NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
PREPARATION OF ANTI-RINDERPEST SERUM.
207
of the serum from animals hyperimmunised by virulent blood
and found that the potency of the serum from animals
injected with artificial
peritoneal fluid was greater
than that of serum from animals injected with virulent
blood.
In all laboratories in which Anti-Rinderpest Serum is made
the cost and the difficulties of getting animals from which viru-
lent material can be obtained for the injection of serum prepar-
ing animals is always a serious problem, and in this laboratory
where over 500,000 doses are annually prepared, the difficulty
of obtaining such animals has been very great and every year
becomes more so. It was, therefore, decided in June 1909 to
hyperimmunise a certain number of animals by artificial
fluid on the lines laid down by Ruediger and to test the result-
ing serum.
THE PREPARATION OF ARTIFICIAL PERITONEAL FLUID.
The animals from which the virulent blood is obtained are
small Himalayan hill bulls—highly susceptible to Rinderpest—
weighing from 100—200lbs., and the average quantity of
blood obtained from each varies from 1,200—2,500 c.c. The
animals from which the artificial peritoneal fluid was to be
collected were inoculated with a small quantity of virulent blood.
When high temperature occurred and vesicles appeared (usually
the 5th day after the inoculation of virulent blood), a sterilised
solution of 0.5 per cent. potassium citrate was injected into
the peritoneal cavity, the solution being injected at blood
heat.
The solution was injected in the right flank, the seat of
injection being an equal distance from the angle of the ileum,
lumbar vertebræ and last rib. The hair was clipped and the
skin was washed and thoroughly cleansed with carbolic solu-
tion. The skin was then cut and the canula of an Ehrlich
injection balloon inserted through the peritoneum into the peri-
toneal cavity and the solution injected. The amount injected
was at the rate of 1,000 c.c. per 100lbs. body weight. After
208
PREPARATION OF ANTI-RINDERPEST SERUM.
injection the skin is sutured. One hour later the animals
were bled and the artificial peritoneal fluid collected into
sterile flasks by pipettes. The average quantity collected
was only ¾ of the amount of blood obtained—only in very few
cases was the quantity of artificial peritoneal fluid collected
equal to the quantity of blood. It was also noticed that bulls
injected with potassium citrate solution gave a smaller
quantity of blood per lb. body weight than those not
injected.
AMOUNT OF ARTIFICIAL PERITONEAL FLUID THAT CAN BE
SAFELY INJECTED.
The weights of the serum making animals vary from 250—
600 lbs., and the amount of virulent blood injected for hyper-
immunising is at the rate of 1,000 c.c. per 100 lbs. body weight,
this amount is well borne and absorbed. It was, therefore, de-
cided to inject the artificial peritoneal fluid at the same rate,
following this a large percentage of the animals injected at this
rate died in from 30 hours to 11 days. At first it was thought
that the deaths might be caused by bacterial contamination
from the peritoneal washing, and a second series of animals
were injected with similar amounts, every care being taken
to exclude as far as possible bacterial contamination, but in
these cases also a similar percentage of deaths occurred. A
number of animals were then injected with varying amounts
of artificial peritoneal fluid. The result of these experiments
showed that if the amount of artificial peritoneal fluid injected
exceeded 800 c.c. per 100 lbs. body weight deaths occurred,
but if the amount injected were less than 800 c.c. per 100 lbs.
body weight, the injections were well borne and absorbed.
In a few cases only sloughing at the seat of inoculation took
place. These accidents were undoubtedly due to the contamina-
tion of the peritoneal washing. For the preparation of
serum by this means the amount to be injected was there-
fore fixed at the rate of 700 c.c. per 100 lbs. body weight
(Charts 4, 5, 6).
PREPARATION OF ANTI-RINDERPEST SERUM.
209
CLINICAL SYMPTOMS OF BULLS THAT DIED AFTER THE LARGE
INJECTIONS OF ARTIFICIAL PERITONEAL FLUID.
In many cases the animals died very quickly and in others
they lingered on for some days. In the former cases the morn-
ing after injection the animals were off feed, lying down, hur-
ried breathing, small rapid pulse, extremities cold, running down
temperature, finally they became comatose and died in 36 to
40 hours (Charts 1 and 3). Amongst those in which death
occurred after some days, except gradual debility, no very pro-
nounced symptoms occurred, the animals fed only fairly, the
respiration was hurried, the pulse somewhat rapid, rumination
was very suspended, and they fell off rapidly in condition and
died on the 10th or 11th day after injection (Chart 2).
TREATMENT.
Various stimulants and hypodermic injections of strych-
nine were tried, but treatment was of little avail. Small and
repeated doses of ammonium carbonate and Nux vomica gave
the best results.
POST-MORTEM APPEARANCES.
The pharynx and trachæ presented congestions and
petechiæ.
The rumen, reticulum and omasum appeared normal.
The abomasum was the seat of the most marked lesions,
presenting acute inflammation, the folds varied from a red to a
deep portwine colour, and in some cases necrotic areas were
seen.
The small intestines showed congestions to a greater or
less amount in different cases, but Peyer's patches showed very
little alteration.
The large intestines were normal.
The rectum presented slight congestion.
The kidneys showed congestions and petechiæ.
The liver was soft and friable.
The spleen appeared normal.
14
210
PREPARATION OF ANTI-RINDERPEST SERUM.
The lungs showed extravasation.
The heart presented petechiæ on the endocardium.
The blood was dark and did not coagulate readily.
CAUSE OF DEATH.
Ruediger does not mention having had any fatal results
after the injection of artificial peritoneal fluid.
Baldrey in the Journal of Tropical Veterinary Science, Vol.
VI, No. 1, 1911, attributes the deaths to Rinderpest toxin,
but in our present state of knowledge regarding Rinderpest,
this theory cannot be accepted until further proof is forthcom-
ing, especially as under certain circumstances, the injection
of normal blood produces somewhat similar results. The
matter is at present being investigated by the Imperial Bacterio-
logist, and the result of his experiments will shortly be pub-
lished.
PREPARATION OF SERUM BY ARTIFICIAL PERITONEAL FLUID
FOR COMPARISON WITH SERUM PREPARED BY VIRULENT
BLOOD INJECTION.
The amounts of artificial peritoneal fluid that could be
safely injected having been determined by the previous experi-
ments, several animals were immunised by the simultaneous
method, of these animals half were hyperimmunised by artifi-
cial peritoneal fluid and the other half by the injection of viru-
lent blood.
The animals were a very fair sample of the bulls usually
obtainable for the preparation of serum, and both lots were
kept under like conditions.
Those hyperimmunised by artificial peritoneal fluid receiv-
ed injections at the rate of 700 c.c. per every 100lbs. body
weight, and those hyperimmunised by virulent blood receiv-
ed injections at the rate of 1,000 c.c. per 100lbs. body weight.
All injections were well borne and rapidly absorbed.
The animals were bled for serum 21 days after in-
jection.
PREPARATION OF ANTI-RINDERPEST SERUM.
211
THE POTENCY OF THE SERUM PREPARED BY INJECTIONS OF
ARTIFICIAL PERITONEAL FLUID (SERUM A), COMPARED
WITH THAT OF THE SERUM PREPARED BY INJECTIONS
OF BLOOD (SERUM B)
Serum A.
2 Bulls were injected at the rate of 36 c.c. per 600lbs. body weight.
2
„
„
„
„ 72 „ „ „
2
„
„
„
„ 108 „ „ „
Serum B.
2 Bulls were injected at the rate of 36 c. c. per 600lbs. body weight.
2
„
„
„
„ 72 „ „ „
2
„
„
„
„ 108 „ „ „
Serum A.
Bull
No.
1
2
3
4
5
6
Dose per
600lbs. body
weight.
36 c.c.
36 „
72 „
72 „
108 „
108 „
Actual
weight in
lbs.
118
150
141
110
113
119
Actual
amount of
serum in-
jected.
7.08 c.c.
9.00 „
16.92 „
13.20 „
20.34 „
21.42 „
Reactions.
Very severe. Died.
„
„
Vesicles.
„ „ „
Slight reaction.
Severe ; vesicles.
Severe.
Recovered.
„
„
„
Serum B.
Bull
No.
1
2
3
4
5
6
Dose per
600lbs. body
weight.
36 c.c.
36 „
72 „
72 „
108 „
108 „
Actual
weight
in lbs.
190
184
190
233
272
273
Actual
amount of
serum in-
jected.
11.40 c.c.
11.04 „
22.80 „
27.80 „
49.14 „
48.96 „
Reactions.
Very severe, vesicles. Recovery.
„ „ „ Died.
Severe temperature reaction. Re-
covered.
Very slight temperature reaction.
Recovered.
No reaction. Recovered.
„ „ „
The 2 controls died on the 6th and 7th day respectively.
The bulls used averaged 1-2 years of age and are highly
susceptible to Rinderpest. The testing animals received 0.5
c.c. virulent blood on one side, and the serum on the other.
212
PREPARATION OF ANTI-RINDERPEST SERUM.
The tests were controlled by 2 animals which died on the 6th
and 7th day respectively.
By reference to the table it is evident that the serum A
is much weaker than serum B. This is the reverse of what
Ruediger found. Had the serum preparing animals been able
to stand larger injections of artificial peritoneal fluid, it is very
probable that the serum would have been very much stronger
as undoubtedly the potency of Anti-Rinderpest Serum depends
to a certain extent upon the quantity of antigen injected. The
artificial peritoneal fluid is not simply a dilution of peritoneal
fluid with the potassium citrate solution. If the peritoneal
fluid be collected from an animal showing a high temperature
and vesicles and diluted down with potassium citrate solution
to give an amount of fluid equal to the quantity of artificial
peritoneal fluid that could be collected from such an animal,
and this diluted peritoneal fluid were used for hyperimmunising,
the potency of the resulting serum would be very low. Such
experiments have been carried out as have also dilutions of blood
and only under certain circumstances can a serum of any prac-
tical value be produced by simple dilutions.
During the time the potassium citrate solution is in the peri-
toneal cavity, vital processes (osmosis effusion of lymph) are
taking place, and the resulting artificial peritoneal fluid contains
a large quantity of antigen and can be used for the preparation
of a serum of practical use.
Conclusions.
1. By using artificial peritoneal fluid the amount of viru-
lent material for hyperimmunising purposes is considerably
increased, and a marked saving in the cost of serum preparation
effected.
2. The serum from animals injected with artificial perito-
neal fluid is usually weaker than the serum prepared by the
injection of blood.
3. Artificial peritoneal fluid injections are well absorbed
and do not produce sloughing.
PREPARATION OF ANTI-RINDERPEST SERUM.
213
BULL NO. I.
BULL NO. II.
214
PREPARATION OF ANTI-RINDERPEST SERUM.
BULL NO. III.
BULL NO. IV.
PREPARATION OF ANTI-RINDERPEST SERUM.
215
BULL NO. V.
BULL NO. VI
ON THE IMMUNE BODIES OCCURRING IN RINDER-
PEST IMMUNE-SERUM.*
PART I.
THE PRECIPITATION OF THE RINDERPEST IMMUNE BODIES
BY DIALYSIS OF THE IMMUNE SERUM.
BY
PERCIVAL HARTLEY, D.Sc. (LONDON), I.C.V.D.,
Physiological Chemist, Muktesar Laboratory.
In recent years numerous experiments have been carried
out with the object of isolating the immune bodies occurring
in the serum of immunised animals and of studying their che-
mical and physical properties. Attempts have been made to
separate these immune bodies from the other constituents of
the serum in which they occur, the question of their protein
character has been studied, comparisons have been made be-
tween the composition of the serum of normal and immune ani-
mals, and certain changes occurring in the serum of animals
during immunisation have been observed. Up to the present
a complete separation of the immune bodies from the other
constituents of the serum has not been effected. The greater
portion of the experiments have dealt with the study of
diphtheria anti-toxin. As far as our knowledge extends at present
it appears that diphtheria anti-toxin is a protein having the char-
acters of a globulin.† It is well established that diphtheria
anti-toxin is precipitated along with the globulins of horse serum.
* Results arrived at in July 1911.
† Proscher (1902) claims to have prepared a diphtheria anti-toxin which gives
none of the usual protein reactions
PREPARATION OF ANTI-RINDERPEST SERUM.
217
These globulins may be precipitated from serum either by dia-
lysis, by means of carbon dioxide and acetic acid or by the
addition of neutral salts. The earlier workers on this subject
adopted one or other of the above methods and discordant re-
sults were obtained; but later investigations on the protein
constituents of normal and immune serum provided an expla-
nation of the varying results obtained by different observers.
Dieudonné (1897), found that the globulins precipitated
by means of carbon dioxide and acetic acid contained no anti-
toxin, that the globulins precipitated by dialysis contained
but little, and that all the anti-toxin was contained in the glo-
bulin precipitate produced by saturation of the serum with
magnesium sulphate. Similar results were obtained by Bel-
fanti and Carbone (1898), and by Seng (1899). Brieger and
Boer (1893), and Astros and Rietsch (1900), precipitated the
globulins and anti-toxin by means of the chlorides of sodium
and potassium. Hiss and Atkinson (1901), saturated the
serum with magnesium sulphate and showed that the anti-toxin
was carried down with the precipitate. Ammonium sulphate
has been widely used for precipitating anti-toxins from serum.
Brieger and Ehrlich (1893), and Wassermann (1894), obtained
precipitates containing anti-toxin from the milk of immunised
animals, and this salt was used by Brodie (1897), Seng (1899),
Pick (1902), Gibson (1906), Ledingham (1907), and others in
studying diphtheria anti-serum Freund and Sternberg (1899),
first precipitated the albumins of the serum by means of potash
alum, and showed that the albumin precipitate contained no
anti-toxin but remained in solution with the globulins. Marcus
(1899), showed that the serum contained two forms of globulin;
one is precipitated by means of carbon dioxide and acetic acid
and by dialysis (insoluble globulin), and the other is not preci-
pitated under these conditions but only on saturation of the
serum with magnesium sulphate, or half saturation with ammo-
nium sulphate (soluble globulin). Seng (1899), showed that
the diphtheria anti-toxin is either associated with, or possesses
similar properties to the soluble globulins, and was thus able to
218
PREPARATION OF ANTI-RINDERPEST SERUM.
explain the absence of anti-toxin in the precipitates obtained
by Dieudonné and by Belfanti and Carbone. Fuld and Spiro
(1900), divided the globulins occurring in horse serum into three
classes—fibrinoglobulin, euglobulin and pseudoglobulin—the
classification being based upon the degree of saturation of am-
monium sulphate required to produce precipitation, and Pick
(1902) showed that the anti-toxin occurring in the serum of
immunised horses is associated or combined with the pseudo-
globulin fraction. Pick makes the interesting observation that
in the case of goats immunised against diphtheria toxin the anti-
toxin occurs in the euglobulin fraction, but Gibson and Collins
(1907), and Ledingham (1907), were unable to confirm this.
Ledingham found that during immunisation of a horse that
ultimately yielded a high grade anti-toxin, the percentage glo-
bulin content of the total protein progressively increased, and
the increase affected the euglobulin more than the pseudoglo-
bulin fraction. In a horse which yielded a low grade anti-toxin
no such increase of globulin was observed.
With regard to the distribution of other immune bodies in
immune serum less information is at hand. Tetanus anti-
toxin was not found in the precipitate obtained from immune
serum either by dialysis or by means of carbon-dioxide and
acetic acid, but occurs in the precipitate obtained by saturation
with magnesium sulphate (Tizzoni and Cattani, 1891). Ac-
cording to Pick tetanus anti-toxin, like diphtheria anti-toxin,
is combined or associated with the pseudoglobulin fraction of
the serum. The same author states that the typhoid agglutinin
of horse serum also occurs in the pseudoglobulin fraction, while
cholera lysin and cholera agglutinin in the horse and goat
occur in the euglobulin fraction. Gibson and Collins (1907).
found that the agglutinins for the dysentery group of organisms,
typhoid, colon and cholera were not confined to either of the
globulin fractions. Landsteiner (1908), showed that the sub-
stance occurring in normal serum which exerts an anti-tryptic
effect is associated with the albumin fraction of the serum,
and this was confirmed by Cathcart (1904). Opie and Barker
PREPARATION OF ANTI-RINDERPEST SERUM.
219
(1907), found that an anti-leucoprotease occurs in serum
albumin.
The experiments described below were begun with the
object of discovering to which fraction of the proteins of Rinder-
pest Immune Serum the Rinderpest Immune Bodies are asso-
ciated, and to see if an economical process could be evolved for
the preparation of the Rinderpest prophylactic in a stable and
convenient form. In this study of the distribution of the Rin-
derpest Immune Bodies in Rinderpest Serum numerous diffi-
culties have been encountered. The methods available are not
so simple as those available for the similar study of diphtheria
anti-toxin. The amount of diphtheria anti-toxin in a given fluid
can be estimated easily and with a considerable degree of accu-
racy. Guinea-pigs serve as excellent biological indicators and
they are cheap and the results are obtained very quickly. In
the case of Rinderpest the amount of Immune Bodies in a given
fluid is determined by balancing—not against a definite amount
of toxin—but against the disease itself. The only unit of
measurement which can be adopted is the amount of serum
which protects a susceptible animal from the disease; the end
point is the death or survival of the animal and the only
indicator available is the susceptible Hill Bull. Although the
means of measurement are not so delicate or refined in the case
of Rinderpest as in Diphtheria, yet it has been found possible,
by careful adjustment of the experimental conditions, to
obtain information regarding the distribution of the Immune
Bodies in Rinderpest serum, and it is hoped that the results
obtained will be capable of practical application.
EXPERIMENTAL PART.
The serum used in the experiments described below was
prepared by the usual routine methods practised in this Labora-
tory. It contained 0.5 per cent. carbolic acid and was of such
a potency that 90 c.c. injected into a susceptible Hill Bull of
600lbs. weight, protected the animal against Rinderpest,
this being the standard unit adopted in the preparation of
220
PREPARATION OF ANTI-RINDERPEST SERUM.
Anti-Rinderpest Serum issued for use in field operations.
Samples from four ''brews'' of serum (69, 73, 76 and
78) were studied.
THE EFFECT OF DIALYSIS OF THE SERUM ON THE RINDERPEST
IMMUNE BODIES.
Experiment 1.—100 c.c. of serum 69 was dialysed in an
ordinary dialysing tube which had previously been sterilised.
The dialysis was continued for six days in all, three days
against tap water and three days against distilled water.
At the end of this time the contents of dialysing tube were
transferred to a flask and filtered. The precipitate was
washed with distilled water and the washings added to the
filtrate. The precipitate on the filter paper was allowed
to drain overnight and then transferred to a sterile graduated
flask, salt solution 0.8 per cent. added until the volume
reached 100 c.c. and the whole thoroughly shaken. It was
observed that only a portion of the solid dissolved, an emulsion
being formed. The whole of this emulsion was injected
subcutaneously into Bull 4359 (age 2 years, weight 150lbs.).
On the other side of the body 0.5 c.c. of virulent Rinderpest
blood was injected. The animal showed no symptoms of
Rinderpest and recovered.
The filtrate from the dialysed serum was injected into Bull
4355 (age 4½ years, weight 210lbs.), 0.5 c.c. of virulent Rinder-
pest blood being injected into the animal on the other side.
The animal died of Rinderpest on the sixth day after in-
oculation.
Experiment 2.—Carried out as described under Experiment
1. 200 c.c. of serum 73 used. The precipitate formed as a
result of dialysis was washed and emulsified in salt solution,
the volume being made up to 200 c.c. For the purpose of dose
calculation and testing of the precipitate it was assumed that
all the Immune Bodies had been precipitated on dialysis, as
the results of Experiment 1 suggest that this takes place.
Bull 5323.—Age 3 years. Weight 240lbs.
PREPARATION OF ANTI-RINDERPEST SERUM.
221
Injected subcutaneously with 36 c.c. of emulsion (one
protective dose) on one side and with 1 c.c. of virulent Rinder-
pest blood on the other.
Result.—Animal recovered. Subsequently received two
massive injections of defibrinated virulent blood (2,000 c.c.
and 2,500 c.c), and was bled for serum.
Bull No. 5320.—Age 2 years. Weight 178lbs.
Injected subcutaneously with 54 c.c. of emulsion (two pro-
tective doses) on one side and 1 c.c. of virulent Rinderpest blood
on the other.
Result.—Animal recovered and was subsequently injected
twice (1,700 c.c. and 2,000 c.c), with virulent blood and
bled for serum.
Bull 5321.—Age 2 years. Weight 186lbs.
Injected subcutaneously with 84 c.c. of emulsion (three
protective doses) on one side and 1.0 c.c of virulent Rinderpest
blood on the other.
Result.—Animal recovered and subsequently received two
massive injections of virulent blood (1,500 c.c. and 1,800
c.c) and bled for serum.
Test of filtrate from dialysis of 200 c.c. of serum 73.
Guided by the result of Experiment 1 it was assumed that
the filtrate contained no Immune Bodies.
Bull 5322.—Age 3½ years. Weight 178lbs.
The whole of the filtrate from Experiment 2 was injected
subcutaneously on one side, and 1 c.c. of virulent Rinderpest
blood on the other. Vesicles appeared on the sixth day after
inoculation and ulcers on the seventh day. Diarrhœa on the
sixth day which persisted until the 15th day, when the animal
died of Rinderpest. Death was somewhat delayed, probably
on account of treatment given in the hope of saving the animal
for serum-making purposes.
From the results of Experiments 1 and 2 it may be conclud-
ed that dialysis of Anti-Rinderpest Serum results in the precipi-
tation of the Rinderpest Immune Bodies. The two animals