Thanks to the work of several investigators this question may now be

answered. Pfeiffer and Marx[459] first supplied important information

concerning the origin of the protective property. Into rabbits they made

subcutaneous inoculations of cholera vibrios, killed by heat (70° C.),

and then examined, most minutely, the protective power of the blood and

of extracts from various organs. Examining, separately, the protective

power of the serum and that of the layer of leucocytes deposited in

tubes, Pfeiffer and Marx were unable to find any marked difference. Nor

did they ever obtain any definite effect with leucocytes collected from

pleuritic exudations. From these observations they concluded that the

leucocytes of the blood could not be regarded as the source of the

protective substance (or “cholera antibody”). At a period when the serum

as yet exhibited an insignificant protective power or none at all, the

extract from the spleen often exerted an action of the most marked

character. In an experiment in which the rabbit was killed 48 hours

after the injection of the vibrios, 0·3 c.c. of the serum was incapable

of preventing fatal infection of a guinea-pig, whereas 0·03 c.c. of an

extract of the spleen exerted a marked protective effect. From this and

similar experiments, Pfeiffer and Marx conclude that the spleen is the

principal source of the protective substance. In order to verify this

observation they injected killed cholera cultures into rabbits which had

previously been deprived of their spleens, but the asplenic rabbits

still produced the same amount of protective substance, and these two

observers were led to conclude that the lymphatic glands and the

bone-marrow might also serve as the sites of origin of this substance.

It is only during the first few days, however, that these organs exhibit

a protective power greater than that of the blood. Three or four days

after the injection of the vibrios the blood serum becomes richer in

protective substance; the organs contain much less of it. This condition

is maintained for some time, after which the blood in turn begins to get

impoverished.

[Sidenote: [307]]

Pfeiffer and Marx put to themselves the question: Is the marked

protective power of the spleen due to the production of preventive

substance by this organ, or is it to be explained by an accumulation in

the spleen of this substance manufactured elsewhere? With the object of

obtaining an answer to this question they injected protective serum from

other individuals into rabbits, when they found that the protective

substance showed not the slightest tendency to accumulate in the spleen.

These authors were compelled to conclude, therefore, that the spleen and

other haematopoietic organs (lymphatic glands and bone-marrow) are the

real seats of the production of the protective substance. We may add

that these organs are also the phagocytic organs _par excellence_, that

is to say, the centres which serve not only for the development of

phagocytes but which contain a large number of the adult elements

capable of exercising the phagocytic function.

Almost simultaneously with Pfeiffer and Marx, A. Wassermann[460], in

collaboration with Takaki, undertook similar researches on the origin of

the substance protective against the typhoid cocco-bacillus. The outcome

of this work was that “it was the bone-marrow, the spleen, and the

lymphatic system, including the thymus gland, which exhibited immunising

power against the bacillus of typhoid fever, whilst the other organs,

the blood, brain, spinal cord, muscles, liver, kidney, etc., did not at

this stage show any marked specific property.”

[Sidenote: [308]]

As these observations on the production of protective substance in the

phagocytic organs was one of essential importance in connection with the

problem of acquired immunity, I asked M. Deutsch[461], working in my

laboratory, to carry out a series of experiments on this subject. Using

guinea-pigs, he injected into the peritoneal cavity cultures of the

typhoid bacillus killed by heat (66° C.). A few days later the serum had

become distinctly protective. At this stage, and even before the

appearance of this property in the blood, Deutsch killed some of his

animals and carefully measured the protective power of the extract of

the various organs. He began by confirming the result obtained by

Pfeiffer and Marx as to the non-production of the protective substance

in the peritoneal exudation. Usually this fluid was insufficient to

protect normal guinea-pigs against typhoid infection. In a few

experiments only was the exudation found to be as protective as the

blood serum; in most of the others, the blood serum was much more active

than the fluid of the exudation. The spleen was the organ which

exhibited the greatest protective power, and in nearly one-half of the

cases it was more active than was the blood. The bone-marrow sometimes

gave analogous though much less marked results. The spleen consequently

must be looked upon as the principal seat of the production of the

protective substance.

Having confirmed this observation of Pfeiffer and Marx and of Wassermann

and Takaki, Deutsch tried to obtain the protective property in

guinea-pigs deprived of their spleens. The experiment was quite

successful, and here again his result agreed with that obtained by

Pfeiffer and Marx. Guinea-pigs from which the spleen had been removed

developed the protective property just as well as did the control

animals; in the former the bone-marrow was found to be specially active.

When Deutsch, instead of removing the spleen from his guinea-pigs before

the injection of the micro-organisms, did so some (3–5) days afterwards,

there often occurred a marked diminution in the amount of the protective

substance produced. We must conclude, therefore, that soon after

inoculation there appears in the spleen a phenomenon which is associated

with the development of the protective power. The most simple

explanation of these facts is that the micro-organisms injected into the

peritoneal cavity and soon afterwards seized by the phagocytes (for the

most part by the microphages), are carried to the phagocytic organs,

particularly the spleen, lymphatic glands, and bone-marrow. In those

animals whose spleens are left intact a large number of these

microphages loaded with micro-organisms make their way into this organ,

a fact confirmed by direct observation. When the spleen is removed the

microphages must necessarily betake themselves to other phagocytic

organs. As the micro-organisms undergo intracellular digestion in the

phagocytes, it is very difficult, if not impossible, to follow them for

any length of time after they have been ingested, but the analogy with

the phenomena of the resorption of red blood corpuscles, described in

Chapter IV, justifies us in concluding that in the case of

micro-organisms matters go on in much the same way. These organisms,

seized at the seat of inoculation by the phagocytes, are transported by

these cells, in their peregrination through the organs, into the general

circulation. The interpretation I have just given has been accepted by

Deutsch.

[Sidenote: [309]]

This observer wished also to come to some conclusion as to the origin of

the agglutinative property so well developed in the fluids of animals

inoculated with the typhoid cocco-bacillus. He did not succeed in

solving this question, but he was able to demonstrate the undoubted

difference between this property and the protective power. The facts

brought forward by Deutsch must, therefore, be ranged alongside the many

others, reported on above, which demonstrate in the most conclusive

fashion that these two powers of the body fluids are essentially

distinct.

Such concordant results obtained by all investigators who have studied

the origin of the protective power warrant the conclusion that it is the

elements of the phagocytic organs, that is to say, the phagocytes

themselves, which produce the protective substance. But it will be

asked: Can we therefore accept the fixative substance or fixative as

being derived from the same source? When the experiments I have just

summarised were carried out the fixatives were not as yet sufficiently

known and were confounded with the protective substances. Nevertheless,

there can be no doubt as to what the answer to the question just put

must be. In the account of the experiments of Pfeiffer and Marx we find

very precise statements as to the granular transformation of the

vibrios. Thus, they observed on several occasions that an extract of the

spleen set up this transformation in a particularly distinct and rapid

fashion at a period when the blood and serum, used in a much stronger

dose, were incapable of producing the same effect. Now, as Pfeiffer’s

phenomenon is a visible manifestation of the action of the specific

fixative, it cannot be doubted that the spleen is really the principal

seat of development of the fixative substance before it makes its

appearance in the blood.

Before concluding this chapter we must review very briefly the principal

phenomena associated with acquired immunity against micro-organisms. The

extracellular destruction of these parasites takes place in the living

animal under special conditions only, when the phagocytes suffer a

temporary injury (phagolysis) and allow their microcytases to escape.

These latter by no means represent attributes of the body fluids, as is

even yet maintained by some writers. These soluble ferments are

connected with the phagocytes and represent the ferments of

intracellular digestion. The cytases undergo no modification during the

process of immunisation and correspond to those which act in natural

immunity.

[Sidenote: [310]]

The agglutinative substance often present in the normal fluids of the

body becomes much more developed in those of immunised animals. It is

truly humoral, as it circulates in the plasmas and passes into the fluid

exudations and transudations. But the part played by it in immunity is

very restricted.

The protective and fixative properties, most often closely connected

with each other, are very markedly developed in an animal enjoying

acquired immunity. They may act upon the micro-organisms which become

permeated by the fixative substance, or upon the infected animal by

stimulating its defensive reaction, but they are incapable of affecting

the vitality or virulence of the micro-organism. The two properties

(protective and fixative) reside in the fluids of the body, but they are

functions of the cell products. The elements of the phagocytic organs

(spleen, bone-marrow, lymphatic glands), or phagocytes, produce the

specific protective and fixative substances which pass thence into the

plasmas.

The phagocytic reaction is very general in acquired immunity. The

phagocytes which have a very imperfect antimicrobial function or none at

all, become, as the result of vaccination, much more active. They

exhibit a very marked positive chemiotaxis and acquire the faculty of

digesting micro-organisms in a greatly intensified degree. It is with

the increase of this digestive power that we have connected the

over-production by the phagocytes of the fixative and protective

substances which are excreted in large quantities by these cells and

pass into the fluids of the animal. As these substances are phagocytic

products it may be readily conceived that in certain examples of

acquired immunity the animal overcomes the micro-organisms without the

protective substances being found in the fluids. It is sufficient that

it is in the possession of the phagocytes, which may retain it within

themselves and not throw it off into the circulation.

[Sidenote: [311]]

From this account it will be seen that the phenomena, in acquired

immunity against micro-organisms, are merely a more or less stereotyped

copy of those that are presented in the animal after the resorption of

cells. There, also, we have intracellular digestion with over-production

of specific fixatives, part of which are excreted and thus pass into the

plasmas. In the resorption of cells there is also a double action of

cytases and fixatives; but in this case the macrocytases intervene,

whilst in the resorption of micro-organisms this function is performed

by the microcytases. The fixatives in the two cases are very different

from the point of view of their action, for they are specific; but the

cells which act in their production belong, in both cases (resorption of

animal cells and of micro-organisms), to the category of phagocytes.

It is often maintained that the theory I have just summarised is

fundamentally opposed to the theory of side-chains or receptors

formulated by Ehrlich[462]. This view I cannot accept. Applied to

acquired immunity against micro-organisms this theory may be summed up

as follows. The micro-organisms, when inoculated in a non-lethal but

immunising dose, combine with certain cells of the animal. The receptors

of the micro-organisms find corresponding receptors in these cells, but,

when once combined, the receptors of the cells become incapable of

fulfilling their normal nutritive function. The cells, thus deprived of

their receptors, reproduce such an enormous quantity of them that a

portion is excreted into the surrounding medium and passes into the

plasmas. These receptors, originating from cells, but which have become

constituent parts of the body fluids, are nothing but the fixatives or

intermediary bodies, or the amboceptors of Ehrlich. On a fresh arrival

of the same micro-organisms, they meet with, in the fluid of the

exudations, numerous amboceptors which combine with the corresponding

receptors of the micro-organisms, without, however, destroying them or

interfering with their vitality. As these amboceptors possess still a

second affinity, that for the molecules of the cytases, or the

“complements” of Ehrlich, the micro-organisms can be placed in contact

with these soluble ferments. Without the intervention of the fixatives,

the combination of the body of micro-organisms with the cytase can never

take place, because the receptors of the micro-organisms are not adapted

to those of the cytases. When the molecules of these ferments are found

in the plasmas in a free state, they can be attacked by the

corresponding group of the amboceptors.

[Sidenote: [312]]

Let us compare the theory we have just sketched with that described

further back. The micro-organisms, inoculated with a non-lethal but

immunising dose, are, as we have seen, ingested by the phagocytes and

afterwards digested within them. This intracellular digestion is

followed by the over-production of the specific fixative, of which a

part is excreted and passes into the plasmas. These are the results of

the well-established experimental data described in this chapter.

Ehrlich’s theory is in no way in opposition to this; it simply attempts

to penetrate more deeply into the mechanism of the phenomena observed as

taking place between the micro-organism and the cell. The act which we

simply term intracellular digestion is divided by Ehrlich into its

constituent parts. According to him there is a combination of the

fixative, on the one hand, with the molecule of the micro-organism, on

the other, with that of the soluble ferment or cytase. According to

Ehrlich it is the amboceptors of the cells which become detached in

order to furnish the fixative that circulates in the plasmas. For us

there is simply an over-production of one of the two ferments of

intracellular digestion, without defining more exactly what constituent

part of this ferment passes into the circulation. The two theories may

supplement each other but are in no way contradictory in principle.

There is only a single important point wherein they do not accord.

Ehrlich thinks that the cytases are always free in the body fluids and

that the cells, in order to exert a digestive action on the

micro-organisms, must previously seize their molecules by means of one

of the groups of their amboceptors. We, on the contrary, have developed

the idea that the cytases are only free in the animal during phagolysis

and that under normal conditions the cytases remain closely bound up

with the phagocytes. This statement is based upon a large number of

well-established experimental facts and must therefore be accepted as

proved. It does not, however, affect any fundamental principle of

Ehrlich’s theory. On the other hand the bases of Ehrlich’s theory affect

none of the main features of the theory I have developed. The doctrine

which regards acquired immunity as a particular case of resorption may

be reconciled with the conception of amboceptors. But it accords equally

well with Bordet’s conception, according to which the fixatives act not

as intermediary substances between the micro-organism and the cytase,

but as substances which sensitise the micro-organisms for the

penetration of the digestive ferment. This delicate question has not yet

been definitely settled, but Bordet’s experiments described in Chapter

IV are greatly in favour of this view.

[Sidenote: [313]]

Neisser and Wechsberg[463] have tried to obtain some idea of the manner

in which the fixatives act on the micro-organisms and have recorded a

series of very interesting facts. They have shown that these substances

only bring about the destruction of bacteria when they are in certain

relations with the cytase. Mixtures of fixatives and cytases in which

the former are found in excess not only do not kill the micro-organisms

but even allow them to develop abundantly. To attain this result Neisser

and Wechsberg mixed constant quantities of bacteria and normal serum

containing cytase with variable quantities of the serum of immunised

animals heated to 56° C. As we know, this specific serum, as the result

of being thus heated, is deprived of its cytases, but may be readily

made active again by the addition of normal, unheated serum. This

paradoxical fact, demonstrated by Neisser and Wechsberg can, in their

opinion, be explained only by Ehrlich’s theory of amboceptors. When

these bodies with double affinities are found in too large quantity as

regards the cytase, it may happen that one part only of those which

combine with the receptors of the micro-organisms succeed in linking to

themselves the molecules of the active ferment. The amboceptor being by

itself incapable of destroying the micro-organism, can be injurious to

it only on condition that it brings cytase. Consequently as the amount

of this cytase is too small for the much larger number of amboceptors we

can readily conceive that the micro-organisms may profit thereby and

remain alive. This interpretation is certainly very ingenious, but

nothing proves that it corresponds with the real state of things.

Neisser and Wechsberg have themselves observed that the serum of the

normal goat can also prevent the bactericidal action of the cytase. In

this case, however, they suggest the intervention of an anticytase of

this normal serum. The same explanation might perhaps serve also to

explain the preventive action of the serum of immunised animals. We know

that anticytases are found frequently enough in the various serums and

that they undergo great variations, according to the conditions present

in the animals furnishing the blood.

[Sidenote: [314]]

In any case, it is evident that the theory of receptors must in no way

be regarded as the antithesis of the theory of phagocytosis. This latter

quite retains its right to affirm that, in acquired immunity against

micro-organisms, phagocytes play the most general and important part.

They hold back the cytases which are capable of ridding the animal of

micro-organisms from destroying them. It is further these same cells

that produce and excrete the fixative and protective substances. The

free fixatives may attack the micro-organisms in the body fluids but

they are incapable of depriving them of life or even of virulence. The

cytases, after escaping from the phagocytes, may certainly, in

collaboration with the fixatives, destroy a certain number of the

micro-organisms, but only in special cases met with, no doubt, but only

rarely, under natural conditions. On the other hand, the phagocytes in

the animal which enjoys acquired immunity constantly fulfil the function

of seizing the micro-organisms and of submitting them in their interior

to the combined action of fixatives and cytases.

Acquired immunity, like natural immunity against micro-organisms,

presents merely special phases of intracellular digestion.

CHAPTER X

RAPID AND TEMPORARY IMMUNITY AGAINST MICRO-ORGANISMS, CONFERRED BY

SPECIFIC AND NORMAL SERUMS, OR BY OTHER SUBSTANCES, OR BY

MICRO-ORGANISMS OTHER THAN THOSE AGAINST WHICH IT IS DESIRED TO PROTECT

AN ANIMAL

Immunity conferred by specific serums.—Analogy of the mechanism of

this immunity with that observed in immunity obtained with

pathogenic micro-organisms and their products.—Part played by

phagocytosis in the immunity conferred by specific

serums.—Influence of opium on the course of immunisation by these

serums.—Stimulant action of specific serums.—Protective and

stimulant action of normal serums.—Influence of fluids, other than

serums: broth, urine, physiological saline solution, etc.

Antagonism between anthrax and certain bacteria.

[Sidenote: [315]]

We have seen how important in the study of acquired immunity against

micro-organisms is the demonstration of the protective power of the body

fluids. Without being absolutely general, this power is, nevertheless,

widely diffused and is found even in certain examples of acquired

immunity against micro-organisms belonging to the animal kingdom. Up to

the present I have refrained from doing more than point out the

presence, in the fluids of the immunised animal, of this protective

property and have studied it exclusively in relation to the animal that

produces it. We must now pass to the question: How do the protective

substances act in the animal which receives them ready formed? This

immunity, which has received from Ehrlich the name of _passive immunity_

against micro-organisms, must now be examined.

[Sidenote: [316]]

The study we now propose to enter upon is rendered much easier from our

study of the data acquired on the phenomena exhibited in the animal

vaccinated with micro-organisms or their products, data already given in

the preceding chapter. There is, indeed, a very striking analogy between

the two kinds of immunity, and though we draw a sharp line of

distinction between them, this is due to the fact that the immunity

conferred by micro-organisms or their products requires some time for

its development and endures for a long period, whilst the immunity due

to the introduction of specific serums into an animal is set up

immediately, but endures for a very short time only.

The diseases of the Invertebrata being seldom due to the micro-organisms

that produce infections in the higher animals, the question as to

whether the Invertebrata can be immunised by means of protective serums

has not yet been studied. Still, we already have certain ideas on the

protection of lower vertebrates by specific serums. Gheorghiewsky[464],

in my laboratory, carried out some experiments on this point. He found

that the serum of mammals (guinea-pig, goat) immunised against the

_Bacillus pyocyaneus_, was under certain conditions capable of

protecting the green frog against a dose of this organism that was

always fatal to the control animals. When injected along with the

_Bacillus pyocyaneus_, the serum did not prevent a fatal infection;

sometimes this infection developed even more rapidly than in the control

frogs. It was only when the protective injection was made 24 or, better

still, 48 hours before the inoculation of the bacilli, that the

protective action became evident. The serum used in these experiments

was not bactericidal for the _Bacillus pyocyaneus_ which grew most

luxuriantly; but it agglutinated a large proportion of the bacilli.

Gheorghiewsky pointed out, however, that frogs injected with cultures

agglutinated by the goat’s serum died just as readily as did the control

animals. As the phagocytic reaction was invariably very active in those

frogs which resisted the virus, after the injection of protective serum,

it is very probable that this fluid exercises a stimulant influence on

the phagocytes.

[Sidenote: [317]]

This idea of stimulation by anti-infective serums in cases of temporary

immunity conferred by these fluids, has already been set forth in my

researches on the immunity of rabbits against the Gentilly

cocco-bacillus, induced by the serum of vaccinated rabbits. This view,

however, has not been favourably received, especially in view of the

discovery of the phenomenon of the transformation of cholera vibrios

into granules. Pfeiffer himself noted that this transformation took

place not only in the peritoneal cavity of vaccinated guinea-pigs but

also in the peritoneal cavity of normal guinea-pigs, into which he had

injected small quantities of specific serum. As this latter fluid, in

Pfeiffer’s hands, was incapable of transforming the vibrios into

granules _in vitro_, he concluded that the cellular elements of the

normal animal were endowed with the power of modifying the inactive

substance of the specific serum into bactericidal substance. According

to this conception the immunity conferred by this serum was not entirely

passive since, in order to prepare the substance which transforms and

kills the vibrios, the co-operation of the living cells was necessary.

My demonstration of the possibility of obtaining Pfeiffer’s phenomenon

_in vitro_ at once turned the balance in favour of the theory that the

immunity induced by the specific serum is due to a direct humoral action

upon the micro-organism. Under these conditions such immunity could only

be interpreted as being purely passive. This view seemed to be finally

established by Bordet’s discovery that a specific serum, inactive by

itself, became capable of producing Pfeiffer’s phenomenon, as soon as a

small quantity of normal, non-specific serum was added to it.

Bordet[465] thus sums up his theory of the immunity conferred by

specific serums: “Passive immunity is due, in part at least, to a

chemical action exerted on the vibrios by two pre-formed substances, the

one present in the animal before any injection is made, the other found

in the serum that is injected; this phenomenon is purely chemical in the

sense that it can go on without the aid of a vital reaction, of any new

cell secretion: indeed it is found to take place in fluids from which

the cells have been entirely removed” (p. 217). These demonstrations led

up to the belief that the organism of the animal remained absolutely

passive when it was subjected to the action of protective or

anti-infective serums, and that the case of the cholera vibrio

represented a kind of schema, which was applicable to the whole of the

group of phenomena met with in passive immunity.

As in the study of the immunity obtained as the result of vaccinations

with micro-organisms or their products, so in “passive immunity” there

was seen only the direct chemical action of two substances on the

micro-organism, and efforts were made to extend this demonstration to a

series of infective diseases.

[Sidenote: [318]]

[Sidenote: [319]]

Pfeiffer and Kolle[466] having observed that the blood serum of persons

convalescent from typhoid fever, as well as that of animals vaccinated

with the typhoid bacillus, exhibited a great protective power for the

guinea-pig, wished to get some idea of the mechanism of this immunity.

They found that in the peritoneal cavity of guinea-pigs, inoculated with

the typhoid cocco-bacillus and simultaneously subjected to the action of

protective serums, the micro-organisms lose their mobility almost

immediately. A little later, they exhibit a degeneration of form, become

less refractile and disintegrate. After the injection of large doses of

specific serum the bacilli, much as in the case of the cholera vibrio,

become transformed into granules. “But,” say these authors, “this last

mode of destruction, that is to say the formation of granules at the

expense of the injected bacteria, does not occur with such remarkable

regularity as it does in Pfeiffer’s phenomenon in the cholera vibrio”

(p. 219). Whilst these changes are going on in the peritoneal fluid, the

leucocytes begin to come up and to ingest the bacilli and their

_débris_. “Phagocytosis, therefore, undoubtedly plays a part in the

destruction of the bacteria. Nevertheless, as most of the injected

bacteria die in the fluid of the exudation, phagocytosis can not be

regarded as the cause of the protective action of the serum” (p. 220).

We see from this description that even in the case of the typhoid

cocco-bacillus the direct action of the fluids of the body is

perceptibly less marked than in the case of the cholera vibrio. Even in

the latter, however, it is necessary to make many reservations. The same

laws apply to the immunity against this micro-organism, conferred by the

serum of immunised animals, as to the immunity due to vaccinations by

the vibrios or their products. To treat this subject fully one would

have to repeat almost textually the two preceding chapters, but I will

simply recall the fact that this transformation, almost general and very

rapid, as we observed _in vitro_ in vibrios placed in contact with fresh

specific serum or with the mixture of this serum, heated to 55°–56° C.,

and normal unheated serum, is only met with in the animal body where

phagolysis appears. Pfeiffer first observed the phenomenon which bears

his name in the peritoneal cavity, and it is best seen in that situation

during the period of the phagolysis of the white corpuscles. Vibrios,

mixed with small doses of specific serum which by itself is able to

render them motionless and agglutinate them, but which is absolutely

unable to transform them into granules, present this transformation

immediately they are introduced into the peritoneal cavity of normal

guinea-pigs. In this case the vibrios, permeated by the fixative of the

specific serum, are affected by the microcytase which has escaped from

the injured phagocytes and is found in the peritoneal fluid. The

preparation of the peritoneal cavity of normal guinea-pigs by means of

an injection of broth or physiological saline solution the day before,

prevents the production of Pfeiffer’s phenomenon, in spite of the

protective serum, just as in vaccinated guinea-pigs. In both cases the

vibrios, without being transformed into granules by the fluid part of

the peritoneal exudation, are ingested by the phagocytes _en masse_ and

with extraordinary rapidity. This experiment was repeated by

Garnier[467] with the typhoid cocco-bacillus. He first injected into the

peritoneal cavity of young guinea-pigs several c.c. of physiological

salt solution, of fresh broth or of some other fluid. The next day he

introduced into the same situation typhoid cocco-bacilli mixed with

blood serum from a donkey that had been for a long time immunised

against this organism. A few minutes (2–4) after this latter injection

the leucocytes, whose phagolysis had been prevented by the previous

day’s preparation, were found crammed with cocco-bacilli. Some of these

bacilli, like those still free in the peritoneal fluid, retained their

normal form, but a very large number of those ingested by the

microphages were already transformed into granules. This experiment

affords fresh confirmation of the hypothesis that the substance which

transforms the cocco-bacilli or the vibrios into granules is the

microcytase. In the phagocytes in their normal condition this

microcytase is found in the microphages, but during phagolysis a portion

of it escapes into the surrounding fluid. In the control experiments

made by Garnier with young normal guinea-pigs not prepared by

preliminary injection, the simultaneous injection of typhoid

cocco-bacilli and specific donkey’s serum set up this attenuated and not

very typical Pfeiffer’s phenomenon described in Pfeiffer and Kolle’s

memoir.

[Sidenote: [320]]

Soon after the discovery of Pfeiffer’s phenomenon I[468] was able to

bring forward a proof that it was produced neither in the subcutaneous

tissue, in the oedemas set up by the arrest of the circulation, nor in

the anterior chamber of the eye of animals when cholera vibrios mixed

with anti-infective specific serum were injected into these situations.

Here the micro-organisms retain their normal form, remain quite alive

and in this condition are ingested by the leucocytes which are brought

up to the points invaded. These cells, attracted by the vibrionic

products, do not undergo any phagolysis and, untrammelled, fulfil their

phagocytic function. Inside them are found vibrios which have kept their

elongated form and others which have become transformed into granules.

The exudations containing these elements still give cholera cultures on

nutrient media, a proof that some at least of the intracellular vibrios

are alive. Here we have no destruction of the micro-organisms in the

fluids of the body, consequently no direct action of the bactericidal

substance. This substance, enclosed in the phagocytes, can only act

through the intervention of these elements.

[Sidenote: [321]]

Mesnil[469] made analogous experiments with the Massowah vibrio, which,

unlike the cholera vibrio, is peculiarly virulent when injected

subcutaneously into guinea-pigs. In spite of this difference, this

micro-organism, when injected along with protective serum into normal

animals, behaves much as does the cholera vibrio proper. Mesnil injected

the Massowah vibrios at the same time as the anti-infective specific

serum, into the subcutaneous tissue of young and adult guinea-pigs and

of young rabbits. In every case he observed the same reaction phenomena

in the animal organism. The vibrios caused the formation of oedema at

the point of inoculation and remained isolated in the fluid. The

majority of these micro-organisms became motionless, but a few remained

motile. Pfeiffer’s phenomenon was never observed. There was sometimes an

aggregation of the vibrios, but this was not comparable with the marked

agglutination brought about by the specific serum _in vitro_. The

vibrios retained their power of reproduction, and Mesnil was able to

observe them in all phases of division. Some hours (6–8) after

inoculation the leucocytes began to come up to the seat of injection and

set to work at once to ingest the vibrios. This phagocytosis became more

and more marked, and finally there was ingestion of the whole of the

micro-organisms. Drops of the exudation containing only intraphagocytic

vibrios, when placed in the incubator, gave abundant cultures. The

leucocytes died outside the animal body, whilst the vibrios continued to

live and grow well under the new conditions. Certain leucocytes became

three times their original size, and their contents were seen to be made

up of vibrios closely packed together. The subcutaneous exudation, when

withdrawn even eight days after the injection of the micro-organisms and

sown on nutrient media, still gave colonies of vibrios.

It is evident, therefore, that the direct action of the protective serum

on the vibrios was reduced to a mere trifle. It rendered them motionless

and brought about a very slight clumping, but it was incapable of

transforming the vibrios into granules or of destroying them. We see,

then, that even in the case of the vibrios, the part played by

Pfeiffer’s phenomenon is very limited. The destruction of the vibrios is

effected with certainty, and completely, under the influence of the

specific serums, not by a direct action of the two antibacterial

substances but through the mediation of the phagocytes. Before the

fixative, introduced with the protective serum, can bring about this

result, the leucocytes, impressed with a special sensitiveness, must

come up to the seat of inoculation, seize the micro-organisms and

secrete around them their cytase. It is only as a result of these

actions, purely vital, that the chemical or physico-chemical reaction of

the substances which intervene in the destruction of the vibrios is

brought about.

[Sidenote: [322]]

Under these conditions it can easily be understood that if the vital

action of the phagocytes is retarded or depressed the injection of

protective serum cannot preserve the life of the animal.

Cantacuzène[470], who had already made a similar demonstration on

guinea-pigs vaccinated against the cholera vibrio by these organisms or

by their products, carried out numerous experiments on the action of

opium on normal guinea-pigs simultaneously inoculated with vibrios and

specific serum. Before injecting this mixture Cantacuzène narcotised his

animals by means of tincture of opium. The great majority (⅘) of the

guinea-pigs so treated died at the end of one or several days. The

transformation of the vibrios into granules, under the influence of the

serum, took place in the peritoneal cavity, but the leucocytes, on

account of the narcotic action of the opium, were tardy in coming up. On

their arrival in the peritoneal cavity they were capable of ingesting

the granules, but absolutely refused to seize entire vibrios, always

fairly numerous in the exudations. In spite of the appearance of a large

number of leucocytes, these cells were still too weak to offer any

adequate opposition to the vibrios, which increased in number and

continued to multiply up to the death of the animal, when the exudation

simply swarmed with very motile vibrios. Sometimes the struggle was

prolonged. The weakened leucocytes allow the vibrios to develop, but,

after a greater or less length of time, they regain their strength and

begin to ingest the micro-organisms vigorously. Complete phagolysis

follows, but the guinea-pig, attacked by the toxic products of the

vibrio, finally succumbs in spite of the absence of free vibrios from

its body.

An analysis of the phenomena observed in the body of an animal treated

with antivibrionic serum, demonstrates that, in spite of a certain

direct action of the substances contained in this fluid, there still

remain a whole series of processes, amongst which the carriers of the

cytases, that is to say the phagocytes, fill the most important _rôle_.

Nevertheless, the cholera vibrio with its allied forms is still the most

sensitive of all the micro-organisms to the bactericidal action of the

fluids of the body. It may, therefore, readily be conceived that the

more resistant micro-organisms are even less subject to the direct

influence of the specific serums. Thus we have seen that the

coccobacillus of typhoid fever presents, in the phagolysed peritoneal

fluid, merely an attenuated form of Pfeiffer’s phenomenon. The other

representatives of the group of bacilli are still less subject to the

direct action of the serums, and Gheorghiewsky[471], in his studies on

the _Bacillus pyocyaneus_, found that normal guinea-pigs, injected

subcutaneously with anti-infective specific serum, and inoculated into

the peritoneal cavity with this organism, present the same phenomena as

those described in Chapter VIII. He never noticed either lysis of the

bacteria in the fluids of the animal or their total transformation into

agglutinated masses outside the phagocytes. The resistance offered by

the animal was always in direct relation to the rapidity of the

appearance and extent of the phagocytic reaction.

[Sidenote: [323]]

In order to determine the relative importance of each of the factors

which act in the preservation of animals subjected to the influence of

the specific serum, Gheorghiewsky repeated Cantacuzène’s experiments on

the effect of narcotisation by tincture of opium. This alkaloid retards

diapedesis, but does not affect the tactile sensibility or the motility

of the leucocytes. The humoral properties, on the other hand, are not in

the least affected by the narcosis. In spite of the fact that in

guinea-pigs, narcotised and treated with anti-infective serum, the

direct action was not interfered with, the animals always died because

the retarded and incomplete phagocytic reaction was insufficient to

destroy the bacilli rapidly enough.

Mesnil[472] studied the action of the specific serum against swine

erysipelas on normal animals into which he had injected it some time

before inoculation of the corresponding bacillus into the peritoneal

cavity. This serum exercises a most marked protective action on the

mouse, an animal very susceptible to the pathogenic action of this

micro-organism. In mice so prepared complete and rapid phagocytosis

takes place. These micro-organisms before being ingested by the

phagocytes show no appreciable change; they are always stained very

uniformly and intensely by Gram’s method, and they never swell up. The

bacilli undergo no agglutination in the body of the mouse, a fact of

which we can convince ourselves by examining hanging drops of the

exudation. The phenomenon which strikes the observer most is the very

pronounced phagocytosis, due principally to the activity of the

microphages. Some hours after inoculation these cells are found to be

crammed with bacilli, a large number of which no longer stain in the

normal fashion. Without being transformed into granules, these

micro-organisms undergo intracellular digestion which at the end of a

few days is complete. This destruction is more rapid and complete in the

microphages, slower in the macrophages. Drops of exudation collected

from these mice, at a stage when the ingestion is completed, produce

fatal septicaemia in untreated mice. This is proof that at the moment

when they were seized by the phagocytes the bacilli still retained their

virulence. Mesnil, as the result of his experiments, concludes that “the

effect of the serum is to stimulate the phagocytes and especially the

polynuclear forms; they ingest more quickly, they digest more quickly.

The serum is, therefore, a stimulant of the cells charged with the

defence of the animal” (p. 496).

[Sidenote: [324]]

We need not describe the phenomena produced in mice inoculated

subcutaneously and treated with protective serum, for even in the

peritoneal cavity neither Pfeiffer’s phenomenon nor any extracellular

destruction of the bacilli can be observed. The micro-organisms, when

subjected to the influence of the specific serum, readily absorb the

fixative, as demonstrated by Bordet and Gengou[473]. This absorption

must certainly favour the action of the intraphagocytic cytases. It is

not, however, sufficient to explain the protective, anti-infective

action of the serum. Such explanation was given by the experiments which

Gengou, at my request, was good enough to make. He inoculated mice with

the bacilli of swine erysipelas, mixed with specific serum heated to 55°

C., to which was added some normal guinea-pig’s serum. The mice so

treated resisted the infection but controls died in a few days. Being

thus assured of the protective action of the serum, Gengou prepared the

same mixtures of swine erysipelas bacilli and of the two serums; but,

instead of injecting the whole of the mixture, he removed the bacilli

from the serums, after a prolonged contact, and injected the bacilli

alone into the mice. The bacilli had become permeated with fixatives,

but, in spite of this, they killed the mice almost as quickly as the

controls. Consequently, it is not the fixative adherent to the

micro-organisms which determines the protective action of the specific

serum. This fluid must contain another substance, one that will

stimulate the phagocytes.

[Sidenote: [325]]

The analysis of the mechanism of the immunity termed passive, that is to

say, communicated to normal animals by the introduction of an

anti-infective specific serum, teaches us that, even when the direct

action of the humoral substances is very limited, the protective effect,

thanks to the stimulant action which brings about the destruction of the

micro-organisms through the mediation of the phagocytic reaction, is

still produced. The result at which we have thus arrived is confirmed by

the examination of the phenomena observed in animals subjected to the

action of anti-anthrax serum. Marchoux[474] first supplied us with

precise details as to the mode of action on the rabbit of the serum of

animals treated with anthrax bacilli. He found that, in the peritoneal

cavity of rabbits injected the day before with anti-anthrax serum, the

inoculated anthrax bacilli almost immediately become the prey of

phagocytes. Within a couple of minutes after the introduction of bacilli

into the peritoneal cavity, the great majority of them are ingested by

the leucocytes; ten minutes later, there are no free bacilli. Not only

the ingestion but also the destruction of these micro-organisms takes

place with great rapidity, and even a few hours after the injection, the

peritoneal exudation, when sown on nutrient media, remains sterile. In

the subcutaneous tissue the phagocytic reaction requires a longer time

than in the peritoneal cavity, nevertheless, it goes on very rapidly.

Thus, when inoculated into the subcutaneous tissue of the ear of rabbits

treated with specific serum, the bacilli are in great part ingested at

the end of half-an-hour. At the end of an hour phagocytosis is usually

complete.

In Marchoux’s experiments the importance of the part played by the

phagocytes becomes still more prominent when it impedes their function

in any way. Rabbits injected with anti-anthrax blood and 24 hours later

inoculated below the skin of the ear with anthrax bacilli always resist

infection, exhibiting the well-marked phagocytosis just mentioned. In

other rabbits, however, prepared in the same way with the serum, but

inoculated the following day into an ecchymosis excited by tapping the

ear lightly, a certain number of the bacilli escape the phagocytes and

succeed in setting up an abundant oedema followed by a fatal anthrax at

the end of a few days. On making a post-mortem examination of these

animals the bacilli were not numerous, but they were found in all the

organs. The same result was obtained in another experiment in which

Marchoux inoculated subcutaneously with anthrax blood which coagulated

_in situ_ rabbits prepared with specific serum. The blood clot attracted

only the macrophages, as pointed out in Chapter IV. The microphages did

not come up until late and then in small numbers. Now, as these are the

phagocytes that are chiefly instrumental in destroying the anthrax

bacillus, their absence allowed the bacilli to multiply and to set up a

fatal anthrax. The rabbits prepared with the same serum but injected

with anthrax blood diluted with broth (which prevents the formation of

clot) completely resisted infection, thanks to the phagocytic reaction

which went on without hindrance.

[Sidenote: [326]]

Sclavo[475] also, who made numerous investigations on the action of the

anti-anthrax serum, is of opinion that this action is not a direct one

upon the bacillus but is produced indirectly through the action of the

animal organism. He maintains that the serum stimulates the function of

the phagocytes and augments the bactericidal action of the body fluids.

But since this bactericidal power enters the cytase as a substance

destroying the micro-organisms, and this cytase is contained in the

phagocytes, we can readily understand what a dominant part in the

process these elements play.

Sobernheim[476], also, has paid much attention to the question now under

discussion. As the result of his researches he comes to the conclusion

that the anti-anthrax serum “cannot exert any effect on the virus by a

direct action of the protective specific substances.” In order that the

serum may be effective, the active intervention of the organism of the

animal is necessary, otherwise, it is impossible to explain why the

serum, used in the same proportion against the same quantity of anthrax

bacilli, should protect one species of animals (the rabbit) and allow

another (guinea-pig, mouse) to succumb. When Sobernheim tried to apply

to anthrax the discovery of the transformation of cholera vibrios into

granules, he got only negative results. There was nothing produced

comparable to Pfeiffer’s phenomenon and the anthrax bacilli usually

underwent no apparent modification. Sobernheim affirms also that the

rapid phagocytosis under the influence of the serum, described by

Marchoux, “does not appear to be produced under all circumstances” (p.

117). As, however, his researches on this subject were made on

guinea-pigs which, in spite of the treatment with specific serum, always

ended by succumbing to anthrax, we readily understand that his results

cannot be compared with those obtained by Marchoux. I was present at the

experiments of this observer and convinced myself of the accuracy of the

facts recorded in his memoir.

[Sidenote: [327]]

Most of the examples here studied justify fully the hypothesis of the

stimulant action of protective serums, a view that I formulated as the

result of my researches on the immunity of rabbits against the Gentilly

cocco-bacillus[477]. In this the first case of anti-infective immunity,

due to the serum elaborated by an immunised animal, I could not find

either a bactericidal action, however slight, or any agglutinative or

attenuating property of the fluids of the body. As, on the other hand,

this serum had no antitoxic power, everything indicated that we must

look for its action, which was _nil_ or very slight on the

micro-organism, as being exerted on the organism of the animal into

which it was injected for protective purposes. A comparative examination

of the course of the phenomena in the subcutaneous tissue of the ear in

rabbits, some of which received an injection of the specific serum into

the veins whilst others were kept as controls, at once showed how widely

different were the two cases. In the control animals, the cocco-bacilli

immediately began to multiply without meeting with any opposition on the

part of the organism of the animal; on the other hand, in the rabbits

treated with serum, the serum became rich in leucocytes which at once

set to work to ingest the micro-organisms. In course of time the latter

gradually diminished in numbers, whilst the leucocytes went on

increasing. The phagocytosis, also, became more and more marked. This

struggle was continued for more than 24 hours, after which the purulent

exudation, containing masses of leucocytes, no longer included any

cocco-bacilli visible under the microscope either outside or inside

cells. Nevertheless, this pus was still capable of producing a fatal

septicaemia in untreated rabbits, clearly proving that it still

contained some living and virulent micro-organisms. These cocco-bacilli

persisted for a long time inside the phagocytes; their presence being

demonstrated by injecting the exudation into unprotected rabbits and

thus setting up a fatal infection. Finally, however, they disappear

completely. On consideration of such facts as these I considered that I

was justified in formulating the following conclusion at the end of my

memoir: “From the facts I have described, taken collectively, we may

draw the conclusion that the preservation of unvaccinated rabbits

treated with serum is due to a superactivity of the phagocytic defence;

and it is allowable to express the opinion that the protective serum of

hog cholera acts in rabbits by stimulating the phagocytes, rendering

them less sensitive to the toxins, and by stimulating them in their

struggle against the bacteria” (p. 310). The facts since collected by

various observers fully justify this hypothesis. Amongst the other

micro-organisms against which a rapid immunisation has been obtained by

means of serum, we must cite the cocco-bacillus of bubonic plague.

Numerous experiments, carried out on several species of animals, have

shown that antiplague serum markedly augments the phagocytic reaction.

[Sidenote: [328]]

In the group of the cocci, the streptococci have been specially fully

studied from the point of view now under discussion. As already stated

in another chapter, success has been attained not only in thoroughly

immunising several species of animals against this dreaded

micro-organism but active serums have been obtained capable of

conferring distinct and certain immunity. The protective action of

Marmorek’s serum, prepared at the Pasteur Institute, has been specially

carefully studied. This serum is obtained from horses that have received

numerous injections of various races of streptococci pathogenic for

animals and for man[478]. At Louvain, Denys and his pupils prepared

several other antistreptococcic serums and studied their protective

effect on laboratory animals.

[Sidenote: [329]]

In collaboration with Leclef, Denys[479] began by vaccinating rabbits

against streptococci and studied the mechanism of the immunity obtained

in these animals. A summary of their researches will be found in the

eighth chapter. Denys and Leclef considered that the serum of vaccinated

rabbits intervenes in two ways, first by directly hindering the

multiplication of the streptococcus and then by exalting the activity of

the leucocytes. They applied these results to the case in which immunity

is conferred upon normal rabbits by the intervention of the serum of the

vaccinated rabbit, but they were unable to furnish any data bearing

directly on this immunity. Somewhat later, Denys[480], in collaboration

with Marchand, published another memoir in which he describes

experiments on the mechanism of the immunity conferred on rabbits by

injections of the blood-serum of vaccinated horses. From these

experiments they draw the conclusion that “the serum of the horse

immunised against the streptococcus possesses no bactericidal

properties, properly so called, against this micro-organism; it does not

affect it directly; but it contains a substance which renders the

phagocytic power of the leucocytes extremely active. Even in the

presence of small quantities of this serum, the white corpuscles rapidly

ingest the streptococci and are capable of stopping all development so

long as they retain their amoeboid movements.” “The action of the serum

upon the leucocyte in its conflict with the streptococcus, is really

derived from the horse immunised against this organism. It exists

neither in the ordinary horse nor in the horse vaccinated against

diphtheria” (p. 15). Against these experiments of Denys and Marchand we

might bring the same objection that we raised against the analogous

experiments of Denys and Leclef, because, in both cases, these writers

lay too much stress on the presence or absence of the phenomena of

phagocytosis in preparations kept outside the body of the animal. Under

these conditions phagocytosis is effected in a fashion too artificial to

be capable of furnishing exact information.

Von Lingelsheim[481] met Denys and Marchand with the fact that, in their

researches, the serum of the horse immunised against the streptococcus

was only feebly bactericidal. After a prolonged contact (6–12 hours)

with a specific serum, the streptococci, when transferred to rabbit’s

blood, showed retarded development as compared with streptococci

subjected to the influence of the antidiphtheritic and antitetanic horse

serum. Von Lingelsheim himself, however, points out that the

bactericidal action of the antistreptococcic serum was feeble and

transient, and required the intervention of the reaction of the animal

cells within the body.

[Sidenote: [330]]

The researches carried out by Bordet[482] in my laboratory are not open

to the objections that we were justified in putting forward against the

experiments made by Denys and Marchand, since he carefully watched the

phenomena of immunity as they developed in the body of the animal

subjected to the action of antistreptococcic horse serum. Bordet began

by studying the properties of this serum and accepted Denys’ and

Marchand’s statement that bactericidal power, however small, was absent.

The streptococcus grows as well in this serum as it does in that of the

untreated horse. In the specific serum, however, markedly longer chains

are produced than in normal serum. This difference is found only in the

earliest period of the growth. The agglutinative power of the

antistreptococcic serum is but feebly marked. The injection of a large

quantity of this serum into a normal rabbit confers no bactericidal

power upon the serum of this animal. “The serum extracted 24 hours after

injection is quite as suitable for use as a culture medium as that

furnished by the blood before the introduction of the serum. In both the

micro-organism grows rapidly and vigorously” (p. 195). Consequently, in

the antistreptococcic serum there is nothing comparable to what we

obtain so readily with antivibrionic serum: nothing which recalls

Pfeiffer’s phenomenon, even of an attenuated nature. We have already

noted the result obtained by Bordet, according to which the

streptococci, developed in the specific horse serum, were found to be

endowed with their normal high virulence. The antistreptococcic serum,

injected into the peritoneal cavity of the rabbit the day previous to

the microbial inoculation, protects the animal absolutely, provided that

the micro-organisms be not too numerous or the quantity of serum not too

small. Under these conditions the virus is ingested pretty rapidly and,

so far as we know at present, completely. The micro-organism is thus

prevented from developing and the animal remains in good health, whilst

the control animal, which has received no serum, dies in a very short

time.

When the number of streptococci is increased the effort of the animal

organism to get rid of them becomes, in spite of the protective serum,

more severe and much more prolonged. Some of the micro-organisms

certainly become the prey of phagocytes, but a sufficient number remain

free in the peritoneal cavity to multiply rapidly. When the number of

streptococci has become sufficiently great a phenomenon, to which Bordet

gives the name of “phagocytic crisis,” is suddenly observed. In the

peritoneal exudation, which has become thick and has taken on the aspect

of a homogeneous and white pus, a most rapid phagocytosis is evidently

set up. In a short time the whole of the streptococci, which were

swarming outside the cells, are ingested by the leucocytes. “The

essential condition for recovery is always this complete ingestion” (p.

203). If the ingestion is not general, the rabbit may die, although much

later than the control animal.

[Sidenote: [331]]

The phases of the struggle between the animal organism, when subjected

to the influence of the protective serum, and the streptococcus, recall

Salimbeni’s experiments on immunised horses. The rabbit, in which

phagocytosis could not take place at once owing to the presence of too

large a number of micro-organisms, exhibits first a stage of free

development of the streptococci, after which the phagocytes begin to

fulfil their antibacterial function. Here it is especially the

macrophages which act; the microphages, although present in fairly large

numbers, are entirely inactive. This first stage of phagocytic reaction

is insufficient. It is followed by a period when the streptococcus

appears to gain the upper hand. Many small chains, having escaped the

phagocytes, multiply and give birth to quite a new generation of

micro-organisms. If a fresh impulse to phagocytosis does not take place

the animal dies from infection. When, however, the protective serum has

been of sufficient strength, a new army of leucocytes arrives on the

scene and these become masters of the situation. Phagocytosis becomes

complete and microphages as well as macrophages devour a large number of

streptococci.

Bordet, who, through his previous investigations, was well acquainted

with the direct action of the protective serum on vibrios, could find

nothing resembling it taking any part in the struggle of the organism of

the animal treated with antistreptococcic serum against the

streptococcus. The most that he could find was that the streptococci

which again begin to swarm in the exudation are smaller in size than the

normal streptococcus. It must be accepted, as indicated by the most

recent researches, that this micro-organism becomes permeated by the

fixative substance of the specific serum. We know already, however, that

this fixation cannot deprive the micro-organisms of their virulence. In

any case, then, a large share in the process must be attributed to the

action of the phagocytes, stimulated by the protective serum, in the

struggle of the animal against the streptococcus.

[Sidenote: [332]]

Having considered this series of examples of immunity against bacteria

conferred by specific serums, we are in a position to form some idea of

the mechanism of this immunity. Before we come to any general

conclusion, it may be useful to glance at an example of this so-called

passive immunity against a micro-organism belonging to the animal

kingdom. Such examples are not numerous, as, in the majority of cases of

acquired immunity against Protozoan parasites, the serum is inactive and

incapable of communicating immunity to normal individuals. We have only

a single example, the _Trypanosoma_ of rats, against which Dr Lydia

Rabinowitch and Dr Kempner[483] have demonstrated the possibility of

immunisation by the blood serum of vaccinated white rats. The mechanism

of this immunity has been studied by Laveran and Mesnil[484], who found

that it was like that described (Chap. VIII) in connection with the

immunity in white rats, conferred by the inoculation of living

_Trypanosomata_. The specific serum does not affect these infusoria

except that it brings about slight agglutination. _Trypanosomata_ placed

in contact with it retain their pristine vitality and motility. This

fact led Mme Rabinowitch and Dr Kempner to advance the hypothesis that

the protective action of the serum must depend upon its antitoxic power.

Since, however, in the infection of rats by the _Trypanosomata_, the

toxic action is very feeble if not nil, it is very difficult to accept

this view. It certainly appears to be much more probable that the serum

acts in this case, as in many others, by stimulating the phagocytic

reaction. The rapidity with which the living _Trypanosomata_ are

ingested by the phagocytes has been shown by Laveran and Mesnil.

[Sidenote: [333]]

Reviewing the whole of the data on immunity produced under the influence

of anti-infective or protective serums, it is evident that they fall

under two main categories. On the one hand there is a direct action of

these serums on the micro-organisms, an action that is either

microbicidal properly so called, agglutinative, or fixative. On the

other hand, a stimulation of the phagocytic defence which leads to the

final destruction of the micro-organisms is set up. This last factor is

general; even in the case where the direct action is most marked

(vibrios in the phagolysed peritoneal cavity), its importance is

considerable. The micro-organisms which can be deeply injured by the

direct action of the specific serum are few in number. In most cases

this action is a feeble one and needs, for its completion, effective

co-operation on the part of the phagocytes. In this respect

micro-organisms present a whole gamut which begins with the cholera

vibrio, the micro-organism most sensitive to the action of the body

fluids, and ends with the _Trypanosoma_ of the rat, a flagellated

Infusorian which cannot have even its motility affected by the direct

action of the fluid elements. In all these cases, of course, the

immunity conferred by the serums is due to the final destruction of the

micro-organisms which invariably resolves itself into the same

fundamental act—digestion by the cytases, a phenomenon which can only be

produced at all quickly by the action of cytases contained in the

protective serums or that have escaped from the phagocytes during

phagolysis. The digestion by the cytases may also, and this is usually

the case, be effected only after the manifestation of a regular series

of vital phenomena on the part of the defensive elements of the body. As

this factor fills such an important _rôle_, it is readily understood

that we can not accept the term passive immunity by which to designate

the immunity conferred by the specific serums. The action of the

cytases, which is necessary to bring about the final result in this

immunity, depends too much on the activity of the cells which contain

the bactericidal ferment. For this reason, when the functional activity

of the phagocytes is in abeyance or is retarded, the animal succumbs, in

spite of the presence in its organism of a more than sufficient quantity

of cytases. In this connection Wassermann’s[485] suggestion of adding

normal serums rich in cytases to the specific serums must be regarded as

very apposite. When protective serums poor in cytases or which have lost

them as the result of heating, of the use of antiseptics, or simply from

the influence of time, are injected, no immunising effect is ever

obtained, simply because of the inactivity of the phagocytes, the cells

in which the cytases are found. If at the same time normal serum rich in

cytases ready prepared be injected, better results should be obtained.

We may recall here an analogous example—the anthrax of the rat. Although

possessing a large quantity of cytase, very effective against the

bacillus, the organism of the rat can make no use of it, because the

phagocytes which contain it do not manifest a sufficient activity. But

the injection into a rat of blood serum from the same species containing

a certain amount of cytase that has escaped during the formation of the

clot, is sufficient to preserve the animal against a fatal infection.

[Sidenote: [334]]

To support his view, sound in principle, Wassermann made an experiment

the interpretation of which presents certain difficulties. He injected

guinea-pigs with protective antityphoid serum, in a dose insufficient to

protect them against a fatal infection. By introducing along with this

serum a certain quantity of normal ox serum which, by itself, is also

incapable of averting a fatal issue, Wassermann obtains an absolute

immunity of his animals. This immunity is due, according to Wassermann,

to the cytase of the ox serum acting along with the fixative of the

specific serum. The united action of the two ferments causes the death

of the micro-organisms. Besredka[486] has justly observed that normal ox

serum contains, in addition to cytases, a substance which exerts a

distinct agglutinative action on the typhoid cocco-bacillus and another

which stimulates the phagocytic action. These two substances resist a

temperature of 55°–60° C., and Besredka shows that with normal ox serum,

deprived of its cytases by heating as above, we can obtain the same

protective effect as with the same serum unheated.

As the result of another series of experiments, Wassermann[487]

recognises the immunising action of normal serum heated to 60° C. and so

entirely deprived of its cytases. Into the peritoneal cavity of

guinea-pigs he injects, mixed with heated normal rabbit’s serum, a dose

of typhoid cocco-bacilli several times greater than the lethal dose. The

guinea-pigs resist this completely. Analysing the mechanism of this

immunity, Besredka (_l.c._ p. 229) attributes it to the combined action

of the agglutinin and of the substance which stimulates the phagocytes.

We have here another proof that the stimulins which play such an

important part in immunity conferred by serums, are found not only in

the specific serums, but also in normal serums, whether unheated or

heated to 55°–60° C.

The protective property of the normal serums of man and animals against

the cholera vibrio has already been referred to. We may now go a little

more deeply into the mechanism by which these serums act. This task is

an easy one thanks to the important work by Issaeff[488] carried out in

R. Pfeiffer’s laboratory. Having confirmed the observation, made by

other investigators, that blood serum from the human subject, whether in

health or affected by any disease, is capable of protecting the

guinea-pig against the cholera vibrio provided that it is injected 24

hours before the micro-organisms, Issaeff studied the phenomena observed

in the peritoneal cavity of the animals experimented upon. By means of

small capillary pipettes he drew off at intervals a small quantity of

fluid from the peritoneal cavity and examined it in hanging drop or in

stained preparations. Some time after the injection this fluid became

more and more rich in leucocytes which seized the vibrios, ingested and

destroyed them. To obtain this protective effect it was necessary to

inject from 0·1 to 5 c.c. of human blood serum. With these doses he

could prevent, not only infection of the guinea-pigs by the cholera

vibrio, but also the lethal effects of other vibrios. The protective

action of normal human serum is general, therefore, and not specific,

such as is the immunity conferred by the serums of vaccinated animals or

of the human subject who has suffered from an attack of cholera.

[Sidenote: [335]]

Shortly afterwards Funck[489] confirmed this result in the case of the

typhoid cocco-bacillus. He observed that normal horse’s serum, injected

as a protective agent in the dose of half a c.c. into the peritoneal

cavity of the guinea-pig, preserved this animal from a fatal infection.

Pfeiffer and Kolle and Chantemesse and Widal obtained the same results

with human serum. The former observers lay special stress on the

non-specific character of this protective action of normal serums. As to

its mechanism, Funck sums it up as follows: “the specific serum brings

about a rapid lysis of the bacilli, normal serum acts in a much more

limited fashion; if the dose is very large and if the animal resists

infection, the phenomena of extracellular degeneration are rarely

appreciable, and it seems that here the specially important factor is

the intracellular destruction of the bacteria, in the phagocytes” (p.

70).

Wassermann has shown the protective action of normal serum against the

experimental disease produced by the staphylococcus. This action,

although not absolutely general, is nevertheless widely distributed.

Wassermann[490], from comparative investigations on this subject, came

to the conclusion that “the serum of a different species of animal acts

by greatly increasing the resistance, whilst the serum of the same

species produces an effect which is not nearly so marked.” As in these

normal serums a stimulating influence on the phagocytes is specially

marked, it may readily be understood that the serum of the same animal

or of the same species does not produce so energetic an effect as the

serum of a different species. As these normal serums possess, not only

the property of exciting phagocytosis, but often also that of rendering

motionless and of agglutinating certain micro-organisms, there might be

some difficulty in interpreting the part played by these serums. It may

be useful, therefore, to pass in review the protective action of fluids

less complicated than blood serums.

[Sidenote: [336]]

Issaeff, in the work already cited, demonstrated that not only normal

serums but a whole series of fluids, such as urine, broth, etc., exert a

protective effect against microbial infections. These fluids must be

injected about 24 hours before the introduction of the bacteria. The

best method consists in injecting them directly into the peritoneal

cavity, after which the animals acquire an immunity against absolutely

fatal doses of cholera vibrios. Funck verified this observation for the

infection caused by the typhoid cocco-bacillus, and Bordet confirmed it

for the streptococcus. The injection of peptonised broth into the

peritoneal cavity of the normal guinea-pig, made 24 hours before an

inoculation of double the fatal dose of the streptococcus, exerts a

distinct protective action; the infection does not kill the animal. This

broth is neither bactericidal, attenuating, nor agglutinative; it forms

a good culture medium for the streptococcus and possesses no fixative

power. Consequently it does not act directly on the vitality or

virulence of the micro-organism; nevertheless, it is distinctly

protective.

According to Issaeff’s researches, the protective substances used by him

must be arranged in the following order as regards their action against

the cholera vibrio. Tuberculin is the most effective; then comes a 2%

solution of nuclein, followed by normal human serum, broth, and urine,

whilst physiological saline solution is the least active. All prevent

infection by the vibrios, but the protection is effective for some days

only; this protective action is exerted against various kinds of

bacteria, being in no sense specific.

Pfeiffer lays so much stress on the great difference between the

protective power of normal serums, as well as of the other fluids

mentioned, and that of the anti-infective specific serums, that he even

proposes to classify the first group as giving rise to _pseudo-immunity_

or _resistance_. This view is certainly an exaggerated one, because it

is difficult to draw a very distinct line between the two groups of

phenomena. There are normal serums, of which 0·1 c.c. is quite

sufficient to confer the protective effect, just as there are specific

serums of which it is necessary to make use of a much greater dose to

attain the same result.

[Sidenote: [337]]

Protective fluids, other than the serums, only manifest their influence

by exciting a great phagocytic “superactivity.” As the result of their

injection into the peritoneal cavity of normal guinea-pigs, first a

transitory phagolysis is induced, this being soon replaced by a very

considerable afflux of leucocytes, which is maintained for 24 hours or

longer, and then gives place to the normal condition. It is during the

period of the greatest leucocytosis of the peritoneal fluid that the

animal exhibits the most marked resistance against infective

micro-organisms. The vibrios are rapidly ingested by the phagocytes,

without having previously been acted upon by the “humours.” Bordet shows

that the same thing happens in the case of the streptococcus inoculated

into guinea-pigs after a protective injection of peptonised broth.

[Illustration:

FIG. 42. Culture of the plague bacillus developed within a macrophage

from guinea-pig.

]

[Illustration:

FIG. 43. Macrophage from guinea-pig filled with plague bacilli.

]

[Illustration:

FIG. 44. Macrophage from guinea-pig containing plague bacilli which

are commencing to escape from the protoplasm.

]

[Illustration:

FIG. 45. Macrophage from guinea-pig which has burst as the result of

the development of plague bacilli within it.

]

[Sidenote: [338]]

We have observed the same phenomenon in guinea-pigs and white rats

inoculated with the cocco-bacillus of plague. Treated with freshly

prepared peptonised broth the day previous to inoculation, these animals

oppose to the micro-organism a much more marked resistance than do the

control animals. The injection of the cocco-bacillus of plague sets up a

marked phagocytosis on the part of the macrophages. These cells ingest

large numbers of micro-organisms which, after a time, have all passed

into the phagocytes. If a drop of the peritoneal exudation is now

withdrawn, we find only intracellular cocco-bacilli (fig. 43). If the

drop be kept for some time outside the animal and at a suitable

temperature the macrophages may be seen to perish and the

micro-organisms to develop in their contents. We thus obtain abundant

cultures which pass from the interior of the macrophages into the fluid

of the exudation (figs. 42, 44, 45). When the animals are not

sufficiently protected the same phenomenon is observed in the peritoneal

cavity of the living animal. The macrophages, crammed with

cocco-bacilli, burst, allowing the micro-organisms to escape. These

multiply in the peritoneal fluid and spread through the animal, which

soon dies.

Wassermann affirms that “the artificially increased resistance is

nothing but an active and reinforced afflux of the complements (cytases)

towards one point in the animal, for the purpose of digestion.”

(_Ztschr. f. Hyg._, Leipzig, 1901, Bd. XXXVII, S. 199.) Wassermann does

not explain how this afflux of cytases is produced. The entirely

concordant researches on this point by Issaeff, Funck, Bordet, and

ourselves, prove that this afflux takes place not through the mediation

of the fluids, but solely through the phagocytes, the carriers of the

cytases. Consequently it is beyond dispute that in the immunity

conferred by physiological saline solution, broth, and several other

fluids, we have to do solely with an augmentation of the phagocytic

reaction. In the immunity conferred by normal or specific serums, this

same stimulating factor still plays the more important part. Along with

it, however, there is an intervention more or less pronounced, according

to circumstance, and more or less frequent, of cytases, brought by the

serums prepared outside the body or that have escaped during phagolysis,

as well as of substances truly humoral, such as the fixatives or the

agglutinins.

[Sidenote: [339]]

Amongst the non-specific substances which are capable of conferring an

immunity more or less stable, must be placed the products of

micro-organisms other than those against which we wish to protect the

animal. Pasteur[491] noted that when the anthrax bacillus, mixed with

other micro-organisms, in themselves inoffensive, is inoculated into

animals, anthrax does not develop and the animals remain well. Later,

Emmerich[492] showed that the streptococcus of erysipelas exerts an

antagonistic influence against the anthrax bacillus. He succeeded in

immunising and even in curing rabbits inoculated with anthrax, by

submitting them to the action of this streptococcus.

These experiments served as the starting-point for several works on the

vaccination of animals against anthrax by means of various

micro-organisms, as well as by their products. Pawlowsky[493],

Watson-Cheyne[494], and Bouchard[495] have proved that bacteria not very

pathogenic and even saprophytes, such as the _Coccobacillus

prodigiosus_, Friedländer’s bacillus, and the _Bacillus pyocyaneus_,

were also capable of preventing infection by the anthrax bacillus.

Freudenreich[496] showed that not only did the bacillus of blue pus

exert an antagonistic action but that the same effect could be obtained

with sterilised cultures of this organism. Woodhead and Cartwright

Wood[497] studied the vaccinating action of these products on rabbits

inoculated with virulent anthrax bacilli. The animals resisted

completely or survived for some time. Analysing the phenomena produced

under such conditions, these two authors came to the conclusion that the

action of sterilised cultures of _Bacillus pyocyaneus_ is “indirect and

as taking place either by opposing itself to the action of the poison

upon the tissues, or by stimulating certain tissues and increasing their

functional activity.” With the object of obtaining an exact

interpretation of this antagonistic influence I suggested to M.

Blagovestchensky[498] that he should investigate in detail the phenomena

which take place in the organism of rabbits inoculated with the anthrax

bacillus and submitted to the action of sterilised cultures of the

_Bacillus pyocyaneus_. At the very outset this observer was met by the

fact that these cultures act directly upon the vitality of the anthrax

bacillus. Thus the association of the former with the anthrax bacillus

_in vitro_ was sufficient to interfere with the development of the

latter. Under these conditions he had to renounce the investigation of

the part played by the cellular elements of the rabbit in the antagonism

of the two bacteria.

[Sidenote: [340]]

Friedländer’s bacillus has been found to be much more suitable for this

line of research as is shown by work carried out by Freiherr von

Dungern[499] in my laboratory. This observer convinced himself that

“anthrax bacilli are weakened neither by the encapsuled bacilli nor by

the substances which they contain.” These micro-organisms do not

interfere in the slightest with the anthrax bacilli either outside or

within the animal, and “when the anthrax infection does not become

generalised it is due to the fact that the anthrax bacilli are ingested

by the phagocytes at the seat of inoculation and destroyed within these

cells” (p. 183).

In this action of foreign micro-organisms upon micro-organisms against

which we wish to protect the animal we have to deal with something

analogous to the condition we obtain when immunising with normal serums

or with any other kind of fluid. In both cases immunity is rapidly

established, but it is very transient and is confined to a stimulation

of the phagocytic resistance. Direct action may also intervene, as in

the case of _Bacillus pyocyaneus_, but it is not indispensable. The

animal whose phagocytes are in a condition of superactivity can do

without this direct action, its own resources being sufficient to arrest

anthrax.

Following the same lines of investigation as those on the antagonism

between the anthrax bacillus and several other micro-organisms,

Klein[500] has demonstrated that, in order to prevent a guinea-pig from

contracting experimental cholera peritonitis, it is only necessary to

inject into it, the day before infection, a culture of Finkler and

Prior’s vibrio or of certain other bacteria. These experiments by Klein

served as the point of departure for Issaeff’s work which led to the

discovery of the stimulating influence of all kinds of fluids injected

into the peritoneal cavity of guinea-pigs.

In this transient immunity obtained with products foreign to the

micro-organism against which one is vaccinating, the most constant and

consequently most important part is again played by the phagocytes. But

there is associated with it an influence, greater or less in degree, of

substances present in the serums, such as the microcytases and

fixatives, which are able to exercise a direct action on the pathogenic

micro-organisms. In all cases known and analysed up to the present, the

intervention of the living organism of the animal is indispensable,

consequently this form of acquired immunity against micro-organisms

cannot be regarded as being really passive.

CHAPTER XI

NATURAL IMMUNITY AGAINST TOXINS

Examples of natural immunity against toxins.—Immunity of spiders and

scorpions against tetanus toxin.—Immunity of the scorpion against

its own poison.—Antivenomous property of the blood of the

scorpion.—Immunity against tetanus toxin in the larvae of

_Oryctes_ and in the cricket.—Immunity and susceptibility of frogs

against this toxin.—Natural immunity of reptiles against tetanus

toxin.—Antitetanic property of the blood of alligators.—Immunity

of snakes against snake venom.—Immunity of the fowl against

tetanus toxin.—Immunity of the hedgehog against poisons and

venoms.—Immunity of the rat against diphtheria toxin.

[Sidenote: [341]]

As in this book we are dealing specially with the immunity against

infective diseases, the question of the resistance of the animal to

poisons interests us only in so far as it is related to immunity against

micro-organisms. Consequently the reader must not expect a treatise on

intoxications properly so called nor one on immunity against all kinds

of poisons. To perform such a task we should have to far overstep the

bounds of the subject that we have chosen and enter upon an examination

of questions which are beyond our sphere. Our chief aim is to present to

the reader a summary of our present knowledge on immunity against

microbial toxins and to establish the relations between this kind of

immunity and immunity against infective micro-organisms. In order to do

this, however, we shall have now and again to go beyond the limits of

our programme and discuss certain problems bearing on the resistance of

the animal organism against poisons not of microbial origin.

[Sidenote: [342]]

The immunity against toxins, like that against the micro-organisms

themselves, may be either natural or acquired. As many poisons have been

known from time immemorial, we are able to collect numerous observations

on the resistance of the animal organism to such substances made when

there was no idea of immunity against infective diseases. The etiology

of intoxications is often much more evident and simple than is that of

infections; this is one of the reasons that the older conceptions on the

subject of immunity against poisons were more advanced than were those

on immunity against infective diseases.

Several examples of natural immunity in the lower animals have already

been cited. Thus, we have seen in previous chapters that the Infusoria

are resistant to poisons that exert a powerful action on a large number

of the higher animals, such as the tetanus and diphtheria toxins and

especially the ichthyotoxin of eel’s serum. We have mentioned the case

of the larva of _Oryctes nasicornis_ which is unaffected by large doses

of the toxins of certain bacteria and which at the same time is very

subject to fatal infections by very small doses of the bacteria that

form the poisons. These larvae, like those of the cockchafer, are,

however, fairly susceptible to the poison of the scorpion. Several other

species of Arthropoda, which have been studied from the point of view of

immunity against toxins, have exhibited analogous features. Thus spiders

and scorpions are refractory to tetanus toxin. In one experiment I

injected into the abdominal cavity of a _Mygale_ from the Congo (which

weighed 7 grm. 5) 1 c.c. of tetanus toxin on two several occasions. This

dose is sufficient to kill, with the symptoms of tetanus, 1000 mice of

double the weight. The spider, kept in the incubator at 36° C., remained

quite well during the two months that the experiment lasted. It

exhibited no symptom, not even transient, of muscular stiffening, nor

any change in its habits and natural functions. The tetanus toxin

disappeared from the blood of the _Mygale_, but this blood at no time

showed the slightest antitoxic power against this poison. This example

of natural immunity cannot, therefore, be ascribed to any antitoxic

property of the fluids and must be regarded as a case of immunity of the

tissues—von Behring’s histogenic immunity. In the present imperfect

state of our knowledge it is impossible to describe precisely the

mechanism of this immunity. When we say that the spider is refractory to

the tetanus toxin because its susceptible elements have no receptors

capable of seizing the haptophore group of this poison, we simply give

expression to a hypothesis which we are not in a position to verify by

experiment.

[Sidenote: [343]]

The scorpion, a well-known representative of the Arachnida with

segmented abdomen, shares with the _Mygale_ in the immunity against

tetanus toxin. The Algerian and Tunisian scorpions (_Scorpio afer_ and

_Androctonus occitanus_) withstand the action of doses of this poison

which are fatal for 1000 mice and more. Taking weight as our standard we

may inject into them, with impunity, more than 5000 times as much toxin

as into mice, without setting up a single morbid symptom. Scorpions,

like the _Mygale_, live well in the incubator at 36° C., where they are

kept whilst submitted to the action of the tetanus poison. Here again we

have to do with a case of histogenic immunity. The fluids of the

scorpion exert no antitoxic action. When blood from the normal scorpion

is mixed with various doses of tetanus toxin and injected into mice

these animals contract tetanus and die just as do the control animals.

In certain exceptional cases some slight retardation was observed, but

the blood of the scorpion is, in most cases, incapable of preventing

tetanus in animals susceptible to this disease.

Scorpions, injected with tetanus toxin, do not retain it in their blood

for long. A few days after the injection of the tetanus poison such

blood, when injected subcutaneously into mice, excites no trace of

tetanus. The preparation of extracts of the different organs of

scorpions treated with tetanus toxin demonstrates that the liver and the

liver only absorbs the poison. It is found there a few days after the

injection of the toxin and it remains there unaltered for some

considerable time. The exudation of the liver of scorpions, killed a

month or more after the introduction of the toxin into the general

cavity, injected into mice sets up a typical and fatal tetanus.

The presence of the tetanus toxin in the organism of scorpions does not

give rise to the production of antitoxin. At any rate a whole series of

experiments on this point carried out by us never gave a positive

result. The scorpions resisted repeated doses of the tetanus toxin and

lived without any difficulty at 36° C., but their blood was never at any

period capable of preventing mice from contracting fatal tetanus.

Nevertheless the scorpion may possess antitoxic power.

[Sidenote: [344]]

Everyone has heard of the supposed suicide of the scorpion. We are told

that when this animal finds itself under conditions in which its death

is inevitable, it stings itself with the end of its tail and dies from

the effect of its own poison. A simple method of reproducing this

experiment is actually described:—Surround the scorpion with a circle of

fire. The animal rushes in all directions to find a way out, and finding

none, deliberately commits suicide. Bourne[501] at Madras carefully

investigated this question in a large species of Indian scorpion and

demonstrated the absolute erroneousness of the story of suicide which,

had it been true, would have afforded a unique example of voluntary

death in animals. On carrying out the classic experiment he observed

that within this ring of fire the scorpion is subjected to a very high

temperature. When the temperature reaches 40° C. the scorpion begins to

grow weak and as the temperature approaches 50° C. it passes into a

comatose condition. Moreover Bourne showed that the scorpion’s poison,

which is fatal for large spiders, insects, and vertebrates, was

innocuous for individuals of the species furnishing it.

I can confirm all the statements of this English observer. When I was

studying the embryology of the scorpion I repeatedly tried the

experiment but the animal never committed suicide. Further, I repeatedly

assured myself of the innocuousness of the scorpion’s poison when

injected into individuals of the same species, and I was able to

demonstrate most conclusively that the blood of the scorpion is endowed

with undoubted antitoxic power. The addition of 0·1 c.c. of this blood

to a dose of poison which kills mice in half-an-hour is sufficient to

enable a mouse injected with the mixture to resist it completely. This

antitoxic power is the same in the _Scorpio afer_ and in the Algerian

_Androctonus_. An emulsion of the liver of the scorpion, however, is

absolutely incapable of preventing fatal intoxication of mice.

[Sidenote: [345]]

This case of antitoxic action is the only one I have been able to

demonstrate in an invertebrate. Must we regard it as a case of natural

innate antivenomous power or as something acquired during the life of

the animal? It is not easy to settle this question by experiment. We can

certainly procure new-born scorpions and rear them for some time, but

the quantity of blood that can be got from them is insufficient for

injection for protective purposes. Scorpions do not love one another and

when kept together we often find them engaged in fierce and mortal

combat, the stronger killing the weaker and sucking their blood. It is

therefore possible that, in some stage of their life, the scorpions find

means of vaccinating themselves against their own poison either through

the intestine or as the result of punctures caused by the point of the

tail. It would be very interesting to study this question under

favourable conditions, because it is capable of throwing light on the

problem of the origin of antitoxins, from a general point of view.

Whichever view be taken, the acquisition of any antitoxic property by

the blood in the Invertebrata must take place slowly and with great

difficulty as is shown by our want of success with tetanus toxin.

Insects are, as a rule, very tolerant of this latter poison. As,

however, the tetanus toxin (we shall illustrate this later) only acts

well and in small doses at a high temperature (about 30° C.) and as most

insects do not readily adapt themselves to this temperature, it was

necessary to choose species capable of living at these high temperatures

and for this line of study the larva of _Oryctes_ is most suited. It

flourishes well at a temperature of 30°–36° C., and under these

conditions exhibits a much greater resistance to infection by _Isaria_

than at lower temperatures. It can be kept in the incubator for months

if placed in glass jars filled with earth mixed with tanner’s bark. The

injection of enormous quantities of very active tetanus toxin directly

into the blood has not the slightest effect on these larvae. Whilst,

however, the blood fluid of the Arachnida rapidly gets rid of the

poison, that of _Oryctes_ retains it for a very long period. If a small

quantity of blood be taken from larvae several months after injection

and then injected into mice, these animals contract typical tetanus and

quickly succumb.

The toxin, however, finally disappears from the blood though a certain

portion of it may still be found in the pericardial cells and especially

in the fat-bodies.

Never, under any circumstances, was I able to observe that the blood of

the larvae of _Oryctes_ exerted any antitoxic action. At the stage when

this fluid no longer gives tetanus to mice, it is absolutely incapable

of preventing intoxication when mixed, before injection, with tetanus

toxin.

Amongst adult insects the cricket is best adapted for researches on

tetanus. The field cricket will bear a temperature even higher than 30°

C. It is completely resistant to injections of tetanus toxin, but it

showed no more capacity than did the larvae of _Oryctes_ or the

Arachnida of producing any tetanus antitoxin.

[Sidenote: [346]]

All the Invertebrata that I have been able to study have exhibited a

remarkable resistance against the known bacterial toxins, but the

mechanism of this natural immunity could not be exactly made out owing

to the difficulty met with in investigating the toxins in the organs and

following their modifications. The idea of making use of these lower

animals for the purpose of solving the problem of the origin of

antitoxins is not realisable, from the fact that the Invertebrata that

have been studied have never, in my experience, produced any of these

substances as the result of injections, whether single or repeated, of

toxins.

The natural immunity of the Invertebrata against bacterial toxins cannot

therefore be regarded as an example of humoral immunity. It must be

placed in the category of histogenic immunity, although we are not in a

position to define accurately the part played by the cellular elements

in the defence of the animal against these poisons. We must, therefore,

go higher up in the animal scale if we are to solve the principal

questions in regard to antitoxic immunity.

[Sidenote: [347]]

The lowest Vertebrata, the fishes, are not well-suited for this kind of

research. The best known bacterial toxins act specially on warm-blooded

animals and require the co-operation of high temperatures. Fishes do not

live well in captivity except at relatively low temperatures and soon

die if placed in an incubator kept at 30° C. or higher. It is necessary,

therefore, to have recourse to the Amphibia, which are much more easily

acclimatised to these temperatures. The Axolotl, coming from Mexico, is

naturally capable of withstanding great heat. These animals will live

for long at a temperature of 30°–37° C. They possess the drawback,

however, of being very susceptible to the tetanus toxin, very small

doses of it being fatal. The green frog (_Rana esculenta_) is the most

suitable for our purpose. It readily adapts itself to optimum

temperatures (30°–36° C.) and exhibits at least a certain degree of

immunity against various bacterial toxins. We have stated in a preceding

chapter that the green frog is unaffected by considerable quantities of

diphtheria toxin. It is resistant also to tetanus toxin, but this

natural immunity appears to be connected with special conditions.

Courmont and Doyon[502] were the first to draw attention to the fact

that beyond 20°–25° C. green frogs may contract tetanus. Refractory in

winter they become susceptible in summer. These observers afterwards

found that of frogs inoculated with the same dose of toxin and divided

into two sets, one set kept at a temperature of about 10° C. remained

quite well whilst the other set subjected to one of 30°–39° C.

contracted tetanus after five days’ incubation. This experiment has been

confirmed by several observers, and indicates that the tetanus poison

demands, for the manifestation of its toxic action, a favourable and

fairly high temperature. This result must, however, be accepted with

some reserve. Undoubtedly the doses of tetanus toxin which induce fatal

tetanus in frogs kept at a high temperature are innocuous when these

animals are living at low temperatures. But we can, by increasing the

dose, produce tetanus in frogs even when the temperature is not very

high. Thus Marie[503] was able, during the whole of the winter, to

tetanise both green and brown frogs living in water the temperature of

which oscillated between 13° and 18° C. The incubation period in this

case is very much longer (sometimes extending to 25 days) than in frogs

kept at higher temperatures.

Temperature, therefore, is an important factor in the poisoning by the

tetanus toxin and in the resistance of the frog, but, in the long run,

this poison can exert its specific action even at relatively low

temperatures.

[Sidenote: [348]]

Morgenroth[504] endeavoured to analyse the mechanism of this resistance

and of the susceptibility of the green frog when maintained at various

temperatures. He demonstrated that the tetanus toxin is fixed in the

central nervous system, even at low temperatures, near 8° C.; under

these conditions, however, it is incapable of causing the slightest

tetanic symptom. When placed in an incubator kept at 32° C. the frogs

contract tetanus after a period of incubation of some (2 to 3) days.

During the first 24 hours of this period the frogs manifest no sign of

tetanus, and if they are again put in a cool place they continue in good

health. If, however, after a not too prolonged stay in the cold, these

animals are subjected a second time to the higher temperature, they

become tetanic, after a shortened incubation period. Cold, therefore,

may arrest tetanus even at a stage when the toxin has already produced

certain latent but permanent modifications of the nervous system.

Frogs injected with tetanus toxin and kept in a cold place finally get

rid of the poison. When transferred to a warm chamber after a certain

lapse of time they no longer contract tetanus. We have found that the

greater part of the tetanus toxin continues for some time in the blood

of frogs injected and kept at a low temperature. A small quantity of

this blood withdrawn almost two months after the last injection produced

fatal tetanus in a mouse. We do not know how frogs eliminate the toxin,

but it has been demonstrated that in this case it causes no production

of antitoxin. Morgenroth has confirmed this result.

[Sidenote: [349]]

Reptiles must be regarded as vertebrates exhibiting a most pronounced

natural immunity against tetanus. They show an unlimited resistance to

enormous doses of tetanus poison, and this at low, medium, or high

temperatures (30°–37° C.). Green lizards withstand considerable doses of

tetanus toxin. Although they do not contract tetanus, they get rid of

the poison exceedingly slowly. Thus, a lizard kept at a temperature of

20° C., and injected with an amount of toxin sufficient to kill 500

mice, at the end of two months still retains in its blood such an amount

of the poison that one-tenth of a c.c. will cause fatal tetanus in a

mouse. Turtles present an analogous case. The marsh turtle, _Emys

orbicularis_, tolerates very large amounts of tetanus toxin, injected

subcutaneously, and this at both low and high temperatures, at 30° C.

and beyond (36°–37° C.). The toxin passes quickly into the blood and

remains localised there for a very long time. In a turtle kept in an

aquarium at the laboratory the blood was tetanigenic for the mouse even

four months after an intraperitoneal injection of the toxin. In another

turtle which lived at incubator temperature (36°–37° C.), the blood was

still toxic two months after a subcutaneous injection of tetanus toxin

in quantity fatal for 500 mice. In turtles kept at 36° C. I observed

abundant transudations into the peritoneal cavity, and the fluid, very

poor in formed elements, was found to be very tetanigenic. It must be

accepted, therefore, that the toxin is retained in the blood plasma with

which it passes into the transudation. Every kind of cell must exhibit a

very marked negative chemiotaxis against tetanus toxin for this poison

to be retained so long in the body fluids. Under these conditions it is

not surprising that in turtles I was never able to observe the slightest

antitoxic power in the blood. Their great natural immunity must be due

to some other factor.

The alligator (_Alligator mississipiensis_) has also been found to be

quite refractory to tetanus both at low and at high temperatures.

Outwardly alligators behave exactly as do turtles, that is to say, after

the injection of various and sometimes very large doses of toxin they

exhibit no morbid symptom either general or tetanic. But the particular

changes which occur in their organism differ essentially from those met

with in the turtle. The toxin is rapidly eliminated from the blood of

the alligator, even when it is kept at a relatively low temperature (20°

C.). Under these conditions of temperature, however, the blood does not

become antitoxic although it has lost its tetanigenic property. When,

however, the alligators are kept at a higher temperature (32°–37° C.),

antitoxic power is developed in their blood, often with very great

rapidity. Quite young alligators (weighing about 500 grammes) are

capable of producing antitoxin, though somewhat slowly. A month after

the first injection of the tetanus toxin their blood is incapable of

causing tetanus in mice, but is not yet antitoxic. A month later,

however, it never fails to prevent an attack of tetanus when mixed with

fatal doses of the toxin and injected into mice.

Older alligators develop antitoxic power much more rapidly, and on

several occasions we have found, to our great astonishment, that, as

early as 24 hours after injection of the toxin, their blood was

distinctly antitetanic. The blood of the same alligators, tested before

the injection of the toxin, like the blood of normal alligators

generally, exhibited no antitoxic property.

In several experiments we took the rectal temperature of our animals and

were never able to observe the slightest rise corresponding to the

temperature of the water in which the alligators lived.

[Sidenote: [350]]

It cannot be doubted then, that, in spite of the facility with which

these reptiles produce tetanus antitoxin, their immunity does not depend

on this antitoxic property. Thus, young alligators which have resisted a

single dose of toxin sufficient to kill 6000 mice must owe their

immunity to some other cause than the antitoxic power of the body

fluids, for their blood does not begin to exhibit this property until

two months after injection.

These same reptiles are also very refractory against cholera toxin, even

in large doses; they react to the injection by the development of the

corresponding antitoxin. On the other hand they are very susceptible to

diphtheria toxin, small quantities of which are quite sufficient to

bring about a fatal intoxication.

Snakes, like other reptiles, are refractory against tetanus toxin. In

the study of their natural immunity, however, we are confronted by the

difficulty that their blood is naturally toxic for laboratory animals.

This toxin, analogous to the ichthyotoxin of eel’s serum, has been

compared with snake venom against which the snakes themselves enjoy a

very marked immunity.

[Sidenote: [351]]

Not venomous snakes only exhibit immunity against their own poison. Long

ago Fontana[505] observed that non-venomous snakes resist the bite of

the viper and even subcutaneous inoculation of its venom. Phisalix and

Bertrand[506] confirmed these observations and were able to show that a

non-venomous snake (_Tropidonotus_) will withstand a dose of venom

capable of killing from 15 to 20 guinea-pigs. Seeking for the cause of

this natural immunity, these observers came to the conclusion that it is

due to the presence in the blood of toxic substances analogous to those

of the venom of the viper. These same substances are found also in the

labial glands of the upper jaw of the _Tropidonotus_ and can from

thence, according to the view of Phisalix and Bertrand, pass into the

blood as an internal secretion. Calmette[507] has shown that the blood

of snakes, injected in a non-toxic dose, vaccinates certain mammals

against snake venom, and Phisalix and Bertrand have even obtained an

antitoxic effect by injecting a mixture of snake’s blood, heated to 58°

C., with lethal doses of venom. There is, then, in this example

something analogous to what we have described in scorpions, with this

difference, however, that the blood of these Arachnids is already

antitoxic, to a certain degree, whilst that of snakes only becomes so

after it has been modified by heat.

The classic example of immunity against a bacterial toxin amongst Birds

is that of the fowl, which is highly refractory against the tetanus

toxin. In the very earliest researches on this poison injections were

made into vertebrates of very different kinds, and a very striking

feature was the facility with which fowls resist very large quantities

of tetanus toxin. However, as is almost always the case, this immunity

has been found not to be absolute. By means of enormous doses, injected

subcutaneously or into the muscular tissue, tetanus of the most typical

kind, ending in death, has been induced in fowls, and in fowls weakened

by cold, tetanic intoxication, even with smaller doses, has been set up.

By injecting the toxin directly into the brain, according to Roux and

Borrel’s method, the fowl may be still more easily tetanised. Thus, von

Behring[508] observed that by injecting one milligramme of the toxin

into the brain of a fowl, weighing one kilo, tetanus may infallibly be

produced.

After the brilliant and fruitful discovery of the antitoxic property of

the blood, made by von Behring in collaboration with Kitasato, we were

justified in concluding that immunity against toxins and, amongst

others, natural immunity, might depend on the power of the body fluids

to neutralise the toxins. This hypothesis has been formulated at various

times, but it was for the first time subjected to experimental control

by Vaillard[509], and specially in connection with tetanus in the fowl.

The blood or blood serum of these birds, when mixed in varying doses,

small, medium, and large, with tetanus toxin, was never found to be

capable of preventing susceptible animals (mice, guinea-pigs, rabbits)

from contracting tetanus: these animals so treated behaved just as did

the controls inoculated with toxin only.

[Sidenote: [352]]

The great resistance of the fowl against tetanus,—one of the most

typical examples of natural immunity against a microbial poison,—cannot,

therefore, be explained by the presence in the body fluids of an

antitoxin capable of neutralising and rendering innocuous the tetanus

toxin. On the other hand, we are not justified in attributing it simply

to the absence of corresponding receptors in the sensitive nerve cells.

Since the fowl readily contracts tetanus when the toxin is injected

directly into the brain or when the fowl is weakened by cold, it is

evident that the sensitive elements never fail to absorb and fix any

poison that is presented to them. In ordinary cases, however, when the

fowl exhibits its remarkable resisting power against the toxin injected

in very large quantity, subcutaneously, into the muscles or into the

peritoneal cavity, the poison does not reach the sensitive cells, being

arrested and rendered innocuous whilst circulating in the tissues of the

organism.

Von Behring[510] is of opinion that in examples of natural immunity,

such as the one just examined, the principal cause of the refractory

condition depends upon the impermeability to the toxin of the capillary

wall of the vessels. It is, however, difficult to maintain this thesis

in regard to tetanus in the fowl, when it is remembered how readily

tetanus toxin passes through filters and membranes, and especially in

view of the fact that weakening of the fowl by means of cold renders it

susceptible to doses of toxin which are tolerated without inconvenience

by normal fowls.

We are, therefore, compelled to place the natural immunity of the fowl

against tetanus toxin in the category of cell immunities. This toxin, as

we have said, must be arrested _en route_ before it reaches the cells of

the nerve centres. But where and how does this beneficent arrest take

place? Ten years ago Vaillard demonstrated that the blood of fowls that

have received an injection of tetanus toxin causes typical tetanus in

susceptible animals. This tetanigenic property of the blood persists for

a certain number of days. When it is measured by the quantitative

method, it is found that all or almost all the tetanus toxin injected

into the peritoneal cavity of the fowl passes into the blood and remains

there intact for a variable number of days. From a morphological point

of view the blood, immediately after the injection of the toxin,

exhibits a hyperleucocytosis of greater or less duration.

[Sidenote: [353]]

When the fowls are killed at the stage when their blood becomes

tetanigenic (as the result of the injection of the toxin into the

peritoneal cavity), it can be demonstrated that their viscera are not

capable of producing tetanus in susceptible animals except in so far as

they contain blood. It is only the vascular organs, rich in blood, such

as the spleen, liver, kidneys, thyroid gland and bone-marrow, that

impart tetanus and then only in so far as they have not been freed from

blood. Of the various organs only the genital glands, ovaries or testes,

absorb a certain amount of the injected toxin. Very young testes or the

smallest ovarian ova containing as yet no trace of yellow yolk, when

injected into mice, produce a fatal tetanus.

In fowls, insusceptible to tetanus toxin, this toxin is found, then, in

the sexual glands and in the blood. When, in order to ascertain the

exact localisation of this toxin, we measure the tetanigenic power of

the whole blood as compared with that of the aseptic exudations induced

by the injection of gluten-casein, and necessarily much richer in

leucocytes, we get the result that the exudations contain more tetanus

toxin than does the blood. We are led, therefore, to the conclusion that

this poison is absorbed, at least in part, by the leucocytes, and it is

in these elements and in the genital cells that we must look for the

factors which arrest the toxin and prevent its reaching the nerve

centres.

Cellular or histogenic immunity is often contrasted with chemical

immunity without taking into consideration the real analogies and

differences to be found between them. It is evident that in both groups

the organism of the animal modifies the introduced toxins and that this

modification is a chemical process. In cellular immunity, however, this

act is preceded by certain biological phenomena, such as the reaction of

the formed elements and the absorption of the noxious substance.

Immunity in these cases is more complex than in the example where the

toxin is neutralised by a direct action of the body fluids, but

ultimately it always resolves itself into a chemical or perhaps

physico-chemical action of the substances of the organism of the animal

on the toxic substances of the poisons.

[Sidenote: [354]]

In Mammals examples of natural immunity against certain poisons are not

rare. Almost a century ago Oken made the observation that a person who

tried to poison a hedgehog with opium, hydrocyanic acid, arsenic or

mercury bichloride usually failed in his attempts because of the great

resisting power of this animal. Harnack demonstrated that the hedgehog

will withstand a dose of potassium cyanide six times as great as that

necessary to kill a cat in a few minutes (0·01 grm.). In Lewin’s[511]

experiments the hedgehog was found to resist the injection of powdered

cantharides in a quantity seven times as great as that which infallibly

kills a dog and greater also than the lethal dose for man. The same

observer also confirms the observation that a much larger dose of

alcohol must be used in order to intoxicate a hedgehog than is required

to obtain the same effect in the rabbit or even in the dog. Horvath[512]

fed hedgehogs for a fairly long period with living cantharides. These

Insectivora devour their venomous prey without showing any sign of

illness except a certain degree of emaciation. When Lewin tried to

ascertain the cause of this natural immunity of the hedgehog he examined

the blood of this animal for a substance antitoxic to cantharidine. His

experiments were all negative; but it is difficult to come to any

definite conclusion in this matter from the fact that the blood and

blood serum of the normal hedgehog are toxic for the small laboratory

animals. A similar objection had already been brought forward by

Phisalix and Bertrand in connection with their experiments, analogous to

those of Lewin, on the immunity of the hedgehog against the venom of the

viper.

[Sidenote: [355]]

It has long been known that hedgehogs have a liking for certain reptiles

and wage an implacable war on snakes in general and on the viper in

particular. In its attack the hedgehog tries to avoid being bitten, but

when, as often happens, it fails to evade a bite the inoculation of the

viper’s venom appears to be well borne. This observation has been

confirmed experimentally. Phisalix and Bertrand[513] have shown that the

resistance of the hedgehog to the viper’s venom is about forty times as

great as that of the guinea-pig, that is to say the hedgehog, though far

from possessing an absolute immunity, nevertheless exhibits a much

greater resistance than do most animals. Lewin[514] convinced himself of

this fact as regards adult hedgehogs, though young animals, according to

him, are much more susceptible. Thus, he has seen a young hedgehog that

had been bitten by a viper die after nine days’ illness. This

observation speaks in favour of the conclusion that the immunity of the

hedgehog might be naturally acquired rather than a really natural

immunity. The hedgehog, hunting all kinds of small animals, might often

be bitten by vipers and in this way acquire its immunity against the

venom. Under these conditions we can readily conceive that the blood of

this “insectivoran” might be placed in a position to develop a specific

antitoxic property.

When Lewin tried to satisfy himself of the existence of this property by

direct experiment he could only show that the blood of the hedgehog was

powerless to prevent the lethal effect of the viper’s venom on small

animals. But here, as in his researches on cantharidine, he did not take

into account the inherent toxicity of the blood of the hedgehog.

Phisalix and Bertrand[515], who have also studied this question, have

obtained results at variance with those of Lewin. They demonstrated

first of all that the blood of normal hedgehogs was capable of

intoxicating and even of killing laboratory animals such as the

guinea-pig. It is quite natural, therefore, that the mixture of this

fluid with viper’s venom could not be tolerated. It was, however,

sufficient to heat the blood of the hedgehog to 58° C. for it to become

not only innocuous of itself, but even for it to exhibit an antitoxic

action against snake venom. Thus, guinea-pigs which had received 8 c.c.

of heated hedgehog’s serum into the peritoneal cavity, were at once in a

condition to resist double the lethal dose of viper’s venom. Phisalix

and Bertrand conclude, therefore, that “the natural immunity of the

hedgehog against the viper’s venom is due to the presence in its blood

of an immunising substance.” The same observers[516] satisfied

themselves that horse’s serum and even that of the guinea-pig exercise

an undoubted antivenomous action; yet these animals are anything but

insusceptible to snake venom. Moreover, the necessity to heat the blood

to 58° C., as a preliminary measure, deprives this conclusion of the

degree of certainty one would like to have in such a matter. On the

other hand, the greater susceptibility of young hedgehogs prevents us

from putting the immunity of the adult in the category of natural

immunity properly so called.

[Sidenote: [356]]

Analogous considerations apply in the case of the mongoose (_Herpestes

ichneumon_), carefully studied by Calmette[517], according to whose

researches the Antilles mongoose is not very susceptible to snake venom;

it readily withstands doses very large relatively to its size, but its

immunity is not absolute. It owes much of its mastery in its fights with

venomous snakes to its extraordinary agility. The blood of the mongoose,

mixed with venom, exhibits an undoubted antitoxic power, though this is

not sufficient to prevent the death of susceptible animals. We have no

data to enable us to explain the origin of this antitoxic property, but

it is probable that here again we have an example of relative immunity,

acquired during life. Calmette points out, however, that his ichneumons

came from Guadeloupe, where no venomous snakes are found. We may, of

course, suppose that the feebly antitoxic power of the blood of these

mammals might be due to other snakes or to species of animals whose

blood possesses a certain venomous property[518].

[Sidenote: [357]]

We have far more exact data on the natural immunity of certain mammals

against toxins of microbial origin. The example most thoroughly studied,

one which has become, one might say, classic, is that of the rat against

diphtheria toxin. Since the discovery of this toxin, the first

well-studied bacterial poison, a discovery made by Roux in collaboration

with Yersin, it has been recognised that mice and rats tolerate large

quantities of diphtheria cultures or of their filtered products. A rat

resists a dose of the diphtheria poison capable of killing several

rabbits. To explain this great natural immunity it was suggested that

the antitoxic property of the body fluids could be called in. It was

supposed that the rat’s blood was, by its very nature, endowed with the

power of neutralising the toxin of diphtheria. But, as in the tetanus of

fowls, it was not long before facts rendered this hypothesis untenable.

Kuprianow[519] studied this question under the direction of Loeffler and

gave an account of the results of his experiments, which proved that the

blood of the sewer rat, which is very refractory against diphtheria,

contains no substance that will neutralise the morbific action of

diphtheria toxin on susceptible animals, especially the guinea-pig.

It was necessary to seek some other explanation, and the idea that the

immunity of the rat depends on the insusceptibility of its living cells

to the diphtheria poison was seized upon. The experiments carried out by

Roux and Borrel[520] demonstrated the incorrectness of this hypothesis.

The immunity of rats to subcutaneous or intraperitoneal injection of

diphtheria toxin is very marked. But a very small dose (0·1 c.c.) of

this poison, introduced directly into the cerebral substance of the rat,

produces a complete paralysis, which lasts for several days, and ends in

the death of the animal. Roux and Borrel conclude from this “that the

brain of the rat is specially sensitive to the action of the diphtheria

poison, and that as this animal does not die as the result of the

injection of large quantities of toxin into the subcutaneous tissue, it

is because the toxin does not reach the brain.” These authors have

pointed out analogous facts in connection with other examples of natural

immunity. The rabbit, which withstands a hypodermic injection of 30

centigrammes of chlorhydrate of morphia, is killed by 1 milligramme only

of this salt, introduced directly into the brain. Here, again, neither

the cellular insusceptibility nor the antitoxic property of the blood

(no “antialkaloidal” power could ever be demonstrated) can explain the

immunity, which appears to be due rather to the factor which arrests the

poison on its way to the nerve centres.

[Sidenote: [358]]

In spite of the insufficiency of our knowledge as regards natural

immunity against soluble poisons we are quite justified in affirming

that this category of phenomena comes mainly into the domain of the

cells. The body fluids of animals which exhibit this immunity have been

found to be antitoxic in a few species only (scorpion, snake, hedgehog,

mongoose). And for the majority of these it is possible to invoke

special causes, such as the internal secretion of snake and scorpion

venoms by the glands which manufacture them, or the acquisition of an

antitoxic power during life resulting from wounds or from the absorption

of venomous food. The theory of the insusceptibility of the cells of

animals naturally refractory to toxins must also be rejected; it is

incompatible with well-established facts. Nothing remains, then, but to

assume that the formed elements are the principal factors in this

natural immunity, and that they interpose to prevent the passage of the

poisons towards the very susceptible nerve cells.

CHAPTER XII

ARTIFICIAL IMMUNITY AGAINST TOXINS

Adaptation to poisons.—Artificial immunity against bacterial and

vegetable toxins and against snake venom.—Principal

methods of immunisation.—Immunisation by toxins and

toxoids.—Inoculation against diphtheria toxin.—Phenomena produced

in the course of vaccination against toxins.—Rise of

temperature.—Leucocytosis.—Development of antitoxic

power.—Properties of antitoxins.—Mode of action of

antitoxins.—Action of antitoxins _in vitro_.—Their action in the

organism.—Influence of living elements on the combination of

antitoxin with toxin.—Antitoxic action of non-specific serums, of

normal serums and of broth.—Immunity against toxins is not in

direct ratio to the amount of antitoxins in the body

fluids.—Hypersensitiveness of an animal treated with

toxin.—Diminution of the susceptibility of the organism immunised

against toxins.

Hypotheses as to the nature and origin of antitoxins.—Hypothesis of

the transformation of toxins into antitoxins.—Hypothesis

of receptors detached from cells as the source of

antitoxins.—Hypothesis of the nervous origin of tetanus

antitoxin.—Fixation of tetanus toxin by the substance of

the nerve centres.—The relations between saponin and

cholesterin.—Anti-arsenic serum.—Part played by phagocytes in the

struggle of the animal against poisons.—Probable part played by

phagocytes in the production of antitoxins.

[Sidenote: [359]]

Although scientific men succeeded only a little more than ten years ago

in vaccinating against poisons by artificial methods, savage races and

ancient peoples at a very remote period undoubtedly possessed methods of

counteracting the effects of certain venomous substances. The frequent

observation of cases in which doses of poisons, insufficient to cause

death, brought about a more or less durable resistant condition, must

result in the elaboration of artificial means of preventing the

intoxications.

Von Behring[521] points out that analogous facts must have been known to

the physicians of ancient times; and it is in such knowledge that we

must look for the source of the dogma put forward by Hippocrates, that

the factor which produces a disease is also capable of curing it.

[Sidenote: [360]]

To Pliny we are indebted for the now well-known story, that Mithridates

of Pontus possessed the means of protecting himself against various

poisons by a process of adaptation, and, amongst others, by the use of

the blood of Pontine ducks to which he had given poisons by the mouth.

The adaptation of horses and of the highlanders of Styria to arsenic, as

well as that of the many morphinomaniacs to morphia, is known to

everybody. A man, habituated to morphia, is able to consume daily a dose

several times the fatal one; indeed, cases have been known of people

acquiring the power of consuming two, and even three, grammes of morphia

per diem. Man may acquire an adaptation to toxic substances of the most

diverse character, such as arsenic, alcohol, morphia, nicotine, etc.

[Sidenote: [361]]

Even when we had obtained much information concerning acquired immunity

against micro-organisms we still knew nothing of the mechanism of such

adaptation, or as to the possibility of acquiring a special immunity

against bacterial poisons. Charrin and Gamaleia’s discovery that animals

vaccinated against a micro-organism are just as susceptible to its toxic

products as normal animals, led Bouchard[522], in whose laboratory it

was made, to say that the idea of the adaptation of cells to bacterial

poisons must be dropped. He developed this thesis at the International

Congress at Berlin in 1890, and formulated it as follows: “When we

inject a healthy animal and a vaccinated one with the soluble products

of the micro-organism which has been used for the vaccination, the dose

required to kill each animal is exactly the same. Let us not speak,

then, of the training of the leucocytes, and of the adaptation of the

nerve cells to bacterial poisons: it is pure rhetoric.” At this time we

had only just commenced to acquire exact knowledge concerning the toxins

of micro-organisms. For a considerable period they were sought for

amongst the ptomains, very stable substances allied to the alkaloids;

here, however, we were working in a wrong direction. It was not until

the classic researches of Roux and Yersin[523] on diphtheria toxin,

published in 1888 and 1889, that the true nature of bacterial poisons

was revealed. It was found that we were not dealing with ptomains, but

with soluble ferments, substances of indeterminate chemical composition,

allied to the albuminoids, and, like them, unstable. The methods adopted

by Roux and Yersin in their study of diphtheria toxin enabled other

investigators to discover the analogous toxins of several other

bacteria. Knud Faber[524] and Brieger and Fränkel[525] soon succeeded in

separating the toxin from the tetanus bacillus, a toxin capable of

producing in animals tetanic contractions as typical as those obtained

with cultures of the tetanus bacillus.

These investigations inaugurated a new era in microbiology and enabled

us to attack the problem of acquired immunity against bacterial toxins

scientifically. Within a few months of the declaration made by Bouchard

at the Berlin Congress, there appeared, almost simultaneously, the

earliest publications on the possibility of vaccinating laboratory

animals against the toxins of diphtheria and tetanus by artificial

methods. Immediately after the discovery of these poisons, the attempt

was made to immunise various species of animals against them, but here

very great difficulties were met with; the animals, after receiving

increasing doses of toxin, became thin and ultimately died. It occurred

to Fränkel[526] that the toxic action of the diphtheria poison might be

weakened by subjecting it to a temperature of 60° C. Independently, von

Behring and Kitasato[527] used chemical substances, especially iodine

trichloride, to attenuate the action of the tetanus and diphtheria

toxins. The animals which resisted these modified poisons were found to

be capable of tolerating gradually increasing doses of unaltered and

very active toxins. By the use of these methods it was found possible to

obtain a definite and lasting immunity against these microbial products.

[Sidenote: [362]]

The discovery of the possibility of vaccinating against bacterial toxins

was soon followed by the demonstration of the antitoxic power of the

blood of animals that had acquired such artificial immunity against

these poisons. Everyone knows of and appreciates von Behring and

Kitasato’s great discovery. It opened up a new and fruitful field of

research from most diverse points of view. Ehrlich[528] was able to

apply it to the vaccination of animals against the vegetable poisons

ricin, abrin and robin, and thus to establish rigorous methods of

immunisation and to obtain very important results concerning immunity

against toxins in general. He also succeeded in demonstrating that

animals vaccinated against these vegetable poisons, which, by their

nature, approximate to the microbial toxins, develop in their blood a

most marked antitoxic property.

Some years later, the discovery of antitoxins was extended to snake

venoms, poisons of animal origin which, like the vegetable poisons

studied by Ehrlich, present a chemical composition analogous to that of

the microbial toxins. Phisalix and Bertrand[529] and Calmette[530],

working independently, discovered methods of vaccination against snake

venom and were able to demonstrate the existence of an antitoxic power

of the blood in immunised animals.

The works above briefly referred to gave us the fundamental basis of our

present knowledge on acquired immunity against toxins.

It would be very interesting to be able to determine whether the lower

animals can be vaccinated against the toxic substances to which they are

susceptible. Unfortunately in the study of this problem we encounter

very great difficulties. Making use of various methods I have often

tried to solve it. The crayfish is susceptible to snake venom and to the

ichthyotoxin of eel’s serum, and I have tried at various times to

vaccinate it against these poisons. The results, however, were so

inconstant and even contradictory that I was unable to draw any definite

conclusion from them.

[Sidenote: [363]]

It is, indeed, very difficult to vaccinate the lower vertebrata against

poisons. Several attempts have been made in my laboratory to immunise

frogs against tetanus toxin, but without success. Calmette and

Deléarde[531] obtained the best results with abrin. They succeeded in

vaccinating frogs—which are not very susceptible to this vegetable

toxin, though they are far from presenting a real natural

immunity—against doses which are absolutely fatal for the control

animals. These observers, however, had to proceed very cautiously, and

they allowed a very long interval between each injection of abrin. The

blood of their vaccinated frogs not only did not prove to be antitoxic

against abrin, when injected into mice, but for long retained sufficient

of this toxin to poison normal mice. This experiment certainly tells

against the hypothesis that the acquired immunity of frogs is due to the

development of a specific antitoxic power in their body fluids, but it

does not settle the question definitely since it may be objected that

the blood, whilst toxic for mice, might, still, be antitoxic for the

frog. The antitoxin of this blood might merely be incapable of

neutralising all the abrin present. Fresh investigations, then, are

necessary.

Even in the higher vertebrata, it is often very difficult to obtain a

real vaccination against the various toxins. In the small mammals, which

exhibit a great susceptibility to these poisons, it is specially

difficult to obtain an artificial immunity. As Vaillard and von Behring

have demonstrated, it is possible to vaccinate such animals by means of

gradually increasing doses of unmodified toxins, but this method demands

much time, is often dangerous, and hence is not very practical. Poisons

that act through the alimentary canal are the most serviceable for

vaccination, as has been demonstrated by Ehrlich. This investigator had

to abandon the vaccination of mice by means of subcutaneous injections

of ricin on account of the sloughing set up at the point of inoculation.

He then had recourse to vaccination by way of the mouth, which gave very

good results, not only with ricin but also with abrin. This mode of

vaccination, however, is applicable to a small number of poisons only.

We can also vaccinate mammals, even laboratory rodents, such as rabbits

and guinea-pigs, by means of unmodified snake venom, but this method is

a very delicate one and must be carefully watched. It is necessary to

begin with very small doses of venom, continue them for some time, and

increase the amount of venom injected very slowly. Calmette[532]

modified this method by inserting, below the skin and leaving it there,

a piece of chalk impregnated with small quantities of venom and

surrounded by collodion through which the venom diffuses very slowly and

continuously.

[Sidenote: [364]]

Large mammals, sheep, oxen and horses, can be more easily vaccinated by

means of unmodified toxins, but they also require to be treated with

very special precaution. Salomonsen and Madsen[533] have given the

history of their horse, immunised with diphtheria toxin. Into a mare

weighing 665 kilos they were able to inject at the commencement only 1

c.c. of this toxin, and the dose had to be increased very carefully.

In the presence of all these difficulties in the use of unmodified

toxins for vaccination, a different method is now generally adopted in

the immunisation of animals, small or large, for the purpose of

scientific research or for the preparation of toxins on a commercial

scale. Vaccination is commenced with toxins modified by heat or by

chemical substances. The diphtheria and tetanus toxins, those most

employed in the serotherapeutic industry, are subjected to various

degrees of heat. Fränkel[534] was the first to make use of this method

for vaccination against diphtheria, and Vaillard[535] for vaccination

against tetanus. It consists in introducing large doses of filtered

cultures, heated to progressively lower degrees of temperature, 60°,

55°, 50° C., and then giving gradually increasing quantities of filtered

cultures whose toxicity is unaltered. This method is very convenient for

small animals, but for large mammals it is greatly simplified by

injecting for a certain period toxins heated to 60° C., and, later,

replacing these by unmodified toxin.

[Sidenote: [365]]

Phisalix and Bertrand[536] applied an analogous method to the

vaccination of the guinea-pig against the venom of the viper. This

poison, which resists much higher temperatures than do the tetanus and

diphtheria toxins, received a preliminary heating to 80° C. in order

that it might be inoculated without danger into small animals. Under

these conditions it confers a certain immunity, but even when heated to

80° C. it, in many cases, still remains sufficiently active to produce

fatal results. For this reason, in the vaccination of animals for the

preparation of antivenomous serum on a large scale, Calmette had

recourse to another method, that of attenuating the venom by means of

chemical substances.

Von Behring and Kitasato[537] were the first to make use of iodine

trichloride in the vaccination of animals against the toxins of tetanus

and diphtheria. In their early experiments this substance was injected

before the toxins were introduced. Later, the mixture was made _in

vitro_ and then injected into the animals. Roux devised another method

which had the advantage of being simple, certain, and easily employed,

for which reason it was soon introduced into commercial and scientific

practice. It consists in the injection of mixtures of the tetanus or

diphtheria toxins with Lugol’s iodo-ioduretted solution. The iodine, in

small doses, instantly neutralises or modifies these poisons and is

itself borne well, even by small animals. By employing progressively

increasing doses of these mixtures, in which the amount of iodised

solution becomes smaller and smaller compared with that of the toxin, we

are able, without difficulty, to vaccinate the most susceptible animals

and enable them to withstand considerable doses of the pure toxin. By

this method it is possible to immunise guinea-pigs against the most

active tetanus toxin. The method serves equally well for the preparation

of horses for injections of unmodified toxins. For a longer or shorter

time (according to the susceptibility of the horse) toxins which are

mixed with Lugol’s iodised water are injected. Having made sure of the

resistance of the horse, larger and larger quantities of pure,

unmodified toxin may be introduced with impunity.

For the immunisation of mammals of all sizes (guinea-pigs, rabbits,

dogs, horses) against snake venom, Calmette, in his work at Lille, also

makes use of venom modified by chemical substances, but his method

differs from those we have just described. During several weeks he

injects increasing quantities of venom, mixed with decreasing quantities

of a solution of 1:60 of hypochlorite of lime. After this treatment the

animals become capable of tolerating fatal doses of unmodified venom and

can be injected with larger and larger doses.

[Sidenote: [366]]

In recent years a method of vaccinating horses against certain microbial

toxins, and especially against the diphtheria toxin, by means of

mixtures of toxin and antitoxic serum, or with these two products

successively, has been introduced. Babes[538] was the first to extol

this method as the best for obtaining a high and durable immunisation.

Afterwards, several other observers, amongst whom I may cite Pawlowsky

and Maksutow[539], Palmirsky, and especially Nikanoroff[540], took up

this question, and communicated very encouraging results. Von

Behring[541] also found it very useful in certain cases. Thus, for the

vaccination of guinea-pigs against tetanus toxin, he recommends the

injection of a mixture containing antitoxin and an unneutralised excess

of toxin. Under these conditions he easily succeeds in immunising these

small animals in cases where all other methods fail. As a general method

of vaccination against toxins, however, this method has not fulfilled

its promise, and Roux, who tried it several times, was not at all

satisfied with it.

[Sidenote: [367]]