moreover,
judging from the absence of reaction in the other fowls inoculated by other
routes with the same vaccine, the birds treated intramuscularly in all pro-
bability, picked up infection from the neighbouring coops and apparently, there
was no protection against contact infection conferred as a result of the vaccine-
treatment. The remaining four birds which survived the injection of the two
doses of the vaccine, died when tested for immunity 13 days after the last
dose of the vaccine.
Therefore, organ vaccine made by using formalin has no apparent protec-
tive value. While doing this work, the technique of Nakamura et al. [1937]
was followed as far as possible, as these workers reported favourable results
with a formalized vaccine. They found that the intravenous route was most
efficacious and double intravenous injections at an interval of five to seven
days gave immunity in 54 out of 87 birds 14 days after the last dose of
vaccine, whereas all controls died. These authors state that in spite of careful
preparation, the efficiency of the different batches of vaccine varied. The
success claimed by the above workers could not be obtained in the present
experiment.
TABLE I
Liver and spleen-formalin vaccine and comparative value of different routes
of inoculation. 5 c.c. inoculated on each occasion
Route
Subcutaneous .
Intravenous . .
Intramuscular .
Fowl No.
1895
1891
1896
1893
1894
1892
Dates of vaccination
16-11-39 and 23-11-39 .
Ditto . .
Ditto . .
Ditto . .
Ditto . .
Ditto
Result
Survived, tested immimity
on 6-12-39, died 5th
day
Ditto
Ditto
Ditto
Died 4-12-39, perhaps from
contact infection
Died 30-11-39, perhaps
from contact infection
(ii) Formalized brain vaccine. One part of 2 per cent formalin was added
to nine parts of 1 per cent brain suspension in saline representing fowl-passage
virus and the mixture was attenuated at 37°C. for three days. Soon after the
above period the vaccine was tested in fowl No. 2009. This fowl survived. An
attempt was made to reinforce the immunity, if any, by treating the bird with
0.1 c.c. of fowl-passage virus containing about 10 million M. L. D. on the 11th
day after vaccination. The bird died on the 5th day.
S.
GANAPATHY
IYER
13
was made by digestion with hot water, as the dye is difficult to dissolve. The
solution, after sterilizing in steam by the intermittent method, was stored in a
glass stoppered bottle at about 20°C. to avoid deposition of crystals. It was
seldom found necessary to filter the solution.
Buffer solution. Anhydrous dibasic sodium phosphate (Na 2 HPO 4 ) was
freed from moisture by freshly drying to constant weight at 110°C. A 3 per
cent aqueous solution was prepared and sterilized by autoclaving at 5 lb.
pressure for 15 minutes.
Vaccine mixture. With sterile precautions, one part of the crystal violet
solution and one part of the buffer solution were stirred into eight parts of
virus suspension. The mixture was inactivated at 37°C. for different periods
(Table II) ranging from 1 to 96 hours. The mixture was agitated by gentle
shaking twice a day during incubation to distribute the sediment uniformly.
Tests. The purity of the vaccine was determined by appropriate cultural
tests after diluting the vaccine so as to reduce the amount of the dye which
would inhibit bacteria especially Gram positive organisms. The safety and
potency of the different lots of vaccine mixture were determined in fowls.
All fowls which survived the vaccine-treatment were tested for immunity after
varying periods by inoculation with fowl-passage virus, or by coop-contact
infection. In a few cases, the presence of immunity in fowls was verified by
serological tests involving the injection of a susceptible fowl or developing chick
embryo with serum-virus mixtures.
TABLE II
Effect of crystal violet on Newcastle virus from fowl, by contact at 37°C. for
different periods
Expt
No.
I
II
III
IV
V
Contact
hours
24
48
72
96
96
1
6
18
24
24
18
24
42
48
48
44
46
48
46
Date of
inoculation
31-10-39
1-11-39
2-11-39
3-11-39
3-11-39
22-11-39
22-11-39
23-11-39
23-11-39
23-11-39
21-12-39
21-12-39
22-12-39
22-12-39
22-12-39
30-12-39
30-12-39
30-12-39
2 doses
25-1-40
and
1-2-40
Dose
1 c.c.
„ „
„
„
1 c.c.
„ „
„
„ 5 c.c.
„ „
„
„ „
„
1 c.c.
5 c.c.
Fowl
No.
1870
1829
1831
1884
1881
2001
1879
1873
2004
1872
2057
2056
2066
2068
2067
2075
2076
2077
2120
2121
2122
2124
2125
2123
2126
Result
Lived . .
Do . .
Do . .
Do . .
Do . .
Died 7th day .
Died 5th day .
Lived . .
Died 8th day .
Died 7th day .
Died 5th day .
Do . .
Died 4th day .
Lived . .
Died 3rd day .
Lived . .
Do . .
Died 3th day .
Died 9 March
1940
Died 11 March
1940
Died 4 March
1940
Died 9 March
1940
Died 13 Sep¬
tember 1940
Lived .
Died 5th day .
Immunity test
Date
17-11-39
17-11 39
17-11-39
17-11-39
17-11-39
...
... 30-11-39
... ...
... ...
... 18-1-40
... 18-1-40
18-1-40
...
...
...
...
...
...
18-3-40
...
Result
Died 4th day
Do.
Died 5th day
Died 3rd day
Died 4th day, control (virus plus
saline)
Died 5th day
(control, virus plus saline)
Died, 12th day (contact infection)
(control, virus plus saline)
(contact infection, died 9th day)
(contact infection, died 6th day)
(control, virus plus saline)
(perhaps contact infection)
Do
Do
Do
Do
Died 3rd day
(control, virus plus saline.)
14
Studies on Newcastle (Ranikhet) Disease Virus
Results
Experiment I. The results were nullified by the fact that the control
fowl (No. 1881) injected with the virus incubated for 96 hours survived and
later proved to be susceptible. Indeed, Newcastle virus seldom remains viable
for longer than 72 hours at 37°C. [Doyle, 1927 ; Iyer, 1939]. Treatment
with inert virus appears to confer no immunity.
Experimnet II. The results were not uniform. The fowl injected with
the vaccine-mixture incubated for 18 hours lived but later died when tested
for immunity ; whereas the fowl treated with the vaccine subjected to 24
hours contact died. This indicates, that a period of more than 24 hours
at 37°C. is necessary to attenuate the virus. A dose of 1 c.c. of the vaccine
was injected subcutaneously in this and in the previous experiment.
Experiment III. A dose of 5 c.c. of the vaccine was injected into each
fowl. The vaccine mixture, depending upon the length of exposure to in-
cubator temperature, either killed the fowl or failed to confer any definite
protection against infection by coop-contact. A period of contact at 37°C.
from 42 to 48 hours is perhaps the optimal time for the inactivation of the
virus in the presence of crystal violet solution.
Experiment IV. In this experiment, fowls which survived vaccination
proved susceptible to experimental infection by coop-contact. The actual
period when the virus changes from virulency to avirulency may be between
42 and 44 hours.
Experiment V. The vaccine was tested in six fowls. Two doses of 5 c.c.
each, per bird, were given at a week's interval. The control fowl which
was injected with the incubated virus without crystal violet died. Five
of the vaccinated fowls died over a month after the inoculation of the second
dose of the vaccine, presumably as a result of Newcastle disease. It was
believed that these fowls might have picked up infection from other experi-
mental birds which were all housed together, though in different coops. Fowl
No. 2123, which survived the vaccine treatment, died on immunity test.
There is little doubt, therefore, that the vaccine as used in this experiment
is valueless as an immunizing agent.
(ii) Effect of crystal violet on Newcastle disease virus at room atmospheric
temperature. The vaccine mixture was made as in the previous experiments
in which attenuation was effected at 37°C. In this experiment the vaccine
was stored inside a cupboard in a room where the atmospheric temperature
was about 70°F. Fowls were inoculated after 24, 48, 72 and 96 hours storage
of the vaccine. The results were irregular (Table III) in fowls surviving
vaccination as no immunity could be demonstrated against inoculation with
virus. Two fowls died of Newcastle disease following the vaccine treatment.
S.
GANAPATHY
IYER
15
TABLE III
Inactivation of Newcastle disease virus by crystal violet at room temperature
Contact
hours
24
48
72
96
96
Date of
Inocn.
31-10-39
1-11-39
2-11-39
3-11-39
3-11-39
Fowl
No.
1869
1828
1830
1880
1882
Result
Lived . .
Died 5th day .
Lived . .
Died 6th day .
Died 10th day .
Immunity test
Date
17-11-39
..
17-11-39
..
..
Result
Died 6th day
Died 4th day
Control, inoculated virus
plus saline
(iii) Crystal violet vaccine using brain tissue virus. As it was recogni-
zed that the nature of the tissues incorporated in the vaccine may
influence their immunizing value, one lot of vaccine was prepared from
brain virus. To eight parts of one per cent saline suspension of brain
tissue, from a fowl which had died of experimental disease, was added one
part of three per cent aqueous dibasic sodium phosphate solution and one
part of 0.5 per cent aqueous solution of crystal violet. The vaccine-mixture
was attenuated at 37°C. for 24 hours and then inoculated subcutaneously in
fowl No. 1874 on 23 November 1939. This fowl remained healthy until the
7th day ; when 0.1 c.c. of fowl-passage virus, representing about 10 million
M.L.D. was injected with a view to reinforcing the partial immunity if such
existed, the fowl died on the 4th day.
(c) Preparation of a vaccine by employing the chorio-allantoic technique
Rous and Murphy [1911] in their study of Rous sarcoma were the first
to make use of the chorio-allantoic membrane of the developing egg. Wood-
ruff and Goodpasture [1931] adopted this technique for the propagation of
viruses on the chorio-allantoic membrane of the hen's egg and were successful
in growing fowl pox virus. Burnet and Ferry [1934] have shown that the New-
castle virus is highly infective for the developing embryo. The virus after
having undergone at least twelve passages through eggs during three months
was found to be fully virulent for fowls.
Burnet [1933, 1934, 1935 and 1936] has given a detailed description of
an improved technique of chorio-allantoic membrane inoculation and of the
method of examination of the developed lesions.
This method of virus culture has been reported on during recent years
by Goodpasture, Woodruff and Buddingh [1932] and Kunert [1935], for
vaccinia ; Paschen [1936] for ectromelia ; Higbie. and Howett [1935], Beard
et al. [1938] and Lyon and Wyckoff [1938] for equine encephalomyelitis ;
Bengston and Dyer [1935] for Rocky Mountain spotted fever ; Brandly [1937]
and Beaudette and Hudson [1939] for infectious laryngo-tracheitis ; Gallo-
way [1937] for foot-and-mouth disease ; Peragallo [1937 and 1937, 1] for rabies
virus ; Hoffstadt and Pilcher [1938] for myxomatosum ; Kunert [1938] for
rinderpest ; Beaudette and Hudson [1938], Glover [1939] and Brandly and
Dunlop [1939] for bird pox virus; and Stevenson and Butler [1939] for the
preparation of a vaccine against smallpox.
18
Studies on Newcastle (Ranikhet) Disease Virus
made and centrifuged ; and the supernatant fluid removed and filtered through
Seitz discs at 10 lb. pressure. The filtrate was frozen and desiccated and
stored at 4°C. For vaccination this dried virus was rehydrated immedi-
ately before use. Reaction to the vaccine when it occurred was mild,
generally consisting of headache, low-grade fever and pains on the 6th or
7th day. No instance of delayed reaction to the vaccination has been
encountered. Antibodies may appear after the 7th and before the 21st days
and persist for at least one year. Under field conditions about 95 per cent
of vaccinated persons are reported to have acquired the immunity.
One of the questions to which Smith et al. [1938] gave consideration
in using any live virus for yellow fever vaccination was that of a possible
transmission of such virus by mosquitos, with perhaps a resulting increase
in virulence with repeated passage. Studies on monkeys and subsequent
observations on man, carried out in areas free from known vectors of yellow
fever, however, showed that the virus circulates in minimal quantities in the
blood, and that, in many cases, no circulating virus at all could be demons-
trated by the mouse test.
As a result of the extended observations with the egg cultivated New-
castle virus, there are indications which point to the fact that, owing to the
unknown nature and variation of the individual susceptibility of fowls, it is
logical to expect a small percentage of reactors which may occasionally be
fatal, especially among birds under 12 weeks of age. This point will be given
consideration in any further work.
Cultured Newcastle virus does not appear to revert to original virulence.
Referring to Newcastle disease, only in one instance was there at this stage
an indication to show that the cultured Newcastle virus did not contain any
harmful virus capable of returning to its original virulence and able to kill
fowls (Table IV). Fowl No. 2286 was killed five days after inoculation with
the egg cultivated virus and its spleen was sub-inoculated into a susceptible
fowl to ascertain whether or not the apparently modified virus could, under
favourable conditions, revert to its original virulence. This fowl remained
well and was shown to be successfully immunized.
Titration of egg virus. In a few limited observations, the end-point of
the egg cultivated virus was determined by the inoculation of eggs. It is
noteworthy that a dilution of 0.05 × 10 —6g killed the embryo in 48 hours.
TABLE IV
Infectivity of Newcastle disease virus cultivated through developing chicken
eggs for fowl (dose 1—5 c.c.)
Line I
Date of
inoculation
5-1-40
29-1-40
5-2-40
Fowl
No.
2025
2090
2130
2138
Inoculum
Passage 3, embryo emulsion
Passage 10, embryo emulsion
Passage 12, embyro incubated
72 hours 37°C.
Result and remarks
Died 5th day
Died 4th day
Died 4th day
S.
GANAPATHY
IYER
19
TABLE IV—contd
Date of
inoculation
7-3-40
2-5-40
6-5-40
23-5-40
23-5-40
27-5-40
24-6-40
10-7-40
10-7-40
22-7-40
22-7-40
26-8-40
26-8-40
26-8-40
26-8-40
26-8 40
26-8-40
26-8-40
26-8-40
26-8-40
26-8-40
Fowl
No.
2831
2830
2279
2290
2285
2286
2335
2253
2336
2338
2339
2341
2387
2388
2402
2404
2515
2516
2517
2518
2519
2520
2521
2522
2523
2524
Inoculum
Passage 19, embryo emulsion
Berkefeld V filtrate
Passage 19, embryo emulsion
Berkefeld V unfiltered
Passage 33, embryo emulsion
Passage 36, fresh embryo
emulsion
Passage 41, fresh embryo
emulsion
Passage 41, fresh embryo
emulsion.
Passage 41, incubated 72
hours 37 °C. and 24 hours at
0°C.
Passage 48, embryo emulsion
Passage 48, refrigerated 7
days
Passage 50, embryo emulsion
Passage 50, embryo emulsion
Passage 51, embryo emulsion
Ditto . . .
Passage 56, embryo emulsion
Ditto . . .
Ditto . . .
Ditto . . .
Ditto . . .
Ditto . . .
Ditto . . .
Ditto . . .
Ditto . . .
Ditto . . .
Result and remarks
Died 5th day
Died 9th day
Lived, tested immunity 16
May 1940, remained well
Lived, tested immunity 23
May 1940, remained well
Lived, tested
immunity 7
June 1940, remained well
Remained well, killed 5th day,
spleen sub-inoculated into a
healthy fowl which also re-
mained well, and proved
immune against fowl virus
test
Remained well, tested immunity
11 June 1940. Survived
Remained well, tested immunity
9 July 1940. Survived
Killed owing to fowl pox infec-
tion
Remained well, tested immunity
25 July, 1940. Survived
Died 8th day, other causes
Remained well, tested immunity
5 August 1940. Survived
Ditto
Remained well, tested immunity
12 September 1940.
Im-
mune
Ditto
Ditto
Ditto
Ditto
Ditto
Ditto
Died 7th day but no lesions of
Newcastle disease
Remained well, tested immunity
12 September 1940. Immune
Died 10th day but no lesions of
Newcastle disease
20
Studies on Newcastle (Ranikhet) Disease Virus
TABLE IV—contd
Line II
Date of
inoculation
24-6-40
10-7-40
22-7-40
12-9-40
12-9-40
12-9-40
12-9-40
12-9-40
12-9-40
12-9-40
12-9-40
12-9-40
12-9-40
Fowl
No.
2366
2389
2403
2405
2581
2582
2583
2584
2585
2587
2588
2590
2591
2592
Inoculum
Passage 12, embryo emulsion
Passage 14, embryo emulsion
Passage 16, embryo emulsion
Passage 21, embryo emulsion
Result and remarks
Died 7th day
Remained well, tested immunity
25 July 1940. Survived
Remained well, tested immunity
5 August 1940. Survived
Remained well, tested immunity
26 September 1940. Survived
Note. The control fowls inoculated with fowl virus at the time of the tests died
on the 3rd or 4th day.
(ii) Crystal violet vaccine for Newcastle disease using egg cultivated virus
Virus. The virus was represented by 10 per cent tissue suspension of
infected embryos in normal saline solution. Egg virus from different passages
was treated with crystal violet. As a result, while the strain of virus was the
same, the number of egg passages of the virus in the different lots of vaccine
varied. The whole embryo from two or more eggs was taken for the preparation
of the vaccine. The infection in egg takes what may be considered as a
generalized form and considerable quantities of egg cultivated virus became
available. Only eggs showing the characteristic lesions of Newcastle disease,
on the embryo in the form of minute, sharply outlined haemorrhages (Plate
IV, fig. 1) were selected for immunological work. The virus solution was
rendered cell free by centrifuging at 500 revolutions per minute for three
minutes and pipetting off the supernatant fluid. Only fresh virus was used
in all the lots of vaccine.
Vaccine mixture. With sterile precautions one part of 0.5 per cent
aqueous crystal violet solution and one part of 3 per cent sodium phosphate
solution were stirred into eight parts of virus suspension. The mixture
was inactivated at 37°C. for 72 hours. The vaccine-mixture was agitated
twice a day during incubation to distribute the sediment uniformly. The
method of preparation of the different lots of vaccine was identical.
Tests. The safety and the potency of the different lots of vaccine were
determined by inoculation in healthy fowls.
Immunity test. Fowls which did not die as a result of the vaccine treat-
ment were tested for active immunity against fowl-passage virus of known
virulence by inoculation, or by coop or pen-contact infection.