NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
222
PREPARATION OF ANTI-RINDERPEST SERUM.
injected with the filtrate from the dialysed serum were not
protected. In the case of Bull 5322 (Experiment 2) the amount
of serum originally taken (200 c.c.) contained for this animal
about 7½ protective doses. The filtrate from this volume of
serum after dialysis did not protect; that is, it contained less
than one protective dose. On the other hand, the test on Bulls
5323, 5320 and 5321 show that practically all the Immune
Bodies are present in the precipitate produced by dialysis,
that the emulsion protects in the same way as the original
serum, and that little or no loss of Immune Bodies has occur-
red.
Experiment 3.—Carried out in a similar manner to Experi-
ments 1 and 2. 200 c.c. of serum 73 used.
The precipitate was thoroughly washed with distilled
water and dried on the filter paper in vacuo over sulphuric acid.
When quite dry it was scraped off the filter paper, ground to a
fine powder, and weighed (10.365 grammes).
The dried powder was tested as follows :—
Bull 5438.—Age 8 months. Weight 100lbs.
Injected subcutaneously with 1 gramme of the powder,
emulsified in salt solution, on one side and 0.5 c.c. virulent
Rinderpest blood on the other.
Result.—Animal recovered. 22 days after inoculation
given 900 c.c. virulent blood, and bled for serum 21 days after-
wards.
Bull 5466.—Age 4 years. Weight 100lbs.
Injected subcutaneously with 1 gramme of powder emulsi-
fied in salt solution on one side and 0.5 c.c. virulent
Rinderpest blood on the other.
Result.—Animal lived. As the animal was in poor condi-
tion and useless for serum-making purposes it was destroyed,
29 days after inoculation.
Bull 5467.—Age 1 year. Weight 100lbs.
Injected subcutaneously with 1 gramme of powder,
emulsified in salt solution on one side and 0.5 c.c. virulent
Rinderpest blood on the other.
PREPARATION OF ANTI-RINDERPEST SERUM.
223
Result.—Animal contracted Rinderpest and died on the
ninth day after inoculation.
Bull 175.—Age 2 years. Weight 207lbs.
Injected subcutaneously with 2 grammes of powder
emulsified in salt solution on one side and 0.5 c.c. virulent
Rinderpest blood on the other.
Result.—Animal lived and was used for serum making,
Test of Filtrate from Dialysis of 200 c.c. Serum 73.
All the filtrate from the dialysis of 200 c.c. serum, 73 was
injected subcutaneously on one side and 0.5 c.c. virulent
Rinderpest blood on the other. (Bull 5358. Age 2 years.
Weight 100lbs.)
Result.—Animal died of Rinderpest nine days after inocu-
lation.
The results of Experiment 3 confirm those of Experiments
1 and 2. The fluid remaining after dialysis contains no Immune
Bodies. In the preparation of the dried powder there was a
small but undetermined loss due to an accident. Assuming
that no loss had occurred and that all the Immune Bodies had
been precipitated by dialysis, then one protective dose for a
1001b. bull should be contained in 0.85 gramme of powder. On
account of the accidental loss mentioned above the powder
was tested in slightly increased doses. The result shows that
of four bulls used for testing the immunising properties of the
powder three lived. Three of these bulls were very small
animals, weak and poor conditioned : by using these animals
the immunising properties of the powder were put to a very
severe test.
Experiment 4.—200 c.c. of serum 76 dialysed for two days
against tap water and two days against distilled water. The
precipitate was dried in vacuo, powdered and weighed (9.79
grammes). The precipitate was tested as in Experiment 3. As
before, it was assumed that all the Immune Bodies are present
in the powder.
Bull 101.—Age 1 year. Weight 122lbs.
224
PREPARATION OF ANTI-RINDERPEST SERUM.
Injected subcutaneously with 0.9 gramme of powder, emul-
sified in salt solution on one side and 0.5 c.c. virulent Rinder-
pest blood on the other.
Result.—Animal died of Rinderpest eleven days after in-
oculation.
Bull 102.—Age 1 year. Weight 99lbs.
Injected subcutaneously with 0.92 gramme of powder, emul-
sified in salt solution on one side and 0.5 c.c. virulent Rinder-
pest blood on the other.
Result.—Animal lived and used for serum making.
Bull 103.—Age 23/4 years. Weight 147lbs.
Injected subcutaneously with 1.62 gramme of powder, emul-
sified in salt solution on one side and 0.5 c.c. virulent Rinderpest
blood on the other.
Result.—Animal lived and used for serum making.
Bull 1.00.—Age 1 year. Weight 109lbs.
Injected subcutaneously with all the filtrate from 200 c.c.
dialysed serum on one side and 0.5 c.c. virulent Rinderpest
blood on the other.
Result.—The animal had a very severe attack of Rinder-
pest. The temperature rose to 41.3º C. on the ninth day after
inoculation and mouth lesions appeared on the following day.
Profuse diarrhœa for nearly three weeks and during this time
the animal fed very slowly and became very emaciated. The
weight decreased to 80lbs., 41 days after inoculation. The
animal was immune to Rinderpest as he withstood an injection of
500 c.c. of virulent blood and was bled for serum 16 days later,
his weight having increased to 98lbs.
The result of Experiment 4 differs somewhat from those of
Experiments 1, 2 and 3. In this experiment the dialysis was
only continued for four days, as compared with six days in the
other experiments, and this fact may explain why the filtrate
from the dialysed serum contained one protective dose for Bull
100. That all the Immune Bodies have not been precipitated
is also shown by the fact that Bull 101, which received ex-
actly one protective dose of powder, on the assumption that
PREPARATION OF ANTI-RINDERPEST SERUM.
225
all the Immune Bodies had been precipitated, died of rinder-
pest. Bull 102 which received 1¼ doses on the same assump-
tion showed no symptoms of rinderpest and was subsequently
found to be immune.
Experiment 5.—200 c.c. of serum 78 was dialysed, three
days against tap water and three days against distilled water.
The precipitate was separated by filtration, washed with dis-
tilled water and dried in vacuo over sulphuric acid. The dried
solid was powdered and weighed (9.682 grams). The immunis-
ing properties of the powder were tested on four bulls. The
powder was dissolved in a faintly alkaline solution before in-
jection. For the purpose of dose calculation it was assumed,
as before, that all the rinderpest immune bodies had been pre-
cipitated by dialysis of the serum.
Bull 297.—Age 16 months. Weight 128lbs.
Inoculated subcutaneously with 0.96 gram of powder on
one side, and 0.5 c.c. virulent rinderpest blood on the other.
Result.—Animal lived and was used for serum making.
Bull 299.—Age 18 months. Weight 150lbs.
Inoculated subcutaneously with 1.7 gram of powder on
one side, and 0.5 c.c. virulent rinderpest blood on the other.
Result.—Animal lived and was used for serum making.
Bull No. 300.—Age 4 years. Weight 174lbs.
Inoculated subcutaneously with 2.6 grams of powder
on one side, and 0.5 c.c. virulent rinderpest blood on the other.
Result.—Animal lived and was used for serum making.
Bull 298.—Age 6 years. Weight 244lbs.
Inoculated subcutaneously with 3 grams of powder on one
side, and 0.5 c.c. virulent rinderpest blood on the other.
Result.—Animal lived and was used for serum making.
Bull 271.—Age 2 years. Weight 146lbs.
Inoculated subcutaneously with the filtrate and washings
from 200 c.c. of dialysed serum 78 on one side, and 0.5 c.c. viru-
lent rinderpest blood on the other.
Result.—Temperature rose to 41.0ºC on the evening of
the fifth day after inoculation, and remained at about this
15
226
PREPARATION OF ANTI-RINDERPEST SERUM.
level until the ninth day when mouth lesions appeared and the
animal died of rinderpest on the tenth day.
The results of Experiment 5 are in agreement with those
obtained in the four previous experiments; the rinderpest
immune bodies are completely precipitated when the Immune
serum is subjected to dialysis.
The results obtained in the above experiments suggest
that the rinderpest immune bodies differ in one important
respect from diphtheria and tetanus anti-toxins. It has been
shewn repeatedly that the anti-toxins are associated with, or
similar to, the so-called soluble globulins or pseudo-globulins,
that is, those globulins which are not precipitated on dialysis.
As shown above, when rinderpest immune serum is dialysed,
the filtrate from the dialysed serum, which contains albumin
and pseudo-globulin, contains no rinderpest immune bodies.
The rinderpest immune bodies appear to be associated with,
or similar to, the so-called insoluble globulins or euglobulins—
the globulins which are precipitated on dialysis.
In the following experiment a quantity of rinderpest im-
mune serum was fractionated according to the method of
Gibson : three fractions were obtained, viz., albumin, pseudo-
globulin and euglobulin, and each fraction was tested for the
presence of immune bodies.
Experiment 6.—50 c.c. of serum 78 was precipitated with
an equal volume of saturated ammonium sulphate solution.
The precipitate, consisting of pseudo and euglobulin, was
separated by filtration and the filtrate, containing the albumin,
was completely saturated with powdered ammonium sulphate.
The precipitated albumin was separated and dialysed. The
solution obtained contains the albumin originally present in
50 c.c. rinderpest immune serum. The precipitate of globulin
was extracted with 50 c.c. of saturated sodium chloride solution.
The pseudo-globulins pass into solution and the euglobulins
remain undissolved. The latter were separated by filtration
and dialysed and finally emulsified in 50 c.c. of sterile salt
solution. The filtrate containing the pseudo-globulins was
PREPARATION OF ANTI-RINDERPEST SERUM.
227
precipitated with an equal volume of saturated ammonium
sulphate solution, filtered, and the pseudo-globulin dialysed.
The three fractions thus obtained were tested as follows :—
Bull 281.—Age 1 year. Weight 99lbs.
Inoculated subcutaneously with the albumin fraction on
one side, and 0.5 c.c. virulent rinderpest blood on the other.
Result.—Animal died of rinderpest eight days after inocu-
lation.
Bull 283.—Age 1 year. Weight 138lbs.
Inoculated subcutaneously with the pseudo-globulin frac-
tion on one side, and 0.5 c.c. virulent rinderpest blood on the
other.
Result.—Animal died of rinderpest ten days after inocula-
tion.
Bull 284.—Age 1¼ year. Weight 153lbs.
Inoculated subcutaneously with the euglobulin fraction
on one side, and 0.5 c.c. virulent rinderpest blood on the other.
Result.—Animal lived and was used for serum making.
The previous results are confirmed, viz., the rinderpest
immune bodies are associated with, or similar to, the euglobulins.
Several questions of theoretical interest and practical
importance arise from a consideration of the experimental
results given in the preceding pages. The rinderpest immune
bodies are precipitated on dialysis of the serum which suggests
that they are allied to the euglobulins, and this conclusion is
supported by the result of Experiment 6. Diphtheria and
tetanus anti-toxins are not precipitated by dialysis, and all the
evidence available goes to show that the anti-toxins are allied
to the pseudo-globulins. The difference may be due to the fact
that the rinderpest anti-serum is obtained from bovines and
diphtheria anti-toxin is usually obtained from equines ; that is,
the species of animal used for the production of immune sera
may be a factor in producing the different results. In this con-
nection the results of Pick, Gibson and Ledingham may be re-
ferred to. It would be interesting to determine the distribution
of the diphtheria anti-toxin in the serum of a bull immunised
228
PREPARATION OF ANTI-RINDERPEST SERUM.
against diphtheria-toxin. On the other hand, there is little
similarity between the preparation of diphtheria and tetanus
anti-toxins and rinderpest immune serum. The anti-toxins
are prepared by the repeated injection of gradually increasing
doses of toxin; rinderpest immune serum is prepared by the
injection of one massive quantity of virulent material into ani-
mals which have been previously immunised against the disease.
In the former case the response of the cells involved is to a toxin
stimulus ; in the latter the response is to a stimulus of the
nature of a living organism. And it is conceivable that in the
two cases different mechanisms are involved resulting in the
production of immune bodies which, though possessing many
common characteristics, possess also points of difference which
are manifested in such a property as that of solubility.
It has been shown that the rinderpest prophylactic can be
prepared in the dry condition. Up to the present the opera-
tions have been carried out on a small scale, experimentally,
and experiments are now in progress the object of which is to
discover, if possible, other methods for the preparation of the
rinderpest prophylactic in a dry condition. By the method
described above, about 50 per cent. of the protein constituents of
rinderpest immune serum, consisting of the albumin and soluble
globulin which take no part in the immunising process, are elim-
inated. The residue containing the active principle is obtained
finally in the dry condition. The preparation of the rinderpest
prophylactic in the dry state has many practical advantages.
It is well known that immune sera deteriorate on exposure
to light and increased temperature and with age, and even when
preserved under the most favourable conditions a diminution
in potency always takes place. In the tropics immune sera
tend to deteriorate more rapidly than in cooler regions, and
Lingard has shown that rinderpest immune serum when ex-
posed to the temperatures experienced in the Plains of India,
loses from 40 to 66.6 per cent. of its protective power. Serum in
the dried condition, however, is much more resistant to such
adverse influences as age, light and increased temperature, and
PREPARATION OF ANTI-RINDERPEST SERUM.
229
if preserved in this condition it retains its original potency
almost indefinitely. There are occasions when the use of the
prophylactic in some such form as the dried globulin described
above would be preferable to the use of the fluid serum, as,
for example, during military operations when questions of trans-
port are of prime importance, and other practical applications
for such a product would doubtless be found by officers in charge
of field operations.
CONCLUSIONS.
1. When rinderpest immune serum is dialysed, the immune
bodies are precipitated. The filtrate from the dialysed serum
fails to protect susceptible animals from the disease.
2. By this process the rinderpest prophylactic can be
prepared in the dry condition.
3. The tests of the dried powder which have been carried
out show that the loss of immune bodies is inappreciable and
that the dried powder, when dissolved or emulsified, and inject-
ed into susceptible animals, protects from a simultaneous
inoculation of virulent rinderpest blood.
4. By this method about half of the total proteids, which
take no part in the immunising process, are eliminated.
5. The rinderpest immune bodies differ from diphtheria
and tetanus anti-toxins. The former appear to be similar to
the insoluble globulins (euglobulins) ; the latter are similar to
the soluble globulins (pseudo-globulins).
230
PREPARATION OF ANTI-RINDERPEST SERUM.
REFERENCES
Dieudonne.—Arbeiten aus dem Kaiserlischen Gesundheitsamte. 1897. Vol. XIII,
p. 293;
Belfanti & Carbone. — Centralblat für Baktcriologie Ref. 1898. Vol. XXIII, p. 906.
Seng.—Zeitschrift für Hygiene, 1899. Vol. XXXI, p. 573.
Brieger & Boer.—Zeitschrift für Hygiene, 1896. Vol. XXI, p. 259.
Astros & Rietsch.—Comptes Rendus Soc. Biologie, 1900. Vol. III, p. 337.
Hiss & Atkinson.—Journal of Experimental Medicine 1901. Vol. V, pp. 47 & 67.
Brieger & Ehrlich.—Zeitschrift für Hygiene, 1893. Vol. XIII, p. 336.
Wassermann.—Zeitschrift für Hygiene, 1894. Vol. XVIII, p. 236.
Brodie.—Journal of Pathology & Bacteriology, 1897. Vol. IV, p. 460.
Pick.—Beit. zur Chem. Physiologie and Pathologie 1901. Vol. I, p. 351.
Gibson.—Journal of Biol. Chemistry, 1906. Vol. I, p. 161.
Ledingham.—Journal of Hygiene, 1907. Vol. VII, p. 65.
Tizzoni & Cattani.—Centralblatt für Bakteriologie, 1891. Vol. IX, p. 685.
Freund & Sternberg.—Zeitschrift für Hygiene, 1899. Vol. XXXI, p. 429.
Mercus.—Zeitschrift für Phys. Chemie, 1899. Vol. XXVIII, p. 559.
Fuld & Spiro.—Zeitschrift für Phys. Chemie, 1900. Vol. XXXI, p. 132.
Gibson & Collins.—Journal of Biol. Chemistry, 1907. Vol. III, p. 233.
Landsteiner.—Centralblatt für Bakteriologie Ref. 1900. Vol. XXVII, p. 357.
Cathcart.—Journal of Physiology, 1904. Vol. XXXI, p. 497.
Opie & Barker.—Journal of Exp. Medicine, 1907. Vol. IX, p. 207.
Lingard.—Report on Rinderpest, 1905, p. 21.
REPORT ON THE PREPARATION OF RINDER-
PEST ANTI-SERUM BY MEANS OF
DILUTED VIRULENT FLUIDS.*
BY
PERCIVAL HARTLEY, D.Sc. (LONDON), I.C.V.D.,
Physiological Chemist, Muktesar Laboratory.
The experiments described in the following notes were
carried out according to instructions issued by the Imperial
Bacteriologist. The various seras described were prepared
at Muktesar in November and December 1910 and January
1911. The serum tests were carried out at Bareilly in February
and March 1911.
The experiments were designed to investigate several
different points, one of the chief of these being to study the
nature of the serum produced by the injection of diluted virulent
fluids. In a series of experiments carried out at Bareilly in
the cold weather of 1909-10 by the Imperial Bacteriologist
it was noticed that subcutaneous injections of virulent blood,
diluted with potassium citrate solution 0.5 per cent. were very
rapidly absorbed. On the other hand, massive injections of defi-
brinated virulent blood are absorbed relatively slowly, and in
many cases the swellings created by the injection often persist
for many days. It was considered, on theoretical grounds,
that rapid absorption of the injected virulent material is proba-
bly a favourable factor in the production of seras of high potency
and the experiments were designed to investigate this point.
Another subject which was studied at the same time deals
with the question of the potency of the serum prepared by the
injection of virulent peritoneal washings. It has been stated
that the peritoneal washings taken from an animal at the height
* Results arrived at in July 1911.
232
PREPARATION OF ANTI-RINDERPEST SERUM.
of an attack of rinderpest are as virulent—or more so—as the
blood, and that the serum prepared by the injection of peri-
toneal washings is as powerful as that resulting from the injec-
tion of virulent blood. The method of preparing the perito-
neal washings is as follows :—A quantity of potassium citrate
solution 0.5% is injected into the peritoneal cavity of a rinder-
pest " Control." An hour or more afterwards the Control
is bled from the vein and afterwards the fluid in the perito-
neum is withdrawn and injected into an immunised bull in the
usual way. The amount of fluid normally present in the peri-
toneal cavity is never very large and probably averages about
100 c.c. About half of the potassium citrate solution injected
is absorbed, and the peritoneal washings finally obtained may
be regarded, perhaps, as a solution consisting of the original
peritoneal fluid strongly diluted in potassium citrate solution.
If this view is correct, then it is reasonable to suppose that a
high grade serum should result from the injection of artificially
prepared peritoneal washings, and that a high grade serum
should also be produced by the injection of other virulent fluids
(e.g., blood) diluted in vitro in potassium citrate solution.
When virulent blood is mixed with 0.5% potassium citrate,
complete hæmolysis occurs and the hæmolysed, diluted fluid
is very rapidly absorbed after injection.
In order to compare the seras made in the usual way with
those made by the injection of diluted virulent fluids the follow-
ing seras were prepared :—
Serum A—prepared by two injections of virulent peritoneal washings.
Serum B—prepared by two injections of defibrinated virulent blood.
Serum C—prepared by two injections of virulent peritoneal fluid diluted in Potas-
sium Citrate Solution.
Scrum D—prepared by two injections of defibrinated virulent blood diluted with
Potassium Citrate Solution.
Serum E—prepared by two injections of virulent blood, run directly from the
vein into Potassium Citrate Solution.
From the above series, comparison can be made between—
(1) Serum A (routine Peritoneal Washing Serum) and
Serum C (Peritoneal Washings made in vitro).
(2) Scrum B (routine virulent blood serum) and
Seras D and E (diluted virulent blood seras).
(3) Serum A and Serum B (Peritoneal Washing Serum and Virulent Blood Serum).
PREPARATION OF ANTI-RINDERPEST SERUM.
233
In the course of the preparation of these seras it was
noticed that the fluids diluted with potassium citrate were,
without exception, exceedingly rapidly absorbed. Twenty-four
hours after injection only a thickening remained and this
disappeared after another 24 hours.
Hill Bulls were used throughout, both for providing the
virulent material and for making the seras. Four bulls were
used for making each of the seras B, D and E ; three were used
for making Serum C and two bulls for preparing Serum A.
Serum A.—
Two injections of virulent Peritoneal Washings.
Two Bulls used. 5370—Age 4 years—Weight 266 lbs.
5371— „ 2 „ — „
178 „
Immunised with V.B., and Serum, November 26th, 1910.
Injections. 5370 Injected with 1500 c.c. Peritoneal Washings, December 21st, 1910.
Injected with 2000 c.c. Peritoneal Washings, January 16th, 1911.
Bled 1200 c.c, February 3rd, 1911.
5371. Injected with 1500 c.c. Peritoneal Washings, December 20th, 1910.
Injected with 1800 c.c. Peritoneal Washings, January 12th, 1911.
Bled 1200 c.c., February 1911.
Serum from 5370 and 5371, mixed and tested.
Results of Serum Testings. (A).
First Series—
No. of
Bull.
321
379
398
377
371
383
Weight
in lbs.
112
147
149
154
154
263
Actual dose
of Serum
injected.
1.68 c.c.
2.20 „
4.47 „
4.62 „
9.24 „
15.78 „
Rate of
Serum
testing per
600 lbs.
9 c.c.
9 „
18 „
18 „
36 „
36 „
Result.
H. T. R. Vesicles 6th day. Ulcers
7th day. Died 10th day.
H. T. R. Vesicles 8th day. Ulcers
9th day. Recovered.
H. T. R. Vesicles 7th day. Ulcers
9th day. Died 12th day.
Weak animal with F. & M. disease.
Died 3rd day from weakness.
Slight T. R. No lesions. Recovered.
H. T. R. Vesicles 9th day. Ulcers
10th day. Recovered.
H. T. R. High Temperature Reaction.
V. B. Virulent Blood.
F. & M. Foot and Mouth Disease.
P. F. Peritoneal Fluid.
234
PREPARATION OF ANTI-RINDERPEST SERUM.
Second Series of Testings. (A).—
No. of
Bull.
413
427
450
448
429
428
Weight
in lbs.
129
135
141
158
172
206
Actual dose
of Serum
injected.
23.22 c.c.
24.30 „
33.84 „
37.60 „
51.60 „
61.80 „
Rate of
Serum
testing per
600 lbs.
108 c.c.
108 „
144 „
144 „
180 „
180 „
Result.
Slight T. R. No lesions. Recovered.
„
„
„
„
„
„
„ „ „ „ „ „
Weak animal with F. and M. disease.
No lesions. Died from weakness 12th
day.
No T. R. No lesions. Recovered.
„ „ „ „ „
Summary of Serum Testings —
Two Bulls tested at 9 c.c. ..
„ 18 „
..
„ 36 „ ..
„ 108 „ ..
„ 144 „ ..
„ 180 „ ..
One died, one recovered.
Both died—one from Rinderpest and one
from weakness and foot and mouth
disease.
Both recovered.
Both recovered.
One recovered ; one died from weakness and
foot and mouth. No Rinderpest le-
sions.
Both recovered.
Approximate Protective dose of Serum A = 50 c.c. per 600 lbs.
Serum B.—
Serum prepared by two injections of defibrinated virulent
blood (" Routine V. B.").
Four Bulls used. 5358—Age 4½ years—Weight
5360— „ 3 „ —
„
5364— „ 12 „ —
„
5369— „ 7 „ —
„
Immunised with scrum and Virulent Blood
First injection, 1500 c.c. defibrinated V. B.
Second injection, 2000 c.c. defibrinated V. B.
Bled for serum
215 lbs
201 „
287 „
271 „
..
..
..
..
Serum from 5358, 5360, 5364, 5369, mixed and tested.
.
.. November 26th, 1910.
.. December 15th, 1910.
.. January 1st, 1911.
.. January 18th, 1911.