NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
September, 1958]
M. R. DHANDA et
al.
141
TABLE II
Results of potency tests in buffalo-bulls
Brew
No.
11
No. of
animals
2
5
4
7
18
Details of
vaccina ion
3 ml. intra-
muscularly
do.
do.
do.
Un-vaccinated healthy buffalo-bulls
2
Interval
between vacci-
nation and
challenge
85 days
67 days
30 days
19 days
Challenge dose
5 ml. of an 18
hour
old
broth cul-
ture of viru-
lent P. sep-
tica
do.
do.
do.
No. of
animals
survived
2
5
4
6*
17
0
Percentage
survival
100.0
100.0
100.0
100.0
0
Result
of bac-
teriolo-
gical exa-
mination
on animals
dying
after
challenge
—
++
REMARKS
One animal died
of causes other
than pasteurel-
losis.
Notes. 1. + = Positive for P. septica on bacteriological examination.
2. — = Negative for P. septica on examination.
3. An 18-hour old broth culture of virulent P. septica was used for challenge purposes.
Of this 1 ml. was estimated to contain 10,000 million mouse M. I. D
4. Adult buffalo-bulls were used throughout the above study.
5. All the 18 vaccinated buffalo-bulls were challenged simultaneously.
(a) Studies on the vaccine stored at Mukteswar at room temperatures
The potency of two different brews that had remained at room temperature
at Mukteswar for 11 months and one year respectively was tested in hill-bulls.
The results are presented in Table IV. These brews had also been earlier tested
on hill-bulls immediately after their preparation and the results obtained were later
utilized for comparison with those obtained in connection with tests carried out
for studying the keeping quality of the vaccine. It would be seen that although the
two tests were not strictly comparable owing to a difference in the dose of the vaccine
as well as of the virulent culture used for challenge and the varying periods of
interval between vaccination and challenge, yet the tests would furnish very useful
information regarding the comparative protective properties of the vaccine, especial-
ly, as in most of the later tests, a higher challenge dose was used resulting in the
stored vaccine being put to more rigorous tests.
142
IMMUNOLOGICAL STUDIES ON PASTEURELLA [Vol. XXVIII, Part III,
TABLE III
The results of potency test in mice
Brew No.
1
2
Un-vaccinated con-
trols
3
4
Un-vaccinated con-
trols
5
6
Un-vaccinated con-
trols
7
8
Un-vaccinated con-
trols
No. of
mice
used
40
40
10
15
15
6
15
15
5
15
15
5
Details of vaccination
0.25 ml. intramus-
cularly.
do.
do.
do.
do.
do.
do.
do.
Interval
between
vaccination
and
challenge
23 days
do.
21 days
do.
do.
do.
do.
do.
Challenge dose
100 M.I.D.
1000 M.I.D.
100 M.I.D.
100 M.I.D.
100 M.I.D.
1000 M.I.D.
100 M.I.D.
do.
do.
do.
do.
do.
do.
do.
do.
Result on
the
7th day
8 18
0 16
7 17
0/16
0/5
0/5
11/14
9/12
0/6
6/10
6/12
0/5
4/11
5/12
0/5
Percentage
survival
on the 7th
day
44.4
0.0
41.2
0.0
0.0
0.0
78.6
75.0
0.0
60.0
50.0
0.0
36.4
41.6
0.0
Result of
bacteriological
examination on
mice dying after
challenge
+ + + + + + + + + +
+ + + + + + + + + + +
+ + + + +
+ + + + + + + + + +
+ + + + + + + + + + +
+ + + + +
+ + + + +
+ + + + +
+ + +
+ + +
+ + + + + +
+ + + +
+ + + + + +
+ + + + +
+ + + + + + +
+ + + + + + +
+ + + + +
6
died
challenge
7
died
challenge
1
died
challenge
3
died
challenge
5
died
challenge
3
died
challenge
4
died
challenge
3
died
challenge
before
before
before
before
before
before
before
before
September, 1958] M. R. DHANDA et al.
143
9
10
Un-vaccinated con-
trols
11
Un-vaccinated controls
20
20
6
20
6
do.
do.
0.25 ml. intramus-
cularly
26 days
do.
21 days
do.
do.
do.
100 M.I.D.
Do.
4/15
6/17
0/6
9/19
0/6
26.7
35.3
0.0
47.4
0.0
+
+ + +
+ + + +
+ + +
+ +
+ +
+ +
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
5 died
challenge
3
died
challenge
1
died
challenge
before
before
before
Notes. 1. + = Positive for P. septica on bacteriological examination.
2. Numerators denote survivals. Denominators denote total number challenged.
3. An 18-hour old broth culture of virulent P. septica was determined to contain 10,000 million mouse M.I.D. Cre ml. of the required dilution was used for
challenge purposes.
4. Swiss mice about 12 weeks of age were used for testing all the 11 brews.
5. The mice were inoculated with the different brews within three to four days of preparation of each brew.
144
IMMUNOLOGICAL STUDIES ON PASTEURELLA [Vol. XXVIII, Part III,
TABLE
IV
The comparative results of potency tests carried out before and after storage of the adjuvant vaccine at room
temperature
Total
no. of
animals
used
38
Brew no.
4 & 5/55
(Mixed)
17
8/55
Details regarding
vaccination
2 ml. intremuscularly
immediately
after
preparation.
(No
storage)
2 ml. intramuscularly
after storage for 11
months at a tempera-
ture range of mini-
mum —2.2° C and
maximum 27.8°C.
3 ml. intramuscularly
immediately
after
preparation
(No
storase)
2 ml. intramuscularly
after storage for one
year at a tempera-
ture range of mini-
mum 2.2°C and maxi-
mum 20.5ºC.
Interval between
vaccination and
challenge
21 days
2½ months
5 months
6 months
1½ months
6½ months
3½ months
2½ months
Challenge dose
50 million mouse
M.I.D.
do.
do.
1000
million
mouse M.I.D.
500 milion mouse
M.I.D.
1000
million
mouse M.I.D.
1000
million
mouse M.I.D.
1000
million
mouse M.I.D.
Result
3/5
3/4
5/5
3/4
14/1S
4/4
2/3
6/7
2/3
5/6
Percentage
survival in
vaccinated
animals
60.0
75.0
1000
75.0
77.7
100.0
66.7
85.7
66.7
33.3
Reaction in
un-vacci-
nated
animals
(controls)
0/2
0/2
0/3
0/2
0/9
0/2
0/2
0/4
0/2
0/6
Percentage
survival
in controls
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Result of bacteriological
examination of animals
dying after challenge
Vaccinated
animals
+ +
+
+
+
+
+
Controls
+ +
+ +
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+
Notes. 1. +=Positive for P. septica on bacteriological examination.
2. Numerators denote survivals. Denominators denote total number challenged.
3. An 18-hour old broth culture of P. septica was used for challege purpose. Of this one ml. of 10- 1 was determined to contain 1.000 million mouse M.I.D.
4. Adult hill-bulls were used throughout the experiment.
146
IMMUNOLOGICAL STUDIES ON PASTEURELLA [Vol. XXVIII, Part III,
TABLE
V
Month-wise average daily temperatures
Month
March, 1955
April, 1955
May, 1955
June, 1955
July, 1955
August, 1955
September, 1955
October, 1955
November, 1955
December, 1955
January, 1956
February, 1956
March, 1956
April, 1956
May, 1956
June, 1956
July, 1956
August, 1956
Maximum
18.1°C
18.8°C
22.9°C
23.9°C
20.5°C
19.4°C
19.5°C
16.6°C
16.3°C
11.7°C
10.2°C
13.9°C
15.7°C
21.1°C
24.0°C
21.9°C
19.9°C
20.2ºC
Minimum
8.3°C
9.1°C
13.0°C
15.1°C
14.8°C
14.2°C
13.3°C
9.5°C
6.6°C
2.8°C
1.7°C
4.0°C
6.8°C
11.6°C
14.8°C
14.6°C
14.1°C
14.3°C
Note. Mixture of brews 4 and 5 was stored at room temperature from March. 1955 to January,
1956, and brew No. 8 was stored at room temperature from September, 1955 to August, 1956.
(b) Studies on the vaccine exposed to high temperatures
This problem was taken up in two parts ; firstly, the effect of exposure to high
temperatures in an electrically heated incubator was investigated and secondly the
vaccine after being transported over long distances during the hot summer months
was used for vaccination of animals in several endemic areas of haemorrhagic sep-
ticaemia, and the potency of the vaccine was judged by the protection afforded to
the vaccinated animals during outbreaks of the disease. The details of the investi-
gation are given below :
(i) Studies on the vaccine exposed to temperatures of 37°C and 40°C in an electrically
heated incubator. The effect of storage of the vaccine at temperatures of 37°C and
40°C for a period of ten days was investigated (Table VI). It would be seen that
exposure to temperatures of 37°C and 40°C for a period of ten days, the maximum
period for which the tests were carried out, did not indicate any signs of deteriora-
tion in its immunogenic properties.
September, 1958]
M. R. DHANDA, et al.
147
TABLE
VI
The comparative results of potency test carried out before and after exposure of the adjuvant
vaccine to temperatures of 40°C and 37°C
Total
no. of
ani-
mals
used
49
Brew
No.
11/56
Details regarding
vaccination
2 ml. intramuscularly
immediately after pre-
paration
*2 ml. intramucularly
after storage at ±4ºC
for two months and
ten days
*2 ml. intramuscularly
after storage for two
months at ±4 C and
for ten days at 40ºC
*2 ml. intramuscularly
after storage for two
months at ±4ºC and
for ten days at 37°C
Interval
between
vaccination
and
challenge
One month
Two months
do.
do.
Challenge
dose
1,000 million
mouse
M.I.D.
do.
do.
do.
Result
3/3
6/6
6/6
6/6
Percentage
survival in
vaccinated
animals
100.0
100.0
100.0
100.0
Reaction in
un-vacainated
animals
(controls)
0/5
0/6
Percentage
survival in
controls
0.0
Result of bacteriological
examination on animals
dying after challenge
Vaccinated
animals
Controls
+
+ +
+
+
+ +
+ +
+
+
NOTE 1. + = Positive for P. septica on bacteriological examination.
2. Numerators denote survivals. Denominators denote total number challenged.
3. *= challenged simultaneously and so common controls were used.
4. An 18 hour old broth culture of virulent P. septica was used for challenge purposes. Of this 1 ml. of 10— 1 dilution was determined
to contain 1,000 million mouse M. I. D.
5. Adult hill-bulls were used throughout the experiment.
6. Amber coloured bottles of 350 ml. capacity were filled in with 300 ml. of the adjuvant vaccine and stored in an electrically heated
incubator regulated to maintain the required temperature.
15 AR/58
7
September, 1958] M. R. DHANDA, et. al.
149
TABLE
VII
The results of field trials
Serial
No.
1
2
3
4
5
6
7
8
9
Place
Military Farm, Pimpri
Military Farm, Seeunderabad
Aarey Milk Colony
Military Farm. Kirkee
Six villages in tehsil Ranbirsin-
ghpora, Jammu State
Ten villages in Ludhiana Dis-
trict (Punjab)
Eight villages in Jullundur Dis-
trict (Punjab)
Eight villages in Hoshiarpur Dis-
trict (Punjab)
Six villages in Amritsar Dis-
trict (Punjab)
Date of vaccination
30-5-56 and 31-5-56
5-6-56
8-6-56 to 30-6-56
20-6-56
22-6-56 to 26-6-56
23-6-56 to 12-7-56
5-7-56 to 11-7-56
7-7-56 to 11-7-56
17-7-56 to 20-7-56
No. of
animals
vaccinated
929
769
16,687
948
1,892
1,743
1,893
1,931
1,739
Description of animals
Cattle and buffaloes,
including
young
stock
do.
do.
Cattle and buffaloes,
including European
and
European
cross-bred animals
Cattle and buffaloes,
including bulls and
young stock
Cattle and buffaloes,
including
young
stock
do.
do.
do.
Observation period
30-5-56 to 31-12-56
5-6-56 to 31-12-56
30-6-56 to 31-12-56
20-6-56 to 31-12-56
26-6-56 to 28-2-57
12-7-56 to 31-12-56
11-7-56 to 31-12-56
11-7-56 to 31-12-56
20-7-56 to 31-12-56
No. of deaths due to pasteurellosis
among the animals vaccinated with
the adjuvant vaccine
No deaths reported due to pasteurellosis
No deaths reported due to pasteurellosis
Only one death reported due to pasteu-
rellosis in a buffalo calf which was
about seven months old at the time of
vaccination
No deaths reported due to pasteurellosis
No deaths reported due to pasteurellosis
No deaths reported due to pasteurellosis
No deaths reported due to pasteurellosis
No deaths reported due to pasteurellosis
Only one death due to pasteurellosis was
reported
September, 1958]
M.R.DHANDA,
et.
al.
151
to be very satisfactory. The comments of the Chief Veterinary Officer of one of
the farms, where as many as 16,687 animals were vaccinated this year are reproduced
below :
" Last year also we were very lucky not to have any incidence of the disease
but previous years were fraught with frequent appearance of haemorr-
hagic septicaemia. I am inclined to believe that the use of adjuvant
vaccine has yielded substantial dividend. So far as the Colony is con-
cerned I wish to adopt this vaccine for routine use hereafter in pre-
ference to the conventional broth vaccine. "
Field trials under village conditions were carried out in the States of Jammu
and Kashmir and the Punjab. In the State of Jammu a total of 1,892 animals
(cattle, buffaloes and young stock) in six villages of tehsil Panbirsinghpora were
vaccinated with the adjuvant vaccine and another group of 1,245 animals with the
commercial broth vaccine. During an observation period of eight months, no deaths
due to pasteurellosis were reported from either group.
In the Punjab, 32 villages in the endemic areas of haemorrhagic septicaemia
were selected and divided into two groups, group I consisting of villages where all
the animals were vaccinated with the new adjuvant vaccine and group II consisting
of villages where some animals were vaccinated with the new adjuvant vaccine and
the remaining animals with the commercial broth vaccine prepared at Hissar.
Incidently, two more groups also got formed viz. group III comprising animals
in villages where some animals were vaccinated with the new adjuvant vaccine and
the rest remained un-vaccinated as the owners were not willing to get their animal
immunized and group IV consisting of animals in villages where all the animal
remained un-vaccinated due to lack of cooperation on the part of the owners. In
all, as many as 7,306 animals in 32 villages of the districts Ludhiana, Jullundur,
Hoshiarpur and Amritsar were vaccinated with the new adjuvant vaccine (Table
VII).
The value of the new adjuvant vaccine was judged from the fortnightly reports
received from the District Animal Husbandry Officers of the districts concerned
regarding the number of deaths in the various groups due to pasteurellosis. The
reports were examined by Dr. Tulsa Ram, Veterinary Investigation Officer, Punjab,
who also personally investigated the efficacy of the new adjuvant vaccine. The
observations made by him till the end of December, 1956 i.e. over a period of five
months during which most outbreaks of haemorrhagic septicaemia usually occur,
in these areas are reproduced below :
(a) Veterinary Hospital, Phillaur. Four villages (Raipur Araian, Selkiana,
Kot Palow, Jhugian Mansingh) remained free of haemorrhagic septicaemia out-
breaks. In these villages almost all animals were vaccinated with the adjuvant
vaccine.
In the village Lassara, only 126 animals were vaccinated out of more than 2,000
animals. Four deaths due to haemorrhagic septicaemia occurred among un-vaccinat-
ed animals during this period. Fourteen deaths due to haemorrhagic septicaemia
152
IMMUNOLOGICAL STUDIES ON PASTEURELLA [Vol. XXVIII, Part III,
were reported from three villages out of five where vaccinations were not carried
out. These were situated within a radius of two miles of above mentioned villages.
In three villages within this radius where broth vaccine (Hissar) was used, three
deaths were reported to have occurred in two villages among the un-vaccinated
animals.
(b) Veterinary Hospital, Shahkot. Three villages where adjuvant vaccine was
used remained free of the disease. Out of 14 surrounding villages within a radius
of two miles, in which no vaccination was carried out, deaths due to haemorrhagic
septicaemia occurred in 11 villages.
(c) Veterinary Hospital, Hoshiarpur. Villages Nasrala and Ghabawal. In
these villages about half the number of animals were vaccinated and half were
left out.
In the former village two and in the latter four deaths occurred among the un-
vaccinated stock. Deaths due to this disease were also reported from 14 out of 26
surrounding villages within a radius of two miles. In six other villages, where almost
all cattle and buffaloes were vaccinated with the adjuvant vaccine no deaths due
to the disease were reported.
(d) Amritsar district. No deaths due to haemorrhagic septicaemia occurred
in villages Wallah, Tungpain, Kot Khalsa, Tandel and Verka among the stock
vaccinated with the new adjuvant vaccine, but six deaths occurred among un-
vaccinated animals in village Wallah. In village Bhopa Rai, two deaths due to
haemorrhagic septicaemia occurred after vaccination. One of these was an un-
vaccinated animal left out due to owner's carelessness while the rest of his stock
was immunised. Among the vaccinated stock also one death occurred within two
months after vaccination. It was verified on the spot that this animal showed
clinical symptoms of the disease and died within 12 hours after the onset of the
disease. However, no laboratory confirmation was obtained.
Judging from the above investigations carried out on animals on organised
farms as well as under village conditions in areas where haemorrhagic septicaemia
is known to be endemic, it would appear that the new adjuvant vaccine is quite
satisfactory in affording protection to cattle and buffaloes against haemorrhagic
septicaemia and that in this respect it is far superior to the commercial broth vaccine.
The fact that only one injection of the new adjuvant vaccine is enough to protect
animals for a full year as against two or three of the commercial broth vaccine as
repeated inoculations of the latter vaccine are often recommended in order to get
a more lasting immunity, coupled with the fact that the immunity conferred by the
new adjuvant vaccine is much stronger, has stimulated investigations of its large-
scale production to meet the country's demand of the product. Consequently,
the Indian Council of Agricultural Research has sanctioned a scheme for this investi-
gation to be undertaken at Izatnagar.
SUMMARY
1. A procedure for the manufacture and standardisation of haemorrhagic
septicaemia adjuvant vaccine has been laid down.
September, 1958]
M.R.DHANDA,
et.
al.
153
2. Large scale field and laboratory trials have shown that the adjuvant vaccine
is absolutely safe for cattle and buffaloes of all ages and that in its immunizing
properties it is far superior to the broth vaccine.
3. The studies carried out on the keeping quality of the adjuvant vaccine
indicate that it does not deteriorate in its protective properties for at least one year
when stored at room temperatures ranging from — 2.2ºC to 29.5°C. The vaccine
can also withstand fairly high temperatures and can be safely transported under
ordinary conditions of rail and bus journey, to far off places covering a journey
period of about ten days during the hot summer months.
4. An immunity test carried out on five animals at an interval of nine months
and 21 days following vaccination showed that all the animals were solidly immune.
ACKNOWLEDGEMENT
The authors thank Shri P. C. Gangulee for technical assistance, and Dr. Tulsa
Ram, Veterinary Investigation Officer, Punjab, Brigadier H. L. Bhandari and Col.
Nayar of the Remount, Veterinary and Farms Corps, Shri K. N. Trisal, Veterinary
Investigation Officer, Jammu and Kashmir, Mr. D. N. Khurody, Milk Commissioner,
Bombay and S. S. M. R. Marathe and N. R. Adkar of the Aarey Milk Colony for
co-operation in field trials. They are also thankful to Shri C. Seetharaman, Head
of the Division of Biological Products, Indian Veterinary Research Institute, Izat-
nagar, for arranging immunity trials with the vaccine on buffalo-bulls and to Shri
S. Sen for the statistical analysis of the data.
APPENDIX I
Procedure for the preparation of haemorrhagic septicaemia adjuvant vaccine
1. Strain. Culture of P. septica stock strain 52 in phase I will be used as
seed.
To maintain the culture in phase I the stock strain is passaged through a sus-
ceptible bovine or a buffalo calf.
(i) Inoculate the animal subcutaneously with one ml. of 18—24 hours old broth
culture.
(ii) Isolate the strain from heart blood of the animal after death, which should
occur within 36-48 hours, by plating on blood agar or tryptose agar petri plate.
(iii) Transfer discrete colonies having a greyish white colour to blood agar or
tryptose agar slants. Such cultures should have their morphological, cultural and
biochemical characters checked which should conform to P. septica, Roberts
154
IMMUNOLOGICAL STUDIES ON PASTEURELLA [Vol. XXVIII, Part III,
group I. Such slants serve as "Seed Bank". Stored in a refrigerator, the culture
on blood agar or tryptose agar slants will remain viable for about two months while
in a deep freeze cabinet, it will remain viable for upto six months.
2. Media. The following formula provides a medium which gives an abundant
growth of the organism :—
Lab Lemco.
Bacto-Peptone, Difco.
Yeast extract (Bacteriological)
Creatinine
Sodium chloride
Agar
Distilled water
3 gm.
3 gm.
1.5 gm.
0.3 gm.
1.5 gm.
9 gm.
300 ml.
Dissolve Lab Lemco in the distilled water. Add peptone, sodium chloride,
agar and melt in running steam for ½ hour. While hot, adjust pH to 7.6. Allow
to cool at 45°C to 50°C. Add white of two eggs per litre of the medium. Transfer to a
steamer and keep for ½ hour. Take out, filter through filter paper, keeping the
container in the autoclave under running steam when a clear filtrate is obtained.
Adjust pH to 7.6, add creatinine and yeast extract and mix. Usually there is no
shift in pH, but, if necessary, adjustments may be done. Transfer 110-120 ml. per
roux flask of one litre capacity.
Autoclave at 15 lb. for ½ hour. The final pH of the medium should be 7.4.
3. Seed culture. (i) Inoculate nutrient broth tubes with phase I culture from
'Seed Bank' tubes.
(ii) Incubate for 18 hours at 37°C and check for purity by smear examination.
4. Inoculation of roux flasks.—(i) With a pipette, transfer 1-2 ml. of broth seed
culture into each roux flask and spread uniformly by a to and fro movement.
(ii) Transfer the flasks to an incubator and stack with agar surface facing down-
ward.
(iii) Incubate at 37°C for 18 hours.
5. Harvesting. (i) Before harvesting examine growth in each flask and discard
those showing evidence of contamination.
(ii) Transfer about 10 ml. of 0.5 per cent formol-saline to each flask; wash the
growth by rolling the flask to and fro until all the growth has gone into suspension.
(iii) Pipette off the bacterial suspension from each flask into a sterile measuring
cylinder.
(iv) Dilute the pooled suspension with more of 0.5 per cent formal saline till an
opacity comparable to 6-7 of the Burroughs Wellcome Standard opacity tubes is
obtained.
(v) Keep overnight at room temperature. The temperature of the room must
not exceed 25°C.