NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
86
Scientific Memoirs by
Examination of the spirillum blood withdrawn from the peritoneal cavity at
intervals of 10 minutes was made with the following results :—
. . . . . .
Interval between injection into and withdrawal from peritoneal cavity.
. . . . . .
10 minutes.
20 minutes.
30 minutes.
Hanging drop .
. One motile and one
dead spirillum.
No spirilla.
No spirilla.
Stained preparation . No spirilla. . No spirilla
. . No spirilla.
(2) Injection into peritoneal cavity of monkey S-9.— This monkey had a
typical attack of spirillum fever lasting for 3 days. The experiment recorded
was made immediately after the crisis of this attack.
The result of the examination of the spirillum blood removed from the peri-
toneal cavity at intervals of 10 minutes may be tabulated as follows :—
......
Interval between injection into and withdrawal from peritoneal cavity.
. . . . . .
10 minutes.
20 minutes.
3o minutes.
Hanging drop
.
. Few spirilla with slug-
gish movement.
No spirilla
. .
No spirilla.
Stained preparation. . Few spirilla which
stain badly.
No spirilla
. . No spirilla.
(3) Injection into peritoneal cavity of monkey S-23. —This animal had a
typical attack of spirillum fever of 3½ days' duration.
The experiment recorded was made 6 days after the crisis of this attack.
The examination made of the spirillum blood withdrawn from the peritoneal
cavity at intervals of 10 minutes gave the following result
:—
. . . . . .
Interval between injection into and withdrawal from peritoneal cavity.
. . . . . .
10 minutes.
20 minutes.
30 minutes.
Hanging drop. . . No definite spirilla ;
some granular dis-
torted spirilla.
Some granular clumps.
No spirilla.
No spirilla.
Stained preparation . A few spirilla which
stain well.
A very few spirilla
which stain well.
No spirilla.
Medical Officers of the Army of India.
87
(4) Injection into peritoneal cavity of fresh control monkey. —A specimen of
the spirillum blood withdrawn an hour after injection contained actively motile
spirilla in great numbers. These spirilla in the dry preparation were seen to take
the stain well and evenly.
It is evident, therefore, that there are present in the lymph of the peritoneal
cavity, both of highly immunized animals and of animals recently recovered from
an attack of spirillum fever, substances which rapidly kill and completely dissolve
up the spirilla when brought in contact with them in vivo. The granular clumps
which were noted in the case of monkey S-23, point to agglutination and
spherulation being steps in this process of complete dissolution.
We may now pass on to the consideration of several experiments which were
undertaken with a view of demonstrating in vitro, the presence of these bacterio-
lytic substances both in the blood serum of monkeys which had been highly
immunized and in the blood serum of animals which had recently recovered from
an attack of the specific fever. I shall preface the record of this investigation
by a short account of the method employed.
The blood of the animal under experiment, taken from the ear, was collect-
ed in capsules such as were devised by Professor Wright for the technique
of serum diagnosis.7 The capsules were then allowed to remain in
the dark at laboratory temperature for about 1 to 2h o u r s , t i l l a c l e a r s e r u m
had separated out.
The serum was then mixed in varying proportions, indicated in the results
detailed below, with spirillum blood freshly drawn from a monkey during the
attack and prevented from clotting by precipitating the lime salts with citrate
of soda. 8
Hanging drop preparations of the mixtures were at once made. These,
sealed with vaseline, were kept at room temperature (26º to 30º C.) and
examined at intervals under the microscope. The rest of the material was
stored in sealed capillary tubes, which were also kept at the temperature of the
laboratory. Stained slide preparations were made at intervals with the contents
of these tubes. The hæmoglobin was in all cases dissolved out with acetic
acid before staining. These preparations were examined microscopically at leisure.
In all instances control experiments were made, the serum of a fresh monkey,
procured in the manner described above, being substituted in the same propor-
tions for the serum of the immune animal.
Employing this method I made the following observations, which are here
detailed in chronological order :—
Experiment 1.—Serum of monkey S-2. This monkey had a severe attack
of spirillum fever with relapse. Seven days after the relapse it received 2 c. c. of
X
88
Scientific Memoirs by
spirillum blood. The experiment . now recorded was made on the eighth day after
this inoculation. The spirillum blood was got from a monkey on the first day of
fever ; its temperature was 105.6° F. and its blood was full of spirilla. The serum
and spirillum blood were mixed in three different proportions :—(1) three parts of
serum to one part of spirillum blood; (2) two parts of serum to one part of
spirillum blood ; and (3) equal parts of serum and spirillum blood.
The following were the results obtained :—
(1) Three parts of serum and one part of spirillum blood
(a) Hanging drop preparation.— Thirty minutes after mixing no normal
spirilla could be seen ; there were a number of granular puckered-
up bodies, without movement, which were probably degenerated
spirilla, One hour after mixing no trace of spirilla could be found.
(b) Stained preparations.—One and a half hours after mixing no trace of
spirilla could be seen.
(2) Two parts of serum and one part of spirillum blood
—
(a) Hanging drop preparation.— One hour after mixing no trace of
spirilla could be seen.
(b) Stained preparations :—No examination was made till 2 hours after
mixing. At this time no spirilla were found.
(3) Equal parts of serum and spirillum blood—
(a) Hanging drop preparation.— One and a half hours after preparation
no motile spirilla could be seen ; there were present a number of
bodies granular and spirillum-like but without motility.
(b) Stained preparations.—Two hours after mixing this showed a few
granular spirilla which stained very faintly and unevenly.
Control experiments were made with normal monkey's serum mixed with
the spirillum blood in the same proportions as noted above. In all these mixtures
the hanging drop preparations contained at the end of 4 hours, actively motile
spirilla : after 23 hours the spirilla were quite motionless. The stained
slide preparations 24 hours after mixing contained abundant spirilla which
stained well and evenly and showed no signs of degeneration.
Experiment 2.—Serum of monkey S-1. This animal had received two
inoculations of spirillum blood after recovery from a severe attack of fever. The
experiment recorded was made 7 days after the last inoculation.
The spirillum blood was got from a monkey on the first day of fever. Its
temperature was 106.4° F. Spirilla were plentiful although not abundant in this
blood.
Medical Officers of the Army of India.
89
The only proportion in which the serum and blood were mixed was that of
equal parts.
T h e f o l l o w i n g r e s u l t w a s o b t a i n e d : —
(a) Hanging drop preparation.—Forty minutes after preparation a few
sluggishly motile spirilla were seen. One hour after mixing motility
had completely ceased, while only one dead spirillum could be
found. Two hours after mixing no trace of spirilla could be seen.
(b) Stained preparation—Forty minutes after mixing a few isolated
spirilla and some clumps of spirilla were seen ; the spirilla took the
stain well. One hour after mixing no normal spirilla could be found,
while a few spirillum-like bodies which stained badly were demon-
strable. Two hours after preparation not a trace of spirilla could be
seen.
On the other hand, the control preparations made with equal parts of normal
serum and the same spirillum blood contained at the end of 2 hours very
actively motile spirilla, which stained well and evenly.
Experiment 3.—Serum of monkey S-9. This animal had an attack of
spirillum fever which lasted 3 days. Three days after the crisis it received
3 c. c. of spirillum blood. The experiment now detailed was made 3 days
after this last inoculation. The spirillum blood was the same as that employed
in experiment 2. The, serum and blood were mixed only in equal proportions.
The following observations were made :—
(a) Hanging drop preparation.— Fifteen minutes after mixing, some
motile spirilla were seen. Forty-five minutes after mixing a few
spirilla with slight movement and a few dead ones were found. One
and a half hours after preparation no motile organisms could
be seen, and only one dead spirillum was demonstrable.
(b) Stained preparation.—Fifteen minutes after mixing, spirilla which took
the stain badly were found both isolated and collected together in
clumps. Forty-five minutes after preparation only a few granular
puckered-up spirilla which stained badly could be found. After one
and a half hours no trace of any spirilla could be seen in these pre-
parations.
The control preparations made with normal serum and the same spirillum
blood contained at the end of two hours actively motile spirilla, which were seen
in the dry film preparation, made after the same interval of time, to take the
stain well and evenly.
90
Scientific Memoirs by
Experiment 4.—Serum of monkey S-10. This monkey had suffered from
a mild attack of spirillum fever of 2 days' duration. The experiment was
made 3 days after the crisis of this attack. The same spirillum blood was
employed as in experiments 2 and 3 detailed above. The only preparation made
was that in which the serum and spirillum blood were mixed in equal parts.
The following results were obtained :—
(a) Hanging drop preparation.— Fifteen minutes after mixing spirilla were
still actively motile. After half an hour no motile spirilla could be
seen, although the corpuscles still showed slight movement. One
and a half hours after preparation no motile spirilla could be found,
but some round granular bodies, which might have been degen-
erated spirilla, were seen.
(b) Stained prepraration.—Twenty minutes after mixing, spirilla, both isolated
and in clumps were seen; some took the stain well, others very
faintly. One hour after preparation a few granular forms were seen.
After two hours not a trace of spirilla could be found.
In the control preparations made with normal serum and the same spirillum
blood, at the end of 2 hours the spirilla were actively motile; while the stained
specimen prepared after the same lapse of time contained plentiful well-stained
spirilla.
Experiment 5.—Serum of monkey S-13. This monkey had suffered from
a mild attack of spirillum fever of two days' duration. The serum used for this
experiment was got a few hours after the crisis.
The spirillum blood employed was taken from a monkey on the first day of
fever with a temperature of 105.4° F.; it contained numerous but not abundant
spirilla. The only proportion in which the serum and blood were mixed was that
of equal parts.
The following observations were recorded :—
(a) Hanging drop preparation.— Fifteen minutes after mixing some motile
spirilla and others with no movement were seen. One hour after
preparation no normal spirilla, but several puckered-up granular
spirillum-like bodies with slight movement were found. After two
hours no spirilla were seen, only a few motionless rounded granular
bodies.
(b) Stained preparation.— One hour after mixing not a single spirillum
could be found. The same negative result was obtained with the
slides made 2 hours after mixing.
Medical Officers of the Army of India. 9 1
Experiment 6 .—Serum of monkey S-23. This monkey had suffered from an
attack of spirillum fever of 3 days' duration. The blood serum which was
used for this experiment was got immediately after the crisis.
The spirillum blood employed was taken from a monkey on the second day of
fever with a temperature of 104.2° F. Spirilla were present only in small number
in this blood. Only one preparation was made : in this preparation the serum and
spirillum blood were mixed in equal parts.
The following observations were recorded :—
(a ) Hanging drop preparation.—Twenty minutes after mixing no trace of
spirilla could be found.
( b ) Stained preparation.— In the specimen, made 40 minutes after
mixing the serum and blood, no spirilla could be seen. Unfortunate-
ly no earlier specimen was prepared.
From the data collated above it is obvious that, as a result of an attack of
spirillum fever, specific anti-bacterial substances make their appearance in the
blood and lymph of the infected organism and that these substances are demon-
strable both 'i n
v i v o '
a n d '
i n v i t r o . '
I t i s f u r t h e r c e r t a i n t h a t u n d e r t h e i n f l u e n c e
of these anti-bacterial substances the spirilla undergo immobilization, distortion,
granulation and ultimately complete dissolution. Moreover, I have evidence to
show that agglutination of the spirilla is a step in the process of dissolution when
the spirilla are brought in contact with a specific serum more dilute than was
used in the experiments recorded above.
We have already seen that an attack of spirillum fever in the monkey
•
gives protection against a second infection for a period of about a month. In con-
nexion with the subject now under discussion, it is important and interesting to re-
cord that a similar immunity is also enjoyed by an animal, the spleen of which has
been removed shortly after the crisis of the primary attack. The experiments
which I have made to demonstrate this point are detailed as follows
: —
Experiment 1.—Monkey S-8 had a severe attack of spirillum fever of four
days' duration. Immediately after the crisis the spleen was removed. Three
weeks afterwards it received 4.5 c.c. of spirillum blood taken from a monkey on
the second day of fever. This blood was full of spirilla. No attack resulted
from this inoculation.
A fresh control monkey received 4 c. c. of the same material. It suffered
as a result of this injection from an attack of spirillum fever which lasted for
two days.
Experiment 2.— Monkey S-12 had an attack of spirillum fever of three
days' duration. The spleen was removed immediately after the crisis. Six days
Y
92
Scientific Memoirs by
afterwards it received 2 c. c. of blood taken from a monkey on the third, day of
fever. This blood contained spirilla in enormous numbers. No attack resulted
from this injection.
A fresh control monkey also received 2 C. c. of the same material. As a
result it suffered from an ordinary attack of spirillum fever of three days' duration.
Experiment 3.—Monkey S-14 had an attack of spirillum fever of two days'
duration. The spleen was removed immediately after the crisis. Ten days
afterwards it received 2 c. c. of blood taken from a monkey on the first day of
fever. This blood contained numerous spirilla. This injection was not followed
by an attack of spirillum fever.
A fresh control monkey received 2. c. c. of the same material. As a result
this animal suffered from a typical attack of spirillum fever of two days' duration.
Experiment 4.—Monkey S-17 had a severe attack of spirillum fever of
four days' duration. The spleen was removed the day after the crisis. Three
weeks afterwards it received 5 c. c. of spirillum blood taken from a monkey on the
second day of fever. This blood contained abundant spirilla. No fever resulted
from this inoculation.
A fresh control monkey received 4. c. c. of the same material. This
inoculation was followed by a typical attack of spirillum fever.
Experiment 5.—Monkey S-19 had suffered from a characteristic attack of
spirillum fever of three days' duration. The spleen was removed immediately
after the crisis. Twelve days afterwards it received 4 c. c. of spirillum blood
taken from a patient on the 4th day of the primary attack. This blood contained
spirilla in large quantity. No fever followed this inoculation.
A fresh control monkey received 2 c. c. of the same material. As a con-
sequence it suffered from a typical attack, of spirillum fever of four days' duration.
Before proceeding to critically sift the data which have been placed on
record above, it would be well to further extend these data by detailing a single
experiment made with a view to demonstrating the protective influence of the
serum of an immunized monkey, when this serum is injected at the same time
as the spirillum material into a monkey from which the spleen had been previous-
ly removed.
This experiment was as follows :—
Monkey S-26 had its spleen removed on the 1st February. Three weeks
afterwards (by which time the wound had completely healed), it received :--
(1) 2 c. c. of spirillum blood taken from a monkey on the second day of
fever. This blood contained abundant spirilla.
(2)18c.c. of blood plasma of monkey S-2. This animal had been
immunized to a high degree in the manner detailed on page 84.
Medical Officers of the Army of India.
93
The plasma was injected subcutaneously in several places. No attack of
spirillum fever followed.
The control monkey, S-25, had its spleen removed also on February 1st.
It received 2 c. c. of the same spirillum blood as monkey S-26. As a result it
suffered from a typical attack of spirillum fever. This attack lasted for three days
and was terminated by crisis in the ordinary way, the spirilla at the same time
disappearing completely from the blood.
It may be noted in passing that this recovery of a spleenless monkey does
not confirm the observations of Soudakewitch 9. This observer produced the
disease in two monkeys from which the spleen had been previously removed. In
these cases the spirilla did not disappear from the blood at the usual time of
crisis, but rather increased in number and a fatal result followed.
Further I have notes of another monkey which was inoculated with spirillum
material 5 days after its spleen had been removed. This animal received
1 c.c. of the spirillum blood by intra-peritoneal injection and 4 c. c. under the skin.
As a result it suffered from a characteristic attack of fever with spirilla in the blood.
This attack lasted for 3 days and terminated by crisis, the spirilla at the
same time disappearing from the blood.
The conclusions which appear to be justified from the data put on record
above can conveniently be stated as follows :—
(1) One attack of spirillum fever in the monkey protects from a second in-
fection. This immunity, however, lasts only for a short period of
time. Monkeys from which the spleen has been removed shortly
after the crisis of an attack are also immune to a second infection
for at least a short period of time.
(2) In the blood and lymph both of highly immunized monkeys and of
monkeys recently recovered from an attack of spirillum fever, anti-
bacterial substances are present and can be demonstrated both in
vivo and in vitro.
(3) The blood plasma of a highly immunized animal confers protection on
a spleenless monkey when inoculated at the same time as the
spirillurn material.
(4) Fresh monkeys from which the spleen has been previously removed
may pass through an ordinary attack of spirillum fever, ending in
recovery by crisis and complete disappearance of the spirilla from
the blood .
From these facts it would, therefore, appear certain that there must be some
relation between the production of anti-bacterial substances and the production.
94
Scientific Memoirs by
of the immunity which I have shown is enjoyed by the monkey as a result of an
attack of spirillum fever, and it would also appear certain that the spleen has not
the important function which has been assigned to it by some observers in the
destruction of the spirillum organism.
The mental picture of the process which these observations suggest to me
may be briefly stated as follows :
As a result of spirillum infection in the monkey, anti-bacterial substances make
their appearance in the blood and lymph from an early date in the course of the
attack : these substances increase rapidly in quantity and ultimately at the time of the
crisis are present in the blood in such amount as to be able to destroy the vitality of
the spirilla circulating there. The spirilla partially, if not completely, devitalised
in conformity with the general rule which obtains when either bacteria or inert
particulate material, such as carmine particles, is introduced into the blood stream,
become deposited in the spleen and other internal organs. There, such as have
not been completely dissolved by the bacteriolytic substances, are taken up and
digested by the polynuclear white cells. It has been shown by Metchnikoff1 0
and other observers that for a period of some hours after the spirilla have com-
pletely disappeared from the blood, they are still alive in the spleen and in a
condition able to give rise to infection in a fresh animal. Experiments, to be
detailed later, point to the fact that the anti-bacterial substances immediately
after the crisis are present in greater amount in the blood serum than in the
splenic serum. Our theory, therefore, is not upset by those observations.
For a short time after the crisis, the anti-bacterial substances present in the
blood may possibly undergo an increase in quantity. The experiments of
Bardach1 1
p o i n t t o t h i s . S u c h a n i n c r e a s e w o u l d b e i n c o n f o r m i t y w i t h t h e
well-known fact that a considerable increase of the specific agglutinins takes
place in some cases of typhoid fever after the defervescence of the fever. It is, how-
ever, only reasonable to suppose that these anti-bacterial substances must under-
go, before the Organism can be re-infected, such a diminution in amount
as to permit of the spirilla again living and cultivating themselves in the blood
and lymph of the body. That they do not completely disappear is evident from the
fact that they can be demonstrated in vitro practically up to the time when re-in-
fection is possible. Now this is a question which at first sight would appear
capable of being resolved by a quantitative determination of the anti-bacterial
substances contained in the blood serum or lymph at varying intervals after the
crisis. A moment's consideration, however, will show that results comparable
among themselves cannot be obtained in the case of any one monkey. For the
bactericidal power of any serum or lymph may a priori be expected to vary