CHAPTER XVII

SUMMARY 544

Means of defence of the animal against infective

agents.—Absorption of micro-organisms.—Phagocytes, and their

function in inflammation.—The action of phagocytes in the

absorption of micro-organisms.—The cytases, phagocytic

ferments.—The cytases are closely bound up with the

phagocytes.—The fixatives and their function in acquired

immunity.—The fixatives are excreted by the phagocytes and pass

readily into the fluids of the body.—Essential mechanism of the

action of the fixatives.—Adaptation of phagocytes to destroy

micro-organisms in acquired immunity.—Difference between the

fixatives and the agglutinins.—Antitoxins and their analogy with

the fixatives.—Hypotheses as to the origin of

antitoxins.—Cellular immunity is a fact of general

import.—Susceptibility and its rôle in immunity.—Applications of

the theory of immunity to medical practice.

INDEX 571

INTRODUCTION

Importance of the study of immunity from a general point of view.—Part

played by parasites in infective diseases.—Intoxications by the

products of microorganisms.—Resistance of the organism to the

invasion of micro-organisms.

Natural immunity and acquired immunity.

Immunity to micro-organisms and immunity to toxins.

[Sidenote: [1]]

The problem of immunity in relation to infective diseases is one that

not merely concerns general pathology but has a very important bearing

on all branches of practical medicine, such as hygiene, surgery and the

veterinary art. The prevention of disease by the production of an

acquired immunity is daily assuming greater importance. With the object

of arresting the multiplication and dissemination of morbific germs, we

are seeking, by artificial means, to render individuals, who may come in

contact with them, refractory to their pathogenic action. Patients who

have just undergone a surgical operation and women in child-bed are

frequently in danger of acquiring a post-operation disease or a

puerperal affection; we are, therefore, striving to protect them by

conferring upon them an artificial immunity.

The immunisation of animals useful to man is likewise a question of such

great importance to agriculture and to industry as to have now become

the object of legislation.

[Sidenote: [2]]

This question of immunity is, however, apart from its practical aspect,

intimately connected with problems of pure theory. There can be no

question that the marked pessimism developed during the century just

closed was in a large measure prompted by the dread of disease and

premature death, scourges against which humanity is as yet powerless. It

is recognised that Byron and Leopardi, the great poets of pessimism,

both suffered from congenital anomaly and from incurable disease and

that these maladies cast a gloom over their poetry. Schopenhauer, the

founder of the pessimistic school in modern philosophy, was noted for

his exaggerated fear of disease.

During the greater part of the nineteenth century our knowledge as to

immunity has been limited to certain practical methods, often

efficacious it is true, but purely empirical, such as those employed in

immunising man against small-pox and certain domestic animals against

sheep-pox or pleuro-pneumonia.

So long as the nature of the viruses was unknown no really scientific

study of their action or of immunity from them could be made. The

revelation of the organised nature of the infective viruses opened up

the way for these researches. This discovery, the outcome of the

demonstration by Pasteur of the organised nature of the ferments, has

enabled us to establish the part played by living agents in a great

number of infective diseases, and, linked with the names of Davaine,

Obermeyer, and above all with that of Robert Koch, it has very greatly

advanced the study of susceptibility and of natural immunity in certain

infections.

A considerable forward step was made with the discovery, by Pasteur and

his collaborators Chamberland and Roux, that it was possible, in certain

infective diseases, to confer immunity by means of micro-organisms which

had had their virulence attenuated. Thanks to this discovery, science

was now in a position to take up the thorough study of acquired

immunity. The field of research was still further enlarged by the

demonstration of the immunising power of the culture products of

pathogenic micro-organisms and above all by the discovery that the blood

of immunised animals is capable of conferring immunity upon susceptible

animals.

Before taking up in detail the problem of immunity as it is revealed to

us as a consequence of these discoveries, it is essential to cast a

glance at infective and allied diseases as a whole and to indicate in

what light we look upon them in view of the present state of our

knowledge.

[Sidenote: [3]]

It has been definitely established that many infective diseases of man

and animals are due to the invasion of small parasitic organisms,

sometimes of animal nature (as in itch, trichinosis, malaria, Texas

fever, nagana, or surra and the allied condition “dourine” in horses),

sometimes belonging to the vegetable kingdom like the Moulds

(aspergillosis), the Hyphomycetes (actinomycosis, Madura foot disease,

bovine farcy) and the Yeasts (disease of the _Daphniae_, some

pseudomyxomas and septicaemias, pseudolupus). But by far the greater

number of infective diseases are due to the development in the organism

of plants of the simplest structure, Bacteria. These Bacteria produce

the gravest and most destructive infections, such as tuberculosis,

bubonic plague, diphtheria, cholera, anthrax, the pneumonias,

suppuration, erysipelas, tetanus, glanders, leprosy, &c. Among these

bacteria some are too small to be resolved individually under the

highest magnifying powers and can only be made out _en masse_. Such is

the micro-organism of the contagious pleuro-pneumonia of cattle. To this

minuteness of certain pathogenic Bacteria is very probably due the fact

that in a considerable number of infections, amongst which are

scarlatina, measles, rabies, syphilis, aphthous fever and small-pox, it

has been impossible, up to the present, to recognise any specific

micro-organisms.

It is probable that we shall succeed in discovering parasites, not only

in the diseases I have just cited, which present the characters of

infective and virulent diseases, but also in diseases of entirely

different types. In spite of the failure of various attempts to

demonstrate the parasite of malignant tumours, it may be hoped that,

with improvement in scientific methods, such a parasite will be

unequivocally demonstrated. In many other conditions which are at

present considered as not dependent on micro-organisms, an intimate

connection with such organisms will probably be established. Such are

the atrophic diseases and certain diseases of nutrition in which the

parasites, without playing a direct or immediate _rôle_, act by means of

their products, or by the changes which they set up in the affected

organism. To give an idea of this possibility it will be useful to cast

a glance at the various modes of action of the numerous etiological

agents in infective diseases. The parasites which produce them have, as

a common feature, their small dimensions; they can only be recognised

with precision by the employment of high powers of the microscope. They

are likewise distinguished by a great variability, which is not

astonishing, since among infective agents are found on the one hand

animals of high structure (such as the Acari of itch) and on the other

plants of the simplest character such as the Gonococci or the various

Cocco-bacilli.

[Sidenote: [4]]

The Acari are capable of perforating the epidermis by the mechanical

action of their feet and mouth-parts. They excavate channels in the skin

and thus provoke the irritation so characteristic of itch. The larvae of

the Trichinae in like manner produce marked lesions by the mere

mechanical act of penetration and migration in the striped fibre of

muscular tissue. In human trichinosis, however, the disease picture is

more complicated than in itch and leads us to assume that there is some

additional action of the excreta of the larvae in the production both of

the febrile state and of certain general phenomena. In the nagana

disease (transmitted by the Tsetse fly) there is equal reason to admit

the preponderating _rôle_ of the mechanical action of the flagellated

parasite (_Trypanosoma_) which obstructs the vessels of the nervous

centres.

In the diseases which are set up by Fungi, such as ringworm and

aspergillosis, the purely mechanical element still appears to play the

more important part. Even certain of the bacterial infections manifest

this same character. Thus, there is no doubt that in chronic

tuberculosis in the guinea-pig, Koch’s bacillus brings about a

substitution of tuberculous elements for the normal tissues, and this to

such a degree that, at the termination of the disease, there may remain

merely traces of the liver and of the lungs, and the animal dies for

want of these organs, whose normal action is no longer possible. In the

tuberculous guinea-pig the phenomenon of intoxication by the bacillary

poisons plays but a secondary _rôle_; yet there are examples of

tuberculosis (as in acute miliary tuberculosis in man or experimental

tuberculosis in cattle, obtained by Nocard’s method of inoculation into

the milk ducts), where the poisoning assumes much greater importance.

Among the bacterial diseases of man, leprosy may be cited as one in

which the intoxication is relegated to a subsidiary position, yielding

place to the mechanical substitution of the specific granuloma for the

normal tissues. It is only in the acute leprous exacerbations that we

perceive any signs of intoxication by the products of the leprosy

bacillus.

[Sidenote: [5]]

All the instances cited, however, constitute but a feeble minority which

is completely thrown into the shade in the presence of very numerous

infections in which the toxic element dominates the situation. Even in

carbuncular diseases an exact analysis of their morbid phenomena has

compelled us to recognise the marked influence of the poison produced by

the bacterium. The majority of the micro-organisms act as poisoners

which introduce themselves into the organism where they can secrete

toxins capable of provoking general disorders of very diverse natures.

Indeed in infective diseases a whole gamut of very remarkable variations

is produced. Thus many of the micro-organisms capable of setting up

septicaemias must multiply abundantly in the organism and be distributed

in the blood, before they can produce a general morbid condition. The

spirillum of human recurrent fever is an example of this. It multiplies

for some days and produces several generations without provoking the

least malaise; then, however, their appearance in the blood suddenly

produces intense fever and constitutional phenomena of the most

pronounced character.

On the other hand there are certain bacteria which are distinguished by

a very much feebler reproductive power, but a more marked toxic

activity. Incapable of spreading through the organism, these bacteria

remain localised at the point of entrance, where they secrete their

poisons and thus frequently set up a fatal intoxication. Some of these

bacteria, such as the bacilli of tetanus and of diphtheria, penetrate

more or less deeply into the living tissues of the affected animal.

Others can manifest their toxic action so to speak at a distance or by

simple contact with the living elements. Into this category comes the

organism of Asiatic cholera. Koch’s vibrio, once established in the

intestine, there secretes its poison; this, absorbed by the apparently

intact intestinal mucous membrane, sets up a fell disease, purely toxic

in character. It is probable that in the case of those intestinal

diseases whose etiology is still unknown, such as infantile choleras,

the poisoning by the products of micro-organisms constitutes the

essential phenomenon. The micro-organisms do not make their way into the

blood or tissues; they remain in the contents of the intestine and

thence set up their deadly intoxication.

[Sidenote: [6]]

Instances do exist in which the pathogenic micro-organism disappears

from the body, leaving there a toxin which, alone, is responsible for

death. Thus in the spirillar septicaemia of geese, the birds die at a

stage when not a single living spirillum can be found in the body. The

poisoners have been destroyed before the toxin produced by them had

completed its work. In other instances, e.g. typhoid fever of the horse,

the specific micro-organism likewise disappears before the death of the

animal; but at the period when the poison of this bacterium finishes its

fatal work, there is a secondary invasion of other micro-organisms which

have nothing to do with the typhoid fever proper of the horse.

This great variability in the action of the different pathogenic agents

is still further increased through the differing relations between the

parasites and the affected organism. Certain micro-organisms are capable

of producing a typical disease, whatever may be the mode and seat of

invasion of the organism. But these are comparatively few in number. The

bacillus of tuberculosis belongs to this minority. Whether it enters

subcutaneously, by the eye or by the respiratory, digestive or

genito-urinary passages, it invariably produces tubercular lesions more

or less grave and more or less capable of generalisation. On the other

hand, a very large number of micro-organisms only exert their pathogenic

action when they invade the organism at definite points. The anthrax

bacillus, when introduced through the slightest lesion of the skin or of

the mucous membranes, produces in man, and in a large number of mammals,

a very grave and usually fatal disease; when absorbed in the vegetative

state with food, it is almost always innocuous. With the cholera vibrio

we have an exactly opposite condition of affairs. When inoculated, even

in large numbers, below the skin in the human subject, it rapidly

disappears, producing merely insignificant disturbances; but when the

same vibrio is introduced into the digestive canal it develops and

produces Asiatic cholera, a disease so often terminating in death.

All these variations and peculiarities associated with the nature of

infective agents are of great importance from the point of view of

immunity.

[Sidenote: [7]]

Do diseases come from without or do their causes arise within the

organism? is a pressing question, long discussed by pathologists. Those

who have discovered most of the pathogenic micro-organisms have ranged

themselves on the side of the former hypothesis. For the majority of

them the essential etiological factor in the causation of infective

diseases consists in the invasion of the patient by the pathogenic

micro-organism from the outer world. This theory is in perfect harmony

with many of the admitted facts of epidemiology, according to which the

viruses of the most deadly epidemic diseases, such as Asiatic cholera,

yellow fever, and bubonic plague, must be imported into a country

previously free from the disease before an epidemic can be developed. In

anthrax and trichinosis it is recognised that the parasites must come

from without. Hence, in the study of pathogenic micro-organisms one

always follows the rule that it is essential to find the specific

micro-organism in all cases of the disease in question and to prove its

absence in healthy individuals or in those who are affected with other

diseases. Thus, Koch[1], in his classical researches on Asiatic cholera,

insisted on the fact that the cholera vibrio was always found in cases

of this disease but never in healthy persons. Almost simultaneously

Loeffler[2], in the course of his work on the etiology of diphtheria,

demonstrated the presence of a specific bacillus not only in a large

number of cases of this disease but also in the throat of a healthy

child; and this fact at first prevented him from accepting this bacillus

as the real cause of diphtheria.

This view accepted by two such eminent bacteriologists cannot however be

maintained. It is impossible to assume that each time that a pathogenic

micro-organism makes its way into a susceptible species its presence

must inevitably be followed by the production of the specific disease.

Although the discovery by Loeffler of the diphtheria bacillus in the

throat of healthy individuals has repeatedly been confirmed, it is

impossible to doubt the etiological _rôle_ of this organism in

diphtheria. Moreover, it has been established that Koch’s vibrio,

although undoubtedly the etiological factor in the production of Asiatic

cholera, has nevertheless been recognised in the digestive canal of

perfectly healthy persons.

[Sidenote: [8]]

As soon as he is born, man becomes the habitat of a very rich microbial

flora. The skin, the mucous membranes, and the gastrointestinal contents

become stocked with such a flora, but a very small number of these

micro-organisms have up to the present been recognised or described. The

buccal cavity, the stomach, the intestines and the genital organs offer

a feeding ground for Bacteria and inferior Fungi of various kinds. For

long it was thought that in healthy individuals all these

micro-organisms were inoffensive and sometimes even useful. It was

supposed that when an infective malady was set up a specific pathogenic

micro-organism was added to this benign flora. Exact bacteriological

researches have, however, clearly demonstrated that as a matter of fact

the varied vegetation in healthy persons often includes representatives

of noxious species of bacteria. Besides the diphtheria bacillus and the

cholera vibrio, which have repeatedly been found in a virulent form in

perfectly healthy individuals, it has been demonstrated that certain

pathogenic micro-organisms, e.g. the _Pneumococcus_, staphylococci,

streptococci and the _Bacillus coli_, are always, or almost constantly,

found among the microbial flora of healthy persons.

This observation has necessarily led to the conclusion that in addition

to the micro-organism there exists a secondary cause of infective

diseases—a predisposition, or absence of immunity. An individual in whom

one of the above-mentioned pathogenic species is present, manifests a

permanent or transitory refractory state as regards this specific

organism. As soon however as the cause of this immunity ceases to act,

the micro-organism gets the upper hand and sets up the specific disease.

It is thus in diabetic persons that boils make their appearance as the

result of the development of _Staphylococcus pyogenes_, a micro-organism

that is almost always found in abundance on the skin and mucous

membranes of the human subject. The diabetes is, in these cases, the

cause of the suspension of the immunity which exists in the healthy

individual.

People who carry the _Pneumococcus_ on their mucous membranes may remain

for long without being attacked by fibrinous pneumonia or any of the

other maladies due to this micro-organism. But often, in consequence of

some special circumstance, a cold for example, the refractory state

gives way to a more or less marked susceptibility.

It is unnecessary to multiply the number of such examples; they

demonstrate in the clearest fashion that, in addition to the causes of

disease which come from the outer world and which are represented by the

micro-organisms, there are yet other causes which lie within the

organism itself. When these internal factors are powerless to prevent

the development of the morbific germs, a disease is set up; when, on the

other hand, they resist the invasion of the micro-organisms properly,

the organism is in a refractory condition and exhibits immunity.

[Sidenote: [9]]

Diseases in general and infective diseases in particular were developed

on the earth at a very remote epoch. Far from being peculiar to man,

animals and the higher plants, they attack inferior forms and are widely

distributed among unicellular organisms, Infusoria and Algae. Diseases

undoubtedly play an important _rôle_ in the history of life on our

planet, and it is very probable that they have contributed in a marked

degree to the extinction of certain species. When we observe the ravages

produced by parasitic Fungi among the young fish which we are trying to

rear, or the destruction of crayfish in certain countries in consequence

of the rapid increase of epizootic germs, we are involuntarily led to

the conclusion that pathogenic micro-organisms must have brought about

the disappearance of certain animal and vegetable species.

Darwin[3], in the chapter on the extinction of species in his book _On

the Origin of Species_, states upon the authority of several observers

that insects so annoy elephants that these large mammals become

incapable of reproducing themselves in sufficient numbers. Now it is

proved that many Insects inoculate pathogenic micro-organisms and thus

transmit destructive diseases. A most formidable epizootic disease,

provoked by a flagellated Infusorium, the _Trypanosoma brucei_, is

inoculated into large mammals in South Africa by a fly, the Tsetse fly;

in certain districts this disease is so widespread and so destructive

that the rearing of domestic animals becomes impossible.

Parasites strike then with great intensity, bringing about the

destruction of numerous human beings, animals and plants. Nevertheless,

in spite of the disappearance of a large number of species, the world

continues well populated. This fact proves that, by the special means at

the disposal of the organism, without any aid of the medical art or

special human intervention, many living species have held their own

throughout the ages. Everybody has seen how dogs lick their wounds,

moistening them with a saliva full of micro-organisms. These wounds heal

well and quickly without dressings or antiseptics.

In all these examples the resistance of the organism depends on

immunity, a condition very general in nature. This immunity against

infective diseases is very complex and its thorough study could only be

undertaken after we had acquired an extended knowledge of these

diseases, and after adequate methods of research had been devised.

[Sidenote: [10]]

By immunity against infective diseases we understand the resistance of

the organism against the micro-organisms which cause these diseases. We

have here to do with an organic property of living beings and not with

the immunity which belongs to certain countries or localities. For this

reason information on the causes of the immunity in Europe and in

mountainous regions from yellow fever will not be found in this book,

nor why the majority of Europeans do not take recurrent fever. The

inhabitants of our continent do not possess organic immunity against

either the virus of yellow fever or Obermeyer’s spirillum of recurrent

fever. Indeed they are very susceptible to these diseases. It is solely

the conditions of life, in the majority of European countries, that

prevent the invasion by the specific germs and the creation of epidemic

foci. The same point of view ought also to be applied to animals. Our

small laboratory rodents, mice and guinea-pigs, are much more

susceptible to anthrax, whether inoculated beneath the skin or in any

other part of the body, than are the large domestic mammals such as the

ox and the horse. And yet these latter are very liable to epizootic

anthrax, whilst the rodents mentioned are seldom, if ever, attacked by

spontaneous anthrax. This apparent immunity in no way depends on the

existence of a true immunity of the organism, but solely on the

conditions under which mice and guinea-pigs live.

We shall therefore in this volume treat only of the phenomena of

_organic_ immunity in living beings, and the problem, even restricted

within these limits, still appears sufficiently complex. With the object

of rendering its study as easy as possible, it will be useful to

commence by giving an account of the phenomena of immunity in the lowest

organisms.

Immunity against infective diseases should be understood as the group of

phenomena in virtue of which an organism is able to resist the attack of

the micro-organisms that produce these diseases. It is impossible, at

present, to give a more precise definition, and useless to insist upon

it. Some have thought it necessary to distinguish between immunity

properly so called, that is to say a permanent refractory state, and

“resistance,” or a very transient property of opposing the invasion of

certain infective micro-organisms. It is not possible to maintain this

distinction, for in reality the limits between these two groups of

phenomena are far from being constant.

[Sidenote: [11]]

Immunity may be inborn or acquired. The former is always natural, that

is to say, independent of the direct intervention of human art. Acquired

immunity is also often natural, from the fact that it is established as

the result of the spontaneous cure of an infective disease. But in a

great number of cases acquired immunity may be the result of direct

human intervention as in the practice of vaccination.

For a long time all the phenomena of immunity against infective diseases

were collected into a single group. Later, it was recognised, as the

result of the demonstrations summarised at the beginning of this

chapter, that it is necessary to distinguish sharply between immunity

against the pathogenic micro-organisms themselves and that against

microbial poisons. Hence the idea of antimicrobial and antitoxic

immunities. In the course of this work this essential distinction must

always be borne carefully in mind.

CHAPTER I

IMMUNITY IN UNICELLULAR ORGANISMS

[Sidenote: [13]]

Infective diseases of unicellular organisms.—Intracellular digestion

in the Protozoa.—Amoebo-diastase.—Part played by digestion in the

defence of the Protozoa against infective parasites.—Defences of

the _Paramaecia_ against micro-organisms.—Part played by

irritability in defence in the lower organisms.

Immunity of unicellular organisms to toxins.—Acclimatisation of

Bacteria to toxic substances.—Protective secretion of membranes by

Bacteria.

Adaptation of the Protozoa to saline solutions—of yeasts to poisons—of

yeasts to milk-sugar.

Irritability of unicellular organisms and Weber-Fechner’s

psycho-physical law.

[Sidenote: [14]]

The immunity of unicellular organisms against infective diseases and

against toxic agents is as yet very imperfectly understood.

Nevertheless, it will be very useful for us to begin our study of the

problem of immunity on these lower organisms, because of their greater

general simplicity. It may be affirmed that if the line of comparative

pathology had been followed in our study of the etiology of diseases of

man and the higher animals, the parasitic nature of these infections

would have been established considerably earlier than was the case.

Thus, at a period when medical men and veterinary surgeons were content

to record the presence of Bacteria in the blood of their patients,

without attributing to them the slightest etiological _rôle_, botanists

and zoologists had already proved most definitely that many plants and

lower animals were subject to epidemic diseases undoubtedly set up by

the parasitism of various exceedingly simple organisms. In the same

year, 1855, that Pollender[4] published his first observations on the

bacterium found in the blood of animals affected by anthrax though he

could not trace the slightest relation between the presence of this

organism and the etiology of the disease, the celebrated botanist

Alexander Braun[5] issued his work on the genus _Chytridium_, in which

he demonstrated the fact that certain plants and flagellated Infusoria

suffer from the invasion of a small mobile parasite which, attaching

itself to their body wall, absorbs the contents and so destroys its

hosts, causing a very great mortality among them. The cycle of

development in the _Chytridia_, established by Braun, left no doubt as

to the accuracy of his view and even renders it possible for us to

interpret more accurately the earlier observations of Stein, on the

supposed evolution of certain Infusoria, by showing that the changes

observed in these organisms were in reality due to an invasion by

_Chytridia_.

Since these observations were made it has been clearly demonstrated that

among the unicellular organisms, certain Flagellata and ciliated

Infusoria are subject to infective maladies the result of parasitism of

the Chytridiaceae, a group of the lower Fungi. Small, mobile, colourless

cells attach themselves to the surface of the Protozoa, penetrate into

their interior and absorb the greater part of their living content.

Sometimes these parasites multiply in a most extraordinary fashion and

destroy enormous numbers of the Infusoria. Thus, Nowakowski,[6] who has

given a very detailed description of _Polyphagus euglenae_, the

Chytridium of the common green fresh-water _Euglena_, records the

disappearance of the _Euglenae_ from his aquaria glasses: the parasites

“were reproduced in such great abundance that ultimately they had

completely replaced the _Euglenae_.”

[Sidenote: [15]]

[Sidenote: [16]]

The Flagellata, subject to infection by _Chytridia_, are found almost

exclusively amongst those genera (_Cryptomonas_, _Chlamydomonas_,

_Haematococcus_, _Phacus_, _Volvox_, etc.) which are nourished after the

fashion of vegetables, that is by the absorption of substances dissolved

in the surrounding fluids. It is very remarkable that in the group of

ciliated Infusoria this parasitism of the _Chytridia_ is observed almost

solely in the encysted forms, that is to say, at a stage when the

animalcules, surrounded by their envelope, do not take any nourishment.

The invasion by the _Chytridia_ has been demonstrated in the case of the

cysts of the Vorticellina, Oxytrichinina, _Nassula_, etc.[7] These facts

indicate that the absence of the digestion of solid aliments, such as

occurs in almost all the ciliated Infusoria, constitutes a condition

favourable to infection by the _Chytridia_. Whilst the growth of

Volvocina, _Euglenae_ and their allies is almost always interfered with

by very destructive parasitic epidemics, the ciliated Infusoria, capable

of seizing and digesting lower organisms, may be cultivated and flourish

for a very long period. Thus Balbiani[8] has watched one of his cultures

of _Paramaecium aurelia_ multiply and thrive in splendid condition for

14 years in succession. Now these Infusoria readily adapt themselves to

ordinary water untreated to render it more hygienic. Such water swarms

with all sorts of lower organisms, among which are the _Chytridia_ and

numerous Bacteria, but the _Paramaecia_ and Infusoria in general feed

upon these organisms and contribute largely to the purification of the

water. Almost the whole body-contents in a ciliated Infusorian is made

up of a digestive protoplasm into which the captured Bacteria and other

lower organisms are conveyed; the nutrient particles becoming surrounded

by transparent vacuoles, in which the ingested organisms are killed and

digested. The food contained in the vacuoles circulates in the endoplasm

of the Infusoria by means of the streaming movements of this layer. The

digestive vacuoles become filled with a fluid having a distinctly acid

reaction. Formerly, in order to demonstrate this reaction, Infusoria

were allowed to ingest small granules of blue litmus which after a

certain time became more or less intensely red; but the use of aniline

colours has much simplified the study of digestion in microscopic

organisms. By introducing a solution of alizarin sulpho-acid into a

liquid containing Infusoria, the yellow staining (characteristic of the

acid reaction) of the digestive vacuoles can be readily made out. When

the Infusoria ingest small clumps of alkaline substances, stained violet

by this reagent, the vacuoles take on a red tint, indicating the acidity

of their contents[9]. Another aniline colour, neutral red (Neutralroth),

introduced into microscopical technique by Ehrlich[10], enables us to

demonstrate the acid reaction in the digestive vacuoles even within a

few minutes. Thus, in _Paramaecia_ treated with a dilute solution of

this reagent, the digestive vacuoles at once assume the deep rose tint,

characteristic of an acid reaction. This coloration is observed during

the life of the Infusorian, but immediately after death the vacuoles

become brownish and then completely lose their colour. This reaction,

easily demonstrated, indicates that neutralisation of the acid of the

vacuoles by the protoplasm and the surrounding water, both of which are

alkaline in reaction, has taken place.

In a medium distinctly acid the Infusoria digest their prey which, in a

very great number of cases, consists of Bacteria. These micro-organisms

are swallowed and carried into the digestive endoplasm in the living

condition; we have evidence of this in the active movements of a certain

number of the bacteria; at first they are found isolated in the interior

of the vacuoles, but later they collect into more or less compact

clumps. These masses of micro-organisms undergoing digestion, when

treated with neutral red assume a very deep rose tint, preserving their

bacillary form to the end, that is to say up to the extrusion of the

effete or waste material. There is, indeed, only very imperfect

dissolution not only of the bacilli as a whole but also of their

contents. _Paramaecia_ placed amongst cholera vibrios swallow them

greedily and in great numbers, digesting them as they would any other

micro-organism. I have never been able to see any conversion of vibrios

into granules going on within the digestive vacuoles.

All the attempts that have been made in my laboratory to extract a

digestive fluid from _Paramaecia_ have failed entirely. Very large

quantities of these Infusoria, obtained by filtration of rich cultures,

and macerated by different methods, have proved inactive even in the

case of those Bacteria which constitute their normal food.

Intracellular digestion in the Infusoria unquestionably takes place as

the result of the action of some diastase; but from the impossibility of

observing the action _in vitro_ the properties of this diastase, except

that it can act in a distinctly acid medium, cannot be determined.

[Sidenote: [17]]

Even less is known concerning the digestion of Rhizopods than concerning

that of Infusoria. It has long been recognised that, in the majority of

cases, _Amoeba_, _Actinophrys_ and Rhizopods in general, absorb a

nourishment composed of lower plants and animals, which are taken into

the protoplasmic body by means of the movements of amoeboid processes,

pseudopodia or lobopodia. Once within the Rhizopod the nutritive

particles are surrounded by a digestive fluid, in which the presence of

acid may be recognised by means of colour reactions. The addition of a

drop of Ehrlich’s neutral red to _Amoebae_ in the act of digesting

Bacteria at once gives the acid colour reaction (Fig. 1 ). Rhumbler[11]

has described very precisely and with much detail the way in which the

_Amoebae_ behave when they are incorporating filaments of _Oscillaria_

very much longer than their own bodies. He has also described the

digestion that these Algae undergo; a process most characteristic in

those cases where a portion only of the filament has been taken into the

interior of the _Amoeba_ and there subjected to the digestive action.

Whilst the free part of the _Oscillaria_ retains its normal properties

and appears of a bluish green colour, the ingested portion progressively

changes colour, assuming first a deep green tint, then becoming light

yellow, orange yellow, brown and finally reddish brown. Simultaneously

the cellulose wall of the Alga begins to soften, and the cells break up

into minute fragments which are soon extruded. The food is seldom

completely digested and there is always an abundant residual material

which is thrown out in the form of solid excreta.

[Illustration:

FIG. 1. An _Amoeba_ treated with neutral red, 1%.

]

[Sidenote: [18]]

Although it is fully recognised that, in the Rhizopods, digestion goes

on in a medium distinctly but feebly acid, and that the intervention of

some soluble ferment is essential, our ideas on this subject were very

vague until the publication of the researches of Mouton[12], carried out

with great care in the Pasteur Institute. In order that he might obtain

exact results Mouton made use of cultures of _Amoebae_ grown on agar, in

association with the _Bacillus coli_ which served them as food. The

bacilli were ingested in large numbers, became enclosed in vacuoles and

were digested by a ferment which Mouton was able to obtain _in vitro_.

To that end he collected large numbers of _Amoebae_, and, after

centrifugalising them in water, treated the deposit with glycerine. On

adding alcohol he obtained a precipitate readily soluble in water.

The fluid thus obtained exerted an undoubted digestive action upon

albuminoid substances. It readily liquefied gelatine and even attacked,

though feebly, albumen coagulated by heat; flakes of fibrin heated to

58° C. remained unaltered. There was present then, in this fluid derived

from _Amoebae_, a proteolytic diastase of feeble activity. On the other

hand, this extract contained neither sucrase, capable of inverting cane

sugar, nor lipase, capable of digesting fatty matters.

The amoebo-diastase of Mouton must be classified with the trypsins. It

is very active in a distinctly alkaline medium and continues the

diastatic action even when the medium becomes weakly acid (a feature

that corresponds to the reaction observed in _Amoebae_ treated with

appropriate staining agents). The amoebo-diastase is affected at as low

a temperature as 54° C. and at 60° C. is rendered completely inactive.

A question of especial importance is that concerning the action of the

amoebo-diastase upon Bacteria. The numerous experiments of Mouton

directed to the solution of this point, and made with living _Bacillus

coli communis_, gave negative results. If, however, these bacilli were

previously killed by heat or by chloroform, they were at once attacked

by the soluble amoebo-ferment. Opalescent emulsions of these dead

bacilli, incapable of undergoing self-digestion of any kind, became

transparent after remaining for some time in contact with the extract of

_Amoebae_. The amoebo-diastase, then, undoubtedly digests dead bacilli

_in vitro_, whereas in the body of the _Amoebae_ the ingested bacteria

are attacked whilst still living. As a result of these observations it

must be concluded that only a fractional part of the diastase is

extracted in the solution prepared by Mouton.

[Sidenote: [19]]

This intracellular digestion in the Protozoa serves not merely for the

nutrition of these organisms, but also as a protection against infective

parasites. The protoplasm of the Infusoria, with its vacuolar

secretions, has a general digestive action on everything that comes

within its reach. If the internal structures, such as the nuclei and the

pulsatile vacuoles, resist this process, it is undoubtedly because they

possess a power of defending themselves against the attack of the

digestive secretions. Thus, as brought out in the beautiful researches

of Maupas[13], the macronucleus of the _Paramaecia_ is, at a certain

stage in the life of the Infusorian, completely digested by the

protoplasm just as is any other nutrient substance introduced from

outside. It must be admitted that in this case the nucleus has ceased to

produce the protective substance which, under ordinary conditions,

interferes with its being digested.

A struggle similar to that observed between the nucleus and the

digestive content of the Protozoa goes on between these latter organisms

and infective microbes. All organisms which, in any way whatever,

penetrate into the body of an Infusorian or Rhizopod, are brought into

contact with the digestive endoplasm of these Protozoa. If the intruders

are killed and partially digested by the digestive secretions, or are

expelled as excrementitious matter, the Protozoon remains uninjured and

continues its normal and routine existence. Here, then, we have an

example of natural immunity, due to intracellular digestion. On the

other hand, when the foreign parasitic organism resists this digestive

action, it instals itself permanently in the body of the Protozoon, and

should it reproduce itself in small numbers merely, excrete no poison

and, in general, exercise no injurious influence upon its host, the

parasite may readily become a commensal. Thus, it is not rare to find in

the contents of Infusoria and Radiolaria small vegetable organisms of

the genera _Zoochlorella_ or _Zooxanthella_ which not only set up no

disease but, owing to their assimilation of carbonic acid, may even be

useful to their hosts. There are cases, however, where the parasites act

in a manner more or less injurious to the Protozoa containing them; in

such cases a true and sometimes fatal infection results.

[Sidenote: [20]]

Among the infective diseases of the Protozoa the one that has been most

thoroughly studied is that set up by several representatives of a

particular genus of micro-organisms discovered by Johannes Müller in

1856 and made the subject of an investigation carried out in my

laboratory by Hafkine[14]. I have already discussed these researches in

my work on the comparative pathology of inflammation[15] and need here

recapitulate only very briefly. _Paramaecia_ are sometimes affected by

needle-shaped or spirillar parasites which penetrate, sometimes into the

macronucleus, sometimes into the micronucleus, reproducing prolifically,

giving rise to a marked hypertrophy of the affected organs. The

Infusorian, in spite of this invasion, may continue to exist and carry

on its reproductive processes; it is, thus, enabled in many cases to

recover from the disease. On the other hand the _Paramaecium_ into whose

body the spores of the parasite are introduced treats them as it would

any other ingested foreign body. Not being able to digest them, owing to

the resistance offered by the membrane of the spore, the _Paramaecium_

expels them just as it would any other excrementitious matter. The

Infusorian behaves in the same way in regard to bacterial endospores.

Hay bacilli, which occur so commonly in the infusions in which the

_Paramaecia_ live, are digested in the endoplasmic vacuoles of the

latter, but the spores of these bacilli, after a more or less prolonged

sojourn in the vacuoles, are expelled with the excrement.

As by far the greater part of the body of a Protozoon is made up of

digestive protoplasm, it is natural that infective epidemics should be

very rare among these animalcules. The Infusoria and Rhizopods,

organisms specially well adapted to live upon the lower Algae and

Bacteria, are, practically, never subject to bacterial diseases. The

infections observed in the Protozoa are due in most cases to the

invasion of the lower Fungi, such as the _Chytridia_, the Microspheres,

the _Saprolegniae_ or the special organisms mentioned as occurring in

the nuclei of _Paramaecia_. Further, these infections are met with most

frequently in Protozoa which are incapable of carrying on true

intracellular digestion or which are in the encysted stage, at which

period the Infusoria, leading a passive existence, neither absorb nor

digest nutriment. As an exception to the above general statement I ought

to mention the epidemic in _Amoebae_ caused by the _Microsphaera_[16]

and the disease in _Actinophrys_ observed by K. Brandt[17] and

attributed to Fungi allied to the genus _Pythium_. In these two

instances we have to do with parasites which live and develop in the

interior of the active protoplasm of these Protozoa. Certainly a

proportion of the parasites are expelled with the excrementa; but there

remain others which instal themselves in the protoplasm, multiply there

and cause the death of their hosts. In these cases the digestive action

of the protoplasm must be neutralised or paralysed by the secretions of

the parasite. This aspect of the question, however, has so far not been

considered.

[Sidenote: [21]]

In addition to intracellular digestion and the expulsion of parasites by

the excretory function, the resistance offered by Protozoa to infective

diseases should, in part, be ascribed to their great irritability.

Anyone who will watch the manœuvres of _Amoebae_ or of certain Infusoria

in the midst of a rich microscopic flora and fauna, will at once be

struck by the preferences which these Protozoa exhibit in the choice of

their food. _Amoebae_ are often seen making search for Diatoms only,

disdaining all other Algae, or again they may single out one species of

Palmellaceae from a very varied flora. The Infusoria also have their

likes and dislikes in the matter of food. Many of the ciliated Infusoria

choose Bacteria to the exclusion of almost everything else; others, as

_Nassula_, have a special partiality for the _Oscillariae_. A most

striking example of this is afforded in _Amphileptus claparedei_, a

voracious Ciliate, which chooses _Vorticellae_ to the exclusion of all

other animalcules; these it devours, and then becomes transformed into a

cyst upon the peduncle of the _Vorticellae_ it has devoured. This

irritability clearly must control and guide the Protozoa in their

relations with other organisms and enable them to escape the invasion of

parasites.

In this connection I must mention a very interesting observation made by

Salomonsen[18] and communicated to the Paris International Medical

Congress in 1900. He was able to demonstrate the fact that almost all

the ciliated Infusoria, on becoming aware of the proximity of dead

bodies of kindred organisms, rapidly draw away, thus manifesting a very

marked negative chemiotaxis. This property must, it is evident, protect

them from any contamination by the parasites contained in the bodies of

Infusoria that have succumbed to infective diseases.

We have, then, quite a number of facts which throw light on the natural

immunity of the Protozoa against the action of pathogenic

micro-organisms. Up to the present, however, we know nothing concerning

the existence or the possibility of an acquired immunity among the lower

animalculae against infective diseases. We are better informed as to the

resistance of unicellular organisms to the action of soluble poisons,

which is, in general, much more easily studied than is immunity against

the micro-organisms themselves.

[Sidenote: [22]]

As a very large number of the higher animals are sensitive to the toxic

action of poisons of bacterial origin, the question has been put, “May

not the Infusoria also be poisoned by these micro-organismal products?”

With the object of answering this question Gengou[19] has studied the

influence of the toxins of tetanus and diphtheria on the ciliated

Infusoria. He was unable, however, to bring forward proof that these

substances exert any special toxic action on the _Paramaecia_. These

Infusoria withstand, perfectly well, doses of cultures of the diphtheria

and the tetanus bacillus grown in broth and deprived of the bacilli by

filtration as large as those of ordinary broth alone in which no bacilli

have been cultivated. Gengou argues from this that the _Paramaecia_

possess a natural and absolute immunity against these two toxins. When

we take into consideration the fact that these poisons act but feebly at

ordinary temperatures and are often innocuous to “cold-blooded” animals

we may perhaps be tempted to attribute the immunity of the Infusoria to

the temperature that was maintained in the incubator whilst Gengou’s

experiments were being carried on. Led by this train of thought Mme

Metchnikoff tried the action of the blood-serum of eels, which is very

toxic, not only for warm-blooded Vertebrates but also for cold-blooded

Vertebrates and the Invertebrates, on the _Paramaecia_, and this at a

low or medium temperature. This eel’s serum, however, exerted no greater

toxic action than did the blood-serum of other animals.

The microbial toxins are innocuous not only to the ciliated Infusoria

but also to many other unicellular organisms. It is now well recognised

that these toxins, exposed to the air, are soon inhabited by quite a

rich flora of micro-organisms, amongst which Bacteria and Yeasts

predominate. I have been able to prove[20] that these organisms are not

only unaffected in their normal life by the presence of the toxins of

diphtheria or tetanus but that they rapidly bring about the more or less

complete destruction of these poisons. Gengou, also, observed that

yeasts thrive luxuriantly in these bacterial toxins. The rapid increase

of micro-organisms and the destruction of these poisons take place at

temperatures varying from 15° to 37° C.

[Sidenote: [23]]

Whilst the lower organisms are refractory to bacterial toxins which in

quite small doses are capable of killing man and the higher animals,

many micro-organisms manifest a special sensitiveness to certain fluids

of animal origin. In a succeeding chapter we shall treat at greater

length of this microbicidal property of the humours. Here it is merely

necessary to indicate certain facts concerning this property, regarding

them solely from the point of view of the immunity of the lower

organisms. The most striking example of the bactericidal power of an

animal fluid is certainly that afforded in the action of the blood-serum

of the rat on the anthrax bacillus. This fact, discovered in 1888 by von

Behring[21], led to the conclusion that the blood of the rat contains an

organic base capable of killing and dissolving a considerable number of

anthrax bacilli. Several observers have confirmed von Behring’s

observation and have supplemented it by the fact that the bacillus can

be readily accustomed to the toxic action of this serum. Thus

Sawtchenko[22], in an investigation carried out in my laboratory, was

able, by successive cultures, to accustom the anthrax bacillus to an

existence in the pure serum of the rat. In this case, therefore, there

has been produced a real acquired immunity of a lower plant against a

toxic substance of animal origin. More recently Danysz has demonstrated

the same thing and has added several other facts which seem to throw

light upon the means by which the bacterium becomes adapted to the

poison. He has shown, in a work carried out in the Pasteur

Institute[23], that the anthrax bacillus protects itself against the

toxic action of the serum by surrounding itself with a thick sheath

composed of a kind of mucus which fixes the toxin of the rat’s blood and

renders it harmless. This same mucus, but in smaller quantity, is

likewise produced in a culture of the bacillus grown in ordinary broth.

When such a culture is freed from the contained bacilli by filtration

through porcelain and a little of this fluid is added to the rat’s

serum, this latter becomes less bactericidal than is a mixture of the

same serum with ordinary broth. Danysz suggests that this is to be

explained by the presence in the filtrate of a certain quantity of the

mucous substance produced by the bacillus, which fixes and neutralises a

portion of the “rat toxin.” If, in place of sowing the ordinary

bacillus, sensitive to this toxin, we inoculate the broth with an

anthrax bacillus which has previously been accustomed to the rat’s

serum, we find that the liquid of this culture when filtered neutralises

a larger proportion of the toxin. Danysz concludes from this that the

acclimatised bacillus has acquired the property of producing more mucus

than does the ordinary bacillus and that, for this reason, a greater

quantity of this protective substance passes into the fluid of the

culture.

[Sidenote: [24]]

The formation of a transparent sheath has several times been observed in

the anthrax bacillus, notably in cases where this organism happens to be

in “a state of defence” against various noxious influences. For example,

this sheath is well developed in the anthrax bacillus which invades the

blood of lizards, animals which are in general very resistant to

anthrax[24]. Under analogous conditions the streptococci which, as a

rule, do not produce a mucous sheath, will develop one of exceptional

size. The guinea-pig is in general very resistant to the streptococcus

against which it exhibits a very effective reaction. Sometimes, however,

this immunity gives way; in such instances, as demonstrated by J.

Bordet[25], the streptococcus, in order to overcome the natural

resistance of the guinea-pig, is found to have surrounded itself with a

sheath of a thickness such as is seldom to be met with in the world of

bacteria (Fig. 2).

[Illustration:

FIG. 2. Streptococcus surrounded by a protective envelope.

]

[Illustration:

FIG. 3. Tubercle bacillus surrounded by a transparent envelope and

enclosed in the giant cell of a gerbil.

]

[Sidenote: [25]]

Analogous facts are also observed in cases where the micro-organism is

defending itself against the action of substances enclosed in animal

cells. I may cite as an example the tubercle bacillus in the interior of

the giant cells of a gerbil (_Meriones shawii_), where, under the

influence of noxious substances contained in these cells, the tubercle

bacillus (Fig. 3) envelops itself in a transparent sheath similar to

that of the bacillus or of the streptococcus. As the action of the giant

cell still does not cease, the tubercle bacillus secretes a second

sheath (Fig. 4) and continues to surround itself with quite a series of

such envelopes (Fig. 5), thus coming to resemble a palmellaceous Alga

surrounded by successive layers of membranes or certain other vegetable

cells whose principal means of defence against all kinds of injurious

influences consists in the production of these protective membranes.

[Illustration:

FIG. 4. Another tubercle bacillus surrounded by two membranes.

]

[Illustration:

FIG. 5. Tubercle bacillus surrounded by a series of concentric layers.

]

[Sidenote: [26]]

Quite recently Trommsdorf[26], in Buchner’s laboratory in Munich, has

carried out a series of experiments on the adaptation of the cholera

vibrio and of the typhoid bacillus to the bactericidal substance found

in the blood of the rabbit. He has been able to confirm the results of

his predecessors and by various experiments has convinced himself that

these two micro-organisms are capable of adapting themselves to

existence in the defibrinated blood and in the blood-serum of the

rabbit.

The immunity, or acclimatisation of injurious organisms to different

toxins, presents an undoubted analogy to the phenomena of adaptation

shown by these organisms to mineral or organic poisons. It has long been

known that the same species of Protozoa are met with in both fresh and

salt water and that it is possible to gradually accustom Infusoria and

_Amoebae_ to tolerate an amount of sea salt which at first is absolutely

fatal to them. This toleration is not acquired unless care be taken to

increase the amount of salt very gradually: too abrupt a rise inevitably

causing death. By this means Cohn[27] accustomed the fresh-water

_Euplotes_ to a life in artificial sea water containing 4% of sodium

chloride. In Balbiani’s experiments[28] the fresh-water Monads

(_Menoidium incurvum_ and _Chilomonas paramaecium_) died very quickly on

the addition of ½% of this salt; but when it was added in small

successive doses (0·05 per day), they readily became accustomed to a

concentration of 1%. In the encysted state the Protozoa are even more

resistant than in the active state to the different salts that may be

added to their normal culture medium. It is probable that the wall of

the cyst interferes with the penetration of these substances into the

endoplasm. If a small quantity of an aniline dye be added to a fluid

containing encysted Infusoria, it is seen that the cyst-membrane becomes

very intensely coloured but the body of the Infusorian remains

unstained. The membrane absorbs a large amount of colouring matter,

after which, being saturated, it ceases to take it up; but it does not

allow the dye to penetrate into the endoplasm.

[Sidenote: [27]]

Balbiani (_loc. cit._ p. 580), having compared the action of the salts

of sodium with that of the salts of potassium and lithium on Infusoria,

comes to the conclusion that the injurious influence of these substances

can only be partially explained by osmotic phenomena. In addition to

these a purely chemical action must be invoked. He bases his opinion on

the fact that the isotonic solutions of the three salts acting on

Infusoria of the same species and same origin exert a different

influence. The salts of potassium and of lithium act in a much more

energetic fashion than do the sodium salts. Consequently, the Protozoa

are able to adapt themselves progressively not only to noxious

influences of a physiological character but also to those of a chemical

nature. Thus Infusoria and Rhizopods can be accustomed to the action of

high temperatures, to an intense light, etc. On the other hand they can

also be habituated to the toxic actions of true poisons. Davenport and

Neal[29] have established the fact that Stentors kept for two days in a

weak solution of corrosive sublimate (0·00005%) acquire a tolerance to a

dose of this poison four times as great as the lethal dose for

individuals previously kept in pure water. The same thing has been

observed in connection with the toxic action of quinine. This immunity

cannot be attributed to the selection and persistence of those Infusoria

which possess a natural resistance to the sublimate. It is really

acquired as the result of a direct and gradual chemical influence on the

protoplasm of the Stentors which, once adapted, all survive doses which

are lethal for the unacclimatised control organisms.

[Sidenote: [28]]

The vegetable micro-organisms, which are much more easily cultivated

than are the Protozoa, frequently manifest most characteristic phenomena

of acclimatisation. The first systematic researches in this direction

were carried out by Kossiakoff[30] in the laboratory of Duclaux. He

studied the antiseptic action of borax, of boracic acid, and of

corrosive sublimate on the anthrax microbe and several other bacilli

(_Bacillus subtilis_, _Thyrotrix scaber_ and _T. tenuis_). He found that

all these micro-organisms can be gradually accustomed to doses which are

absolutely bactericidal to the same species when not so acclimatised.

The acclimatised _Thyrotrix tenuis_ withstands almost double the amount

of bichloride of mercury that the non-acclimatised bacillus will resist.

The ordinary anthrax bacillus will not develop at all if the culture

medium contains more than 0·005 of boracic acid whilst the same

organism, when accustomed by passage through successive cultures in

which this substance is present in gradually increasing proportions,

grows well in spite of the presence of 0·007 of the same antiseptic.

Since these observations were made similar facts have been demonstrated

by several other observers, and the ready acclimatisation of Bacteria to

poisons is now generally admitted. Danysz (_loc. cit._), with the object

of elucidating the mechanism of this adaptation, has studied the action

of arsenic acid on the _Bacillus anthracis_. He has demonstrated that

this bacillus will gradually accustom itself to grow in broth containing

a quantity of arsenic acid which at first inhibited all development.

During this phenomenon of adaptation, which is acquired after a series

of passages through media more and more highly arsenicated, the bacillus

secretes a coating of mucous substance which protects the sensitive

parts of the microbial cell. Here, therefore, is formed something

exactly corresponding to what the same observer has demonstrated in

anthrax bacilli that have acquired a tolerance for rat’s serum. This

analogy extends even to the throwing out of the protective substance

into the culture fluid. When one sows an ordinary unadapted bacillus in

arsenicated broth to which has been added some of the fluid from a

culture of the adapted bacillus, development takes place in a marked

fashion. On the contrary when the same material is “seeded” into

arsenicated broth of the same composition but to which has been added

the filtrate from an unadapted culture, the bacillus does not develop

nearly so well. The difference is explained by the presence, in the

fluid in which the adapted bacillus had grown, of a certain quantity of

the mucous substance which fixes the arsenic and prevents it from acting

on the protoplasm of the micro-organisms.

[Sidenote: [29]]

The Yeasts, also, adapt themselves very readily to antiseptics. This

property has even had a practical application. We know that small doses

of hydrofluoric acid are capable of preventing the proliferation of the

yeast of beer, and Effront[31] has accustomed this plant to live in

media containing an amount of hydrofluoric acid which is absolutely

inhibitory to the unadapted yeast. Under these conditions the adapted

cells undergo a stimulation which causes the production of a greater

quantity of alcohol. The yeast, in adapting itself to antiseptic doses

(300 mm. of hydrofluoric acid per 100 c.c. of beer wort), acquires a

kind of immunity which it did not possess in the first instance.

Moreover this acquired property can be hereditarily transmitted to new

generations developed in ordinary beer wort to which hydrofluoric acid

has not been added. The stimulating action of this substance on the

fermentative property does not depend upon the acid reaction of the

hydrofluoric acid, for other acids which are non-antiseptic, such as

tartaric acid, are incapable of inducing it.

The acquired immunity against hydrofluoric acid is strictly specific,

the yeasts that have been adapted to this substance becoming even more

susceptible to the action of other poisons.

Duclaux[32] has already insisted on the relations which exist between

antiseptics and foods. Formic aldehyde which has a very powerful

coagulative and therefore strongly antiseptic action on protoplasm may

actually serve as a food for micro-organisms. The _Thyrotrix tenuis_,

studied in this connection by Péré[33], adapts itself to the presence of

this aldehyde and utilises it for its nutrition. Here is produced

something that recalls the case of the Protozoa that digest parasitic

organisms.

It is now a current idea in microbiology that Bacteria and Yeasts which

primarily do not make use of certain substances, adapt themselves to use

them as nutrient substances. Dienert[34] has published a detailed work

on the adaptation of the yeasts to milk-sugar. This sugar is usually

disdained by the yeasts that set up the fermentation of glucose; it is

not difficult, however, to adapt them to galactose which they then

attack and transform into alcohol and carbonic acid.

The Protozoa can be progressively accustomed not only to poisons but

also to altered physical conditions. Thus, Dallinger[35] succeeded in

raising the temperature of the water in which flagellated Infusoria were

growing from 15°·5 to 23° C. without causing their death. By prolonging

the experiment over several months, he was even able to habituate them

to an existence at a temperature of 70° C. In the opinion of

Davenport[36], a view which is shared by many other observers, this

resistance to high temperatures was dependent on the abstraction of

water from the protoplasm. Dallinger has also observed that in Infusoria

that are accustomed to life in hot water, the vacuoles become smaller

and smaller and may even actually disappear.

[Sidenote: [30]]

This adaptation, then, is a property that is very general and widespread

in the microcosm of the unicellular organisms. It is connected with the

intracellular digestion of solid food and with the absorption and

transformation of soluble substances. These phenomena, chemical in

character, are intimately linked with the irritability of microscopic

organisms, which represents one of the fundamental properties of living

organisms.

A Protozoon, which is refractory to a parasite, may protect itself by

flight or it may devour and digest the parasite; another, which acquires

a tolerance in regard to a toxin or to a mineral poison, absorbs, fixes

and transforms this substance. Consequently, in all these instances of

immunity there is a reaction of the living elements of the organism,

this being a direct consequence of the irritability of the protoplasm.

Before an Infusorian retreats from the dead body of an allied organism,

before a Protozoon secretes a digestive fluid around the prey it has

ingested, before a Bacterium secretes a glairy layer for its defence,

etc., these unicellular organisms must receive sensations which provoke

the above-mentioned reactions. It is to a celebrated botanist, Pfeffer,

that we owe the most important researches on this irritability of

unicellular organisms, researches which may be summed up in the general

statement that this property is subject to the psycho-physical law of

Weber-Fechner. Pfeffer, by the observation of the movements of Bacteria

under the influence of increasing stimulations, has established the fact

that, conformably to this law, when the stimulus increases in

geometrical ratio, the irritability increases in arithmetical ratio,

that is to say, the reaction is proportional to the logarithm of the

stimulation. In order that a motile bacterium (_Bacterium termo_), grown

in a peptonised solution, may perceive a difference of medium, it is

necessary to place it in a peptone solution of five times the original

concentration; weaker solutions, in which the concentration is but three

or four times greater than the original fluid, do not attract the

bacteria at all; consequently these differences are below their

chemiotactic sensibility.

The different reactions that are exhibited in the immunity of

unicellular organisms, reactions which are dependent on the irritability

of their protoplasm, therefore, come undeniably under the category of

purely cellular phenomena.

CHAPTER II

IMMUNITY IN MULTICELLULAR PLANTS

Infective diseases of plants.—Plasmodia of the Myxomycetes and their

chemiotaxis.—Adaptation of the plasmodia to poisons.—Pathogenic

action of _Sclerotinia_ upon Phanerogams.—The cicatrisation of

plants.—Defence in plants against Bacteria.—Sensitiveness of

vegetable cells to osmotic pressure.—Adaptation of plants to

modifications of osmotic pressure.—Dependence of the chemical

phenomena upon the irritability of the vegetable cells.—The law of

Weber-Fechner.

[Sidenote: [31]]

For several reasons this immunity in the vegetable kingdom cannot be

treated in a satisfactory fashion. Much attention has been devoted to

the pathology of plants and the etiology of a number of vegetable

diseases was well established at a period when we were still groping in

the dark for the causes of infective diseases in man and the higher

animals. In spite of this, the botanist has relegated the study of the

phenomena of immunity to a secondary position, and up to the present no

work specially devoted to this subject has appeared. It is only

incidentally that the question of the resistance of certain plants to

morbific factors capable of infecting or intoxicating them has been

touched upon. We should require, therefore, to carry out special

researches in this direction and to make a very complete study of

botanical literature, before we should be able to present to our readers

a _résumé_ of the question of immunity in the vegetable kingdom. Such a

programme being impossible we must content ourselves with borrowing from

the botanists certain facts which throw light on some aspects of the

general problem in which we are interested.

[Sidenote: [32]]

Many of the higher plants are subject to infective diseases set up by

the lower plants, of which the most important are the Fungi. Whereas in

the animal kingdom the majority of the infections are due to Bacteria,

these micro-organisms rarely occur in plants; moreover when they are

present the part they play is nearly always a secondary one. This

difference is due mainly to the chemical composition of the “humours” in

the two kingdoms, the cell-juice of plants being generally acid; under

this condition the Fungi develop much better than do the Bacteria.

The various modes of defence against infective diseases that have been

met with in unicellular organisms are also found in the multicellular

plants. Whereas in almost all plants the cells are rigid, owing to the

presence of a well-developed membrane, some of the lower plants have

preserved a condition in which the protoplasm is completely naked and

capable of movement. Myxomycetes are specially distinguished by an

amoeboid stage of existence and by the formation of large plasmodia

which protrude protoplasmic processes and exhibit a kind of locomotion

similar to that met with in the Rhizopods and the Sporozoa.

[Sidenote: [33]]

Infective diseases among the Myxomycetes must be very rare since, up to

the present, they have not been noted by a single observer. It is very

probable that the plasmodia get rid of the infective germs, as do the

Protozoa, both by expulsion of the parasites and by means of

intracellular digestion. This latter takes place in a medium which is

distinctly acid and by means of a soluble ferment described by

Krukenberg[37] as a kind of pepsin. I need not here enter into further

detail as I have already treated this subject in my _Lectures on the

comparative pathology of inflammation_. The fact that the Myxomycetes

can ingest living organisms has been demonstrated by Celakovsky,

jun.[38], who has observed that the spores of the various Fungi can

germinate in the interior of the plasmodium. Whilst our conceptions

concerning the resistance of the plasmodia in regard to micro-organisms

are merely based upon analogies and hypotheses, our ideas as to their

immunity against soluble substances rest on well-established

experimental facts. We owe to Stahl[39] our first information as to the

mode by which the plasmodia resist poisons. When they are placed in

contact with solutions of salts, of acids or of sugar in a sufficiently

concentrated form to bring about an injurious action, the plasmodia make

use of their amoeboid power of motion to escape from these fluids. Hence

they exhibit a _negative chemiotaxis_, exactly parallel to that so often

observed in the case of the unicellular organisms. Consequently there is

in the Myxomycetes a natural immunity due to the activity of their

movements. Further, a kind of acquired immunity in these plants has also

been demonstrated by Stahl. The following is the passage in his paper

referring to this subject, a passage very important from a general point

of view[40]: “If we replace the water in a vessel by a 1 or 2% solution

of glucose, we observe either the death of the plasmodia, if the action

is too rapid, or merely their retreat from the glucose solution. Even

solutions of ½ or ¼% are at first avoided by the plasmodia and, should

the action be too rapid, may cause their death. Usually the plasmodia

emigrate into those portions of the substratum remote from the solution,

to return after some time, often only after several days, and immerse

themselves in the solution of glucose as they do in an infusion of tan,

although with more hesitancy. _Consequently the Myxomycetes accommodate

themselves slowly_[41] to a more concentrated solution, probably by

giving up a certain proportion of their water. I was able to observe the

same phenomena with even much more concentrated solutions (2%). A

plasmodium which at the end of several days had adapted itself to a 2%

solution of glucose and had sent out numerous processes into it, found

itself injuriously affected when the sugar solution was suddenly

replaced by pure water. Those that remained alive had retired to a great

distance from the upper layer of the fluid and did not descend again

until the end of the second day. After a fresh change of fluid we were

able to observe first the repulsion and later the attraction of the

plasmodia, but a certain time always elapses before the plasmodia become

accustomed to the change in concentration. We obtain the same result

when we replace a 2% solution, not by pure water, but by a ½ or a 1%

solution” (p. 166).

[Sidenote: [34]]

De Bary[42] had already interpreted these facts as being due to an

immunity acquired by the plasmodia, the result of an adaptation of these

organisms to solutions which they had, at first, carefully avoided. He

threw out the suggestion that a similar adaptation might take place in

regard to solid substances ingested by the Myxomycetes.

As these phenomena of acquired immunity, in organisms so primitive and

of so simple a structure, are of immense importance from the point of

view of Immunity in general I felt bound to submit them to a personal

investigation. I found it an easy matter to accustom the plasmodia of

_Physarum_ to solutions of arsenious acid which at first repelled them

in a very striking manner. This adaptation manifests itself by movements

of the plasmodia and by the change from negative chemiotaxis (repulsion)

to positive chemiotaxis (attraction).

It is impossible in the present state of our knowledge to state

precisely the modifications that the plasmodia undergo during this

process of adaptation. Stahl supposes that they depend “on some special

properties of the plasmodia (probably in a greater or less richness in

water)”; and that it is a case “not of simple phenomena, easy of

explanation, but of extremely complicated phenomena of irritability.”

It is evident that, in this case of acquired immunity, we have not to do

with a question of physical or chemical modification of the solutions

employed but solely with reactive phenomena on the part of the living

plasmodia.

After a phase of active life, during which the Myxomycetes move, feed,

digest and expel waste products as do the lower animals, there comes a

stage when they become immobile and transform themselves into a number

of sporangia filled with rounded spores. Before leaving their animal

aspect for that of true plants, the plasmodia exhibit entirely new

attributes. They reject all nourishment and no longer ingest foreign

bodies; they avoid the moisture which previously attracted them and

cease to shrink from the light.

[Sidenote: [35]]

Having come to maturity, the Myxomycetes declare themselves true plants

and lead a passive life until the new generation comes forth. Most

plants are restricted to this passive phase of the Myxomycetes. In these

latter it persists only for a short period, whereas in almost all plants

it is the permanent condition. It is at this stage that these organisms

are liable to the attack of parasites against which it is necessary for

them to oppose all their means of defence. Our knowledge of these means

of defence is as yet, as I have already stated, very imperfect, and the

example of _Sclerotinia libertiana_ (or _Peziza sclerotiorum_) which has

been the subject of the researches of de Bary[43] remains up to the

present the one that has been most thoroughly studied.

This Fungus, belonging to the group of the Discomycetes, invades many

species of plants and often produces great ravages amongst the

cultivated plants of our fields and gardens, such as colza, hemp

petunias, dahlias, etc. The mycelium of this _Sclerotinia_ develops in

the stems of herbaceous plants and produces sclerotia inside them, forms

of resistance, which in this instance are black and resemble small

particles of mouse excrement.

The spores of the _Sclerotinia_ germinate and form mycelial threads on

the surface of the plants. In order that they may penetrate into the

tissues these threads must attack the cell-membrane and for this purpose

they secrete a fluid, which contains both a digestive ferment and oxalic

acid, the latter being essential for the action of the ferment.

The presence of this “toxin” has been demonstrated by de Bary by

macerating the mycelium of the _Sclerotinia_. The resultant extract has

a well-marked action on the tissues of many plants (carrot, Jerusalem

artichoke, chicory, etc.). Under its influence the protoplasm of the

cells contracts, a genuine plasmolysis is set up, the cell-membrane

swells and its layers between the cells are dissolved. As the result of

this digestive action, the cells become separated and the tissue

softens. This extract, when heated to 52° C., loses its digestive action

on the cellulose membrane, but still retains its power of setting up

plasmolysis. This reaction to temperature confirms the view that the

juice of the Fungus contains a soluble ferment. The results of de Bary’s

researches have been confirmed and in part supplemented by the

experiments of Laurent[44].

[Sidenote: [36]]

It is a fact of common observation that the _Sclerotinia libertiana_

invades for the most part young plants. It may therefore be asserted

that the disease produced by this Fungus is, like scarlatina or measles

in the human subject, an “infantile” disease. De Bary suggested that the

immunity of adult plants must depend on the greater resistance which

their cell-membranes offer to the fluid secreted by the mycelial

filaments. Direct experiments have shown the accuracy of his suggestion.

Whilst the fluid extracted from the _Sclerotinia_ readily digests the

tissue of young plants it leaves intact that of adult plants of the same

species.

In the course of this disease we have a struggle going on between two

plants. The parasite brings into play toxic and digestive secretions

with which it seeks to impregnate its host. The attacked plant defends

itself by the secretion of membranes capable of resisting the action of

the secretions of the Fungus. This struggle by means of chemical

substances is, however, directed by the activity of the living cells of

the two belligerent plants, an activity dependent upon the irritability

of their protoplasm.

The example we have just studied may serve as a type for our examination

of the phenomena of immunity in the vegetable kingdom. The crux is above

all to prevent the access of the parasites to the vital parts of the

plant by opposing to them membranes as resistant as possible.

Consequently the majority of plants, directly the smallest lesion is

produced, react by an abundant cell-proliferation and by the

suberisation of the outer layers. The cell-membranes of the latter

thicken, the cellulose is transformed into suberin; a layer of cork not

very permeable to fluids and gases being thus formed. By suberisation

the plant reacts against grosser lesions, incisions or burns, as well as

against the decay set up by micro-organisms.

Massart[45], in an extremely interesting memoir, has brought together

the known data concerning cicatrisation in plants and has demonstrated

the fact that it is a very variable process. In many leaves after being

damaged there is no attempt to react by forming cicatricial tissue. Many

aquatic and marsh plants react but feebly. Their tissues die and turn

brown, the plants failing to defend themselves by cicatrices, probably

owing to the ease with which the lost parts can be replaced. When,

however, in the same plants, there is produced a lesion of parts which

are of great importance for the preservation of the integrity of the

individual or a lesion of the organs which enable the plant to continue

its existence through the winter, cicatrisation of the wounds takes

place rapidly.

[Sidenote: [37]]

The old or adult parts in most cases react differently from the young

parts. Thus, the young leaves of _Clisia_ (the example selected by

Massart) react to traumatism very promptly and form a genuine callus

which makes good the injury, but the adult leaves merely produce a layer

of cork in the immediate neighbourhood of the lesion.

The essential mechanism of cicatrisation has not yet been satisfactorily

analysed, but it is evident, when all is said and done, that it is

directed by the irritability of the living protoplasm of the vegetable

cells.

Many plants protect their wounds with a kind of dressing, using for that

purpose juices which harden on exposure to the air. Sometimes these

juices, _e.g._ latex, are preformed in the plant and are as it were

always ready for use; at other times they may be formed only as the

result of an injury. In this latter case the resins and gums which serve

to close the wound and to protect the living parts receive the name of

“cicatricial secretions” (Wundsecrete). According to the view first

formulated by de Vries, those juices which harden under the action of

air prove of great service both as natural “dressings” and as safeguards

against the attacks of plants and animals. Indeed many of these

secretions contain essences whose antiseptic and toxic action is now

generally recognised[46].

[Sidenote: [38]]

The suberisation, the formation of a callus, and the secretion of juices

which close the wounds, are all means readily utilised and very potent

in ensuring the resistance of plants against all sorts of injurious

influences which may be set up by a morbid condition. But these

processes are not the only means which plants have at their disposal.

The living elements of plants usually secrete a cell-juice of acid

reaction which plays a very important part in the defence of plants

against pathogenic agents. Laurent[47] has studied this phase of the

immunity of plants against bacterial decay. A variety of the _Bacillus

coli communis_, according to this observer, attacks the potato by means

of its secretions in a fashion analogous to that already described when

discussing _Sclerotinia_. This bacillus produces a soluble ferment which

has the power of digesting the cellulose membrane in the tuber of the

potato, and at the same time secretes an alkaline juice without which

this digestion cannot go on. Heating to 62° C. destroys the soluble

ferment and the fluid thus heated is no longer able to digest the layers

of the cell-membrane between the cells. In spite of exposure to this

temperature, however, it still retains intact one or even several

substances which may continue to cause contraction of the protoplasm and

ultimately kill it.

When Laurent placed cut halves of tubers coming from races of potato

which were most resistant to bacterial decay in the fluid produced by

the _Bacillus coli_ and afterwards inoculated them with the bacillus

itself, he invariably found that the vegetable cells were profoundly

affected.

The alkaline secretions of the bacillus studied by Laurent may be

neutralised by the acid juice of the potato, and when certain races of

tubers prove immune from decay, it is, according to this observer,

because of the production of sufficiently acid cell-juices. Moreover he

actually succeeded in communicating an artificial immunity to varieties

of the potato which were most susceptible to decay by immersing them for

several hours in solutions of certain organic acids. On the other hand,

when he treated varieties endowed with a well-marked natural immunity

with alkaline solutions, the tubers became very susceptible to the decay

set up by the bacillus.

The struggle between the potato and the _Bacillus coli_ reduces itself,

then, to the chemical reaction between the alkaline cell-secretions of

the micro-organism and the acid secretions of the potato. This general

fact, according to Laurent, explains the part played by certain manures

in determining the susceptibility or the resistance manifested by the

potato and many other plants against infective diseases.

We know that the addition of phosphates to the soil increases the

immunity of certain cultivated plants. These substances are greedily

absorbed by the roots and produce acid salts which are dissolved in the

cell-juice. The nitrogenous manures, on the other hand, both potassic

and lime, diminish the resistance of the same plants, probably from the

fact that they bring about a diminution of the acidity of the

cell-juice.

But these manures can act differently on different plants. Thus the same

phosphates which confer immunity on the potato against bacterial decay

render the Jerusalem artichoke more susceptible to the attacks of the

_Sclerotinia_.

[Sidenote: [39]]

Laurent explains this fact as due to the difference in the reaction of

the medium, which favours the action of one or the other of the soluble

ferments of the two parasites. The ferment of the bacillus digests the

cell-membrane in an alkaline or feebly acid medium, whereas the

hyperacidity which results from the absorption of the phosphates

prevents this digestion and consequently aids the plant in its struggle.

On the other hand, the ferment of _Sclerotinia_, as is seen from the

researches of de Bary, will digest cellulose even in a distinctly acid

medium. The hyperacidity, induced by the phosphated manure, in this case

favours the parasite and enables it to gain the upper hand in the

struggle with the tissues of the artichoke.

In addition to neutralising the microbial products the acids of the

cell-juice also act injuriously on most bacteria, which will only

develop in neutral or alkaline media; it is for this reason that

bacterial diseases are so much rarer in plants than in animals.

The secretion of cell-juices is consequently a very important element in

the defence of plants; it will be useful, therefore, to ascertain as

definitely as possible the essential mode of its action. Vegetable cells

are as a rule very sensitive to the influences to which they are

exposed; they distinguish with great precision the changes which take

place in their surroundings. They are, indeed, capable of discerning not

only the physical properties but also the chemical composition of the

medium in which they live.

Vegetable cells estimate very accurately the osmotic pressure of the

fluid which bathes them, and they react towards this solution by

increasing or diminishing their own internal pressure. Van

Rysselberghe[48], in an investigation very carefully carried out,

demonstrated that when vegetable cells (especially the epidermic cells

of certain species of _Tradescantia_) are placed in a solution of

greater density than that to which the cells are accustomed, the

intracellular pressure increases; in a solution of less density the

pressure diminishes. These changes in osmotic pressure are due to

variations in density of the cell-juice, whilst these variations are in

turn determined by chemical transformations. Thus, if the cell be

treated with a too concentrated solution it produces oxalic acid, which

dissolving in the cell-juice, is, owing to the smallness of its

molecule, very osmotic.

[Sidenote: [40]]

With the purpose of confirming this by exact facts van Rysselberghe has

studied the acids of the cell-juice of _Tradescantia_. In the normal

juice he found that malic acid was constantly present and, in rare cases

only, traces of oxalic acid. He then determined the acids present in the

leaves of the same plant after they had been several days in contact

with fairly concentrated solutions of cane sugar. In each analysis he

found oxalic acid in quite appreciable quantity. There is then, in the

plant which adapts itself to more concentrated solutions of the medium,

a production of oxalic acid which serves the purpose of increasing the

pressure of the cell-juice.

The origin of this oxalic acid could not be accurately demonstrated, but

van Rysselberghe considers that it is probably formed at the expense of

the glucose.

According to the researches of Giessler oxalic acid is localised

specially in the epidermis and generally in the peripheral tissues of

plants; it is very probable, therefore, that it fulfils a protective

_rôle_ against all kinds of injurious influences. Botanists hold indeed

that oxalic acid keeps herbivorous animals, especially slugs and plant

lice, from attacking plants that are rich in this substance. It is of

use, also, in retaining the moisture in the superficial cells. It is

very probable that it also plays an important part as a factor in the

maintenance in plants of immunity against bacterial diseases.

The vegetable protoplasm, which is capable of increasing the production

of acids in order to raise the osmotic pressure, can also, in case of

need, cause a diminution.

When the cells of _Tradescantia_ are transferred from a concentrated

solution into one much more dilute there may often be observed a

precipitation, in the cell-juice, of crystals of oxalate of lime; this

brings about a diminution in the osmotic pressure. When the density of

the medium is altered, and the vegetable tissue is again transferred to

a stronger solution, the oxalate crystals are seen to dissolve, as a

result of a new production of acid.

These chemical processes, so important to the life of plants in general

and for ensuring to them immunity against infective agents in

particular, are dependent upon the irritability of the protoplasm.

Imprisoned in its resistant and more or less thick membrane, the living

part of the vegetable cell estimates with nice discrimination every

change that takes place around it.

[Sidenote: [41]]

Massart[49] has shown that the stimulation produced by traumatism is

often propagated a considerable distance and may excite a reaction in

very remote cells. If the mid-rib of a leaf of _Impatiens sultani_ be

cut near the base of the limb the wound does not cicatrise but, a few

days later, the leaf becomes detached from the stem.

Irritability is a fundamental property of all living beings. The plant

may react by rapid movements, as in the case of the _Mimosa pudica_, or

more slowly—by chemical reactions—as in the case of adaptation to

density of medium. These reactions are produced as the result of various

irritabilities which exhibit a specific character.

It is this specificity that determines whether the reaction that is

manifested by the movements shall be produced in this direction or in

that. The stem, owing to the specific irritability of its living parts,

turns to the light; whilst the root, guided by a different irritability,

grows down into the soil.

The irritability of plants, like that of unicellular organisms, is

subject to the psycho-physical law of Weber-Fechner. Pfeffer[50] first

demonstrated this for the motile spermatozoids of the Cryptogams.

Massart[51], by a series of ingenious experiments on the irritability of

a Mould (_Phycomyces nitens_) to light, has shown that the same law

regulates the movements of this plant towards the source of light. This

irritability of the Fungus to light is much more delicate than is the

chemiotaxis of the spermatozoids of the Mosses and the Ferns and than

that of the Bacteria.

Errera concluded from a consideration of the experiments of van

Rysselberghe that the osmotic reaction of plants must also come under

this psycho-physical law. His pupil at his request made systematic

researches on the subject and the results have entirely confirmed his

prevision. According to the data obtained by van Rysselberghe[52], the

cellular osmotic reaction increases in arithmetical progression as the

osmotic stimulation increases in geometrical progression. The osmotic

reaction is therefore proportional to the logarithm of the stimulation.

[Sidenote: [42]]

To sum up, the phenomena of adaptation and of immunity in plants are, as

in the unicellular organisms, very widely distributed. Plants defend

themselves by means of their resistant membranes and by secretions whose

physical and chemical properties they are able to modify. These

phenomena are dependent on the living parts of the cell which regulate

them according to their greatly developed irritabilities. Thanks to this

power, plants can gradually adapt themselves to concentration of the

medium and to the presence of poisons which, at first, set up serious

disturbances. Plants therefore, alongside a natural immunity, possess an

acquired immunity against many pathogenic agents.

CHAPTER III

PRELIMINARY REMARKS ON IMMUNITY IN THE ANIMAL KINGDOM

Examples of natural immunity among the Invertebrates.—Immunity against

micro-organisms and insusceptibility to microbial poisons are two

distinct properties.—The refractory organism does not eliminate

micro-organisms by the excretory channels.—It destroys them by a

process of resorption.—The fate of foreign bodies in the

organism.—The resorption of cells.—Intracellular digestion.—This

digestion effected by the aid of soluble ferments.—Digestion in

Planarians and Actinians.—Actinodiastase.—Transition from

intracellular digestion to digestion by secreted juices.—Digestion

in the higher animals.—Enterokynase and the part it plays

in digestion.—The psychical and nervous elements in

digestion.—Adaptation of the pancreatic secretion to the kind of

food.—Excretion of pepsin in the blood and in the urine.

[Sidenote: [43]]

As shown in the two preceding chapters unicellular organisms and plants

afford evidence of numerous phenomena of immunity. Alongside natural

immunity we find in them undoubted evidence of an adaptation to the

presence of morbific agents, evidence which warrants us in inferring

that cases of acquired immunity are frequent. This being the case it is

quite natural that the animal kingdom should be no exception to the

general rule. Indeed, immunity against pathogenic agents is widely

distributed in animals, and we continually see manifestations of natural

immunity not only against parasites and their toxins, but against

poisons in general. Just as frequently we find cases of acquired

immunity against these morbific agents.

[Sidenote: [44]]

As yet we know but little concerning the phenomena of immunity in the

lower animals belonging to the great group of the Invertebrata. But it

may be affirmed with certainty that these also are often endowed with a

natural immunity against micro-organisms and bacterial toxins. As an

example I may cite the case of the large white larvae of the Rhinoceros

beetle (_Oryctes nasicornis_) frequently met with in tanner’s bark. Very

susceptible to the cholera vibrio—¹⁄₈₀₀₀ of a culture[53] of this

organism being sufficient to set up a fatal septicaemia—these larvae

exhibit a very remarkable natural immunity against the bacilli of

anthrax and diphtheria. A large dose of bacteria of the second anthrax

vaccine, fatal to rabbits, guinea-pigs and mice, is borne without any

inconvenience by the larvae of the Rhinoceros beetle. They are equally

refractory to large doses of the diphtheria bacillus. And yet, there are

not wanting species of insects which are susceptible to these same

micro-organisms. Thus, according to A. Kovalevsky[54], crickets contract

anthrax very readily even at moderate temperatures (22°–23° C.). On the

other hand they are, according to the same author, refractory to the

bacillus of avian tuberculosis. Many of the Invertebrata, studied from

this point of view, present analogous facts, with which, however, we

need not at present occupy ourselves.

In the Vertebrata in general and in Man in particular, natural immunity

against many infective diseases and soluble poisons is so widespread

that we are at no loss to find examples for citation. We have a whole

series of human infections whose study is rendered particularly

difficult simply because of the natural immunity of all other species of

animals from these infections. Such are syphilis, scarlatina, leprosy,

exanthematous typhus, etc. On the other hand, a large number of

diseases, very infective for domestic animals, are quite innocuous to

man. In this group we have cattle plague, strangles, contagious

pleuro-pneumonia, fowl cholera, pneumo-enteritis of pigs, and a number

of other diseases.

As in a very large majority of instances pathogenic organisms act

through the agency of their toxic products, one might believe—and this

has been assumed repeatedly—that natural immunity against infective

diseases is dependent on the insusceptibility of the refractory organism

to the specific poisons.

[Sidenote: [45]]

Such a supposition cannot survive criticism. We have undoubted instances

of a species of animal being resistant both to a micro-organism and to

its toxin. Such instances, however, are rare and usually an organism

that is refractory or only slightly susceptible to the micro-organism

itself is very susceptible to its toxic products. Even those

micro-organisms which come almost constantly in contact with the human

organism without becoming pathogenic, may produce toxins capable of

gravely affecting health. Let us take as an example the bacillus of blue

pus. This organism is most widely diffused in human surroundings.

According to Schimmelbusch[55] it is met with on the skin of the

arm-pits and of the inguinal region of one-half of mankind. From the

skin it very often passes into the dressings of wounds which then assume

the characteristic and so long recognised blue colour. The same bacillus

is also found in the intestines of both sick and healthy persons.

Jakowski[56] has met with it in the faeces coming from intestinal

fistulae in two women who had undergone operations. Now, in spite of

these specially favourable conditions for the production of infection,

the _Bacillus pyocyaneus_ has remained harmless. It is only in children,

and even then rarely, that it can be convicted of exciting disease. Man,

then, usually enjoys a true natural immunity against the _Bacillus

pyocyaneus_. And yet it is not to his insusceptibility to the pyocyanic

toxin that he is indebted for this immunity. Schaffer[57], having

injected himself in the shoulder with half a c.c. of a sterilised

culture of _B. pyocyaneus_, developed fever and an erysipelatous

swelling. Bouchard and Charrin[58] injected pyocyanic toxin into

patients who reacted with more or less fever and by other toxic

symptoms.

[Sidenote: [46]]

Another extremely common saprophyte, the _Micrococcus prodigiosus_, is

incapable of setting up an infective disease, but this does not prevent

its products from exercising a toxic action, often very grave, in man.

The frog, which is refractory to the cholera vibrio, undergoes a fatal

intoxication when cholera toxin is injected. One of the most striking

examples is furnished in the case of the human tubercle bacillus and

tuberculin. Man is much more resistant than is the guinea-pig to the

pathogenic action of this organism, yet he is incomparably more

susceptible to its toxin (tuberculin). According to the researches of

Behring and Kitashima[59], the sheep, of all species of mammals, is most

susceptible to the tubercular poison; the Bovidae and the guinea-pig

occupy an inferior rank in the scale of susceptibility. On the other

hand, the guinea-pig is very susceptible to the tubercle bacillus; the

Bovidae are less so and the sheep is still more resistant to

tuberculosis. It is unnecessary to multiply instances. Immunity against

microbial infection and against intoxication are two distinct

properties, so that it is impossible to reduce the former to an

insusceptibility to toxins. We must therefore consider these two kinds

of immunity separately and we will first consider the resistance of the

animal organism against living infective micro-organisms.

Refractory human beings and animals may be inoculated with a large

number of micro-organisms without being affected. Thus Opitz[60]

injected 10,000,000 organisms into the blood of a dog. Twenty minutes

later he could find no more than 9000. It is then quite natural to ask,

What becomes of these micro-organisms after they have made their way

into the interior of the refractory organism? It has been suggested that

the animal gets rid of the pathogenic germs much as it does of all kinds

of soluble poisons. Certain of these poisons, such as iodine and

alcohol, are in great part eliminated by the kidneys; others, such as

iron, by the alimentary canal. Why, it is asked, should not

micro-organisms also be eliminated by the same channels? Flügge has

adopted this view and has expounded it in his work on ferments and

micro-organisms[61]. Moreover he suggested to Wyssokowitch[62] that he

should carry out a large series of experiments with the object of

verifying this theory. But numerous very careful researches have given a

result quite at variance with the forecast made by Flügge.

Micro-organisms of various species, injected into the blood vessels of

rabbits and dogs, were, in those cases where these animals are

refractory, never eliminated, either by the kidneys or by any other of

the excretory channels which were studied. When bacteria pass into the

secretions, lesions of the tissues, more or less grave, are invariably

present.

[Sidenote: [47]]

This result has been repeatedly confirmed and has been accepted as a

general experience. The elimination of micro-organisms by the urine

indicates not merely the absence of immunity, but implies, also, a

susceptibility of the organism. In many septicaemias, such as those

produced by the anthrax bacillus, the streptococcus and other bacteria,

or in less generalised diseases, such as typhoid fever, bacteria are

found in the urine, often in large numbers. In these cases it is a

question of anything but a refractory condition even of the slightest

degree.

In recent years, however, several works have been published the aim of

which was to demonstrate the inaccuracy of this apparently

well-established thesis. Biedl and Kraus[63] in Vienna took the

initiative and announced in a detailed work that micro-organisms can

readily pass intact into the kidney and that this organ in virtue of its

physiological function eliminates them. The organisms were said to leave

the blood capillaries by the normal process of diapedesis and were then

eliminated with the urine. The liver in a physiological condition,

according to the researches of these authors, is equally capable of

allowing of the passage of micro-organisms; indeed it aids in

discharging them from the system. On the other hand, the pancreas and

the salivary glands were incapable of fulfilling this function. Von

Klecki[64] obtained similar results. He also holds that the kidney is

the principal organ of elimination for micro-organisms which have

penetrated into a refractory organism.

With these contradictions before him, Opitz[65] set himself to study

this question in Flügge’s laboratory at Breslau. Having critically

reviewed the technical methods of his predecessors and carried out a

series of new experiments, he declared categorically “that a

physiological excretion, by the kidneys, of the micro-organisms which

circulate in the blood, does not exist.” For Opitz “the frequent

appearance of micro-organisms in the urine of animals into whose blood,

a short time previously, living bacteria have been injected, is due to

mechanical and chemical lesions of the vessel wall and of the renal

epithelia.”

[Sidenote: [48]]

This question might be looked upon as definitely settled in favour of

the first results obtained by Wyssokowitch were it not that other voices

had been raised in favour of a physiological excretion of the

micro-organisms by the renal channels. Pawlowsky[66] has recently

published a long work on this subject in which he attempts to

demonstrate that certain micro-organisms, even when introduced into the

subcutaneous tissue of animals, pass very rapidly (at the end of a

quarter of an hour) into the uropoietic organs and are eliminated with

the urine.

It was necessary to put an end to these controversies and Métin[67]

undertook a series of researches at the Pasteur Institute with the

object of clearing up this question. He guarded himself against the

objections justly made against his predecessors and conducted his

experiments under unexceptionable conditions. He injected several

species of micro-organisms into the veins of rabbits and into the

subcutaneous tissue of guinea-pigs. At various intervals he performed

laparotomy on these animals, pulled out the bladder and drew off the

urine in such a fashion that no trace of blood could get into it. The

results were most conclusive. Never, when the experiment was conducted

under the rigorous conditions just mentioned, did the micro-organisms

traverse the kidneys of resistant animals nor were they ever met with in

their urine.

Métin’s researches on the passage of micro-organisms through the liver

in refractory animals gave the same results. In no case was he able to

find in the bile any of the organisms that had been injected into the

blood or under the skin. At the end of his memoir Métin sums up his

results as follows: “(1) The kidneys and the liver are impermeable to

bacteria introduced into the organism, subcutaneously or intravenously;

(2) when the culture tubes contain colonies of the injected

micro-organism, it is because there has been a certain amount of blood

in the fluid inoculated, this being an indication of a vascular or

epithelial lesion, either mechanical or chemical.” We were present at M.

Métin’s experiments and can bear witness to their exactitude.

[Sidenote: [49]]

There can no longer be any doubt then on this point. The elimination of

the micro-organisms from the refractory animal takes place, as indicated

in Wyssokowitch’s first investigation, neither by the kidneys nor by the

liver. Some observers have asserted that this elimination may take place

by the sudoriparous glands. Thus, Brunner[68] made experiments with

young pigs and cats into which he had previously injected

micro-organisms, for the most part pathogenic. Then producing a

transpiration by means of pilocarpin, he “cultivated” the sweat and

noted the development of the same bacteria as he had introduced into the

blood. In a single experiment with a saprophyte (_Coccobacillus

prodigiosus_) he obtained a positive result, from which he concludes

that the refractory animal gets rid of bacteria which circulate in its

blood by way of the sudoriparous glands. It is scarcely allowable to

draw any conclusion from this experiment from the fact that the snout of

the pig, the seat of the transpiration, is very liable to small vascular

lesions which might furnish the bacteria that developed on Brunner’s

plates. Nevertheless, even in the case of pathogenic organisms, which

swarm in the blood, the sweat is usually free from them. This has been

shown by Krikliwy[69] in the case of cats inoculated with anthrax whose

sweat, in spite of the passage of numerous bacteria into the

circulation, contained none.

[Sidenote: [50]]

Micro-organisms, then, after their entrance into the refractory animal,

are not eliminated by any of the excretory channels which serve for the

elimination of many of the soluble poisons. It was necessary therefore

to seek some other process capable of affording an explanation of the

disappearance of the micro-organisms which so often and by such varied

means make their way into the interior of a resistant organism. For it

is a well-established fact that in these cases the micro-organisms do

disappear completely. This has been observed so often that it is

unnecessary to offer any demonstration of the fact. Perhaps in the

refractory organism the micro-organisms undergo the fate of the foreign

bodies which penetrate, or which are introduced, into the circulation.

It has long been known, thanks especially to the work of Hoffmann and

Recklinghausen[70], and of Ponfick[71], that particles of carmine or

vermilion when injected into the blood are deposited in several organs.

They are found in the spleen, the lymphatic glands and the bone-marrow.

A certain number of these foreign particles may even be fixed in the

liver and kidneys, but, instead of passing into the bile and the urine,

they remain lodged in the interstitial tissue of the organs. The

observers just cited noted that the coloured granules do not remain long

in either the blood or the lymph but will be found in the interior of

the cellular elements. These granules persist for weeks without any

appreciable modification, differing in this from the micro-organisms

which, as a rule, after several days or even after a few hours,

disappear from the refractory organism. This disappearance might be more

justly compared to the resorption of corpuscular elements which results

in a more or less complete atrophy. The facts concerning the resorption

of pus, of extravasated blood, of the mucosa of the uterus in pregnancy,

etc., have long been known, and it is among these that one should seek

analogies with the disappearance of the micro-organisms. When bacteria

of various species are injected into refractory or not very susceptible

animals, we always observe a local reaction in the form of inflammation,

accompanied by the appearance of white corpuscles. Gradually the

organisms disappear from the point at which they are introduced; the

exudation becomes sterile and ultimately is completely absorbed.

Numerous researches, which will be set forth in the succeeding chapters,

have, indeed, demonstrated the remarkable analogy that exists between

the disappearance of the micro-organisms from the refractory animal and

the resorption of corpuscular elements or of animal cells.

The analysis of the phenomena of this resorption will help us

considerably in our study of immunity against micro-organisms. When in

any part of the animal organism a collection of pus, an effusion of

blood, or any other organic lesion is produced, these lesions are

usually repaired after the lapse of a longer or shorter interval. In

those cases where the cells retain their integrity, they are taken into

the lymphatic vessels and then pass into the circulating blood. In the

course of his researches on the transfusion of blood, Hayem[72] observed

“that blood injected into the peritoneum is absorbed unaltered and

passes with its anatomical elements into the general circulation.” He

was able to demonstrate “that the lymphatic channels play an important

part in this absorption.” Lesage of Alfort[73] confirmed this result. He

found that in the dog “one hour after an abundant haemorrhage into the

peritoneum, induced experimentally, the red corpuscles commenced to pass

freely, without alteration and in very large numbers, into the thoracic

duct.” I have observed a similar resorption of the red blood corpuscles

of the guinea-pig when injected into the peritoneal cavity of other

individuals of the same species. The white corpuscles can also be taken

up by the lymphatic vessels without being modified in any way. At the

end of an inflammatory reaction of feeble intensity, set up in

cold-blooded animals, especially in the tadpole, the direct passage of

leucocytes from the exudation into the lymphatic system may be observed.

[Sidenote: [51]]

The examples I have just cited are, however, quite exceptional. In the

great majority of cases the cellular elements that are undergoing

resorption are seized by the amoeboid cells and are taken into their

substance. Even in the resorption of the red corpuscles, lying free in

the peritoneal cavity of the same species of animal, a certain number of

the globules do not pass directly into the circulation but are first

ingested by the amoeboid elements. This fact is insisted upon by Lesage.

In inflammatory exudations the leucocytes also become the prey of their

fellows. The ingested white corpuscles may be recognised for some time

lying in the interior of other leucocytes; they are soon broken up,

however, and finally disappear completely. When, instead of isolated

cells such as leucocytes, we introduce fragments of tissues or of organs

into any part of the organism, the same mode of resorption may always be

observed. The introduced fragments are first surrounded and infiltrated

by amoeboid cells and are then taken up into their interior.

[Sidenote: [52]]

The mode of absorption just described is very general. It applies to all

kinds of cells and is observed in the absolutely normal organism, as

well as in a large number of pathological conditions. For more than

fifty years, the existence of cells which contain red blood corpuscles

(“blutkörperchenhaltige Zellen” of German writers) has been recognised;

they were met with in the spleen, the lymphatic glands and in many

pathological products. For long we could not explain how the red

corpuscles come to be inside other cells. Virchow[74] thought that they

got there as the result of a mechanical pressure. Later histologists

succeeded in determining the true nature of cells containing red blood

corpuscles and in recognising that the leucocytes had really ingested

the corpuscles. There has been much discussion, also, on the presence of

leucocytes in the interior of large cells in exudations. It was thought

that these were mother-cells which contained a new generation of small

cells. Writers even described a fusion between the large cell and those

found inside it; but Bizzozero[75] first recognised that the former was

an amoeboid cell which had ingested pus corpuscles. Since this

observation was made numerous cases have been described in which

different cell elements have been found in the large cells. There could

no longer be any hesitation in interpreting these cases as instances of

ingestion by leucocytes or similar cells.

The changes that the ingested elements undergo within amoeboid cells may

be compared with those that take place in intracellular digestion. If

the modifications of the particles ingested by the _Amoebae_ be studied

side by side with those which take place in ingested cells in the

process of resorption, a striking analogy may be observed. To establish

this satisfactorily it is essential to begin with a study of

intracellular digestion properly so called, especially as in this

phenomenon we have the fundamental basis of the whole of the theory

developed in this work.

In our first two chapters we have already cited examples of this

intracellular digestion in the Protozoa (_Amoebae_, Infusoria, etc.) and

in the plasmodium stage of the Myxomycetes. In all these cases it goes

on in the organism, in a distinctly acid medium, by the aid of ferments

which could be demonstrated in the _Amoebae_ and Myxomycetes, and which

are analogous sometimes with trypsin, sometimes with pepsin.

In the lower Invertebrata we find the principal source of our knowledge

of intracellular digestion in the digestive organs. This form of

digestion is met with in Sponges, in the whole of the Coelenterates

(Medusae, Siphonophora, Ctenophora, etc.), in the great majority of the

Turbellaria (Planarians, Rhabdocoela), and in certain of the Mollusca

(the lower Gasteropods). In the Invertebrata higher in the animal scale,

intracellular digestion in the digestive organs becomes more and more

rare, and sometimes it manifests itself only in the larval condition

(_Phoronis_); ultimately it gives place permanently to digestion by

juices secreted into the gastro-intestinal canal.

[Sidenote: [53]]

In his sketch of the comparative physiology of digestion, Krukenberg[76]

sought to establish two types: protoplasmic or cellular digestion and

secretory digestion. The former is effected, according to this observer,

by a vital action independently of any production of soluble ferments.

Secretory digestion alone, characteristic of the Vertebrates and of

almost all the higher Invertebrates, is effected by means of these

ferments (diastases or enzymes). Many observers, adopting this view,

maintain that intracellular digestion presents a purely vital phenomenon

essentially different from that of chemical digestion due to juices

containing soluble ferments secreted in the gastro-intestinal canal.

That this theory is absolutely erroneous the succeeding pages of this

work will furnish ample proof.

[Sidenote: [54]]

The Protozoa, from their small size, are unsuitable for researches on

the essential phenomena of intracellular digestion. Amongst animals

higher in the scale the Planarians lend themselves most readily to the

observation of this process. These flat worms are very common in both

fresh and sea water and are easily fed in captivity. They are very

voracious animals and, among other things, devour the blood of man or

animals with avidity. One has merely to allow them to fast for a few

days, and then to give them a drop of blood in order to see their

digestive canal fill itself with this fluid (fig. 6). The white

Planarian, _Dendrocoelum lacteum_, is well adapted for these researches.

In a worm that has sucked blood from a Vertebrate, owing to its great

transparency, the whole length of its intestine with its numerous

ramifications may be seen. For some time this organ remains of a bright

red colour, but gradually the tinge becomes brownish or faintly violet.

These changes of colour recall those observed in effusions of blood in

or under the human skin resulting from contusions. A microscopical

examination of Planarians that have been fed with blood shows that the

coloration of their digestive canal is due to red blood corpuscles in

different stages of digestion. Immediately after the taking in of the

blood by the Planarian all the red blood corpuscles are ingested by the

epithelial cells of the intestine. Connected with the wall by slender

stalks, these elements appear as large amoeboid cells whose free end

projecting into the lumen of the intestine sends out protoplasmic

processes which seize the red blood corpuscles and convey them into the

interior of the cell. This goes on very rapidly, and in a very short

time all the red corpuscles are found within the epithelial cells. As a

result of the increase in volume of these cellular elements the

intestinal cavity is completely occluded.

[Illustration:

FIG. 6. Young Planarian some time after having sucked goose’s blood.

]

[Sidenote: [55]]

[Illustration:

FIG. 7. Intestinal cell of a Planarian, filled with red blood

corpuscles, undergoing digestion, of the goose.

]

Once inside the cells of the intestine the red blood corpuscles exhibit

changes which are readily followed under the microscope. It is better

still to feed the Planarians with the blood of those lower Vertebrates

whose red corpuscles are nucleated. In my researches I have used the

blood of the goose. The red blood corpuscles of this bird, when ingested

by the epithelial cells of the intestine of Planarians, are usually

collected into compact groups (fig. 7), only a few remaining isolated.

The majority of these red corpuscles soon lose their normal appearance

and contour; they become rounded and fused together, but the nucleus and

the haemoglobin enable us to recognise them without any difficulty.

Later the red colouring matter begins to diffuse into the digestive

vacuoles which form around the corpuscles. These corpuscles empty

themselves, retaining their nuclei and capsules, which shrivel more and

more. The nucleus also undergoes almost complete digestion, its

membranous layer alone persisting (fig. 8). Even several days after the

digestion of the blood has begun one can still find _debris_ of

perfectly recognisable red corpuscles, but the red colour has been

replaced by a more or less pronounced brown tint. In the last stage of

the digestive process, as the red corpuscles disappear, the protoplasm

of the intestinal cells becomes filled with round vacuoles, containing

brown irregular concretions—excreta—which are expelled into the

intestinal cavity.

[Illustration:

FIG. 8. Digestion of red blood corpuscles of the goose within an

intestinal cell of a Planarian.

]

This slow digestion of a substance usually so easily assimilable as

blood takes place entirely within the epithelial cells of the intestine.

Continuous microscopical observation demonstrates most clearly the

complete absence of any extracellular digestion of the blood corpuscles

in the intestinal content.

[Sidenote: [56]]

When goose’s blood mixed with blue litmus powder is given to Planarians,

the coloured grains may be found some hours afterwards inside the

epithelial cells of the intestine, but only a few of the blue litmus

granules change colour, taking on a light violet tinge; the great

majority retain their blue coloration. It might be concluded from this

that in Planarians intracellular digestion is effected in a neutral or

nearly neutral medium. If, however, the preparations of intestinal cells

gorged with goose’s blood are treated with a 1% solution of neutral red,

we at once notice that the red corpuscles and the vacuoles which contain

them are stained bright red, assuming a tint similar to that given with

picrocarmine staining (fig. 9). This colour reaction indicates,

according to our researches on neutral red, an acid reaction, more

feeble, however, than that met with in _Paramaecium_ and many other

Protozoa.

[Illustration:

FIG. 9. Portion of an intestinal cell of a Planarian, treated with 1%

neutral red.

]

Macerations of Planarians in normal saline solution to which has been

added a small quantity of the red corpuscles of the goose’s blood

exhibit _in vitro_ a very distinct solvent action on these corpuscles,

which become rounded and lose their haemoglobin, this latter diffusing

into the surrounding fluid, and at the close of the experiment there

remain simply the membranes and the nuclei of the corpuscles.

[Sidenote: [57]]

The study of these Planarians shows us, then, that the food of these

animals undergoes exclusively intracellular digestion in a feebly acid

medium and by means of a soluble ferment, and it furnishes us with proof

that typical intracellular digestion is essentially a chemical process

due to the intervention of enzymes. Now there can be no question, here,

of a protoplasmic action proper, but the branched digestive canal, so

intimately associated with the parenchyma, cannot be completely isolated

from the rest of the Planarian, and it is impossible to study _in vitro_

its digestive action apart from other tissues. To attain this end we

must turn to animals of larger size and those in which the digestive

organs can be isolated more easily. In the Coelenterata intracellular

digestion is general. Many of them are so transparent that they can be

examined _in vivo_. It is easy to observe that the particles of food are

seized by amoeboid processes of the entodermic cells and that they pass

into the substance of these elements there to be digested. For the

systematic study of the digestive phenomena, however, it is not

sufficient merely to examine all that takes place in the living animal.

Experiment _in vitro_ is also necessary. For this purpose the Actinians

or sea-anemones offer us really excellent material. As these animals are

very common in all our seas and are easily kept alive for long periods

in aquaria, they have been used for various researches, among others for

the study of the process of digestion.

[Sidenote: [58]]

The Actinians are easily fed in captivity; they devour morsels of flesh,

of shrimps, of mollusca and other marine animals with avidity. The

ingenious English observers Couch and G. H. Lewes[77] long ago

demonstrated that morsels of food when introduced enclosed in perforated

quills or wrapped in test paper or gutta percha silk and swallowed by

the anemones were afterwards ejected surrounded by mucus but with no

trace of digestion. Having failed in their search for digestive juices

in the large gastric or coelenteric cavity of the Actinians, Lewes

concluded that digestion in these animals is effected in a purely

mechanical fashion. The greatly developed muscles of the Actinians were

supposed to squeeze the food and extract its fluid which is then

absorbed by the walls of the general cavity. It was not until very much

later that the problem of digestion in the Actinians could be resolved

in any accurate and definitive fashion. More than twenty years ago I

demonstrated[78] that the digestion in these polyps is intracellular. In

order that a clear conception of this phenomenon may be obtained it may

be useful to recall in a few words the fundamental features of the

organisation of Actinians. They are cylindrical bodies, sometimes as

large as the fist, attached by their base to stones, shells, or other

submarine objects, and furnished at their free extremity with one or

more series of tentacles. In the middle of this extremity is an

elongated opening, the mouth, which leads into a spacious sac, often

spoken of as the stomach. It is, however, only a kind of oesophagus,

through which the food passes into the large coelenteric cavity which is

divided by septa into numerous compartments lined by the entodermic

epithelium. These septa give origin to many very long and tortuous

filaments, spoken of as mesenterial filaments from their resemblance, a

purely superficial one, to the mesentery of higher animals (fig. 10).

When the Actinian is hungry it protrudes its tentacles in order to seize

marine animals, which it conducts to its mouth. The lips and the

oesophagus are used to estimate the quality of the capture, and if it is

found unsuitable the anemone rejects it, first surrounding it with a

layer of mucus. If however the food is found to be suitable, the

Actinian retains it in its large cavity and throws around it a multitude

of its mesenterial filaments. These penetrate it in all directions, and

as their epithelial cells are capable of sending out amoeboid processes

they seize and ingest the particles, which immediately enter the

protoplasmic content. This work is done with such precision and nicety

that the sea-anemone is able to extract the contents of a shrimp from

the carapace, which latter alone it rejects.

[Illustration:

FIG. 10. Longitudinal section of an Actinian (after Hollard).

]

[Illustration:

FIG. 11. An Actinian in which carmine after absorption has passed into

the mesenterial filaments.

]

[Sidenote: [59]]

The epithelium of the mesenterial filaments is therefore the organ of

digestion in the Actinians. The nutritive parts of their prey pass into

the amoeboid epithelial cells and there undergo a purely intracellular

digestion. If we add to the shrimp-muscle or other food a little carmine

or blue litmus powder, the mesenterial filaments ingest it also and

become pigmented. After eating carmine they assume a very brilliant rose

colour (fig. 11); blue litmus colours them rose violet. This change of

colour in the interior of the cells of the filaments indicates a

decidedly acid reaction of their contents[79]. When one adds to the

mesenterial filaments which are carrying on the process of digestion a

drop of a 1% solution of neutral red they assume various shades of red

(fig. 12).

[Illustration:

FIG. 12. Portion of mesenterial filament of an Actinian, stained with

1% neutral red.

]

This intracellular digestion in the Actinians has been confirmed by

several observers, amongst whom may be cited Chapeaux[80] and

Bjelooussoff[81]. It has often been asserted, however, that, along with

a digestion in the interior of the cells of the mesenterial filaments,

there is, in the Actinians, a secretion in the coelenteric cavity of

their body of fluids which digest nutritive matter by means of a soluble

ferment. A ferment similar to trypsin has been extracted from Actinians

by Léon Frédéricq and Krukenberg. But, in presence of contradictory

assertions, it remained undecided whether, in the enzymatic digestion,

this ferment does its work in the fluid of the coelenteric cavity or

whether it represents the active factor in intracellular digestion.

With the object of definitely elucidating a problem of such general

importance, Mesnil, the superintendent of my laboratory, has been good

enough to carry out a fresh series of experiments on the digestion of

the Actinians and has studied this process not only in animals kept in

captivity in aquaria but also in Actinians living under natural

conditions in the sea[82].

[Sidenote: [60]]

As intracellular digestion is of interest to us specially in connection

with the resorption of formed elements in the tissues and cavities of

animals, Mesnil directed his attention to the digestion of the red

corpuscles of the blood. He made use of the red corpuscles of several

species of Vertebrata, but he made a special study of the digestion of

nucleated red blood corpuscles. These corpuscles are very delicate, and

may even undergo a certain degree of maceration in ordinary sea water.

In spite of this these red corpuscles are not digested in the

coelenteric cavity of the Actinians but, once ingested by the entodermic

cells of the mesenterial filaments, they are completely dissolved by the

intracellular digestion. Mesnil also observed that fibrin is not

digested except in the cells of the filaments. The facts cited by

Chapeaux in favour of an extracellular digestion in the fluid of the

coelenteric cavity in no way support his hypothesis, and reduce

themselves, according to Mesnil, to a digestion by the diastase of blood

itself fixed by the fibrin, after the bleeding, at the moment of the

formation of the clot.

For a certain period the red corpuscles may be met with inside the cells

of the mesenterial filaments. They are ingested in their normal

state—oval red corpuscles with a nucleus. As several hours are required

for the ingestion, it is evident that the fluid of the coelenteric

cavity has been incapable of attacking the red corpuscles. In the

protoplasm of the entodermic cells the red corpuscles become rounded,

their walls become permeable, and the haemoglobin begins to diffuse from

them. It passes first into the vacuoles of the digestive cells and is

then, in part, ejected into the general body cavity. The haemoglobin is

transformed into a green substance which reminds one of biliary pigment.

The membranes and nuclei of the red corpuscles are also digested and

ultimately disappear completely.

The digestive cells of the entoderm ingest not only blood corpuscles or

fibrin, but also fragments of muscular fibre and particles of carmine

and litmus. These latter, as already stated, indicate a marked acid

reaction.

[Sidenote: [61]]

In the Actinians, then, the mesenterial filaments, or rather their

entodermic portion, represent the real organ of intracellular digestion.

There are indeed other regions of the entoderm which also carry on this

function, but in an insignificant degree as compared with the

mesenterial filaments which are capable, however, not only of ingesting

and digesting solid substances, but also of absorbing solutions. Mesnil

has demonstrated this by injecting soluble colouring matters, such as

eosin, carminate of ammonia, etc., into Actinians. These solutions,

although in great part absorbed by the digestive cells of the

mesenterial filaments, can, however, also be retained by other elements,

amongst others, the cells of the ectoderm.

As the digestion of the food-particles goes on within the entodermic

cells of the mesenterial filaments and as these organs can easily be

isolated from the rest of the Actinian, Mesnil was able to study with

great precision and care the phenomena of digestion outside the

organism. With this object he prepared extracts of the filaments in sea

water and studied their action on various nutritive substances. He

confirmed the discovery of a soluble ferment made by Léon Frédéricq and

demonstrated that it is capable of digesting albuminoid substances

(fibrin, coagulated albumen) in media which are neutral, slightly

alkaline or weakly acid. In this respect the _actino-diastase_ (the name

given by Mesnil to the soluble ferment of the Actinians) approaches most

nearly to papain. On the other hand, it is distinguished by its greater

sensitiveness to an excess of acid and also by its more powerful action

on coagulated albumen.

The actino-diastase acts vigorously at any temperature between 15° and

20° C., but the optimum temperature for its digestive action is between

36° and 45° C. Higher temperatures weaken the diastatic power, and

heating to 55–60° C. inhibits it completely. Among the products of the

digestion of albuminoids by actino-diastase, Mesnil, like his

predecessors, found not only a notable quantity of peptone but also

products of the disintegration of the albuminoid molecule, such as

tyrosin and proteino-chromogen. Consequently actino-diastase resembles

Mouton’s amoebo-diastase in certain respects.

[Sidenote: [62]]

The nucleated red blood corpuscles of the lower Vertebrata are very

convenient objects on which to observe the process of intracellular

digestion within the cells of the mesenterial filaments. Mesnil has also

studied them _in vitro_ under the influence of actino-diastase. Under

these conditions the phenomena of digestion recall very clearly those

that have been observed within the digestive cells. The oval red

corpuscles of the fowl and goose become spherical as a result of the

solvent action on their membrane, and the haemoglobin diffuses into the

fluid. The membranes and the nuclei of the corpuscles are, however,

little altered and may be recognised under the microscope. The

difference between this and digestion within the cells reduces itself to

a more feeble digestive action of the aqueous extract. It is evident

that the preparation of this extract is only capable of bringing into

prominence a certain proportion of the actino-diastase contained in the

entodermic cells of the filaments.