344

LVIII. BESREDKA'S METHOD OF VACCINATION.

BY SYDNEY ROWLAND, M.A., M.R.C.S.
Of the Lister Institute.

A METHOD of vaccination that has achieved considerable reputation is that advocated by Besredka of the Pasteur Institute, Paris'.
"V4ccination par les virus sensibilises," as he terms his method,
consists essentially in the use of killed cultures of the organism
concerned. In this it does not differ, so far as concerns plague, from
the method of Haffkine. But Besredka claims that, if the killed
organisms are submitted to a preliminary soaking in a serumn containing the specific antibodies to the organism concerned, these
antibodies neutralise the toxic substances contained in the bacilli,
which can then be used as a vaccine having the advantage of being
atoxic.
The present position of the author of this method will be found
described in the Builletin de l'Institut Pasteur (1910), vol. viii. p. 241,
where he says "le vaccin antipesteux sensibilise est depourvu de toute
action toxique."
Now in the case of the plague bacillus we can extract the specific
endotoxine (for rats), consequently we are in the position of being able
to control this claim. Assuming that a given quantity of plague
bacilli will normally yield a certain definite amount of endotoxine,
then it is obvious that, if Besredka's explanation of the principles
underlying his method be correct, the same quantity of plague bacilli
will, after soaking in the antiserum, yield a smaller amount of endotoxine or none at all.
The following experiments were undertaken to determine
(1) The toxicity of a "whole" vaccine prepared according to
Besredka's method.
(2) The amount of endotoxine that could be extracted from
plague bacilli before and after soaking (sensitising) in antiserum.
1 See the reviews

by Besredka in Bulletin de l'Inetitut Pasteur, vol. viii. (1910), p. 241

and vol. x. (1912), p. 529.

Reports on Plague Investigations in India

345

Experiment I. Determination of the toxicity of a vaccine prepared

according to Besredka's method.
In this experiment the relative toxicity of three vaccines is determined. The three vaccines are comparable in every way except that in
the case of vaccine 1, the bacilli were soaked (sensitised) in an antiplague serum; in the case of vaccine 2 they were soaked in normal
horse serum; and in the case of vaccine 3 they were soaked in physiological salt solution.
The serum employed in the case of vaccine 1 was prepared by immunising a horse by means of the endotoxine of the plague bacillus
as already described (Journal of Hygiene, vol. xi. (1911), supplement,
p. 11). At the time of the experiment this serum was of such a
strength that one cubic centimetre neutralised 150 lethal doses of
endotoxine. The proportion of serum to bacilli was so arranged
that there was present in the quantity of serum employed sufficient
antitoxin to neutralise four times the amount of endotoxine present
in the bacilli.
These relations having been determined by preliminary experiments,
100 Roux bottles of agar were inoculated with plague and incubated
for four days at 340 C. The bottles were then heated to 600 C. for an
hour to kill the bacilli. This is the temperature and duration of heating
employed by Besredka. The growth was washed off in salt solution
(0 8 l/0) and the bacilli twice washed in salt solution in the centrifuge.
After the last washing the bacilli were ag,ain centrifuged and 41
grammes of paste obtained. This paste contained 13-84 0/0 of solids
when dried at 1050 C. Ten grammes of the paste were put in each of
three flasks containing glass beads and there were added 35 c.c. of
antiplague serum, normal serum and salt solution respectively. When
a uniform distribution of the organisms throughout the respective fluids
had been effected the flasks were put aside in a cool place till the
following day. Meanwhile the quantity of bacilli in emulsion 3 was
determined. It was found that the strength of this emulsion as
determined directly corresponded with the strength as calculated from
the analysis of the paste from which it was made. We may take it
then that the paste of organisms was homogeneous, that the process of
emulsification was adequate and that the three emulsions were comparable in the amount of organisms they contained.
The lethal dose of the three emulsions was then determined by
subcutaneous inoculation into rats. Two rats were used for each
dose:

Begredka's Vaccine

346
Dose mg.

Emulsion 1

Emulsion 2

Emulsion 3

10

Died 2 days

,,

Died 2 days
.. ,.
Survived

Died 2 days
Survived
Died 2 days

,,
Survived
Died 3 days

Survived

,,

Died 2 days

Survived

Survived
Died 2 days

,,

S,, urvived

5
..,.

,.

4
2

,.

,.

..

The lethal dose of the three emulsions does not differ to any
great extent. Outside the error of this kind of experiment there is
no difference discoverable between the lethal dose of the organism that
had been soaked (sensitised) in the antiplague serum and those that
had been treated simply with salt solution. A slight apparent difference
is noticed in favour of the organisms that had been sensitised in the
normal horse serum.
A repetition of this experiment showed no diminution of toxicity in
the case of the bacilli treated with serum.
Experiment II. Estimation of the amount and toxicity of the
endotoxine that can be extracted from plague bailli before and after
soaking in antiplague serum, normal horse serum acnd salt solution.
A paste of organisms was prepared as in the last experiment:
32-5 grammes of paste were obtained containing 15-6 0/ solids.
Three suspensions of the paste were made as in the last experiment.
Suspension 1 contained 10 gr. paste in 37'5 c. c. antitoxic serum.
,,
,,

2
3

,,
,,

,,
,,

,,
,,

normal horse
salt solution.

serum.

The three suspensions were left in a cool place until the following
morning. They were then centrifugalised and the paste of organisms
washed free from serum proteins. The final washed paste obtained
was mixed with twice its weight (in each case) of anhydrous sulphate
of soda. The semi-fluid mass thus formed soon set to a solid mass
which was reduced to powder. The full description of this process

Reports on Plague Investigations in India

347

of extracting the endotoxine of the plague bacillus has already been

given in these reports (vol. x. (1910), p. 553). The powder was
dissolved in warm distilled water and in such a volume as to form
a satuirated solution of sodium sulphate. After filtering off the solution
the residue on the filter consisting of plague bacilli, sensitised or not as
the case might be, was extracted with distilled water. The same
amount of water was of course used in each case. Three extracts were
thus obtained which contained the endotoxine from the three portions
of bacilli which had been treated in the three ways indicated. Each
extract was filtered through hardened filter paper to remove the bodies
of the bacilli and the three filtrates examined as follows.
First the amount of nucleoprotein in each was determined by boiling
a portion after acidification with acetic acid, drying and weighing the
precipitate obtained. The weights of nucleoprotein in the three extracts
in milligrammes per c.c. was as follows:
No.1

No.2

No.3

6-62

6-34

6-66

These figures are very similar. There is no appreciable difference

in the weight of endotoxine (nucleoprotein) extracted in the three
cases.
Having thus failed to find any difference in the quantitative yield
of nucleoprotein in the three cases, it remained to ascertain whether
there was any qualitative difference in the three extracts. For this
purpose the lethal doses of the three extracts were determined on
rats. For the purposes of this determination the three extracts were
made up to such a strength that 5 c.c. of each extract contained
1 milligramme of nucleoprotein. The three solutions were inoculated
subcutaneously into rats as follows, two rats being used for each dose:
Dose mg.

Extract 1

Extract 2

Extract 3

1.0

Died 1 day

Died 1 day

Died 1 day

0-8,6
0s6

04
0-2
0.1

,,

Died 2 days
Survived
Died 1 day
Died 2 days
Survived
,,

,,

Died 2 days
Died 1 day
Survived
Died 1 day

Survived

Died 3 days
,,

Survived

The minimal lethal dose of the nucleoprotein obtained from bacilli

that had been sensitised in immune horse serum was thus substantially

348

Besredka's Vaccinie

the same as the minimal lethal dose of the nucleoprotein obtained

from the bacilli that had been treated (sensitised) in normal horse
serum or in saline. The yield of nucleoprotein was also the same in
the three cases.
From this experiment, which was repeated with similar results, it is
concluded that on sensitising the plague bacillus in an antitoxic serum
no permanent neutralisation of the endotoxine takes place.
The control experiment in which the bacilli were treated in salt
solution shows that the effect of sensitising in either anti or normal
serum is nil as regards the amount of nucleoprotein or the toxicity
of the nucleoprotein.
I am therefore unable to confirm Besredka's statement that
sensitised organisms yield an atoxic vaccine, for the whole organisms
after sensitisation are just as toxic as before and no neutralisation of
the endotoxine takes place as the result of the sensitising.
As the immune serum neutralised the endotoxin after extraction
from the bacilli it must be concluded from these experiments that
the antitoxine cannot enter the bodies of bacilli killed by heat.
There is, however, evidence that Besredka's vaccine, is possessed
of good immunising power and users of it have reported favourably
as to the minimum of discomfort following its inoculation. A great
point of this latter property is made by Besredka. Paladino-Blandini
(Annali d'Igiene sperimentale (1905), pp. 295, 411), after an exhaustive examination of several methods of antityphoid vaccination, speaks
in terms of high praise as to this property of Besredka's vaccine.
Dopter (Annales de l'Institut Pasteur (1909), vol. xxiii. p. 677;
C. R. Soc. Biol. vol. LXIV. (1907), p. 379), working with dysentery,
speaks to the same purpose.
Besredka attributes the advantages of his sensitised vaccines firstly
an
to actual neutralisation of the endotoxin and secondly to sensitisation (opsonisation) of the bacteria. For the former I can find no
support from my experiments with plague, but immune horse serum
does contain opsonin. At one time Besredka recommended the use of
normal horse serum in the preparation of his sensitised vaccine as
being equally efficacious. Normal serum, however, can hardly be
supposed to be antiendotoxic or possessed of any considerable specific
opsonising power.
A further possibility is that after soaking in immune serum the
bacilli are more readily lysed when placed under the skin. In this
respect treatment with normal horse serum may be advantageous, for

Reports on Plague Investigation in India

349

whilst engaged in some investigations into the mechanism of plague

immunity I found that in the normal horse a natural amboceptor for
the plague bacillus exists in considerable quantity. It is possible that
in Besredka's method this natural ainboceptor is responsible for sensitising the bacilli so that they dissolve more rapidly after injection.
Whilst at first Besredka employed specific sera for his sensitisation,
later he abandoned these for the use of normal horse serum. Later
still he reverted to the use of specific sera and makes the statement
that it is necessary for these to be highly agglutinating.
The main experiments that led to the recognition of the normal
amboceptor in the serum of normal horses are as follows:
(1) 80,000,000 virulent living bacilli were added to 1 c.c. of fresh
normal horse serum. The serum was kept at 370 C. No multiplication
took place and at the end of 24 hours the serum was sterile.
(2) 83,000,000 living virulent bacilli were added to 1 c.c. of the
same serum as was used in the last experiment. The serum had been
heated to 58 C. for one hour. Multiplication took place and followed
the same curve when the numbers were plotted against time as the
same number of bacilli inoculated into broth.
(3) 10,000,000 living virulent bacilli were added to one c.c. of
the same serum that had been heated. At the same time 0-04 c.c.
of fresh normal rat serum was added to the tube. No multiplication
took place and at the end of four hours only half a million bacilli were
recognised.
The growth and fate of living plague bacilli were traced in horse
serunm by the use of a method of direct microscopical observation.
Specially constructed observation slides were used which, combined
with a very perfect method of dark ground illumination, allowed of
a record of the appearances of the living organisms and of their number
being made as time advanced (see below, p. 362).
The further fact that so far as they have been examined all
antiplague sera prepared by Yersin's or some equivalent method are
strikingly deficient in specific plague antitoxine supports the explanation above given.