NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
385 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, IV
It seemed clear therefore that in this experiment rinderpest failed to spread as
a highly contagious disease as it had been expected to do. The failure might be
explainable as due either to too small a proportion of affected animals; too short a
period of contact; or because the affected animals were not themselves in the most
highly infective stage of the disease. The subject thus clearly required further
study.
During the interval which occurred before a further experiment was started, it
is of interest to record that on two separate occasions single highly susceptible
hill-bred cattle were placed in a position that would seem to ensure natural infec-
tion, if rinderpest were the highly contagious disease it is considered to be.
Rinderpest virus has been kept alive at Muktesar for purposes of serum pro-
duction, " serum-simultaneous" inoculations, and for experimental uses, for a
period of certainly not less than 30 years. It may be noted here that obviously a
single " strain " of virus has not been maintained during this long period of time.
Also that there is no evidence available to indicate that the virus used at Muktesar
behaves differently in any way from " field " strains, except, perhaps, that by con-
stant passage a virus attains a more uniform virulence. It is only possible to
maintain living virus by constant passage in susceptible cattle, and the hill-bulls
used at Muktesar are believed to be as susceptible as it is possible to obtain cattle
in India. Passage inoculations have been made from animal to animal in one par-
ticular shed set aside for the purpose at Muktesar during the whole of the time
mentioned above, and the shed is constantly filled with cattle exhibiting all stages
of rinderpest infection, since they remain in the shed from the time they receive an
injection of virulent blood until they die, or completely recover. The shed is
certainly divided into separate stalls, but the partitions consist only of a very light
wooden framework.
The two hill-bulls referred to were placed at different times in this shed in the
ordinary routine course to receive an injection of virus, going directly into stalls
where the previous occupants have died of acute rinderpest. In both cases, how-
ever, virus injection was withheld for about ten days, and, as no evidence of
natural infection was exhibited by them, they were then injected with rinderpest
virulent blood, when both bulls reacted normally.
These observations added further evidence indicating unexpected difficulty in
setting up rinderpest by natural means, and the experiments recorded below were
accordingly carried out at later dates.
THE FIRST EXPERIMENT.
This experiment was planned to find out definitely whether rinderpest infection
could be transmitted by direct contact alone between affected and healthy cattle
RINDERPEST : TRANSMISSION BY CONTACT.
386
under experimental conditions; and if so, to obtain a preliminary idea of the
number of days of contact necessary.
It started on the 10th August 1929, and the proportion of affected to healthy
animals was adjusted to be an equal number of each. Thus on this date six hill
bulls were injected with rinderpest virus and turned loose in an enclosure within
one of the sheds used ordinarily for such experimental work at Muktesar, and on
the same day another six healthy, uninoculated hill bulls (called Lot A) were
turned loose with them in the same enclosure, and daily observations were made
upon them all. The enclosure consisted simply of a portion of the shed with the
partitions removed to form a " loose box ", in which the cattle were confined only
by posts and rails. They were left untied, and were fed and watered from common
troughs. After three days of this close contact, two of Lot A (Lot A-1) bulls
were removed, and observations were continued upon them thereafter in strict
isolation elsewhere. On the same day, namely, the third day of the experiment,
two new healthy hill bulls, Lot B, were turned into the enclosure to replace the two
which had been removed. Three days later again a similar change was made,
when two more of Lot A (Lot A-2) bulls were removed to isolation, and two new
bulls, Lot C, replaced them. Contact between the artificially infected and the
healthy bulls was maintained for a total period of sixteen days. As soon as one
of the infected animals died, one of the healthy cattle was removed to isolation in
order that an equal number of each should be maintained during the whole
experiment.
The artificially infected animals used in the experiment all reacted to the virus
injection given on the 10th August 1929, although two of them somewhat mildly,
ending in recovery. The other four bulls all showed severe and typical reactions
and died of rinderpest, one after seven days and the other three after eleven days.
Table I summarises the results obtained in the healthy animals that were in-
contact during the experiment, and they proved that it was possible to transmit
rinderpest by the method of close contact alone that had been used. Thus, hill
bull No. 595 of Lot A-2 died of the disease resulting from infection gained while in
contact; and both hill bulls No. 488 of Lot A-3, and No. 475 of Lot B reacted
typically, and, although they recovered from the attack, they were both solidly
immune to a subsequent immunity test when virulent blood was injected.
The results also showed clearly, however, that even with an equal number of
affected and healthy cattle kept in the closest possible contact, rinderpest definitely
failed to develop in as many as half the number of animals used in the experiment.
Failure in this respect was certainly to be expected in the case of the pair Lot A-1,
since they were in contact only for three days of the incubation period in the ex-
387 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, IV
perimentally infected animals. In the case of hill bulls No. 589 of Lot A-2, No. 528
of Lot B, and No. 343 of Lot C, however, which had been in contact for 6, 9 and 10
days respectively and failed to contract the disease although later proved to be
susceptible to it, there is further clear evidence of difficulty in establishing rinder-
pest infection by simple contact.
The results in the case of the two remaining animals are not so clearly apparent
and require some explanation. The mild fever exhibited by hill bull No. 601 of
Lot A-3 was unaccompanied by other symptoms suggesting rinderpest. It was
solidly immune to the subsequent virus injection and the probability is that it was
originally immune before the experiment began. Hill bull No. 516 of Lot C, on the
other hand, exhibited a mild attack of fever during the period of contact which was
accompanied by diarrhœa and thus resembled more nearly a rinderpest reaction.
Since, however, the reaction which occurred later in the immunity test resulted in
death diagnosed post mortem as due to rinderpest, it must be concluded that the
first attack was some disturbance other than rinderpest.
Since the results in these two animals were not clearly decisive they are better
excluded for the main interpretation of the experiment. Excluding also the pair
Lot A-1 already referred to, the results in the remaining six bulls after contact with
an equal number of affected animals can be tabulated as follows:—
Serial
No.
1
2
3
4
5
6
Hill bull
No.
595
488
475
589
528
343
In contact
for days
6
9
10
6
9
10
during period
0—6th
0—9th
3rd—13th
0—6th
3rd—12th
6th—16th
Result
Contracted
Rinderpest
Escaped
infection
Immunity test result
Dead before test
Proved immune
Proved susceptible and develop-
ed Rinderpest
The pair of animals Nos. 1 and 4 in the above Table are identical; and the pair
Nos. 2 and 5 were in contact for the same number of days, yet the chances of in-
fection were in favour of the one (hill bull No. 528) which did not contract the
disease, because the first three days in the case of the other bull have already been
admitted to be incubation period. Since No. 6 in the Table only came into contact
as late as the sixth day it is not exactly comparable with No. 3, but even if exclud-
ed it still leaves the figures showing that forty per cent. of the bulls where decisive
RINDERPEST: TRANSMISSION BY CONTACT. 388
results were obtained failed to contract infection by contact in what would appear
to be ideally favourable conditions.
The three bulls which definitely contracted infection showed symptoms of the
disease after 6, 9 and 10 days of contact, or, deducting an incubation period of three
days, infection probably took place only on the 3rd, 6th, and 7th days of contact.
THE SECOND EXPERIMENT.
The next experiment as originally planned was to extend over a longer period of
time, namely, until all the healthy animals had become infected by contact. No
means of closer contact could be devised, or seemed necessary since they were
already considered to be more continuously intimate than any normal village
conditions.
In this experiment healthy hill bulls were set free in similar enclosures to the
one used in the previous experiment, and artificially infected cattle were brought
to them in order to establish contact. Three such enclosures within separate sheds
were prepared, and six healthy hill bulls were placed in each of the first two, while
three bulls only were put in the third enclosure.
A supply of susceptible animals artificially infected with rinderpest was main-
tained by injecting virus every fifth day into a batch of three hill bulls. The first
batch of these infected bulls was turned loose with the six healthy cattle in the first
shed (Shed A) immediately after having received virus, and they remained there in
contact until the morning of the fifth day. On the fifth day this first batch of
infected cattle were transferred to contact with the second lot of healthy animals in
Shed B, while a newly infected batch of bulls took their place in Shed A. On the
tenth day batch No. 1 of infected bulls moved from Shed B into contact with the
third lot of three healthy bulls, in Shed C, and a similar move was made from Shed
A into Shed B, when the third batch of newly infected bulls came into the first
Shed A. This succession of transfers was carried out in a similar manner on every
subsequent fifth day during the whole course of the experiment. In the last Shed
C only three healthy bulls were kept for the reason that it was expected that the
full number of affected bulls would not survive long enough to reach them. At the
end of each 10th to 15th day period spent in Shed C, the surviving affected bulls
were removed elsewhere for final observations.
The main information sought in this experiment was the most infective period
of rinderpest infection from contact, and the plan adopted divided the experiment
into three periods, as follows :—
1. 0 to 5th day period in Shed A.
2. 5 to 10th day period in Shed B.
3. 10 to 15th day period in Shed C.
389 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, IV
The experiment began on the 30th June 1930, when the first batch of artificially
infected bulls received their injection of rinderpest virus, and ended on the 3rd
September 1930, when selected survivors were tested for immunity, or a total
period of contact lasting for 68 days.
The artificially infected animals used in the whole experiment consisted of
eleven batches of three hill bulls each, or 33 in all. The five-day periods in the
rinderpest syndrome were distinguished as follows :—
Lot A—0 to 5th day period.—Incubation period of three days, and 3 or 2 days of
high fever, without other symptoms.
Lot B—5th to 10th day period.—Falling temperature; impaired appetite; diarrhœa
and the appearance of specific lesions, consisting of vesicles and ulcers on the buccal
mucous membrane. Seven of the 33 bulls died during this period, or 21.2 per cent.
Lot C—10th to 15th day period.—Normal or sub-normal temperature ; loss of
appetite ; diarrhœa; mouth lesions advanced and beginning to heal; death or
commencing convalescence. Eight of the 26 bulls which reached this shed died
during the period of contact, or 30.9 per cent.
Nine of the bulls died subsequently from the 16th to 33rd day, and nine only
finally survived and recovered. Thus, a total number of 24, or 72.7 per cent. of the
33 artificially infected bulls, died during the whole course of the experiment.
The results of the experiment in the healthy animals used are summarised in
Table II.
It is to be noted that the symptoms described above under each five-day period
can only be referred to the time until the first healthy animal in each lot became
affected with rinderpest, because no steps were taken to remove the " healthy "
animals when they showed evidence of having become infected, and the full course
of the disease in them took place in the presence of the remaining healthy animals
in that lot. In view of this fact examination of the first animal to become infected
by contact in each lot is of particular significance. Such examination suggests at
once that the second period, Lot B, is the most infective of the three periods,
since the first animal in that lot became infected as early as the fourth day of contact,
whereas in Lot A infection did not occur until the twelfth day, and no animal at
all became infected in Lot C. That the second five day period is the most infective
is also borne out by the fact that four animals in Lot B became infected before the
twelfth day of contact, that is, before the first animal contracted the disease in Lot
A.
Again in this experiment it is ne-essary to exclude one animal from considera-
tion. Hill bull No. 45 in Lot A showed only mild fever without other symptoms
RINDERPEST : TRANSMISSION BY CONTACT.
390
as late as on the 43rd day of contact. This animal proved to be solidly immune
when submitted to a test with rinderpest virus subsequently, and it'is at least highly
probable that the bull was immune before the experiment began, and the mild fever
was probably due to some cause other than Rinderpest.
If hill bull No. 45, therefore, is excluded from consideration, it is seen that the
last- bull in each Lot A and B became infected after approximately one month (31
and 33 days respectively) of contact. That infection can be so long delayed in
highly susceptible cattle in close contact with others showing typical rinderpest is a
striking fact, and greatly at variance with the usual conception of the disease.
Another particularly striking result is in Lot C, where three highly susceptible
cattle, as proved in the subsequent immunity test, were in close contact continuously
with either one, two or three animals at a time when they were showing advanced
symptoms and, lesions of rinderpest for as long a period as 68 days and not one of
them contracted the disease.
SUMMARY AND CONCLUSIONS.
In the experiments described rinderpest spread by direct contact from artificially
infected to healthy susceptible cattle at a. surprisingly slow rate.
It required contact for a minimum period of three days, but the average length
of contact before infection occurred was far greater. In a total number of fourteen
healthy-cattle used in two experiments which were exposed to various stages of
the disease, the average contact period was just over fifteen days; and the longest
time a susceptible animal remained in contact before becoming infected was 33 days.
The most contagious period in the rinderpest syndrome was found to be between
the 5th and 10th days after artificial infection. Spread of infection was longer
delayed from contact before the 5th day, and from the 10th to 15th day the
disease failed to spread to any one of three susceptible animals exposed to it, even
after 68 days of contact.
Until the results of a further series of controlled experiments are known, in
which the protective effects of " serum alone " immunisation are actually tried out
against rinderpest transmitted by contact, it is perhaps early to draw conclusions
on that point. Since, however, in the experiments described here the disease spread
only as a slowly progressive one, its control by " serum alone " must at best be a
prolonged and therefore tedious and expensive procedure.
F
391
THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [II, IV
TABLE I.
The First Experiment.
Lot
No.
A. 1
A. 2
A. 3
B.
C.
In contact
days
3
6
9
16
9
10
6
10
period
0 to 3rd
0 to 6th
0 to 9th
0 to 16th
3rd to 12th
3rd to 13th
6th to 12th
6th to 16th
Hill bull
No.
586
535
595
589
488
601
528
475
516
343
Estimated
infected day
of contact (a)
.....
.....
4th
7th
7th
.....
6th
1st
.....
Reaction
Nil
Nil
Fever, diarrhœa
and vesicles
Nil
Fever, diarrhœa
and vesicles
Mild fever
Nil.
Fever, diarrhœa
and vesicles
Fever and diarr-
hœa
Nil
Result
Escaped
infec-
tion
Escaped
infec-
tion
Died 12th day—
Rinderpest
Escaped infection
Recovered
Doubtfullv infec-
ted
Escaped infection
Recovered
Recovered
Escaped infection
Immunity Test
Reaction
Fever, diarrhœa
and vesicles
Fever and vesi-
cles
....
Fever, diarrhœa
and vesicles
Nil
Nil
Fever and vesi-
cles
Nil
Fever and diarr-
hœa
Fever and vesi-
cles
Result
Died 17th day—
Rinderpest
Recovered
....
Died 10th day—
Rinderpest and
Redwater
Immune
Immune
Recovered
Immune
Died 10th day—
Rinderpest
Died 11th day—
Rinderpest and
Redwater
(a) i. e., three days before initial rise in temperature
RINDERPEST : TRANSMISSION BY CONTACT.
392
TABLE II.
The Second Experiment.
Hill
bull
No.
Estimated infected (a)
Day of
contact
During batch
Reaction
Result (b)
Immunity test
Lot A—contact 0 to 5th day
18
273
34
40
1703
45
12th
19th
21st
22nd
31st
43rd
2nd day of 3rd batch
4th „
4th „
1st „
5th „
2nd „
5th „
1st „
7th „
3rd „
9th „
Typical with vesi-
cles
ditto .
ditto .
ditto .
ditto .
Mild fever only .
Died 11th day—
Rinderpest
Died 15th day—
Rinderpest
Recovered
Recovered
Died 6th day—
Rinderpest
Doubtfully infect-
ed
No reaction
Immune
Lot B—contact 5th to 10th day
3
227
228
253
250
1980
4th
6th
8th
11th
22nd
33rd
4th day of 1st batch
1st „
2nd „
3rd „
2nd „
1st „
3rd „
2nd „ 5th „
2nd „
7th „
Typical but no
vesicles
Typical with vesi-
cles
ditto .
ditto .
Fever and diar-
rhœa
Typical with vesi-
cles
Died 11th day—
Rinderpest
Died 13th day—
Rinderpest
Recovered
Recovered
Recovered
Died 14th day—
Rinderpest
No reaction
Immune
Lot C—contact 10th to 15th day
15
293
73
..
..
..
....
....
....
Nil ....
Nil . .
Nil .
Escaped infection
Died l1th day—
Rinderpest
Died 20th day—
Rinderpest and
pneumonia
(a) i.e. three days before initial rise in temperature
(b) Day of death after estimated infection
VIABILITY OF ANTHRAX BACILLI AND SPORES AFTER
FIXATION AND STAINING
BY
S. M. SARWAR, M.R.C.V.S.,
Veterinary Investigation Officer, Punjab.
(Received for Publication on 17th March, 1932.)
The great resistance of the bacilli and spores of B. anthracis has been recog-
nised since the early days of Bacteriology.
From time to time various workers have demonstrated the resistance of these
bacilli and spores to heat and chemicals under many diverse conditions.
With this fact in view, experiments were carried out in order to ascertain the
degree of danger to which a laboratory worker is subjected in handling anthrax
slides which have been fixed by heat and stained according to the usual laboratory
methods.
The results were somewhat unexpected as it was demonstrated that anthrax
organisms could be cultivated by inoculating agar tubes with scrapings from fixed
and stained slides.
As a result of this, a series of experiments was carried out to determine the
resistance to the heat of a bunsen flame of anthrax bacilli and spores in the form
of a smear on a glass slide. The experiment is divided into 3 parts.
The first part deals with the resistance of the bacilli to fixation and staining
with aqueous solutions of methylene blue.
The results of this experiment show that it is necessary to pass a blood smear
at least 4-5 times over the bunsen flame, necessitating a temperature of 220°C to
kill the bacilli. The temperature of 220°C can be attained only if the slide is
passed over the flame in such a manner as to take one second for each passage.
This procedure is effective only in the case of blood smears which have been
prepared from the heart blood of an anthrax carcase, in which case the bacilli have
not had an opportunity of forming spores.
The smears should be fixed immediately after preparation by the above method.
Mere drying of the smear does not seem to inhibit anthrax spore formation, as
shown by the following experiment.
A guinea pig was killed by inoculation with anthrax culture. Immediately
after death about 20 blood-smears were prepared from the heart blood. Two of
these 20 smears were fixed by passing over the bunsen flame 5 times, raising the
temperature to about 220°C with a view to kill all anthrax bacilli in the smear
( 393 )
VIABILITY OF ANTHRAX BACILLI AND SPORES.
394
but not the spores. Emulsion from these fixed smears was inoculated in agar tubes
for cultural examination.
No growth was found even after 48 hours incubation, showing that no spores
were present in the blood at the time of fixation. The remaining 18 smears were
kept unfixed in the laboratory.
Each day two of these slides were fixed in similar manner and agar tubes
inoculated with the emulsion from the fixed smear. On the third day there
appeared two colonies of anthrax in one of the two tubes. All subsequent slides
gave some growth in the culture tubes though the number of the colonies was
variable. This experiment was carried out at the Muktesar Institute and has been
repeated here, at the Punjab Veterinary College, many times but with variable
results.
It is quite possible that the uniformity of the results at Muktesar may be due
to the constant presence of certain amount of moisture in the atmosphere.
Experiment No. 2 (Table II) deals with the viability of anthrax spores of a 24
hour old agar culture.
Experiment No. 3 (Table III) with similar spores from a 48 hour old culture.
The results of Experiments Nos. 2 and 3 show that it is necessary to pass the
slide over the bunsen flame at least 9 times, raising the temperature on the upper
surface of the slide to 320°C before the spores can be considered innocuous.
In smears thus fixed and afterwards stained with a saturated solution of
methylene blue, it has been noticed on microscopic examination that the penetra-
tion of the stain towards the centre of the spore is proportionate to the amount of
heat employed.
My thanks are due to Mr. F. Ware, Director, Imperial Institute of Veterinary
Research, Muktesar, for providing all possible facilities for carrying out the experi-
ment and to Messrs. Menon and Naik for their kind assistance.
TECHNIQUE.
The culture used in carrying out these experiments was of a virulent strain.
To obtain uniform thickness of the smears an emulsion of the culture was
prepared. A loop full of this emulsion was used for each smear.
The slides employed were of uniform thickness.
The chief difficulty encountered in carrying out these experiments was the
recording of the temperature on the slides. The method employed to over-come
this difficulty, although crude, proved adequate. A set of thermometers, recording
temperatures to 360°c., was employed.
G