The very precise data that we have just summarised do not seem to agree

with the statements of certain other investigators. Thus Schütze[162],

from his researches on the antihaemotoxic serum of guinea-pigs, directed

against the rabbit’s haemotoxin, has arrived at the conclusion that in

the former an antifixative only is produced. As he merely injected into

his guinea-pigs haemotoxic rabbit’s serum that had been heated to 60° C.

and consequently deprived of the macrocytase, he concluded that in this

serum there remained only the specific fixative capable of provoking the

formation of an antitoxin. This must consequently be an antifixative.

Paul Müller[163] came to a similar conclusion, after injecting rabbits

with the heated haemotoxic serum of fowls. These injections caused the

formation in the rabbit’s serum of an antitoxin that Müller regarded as

an antifixative.

Ehrlich and Morgenroth[164] objected to this interpretation, taking

their stand on experiments made with the serums of normal animals. They

were able to show that these serums, when injected in the fresh state or

after being heated to 60° C., caused the production of a corresponding

antihaemotoxin which is nothing but an anticytase. When Schütze and Paul

Müller concluded that by heating the serums they had entirely deprived

them of cytase elements they did not take into account the possibility

of the cytases being transformed, under the influence of heat, into

other bodies unable to produce haemolysis, but quite capable of

provoking the formation of anticytases. Ehrlich and Morgenroth give to

these new bodies, derived from cytases under the influence of

temperatures between 55°–60° C., the name of _complementoids_; and these

complementoids appear in the experiments of Schütze and Müller to have

caused the production of antitoxins—anticytases.

In all the investigations just summarised the anticytases have been

obtained by the injection into animals of various blood serums, fresh or

heated. Wassermann[165] has discovered another method of arriving at the

same result. He injected into guinea-pigs the leucocytes of rabbits,

carefully deprived of all traces of serum. After some time the blood

serum of guinea-pigs thus treated became weakly but distinctly

anticytatic. From this experiment Wassermann draws the conclusion that,

as has been often affirmed by several observers, the leucocytes really

contain cytases.

[Sidenote: [123]]

How do the anticytases act upon the cytases? On this point all observers

who have studied this question have but one answer, the action of the

anticytases is direct. Bordet thinks that the two substances combine so

intimately that they cannot be again separated by heat. We know that the

cytases are very sensitive to heat and that their haemolytic property is

destroyed at 55° C. The anticytases, on the other hand, as already

noted, are much more resistant to the action of heat. Bordet has

prepared mixtures of haemolytic cytase serum and of antihaemolytic

serum, neutral mixtures, that is to say, inactive for red corpuscles or

with a very feeble action upon red corpuscles that have been

sensibilised by the specific fixative. These mixtures no longer exhibit

antihaemotoxic properties or they exercise this power in a very feeble

degree. If in these mixtures the cytases remain uncombined alongside the

anticytases, it is to be expected that heating them to 55° C. will

restore the antihaemotoxic function of the anticytases; the cytases

being destroyed at 55° C. there will remain in the mixtures only active

anticytase. The experiments made on this point have demonstrated that

the heating of these mixtures does not restore the antihaemotoxic

action, that is to say, the anticytase is definitely combined with the

cytase.

Ehrlich and Morgenroth have satisfied themselves that their

antihaemotoxin exerts no influence, either upon the red corpuscles or

upon the fixative, and is only capable of preventing the action of the

cytase. They introduced red corpuscles of the rabbit into a mixture of

goat’s serum, heated to 56° C. and thus only retaining its fixative, and

anticytase serum. The fluid bathing the red corpuscles was then removed

by centrifugalisation and the corpuscles were mixed with normal

haemolytic horse’s serum. Solution of the red corpuscles took place at

once as the anticytase had been completely removed during

centrifugalisation, being combined with neither the red corpuscles nor

the fixative.

These investigators have obtained various anticytases by injecting serum

of various species of animals into other mammals. They observed,

however, that injections of the serum of an allied species did not bring

about the formation of anticytases. Thus the injection of goat’s serum

into sheep, or of that of sheep into goats, never produced anticytase

serum.

[Sidenote: [124]]

In addition to antihaemotoxic serums several other analogous

anticytotoxic serums have now been obtained. Thus Delezenne has prepared

serums which prevent the action of neurotoxin and of the cell poison

which destroys the liver cells. We[166] have been able to obtain a

rabbit’s serum which prevents the spermatozoa of this rodent being

rendered motionless by the specific spermotoxin of the guinea-pig. More

recently Metalnikoff[167], working in my laboratory, has prepared

another antispermotoxic serum which prevents the specific spermotoxin of

the rabbit from arresting the movement of the guinea-pig’s spermatozoa.

[Sidenote: [125]]

As the history of these antispermotoxins presents certain interesting

general features we may with advantage, perhaps, dwell on some of their

characters. The two antispermotoxins mentioned above are distinguished

by certain peculiarities. When Metalnikoff set to work to inject

rabbit’s spermotoxin into guinea-pigs, he thought that he had an easy

task before him and that after a few injections the guinea-pig’s serum

would become antispermotoxic. This, however, was not the case. The serum

from these animals when mixed with spermotoxic serum was powerless to

prevent the immobilisation of the spermatozoa of the guinea-pig. It was

only when he heated the serum of his treated guinea-pigs to 56° C. that

the antispermotoxic power appeared with the greatest distinctness. The

inefficacy of the unheated serum must therefore depend on the toxic

action of the guinea-pig’s macrocytase, because it is this substance

alone that can have been destroyed by the heating process. Now, in order

that this macrocytase may act, the presence of the fixative is

necessary, which leads us to the conclusion that the serum of the

guinea-pigs injected by Metalnikoff contained no antifixative. This

hypothesis was fully confirmed by experiment. Metalnikoff introduced a

drop of guinea-pig’s serum into a mixture of antispermotoxic serum,

heated to 56° C., with spermotoxic serum. The spermatozoa continued

their movements in normal fashion. But when afterwards he added a few

drops of unheated serum from a normal guinea-pig the motions of the

spermatozoa were arrested almost instantaneously. Consequently there was

present in the mixture rabbit’s macrocytase which had been neutralised

by the anticytase of the prepared guinea-pig’s serum and for that reason

the spermatozoa remained motile. But in the same mixture we had also the

specific fixative, coming from the rabbit’s spermotoxic serum, which

remained free and not neutralised. The motile spermatozoa had become

impregnated with this fixative and a little guinea-pig’s macrocytase

(against which the anticytase was powerless) was sufficient to make them

suddenly cease their movements.

There is no doubt, then, that the serum of guinea-pigs that have been

treated with spermotoxin contains anticytase only and no, or almost no,

antifixative. Such is not the case with the antispermotoxin obtained by

us in rabbits that were treated with spermotoxic toxin of guinea-pigs.

Several consecutive injections were sufficient to render the serum of

the rabbits so treated capable of preventing the action of the

spermotoxic serum of the guinea-pig on the motility of the rabbit’s

spermatozoa. In the mixture of antispermotoxic serum and spermotoxic

serum these spermatozoa continue to move for a considerable time, whilst

in the control mixture prepared with normal rabbit’s serum and

spermotoxic serum they become motionless at the end of a few minutes. To

obtain this marked effect it was not necessary to heat the

antispermotoxic serum as in Metalnikoff’s case. Indeed I have performed

almost all my experiments with fresh serums, unheated. As the rabbit’s

serum contains macrocytase capable of rendering the spermatozoa,

sensibilised by the fixative, motionless and as this macrocytase cannot

be neutralised by the anticytase that is active against the guinea-pig’s

macrocytase, the fact I have just pointed out indicates that the

antispermotoxic serum of my rabbits contains antifixative. The

difference between the antispermotoxic serum obtained by Metalnikoff and

that prepared by me is similar to that observed between the

antihaemotoxic serums. Some contain only anticytase but others

undoubtedly contain antifixative also.

[Sidenote: [126]]

As this result appeared to me to be of far-reaching importance I felt

bound to verify it by another method. I injected certain rabbits with

spermotoxic serum of the guinea-pig and others with normal guinea-pig’s

serum. The amount of cytases being about the same in both, the strength

of the serums obtained as the result of injections of normal serum and

of specific serum should be the same if the antispermotoxic serums

contain anticytase only. Experiment demonstrates just the contrary. The

antispermotoxic serum of rabbits treated with normal guinea-pig’s serum

was on every occasion much less active than the serum of rabbits

injected with the spermotoxic serum of prepared guinea-pigs. The former

contains anticytase only, whilst the latter contains in addition

antifixative. Weichhardt’s[168] experiments carried out in my laboratory

corroborated the conclusion I have just formulated.

Having made ourselves acquainted with the constitution of the

anticytotoxins we may now pass to the question of the origin of these

bodies and of analogous ferments which act in the resorption of

albuminoid substances in the blood and in the tissues.

We have already mentioned that the leucocytes are charged with a soluble

ferment which digests gelatine, and that in animals treated with

injections of gelatine these cells elaborate a much larger amount of the

ferment. Here we have evidence of a kind of education of the leucocytes

to produce a greater amount of digestive ferment, in a manner quite

analogous to that which has been described in Chapter III in connection

with the augmentation of the pancreatic ferments in intestinal

digestion. It is, then, quite permissible to look upon leucocytes, and

probably phagocytes in general, as the source of the soluble ferment

that digests gelatine.

[Sidenote: [127]]

Is this the case with the other substances which take an active part in

the resorption of albuminoid substances in the fluids and tissues of the

organism? Up to the present the origin of precipitins and antiferments,

such as antirennet, has not been studied. The problem being very complex

and difficult, it appears to be impossible at present to solve it. It is

known indeed that the introduction of these substances into the organism

provokes a reaction similar to the one we have described in the case of

the injection of gelatine into the peritoneal cavity of guinea-pigs.

Thus Morgenroth[169] observed that in his goats the subcutaneous

injection of sterile rennet caused the formation of extensive

infiltration at the seat of inoculation, this being accompanied by

fever; we are justified in concluding from this that rennet provokes a

marked leucocytic reaction. Hildebrandt[170] has demonstrated by direct

experiment that rennet, when enclosed in capillary glass tubes and

introduced below the skin of rabbits, induces a marked positive

chemiotaxis. This led to the formation of a leucocytic plug several

millimetres long. Now we know from Briot that the rabbit is capable of

producing antirennet. Hildebrandt has further shown that several other

diastases, or hydrolytic ferments, such as sucrase and emulsin, give

rise to a similar chemiotactic phenomenon. The leucocytic reaction is

consequently a general phenomenon following the introduction into the

tissues of substances of complex chemical composition capable of

provoking the formation of antibodies. We are tempted from this fact to

accept it as a law that the leucocytes are capable of producing these

latter substances. Although this hypothesis may be very probable, the

number of facts at our disposal is not yet sufficient to justify the

statement that its truth is demonstrated.

Since it is the red corpuscles which are affected by the haemotoxins it

might be asked whether it may not be that these elements defend

themselves by the production of antihaemotoxins the overplus of which is

thrown into the blood and fluids in general? The researches that have

been made on this point relate especially to the antihaemotoxin of the

blood serum of rabbits in relation to the ichthyotoxin of eel’s serum.

We must therefore examine the collected evidence bearing on

anticytotoxins and analogous bodies and endeavour to form some idea as

to their probable origin. A large accumulation of exact data bearing on

the antihaemotoxins does not afford us sufficient information as to the

source of these substances.

Let us first examine the question, is it possible to attribute to the

red corpuscles the function of producing the antihaemotoxins? If these

elements are really the source of the antihaemotoxins it is probable

that the red corpuscles of animals whose serum is antihaemotoxic will

exhibit marked resistance to the toxins; thus we know that the white

corpuscles which produce gelatinase digest gelatine much better than

does the serum of the same animals. From the experiments of Tchistovitch

(_l. c. supra_ p. 110) on rabbits that have been immunised against eel’s

ichthyotoxin, it must be accepted that the red corpuscles of these

animals are often very sensitive to the action of the poison at a period

when the blood serum of the same rabbits exhibits a marked

antihaemotoxic power. It is not until later in the process of

immunisation, when the serum loses a great part of this power, that the

red corpuscles become resistant to the ichthyotoxin.

[Sidenote: [128]]

But before we abandon the hypothesis of the production of

antihaemotoxins by the red corpuscles we must see if it cannot be

reconciled with the facts, by the application of Ehrlich’s side-chain

theory[171]. This theory was evolved with the object of explaining the

production of antitoxins and their action on bacterial and vegetable

toxins. Later, Ehrlich has extended it to the cytotoxins, anticytotoxins

and bactericidal substances.

[Sidenote: [129]]

According to Ehrlich the complex molecule of albuminoid substances

contains, besides the central stable nucleus, a number of side-chains,

or “receptors,” which fulfil various accessory functions and serve

especially for the nutrition of the cell. These receptors have a great

affinity for the various substances necessary for the maintenance of the

life of the cell. Under normal conditions these receptors seize

nutritive molecules, as a leaf of _Dionaea_ seizes the fly that serves

it as food. Under special conditions these receptors lay hold of complex

molecules of albuminoid substances, such as the various toxins. In this

case the receptor, instead of combining with a molecule which supports

life, fixes a molecule which poisons the cell. According to Ehrlich’s

theory on the constitution of toxins their molecules contain an atomic

group which poisons—the _toxophore_, and another group which combines

with the receptor—the _haptophore_. The toxic group of a complex poison,

such as ichthyotoxin, cannot penetrate into a red corpuscle except by

the help of the haptophore group and of the corresponding receptor. When

a red corpuscle has absorbed a large number of molecules of

ichthyotoxin, the united action of the toxophore groups renders life

impossible and the corpuscle is dissolved. But when a red corpuscle has

been touched by only a few toxic molecules, too few to compromise life,

there is merely immobilisation of the receptors which are combined with

the haptophore groups of the ichthyotoxin. As these receptors fulfil an

important function in the nutrition of the red corpuscles, the latter

reproduce them in larger numbers than were originally present. We know

that in the phenomena of repair an over-production of the new-formed

parts often takes place and, according to Ehrlich, to this

over-production the presence of antitoxins in the fluids of the body is

due. The receptors, developed in excess by the red corpuscles, fill

these cells, and no longer finding room therein are extruded from them

and overflow into the blood and other fluids of the organism. When a

fresh injection of toxin makes its way to the blood it there meets with

a number of free receptors, endowed with an affinity for the haptophore

group of the molecule of the toxic substance. The chemical combination

between the two substances takes place at once in the plasmas, a fact

which prevents the haptophore group of the toxin from uniting with the

receptor of the red corpuscles and so injuring these cells by

introducing the toxophore group into them. According to this theory the

same receptors which, in the free state in the fluids, fulfil the

_antitoxic_ function become in the interior of the red corpuscles the

vehicles of intoxication and consequently fulfil a _philotoxic_

function. This opposite _rôle_ of the receptors has often been compared

to a lightning-conductor; so long as the receptors are attached to the

molecule of the living protoplasm they attract the toxin just as a

lightning-conductor attracts the lightning when it is badly insulated.

[Sidenote: [130]]

So interpreted, it is easy to conceive that the red corpuscles of

animals whose fluids are antihaemotoxic may be sensitive to the toxic

action of eel’s serum, as has been observed by Tchistovitch. As soon as

the protective fluids have been removed from the red corpuscles of the

immunised organism, the corpuscles when placed in contact with

ichthyotoxin (eel’s serum) attract the haptophore groups of the poison

by means of their numerous receptors. These haptophores in their turn

introduce the toxophore groups which dissolve the red corpuscles without

the slightest difficulty. This theory does not explain the cases, which

are numerous, in which the red corpuscles of rabbits that are vaccinated

against eel’s poison resist this poison. Camus, Gley, and Kossel,

working independently, have arrived at the result that the red

corpuscles of immunised rabbits, from which the serum has been carefully

removed, are not dissolved when submitted to the action of ichthyotoxin,

whilst the red corpuscles of untreated rabbits placed under the same

conditions, undergo a rapid solution. Tchistovitch confirming this fact

has added to it the observation that the resistance of the red

corpuscles of the rabbit is most often found when the serum loses its

antitoxic power. If the receptors of the red corpuscles of immunised

rabbits, owing to their great affinity for the haptophore group of the

ichthyotoxin molecule, only attract the toxophore group of this poison,

as the lightning-conductor when badly insulated attracts the lightning,

the red corpuscles should never manifest resistance. To explain this

contradiction we must not suppose that the red blood corpuscles which

have become resistant have got rid of their receptors. In fact, if these

receptors are so necessary to the nutrition of the cell that their

absence has set up this extraordinary over-production which has

inundated the fluids, it is evident that one cannot admit the existence

of red corpuscles entirely deprived of corresponding receptors.

When examined from different points of view the hypothesis of the

production of antihaemotoxin by the red corpuscles is surrounded with

very great difficulties. It appears to be probable, therefore, that the

source of this antitoxin must be sought for in other cell elements, and

we may be allowed to recall to mind those cells which manifest a general

and local reaction of the most constant kind after each injection of

ichthyotoxin. Tchistovitch has observed that eel’s serum when introduced

into rabbits in non-fatal but immunising doses excites a marked

hyperleucocytosis.

[Sidenote: [131]]

The question of the origin of anticytotoxins being so complicated, it

has been necessary for its elucidation to seek an experimental method of

excluding the organ in which the antibody is supposed to have its

origin. As we cannot think of eliminating the red or white corpuscles,

nor the greater part of the tissues and organs, there remains only one

way of bringing about this result. It is the suppression of the male

genital organs. We know already that the injection of semen readily

excites the production of a spermotoxin, and that this spermotoxin gives

rise to the development of a corresponding antispermotoxin. If it is the

spermatozoa, that is to say the elements having a particular affinity

for the spermotoxin, which elaborate the antitoxin we must conclude that

castrated males would be incapable of producing it. With this in view we

have carried out a great number of experiments which have amply proved

to us that male rabbits when deprived of their sexual organs are fully

as capable of developing antispermotoxin in their fluids as are control

rabbits in which the male genital apparatus remains intact. Doe-rabbits,

and young, sexually immature rabbits of both sexes, also react to

injections of spermotoxin by producing the corresponding

antispermotoxin. The specific elements which are sensitive to the action

of a cytotoxin undoubtedly are not indispensable for the development of

the corresponding anticytotoxin. This result is in complete harmony with

the hypothesis above put forward, that the red corpuscles cannot be

regarded as the source of the antihaemotoxin. In the case of

antispermotoxin this fact can be rigorously established by experiment.

Here arises the following question. We have seen that the anticytotoxins

are composed of two different substances: an anticytase and an

antifixative. The former is an antitoxin capable of neutralising

macrocytase, the soluble ferment which will attack indifferently all

kinds of cell elements. It is not to be wondered at, then, that the

exclusion of the spermatozoa in no way prevents the production of

anticytase by an organism which receives injections of cytotoxins. These

latter, as we have already said, contain cytase along with the specific

fixative; the macrocytase can attack any kind of animal cell provided

that it can find some fixative or any other means to penetrate into the

interior of these formed elements. We have seen that the

antispermotoxin, obtained by Metalnikoff in guinea-pigs, does not

contain any anticytase. Amongst his animals treated with spermotoxin was

a castrated male guinea-pig which also produced anticytase. There is

nothing astonishing in this fact, the injected cytase must have linked

itself to many other cells which were able to develop anticytase.

[Sidenote: [132]]

But the example of the antispermotoxin of the rabbits in my own

experiments is very different. In order that it might manifest its

action the serum of these rabbits did not need to be heated to 56° C.;

it was not necessary to rid it of its own macrocytase which could have

acted under the influence of the fixative, if this latter for want of

antifixative had remained free in the added spermotoxin. This

antifixative, then, is undoubtedly found in the serum of castrated males

which have shown themselves capable of producing not only anticytase,

but also antifixative. This result has been further verified by

comparative experiments on castrated male rabbits, some of which

received spermotoxic guinea-pig’s serum whilst the others received only

normal guinea-pig’s serum. It has been demonstrated that the amount of

cytases remains almost constant in both normal and vaccinated

animals[172]. If, then, the antispermotoxins contain only anticytase,

the injection of specific guinea-pig’s serum and that of normal

guinea-pig’s serum should produce the same result, that is to say the

serums of castrated rabbits, when treated by these two kinds of

guinea-pig’s serum, should exhibit the same antispermotoxic power.

Experiments have, however, proved that this is not the case. The serum

of castrated rabbits that have been injected several times with normal

guinea-pig’s serum becomes distinctly antispermotoxic, but its power to

protect the spermatozoa of the rabbit against being deprived of motility

by the guinea-pig’s spermotoxin is greatly inferior to that which is

developed in the serum of other castrated rabbits that I injected with

spermotoxic guinea-pig’s serum. Of course all the other conditions of

the experiment were the same for the two groups of rabbits.

[Sidenote: [133]]

Several series of facts, then, focus to this fundamental point, that the

organism of an animal that has been deprived of its male sexual organs

is in a condition to produce antispermofixative. Against the argument

that we have drawn from the fact that the antispermotoxic serum of

castrated rabbits that have been treated with spermotoxic serum acts

without being heated, might be cited certain experiments made by Ehrlich

and Morgenroth. The antispermotoxic action in this case, as already

stated, demonstrates that the serum of prepared rabbits contains

antifixative. Otherwise, had the fixative not been neutralised, it would

have allowed the macrocytase of the rabbit’s serum to arrest the

movements of the spermatozoa. Now the two above-named observers have

demonstrated[173] that the injection of different serums into animals is

capable of exciting in their blood the development of anticytases. The

macrocytase of castrated rabbits which, before treatment with the

spermotoxin, was capable of arresting the movements of rabbits’

spermatozoa acted upon by a fixative, might become inert after the

injections of spermotoxic serum of guinea-pigs. To clear up this point I

asked M. Weichardt[174], who has carried out work on this subject in my

laboratory, to try by means of unheated serums of normal animals, to

restore the activity of spermotoxin that had been mixed with

antispermotoxic serum. Spermatozoa of rabbits were put into a definite

mixture of spermotoxic guinea-pig’s serum, heated to 56° C., and

antispermotoxic serum, also heated to 56° C., obtained from castrated

rabbits that had been treated with spermotoxin. The spermatozoa remained

very active in this mixture which contains specific fixative (in the

spermotoxic guinea-pig’s serum) and antispermotoxin. To this mixture is

added a little normal rabbit’s or horse’s serum, unheated. These serums

contain cytases and would be quite capable of arresting the movements of

the spermatozoa if there was found in the mixture any free fixative that

would enable the macrocytase to be linked to the spermatozoa. Under

these conditions the spermatozoa remain motile for a long time. The

fixative, then, was no longer active; it was neutralised by the

antifixative of the antispermotoxic serum of castrated rabbits. A

control experiment was made with the same substances; but the castrated

rabbits’ serum that had been treated with spermotoxic serum was replaced

by the serum of other castrated rabbits treated with normal guinea-pig’s

serum. In these latter mixtures the spermatozoa became motionless at the

end of a very short time; the fixative, not being neutralised, readily

allowed the rabbit’s and horse’s cytases to affect the spermatozoa.

It follows from all this that the antispermotoxic serum of castrated

male rabbits, when treated with normal guinea-pig’s serum, contains

anticytase only; whilst the serum of castrated male rabbits, treated

with specific and spermotoxic guinea-pig’s serum, contains anticytase

and antifixative. The latter, then, has been produced independently of

the sensitive elements,—the spermatozoa.

[Sidenote: [134]]

Having established the fact that antispermotoxin does not come from the

male organs, it was necessary to try to ascertain its true source. With

this object in view we injected spermotoxic serum into young rabbits

(quite capable of producing antispermotoxin) and tried to follow the

fate of the spermotoxin in the organism. When spermotoxic guinea-pig’s

serum is injected into the peritoneal cavity of the rabbit a notable

amount of spermotoxin is found in the thickened portion of the omentum

made up of lymphoid tissue. But the greater portion of the poison passes

into the circulation whence it goes to fix itself in various organs,

especially the spleen. At the moment when the spermotoxin is found in

the blood a certain quantity of this fluid was drawn off into tubes

containing some drops of extract of leeches’ heads. After the blood thus

treated had been centrifugalised the plasma was decanted and its power

of arresting the movements of spermatozoa was compared with that of

serum of the same blood prepared in the usual way. From these researches

it results that the plasma is always richer in spermotoxin than is the

corresponding serum. Sometimes the difference in favour of the plasma is

very great.

A part of the spermotoxin passes into the kidneys and the suprarenal

capsules. It is probable that, as is the case with so many soluble

poisons, a certain proportion of the spermotoxin may be eliminated by

the uropoietic organs. A small quantity of this poison is found also in

the male and female sexual glands of young non-castrated rabbits.

The search for some main centre of origin for the production of

antispermotoxin has as yet led to no positive result. The power of

arresting the movements of spermatozoa first appears in the blood

plasma, and it is this same fluid which, later, is more antispermotoxic

than is any organ. Amongst the tissues which fix spermotoxin the genital

organs play not the slightest part in the production of antispermotoxin.

The experiments with castrated rabbits afford sufficient proof of this.

On the other hand it becomes more and more probable that the phagocytic

system, disseminated in many organs, and especially the leucocytes,

furnish the antispermotoxic substance. The fixation of the spermotoxin

by the leucocytes of the blood, such as the cells of the omentum and of

the spleen, already offers us a valuable indication. The absence of any

particular organ that might have the monopoly of fixing the spermotoxin

and which should later be found charged with a predominant amount of

antispermotoxin also speaks in favour of the phagocytic origin of this

antitoxin.

[Sidenote: [135]]

After a single intraperitoneal injection of spermotoxic guinea-pig’s

serum into young rabbits, the blood of the latter is distinctly

spermotoxic for several days; later it becomes indifferent, but eight or

ten days after the commencement of the experiment the blood begins to

exhibit an antispermotoxic power. In these cases the plasma shows itself

more active than the serum. When the rabbits are killed at this stage of

commencing antitoxic production, it is found that an extract of the

organs is not antispermotoxic or only feebly so. In all cases this

power, when it exists, is more feeble than that of the blood fluid. The

results obtained with extracts of organs are not constant. Sometimes the

spleen possesses more antitoxic activity, whilst the liver, thymus,

omentum, lymphatic glands and genital glands exhibit none of this

property. In other cases the survival of the spermatozoa that are

influenced by the spermotoxin has been longest in the extract of the

suprarenal capsules. Sometimes the extract of the omentum exhibits the

greatest antispermotoxic power. This great variability in the

development of the property of protecting the spermatozoa accords well

with the idea that the elements which produce antispermotoxin are

wandering cells which, under diverse influences, may be localised in

very diverse points of the organism.

We must not deceive ourselves. The facts which have been collected up to

the present do not allow us as yet to form a final opinion on the origin

of anticytotoxins, but we are quite justified in regarding as very

probable the hypothesis that the phagocytes play a most important part

in the process. It is in all cases beyond doubt that the amoeboid cells

which resorb the formed elements play a very important part in the

resorption of fluids of very complex molecular composition.

CHAPTER VI

NATURAL IMMUNITY AGAINST PATHOGENIC MICRO-ORGANISMS

Natural immunity and the composition of the body fluids.—Cultivation

of the bacteria of influenza and pleuro-pneumonia in the fluids of

refractory animals.—Resistance of _Daphniae_ to the

Blastomycetes.—Examples of natural immunity in Insects

and Mollusca.—Immunity of Fishes against the anthrax

bacillus.—Immunity of frogs against anthrax, Ernst’s bacillus, the

bacillus of mouse septicaemia and the cholera vibrio.—Natural

immunity in the cayman.—Immunity of the fowl and pigeon against

anthrax and human tuberculosis.—Immunity of the dog and rat

against the anthrax bacillus.—Immunity of Mammals against anthrax

vaccines.—Immunity of the guinea-pig against spirilla, vibrios,

and streptococci.—Natural immunity against anaerobic bacilli.—Fate

of Blastomycetes and _Trypanosomae_ in the refractory organism.

[Sidenote: [136]]

In the third chapter reference has been made to the frequency of cases

of natural immunity against infective diseases. Examples of this

immunity occur in the lower animals—the Invertebrata—and are widely met

with among the Vertebrata. We have already mentioned that this natural

immunity can be attributed neither to insusceptibility to microbial

toxins nor to the elimination of the micro-organisms by the excretory

channels. Nevertheless the pathogenic agents which have penetrated into

the tissues of the refractory organism disappear, without being

eliminated. To facilitate the study of their disappearance it has been

necessary to pass in review the phenomena that follow the introduction

of foreign bodies into the organism and to present a brief analysis of

the process of resorption of cell elements in its relations to

digestion. We have tried to demonstrate that resorption is nothing more

than a process of digestion which, instead of going on in the intestinal

canal, takes place in the tissues; that it is, indeed, an intracellular

digestion exactly comparable to that which serves for the nutrition of

certain of the lower animals.

[Sidenote: [137]]

A knowledge of all these facts is necessary before we can deal with the

subject to which the present chapter must be devoted—the innate natural

immunity of animals and man against pathogenic micro-organisms. As,

under natural conditions, it is the micro-organism and not its toxic

products which invades the organism, it is clear that we must give the

first place to the study of immunity against the micro-organism. The

more so because this form of immunity is much more frequently met with

than is an insusceptibility to toxins.

Since the animal organism has a very variable composition it might be

concluded that the micro-organisms find in the refractory species simply

a chemical medium in which they cannot live. We cannot go far in the

discussion of this supposition without seeing that it may be rejected.

Among the pathogenic micro-organisms some are distinguished by a great

fastidiousness and sensitiveness as regards the medium in which they are

placed. Such, for example, are the parasites of malaria and their

allies. They live inside the red blood corpuscles of Vertebrata and

appear to be extremely discriminating in regard to their requirements.

All animals, even monkeys, are refractory to human malarial fevers. It

might be concluded from this that here at least the immunity may be due

to the fact that the chemical composition of the contents of the red

corpuscles in the immune animals is different from that of the red

corpuscles of man. But when we see, as was first demonstrated by

Ross[175], that the malaria parasite of Laveran, having made its way

into the digestive canal of certain mosquitos (_Anopheles_), there

develops abundantly, it is difficult to maintain this thesis.

Among other micro-organisms of animal origin we have the _Trypanosoma_,

the parasite of the terrible disease propagated by the Tsetse fly which

commits such ravages amongst mammals. Man alone escapes it, exhibiting a

natural immunity that nothing apparently can overcome. Are we to affirm

that it is the difference in the chemical composition of the human body

which assures to man his immunity against a parasite that attacks

indifferently an herbivorous animal, such as the ox or rabbit, or a

carnivorous animal, such as the dog? In these examples I have chosen

merely those micro-organisms which it has never been possible to

cultivate on any artificial nutrient medium and which are kept alive

with very great difficulty outside the living organism.

[Sidenote: [138]]

What is to be said then of the vegetable micro-organisms which, in this

respect, are much less exacting? The most important of these and the

most numerous of all pathogenic micro-organisms, the Bacteria, can as a

rule be cultivated without difficulty not only in the blood and fluids

of animals that are susceptible or refractory to their morbific action,

but also on all kinds of vegetables and artificial media: broths, fluids

composed of mineral salts and of certain organic substances. It is

really not possible to attribute the natural immunity of the dog and the

fowl against the anthrax bacillus—so fatal to a great number of mammals,

man included,—to its incapacity to feed on the fluids of these animals,

when we see that this same bacillus is capable of killing lower animals,

such as the cricket, and can thrive on carrots, potatoes and other

vegetables.

Even when, among the bacteria, we take those that are most exacting in

the choice of their food, we still find it impossible to explain natural

immunity as being due to the want of power on the part of these

organisms to obtain their nutriment from the juices of refractory

species. The bacillus discovered by R. Pfeiffer[176] in influenza does

not develop on media that are ordinarily employed in bacteriology in the

cultivation of a great number of micro-organisms. It needs a special

food, which is prepared for it by spreading a drop of fresh blood on the

surface of agar. Pfeiffer has established the fact—confirmed by many

observers—that the best species of blood to use for this purpose is that

of the pigeon. We should have to believe, then, did the immunity really

depend on the composition of the fluids, that the pigeon is the least

refractory of all animals. Experiment has demonstrated the erroneousness

of such a supposition: the pigeon is quite as refractory to Pfeiffer’s

bacillus as are most other species of animals.

[Sidenote: [139]]

As a second example the bacterium of bovine pleuro-pneumonia may be

cited. It is the smallest of all known bacteria. The difficulties

surrounding the discovery and identification of this organism were very

great, and the ingenuity of Nocard and Roux[177] was required for the

demonstration of its existence. Very exacting in its choice of nutritive

material, it was first cultivated in the fluids of the rabbit, a species

endowed with an absolute immunity against bovine pleuropneumonia. It is

unnecessary to multiply examples to obtain a general proof that natural

immunity against micro-organisms cannot be explained by the incapacity

of these pathogenic agents to live in the fluids of the refractory

organism.

We must, however, ascertain what takes place in resistant animals

inoculated with micro-organisms. Here, again, it is preferable to begin

with the lower animals of simple organisation. We have already seen that

examples of natural immunity are not rare in the Invertebrata. When

engaged in the study of the disease found in _Daphniae_, small crustacea

so common in fresh water, I was able to show that the special

Blastomycetes which cause it meet with a vigorous resistance on the part

of the organism. As the _Daphniae_ are small, transparent, and

consequently easily observed under the microscope, I was able without

difficulty to establish the main phenomena observable in these

organisms. I can be the more brief in describing these phenomena of

resistance as, in addition to devoting a special memoir to the _Daphnia_

disease[178], I have, in my _Lectures on Inflammation_ (pp.

97–103)[179], described at some length the reaction of their organism to

the _Monospora_. It is nevertheless necessary that I should recall, very

briefly, the mechanism by which these small crustaceans secure immunity.

The spores of the parasite—very delicate and rigid needles—are swallowed

with the food. By means of their sharp points they perforate the

intestine and penetrate into the body cavity, full of blood, where they

find themselves exposed to the attacks of leucocytes. These leucocytes,

guided by their tactile sense, gather around the foreign body, ingest it

completely and destroy it. It is remarkable that the spore, which is

furnished with a very resistant membrane, once in the interior of the

mass of leucocytes, undergoes modifications which afford evidence of the

presence in these cells of an extraordinary digestive power. The surface

of the spore, from being smooth and regular, becomes pitted and sinuous,

the spore breaks up into fragments and is reduced to a mass of _débris_

which, in the form of brown granules, remains indefinitely in the

contents of the leucocytes. From this it is evident that these

phagocytes must produce a ferment which is capable of digesting the

cellulose or analogous substance which forms the membrane of the spore.

Unfortunately, the small size of the _Daphniae_, so useful for the

direct observation of the phenomena of immunity, presents an

insurmountable obstacle to the study of its leucocyte ferments,

especially _in vitro_.

[Sidenote: [140]]

The destruction of the spores of the parasite by the leucocytes secures

to the _Daphnia_ a real immunity. Of a hundred _Daphniae_ taken in my

aquarium and carefully examined under the microscope, fourteen only were

found to be infected by the budding conidia of the parasite, whilst

fifty-nine of the others contained the remains of spores that had been

destroyed by the phagocytes. When transferred to pure water containing

no new source of contagion, these _Daphniae_ flourished and lived a

normal life, giving birth to a numerous progeny.

The immunity of the _Daphnia_, due to the intervention of phagocytes, is

an example of natural, individual immunity. It is not the specific or

racial possession of these crustacea, for when the leucocytes do not

seize the spore, at once, on its penetration into the body cavity, it

commences to germinate and gives rise to a whole generation of budding

cells. These cells, then, secrete a poison which not only repels the

leucocytes, but kills and completely dissolves them. Under these

conditions the _Daphnia_ is disarmed; the parasites grow in the

organism, deprived of its arm of defence, as in a culture tube, and the

animal rapidly succumbs.

Since I first observed this struggle between the _Daphnia_ and its

parasite, some eighteen years ago, no other example has been found that

is so easily observed and so demonstrative of the protective action of

phagocytes in an animal that can be kept under observation, alive, under

the microscope. Cases, however, are not wanting in the Invertebrata in

which the different phases of this struggle may be observed with

sufficient accuracy to warrant the conclusion that in these cases also

the phenomena are analogous to those observed in the case of the

_Daphniae_.

[Sidenote: [141]]

It has already been stated in Chapter III. that the larvae of the

rhinoceros beetle (_Oryctes nasicornis_), although very sensitive to the

cholera vibrio, are very refractory to anthrax and diphtheria. In order

that we may obtain some idea of the mechanism of this immunity let us

inject into the body cavity of these large white grubs a trace of

anthrax culture. In the blood, drawn off the following morning, the

injected bacilli are found, not in the plasma, but inside many of the

leucocytes. Here there has occurred, as in the _Daphnia_, an ingestion

of the parasites which have then been destroyed by the intracellular

digestion of phagocytes. The process is the same, then, as that by which

the resorption of the red corpuscles of the goose takes place when they

are injected into the blood of cockchafer larvae. In both cases the

foreign bodies are ingested and destroyed by the leucocytes of the

blood; this act of resorption, however, taking a very long time.

Although the leucocytes of the larvae of the rhinoceros beetle exhibit a

positive chemiotaxis for the bacillus, these same cells behave in a very

different fashion in presence of the cholera vibrio. Very small

quantities of this vibrio, when injected into the blood of the larvae,

give them a fatal disease: the vibrios excite in the leucocytes a

negative chemiotaxis and flourish without hindrance in the blood plasma.

The larva is soon transformed into a culture vessel and the numerous

vibrios that develop in it cause the death of the animal.

The difference in action of the two bacteria cannot be explained by any

corresponding difference in their mode of life in the blood. Removed

from the organism the blood plasma of the white larvae of the rhinoceros

beetle is a culture medium just as favourable to the growth of the

anthrax bacillus as to that of the cholera vibrio. Moreover, the former

of these micro-organisms is quite capable of setting up a fatal disease

in other representatives of the class of Insects. Kovalevsky[180] has

discovered in the house cricket four phagocytic organs, with a great

appetite for all kinds of foreign particles that may penetrate into its

body. The blood of mammals, when injected below the skin of the cricket,

is rapidly absorbed by the cells of the four “spleens” (for so

Kovalevsky designates these phagocytic organs). The resorption of the

red blood corpuscles goes on within these phagocytes owing to their

power of intracellular digestion. When Kovalevsky kept crickets at a

temperature of 22°–23° C. and injected them with anthrax bacilli he

noted that these bacilli also were ingested by the cells of the spleens.

There was, thus, no manifestation of negative chemiotaxis of these

elements towards the bacillus. The ingestion of the bacilli by the

phagocytes was not sufficient, however, to protect the animal. The

bacilli reproduced themselves rapidly in the blood fluid; the

intracellular lacunae of the spleens were full of them and the crickets

quickly succumbed to the infection.

[Sidenote: [142]]

Nevertheless these crickets are quite capable of resisting certain other

bacteria. Balbiani[181] has shown that they are refractory to a great

number of bacilli belonging to the group of _Bacillus subtilis_. He

observed that when injected into the body of the cricket these bacilli

are devoured and destroyed by the leucocytes of the blood and by the

large cells of the pericardial tissue corresponding to the elements of

the spleens of Kovalevsky. Whilst the crickets and other Orthoptera,

which are rich in phagocytes, exhibit a real immunity against these

bacilli, insects which have very few leucocytes such as butterflies,

flies and Hymenoptera are found to be much more susceptible to infection

by the same bacilli. In this case the direct relation between immunity

and phagocytosis is very marked.

[Sidenote: [143]]

The Mollusca also furnish some interesting examples of natural immunity.

Karlinsky[182] has observed that anthrax bacilli, when injected into the

blood of slugs and snails, soon disappear from their bodies; these

pulmonate Gasteropods are absolutely unaffected by this bacillus so

formidable for many species of animals. From the rapidity of this

disappearance of the bacilli it has even been concluded that it was

impossible for this bacillus to live in the fluids of Mollusca.

Kovalevsky (_l.c._ p. 443) has studied this question with the

carefulness that characterises all his work. He confirms the fact that

snails (_Helix pomatia_) resist the introduction of a large quantity of

anthrax bacilli into their bodies; he notes also that these bacteria

disappear from the blood. But he finds them again in the tissues of the

foot, and especially in the cells which surround the pulmonary vessels.

“The greater number of the bacteria are found in the cells of that part

of the pulmonary region in _Helix_ which adjoins the heart and kidney.

All the bacteria were ingested by the cells and I easily succeeded in

demonstrating this not only in sections but also in bulk” (p. 444). The

snails remained in good health in spite of the presence in their

phagocytes of numerous bacteria which maintained themselves there for

some time. At the end of ten or twelve days and more these bacteria

still presented their usual aspect; this accords well with the slowness

with which intracellular digestion goes on in the majority of the

Invertebrata. These bacteria were, however, no longer living, although

still undigested. Morsels of the pulmonary tissue of the snails that

were injected with anthrax bacilli still gave cultures 48 hours after

injection and contained bacilli capable of giving fatal anthrax to mice.

Later, media seeded with similar particles remained sterile, and mice

inoculated therewith continued to live. From these experiments it may be

accepted that bacteria, living in the blood plasma, become the prey of

phagocytes which render them inoffensive and kill them. This example

demonstrates once again that the organism gets rid of bacteria by the

same mechanism as that which serves for the resorption of any of the

formed elements. The snail reacts to bacteria as it does to the red

corpuscles of the goose.

It is unnecessary to insist further on the natural immunity of the

Invertebrata, and it is useless to multiply examples which always point

in the same direction: to the importance of phagocytic reaction and of

intracellular digestion in resorption and immunity. We must pass on to

the examination of the reaction phenomena of the vertebrate organism

towards pathogenic micro-organisms, following, as hitherto, the

comparative method. We will commence with the study of the natural

immunity of fishes as lower representatives of the great group of the

Vertebrata.

[Sidenote: [144]]

It is well known that fishes are liable to infective diseases and

pisciculture has often to deplore considerable losses brought about

sometimes by certain of the lower Fungi (_e.g. Saprolegniae_), sometimes

by Bacteria. The pathogenic microbes which produce epidemics in fishes

are still little understood; but among the bacteria which kill many of

the higher animals are some which cause fatal maladies in certain

fishes. Thus the anthrax bacillus so virulent for many mammals is

capable also, as we have seen, of producing an infection in the cricket,

and may cause the death of small marine osseous fishes, the

_Hippocampi_. Sabrazès and Colombot[183], who have studied this

question, have demonstrated that the anthrax bacillus, which is virulent

for the rabbit, when inoculated into these fishes first produces

swellings at the seat of inoculation and ultimately becomes generalised

throughout the body, producing a fatal septicaemia. As these experiments

have given this result at a temperature of 14°–16° C., it is quite

evident that the bacillus, in order to manifest its pathogenic effect,

in no way needs the high temperature of the mammalian body for its

action.

Now among fishes there are not wanting species which resist the anthrax

bacillus. Mesnil[184] has, in our laboratory, thoroughly studied the

mechanism of this immunity. He has shown that several fresh-water

fishes, _e.g._ the perch (_Perca fluviatilis_), the gudgeon (_Gobio

fluviatilis_), and the gold-fish (_Carassius auratus_), will resist an

injection of a considerable number of bacilli into the abdomen. When

kept at temperatures of 15°–20° C. or even 23° C., a temperature at

which the bacilli are able to develop very abundantly, these fishes

destroy a large number of the bacteria in their bodies. Soon after the

introduction of the bacilli into the peritoneal cavity, the numerous

leucocytes accumulate around them and ingest them by the same mechanism

that is observed in the Invertebrata or in the same fishes when

absorbing the red blood corpuscles of alien species. In the gudgeon, at

as early as six and a half hours, a very marked, nay, an almost complete

phagocytosis is set up.

It is impossible to doubt the fundamental fact that the bacilli, at the

moment of their ingestion, are in a perfect condition of vitality and

virulence. The fluid of the peritoneal exudation, when withdrawn from

the animal, is of itself incapable of preventing the development of the

anthrax bacilli. The peritoneal lymph of the above-mentioned fishes is,

_in vitro_, even a good culture medium for these bacilli.

[Sidenote: [145]]

When, long after the completion of the phagocytosis by the leucocytes of

the peritoneal exudation, a drop of the exudation is withdrawn and kept

outside the organism under suitable conditions of temperature and

moisture, a number of the ingested bacilli begin to multiply and give an

abundant culture. This experiment proves, indisputably, that the bacilli

are devoured in the living state. If a little of the peritoneal

exudation, withdrawn several (up to nine) days after the injection of

the bacilli, be injected below the skin of guinea-pigs these animals die

from generalised anthrax, a fact which demonstrates that the bacilli,

which have been ingested alive, have retained their virulence a long

time after they have been devoured by the leucocytes. But, if the

peritoneal exudations that have been withdrawn at still longer periods

after injection be examined, it is found that they no longer contain

bacilli capable of developing in culture media or of setting up the

disease in the most susceptible animal. Hence it follows that in the

organism of the refractory fish, the bacteria are not destroyed by the

fluids but by the phagocytes, which take a long time to bring about the

complete intracellular digestion of ingested micro-organisms.

The phagocytes which assure immunity to the osseous fishes that were

studied by Mesnil belong principally to the group of haemomacrophages.

These are leucocytes with abundant protoplasm which stain readily by

basic aniline dyes, mononuclear cells whose nucleus, however, is

sometimes divided into lobes. It is to be noted that in the perch these

are the sole representatives of the motile phagocytes, and that in this

fish not only the eosinophile but every other variety of granular

leucocyte is completely wanting. In the gudgeon, in addition to

haemomacrophages, some microphages whose protoplasm stains faintly with

acid aniline colours are met with. These facts will be useful to us when

we come to study the part played by phagocytes in immunity from a

general point of view.

Another class of cold-blooded animal, the Amphibia, has been much more

frequently studied from the point of view of infection and immunity. The

frog, an animal so convenient for many physiological and pathological

researches, has been much employed for the study of immunity against

pathogenic micro-organisms. Quite a literature, which has been

excellently summarised in the memoir of Mesnil already cited, and to

which we shall have occasion to return more than once, has been

accumulated on the subject.

[Sidenote: [146]]

The immunity of frogs against the anthrax bacillus was early

demonstrated and studied in Robert Koch’s celebrated memoir[185] on

anthrax. This observer, after injecting an emulsion of anthrax spleen

into the lymph sac of the frog, recovered the bacilli from the interior

of round cells which burst readily when transported into water. Koch,

accepting the view then generally held, thought that the bacilli found a

favourable culture medium in the contents of certain cells, but that, in

spite of this, the frog was capable of manifesting a real immunity

against anthrax. Gibier[186] made the interesting discovery that frogs

when subjected to the influence of high temperature (about 37° C.) lose

their natural immunity and readily contract fatal anthrax.

Since that time the mechanism by which the organism of the frog secures

immunity against the anthrax bacillus has repeatedly been studied. In a

memoir which appeared in 1884[187] I insisted that the principal part

played in this immunity belonged to the phagocytes which devour the

injected bacteria and subject them to intracellular digestion. The round

cells described by Koch are nothing but the leucocytes of the lymph sac

which have seized upon the anthrax bacilli. These bacilli instead of

thriving in the cell contents find there a very unfavourable medium, and

perish at the end of a longer or shorter period. When the activity of

the phagocytes is impeded by unfavourable influences, _e.g._ high

temperature, they exhibit a very feeble reaction, incapable of assuring

to the frog that immunity which, under normal conditions, it possesses.

The conclusions I have just summarised have raised very lively

opposition from a large number of observers. Baumgarten[188], with his

pupils Petruschky[189] and Fahrenholtz[190], have endeavoured to

demonstrate that phagocytosis plays no part in immunity and that the

frogs resist anthrax simply because the bacilli are incapable of

maintaining themselves alive in the fluids of this Batrachian.

Nuttall[191], of Flügge’s school, also maintained that frogs resist

anthrax owing to the bactericidal power of their fluids. This view has

been defended by several other observers and appeared for some time to

become quite dominant.

[Sidenote: [147]]

Nevertheless, it is possible to demonstrate that the plasmas of the frog

not only are not inimical to the life of the bacillus, but serve as a

good culture medium for it[192]. All that is necessary for the

demonstration of this fact is to introduce below the skin of frogs

anthrax spores enclosed in a sac of reed pith, or simply enveloped in a

small piece of filter paper. The plasma of the lymph sac at once

permeates the spores and allows them to germinate and produce quite a

generation of bacilli. But, as soon as the leucocytes pass through the

paper, they seize upon the young bacilli, digest them in their substance

and prevent their pathogenic action. The germination of the spores may

take place even where they have been introduced below the frog’s skin

without being protected in any way whatever. But, under these

conditions, only a certain number of the spores germinate, the majority

not having time to do so before the arrival of the leucocytes. The

small, very short bacilli which proceed from the germinated spores, are,

along with the spores that have not germinated, soon ingested by the

phagocytes. But, whilst the rods are in the end digested within these

cells, the ingested spores remain intact for a very long time: they do

not germinate, but they are not destroyed and retain their vitality

indefinitely, in spite of the influence of the phagocytes. It is

sufficient to withdraw from a frog, that has been inoculated with

anthrax spores some time before and kept at a moderate temperature

(15°–25° C.), a little lymph and sow it in any nutrient medium (of those

employed in the culture of bacteria), in order to see the spores

germinate and produce a whole generation of absolutely normal

filamentous bacilli. All these phenomena have been carefully studied by

Trapeznikoff[193] in a work executed in my laboratory. It is obvious

from his experiments that the phagocytes of the frog are quite capable

of protecting the organism against the anthrax bacillus by ingesting and

digesting the bacilli in the vegetative state and by preventing the

germination of the ingested spores. This phagocytic action is very

important in presence of the fact that the plasmas of the frog allow the

spores to germinate and the bacilli to develop and produce abundant

cultures.

[Sidenote: [148]]

The immunity of frogs against the anthrax bacillus that we have just

described and which is guaranteed by the activity of the phagocytes, is

constant under the conditions of temperature above mentioned (15°–25°

C.), conditions which are sufficient, however, to ensure the death of

susceptible cold-blooded animals, such as the cricket or _Hippocampus_,

from anthrax. The edible frog, a species that readily accommodates

itself to a temperature of 35° C., resists, even under these conditions,

infection by the bacillus, as pointed out by Mesnil in a work already

cited when treating of the immunity of fishes. The green frog (_Rana

esculenta_) when kept for a long time at this high temperature, so

suitable for the development of the anthrax bacillus, reacts by the same

phagocytic mechanism. The leucocytes of the lymph and blood, the cells

of the splenic pulp and Kupffer’s stellate cells of the liver, seize the

introduced bacilli and digest them as in any other case of phagocytosis.

The brown frog (_Rana temporaria_) adapts itself but slightly and with

great difficulty to the high temperature and dies whether it has been

inoculated with anthrax or not. Under these conditions the bacteria

develop in the body of the dead or dying frogs, but Mesnil insists on

the fact that a true anthrax infection is not produced, as has been

maintained by Gibier as the outcome of his researches.

[Sidenote: [149]]

Dieudonné[194], however, has found a method of removing the natural

immunity of the frog against the anthrax bacillus, by inoculating it

with an artificial bacterial race which he had adapted to develop fairly

luxuriantly at the low temperature of 12° C. Under these conditions all

the inoculated frogs, even those which had resisted the inoculation with

ordinary bacteria (grown at 37°·5 C.), died within a period of 48 to 56

hours, containing many bacilli in the blood and organs. Dieudonné has

not studied the essential mechanism that accompanies this loss of

immunity; but it is very probable that, for one thing, we have here to

do with a reinforcement, special for the frog, of the bacillus that has

become accustomed to develop at a low temperature. This bacillus must

multiply, in frogs that have been maintained at a low temperature, much

more rapidly and profusely than would the ordinary bacillus. On the

other hand, the susceptibility of Dieudonné’s frogs must depend on a

less resistance of the organism under the conditions of his experiments.

Unfortunately, we cannot find in his memoir sufficient data on these

points; he does not even state the temperature at which the frogs that

had been inoculated with bacteria adapted to cold lived. Dieudonné

invokes the analogy of his results with those obtained in the case of

the immunity and susceptibility of frogs as regards a septicaemic

bacillus.

This bacillus (_Bacillus ranicida_) has been made the subject of an

interesting study by Ernst[195]. It is a small, very slender bacillus,

which, in frogs, produces a fatal malady epidemic in spring, but ceasing

completely during summer. Taking this fact as a basis, Ernst has

succeeded in conferring immunity upon frogs in autumn by placing them in

an incubator at 25° C. In spite of the injection of a considerable dose

of the small bacillus, the frogs living at this temperature remained in

good health, whilst control animals exposed to a low temperature died of

septicaemia. The counter-test was made in summer. Inoculated frogs that

were kept in the laboratory were unaffected, whilst those that had been

kept in a refrigerating apparatus at 6°–10° C. invariably died. It may

be asked, Is this evident influence of temperature on immunity and

receptivity exercised on the organism of the frog or upon the pathogenic

bacillus? In the case where a bacillus can only develop at low

temperatures its harmlessness at the higher temperature may be readily

understood. The experiments of Ernst have demonstrated, however, that

this small bacillus develops much better at 22° C., and even at 30° C.,

than at lower temperatures. It must be concluded, therefore, that the

high temperature which confers immunity acts not by weakening the

bacillus, but rather by reinforcing the resisting power of the organism.

The low temperatures (6°–10° C.) that are favourable to a fatal

infection have a different action; that is to say, they weaken the

reaction of the inoculated frogs.

[Sidenote: [150]]

Although Ernst has not studied the mechanism of this resistance fully,

it is evident, from the data he has supplied, that it consists in a

phagocytic reaction. He was able to demonstrate the ingestion of the

bacilli by the phagocytes in the susceptible refrigerated frogs, as well

as in the refractory frogs, kept at a higher temperature; but in the

former case the phagocytosis was so feeble that 24 hours after

inoculation a considerable number of free bacilli were still found in

the lymph of the dorsal sac, whilst in the refractory frogs the much

more active phagocytosis brought about the disappearance of the free

bacilli during the first day. If, as is very probable, the analogy of

this septicaemia with anthrax in frogs, upon which Ernst insists, really

exists, it must be concluded that the susceptibility of these

Batrachians to the modified race of the bacillus depends on their weak

phagocytic resistance.

Since, in these two examples of natural immunity in the frog, we have

seen that the phagocytic activity exhibits itself in an active form

against bacteria which readily develop in the fluids of the same animal,

we might conclude that the reaction of the phagocytes constitutes a

general mode of defence in cold-blooded animals. But Lubarsch[196], a

very cautious observer, has expressed an opposite view, based on his

studies on the bacillus of mouse septicaemia. He convinced himself that

frogs will resist injections of even considerable quantities of this

bacillus, without any co-operation on the part of the phagocytes. As we

have, here, to do with a matter of fact, Mesnil (_l.c._) set himself to

verify these observations, with the object of establishing whether it

was a case of a real exception or of a simple misunderstanding. He was

able to demonstrate, by irrefutable observations and experiments, that

the bacilli of mouse septicaemia when inoculated into frogs, set up a

very pronounced positive chemiotaxis on the part of the phagocytes,

which seized and digested the bacilli just as they do the anthrax

bacillus. This apparent exception, therefore, becomes transformed into

an additional argument in favour of phagocytic reaction being a general

factor in immunity. In support of this hypothesis I may adduce a further

example, already mentioned in a preceding chapter when discussing

another question. The frog is very refractory against the cholera

vibrio. When these vibrios are inoculated into the dorsal lymphatic sac

or into any other part of the body the animal retains its health

unimpaired. An examination of the exudation at the point of inoculation

demonstrates that the vibrios meet with a vigorous opposition on the

part of the phagocytes, which ingest and completely digest them. This is

of special interest from the fact that the frog is very sensitive to the

toxin of the cholera vibrio. When injected in a weak dose it kills the

frog very quickly. Two small frogs died in less than an hour from the

effect of 0·5 c.c. of cholera toxin.

[Sidenote: [151]]

The natural immunity of the frog against the cholera vibrio affords,

then, an example in which the organism, destroying the vibrio by

phagocytosis, prevents the production of the poison, which, otherwise,

would infallibly kill it.

Having demonstrated that phagocytic reaction manifests itself in the

frog in all cases of natural immunity that have been sufficiently

studied, we must dwell for an instant on the question of the condition

of the bacteria at the moment of their ingestion by the phagocytes. It

is very evident that this phagocytic defence is only efficient on

condition that it is exercised against bacteria which, in its absence,

might injure the organism by their multiplication and their virulence.

For this reason the question as to whether the micro-organisms, before

being ingested, were living and capable of producing their pathogenic

action has been widely discussed. It has even been suggested that the

phagocytes are only capable of ingesting the dead bodies of

micro-organisms that have been killed by other agents. Frogs are very

suitable for a study of this question. When a drop of the exudation is

removed some time after inoculation with a motile organism, such as the

_Bacillus pyocyaneus_ or the cholera vibrio, the organism was often

found moving rapidly within the vacuoles inside leucocytes. The

experiment will succeed even more completely if a drop of frog’s lymph

be mixed, on a slide, with a trace of a culture of these motile

micro-organisms, the latter being soon found in the clear vacuoles

included in leucocytes and executing extremely rapid movements.

Besides this direct proof we can assure ourselves of the living

condition of the micro-organisms in another way. Withdraw a drop of the

exudation at an advanced stage of the process when there are no longer

any free micro-organisms; inside the phagocytes a few scattered

bacteria, more or less well preserved, can still be seen. It is

sufficient to keep a hanging drop of such an exudation at a temperature

of about 30° C., care being taken to keep it from drying, but without

adding to it any nutrient medium. Under these conditions the leucocytes

die more or less rapidly, but the bacteria regain vigour: they begin to

multiply, and at the end of a short time produce a generation of

bacteria within the dead leucocyte. The multiplication of the bacteria

goes on progressively and the hanging drop is transformed into a real

pure culture. Mesnil was able to confirm these data with the exudations

of frogs that had been inoculated with either the bacilli of anthrax or

of mouse septicaemia.

[Sidenote: [152]]

The bacteria, ingested in the living state by phagocytes, retain their

original virulence. Some authors think, and I was formerly of this

opinion, that at the end of a more or less prolonged sojourn within the

leucocytes, anthrax bacilli undergo an attenuation in their virulence.

Later, numerous researches have, however, demonstrated that this opinion

is incorrect, and that the virulence is maintained in the bacteria

included in the phagocytes of frogs the whole time that these bacteria

remain alive. Dieudonné has insisted on this fact as regards the anthrax

bacillus. Mesnil has confirmed it for this same species and for the

bacillus of mouse septicaemia. It is impossible, therefore, to doubt

this general result, that frogs which are refractory against certain

bacteria resist because of the phagocytosis which is exercised against

living and virulent micro-organisms.

We have insisted sufficiently on the analysis of the natural immunity of

the frog, and need not tarry over the facts relating to other amphibia

which, moreover, have been much less studied. The reptiles, those higher

representatives of the Vertebrata called cold-blooded, often present

examples of really remarkable immunity. Thus alligators will resist

enormous doses of various bacteria, such as the anthrax bacillus, that

of human tuberculosis or the cocco-bacillus of typhoid fever. When, some

time after an injection is made, the exudation at the point of

inoculation is withdrawn there is found a large number of leucocytes,

amongst which may be recognised many eosinophile microphages, though the

majority are macrophages with one, two or more nuclei. Really giant

cells are found in the exudation. It is the macrophages which specially

manifest phagocytosis and they are often found crammed with the injected

bacteria, as I was able to assure myself after injections of typhoid

cocco-bacilli. The natural immunity of alligators (_Alligator

mississipiensis_) persists not only at the temperature of the incubator

(37° C.), but also at room temperature (20°–22° C.).

[Sidenote: [153]]

Passing in review the animal kingdom we must pause for a moment to

consider the natural immunity of birds or lower warm-blooded

Vertebrates. The classic example of this immunity is that of the fowl

against anthrax. It has long been known that birds resist inoculation

with anthrax or only exhibit a feeble receptivity; though smaller birds

are for the most part susceptible to anthrax, the pigeon is much less so

and the fowl presents a case of the most pronounced immunity. It was

believed to be absolutely refractory until the experiments of Pasteur

and Joubert[197], who found a sure method of suppressing this immunity.

Fowls that had been inoculated with the bacillus were immersed up to the

thighs in cold water in order to bring down their temperature. It was

found that, under these conditions, the anthrax bacillus developed at

the seat of inoculation and later became generalised in the blood, and

invariably caused death. It was concluded from this that the natural

immunity of the fowl was dependent on its very high normal temperature

(41°–42°) which interfered with the pathogenic functions of the anthrax

bacillus.

Hess[198] studied the mechanism of this immunity of the fowl and pointed

out the important part that phagocytosis plays in the destruction of the

inoculated bacteria.

These researches were resumed in my laboratory by Wagner[199]. Having

established that the anthrax bacillus develops readily in the blood and

the blood serum of fowls, outside the organism, at high temperatures

(42°–43° C.), he came to the conclusion that the lowering of the

temperature of the body of the fowls by immersing them in water

produced, not a reinforcement of the bacillus, but a weakening of the

resisting power of the animal. He was able to convince himself that this

resistance exhibits itself in the activity of the phagocytes which

ingest and destroy the anthrax bacillus in its vegetative state. In the

normal fowl the phagocytosis is rapid and very pronounced, whilst in a

fowl that has been refrigerated this reaction is very slight or absent.

To corroborate this general conclusion, Wagner, instead of lowering the

temperature by means of cold water, made use of antipyrin and chloral.

The application of this treatment likewise caused enfeeblement of the

natural defence of the organism and suppressed the immunity of the fowl

against anthrax.

[Sidenote: [154]]

Trapeznikoff[200] has studied carefully the fate of anthrax spores when

injected into fowls. He observed that most of them are devoured by the

leucocytes. Some of the spores were first transformed into small rods,

sometimes growing into real bacilli, but finally they all became the

prey of phagocytes and perished in their interior. Those in the

vegetative condition are soon digested, the spores, however, persist for

some time inside the phagocytes, but ultimately disappear. The

phagocytosis in fowls inoculated with spores is very marked, and

preparations, stained by Ziehl’s method, demonstrate most clearly the

reality of this reaction phenomenon. These preparations have for long

been used in the course in bacteriology at the Pasteur Institute for the

demonstration of phagocytosis.

[Sidenote: [155]]

In the face of these facts, well established and confirmed many times,

it is impossible to accept Thiltges’[201] denial of the ingestion of

these bacteria by the phagocytes of the fowl. Some fault of technique,

which I am not at the moment in a position to indicate exactly, has

evidently slipped into this author’s work. The positive data, however,

on phagocytosis in the fowl, obtained by Hess, Wagner, and Trapeznikoff,

data confirmed by myself, render unnecessary any fresh researches for

the purpose of explaining the negative results obtained by Thiltges. As

regards his experiments on the bactericidal action of defibrinated blood

and of blood serum of fowls against the bacillus and its spores,

experiments whose results are opposed by those of Wagner, the

contradiction may be explained pretty easily, at least in part. Thiltges

mentions several times that the bacilli, when sown in the blood serum of

the fowl, were aggregated in clumps. Nevertheless, he has failed to

guard against this source of error and has attributed the diminution in

number of the colonies on plates to the destruction and not to the

agglutination of the bacilli. Thiltges gives so few particulars of the

conditions under which his experiments were performed that we do not

even know at what temperature he kept his tubes containing blood and

serum sown with bacilli. As Wagner kept his at 42°–43° C., a temperature

which corresponds to that of the body of the fowl, I asked M. Gengou to

make a series of experiments on the bactericidal power of the plasma and

blood serum of fowls on the anthrax bacillus, keeping his tubes at 37°

C. The result of his experiments was in complete accord with those of

Wagner. Under the conditions that I have just stated the fluids of the

fowl are no more bactericidal than they are under the conditions

maintained in Wagner’s experiments.

In summing up these data on the natural immunity of fowls against

anthrax, we are certainly justified in concluding that it is due to the

phagocytosis and not to any bactericidal property of the “humours.”

The pigeon is more susceptible than the fowl to the action of the

anthrax bacillus, still it manifests a certain degree of resistance

against the microbe. After what we have said on the subject of the fowl

we need make but few remarks on the pigeon, in spite of the very

animated discussions that have taken place on the mechanism of its

immunity. When Baumgarten was offering a systematic opposition to the

part played by phagocytic reaction in immunity, he set his pupil

Czaplewski[202] to investigate the resistance of pigeons against

anthrax. The results of this investigation were absolutely negative as

regards phagocytosis. The latter was said to have no importance in the

defence of the organism, which resisted simply because it was impossible

for the bacillus to live in the body of the pigeon. I then set myself to

study this question[203], and I was able to demonstrate that the anthrax

bacillus is quite capable of keeping alive in the pigeon, that it can

develop in its fluids, but that it is unable to defend itself against

the aggression of the phagocytes which ingest it and completely digest

it. By isolating the phagocytes that had ingested the bacilli injected

into the body of the pigeon, I was able to prove that a number of these

bacilli were still alive. The enfeeblement and death of the phagocytes

when outside the body allowed the anthrax bacilli again to get the upper

hand in this struggle, to develop and to give virulent cultures. The

part played by phagocytes in this example of natural immunity was thus

placed beyond doubt.

[Sidenote: [156]]

Later, Czaplewski[204] himself became convinced that his previous

negative results would not stand criticism, and Thiltges, in his work

already mentioned, when discussing the fowl, was able to confirm the

importance of phagocytosis in the defence of the organism of the pigeon

against anthrax. He was struck by the difference between these two

species of birds. In the pigeon it was easy for him to prove that in the

individuals that succumb to anthrax the phagocytic reaction is very

feeble, whilst in those which ultimately resist the bacillus it is very

pronounced. Thiltges likewise observed that the blood and blood serum of

pigeons when sown _in vitro_ with the anthrax bacillus, manifest only an

insignificant bactericidal power, a fact that further warrants him in

attributing great importance to phagocytosis in the maintenance of the

natural immunity of the pigeon. It is remarkable that, in presence of

these facts, it did not occur to the author to ask whether this

fundamental difference in the mechanism of the resistance, which he

thought possible in two birds so closely allied as are the pigeon and

the fowl, really did exist in nature. I infer that his experiments on

the fowl were made before those on the pigeon, and that the difference

in his results depended specially on the fact that he had acquired

greater skill in executing his later experiments.

Having observed that frogs die readily when inoculated with an anthrax

bacillus that was adapted to develop at a low temperature, Dieudonné

(_l.c._) endeavoured to suppress the immunity of the pigeon by using

bacilli adapted to a high temperature. But the inoculation of a second

generation of the anthrax bacillus, cultivated at 42° C., was borne by

five pigeons without inconvenience. Even bacilli that were rendered

capable, by cultivation through sixteen generations, of developing at

this temperature were not in a condition to kill more than five pigeons

out of thirteen inoculated. These attempts to explain immunity as due to

the properties of the bacilli rather than to those of the organism of

the pigeon, have therefore led to a result very different from that

anticipated by Dieudonné.

[Sidenote: [157]]

The pigeon is further of interest to us because of its natural immunity

against the bacillus of human tuberculosis. It resists considerable

doses of this bacillus, so virulent for man and for the majority of

mammals, and even for some birds (canaries and parrots). Dembinski[205],

studying the mechanism of this immunity, was able to prove that the

bacilli of human tuberculosis encounter in the organism of the pigeon a

very great resistance from the phagocytes, especially from the

macrophages. These cells fuse together around masses of bacilli and

imprison them within real giant cells or polynucleated macrophages (Fig.

21). The microphages in this struggle play only a secondary part, but

the resistance offered by the macrophages is a most effective one.

Incapable of completely destroying the bacilli, these phagocytes

exercise over them an unfavourable influence and prevent them from

multiplying and exhibiting their noxious action. The importance of the

defence by the macrophages comes out still more clearly when compared

with what takes place if, instead of the bacillus of human tuberculosis,

we inoculate into pigeons the bacillus of avian tuberculosis. In the

latter case the microphages certainly promptly seize the bacilli, but

being powerless against them they perish, whilst the macrophages only

intervene later on and in small numbers. The result is that in the

pigeon the avian bacillus becomes generalised in the organism and sets

up a fatal tuberculosis.

[Illustration:

FIG. 21. Reaction of the phagocytes of the pigeon against the bacilli

of human tuberculosis.

]

It must be admitted, then, that the immunity of the pigeon against the

bacillus of human tuberculosis is due to the defence by the macrophages.

This conclusion is corroborated by the fact that in the fowl—equally

refractory against the same bacillus—there is also observed a very

strong macrophagic reaction.

[Sidenote: [158]]

Nocard[206], who for several years has been carrying on studies on the

relations between the bacilli of human and avian tuberculosis, conceived

the idea of adapting the former to the organism of the fowl. With this

object he enclosed a culture of the bacillus of human tuberculosis in a

sac of collodion which he then introduced into the peritoneal cavity of

fowls. Under these conditions the bacillus, protected against the

aggression of phagocytes, continued to live inside the sac through whose

walls the fluid part of the peritoneal lymph could diffuse. After

several passages from sac to sac the human bacillus becomes acclimatised

to the body of the fowl and is transformed into a variety quite

comparable to the bacillus of avian tuberculosis. This experiment has

definitely settled the question so long under discussion of the specific

difference between the two tubercle bacilli. It has resolved it in the

sense of affirming their unity; the avian bacillus is only a modified

race of the same bacillus which sets up tuberculosis in man and other

mammals.

In spite of the great difference between the anthrax bacillus and that

of human tuberculosis, the immunity against these two bacteria, which is

shown in birds, depends in every case upon the reaction of the

phagocytic system.

Having rapidly glanced at natural immunity as we ascend the scale of the

animal series we now come to it as it presents itself in the highest

class, Mammals, a question on which it is necessary to dwell at greater

length because of its great importance, and also because of the fuller

study that has been given to it.

[Sidenote: [159]]

As the immunity of the Invertebrata and of the lower Vertebrata against

the anthrax bacillus has furnished us with several important indications

we will first endeavour to throw light on the mechanism of the

resistance offered to anthrax by certain mammals. The representatives of

this class being, however, for the most part extremely susceptible to

this disease, examples of true natural immunity are very rare. The first

place among resistant mammals is occupied by the dog. Although young

dogs, as demonstrated by Strauss[207], readily take fatal anthrax, the

canine species may nevertheless be regarded as possessing a real

immunity, as adult dogs withstand, without inconvenience, the

inoculation of large quantities of bacilli. When introduced beneath the

skin these bacilli excite a local inflammation, accompanied by a very

marked diapedesis of white corpuscles which at once begin to devour the

bacilli. This phagocytosis has already been observed by Hess[208],

Malm[209], myself, and several other investigators, so that its

existence cannot be doubted. Recently, Martel[210] has demonstrated a

very distinct phagocytic reaction in all those cases where he has had to

deal with dogs that were refractory or not very susceptible. This

reaction is shown by the ingestion of the bacteria and by the large

accumulation of leucocytes at the seat of inoculation. His researches

are of special interest by reason of the counter-test that he was able

to make upon dogs that were susceptible to anthrax. It was demonstrated

some years ago that the natural immunity of the dog against the

bacillus, although very real, is, nevertheless, relative and limited.

Thus Bardach[211] established the fact that dogs from whom the spleen,

an organ full of phagocytes, had been removed, became susceptible to

anthrax. Even dogs into whose veins he injected fine wood-charcoal

powder suspended in water, with the purpose of “diverting” the

phagocytosis, readily succumbed to anthrax.

Martel endeavoured to suspend the natural immunity of dogs by injecting

into them phloridzin or pyrogallic acid. But he obtained much more

constant results by inoculating the bacillus into rabid dogs. The

organism, weakened by this terrible disease, became very susceptible to

anthrax, and the rabid animal succumbed to anthrax before the rabies had

completed its evolution. By its passage through the rabid dog the

anthrax virus is so augmented in virulence that it becomes fatal for

normal dogs. Martel succeeded also in reinforcing the bacillus isolated

from a cow affected with anthrax. In all these cases where the

reinforced bacilli set up a severe and rapidly fatal infection, Martel

could demonstrate only a feeble phagocytic reaction.

Researches on the phagocytosis of dogs, inoculated with the anthrax

bacillus, have always demonstrated a regular and constant relation

between this reaction and the resistance of the organism. On the other

hand, experiments undertaken for the purpose of establishing the part

played by the body fluids in this immunity, have always given negative

results.

[Sidenote: [160]]

As the dog, of all mammals, exhibits the greatest natural immunity from

anthrax, it is very natural that in the bactericidal property of its

blood the key to the enigma has been sought. Thus Nuttall[212] concludes

from his experiments that the anthrax bacillus is readily destroyed by

defibrinated dog’s blood. But, as this result was not in accord with my

observations[213] that the bacillus is easily cultivated in dog’s blood,

and as several observers, especially Lubarsch[214], had arrived at

conclusions opposed to those of Nuttall, systematic researches were made

for the purpose of solving this complicated problem. Denys and

Kaisin[215] sought to remove the objections formulated against the

explanation of the immunity of the dog as due to the bactericidal

property of its blood by affirming that this power, which is absent in

the inoculated dog, develops whilst the animal is under the influence of

the bacillus. Immunity is reduced, then, in this case to the

establishment of a new property in the fluids during the course of the

struggle of the organism against the inoculated bacillus. None of the

observers, however, who have repeated these experiments, _e.g._

Lubarsch[216] and Bail[217] were able to confirm the results of the

Belgian observers. Denys himself, indeed, having resumed this study with

Havet[218], had to reject the conclusions of his former work executed in

collaboration with Kaisin. He is persuaded that their error was due to

the fact that in their experiments _in vitro_, the living leucocytes

ingested the bacilli and prevented their development. As the result of

these new researches Denys and Havet have come to the conclusion “that

the main, the predominating part of the bactericidal power of the dog’s

blood must be ascribed to the leucocytes acting as phagocytic elements”

(_loc. cit._ p. 15).

[Sidenote: [161]]

As a result of the investigations I have summarised the conclusion is

forced upon us that the natural immunity of the dog from anthrax is a

function of the phagocytes. In presence of this uniformity of the

experimental results it becomes very important to make a more profound

study of the phenomena that manifest themselves during the destruction

of the bacilli by the phagocytes of the dog. What are the phagocytic

elements which play the principal part in this struggle, and by what

means do they attain this result? Gengou[219] undertook a detailed

investigation in my laboratory to answer these questions. He was able to

convince himself, in agreement with the statements of his predecessors,

that not only was the serum of dog’s blood not bactericidal for the

anthrax bacillus, but that the plasma of the blood is no more so. The

fluid of the aseptic pleural exudation obtained after injection of

gluten-casein, was likewise incapable of killing the anthrax bacillus.

When Gengou, by means of centrifugalisation, isolated the leucocytes

from these exudations, washed them in physiological salt solution, froze

them, and then macerated them in broth, he obtained suspensions of white

corpuscles, to which he added bacilli. He was able to demonstrate that

when the exudations contained macrophages principally, as is observed in

exudations taken at the end of two or three days, the bactericidal power

of the suspensions was _nil_ or insignificant. When, on the other hand,

the leucocytes came from exudations only twenty-four hours old and were

composed almost exclusively of microphages, the destructive action on

the bacilli of the extract of the microphages in broth was most marked.

Now it is fully demonstrated that in the exudation set up in the

refractory dog by the injection of anthrax bacilli, it is especially the

microphages which exhibit the phagocytic reaction against this bacillus.

This is how the question of the immunity of the dog from anthrax stands

at present. The natural immunity of this species, which although not

unlimited, is very real, depends on the activity of phagocytes. These

elements, under the stimulus of the bacillus and its products, exhibit a

positive chemiotaxis of the most marked character, they approach the

bacilli, ingest them by a physiological act, and destroy them by means

of a substance which is not found in either the plasma or the blood

serum, but which can be demonstrated in an extract of the microphages.

[Sidenote: [162]]

In spite of the uniformity and precision of these data, it is impossible

to rest satisfied with describing, as an example of natural immunity

from anthrax, the single case of the dog. If the resistance of the rat

against this disease was merely of historical interest because of the

large number of works devoted to this question, we might relegate it to

the chapter reserved for the history of our knowledge on immunity. But

it is not so. The anthrax of rats is a subject full of very valuable

instruction, and von Behring was quite justified in saying that whoever

wished to get a true conception of natural immunity from a virus should

pay special attention to this example.

As a matter of fact, it may be stated that the grey rat (_Mus

decumanus_), the black rat (_Mus rattus_), and white rats are far from

enjoying a true immunity from anthrax. They, nevertheless, exhibit a

more or less marked resistance against this disease and are always less

susceptible than are the other laboratory rodents: mice, guinea-pigs and

rabbits. Rats resist attenuated bacilli (anthrax vaccines) better than

do these three species, and in order to induce in them fatal anthrax it

is necessary to inoculate a much larger number of virulent bacilli. On

the other hand, rats are distinguished by a great irregularity in the

resistance they offer to the bacillus. At times they resist very

virulent bacilli; at others they contract a fatal disease after an

injection of very attenuated bacilli (Pasteur’s first vaccine).

[Sidenote: [163]]

In my first memoir on anthrax[220] I noted the fact that in rats the

phagocytosis against the bacillus when injected subcutaneously was more

marked than after the same inoculation into the rabbit and guinea-pig.

Later, this fact was disputed by several observers, who refused to

accept the extent and importance of the phagocytic reaction in the rat.

This opposition was strengthened by a very interesting discovery made by

von Behring[221], namely, that the blood serum of the rat possessed a

remarkably destructive power for the anthrax bacillus. When this

observer added a certain quantity of anthrax bacilli to some blood serum

of the rat, instead of elongating into filaments and dividing they

underwent a change, lost their normal refraction and took on staining

reagents very imperfectly. The membranes alone remained of the bacilli

thus treated. Von Behring considered that this bactericidal action of

the serum depends on the presence of an organic base dissolved in the

blood fluid. He had merely to neutralise the serum by means of an acid,

and there was at once a very abundant development of the bacillus. From

these researches von Behring came to the conclusion that the natural

immunity of the rat from anthrax can be reduced to terms of the chemical

action of the blood on the bacillus.

In one of his most recent publications this author[222] returns to the

question of anthrax in rats and sums up his present point of view as

follows. He regards the immunity of these rodents as being relative, not

absolute. “The anthrax bacilli”—he says—“die in rat’s serum _in vitro_;

and in the cases where the inoculation of these animals with the anthrax

virus is not fatal, it is at least reasonable to assume that the blood

fluid likewise produces this protection in the organism of the living

rat. Now, an immunity that manifests itself without the aid of any

activity of the cell must undoubtedly be regarded as being of a humoral

character” (_loc. cit._ p. 202).

[Sidenote: [164]]

Let us begin by analysing the facts as presented in rats into whose

subcutaneous tissue we have injected anthrax virus. A certain number of

them resist, without exhibiting any lesion other than a certain

exudative inflammation at the seat of inoculation. The exudation is, in

this case, very rich in leucocytes which quickly exert their phagocytic

function and destroy the ingested bacilli. In this reaction it is the

microphages that play the chief part, the macrophages intervening later

and in a much less pronounced fashion. Usually, however, the inoculated

rats exhibit a more serious illness: the bacilli multiply at the point

of inoculation and excite the formation of an extensive oedema, rich in

serous fluid, transparent, and very poor in leucocytes. It is only later

that these cells intervene in any considerable number. The exudation

becomes thicker and turbid, the numerous white corpuscles devour the

bacilli and cause their disappearance. Under the influence of this

marked reaction the animals in most cases recover, as has already been

established by Frank[223]. But even in those individuals which succumb

to anthrax death occurs more or less tardily, an examination of the

internal organs then revealing a considerable phagocytic reaction. The

spleen, often of enormous size, contains numerous macrophages which are

filled with normal or more or less altered bacilli. In the liver these

macrophages, which have devoured several microphages and some bacteria,

are also found (Figs. 22 and 23).

When instead of bacteria in the condition of rods, anthrax spores are

inoculated subcutaneously or into the anterior chamber of the eye, we

can observe their germination. There is developed a whole generation of

bacilli which behave like those we have already described, that is to

say, they excite an exudation and are ultimately digested within the

phagocytes (Figs. 24 and 25). All these phenomena of phagocytosis I

described in detail more than ten years ago in my memoir on the anthrax

of rats[224]. Since then not a single fact has been brought forward to

invalidate the results there set forth.

[Illustration:

FIG. 22. Macrophage from the liver of a rat affected with anthrax.

]

[Illustration:

FIG. 23. Macrophage containing bacilli, from the liver of a rat

affected with anthrax.

]

[Illustration:

FIG. 24. Microphage of rat filled with bacilli.

]

[Illustration:

FIG. 25. Two microphages of rat that have ingested bacilli.

]

[Sidenote: [165]]

How is this paradoxical fact to be explained, that anthrax which grows

in the body of the rat, there setting up a disease more or less grave

and sometimes fatal, is so readily destroyed by the serum and blood when

removed from the organism? From numerous experiments, carried out by

Hankin[225] and by Roux and myself[226], it has been demonstrated that

the bactericidal power of the fluids of the rat cannot be invoked as the

cause of the animal’s resistance to anthrax. Those rats which show

themselves very susceptible to this disease and die from anthrax

infection, furnish, nevertheless, a serum that will prevent anthrax in

other rats, and which will protect even mice into which the bacilli have

been injected. Rats into which we inoculate on one side of the body a

little anthrax culture, and on the other side the same quantity of

bacilli mixed with blood serum from the same animal, manifest oedema at

the former place only. It is from this latter point that the general

infection takes place, the side where the anthrax bacilli mixed with

serum was introduced remaining unaffected. Sawtchenko[227], who has

investigated the immunity of the rat in my laboratory, has to the facts

just mentioned added the observation that when the injection of bacilli

causes haemorrhage the rat survives. When, on the contrary, the

injection is made with a fine needle and without effusion of blood, the

rat contracts a fatal anthrax.

[Sidenote: [166]]

[Sidenote: [167]]

It follows from these facts that the blood, immediately it has escaped

from the vessels, undergoes a change in its composition and becomes

bactericidal for the anthrax bacillus, whilst, when it is circulating in

the organism, it exhibits no such power. Sawtchenko has studied the

substance in the serum which kills the bacilli and has demonstrated that

it will resist heating to 56° C.; even when heated to 61° C. the serum

still exercises a certain amount of bactericidal power for very

attenuated bacilli (Pasteur’s first vaccine). Researches on the

distribution of this bactericidal power in the living rat have convinced

Sawtchenko that none of it passes into the fluid of the passive oedema

set up by the slowing of the circulation, nor into that of the active

oedema developed as the result of the inoculation of anthrax bacilli. He

observed that even the bacillus of Pasteur’s first vaccine grows

abundantly in the oedematous fluid produced by the injection of virulent

bacilli. The peritoneal lymph, however, exerts a very marked

bactericidal action on the bacilli. Having demonstrated this fact

Sawtchenko put to himself the question: May not the great difference

between the action of these fluids depend on the fact that the lymph is

rich in leucocytes, whilst in the fluid of the oedema they are almost

absent? Pursuing this question, Sawtchenko made a comparative study of

the bactericidal power of the serum, prepared outside the body, and of

the blood plasma obtained by means of an extract of the heads of

leeches, and he concluded from his researches that the bactericidal

substance circulates in the plasma of the living rat and that it is not

derived from the microphages, but must be looked upon rather as a

secretion of the macrophages in the blood and of endothelial cells. This

result was not confirmed by Gengou[228], who also took up the study of

this important question in my laboratory. Instead of preparing the

plasma by means of the addition of an extract of leeches he made use of

a method much more perfect and free from sources of error. He introduced

no foreign substance capable of affecting the results of his

experiments. Collecting the rat’s blood in paraffined tubes, and

centrifugalising it in similar tubes, he obtained a fluid which

approaches much more closely the plasma of circulating blood than does

serum. This fluid, however, will coagulate at the end of a fairly long

interval, which proves that it cannot be looked upon as blood plasma.

Gengou examined the bactericidal power of the fluid portion of the

“plasma,” obtained by the process just described, on the anthrax

bacillus, and also that of serum prepared in tubes in the ordinary way.

The difference between the two fluids is very marked; whilst the serum

destroys the bacilli sown in it very rapidly and dissolves their

contents, the fluid of the “plasma” has no similar action. These

results, confirmed several times, demonstrate very definitely that the

plasma of the circulating blood does not contain any bactericidal

substance. This, during the life of the animal, is found inside

leucocytes and only escapes from them when the cells burst or undergo

profound lesions, this taking place when the clot is formed and when the

serum is prepared outside the organism, or in the effused and coagulated

blood, or again in the peritoneal lymph during phagolysis. This

phagolysis is inevitably produced as a result of rapid injection of

foreign fluids into the peritoneal cavity, _e.g._ of broth or of

physiological salt solution, containing bacteria in suspension.

[Sidenote: [168]]

The facts we have brought together on the subject of anthrax in rats

form a whole whose several parts are in complete harmony. The phagocytes

of this species of rodent contain a bactericidal ferment, a kind of

cytase, which resists temperatures approaching 60° C. This cytase is

very active against the bacilli, but in the living animal it can only

act within the phagocytes, or, in a transitory and incomplete fashion,

outside these cells, when phagolysis is taking place in the peritoneal

cavity. The resistance offered by the rat to anthrax depends, then, on

this phagocytic activity. For its manifestation it is necessary, first

of all, that the phagocytes should manifest a positive chemiotaxis for

the bacilli, and then that they should seize and ingest these organisms.

These are the vital acts that decide the result of the struggle. When

the phagocytes show themselves inactive the bacilli multiply in the

oedematous fluid which contains no bactericidal cytase, and pass into

the plasmas of the lymph and of the blood, which also are incapable of

killing these bacteria. The animal may, then, die of anthrax, in spite

of the presence in its body of a large quantity of bactericidal cytase

which is to be found in situations to which the bacilli have not

penetrated. In those cases, on the other hand, where the phagocytes

accomplish their function, where they rush up to the menaced point and

devour the inoculated bacteria, these bacilli are placed in contact with

the intracellular cytase and undergo complete digestion. The organism in

this case gets rid of its enemies and victoriously resists infection.

[Sidenote: [169]]

Anthrax in rats, then, presents one of the most instructive examples of

natural immunity. But the detailed analysis of the mechanism of this

resistance demonstrates very clearly the great part played by the

phagocytes in this process. In this respect the organism of the rat

presents, in a general fashion, a great analogy to the natural immunity

of the dog, of birds, and of other representatives of the animal kingdom

that we have examined. Under these conditions it is useless to insist at

any length on other examples of resistance against anthrax which,

moreover, have relation much more often to a natural immunity against

attenuated bacilli than to one against true anthrax virus. Rabbits and

guinea-pigs, so sensitive to this virus, often resist the inoculation of

Pasteur’s vaccines. The rabbit is, in general, refractory to the first

anthrax vaccine; it may even resist the second vaccine. The guinea-pig,

a more sensitive animal, does not exhibit any natural immunity except

against the first vaccine. In all these cases the mechanism is similar

to that which the rat and the dog oppose to virulent anthrax. The

bacilli, into whatever part of the body they are injected, set up an

exudative inflammation which brings up a large number of leucocytes to

the point menaced. These cells readily exert their phagocytic function

and rid the organism of the introduced bacteria. In order to obtain a

complete grasp of the part played by this reaction it will be found

useful to inject beneath the skin of one ear of a rabbit a little

anthrax vaccine and beneath the skin of the other the same quantity of

virulent bacilli. The difference between the reaction in the two cases

is very striking. The ear inoculated with vaccine soon becomes the seat

of a circumscribed inflammation with a purulent exudation, all the

bacilli in which have been devoured by the leucocytes. The other ear, on

the contrary, presents, around the injected virus, only a serous or

blood-tinged exudation containing no, or few, leucocytes; the bacilli

are found free in the liquid and multiply without let or hindrance.

Meeting with no opposition the virus becomes generalised throughout the

organism and brings on death by anthrax septicaemia. Rabbits, into which

anthrax vaccines only are introduced, oppose to the invasion of the

bacilli a leucocytic barrier which arrests their extension. The natural

immunity of the sheep, rabbit and guinea-pig is also a phagocytic

immunity, but it is only capable of being exercised against bacilli

previously attenuated in virulence. The researches of Mme

Metchnikoff[229] on the reaction of the phagocytes of these animals to

the bacilli of Pasteur’s two anthrax vaccines have demonstrated the

importance of the destruction of these bacilli by the leucocytes. All

the other examples of natural immunity against anthrax are also merely

relative. The fowl that resists an anthrax virus strong enough to kill

an ox or a horse, succumbs to a special variety of anthrax cultivated by

Levin[230]. The dog, as we have seen, in spite of its pronounced natural

immunity against anthrax, is killed by the special anthrax bacillus

prepared by Martel.

In this immunity against anthrax we have to deal with a bacillus capable

of living and reproducing itself in extremely varied media. Hence the

reason, it may be said, that the bactericidal influence of the fluids is

so little pronounced in this case. To bring it into relief we must,

therefore, choose a bacterium less capable of adapting itself to the

chemical composition of various culture media. In this matter we cannot

do better than select pathogenic spirilla of extremely delicate nature

and analyse the mechanism of the natural immunity of certain species of

animals with respect to them. It must not be forgotten, however, that

here we are making use of representatives of an infinitely small

minority of pathogenic bacteria, the majority resembling the anthrax

bacillus in the facility with which they can be cultivated in all sorts

of nutritive media.

[Sidenote: [170]]

[Sidenote: [171]]

[Illustration:

FIG. 26.—Leucocyte of guinea-pig in the act of ingesting two spirilla.

]

[Illustration:

FIG. 27.—The same leucocyte, half-an-hour later.

]

[Illustration:

FIG. 28.—The same leucocyte, ten minutes later than Fig. 27.

]

[Illustration:

FIG. 29.—Leucocyte of guinea-pig in the act of ingesting a very active

spirillum.

]

[Illustration:

FIG. 30.—The same leucocyte, forty minutes later.

]

[Illustration:

FIG. 31.—The same leucocyte, half an hour later than Fig. 30.

]

The spirillum of recurrent fever of man (_Spirochaete obermeyeri_) was

the first pathogenic microbe found in an infective disease distinctly

human. Discovered a third of a century ago, it has passed through the

hands of the most skilful bacteriologists, who have tried all possible

methods of cultivating it outside the body. Koch himself tried to solve

the problem, but, in spite of his incomparable skill, did not succeed.

Later, Sakharoff[231], at Tiflis, discovered a spirillum very similar in

appearance which produced a fatal septicaemia in the goose. He, also,

tried to cultivate it, but in vain. His successors have not been more

fortunate in this respect. Here, then, are two micro-organisms, against

which natural immunity should be easily obtainable and in a fashion

quite other than that against anthrax. Nothing, indeed, is more frequent

than examples of very stable natural immunity against the spirilla of

Obermeyer and of Sakharoff. As I wished to obtain a clear idea of the

mechanism by which the guinea-pig resists injections of the spirillum of

goose septicaemia (_Spirochaete anserina_) I made injections of goose’s

blood, containing a quantity of these organisms, into the peritoneal

cavity of guinea-pigs. This injection, as usual, causes the

disappearance of most of the leucocytes, as the result of a very marked

phagolysis. We know that, under these conditions, the damaged leucocytes

allow a certain quantity of the bactericidal cytase to escape. In spite

of this the spirilla remain intact and exhibit very active movements in

the peritoneal exudation. This exudation, after a period of phagolysis,

which lasts for two or three hours, begins to be stocked again with

leucocytes which come up in increasing numbers, a fact that does not

prevent the spirilla moving about with great rapidity. Even seven hours

after the injection of goose’s blood we still find many extremely active

spirilla among a large number of recently migrated leucocytes, some of

which even at this stage contain red corpuscles of the blood of the

goose. It is not until later that the ingestion of these spirilla by the

leucocytes commences, the leucocytes at last damaging and completely

destroying them. This act of phagocytosis may be readily observed in

hanging drops of the peritoneal exudation of inoculated guinea-pigs. The

attention of the observer is drawn to certain macrophage leucocytes

which throw out one or two conical-looking processes (Figs. 26–28).

These pseudopodia attach themselves to spirilla which exhibit very

violent movements as though wishing to extricate themselves from the

grasp of the leucocyte. Sometimes the spirillum succeeds in escaping,

but usually it becomes surrounded by the protoplasm and sinks more and

more deeply into the substance of the leucocyte. Even when almost

surrounded the free part of the spirillum still continues to move (Figs.

29–31). These movements cease only after the complete ingestion of the

spirillum. Once inside the phagocyte the spirillum is digested and soon

becomes unrecognisable.

[Illustration:

FIG. 32.—Macrophage of guinea-pig filled with spirilla of recurrent

fever (after Sawtchenko).

]

[Illustration:

FIG. 33.—Macrophage of guinea-pig containing three _Spirochaete

obermeyeri_ (after Sawtchenko).

]

[Sidenote: [172]]

Recently, Sawtchenko[232] took advantage of an epidemic of recurrent

fever at Kazan to make similar investigations on the natural immunity of

the guinea-pig against Obermeyer’s spirillum. He observed that these

organisms, when injected into the peritoneal cavity, remained there,

alive, for 24 and even 30 hours, whilst these same spirilla, when kept

at 37° C. outside the organism in their natural medium, died at the end

of some (4–7) hours. The injection of human serum containing spirilla

into the peritoneal cavity of guinea-pigs set up a phagolysis succeeded

by a considerable afflux of leucocytes. In spite, however, of the

arrival of quite an army of these cells, the spirilla continued to move

rapidly; for a long time they evaded the phagocytes which, however, in

the end always ingested them. But it is only the macrophages which

fulfil their phagocytic function (Figs. 32 and 33); the microphages

obstinately exhibit an absolutely negative chemiotaxis. Now, as the

macrophages do not make their way into the peritoneal cavity until after

the microphages have appeared, it is easy to understand that

phagocytosis can only take place at a late period. Sawtchenko came to

the conclusion that “in the peritoneal cavity of animals naturally

refractory, the spirochaetes perish as the result of a slow phagocytosis

and not from the action of the bactericidal substances of the fluids.”

In conformity with this result this observer has often noted the

ingestion of living spirilla by the macrophages, in hanging drops of the

peritoneal exudation of inoculated guinea-pigs. The phenomenon

corresponds exactly to that described in connection with the spirillum

of the goose.

In spite of the great difference between the spirillum and the anthrax

bacillus from the point of view of their adaptation to surrounding

media, the general result is the same with both these microbes: animals

endowed with natural immunity get rid of them through the agency of

their phagocytes.

[Sidenote: [173]]

[Sidenote: [174]]

It would be impossible and even useless here to pass in review all the

cases of natural immunity against infective micro-organisms. We must

consequently limit ourselves to several examples which may have an

interesting bearing on the study of the problem as a whole. The

spirilla, whose history we have just recorded, remain in the peritoneal

fluid, without change of form, up to the moment when they are captured

by the macrophages. Let us see by what mechanism the natural immunity

against micro-organisms, characterised by a very special sensitiveness

to external influences and by a considerable change of shape, is

produced. The cholera vibrio and its allies best satisfy this postulate.

When they find themselves placed under unfavourable conditions, these

vibrios immediately become transformed into small spherical bodies which

are much more like cocci than vibrios. The cholera vibrio is pathogenic

for the laboratory rodents, especially for the guinea-pig, when a fairly

large quantity of a culture is injected into the peritoneal cavity.

Against smaller doses, however, the natural immunity is a most marked

one. If we take a race of the cholera vibrio of medium virulence, and

inject into the peritoneal cavity of guinea-pigs a sublethal dose of a

culture, the following phenomena may be observed[233]. The inoculated

vibrios move actively in the peritoneal fluid, from which almost all the

leucocytes have disappeared. There remain only a few lymphocytes which

appear to be indifferent to the influences that set up a real

phagolysis. But, little by little, fresh leucocytes come into the

exudation and engage in a struggle with the vibrios which, so long as

they are free, retain their curved form and complete motility. The

microphages, especially, swarm into the peritoneal cavity. Some of them

begin to ingest vibrios, but this phagocytosis is at first slight. Later

it becomes much more active. The microphages and macrophages seize

vibrios that are evidently living and uninjured, which, sometimes, may

be observed inside the vacuoles of the leucocytic contents exhibiting

very lively movements. Once ingested, however, many of the vibrios

become transformed into round granules. This change of shape is constant

when inside microphages, but is completely absent when inside

macrophages (Figs. 34 and 35). Finally, the phagocytosis becomes

complete, and the organism gets rid of the vibrios solely by means of

this reaction. Even seven hours after injection of the vibrios, when the

peritoneal fluid, crammed with leucocytes, has become thick and turbid,

there still remain a few scattered vibrios which always retain their

shape and their normal activity. A drop of this exudation, maintained at

38° C. outside the organism, gives, in a few hours, an abundant culture

of very active vibrios. It must, therefore, be concluded that the fluid

part of the exudation was powerless to destroy the vibrios or even to

render them motionless, whilst the living leucocytes have shown

themselves capable of ingesting and digesting them. The peritoneal

exudation, withdrawn at a period when it no longer contains any free

vibrios, still gives cultures of the organism for some time. Soon,

however, there comes a period when the inoculated exudation remains

sterile, this proving that the vibrios, ingested in a living state by

the phagocytes, have at length been killed by the microphages and

macrophages.

[Illustration:

FIG. 34.—Microphage of guinea-pig filled with cholera vibrios, the

majority of which are transformed into granules.

]

[Illustration:

FIG. 35.—Macrophage of guinea-pig filled with cholera vibrios not

transformed into granules.

]

[Sidenote: [175]]

When, instead of cholera vibrios of medium virulence, we take a variety

completely deprived of pathogenic activity, it is sometimes observed

that certain of these organisms, when injected into the peritoneal

cavity of the normal guinea-pig, become transformed into spherical

granules in the fluid of the exudation without any direct co-operation

of the phagocytes. This transformation into granules was first studied

by R. Pfeiffer[234] and hence has been termed Pfeiffer’s phenomenon. It

is of limited occurrence in natural immunity and is produced, as I have

been able to demonstrate, only under certain well defined conditions.

Pfeiffer’s phenomenon is observed in the peritoneal fluid. It commences

soon after the injection of the vibrios and takes place during the

period of phagolysis. In other parts of the body of the guinea-pig,

notably in the subcutaneous tissue and in the anterior chamber of the

eye, Pfeiffer’s phenomenon does not manifest itself; the animal, none

the less, resists the inoculation of the vibrios. Even in the peritoneal

cavity, moreover, it is easy to check the granular transformation of the

vibrios by means which prevent the production of phagolysis. When we

inject into the peritoneal cavity of a guinea-pig a foreign fluid,

capable of exciting the phagocytic action, e.g. veal broth,

physiological salt solution, urine, etc., we first excite a transitory

phagolysis. To this stage succeeds another in which the leucocytes

become very numerous and much more resistant than before. If we take

advantage of this period of leucocytic stimulation to inject vibrios

which have been attenuated as much as possible, we shall observe that

they soon become the prey of the peritoneal phagocytes, without

manifesting any sign whatever of Pfeiffer’s phenomenon.

It is evident, then, that this extracellular destruction of the vibrios,

sometimes observed in the peritoneal cavity, is really the work of the

microcytase that has escaped from the phagocytes during their period of

transient injury.

[Sidenote: [176]]

[Sidenote: [177]]

[Illustration:

FIG. 36.—Peritoneal exudation from guinea-pig showing free

streptococci and microphages that have ingested _Proteus_ bacilli.

]

Having analysed the mechanism of natural immunity against certain

bacilli, spirilla and vibrios, it will be interesting to determine

whether the same rules are to be applied in the case of the cocci.

Choice is not difficult since we may equally well fix upon the

staphylococci, the pneumococci, streptococci or gonococci. Should we

decide upon the streptococcus it is solely because the natural immunity

against this micro-organism has attracted the special attention of

several observers. A second advantage of the streptococcus, however, is

the high degree of natural immunity manifested against it by a

laboratory animal so convenient as the guinea-pig. Dr Jules Bordet[235]

studied this subject in my laboratory. He observed that the injection of

streptococci into the peritoneal cavity sets up a marked leucocytosis

which ends in a complete destruction of the micro-organisms. The

leucocytes rapidly ingest the great majority of the streptococci and

destroy them; there remain only a few isolated and free individuals

which are protected by a clear zone (aureola) which develops around

them, but in the end they also become the victims of the voracity of the

phagocytes. When we increase the dose of streptococci injected,

phagocytosis still goes on, but some of the streptococci succeed in

escaping, and we see a new generation produced which is distinguished by

the thickness of the protective aureola. In spite of the afflux of a

large number of leucocytes, they no longer ingest the streptococci and

generalisation of the infection results, followed by the death of the

animal. Natural immunity, then, can be suppressed under certain definite

conditions. Dr Jules Bordet[236] wished to satisfy himself whether the

leucocytes failed to fulfil their phagocytic function because of the

paralysis of their movements, or as the result of some other weakness.

With this object he injected into the peritoneal cavity of guinea-pigs,

at the moment when the streptococci begin to get the upper hand of the

leucocytes, a definite quantity of a culture of _Proteus vulgaris_.

These small bacilli in a short time become the prey of phagocytes which,

however, still refuse to ingest streptococci (fig. 36). There is thus in

the peritoneal cavity a kind of selective process as regards the

ingestion of these microbes. The _Proteus_ disappears as the result of

phagocytosis, whilst the streptococci thrive in the fluid of the

exudation and continue to multiply. This experiment, which readily

succeeds, demonstrates very clearly the difference between the positive

susceptibility of the leucocytes (with respect to the _Proteus_) and the

negative (with respect to the streptococcus). Bordet, in accordance with

the view now generally accepted, regards this sensitiveness as a

chemiotaxis, that is to say a perception of the chemical composition of

the surrounding medium. It must be admitted that the substance which

excites the chemiotaxis of the leucocytes does not readily diffuse and

may not, therefore, be found in a state of solution in the plasma of the

peritoneal exudation. Otherwise the leucocytes would refuse to ingest,

not only the streptococci, but also the small _Proteus_ bacilli, bathed

in the same repellent fluid. It is more probable that the substance

which excites the negative chemiotaxis is contained in the aureola that

surrounds the streptococci, from which it only escapes with difficulty

and for a short distance.

[Sidenote: [178]]

Marchand[237] continued the investigation of the same subject in Denys’

laboratory at Louvain. He studied the natural resistance of the

guinea-pig, rabbit and dog against the streptococcus. He, also, came to

the conclusion that phagocytosis constitutes the principal means of

defence of these mammals in their struggle against one of the most

formidable of the pathogenic micro-organisms. Starting from a single

colony, Marchand obtained two distinct races, one very virulent for the

rabbit, the other encountering a most effective natural resistance. This

resistance is due to the activity of the phagocytes which destroy the

streptococci in the ordinary fashion. He states as the general result of

his investigation that “an attenuated streptococcus is a streptococcus

readily devoured by phagocytes” whilst “a very virulent streptococcus is

a microbe that is not attacked by the leucocytes,” and he adds that “a

streptococcus is virulent because it is not devoured by phagocytes”

(_l.c._ p. 270). Up to this point the views of Marchand are in accord

with those of Bordet; but here they diverge, in fact as soon as it

becomes a question of the explanation of the origin of the difference in

the behaviour of the leucocytes. Marchand refuses to apply the theory of

chemiotaxis and asserts “that the phagocytosis depends on some physical

property of the streptococcus and is consequently dependent on the

tactile functions of the leucocytes” (p. 292). The experiments upon

which he founds his conclusion cannot, however, be regarded as

absolutely demonstrative. Thus, Marchand observed that the attenuated

streptococci, when conveyed in the culture-fluid of the virulent

variety, are as readily devoured by the phagocytes as when they were

injected alone. According to him, therefore, there was in the

culture-fluid of the virulent streptococcus no soluble substance capable

of exciting the negative chemiotaxis of the leucocytes. But is it quite

proved that this substance must necessarily pass into the filtrate of a

virulent culture? If it adheres closely to the glairy aureola, as we

have suggested, may it not remain behind with the bodies of the

streptococci, without passing through the filter in any appreciable

amount? The question cannot be regarded as definitely settled, but

probability appears to be on the side of the theory of chemiotaxis.

Marchand also investigated whether the immunity against the attenuated

streptococcus might not be explained by the bactericidal activity of the

fluids of refractory animals. His results were unvarying and definite.

The blood serum of his animals never exhibited any bactericidal power

against the streptococcus, and the attenuated race, like the virulent

one, grew well in the serums of the rabbit, dog and guinea-pig.

More recently, Wallgren[238] has taken up the study of the immunity and

susceptibility of rabbits with respect to the streptococcus. His

conclusions are, on the whole, in accord with those of his predecessors.

He found that if the injected streptococci were not very virulent

phagocytosis began immediately after the injection into the peritoneal

cavity and continued as long as there were any streptococci to be

attacked. In those cases, on the other hand, where the streptococcus was

endowed with a greater virulence, a transitory phagocytosis took place

at the beginning of the infection; but the streptococci soon succeeded

in adapting themselves to the struggle with the leucocytes and kept them

at a distance. The multiplication of the streptococci could then go on

without restraint and the animal soon succumbed to a generalised

infection. Wallgren considers that, in the defence of the organism

against the streptococcus, the products of the destroyed leucocytes may,

sometimes, play a part.

[Sidenote: [179]]

As the mechanism of natural immunity against the groups of

bacteria—bacilli, spirilla (and vibrios) and cocci—presents a very great

analogy in all three, it might be considered superfluous to continue our

analysis of this phenomenon. Our review, however, would be incomplete if

we omitted to take note of the natural immunity of the animal organism

against micro-organisms which are distinguished by an exceptional

toxicity. The first place in this group must undoubtedly be assigned to

the bacillus of tetanus.

[Illustration:

FIG. 37.—Leucocytes of rabbits filled with tetanus spores.

]

[Sidenote: [180]]

It may appear very inconsequent to be told that animals very susceptible

to tetanus, such as the guinea-pig and rabbit, are endowed with a

natural immunity against the tetanus bacillus. And yet this fact,

paradoxical as it may seem, has been demonstrated beyond doubt by

Vaillard and his collaborators Vincent and Rouget[239]. When a small

quantity of a culture of the tetanus bacillus was injected into one of

the animals just mentioned, tetanus was not long in declaring itself.

After a period of incubation, certain muscles became stiff and a

tetanus, local at first, soon became general and had a fatal issue. Now,

when much larger quantities of bacilli are inoculated, but care is taken

to rid them of the tetanus poison elaborated in the culture-fluid, the

animals resist without exhibiting any trace of tetanus. This experiment,

repeated many times, always with the same result, demonstrates that the

tetanus bacillus, when deprived of the co-operation of the toxin,

encounters, in these animals so susceptible to the latter, a most

effective opposition. Why is this? It was supposed that, in diseases

like tetanus so markedly toxic in character, the resistance was in no

way dependent on the phagocytic function. Thus Vaillard and Vincent were

quite prepared to attribute no share to the phagocytes in the example of

natural immunity which they had discovered. A detailed analysis of the

facts convinced them, however, that in this they were in error.

Guinea-pigs and rabbits do not contract tetanus, after the inoculation

of a quantity of spores and bacilli of tetanus deprived of their toxin,

solely because of the occurrence of very pronounced phagocytosis. Such

an injection is soon followed by a very marked invasion of leucocytes

which cram themselves with spores and bacilli without being in any way

inconvenienced thereby (Fig. 37). Once the phagocytes have devoured all

these organisms, the latter become incapable of producing their morbific

effect. The spores cannot germinate within the phagocytes, but there

undergo a marked degeneration and finally, after a longer or shorter

interval, disappear.

When, on the other hand, the tetanus bacilli or their spores are

accompanied by the pre-formed toxin, the latter, according to Vaillard,

excites a negative chemiotaxis of the leucocytes which keep away from

the organisms and which are thus allowed to multiply and to secrete

fresh quantities of toxin. The natural immunity of the animal’s organism

against the tetanus bacillus can be suppressed whenever the phagocytic

defence is hampered in any way. Under natural conditions it is usually

the adjuvant micro-organisms that aid the tetanus infection by hindering

the phagocytes from seizing the spores with sufficient rapidity to

prevent their germination. This fundamental result, established by

Vaillard and Vincent, has often been gainsaid on the evidence of

insufficient experiments (Sanchez-Toledo, Klipstein, Roncali), but,

ultimately, its accuracy has been completely confirmed. Cases have been

cited in which the tetanus spores, deprived of their toxin, still set up

a fatal tetanus. When a small fragment of an agar culture of tetanus,

previously heated to 85° C. for the purpose of destroying the toxin, is

inoculated, we produce tetanus. Vaillard and Rouget have demonstrated

that, under these conditions, the leucocytes penetrate merely into the

superficial layer of the agar, the spores germinating and the bacilli

multiplying in the deeper part. We can also set up a fatal tetanus in

animals by inoculating, along with sterilised earth, spores deprived of

their toxin by means of heat. The particles of soil protect the spores

against the aggression of the phagocytes, allow them to germinate and

then to poison the organism. Lactic acid produces an analogous effect,

by destroying or weakening the phagocytes. Micro-organisms, most often

inoffensive in themselves, also prevent the phagocytosis of the tetanus

spores and thus aid the intoxication.

[Sidenote: [181]]

The facts above summarised have been demonstrated to be the rule for

several species of anaerobic pathogenic bacteria. Thus, Besson[240]

showed that the septic vibrio is, by itself, incapable of setting up

septicaemia; in order to do this it needs the co-operation of other

micro-organisms. Leclainche and Vallée[241] have extended the same rule

to the bacillus of symptomatic anthrax (_Bacillus chauvaei_), so

important as being the cause of an epizootic disease of the Bovidae. The

spores of this bacillus when heated to 80°–85° C. lose the preformed

toxin and at once become incapable of producing infection.

In this case also, these spores soon after injection become the prey of

phagocytes, which seize them, prevent their germination and check their

pathogenic action. If to these heated spores, however, we add a small

quantity of toxin, they are enabled to germinate in the tissues and set

up a typical infection. If heated spores are mixed with sterile sand,

and the mixture introduced into guinea-pigs, these animals almost

invariably acquire a fatal symptomatic anthrax. The spores in the

superficial part of the sandy mass are readily devoured by the

phagocytes; but those which are included within the central part of the

mass, being protected for some time against these cells, germinate as

soon as they become permeated with the fluids of the animal organism. If

we envelope the sand in a paper sac the protection against the

phagocytes is still more complete and allows almost all the spores to

germinate and in every case to set up a fatal infection. Leclainche and

Vallée conclude from their experiments “that we only require to protect

the spore _mechanically_ in order to see an infection produced; here we

cannot allege an increase of its virulence, as when we associate a

chemical substance with the virus, and the exclusive part played by the

phagocytosis in the protective process stands out clearly” (p. 221).

The history of these three anaerobic organisms clearly proves that the

natural immunity against them cannot be made dependent on either the

bactericidal power of the fluids, or on any antitoxic property, or on

the incapacity of the micro-organism to secrete its toxin in the fluids

of the refractory animal. The cause of this immunity resolves itself

into the reaction of the phagocytes which prevent the micro-organisms

from producing their poisons.

[Sidenote: [182]]

All that has been said on the subject of the natural immunity of the

Vertebrates has had reference to cases of resistance against Bacteria.

But may not the immunity against micro-organisms belonging to other

groups depend on other factors with which the reader has not yet been

made sufficiently acquainted? Amongst the lower plants there are

Blastomycetes (_Torulae_ and Yeasts) which are capable of producing

infections, e.g. the disease amongst the _Daphniae_.

[Sidenote: [183]]

Some observers, no doubt, have come to the conclusion that the various

Blastomycetes, when introduced into a refractory organism, undergo

complete destruction within a few hours without any intervention of

phagocytosis. Thus Jona[242] explains the disappearance of yeast-cells

injected into the veins or peritoneal cavity of the rabbit as due to the

sole influence of the microbicidal property of the bloodfluid.

Gilkinet[243] looks at it from the same point of view. He injected beer

yeast (_Saccharomyces cerevisiae_) into a rabbit and observed that it

had disappeared shortly afterwards. The destruction of the yeast-cells,

according to this observer, “is effected by means of plasmatic juices”

and “is due to a specific property of the organic fluids” whose nature

is “quite unknown as regards its essential principle.” Phagocytosis is

said to play no part in this phenomenon. Let us hasten to say that

before the publication of the two works just cited, a memoir by

Schattenfroh[244] had appeared on the same subject. This observer, who

carried out his experiments in Buchner’s laboratory at Munich,

accurately observed and described the destruction of injected yeasts by

phagocytes, whilst his experiments on the microbicidal power of the

blood and serum failed. This testimony is the more important that it

emanates from a school by whom the microbicidal power of the “humours”

is regarded as the principal factor in the defence of the animal

organism. The facts described by Schattenfroh are perfectly accurate and

have been confirmed in my laboratory by Skchiwan[245], who did not

restrict himself to injecting ordinary yeasts (pink yeast,

_Saccharomyces pastorianus_) but inoculated guinea-pigs with pathogenic

yeast-cells, isolated by Curtis[246] from a case of myxomatous tumour in

man. The guinea-pig is refractory to small doses of this yeast but

succumbs to injections of larger quantities: Skchiwan convinced himself

that the ingestion of the non-pathogenic yeast-cells takes place with

great rapidity. Thus the _Saccharomyces pastorianus_, in the peritoneal

cavity of the guinea-pig, is ingested almost exclusively by microphages

at the end of two hours. Some (3–4) hours after injection, “sowings” of

the peritoneal exudation no longer yield growths. On the other hand

Curtis’ pathogenic yeast-cells resist the action of the phagocytes for a

much longer time. After a period of phagolysis in the peritoneal cavity,

the leucocytes that have just arrived in large numbers begin to seize

the yeast-cells. Usually several macrophages fuse around the same yeast

globule forming a very characteristic kind of rosette. Sometimes the

macrophages run together to produce a giant cell, whose centre contains

the yeast-cell. This latter defends itself against phagocytosis by

secreting a fairly thick membrane. The struggle between the two living

elements is a fairly prolonged one; 24 to 48 hours after inoculation all

the yeasts are surrounded by phagocytes, amongst which microphages are

exceptional. But the parasites remain alive for 4–6 days after their

injection into the peritoneal cavity, as proved by the cultures that are

obtained from the exudation when the fluid is “seeded” out. It must be

concluded, therefore, that the yeast-cells were surrounded by the

phagocytes whilst still presenting all the signs of life. Skchiwan was

no more successful than Schattenfroh in demonstrating any kind of

microbicidal action of the fluids on the Blastomycetes.