NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ III, II
Of the animals which completed the experiment, 53 showed no definitely
appreciable reaction, either at the time of inoculation or after the test dose, and
it is presumed that at the time of the initial inoculation the virus in most, if not
all, of these animals was capable of multiplication and diffusion to a sufficient
extent to stimulate a durable immunity, although the effect was not sufficient to
produce symptoms appreciable to the ordinary methods of clinical examination.
Support is lent to this view by the fact that the great majority of the animals in
this group were immunised between the ages of 4—8 months, at which period we
have generally found that calves are definitely susceptible to rinderpest, although,
as it is the custom to wean calves about 4—6 months old, the susceptibility at this
age is not as great as at later periods.
Unfortunately no subinoculations of blood were carried out from any of the
calves at the time of serum-simultaneous inoculation so that definite evidence of
the presence of the virus in the blood at this time is lacking.
Thirty one calves, consisting of 10 indigenous, of which 7 were of Sahiwal
breed, and 21 European crosses, gave a more or less definite reaction at the time of
immunisation and these animals, with three or four exceptions, gave no reaction to
the subsequent test dose.
In the exceptions noted, the reactions to the test dose were very indefinite,
and in the absence of control subinoculations, it is not possible to state that either
the original reaction or the " test " reaction was due to rinderpest. In any case,
if such double reactions do occur, they would appear to be very rare.
A point of considerable interest in this group is that although the majority
of the calves (approximately two-thirds) were six months of age or over, the
remainder under six months gave evidence of a definite reaction, which was very
much more marked in the case of the animals lent by the military dairies than in
the case of the locally bred calves, and we presume that this difference is due to a
possible difference in the immunity of the parents.
Finally there is a group of 14 calves which reacted in varying degree to the test
dose, indicating that the diffusion or stimulation of immune response on the part
of the original virus was incomplete or in some cases entirely absent. The distri-
bution of these calves according to breed, age and interval of test is given in
Tables II and III.
IMMUNITY IN CALVES AGAINST RINDERPEST
143
TABLE II.
Distribution of X European calves reacting to test dose, according to age at immunisation, and interval of test.
Age at time of
immunisation
Up to 3 months
4—6 months .
7—9 months .
10—12 months.
13—24 months.
Total .
Interval between Immunisation and test
Up to 12
months
No.
immu-
nised
2
13
..
..
1
16
No.
reacted
to test
1
..
..
..
..
1
13—24 months
No.
immu-
nised
..
6
11
3
4
24
No.
reacted
to test
..
1
..
..
..
1
25—36 months
No.
immu-
nised
3
6
1
3
2
15
No.
reacted
to test
3
3
1
..
..
7
37—48
months
No.
immu-
nised
3
6
2
1
..
12
No.
reacted
to test
2
..
..
..
..
2
Total.
No.
immu-
nised
8
31
14
7
7
67
No.
reacted
to test
6
4
1
..
..
11
Percentage
reacted
to test
75
13
7
..
..
16
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INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ III, II
TABLE III.
Distribution of indigenous calves reacting to test dose according to age at immunisation, and interval of test.
Age at time of
Immunisation
Up to 3 months
4—6 months .
7—9 months .
10—12 months
13—24 months
Total .
Interval between Immunisation and test
Up to 12 months
No.
immu-
nised
..
7
2
3
1
13
No.
reacted
to test
..
..
..
..
..
..
13-24 months
No.
immu-
nised
..
5
..
1
..
6
No.
reacted
to test
..
..
..
1
..
1
25-36 months
No.
immu-
nised
4
3
1
1
1
10
No.
reacted
to test
1
..
1
..
..
2
37-48 months
No.
immu-
nised
..
1
..
..
..
1
No.
reacted
to test
..
..
..
..
..
..
Total
No.
immu-
nised
4
16
3
5
2
30
No.
reacted
to test
1
..
1
1
..
3
Percentage
reacted
to test
25
..
33
20
..
10
IMMUNITY IN CALVES AGAINST RINDERPEST
145
The relation of age to lack or loss of immunity is particularly well marked in the
European crossbred animals, although not so evident in the calves of indigenous
breeds, probably on account of the smaller numbers involved in the age groups in
which breakdowns occurred.
Further there is with increasing age at the time of inoculation a decrease in the
severity of the reaction to the test dose. The only animal in the whole series which
died as a direct result of rinderpest was inoculated when 14 days old and tested 31
months later, which would indicate quite a wide range in the degree of immunity
conferred by the original inoculation. Owing to inequality of numbers in the various
age groups as compared with the period of test it is not possible to decide whether
the immunity conferred originally wanes proportionately to the interval allowed
before test or whether the evidence of incomplete immunity at the time of test
indicates that only this degree of immunity was conferred at the time of the
first inoculation.
We are inclined to believe that the immunity conferred by the first inocula-
tion does not remain constant but decreases more or less rapidly, depending on the
individual, until the stage of complete susceptibility is once more reached.
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INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ III, II
TABLE IV.
Distribution of calves reacting to test dose arranged in approximately equal numerical groups according to age at
immunisation and interval of test.
Age at
time of
Immunisa-
tion
Up to 5
months.
6—8
months.
9—24
months.
Total .
Up to 12 months
No. immu-
nised
12
13
5
30
No. reacted
to test
1
...
...
1
Interval between immunisation and test
13—24 months
No. immu-
nised
5
14
11
30
No. reacted
to test
1
...
1
2
25—36 months
No. immu-
nised
13
4
8
25
No. reacted
to test
5
3
1
9
37—48 months
No. immu-
nised
7
5
1
13
No. reacted
to test
2
...
...
2
Total
No. immu-
nised
37
36
25
98
No. reacted
to test
9
3
2
14
Percentage
reacted
to test
24
8
8
14
IMMUNITY IN CALVES AGAINST RINDERPEST
147
In Table IV we have attempted to place the calves in groups, as numerically
equal as possible, according to their age at the time of immunisation and the
interval of time between immunisation and test, and we consider that the figures in
the " total " columns, vertical and horizontal, of this Table indicate the relation-
ship of these two factors to the duration of immunity, and also support our belief
stated above.
In general the results obtained in these experiments are in conformity with
those obtained by other workers, and we conclude that in India calves under 4
months should not be submitted to the serum-simultaneous method of inoculation
as the immunity conferred, if any, is too short to be of any practical value.
In calves over this age, however, the method is applicable as the economy
effected in serum, and the early reduction of the number of unprotected animals in
a herd, will more than counterbalance the small percentage of cases of incomplete
immunity of short duration.
As a further precaution to eliminate these doubtful cases, it appears to us
advisable to re-inoculate all animals immunised in calf-hood with a dose of virus—
the cost of which is negligible in comparison with serum—about 12-18 months after
the first inoculation.
With regard to the relationship of the degree of reaction to the subsequent dura-
tion of immunity we have observed that in those cases where a definite rinderpest
reaction has occurred the duration of complete immunity has always been longer
than the test period—the maximum test period in this experiment being 4 years.
A durable immunity can, however, be conferred without the appearance of any
appreciable symptoms, provided that the virus is capable of diffusing throughout
the body in spite of the inhibiting influence exerted either by the antibodies of the
simultaneously inoculated serum, or by a natural resistance on the part of the
tissues of the inoculated animal. In cases where either or both of these factors
prevent or limit the contact between virus and reacting tissue, little or no immunity
is developed. From this it appears that the severity of the reaction, as judged by
the presence or absence of symptoms, does not give a useful indication of the
probable duration of immunity.
In this experiment the greater resistance to rinderpest infection of indigenous
breeds, except the Sahiwal, as compared with European × indigenous cattle is
evident, but we do not consider that we have sufficient data to enable us to formu-
late an opinion upon the relative duration of immunity in Indian breeds as
compared with European × indigenous breeds, or with other exogenous breeds.
C
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INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ III, II
SUMMARY.
1. The results of serum-simultaneous inoculation carried out in 98 calves of
various indigenous and European × indigenous breeds are reported together with
the results of immunity tests carried out at various intervals.
2. The serum-simultaneous inoculation of calves under 4 months old is con-
sidered to be of little practical value.
3. The serum-simultaneous inoculation of calves four months old and upwards
is recommended as economical. Better results can be obtained if the virus inocula-
tion is repeated 12-18 months after the serum-simultaneous inoculation.
4. The severity of the reaction at the time of immunisation as judged by the
presence or absence of clinical symptoms did not give any indication of the dura-
tion of immunity within the maximum test period (4 years). All calves showing a
reaction which could be definitely attributed to rinderpest were immune for this
period.
ACKNOWLEDGMENTS.
The experiments recorded in this article were planned and carried out under
the direction of Dr. J. T. Edwards, D.Sc., M.R.C.V.S., late Director of this
Institute, to whom we tender our acknowledgments.
We also desire to thank Mr. H. Cooper, M.R.C.V.S., for guidance in the
execution of the work, and Mr. J. R. Haddow, B.Sc., M.R.C.V.S., D.V.S.M.,
for assistance in the preparation of the records.
REFERENCES.
Askar, Mohammad Bey (1924). Experiments to determine the susceptibility of weaned calves to
Cattle Plague. Ministry of Agriculture, Egypt, Bull. No. 42.
Croveri, P. (1919). Bull Soc. Path. Exot., 12, 2, 65-71.
Doutressoulle (1924). Rec. Med. Vet., 100, 464-468.
Edwards, J. T. (1927). Trans. Seventh Cong. Far East Assn. Trop. Med., Calcutta, 3,
707-717.
Montgomery, R. E. (1915-16). Ann. Rep. Dept. Agr., British East Africa, p. 66.
Pool, W. A. (1921). The Serum Simultaneous Method of Inoculation against Rinderpest. Agr.
Res. Inst., Pusa, Bull. No. 120.
Rabagliati, D. S. (1924). J. Comp. Path. and Ther., 37, 1-18.
Walker, J. (1921). Experiments and Observation in connection with (1) Rinderpest Immunisa-
tion, (2) Pleuro-pneumonia Contagiosa Bovum (lung-sickness), and the
Preventive Method of Inoculation. Dept. Agr. Kenya Bull. No. 2.
(1929). Rinderpest Research in Kenya. Dept. Agr. Kenya Bull. No. 8-A.
ON THE FRACTIONAL PRECIPITATION OF THE ANTIBODY
CONTAINING PROTEIN OF ANTI-RINDERPEST SERUM:
PART I, PRELIMINARY COMMUNICATION
BY
J. R. HADDOW, B.Sc., M.R.C.V.S., D.V.S.M., I.V.S.,
K. C. SEN, D.Sc., AND A. C. ROY, M.Sc.,
Imperial Institute of Veterinary Research, Muktesar.
(Received for publication on 1st February 1933.)
INTRODUCTION.
The nature of the antibodies occurring in the serum of immunised animals has
been the subject of much study in recent years, and numerous experiments have
been carried out to separate the particular fraction of the serum with which anti-
bodies are associated from other serum proteins which appear to be inert in this
respect. Many of these experiments have been undertaken with the object of
isolating a particular antibody and of studying its chemical and physical properties.
A review of this aspect of the subject has been given by Hartley [1931], and al-
though a complete separation of antibodies free from proteins has not been effected
up till now, Frankel and Olitzki [1930] and Frankel [1932] being probably the only
workers claiming to have done so, these fractionation experiments have been
utilised in concentrating an immune serum by discarding other fractions which do
not appear to contain any antibody. The greater part of these experiments has
been done on antitoxic sera, and a number of investigators have shown that the
protective substances or antibodies present in immune sera are associated with the
globulin fraction of the serum, either with the so-called euglobulin, with the
pseudoglobulin, or with both, depending on the antigen and also on the experimental
animal. Thus Seng [1899] demonstrated that diphtheria antitoxin was associated
with the soluble globulin or pseudoglobulin fraction of horse serum, and this obser-
vation of Seng was supported by Pick [1901]. The latter worker, however, showed
that in goats immunised against diphtheria toxin the euglobulin fraction instead
of the pseudoglobulin fraction contained the antitoxin, and that tetanus antitoxin
( 149 )
C 2
PRECIPITATION OF ANTIBODY FROM ANTI-RINDERPEST SERUM
151
be observed from this general review of the literature that a large amount of work
has been done on the fractionation of immune serum and more or less definite
knowledge has been gained as to the nature of the protein fractions with which the
antibodies in different types of anti-sera are associated. Since, however, the
nature of the different precipitated fractions of the protein depend on the nature
of the precipitant used, it is considered advisable to give a short summary of the
precipitation methods hitherto used before the object of the present research is
detailed.
One of the earliest workers on this subject was Dieudonné [1897], who found
that globulins precipitated by means of carbon dioxide and acetic acid contained
no antitoxin, but all the antitoxin was precipitated on saturation with magnesium
sulphate. This work was done on anti-diphtheria serum. Belfanti and Carbone
[1898] and Seng [1899] used the same method of precipitation. Brieger and
Boer [1893], and Astros and Rietsch [1900] precipitated the globulins and antitoxin
by means of the chlorides of sodium and potassium. Hiss and Atkinson [1901] used
magnesium sulphate. Ammonium sulphate is, however, probably the most widely
used salt for precipitating euglobulin and total globulin, and some of the early
users of this substance were Brieger and Ehrlich [1893], Wasserman [1894], Brodie
[1897], Pick [1901], Gibson [1906], Ledingham [1907], Gibson and Benzhaf [1910],
and Hartley [1914]. Among recent workers with this method may be mentioned
the names of Findlay, and Ledingham, Morgan and Petrie, reference to whose work
has already been made. Most of these authors used a solution of ammonium
sulphate, but Barr, Glenny, and Pope [1931] have recently pointed out the supe-
riority of using solid ammonium sulphate in many of these fractionations. Felton
(loc. cit.) has introduced a modification of the older method of acid precipitation,
and both he and Goodner (loc. cit.) found that more than 80 per cent. of the immune
bodies are precipitated by this method. The principle of the method is that the
anti-serum is brought down to a certain hydrogen ion concentration at a dilution of
1: 10, and the resulting precipitate containing most of the immune bodies is
filtered off.
OBJECT OF THE PRESENT INVESTIGATION.
Although, as already mentioned, Felton's experiments were carried out with
anti-pneumococcus serum, it seemed to us that his methods might be applicable to
anti-rinderpest serum. According to Hartley [1914] the antibodies in anti-rinder-
pest serum are mainly associated with the water insoluble protein, and it was
thought probable that most of the antibodies would be precipitated with the so-
called Felton fraction. In large scale work on the separation of antibodies, this
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INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ III, II
would be advantageous because, apart from introducing another salt into the
system, the trouble of dialysing the added ammonium sulphate, when this method
is employed, is removed. Nothing is, however, known about the relative efficiency
of these two methods in the precipitation of antibodies from anti-rinderpest serum,
so a comparative study was first required, and it was considered desirable to obtain
some data on the following points :—
A. Whether the protein fraction containing the protective bodies of anti¬
rinderpest serum can be separated by the water-precipitation
method which has been found very efficient by Felton in the case of
anti-pneumococcus serum, and if so, what would be the extent of
separation of the antibodies, and what is the optimal pH, or the
most suitable pH range, for antibody precipitation by this method ?
B. What is the relative efficiency of the above method as compared with
the precipitation of either euglobulin or total globulin by ammonium
sulphate so far as the separation of the antibodies is concerned ?
In the course of the experiments another question of great practical import-
ance arose, namely :—
C. Whether anti-serum of low potency can be rendered fit for field use
by fractionating the antibodies and thus concentrating the serum by
redissolving the precipitates obtained in a smaller volume than that
of the original serum used ?
In the following pages, an account of the preliminary observations made by us
are given.
EXPERIMENTAL PART.
In carrying out the precipitations, four different samples of anti-rinderpest
serum have been used. Sample 1 was a serum of exceptionally high potency,
designated as class I at this Institute and used exclusively for serum-simultaneous
immunisation of the most susceptible cattle, such as imported European cattle.
Sample 2 was of the same class although not quite as potent as sample 1. Sample
3 was a serum of low grade potency, much below the average potency of sera
designated as class P, which is issued for preventive (serum alone) inoculations only.
Sample 4 was a mixture of various sera, which had previously been shown by animal
inoculation to be practically valueless for protective purposes.
PRECIPITATION OF ANTIBODY FROM ANTI-RINDERPEST SERUM
153
From these four sera the following different fractions were prepared:—
Serum used.
Sample 1 . . . .
Sample 1a . . . .
Sample 1b . . . .
Sample 2 . . . .
Sample 2a . . . .
Sample 2b . . . .
Sample 2c . . . .
Sample 2d . . . .
Sample 3 . . . .
Sample 3a . . . .
Sample 3b . . . .
Sample 4 . . . .
Sample 4a . . . .
Sample 4b . . . .
Protein fraction.
Euglobulin .
.
.
.
„ . . . .
Euglobulin .
.
.
.
„ . . . .
„
.
.
.
.
Total globulin . . .
Euglobulin .
.
.
.
Total globulin . . .
Euglobulin
.
.
.
.
Total globulin . . .
Method of precipitation.
Untreated.
Water precipitation, pH 6.4.
Ammonium sulphate.
Untreated.
Water precipitation, pH 6.8.
„
„
„ 6.4.
Ammonium sulphate.
„ „
Untreated.
Ammonium sulphate.
„ „
Untreated.
Ammonium sulphate.
„ „
In the case of the fractions obtained from samples 1 and 2 the total volume
was brought up to that of the original serum used, as these experiments were
made mainly for the study of the relative antibody content of the various
fractions, and not for a study of methods of concentration.
The methods used by us for preparing the three different fractions may be
briefly given as follows :—
(a) Euglobulin by ammonium sulphate.
100 c.c. of the serum was mixed with an equal volume of normal saline and
to this mixture an equal volume of 66.6 per cent. saturated ammonium sulphate
solution was added. The whole was well mixed, and then allowed to stand for
3 to 4 hours for complete precipitation. The precipitate was then filtered off with
a Buchner funnel under suction and removed to a parchment dialyser and dialysed
for six days, during the first four days against running tap water, and the remain-
ing two days against distilled water. One or two drops of toluene were added
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everyday to prevent bacterial decomposition. When the precipitate was free from
ammonium sulphate, it was redissolved in normal saline and the volume made
up to the original volume of the serum used. The pH was adjusted to 7.6—7.8
by the addition of sodium hydroxide.
(b) Total globulin by ammonium sulphate.
The method was the same as above except that an equal volume of saturated
solution of ammonium sulphate instead of 66.6 per cent. saturated solution was
added to the mixture of serum and saline. The rest of procedure was identical
with that given in the case of euglobulin.
(c) Euglobulin by water-precipitation.
This fraction, most of which is euglobulin but may be better termed as water-
insoluble protein, was obtained by a method similar to that used by Felton in the
case of anti-pneumococcus serum. 160 c.c. of the serum was poured into 1,440 c.c.
of distilled water containing an amount of phosphoric acid necessary to
bring down the pH of the resulting mixture to a definite value. The amount of
acid was determined previously by separate experiment.
The total volume of the mixture was 1,600 c.c., i.e., the final dilution was 1 in
10. The serum was mixed with the acidulated water in a long cylinder and the
whole allowed to stand for 18 hours. It was observed that a larger amount of
precipitate could be obtained when the cylinder was allowed to stand inside a
refrigerator at 5°-C. instead of at room temperature, and when allowed to sediment
in this way it is easy to syphon off the supernatant fluid. The precipitate can,
however, be filtered with a Buchner funnel, and then dissolved in normal saline
to which sufficient sodium hydroxide is added to make the final pH about 7.6.
The selected test pH for precipitation was 6.4 in two cases and 6.8 in one case.
For the present no other pH has been tested, but in a future communication the
effect of variation in the pH on the precipitation of the antibodies will be con-
sidered. The pH for precipitation was selected to accord with the results of
Felton who has shown that the maximum precipitation of immune bodies in
anti-pneumococcus serum occurs at or above a pH of 6.0. Hartley has also
shown that 0.2 per cent. hydrochloric acid destroys the immune bodies in anti-
rinderpest serum in a short time.
The fractions obtained from samples 1 and 2 as well as the original sera were
then tested on hill bulls to obtain an indication of their relative potency.
The method of estimating the relative potency was that commonly used in
this Institute. The principle in brief is that anti-serum, usually at varying doses