Charles Bradfield Morrey

Pathogenic Bacteriology treats of the unicellular microÖrganisms which are responsible for disease conditions, i.e., pathological changes in other organisms. Hence not only are bacteria considered, but also other low vegetable forms, as yeasts and molds, likewise protozoa in so far as they may be pathogenic. For this reason the term pathogenic “Microbiology” has been introduced to include all these organisms. It is largely for the reason that the methods devised for the study of bacteria have been applied to the investigation of other microÖrganisms that the term “bacteriology” was extended to cover the entire field. The general discussion in this chapter is intended to include, therefore, microÖrganisms of whatever kind pathogenic to animals.

The term pathogenic as applied to an organism must be understood in a purely relative sense, since there is no single organism that can cause disease in all of a certain class, but each is limited to a more or less narrow range. Some form of tuberculosis attacks nearly all vertebrates, but no other classes of animals and no plants. Lockjaw or tetanus attacks most mammals, but not any other vertebrates naturally. Typhoid fever affects human beings; hog cholera, swine, etc. This point is more fully discussed in Chapter XXIII but can not be too greatly insisted upon.

“The greatest enemy to mankind is man.”

Exceptions to this statement do occur and are important and must be considered in efforts to protect completely human beings from disease (tuberculosis from cattle, glanders from horses, poisoning from spoiled canned goods, anthrax from hair, hides, wool, of animals dead of the disease), but the most common human diseases are derived from other human beings directly or indirectly.

Diseases which are due to unicellular pathogenic microÖrganisms are called infectious diseases, while if such diseases are transmitted under natural conditions from organism to organism they are spoken of as contagious diseases. Most infectious diseases are contagious but not all. Tetanus is a good illustration of a non-contagious infectious disease. There are very few such diseases.

When a unicellular microÖrganism gains entrance into the body and brings about any pathological changes there, the result is an infection. Undoubtedly many pathogenic organisms get into the body but never manifest their presence by causing disease conditions, hence do not cause an infection. It is the pathological conditions which result that constitute the infection, and not the mere invasion.

The time that elapses between the entrance of the organism and the appearance of symptoms is called the period of incubation and varies greatly in different diseases.

The term infestation is used to denote pathological conditions due to multicellular parasites. Thus an animal is infested (not infected) with tapeworms, roundworms, lice, mites, etc. Many of these conditions, probably all, are contagious, i.e., transmissable naturally from animal to animal. The word contagious has been used in a variety of ways to mean communicated by direct contact, communicated by a living something (contagium) that might be carried to a distance and finally communicable in any manner, transmissable. The agency of transmission may be very roundabout—as through a special tick in Texas fever, a mosquito in malaria, etc.,—or by direct personal contact, as generally in venereal diseases. After all, though exactness is necessary, it is better to learn all possible about the means of transmission of diseases, than quibble as to the terms to be used. An infectious disease may be acute or chronic. An acute infection is one which runs for a relatively short time and is “self-limited,” so-called, i.e., the organisms cease to manifest their presence after a time. In some acute infections the time is very short—German measles usually runs five or six days. Typhoid fever may continue eight to ten weeks, sometimes longer, yet it is an acute infectious disease. It is not so much the time as the fact of self-limitation that characterizes acute infections.

In chronic infections there is little or no evidence of limitation of the progress of the disease which may continue for years. Tuberculosis is usually chronic. Leprosy in man is practically always so. Glanders in horses is most commonly chronic; in mules and in man it is more apt to be acute.

Many infections begin acutely and later change to the chronic type. Syphilis in man is a good illustration.

The differences between acute and chronic infections are partly due to the nature of the organism, partly to the number of organisms introduced and the point of their introduction and partly to the resistance of the animal infected.

An infectious disease is said to be specific when one kind of organism is responsible for its manifestations—as diphtheria due to the Corynebacterium diphtheriÆ, lockjaw due to Clostridium tetani, Texas fever due to the Piroplasma bigeminum, etc. It is non-specific when it may be due to a variety of organisms, as enteritis (generally), bronchopneumonia, wound infections.

Henle, as early as 1840, stated certain principles that must be established before a given organism can be accepted as the cause of a specific disease. These were afterward restated by Koch, and have come to be known as “Koch’s postulates.” They may be stated as follows:

1. The given organism must be found in all cases of the disease in question.

2. No other organism must be found in all cases.

3. The organism must, when obtained in pure culture, reproduce the disease in susceptible animals.

4. It must be recovered from such animals in pure culture and this culture likewise reproduce the disease. These postulates have not been fully met with reference to any disease, but the principles embodied have been applied as far as possible in all those infections which we recognize as specific, and whose causative agent is accepted. In many diseases recognized as infectious and contagious no organism has been found which is regarded as the specific cause. In some of these the organism appears to be too small to be seen with the highest powers of the microscope, hence they are called “ultramicroscopic” organisms. Because these agents pass through the finest bacterial filters, they are also frequently called “filterable.” The term “virus” or “filterable virus” is likewise applied to these “ultramicroscopic” and “filterable” agents.

The term primary infection is sometimes applied to the first manifestation of a disease, either specific or non-specific, while secondary refers to later developments. For example, a secondary general infection may follow a primary wound infection, or primary lung tuberculosis be followed by secondary generalized tuberculosis, or primary typhoid fever by a secondary typhoid pneumonia. The terms primary and secondary are also used where the body is invaded by one kind of an organism and later on by another kind; thus a primary measles may be followed by secondary infection of the middle ear, or a primary influenza may be followed by a secondary pneumonia, or a primary scarlet fever by a secondary nephritis (inflammation of the kidney). Where several organisms seem to be associated simultaneously in causing the condition then the term mixed infection is used—in severe diphtheria, streptococci are commonly associated with the Corynebacterium diphtheriÆ. In many cases of hog-cholera, mixed infections in the lungs and in the intestines are common. Wound infections are usually mixed. Auto-infection refers to those conditions in which an organism commonly present in or on the body in a latent or harmless condition gives rise to an infectious process. If the Bacterium coli normal to the intestine escapes into the peritoneal cavity, or passes into the bladder, a severe peritonitis or cystitis, respectively, is apt to result. “Boils” and “pimples” are frequently autoinfections. Such infections are also spoken of as endogenous to distinguish them from those due to the entrance of organisms from without—exogenous infections. Relapses are usually instances of autoinfection.

Those types of secondary infection where the infecting agent is transferred from one disease focus to another or several other points and sets up the infection there are sometimes called metastases. Such are the transfer of tubercle bacilli from lung to intestine, spleen, etc., the formation of abscesses in internal organs following a primary surface abscess, the appearance of glanders nodules throughout various organs following pulmonary glanders, etc.

The characteristic of a pathogenic microÖrganism which indicates its ability to cause disease is called its virulence. If slightly virulent, the effect is slight; if highly virulent, the effect is severe, and may be fatal.

On the other hand, the characteristic of the host which indicates its capacity for infection is called susceptibility. If slightly susceptible, infection is slight, if highly susceptible, the infection is severe.

Evidently the degree of infection is dependent in large measure on the relation between the virulence of the invading organism and the susceptibility of the host. High virulence and great susceptibility mean a severe infection; low virulence and little susceptibility a slight infection; while high virulence and little susceptibility or low virulence and great susceptibility might mean a moderate infection varying in either direction. Other factors influencing the degree of infection are the number of organisms introduced, the point where they are introduced and various conditions. These will be discussed in another connection (Chapter XXV).

The study of pathogenic bacteriology includes the thorough study of the individual organisms according to the methods already given (Chapters XVIII–XXI) as an aid to diagnosis and subsequent treatment, bacteriological or other, in a given disease. Of far greater importance than the treatment, which in most infectious diseases is not specific, is the prevention and ultimate eradication of all infectious diseases. To accomplish these objects involves further a study of the conditions under which pathogenic organisms exist outside the body, the paths of entrance into and elimination from the body and those agencies within the body itself which make it less susceptible to infection or overcome the infective agent after its introduction. That condition of the body itself which prevents any manifestation of a virulent pathogenic organism after it has been once introduced is spoken of as immunity in the modern sense. Immunity is thus the opposite of susceptibility and may exist in varying degrees.

That scientists are and have been for some years in possession of sufficient knowledge to permit of the prevention and eradication of most, if not all, of our infectious diseases can scarcely be questioned. The practical application of this knowledge presents many difficulties, the chief of which is the absence of a public sufficiently enlightened to permit the expenditure of the necessary funds. Time and educative effort alone can surmount this difficulty. It will probably be years yet, but it will certainly be accomplished.

Pathogenic bacteria may exist outside the body of the host under a variety of conditions as follows:

1. I. In or on inanimate objects or material.
   1. (a) As true saprophytes.
   2. (b) As facultative saprophytes.
   3. (c) Though obligate parasites, they exist in a latent state.
2. II. In or on other animals, or products from them:
   1. A. Susceptible to the disease.
      1. (a) Sick themselves. (As far as human beings are concerned these are mainly:
         1. 1. Other human beings for most diseases.
         2. 2. Rats for plague.
         3. 3. Dogs for rabies.
         4. 4. Horses for glanders.
         5. 5. Cattle, swine, parrots for tuberculosis).
      2. (b) Recovered from illness.
      3. (c) Never sick but “carriers.”
   2. B. Not susceptible.
      1. (d) Accidental carriers.
      2. (e) Serving as necessary intermediate hosts for certain stages of the parasite—this applies to protozoal diseases only, as yet.

I.

(a) The bacilli of tetanus, malignant edema and the organisms of “gas gangrene” are widely distributed. There is no evidence that their entrance into the body is at all necessary for the continuation of their life processes, or that one case of either of these diseases ever has any connection with any other case; they are true saprophytes. Manifestly it would be futile to attempt to prevent or eradicate such diseases by attacking the organism in its natural habitat. Clostridium botulinum, which causes a type of food poisoning in man, does not even multiply in the body, but the disease symptoms are due to a soluble toxin which is produced during its growth outside the body.

(b) Organisms like the bacterium of anthrax and the bacillus of black-leg from their local occurrence seem to be distributed from animals infected, though capable of a saprophytic existence outside the body for years. These can no more be attacked during their saprophytic existence than those just mentioned. Doubtless in warm seasons of the year and in the tropics other organisms pathogenic to animals may live and multiply in water or in damp soil where conditions are favorable, just as the cholera organism in India, and occasionally the typhoid bacillus in temperate climates do.

(c) Most pathogenic organisms, however, when they are thrown off from the bodies of animals, remain quiescent, do not multiply, in fact always tend to die out from lack of all that is implied in a “favorable environment,” food, moisture, temperature, light, etc. Disinfection is sometimes effective in this class of diseases in preventing new cases.

II. A.

(a) The most common infectious diseases of animals are transmitted more or less directly from other animals of the same species. Human beings get nearly all their diseases from other human beings who are sick; horses, from other horses; cattle, from other cattle; swine, from swine, etc. Occasionally transmission from one species to another occurs. Tuberculosis of swine most frequently results from feeding them milk of tuberculous cattle or from their eating the droppings of such cattle. Human beings occasionally contract anthrax from wool, hair and hides of animals dead of the disease or from postmortems on such animals; glanders from horses; tuberculosis (in children) from tuberculous milk; bubonic plague from rats; rabies practically always from the bites of dogs and other rabid animals, etc. The mode of limiting this class of diseases is evidently to isolate the sick, disinfect their discharges and their immediate surroundings, sterilize such products as must be handled or used, kill lower animals that are dangerous, and disinfect, bury properly, or destroy their carcasses.

Classes of the sick that are especially dangerous for the spread of disease are the mild cases and the undetected cases. These individuals do not come under observation and hence not under control.

(b) This class of carriers offers a difficult problem in the prevention of infectious diseases since they may continue to give off the organisms indefinitely and thus infect others. Typhoid carriers have been known to do so for fifty-five years. Cholera, diphtheria, meningitis and other carriers are well known in human practice. Carriers among animals have not been so frequently demonstrated, but there is every reason for thinking that hog-cholera, distemper, roup, influenza and other carriers are common. Carriers furnish the explanation for many of the so-called “spontaneous” outbreaks of disease among men and animals.

It is the general rule that those who are sick cease to carry the organisms on recovery and it is the occasional ones who do not that are the exceptions. In those diseases in which the organism is known it can be determined by examination of the patient or his discharges how long he continues to give off the causative agent. In those in which the cause is unknown (in human beings, the commonest and most easily transmitted diseases, scarlet fever, measles, German measles, mumps, chicken-pox, small-pox, influenza), no such check is possible. It is not known how long such individuals remain carriers. Hence isolation and quarantine of such convalescents is based partly on experience and partly on theory. It is highly probable that in the diseases just mentioned transmission occurs in the early stages only, except in small-pox and chicken-pox where the organism seems to be in the pustules and transmission by means of material from these is possible, though only by direct contact with it. The fact that such individuals are known to have had the disease is a guide for control. The methods to be used are essentially the same as for the sick, (a), though obviously such human carriers are much more difficult to deal with since they are well.

(c) Another class of carriers is those who have never had the disease. Such individuals are common and are very dangerous sources of infection. Many of them have associated with the sick or with convalescents and these should always be suspected of harboring the organisms. Their control differs in no way from that of class (b). Unfortunately a history of such association is too often not available. Modern transportation and modern social habits are largely responsible for the nearly universal distribution of this type of carrier. Their detection is probably the largest single problem in the prevention of infectious diseases. A partial solution would be universal bacteriological examination. In our present stage of progress this is impossible and would not detect carriers of diseases of unknown cause.

The various classes of carriers just discussed are in a large part responsible for the continued presence of the commoner diseases throughout the country. The difficulties in control have been mentioned. A complete solution of the problem is not yet obtained. The army experience of the past few years in the control of infectious diseases shows what may be done.

There is another class of carriers which might be called the “universal carrier,” i.e., there are certain organisms which seem to be constantly or almost constantly present in or on the human body. These are micrococci, streptococci and pneumococci, all Gram positive organisms. They are ordinarily harmless parasites, but on occasion may give rise to serious, even fatal, infection. Infected wounds, pimples, boils, “common colds,” most “sore throats,” bronchitis, pneumonia are pathological conditions that come in this class. Such infections are usually autogenous. There is a constant interchange of these organisms among individuals closely associated, so that all of a group usually harbor the same type though no one individual can be called the carrier. Whenever, for any reason, the resistance of an individual (see Chaps. XXV et seq.) is lowered either locally or generally some of these organisms are liable to gain a foothold and cause infection. It sometimes happens that a strain of dangerous organisms may be developed in an individual in this way which is passed around to others with its virulence increased and thus cause an epidemic. Or, since all of the group are living under the same conditions the resistance of all or many of them may be lowered from the same general cause and an epidemic result from the organism common to all (pneumonia after measles, scarlet fever and influenza in camps). Protection of the individual is chiefly a personal question, i.e., by keeping up the “normal healthy tone” in all possible ways: The use of protective vaccines (Chap. XXX) appears to be advisable in such instances (colds, pneumonia after measles and influenza, inflammation of throat and middle ear following scarlet fever and measles). Results obtained in this country during the recent influenza epidemic have been conflicting but on the whole appear to show that preventive vaccination against pneumonia liable to follow should be practiced.

It would seem that among groups of individuals where infection may be expected the proper procedure would be to prepare autogenous vaccines (Chapter XXX) from members of the group and vaccinate all with the object of protecting them.

II. B.

(d) In this class come the “accidental carriers” like flies, fleas, lice, bed-bugs, ticks, and other biting and blood-sucking insects, vultures, buzzards, foxes, rats, and carrion-eating animals generally; pet animals in the household, etc. Here the animals are not susceptible to the given disease but become contaminated with the organisms and then through defilement of the food or drink or contact with individuals or with utensils pass the organisms on to the susceptible. Some biting and blood-sucking insects transmit the organisms through biting infected and non-infected animals successively. The spirilloses and trypanosomiases seem to be transmitted in this way, though there is evidence accumulating which may place these diseases in the next class. Anthrax is considered in some instances to be transmitted by flies and by vultures in the southern United States. Transmission of typhoid, dysentery, cholera and other diseases by flies is well established in man. Why not hog-cholera from farm to farm by flies, English sparrows, pigeons feeding, or by turkey buzzards? Though this would not be easy to prove, it seems reasonable.

Preventing contact of such animals with the discharges or with the carcasses of those dead of the disease, destruction of insect carriers, screening and prevention of fly breeding are obvious protective measures.

(e) In this class come certain diseases for which particular insects are necessary for the parasite in question, so that certain stages in its life history may be passed therein. The surest means for eradicating such diseases is the destruction of the insects concerned. Up to the present no bacterial disease is known in which this condition exists, unless Rocky Mountain spotted fever and typhus fever shall prove to be due to bacteria. Such diseases are all due to protozoa. Among them are Texas fever, due to Piroplasma bigeminum in this country which has been eradicated in entire districts by destruction of the cattle tick (Margaropus annulatus).

Piroplasmoses in South Africa among cattle and horses, and in other countries are transmitted in similar ways. Probably many of the diseases due to spirochetes and trypanosomes are likewise transmitted by necessary insect intermediaries. In human medicine the eradication of yellow fever from Panama and Cuba is due to successful warfare against, a certain mosquito (Stegomyia). So the freeing of large areas in different parts of the world from malaria follows the destruction of the mosquitoes. The prevention of typhus fever and of trench fever by “delousing” methods is familiar from recent army experience though for typhus this method has been practiced in Russia for more than ten years to the author’s personal knowledge. The campaign against disease in animals and man from insect sources must be considered as still in its infancy. The full utilization of tropical lands depends largely on the solution of this problem.

A. The Skin.—If the skin is healthy there is no opportunity for bacteria to penetrate it. It is protected not only by the stratified epithelium, but also in various animals, by coats of hair, wool, feathers, etc. The secretion pressure of the healthy sweat and oil glands acts as an effective bar even to motile bacteria. Nevertheless a very slight injury only is sufficient to give normal surface parasites and other pathogenics, accidentally or purposely brought in contact with it, an opportunity for more rapid growth and even entrance for general infection. Certain diseases due to higher fungi are characteristically “skin diseases” and rarely become general—various forms of favus, trichophyton infections, etc. A few disease organisms, tetanus, malignant edema, usually get in through the skin; others, black-leg, anthrax, quite commonly; and those diseases transmitted by biting and blood-sucking insects, piroplasmoses, trypanosomiases, spirilloses, scarcely in any other way. Defective secretion in the skin glands from other causes, may permit lodgment and growth of bacteria in them or in the hair follicles. “Pimples” and boils in man and local abscesses occasionally in animals are illustrations. Sharp-edged and freely bleeding wounds are less liable to be infected than contusions, ragged wounds, burns, etc. The flowing blood washes out the wound and the clotting seals it, while there is less material to be repaired by the leukocytes and they are free to care for invading organisms (phagocytosis). Pathogenic organisms, especially pus cocci, frequently gain lodgment in the milk glands and cause local (mastitis) or general infection.

B. MucosÆ directly continuous with the skin and lined with stratified epithelium are commonly well protected thereby and by the secretions.

(a) The external auditory meatus is rarely the seat even of local infection. The tympanic cavity is normally sterile, though it may become infected by extension through the Eustachian tube from the pharynx (otitis media).

(b) The conjunctiva is frequently the seat of localized, very rarely the point of entrance for a generalized infection, except after severe injury. Those diseases whose path of entrance is generally assumed to be the respiratory tract (see “Lungs” below) might also be admitted through the eye. Material containing such organisms might get on the conjunctiva and be washed down through the lachrymal canal into the nose. Experiment has shown that bacteria may pass in this way in a few minutes. In case masks are worn to avoid infection from patients suffering with these diseases, the eyes should therefore be protected as well as the nose and mouth.

(c) The nasal cavity on account of its anatomical structure retains pathogenic organisms which give rise to local infections more frequently than other mucosÆ of its character. These may extend from here to middle ear, neighboring sinuses, or along the lymph spaces of the olfactory nerve into the cranial cavity (meningitis). Acute coryza (“colds” in man) is characteristic. Glanders, occasionally, is primary in the nose, as is probably roup in chickens, leprosy in man. The meningococcus and the virus of poliomyelitis pass from the nose into the cranial cavity without local lesions in the former.

(d) The mouth cavity is ordinarily protected by its epithelium and secretions, though the injured mucosa is a common source of actinomycosis infection, as well as thrush. In foot-and-mouth disease no visible lesions seem necessary to permit the localization of the unknown infective agent. (e) The tonsils afford a ready point of entrance for ever-present micrococci and streptococci whenever occasion offers (follicular tonsillitis, “quinsy”), and articular rheumatism is not an uncommon sequel. The diphtheria bacillus characteristically seeks these structures for its development. Tubercle and anthrax organisms occasionally enter here.

(f) The pharynx is the seat of localized infection as in micrococcal, streptococcal and diphtherial “sore throat” in human beings, but both it and the esophagus are rarely infected in animals except as the result of injury.

(g) The external genitalia are the usual points of entrance for the venereal organisms in man (gonococcus, Treponema pallidum, and Ducrey’s bacillus). The bacillus of contagious abortion and probably the trypanosome of dourine are commonly introduced through these channels in animals.

C. Lungs.—The varied types of pneumonia due to many different organisms (tubercle, glanders, influenza, plague bacilli, pneumococcus, streptococcus, micrococcus and many others) show how frequently these organs are the seat of a localized infection, which may or may not be general. Whether the lungs are the actual point of entrance in these cases is a question which is much discussed at the present time, particularly with reference to tuberculosis. The mucous secretion of the respiratory tract tends to catch incoming bacteria and other small particles and the ciliary movement along bronchial tubes and trachea tends to carry such material out. “Foreign body pneumonia” shows clinically, and many observers have shown experimentally that microÖrganisms may reach the alveoli even though the exchange of air between them and the bronchioles and larger bronchi takes place ordinarily only by diffusion. The presence of carbon particles in the walls of the alveoli in older animals and human beings and in those that breathe dusty air for long periods indicates strongly, though it does not prove absolutely, that these came in with inspired air. On the other hand, experiment has shown that tubercle bacilli introduced into the intestine may appear in the lungs and cause disease there and not in the intestine. It is probably safe to assume that in those diseases which are transmitted most readily through close association though not necessarily actual contact, the commonest path is through the respiratory tract, which may or may not show lesions (smallpox, scarlet fever, measles, chicken-pox, whooping-cough, pneumonic plague in man, lobar and bronchopneumonias and influenza in man and animals, some cases of glanders and tuberculosis). On the other hand, the fact that the Bacterium typhosum and Bacterium coli may cause pneumonia when they evidently have reached the lung from the intestinal tract, and the experimental evidence of lung tuberculosis above mentioned show that this route cannot be excluded in inflammations of the lung.

D. Alimentary Tract.—The alimentary tract affords the ordinary path of entrance for the causal microbes of many of the diseases of animals and man, since they are carried into the body most commonly and most abundantly in the food and drink.

(a) The stomach is rarely the seat of local infection, even in ruminants, except as the result of trauma. The character of the epithelium in the rumen, reticulum and omasum in ruminants, the hydrochloric acid in the abomasum and in the stomachs of animals generally are usually sufficient protection. Occasionally anthrax “pustules” develop in the gastric mucosa. (The author saw nine such pustules in a case of anthrax in a man.)

(b) The intestines are frequently the seat of localized infections, as various “choleras” and “dysenteries” in men and many animals, anthrax, tuberculosis, Johne’s disease. Here doubtless enter the organisms causing “hemorrhagic septicemias” in many classes of animals, and numerous others. These various organisms must have passed through the stomach and the question at once arises, why did the HCl not destroy them? It must be remembered that the acid is present only during stomach digestion, and that liquids taken on an “empty stomach” pass through rapidly and any organisms present are not subjected to the action of the acid. Also spores generally resist the acid. Other organisms may pass through the stomach within masses of undigested food. The fact that digestion is going on in the stomach of ruminants practically all the time may explain the relative freedom of adult animals of this class from “choleras” and “dysenteries.”

MECHANISM OF ENTRANCE OF ORGANISMS.

In the preceding chapters statements have been made that “bacteria enter” at various places or they “pass through” different mucous membranes, skin, etc. Strictly speaking such statements are incorrect—bacteria do not “enter” or “pass through” of themselves. It is true that some of the intestinal organisms are motile, but most of the bacteria which are pathogenic are non-motile. Even the motile ones can not make their way against fluids secreted or excreted on free surfaces. Bacteria cannot pass by diffusion through membranes since they are finite particles and not in solution.

In the case of penetrating wounds bacteria may be carried mechanically into the tissues, but this is exceptional in most infections. Also after gaining lodgment they may gradually grow through by destroying tissue as they grow, but this is a minor factor. Evidently, there must be some mechanism by which they are carried through. The known mechanisms for this in the body are ameboid cells, especially the phagocytes. It is most probable that these are the chief agents in getting bacteria into the tissues through various free surfaces. The phagocytes engulf bacteria, carry them into the tissues and either destroy them, are destroyed by them, or may disgorge or excrete them free in the tissues or in the blood.

DISSEMINATION OF ORGANISMS.

Dissemination of organisms within the tissues occurs either through the lymph channels or the bloodvessels or both. If through the lymph vessels only it is usually much more restricted in extent, or much more slowly disseminated, while blood dissemination is characterized by the number of organs involved simultaneously.

PATHS OF ELIMINATION OF PATHOGENIC MICROÖRGANISMS.

I. Directly from the point, of injury. This is true in infected wounds open to the surface, skin glanders (farcy), black-leg, surface anthrax, exanthemata in man and animals (scarlet fever (?), measles (?), smallpox; hog erysipelas, foot-and-mouth disease): also in case of disease of mucous membranes continuous with the skin—from nasal discharges (glanders), saliva (foot-and-mouth disease), material coughed or sneezed out (tuberculosis, influenza, pneumonias), urethral and vaginal discharges (gonorrhea and syphilis in man, contagious abortion and dourine in animals), intestinal discharges (typhoid fever, “choleras,” “dysenteries,” anthrax, tuberculosis, Johne’s disease). Material from nose, mouth and lungs may be swallowed and the organisms passed out through the intestines.

II. Indirectly through the secretions and the excretions where the internal organs are involved. The saliva of rabid animals contains the ultramicroscopic virus of rabies (the sympathetic ganglia within the salivary glands, and pancreas also, are affected in this disease as well as the cells of the central nervous system). The gall-bladder in man is known to harbor colon and typhoid bacilli, as that of hog-cholera hogs does the virus of this disease. It may harbor analogous organisms in other animals, though such knowledge is scanty. The kidneys have been shown experimentally to excrete certain organisms introduced into the circulation within a few minutes (micrococci, colon and typhoid bacilli, anthrax). Typhoid bacilli occur in the urine of typhoid-fever patients in about 25 per cent. of all cases and the urine of hogs with hog cholera is highly virulent. Most observers are of the opinion, however, that under natural conditions the kidneys do not excrete bacteria unless they themselves are infected.

The milk both of tuberculous cattle and tuberculous women has been shown to contain tubercle bacilli even when the mammary glands are not involved. Doubtless such bacteria are carried through the walls of the secreting tubules or of the smaller ducts by phagocytes and are then set free in the milk.

SPECIFICITY OF LOCATION OF INFECTIVE ORGANISMS.

It is readily apparent that certain disease organisms tend to locate themselves in definite regions and the question arises, Is this due to any specific relationship between organism and tissue or not? Diphtheria in man usually attacks the tonsils first, gonorrhea and syphilis the external genitals, tuberculosis the lung, “choleras” the small intestine, “dysenteries” the large intestine, influenza the lungs. In these cases the explanation is probably that the points attacked are the places where the organism is most commonly carried, with no specific relationship, since all of these organisms (Asiatic cholera excepted) also produce lesions in other parts of the body when they reach them. On the other hand, the virus of hydrophobia attacks nerve cells, leprosy frequently singles out nerves, glanders bacilli introduced into the abdominal cavity of a young male guinea-pig cause an inflammation of the testicle, malarial parasites and piroplasms attack the red blood corpuscles, etc. In fact, most pathogenic protozoa are specific in their localization either in certain tissue cells or in the blood or lymph. In these cases there is apparently a real chemical relationship, as there is also between the toxins of bacteria and certain tissue cells (tetanus toxin and nerve cells). Whether “chemotherapy” will ever profit from a knowledge of such chemical relationships remains to be developed. It appears that a search for these specific chemical substances with the object of combining poisons with them so that the organisms might in this way be destroyed, would be a profitable line of research.

Immunity, as has already been stated, implies such a condition of the body that pathogenic organisms after they have been introduced are incapable of manifesting themselves, and are unable to cause disease. The word has come to have a more specific meaning than resistance in many instances, in other cases the terms are used synonymously. It is the opposite of susceptibility. The term must be understood always in a relative sense, since no animal is immune to all pathogenic organisms, and conceivably not entirely so to anyone, because there is no question that a sufficient number of bacteria of any kind might be injected into the circulation to kill an animal, even though it did it purely mechanically.

Immunity may be considered with reference to a single individual or to entire divisions of the organic world, with all grades between. Thus plants are immune to the diseases affecting animals; invertebrates to vertebrate diseases; cold-blooded animals to those of warm blood; man is immune to most of the diseases affecting other mammals; the rat to anthrax, which affects other rodents and most mammals; the well-known race of Algerian sheep is likewise immune to anthrax while other sheep are susceptible; the negro appears more resistant to yellow fever than the white; some few individuals in a herd of hogs always escape an epizoÖtic of hog cholera, etc.

Immunity within a given species is modified by a number of factors—age, state of nutrition, extremes of heat or cold, fatigue, excesses of any kind, in fact, anything which tends to lower the “normal healthy tone” of an animal also tends to lower its resistance. Children appear more susceptible to scarlet fever, measles, whooping-cough, etc., than adults; young cattle more frequently have black-leg than older ones (these apparently greater susceptibilities may be due in part to the fact that most of the older individuals have had the diseases when young and are immune for this reason). Animals weakened by hunger or thirst succumb to infection more readily. Frogs and chickens are immune to tetanus, but if the former be put in water and warmed up to and kept, at about 37°, and the latter be chilled for several hours in ice-water, then each may be infected. Pneumonia frequently follows exposure to cold. The immune rat may be given anthrax if first he is made to run in a “squirrel cage” until exhausted. Alcoholics are far less resistant to infection than temperate individuals. “Worry,” mental anguish, tend to predispose to infection.

The following outlines summarize the different, classifications of immunity so far as mammals are concerned for the purposes of discussion.

1. I. Natural
   1. A. Congenital
      1. 1. Inherited through the germ cell or cells.
      2. 2. Acquired in utero.
         1. (a) By having the disease in utero.
         2. (b) By absorption of immune substances from the mother.
   2. B. Acquired by having the disease.
2. II. Artificial—acquired through human agency by:
   1. 1. Introduction of the organism or its products.
   2. 2. Introduction of the blood serum of an immune animal.

1. I. Active—due to the introduction of the organism or due to the introduction of the products of the organism.
   1. A. Naturally by having the disease.
   2. B. Artificially.
      1. 1. By introducing the organism:
         1. (a) Alive and virulent.
         2. (b) Alive and virulence reduced by
            1. 1. Passage through another animal.
            2. 2. Drying.
            3. 3. Growing at a higher temperature.
            4. 4. Heating the cultures.
            5. 5. Treating with chemicals.
            6. 6. Sensitizing.
            7. 7. Cultivation on artificial media.
         3. (c) Dead.
      2. 2. By introducing the products of the organism.
2. II. Passive—due to the introduction of the blood serum of an actively immunized animal.

Immunity present in an animal and not due to human interference is to be regarded as natural immunity, while if brought about by man’s effort it is considered artificial. Those cases of natural immunity mentioned above which are common to divisions, classes, orders, families, species or races of organisms and to those few individuals where no special cause is discoverable, must be regarded as instances of true inheritance through the germ cell as other characteristics are. All other kinds of immunity are acquired. Occasionally young are born with every evidence that they have had a disease in utero and are thereafter as immune as though the attack had occurred after birth (“small-pox babies,” “hog-cholera pigs”). Experiment has shown that immune substances may pass from the blood of the mother to the fetus in utero and the young be immune for a time after birth (tetanus). This is of no practical value as yet. It is a familiar fact that with most infectious diseases recovery from one attack confers a more or less lasting immunity, though there are marked exceptions.

Active Immunity.—By active immunity is meant that which is due to the actual introduction of the organism, or in some cases of its products. The term active is used because the body cells of the animal immunized perform the real work of bringing about the immunity as will be discussed later. In passive immunity the blood serum of an actively immunized animal is introduced into a second animal, which thereupon becomes immune, though its cells are not concerned in the process. The animal is passive, just as a test-tube, in which a reaction takes place, plays no other part than that of a passive container for the reagents.

In active immunity the organism may be introduced in what is to be considered a natural manner, as when an animal becomes infected, has a disease, without human interference. Or the organism may be purposely introduced to bring about the immunity. For certain purposes the introduction of the products of the organism (toxins) is used to bring about active immunity (preparation of diphtheria and tetanus antitoxin from the horse). The method of producing active immunity by the artificial introduction of the organism is called vaccination, and a vaccine must therefore contain the organism. Vaccines for bacterial diseases are frequently called bacterins. The use of the blood serum of an immunized animal to confer passive immunity on a second animal is properly called serum therapy, and the serum so used is spoken of as an antiserum, though the latter word is also used to denote any serum containing any kind of an antibody (Chapters XXVII–XXXI). In a few instances both the organism and an antiserum are used to cause both active and passive immunity (serum-simultaneous method in immunizing against hog cholera).

In producing active immunity the organism may be introduced (a) alive and virulent, but in very small doses, or in combination with an immune serum, as just mentioned for hog cholera. The introduction of the live virulent organism alone is done only experimentally as yet, as it is obviously too dangerous to do in practice, except under the strictest control (introduction of a single tubercle bacillus, followed by gradually increasing numbers—Barber and Webb). More commonly the organisms are introduced (b) alive but with their virulence reduced (“attenuated”) in one of several ways: (1) By passing the organism through another animal as is the case with smallpox vaccine derived from a calf or heifer. This method was first introduced by Jenner in 1795 and was the first practical means of preventing disease by vaccination. This word was used because material was derived from a cow—Latin vacca. (2) By drying the organism, as is done in the preparation of the vaccine for the Pasteur treatment of rabies, where the spinal cords of rabbits are dried for varying lengths of time—one to four days, Russian method, one to three days, German method, longer in this country. (It is probable that the passage of the “fixed virus” through the rabbit is as important in this procedure as the drying, since it is doubtful if the “fixed virus” is pathogenic for man.) It would be more correct to speak of this as a preventive vaccination against rabies, since the latter is one of the few diseases which is not amenable to treatment. The patient always dies if the disease develops. (3) The organism may be attenuated by growing at a temperature above the normal. This is the method used in preparing anthrax vaccine as done by Pasteur originally. (4) Instead of growing at a higher temperature the culture may be heated in such a way that it is not killed but merely weakened. Black-leg vaccines are made by this method. (5) Chemicals are sometimes added to attenuate the organisms, as was formerly done in the preparation of black-leg vaccine by Kruse’s method in Germany. The use of toxin-antitoxin mixtures in immunizing against diphtheria and in the preparation of diphtheria antitoxin from horses is an application of the same principle, though here it is the product of the organism and not the organism whose action is weakened. (6) Within the past few years the workers in the Pasteur Institute in Paris have been experimenting with vaccines prepared by treating living virulent bacteria with antisera (“sensitizing them”) so that they are no longer capable of causing the disease when introduced, but do cause the production of an active immunity. The method has been used with typhoid fever bacilli in man and seems to be successful. It remains to be tried out further before its worth is demonstrated (the procedure is more complicated and the chance for infection apparently much greater than by the use of killed cultures). The term sero-bacterins is used by manufacturers in this country to designate such bacterial vaccines. (7) Growing on artificial culture media reduces the virulence of most organisms after a longer or shorter time. This method has been tried with many organisms in the laboratory, but is not now used in practice. The difficulties are that the attenuation is very uncertain and that the organisms tend to regain their virulence when introduced into the body.

In producing active immunity against many bacterial diseases the organisms are introduced (c) dead. They are killed by heat or by chemicals, or by using both methods (Chapter XXX).

When the products of an organism are introduced the resulting immunity is against the products only and not against the organism. If the organism itself is introduced there results an immunity against it and in some cases also against the products, though the latter does not necessarily follow. Hence the immunity may be antibacterial or antitoxic or both.

Investigation as to the causes of immunity and the various methods by which it is produced has not resulted in the discovery of specific methods of treatment for as many diseases as was hoped for at one time. Just at present progress in serum therapy appears to be at a standstill, though vaccines are giving good results in many instances not believed possible a few years ago. As a consequence workers in all parts of the world are giving more and more attention to the search for specific chemical substances, which will destroy invading parasites and not injure the host (chemotherapy). Nevertheless, in the study of immunity very much of value in the treatment and prevention of disease has been learned. Also much knowledge which is of the greatest use in other lines has been accumulated. Methods of diagnosis of great exactness have resulted, applicable in numerous diseases. Ways of detecting adulteration in foods, particularly foods from animal sources, and of differentiating proteins of varied origin, as well as means of establishing biological relationships and differences among groups of animals through “immunity reactions” of blood serums have followed from knowledge gained by application of the facts or the methods of immunity research. Hence the study of “immunity problems” has come to include much more than merely the study of those factors which prevent the development of disease in an animal or result in its spontaneous recovery. A proper understanding of the principles of immunity necessitates a study of these various features and they will be considered in the discussion to follow.

Pasteur and the bacteriologists of his time discovered that bacteria cease to grow in artificial culture media after a time, because of the exhaustion of the food material in some cases and because of the injurious action of their own products in other instances. These facts were brought forward to explain immunity shortly after bacteria were shown to be the cause of certain diseases. Theories based on these observations were called (1) “Exhaustion Theory” of Pasteur, and (2) “Noxious Retention Theory” of Chauveau respectively. The fact, soon discovered, that virulent pathogenic bacteria are not uncommonly present in perfectly healthy animals, and the later discovery that immunity may be conferred by the injection of dead bacteria have led to the abandonment of both these older ideas. The (3) “Unfavorable Environment” theory of Baumgartner, i.e., bacteria do not grow in the body and produce disease because their surroundings are not suitable, in a sense covers the whole ground, though it is not true as to the first part, as was pointed out above, and is of no value as a working basis, since it offers no explanation as to what the factors are that constitute the “unfavorable environment.” Metchnikoff brought forward a rational explanation of immunity with his (4) “Cellular or Phagocytosis Theory.” As first propounded it based immunity on the observed fact that certain white blood corpuscles, phagocytes, engulf and destroy bacteria. Metchnikoff has since elaborated the original theory to explain facts of later discovery. Ehrlich soon after published his (5) “Chemical or Side-chain Theory” which seeks to explain immunity on the basis of chemical substances in the body which may in part destroy pathogenic organisms or in part neutralize their products; or in some instances there may be an absence of certain chemical substances in the body cells so that bacteria or their products cannot unite with the cells and hence can do no damage.

At the present time it is generally accepted, in this country at least, that Ehrlich’s theory explains immunity in many diseases as well as many of the phenomena related to immunity, and in other diseases the phagocytes, frequently assisted by chemical substances, are the chief factors. Specific instances are discussed in Pathogenic Bacteriologies which should be consulted. It is essential that the student should be familiar with the basic ideas of the chemical theory, not only from the standpoint of immunity, but also in order to understand the principles of a number of valuable methods of diagnosis.

The chemical theory rests on three fundamental physiological principles: (1) the response of cells to stimuli, in this connection specific chemical stimuli, (2) the presence within cells of specific chemical groups which combine with chemical stimuli and thus enable them to act on the cell, which groups Ehrlich has named receptors, and (3) the “over-production” activity of cells as announced by Weigert.

1. That cells respond to stimuli is fundamental in physiology. These stimuli may be of many kinds as mechanical, electrical, light, thermal, chemical, etc. The body possesses groups of cells specially developed to receive some of these stimuli—touch cells for mechanical stimuli, retinal cells for light, temperature nerve endings for thermal, olfactory and gustatory cells for certain chemical stimuli. Response to chemical stimuli is well illustrated along the digestive tract. That the chemical stimuli in digestion may be more or less specific is shown by the observed differences in the enzymes of the pancreatic juice dependent on the relative amounts of carbohydrates, fats, or proteins in the food, the specific enzyme in each case being increased in the juice with the increase of its corresponding foodstuff. The cells of the body, or certain of them at least, seem to respond in a specific way when substances are brought into direct contact with them, that is, without having been subjected to digestion in the alimentary tract, but injected directly into the blood or lymph stream. Cells may be affected by stimuli in one of three ways: if the stimulus is too weak, there is no effect (in reality there is no “stimulus” acting); if the stimulus is too strong, the cell is injured, or may be destroyed; if the stimulus is of proper amount then it excites the cell to increased activity, and in the case of specific chemical stimuli the increased activity, as mentioned for the pancreas, shows itself in an increased production of whatever is called forth by the chemical stimulus. In the case of many organic chemicals, the substances produced by the cells under their direct stimulation are markedly specific for the particular substance introduced.

2. Since chemical action always implies at least two bodies to react, Ehrlich assumes that in every cell which is affected by a chemical stimulus there must therefore be a chemical group to unite with this stimulus. He further states that there must be as many different kinds of these groups as there are different kinds of chemicals which stimulate the cell. Since these groups are present in the body cells to take up different kinds of chemical substances, Ehrlich calls them receptors. Since these groups must be small as compared with the cell as a whole, and must be more or less on the surface and unite readily with chemical substances he further speaks of them as “side-chains” after the analogy of compounds of the aromatic series especially. The term receptors is now generally used. As was stated above, the effect of specific chemical stimuli is to cause the production of more of the particular substance for which it is specific and in the class of bodies under discussion, the particular product is these cell receptors with which the chemical may unite.

3. Weigert first called attention to the practically constant phenomenon that cells ordinarily respond by doing more of a particular response than is actually called for by the stimulus, that there is always an “overproduction” of activity. In the case of chemical stimuli this means an increased production of the specific substance over and above the amount actually needed. The student will better understand this theory if he recalls his fundamental physiology. Living substance is characterized, among other things, by irritability which is instability. It is in a constant, state of unstable equilibrium. Whenever the equilibrium becomes permanently stable the substance is dead. It is also continually attempting to restore disturbances in its equilibrium. Whenever a chemical substance unites with a chemical substance in the cell, a receptor, the latter is, so far as the cell is concerned, thrown out of function for that cell. The chemical equilibrium of the latter is upset. It attempts to restore this and does so by making a new receptor to take the place of the one thrown out of function. If this process is continued, i.e., if the new receptor is similarly “used up” and others similarly formed are also, then the cell will prepare a supply of these and even an excess, according to Weigert’s theory. Whenever a cell accumulates an excess of products the normal result is that it excretes them from its own substance into the surrounding lymph, whence they reach the blood stream to be either carried to the true excretory organs, utilized by other cells or remain for a longer or shorter time in the blood. Hence the excess of receptors is excreted from the cell that forms them and they become free in the blood. These free receptors are termed antibodies. They are receptors but instead of being retained in the cell are free in solution in the blood. One function of the free receptor, the antibody, is always to unite with the chemical substance which caused it to be formed. It may have additional functions. The chemical substance which caused the excess formation of receptors, antibodies, is termed an antigen for that particular kind of antibody.

To recapitulate, Ehrlich’s theory postulates specific chemical stimuli, which react with specific chemical substances in the body cells, named receptors, and that these receptors, according to Weigert, are produced in excess and hence are excreted from the cell and become free receptors in the blood and lymph. These free receptors are the various kinds of antibodies, the kind depending on the nature of the stimulus, antigen, the substance introduced. Any substance which when introduced into the body causes the formation of an antibody of any kind whatsoever is called an antigen,23 i.e., anti (body) former.

The foregoing discussion explains Ehrlich’s theory of immunity. According to this theory the manner of formation of all antibodies is the same. The kind of antibody and the manner of its action will differ with the different kinds of antigens used.

The succeeding chapters discuss some of the kinds of antibodies, the theory of their action and some practical applications. It must be borne in mind throughout the study of these, as has been stated, that every antibody has the property of uniting with its antigen whether it has any property in addition or not.

Just what antibodies are chemically has not been determined because no one has as yet succeeded in isolating them chemically pure. To the author they appear to be enzymes.

Antigens were considered by Ehrlich to be proteins or to be related to proteins. Most workers since Ehrlich have held similar views. Dr. Carl Warden of the University of Michigan has been doing much work in recent years in which he is attempting to show that the antigens are not proteins but are fats or fatty acids. Mr. E.E.H. Boyer, in his work (not yet published) in the author’s laboratory for the degree of Ph.D., received in June, 1920, succeeded in producing various antibodies from Bacterium coli antigens. In these antigens he could detect only fatty acids or salts of fatty acids. If the work of these men is confirmed, it will open up a most interesting and extremely important field in immunity and in preventive medicine. It is not apparent that the nature of the antigen would affect Ehrlich’s theory of the formation of antibodies.

The author has no doubt that eventually the formation of antibodies and the reactions between them and their antigens will be explained on the basis of physical-chemical laws, but this probably awaits the discovery of their nature.

The general characteristics of toxins have been described (Chapter XII). It has been stated that they are more or less specific in their action on cells. In order to affect a cell it is evident that a toxin must enter into chemical combination with it. This implies that the toxin molecule possesses a chemical group which can combine with a receptor of the cell. This group is called the haptophore or combining group. The toxic or injurious portion of the toxin molecule is likewise spoken of as the toxophore group. When a toxin is introduced into the body its haptophore group combines with suitable receptors in different cells of the body. If not too much of the toxin is given, instead of injuring, it acts as a chemical stimulus to the cell in the manner already described. The cell in response produces more of the specific thing, which in this instance is more receptors which can combine with the toxin, i.e., with its haptophore group. If the stimulus is kept up, more and more of these receptors are produced until an excess for the cell accumulates, which excess is excreted from the individual cell and becomes free in the blood. These free receptors have, of course, the capacity to combine with toxin through its haptophore group. When the toxin is combined with these free receptors, it cannot combine with any other receptors, e.g., those in another cell and hence cannot injure another cell. These free receptors constitute, in this case, antitoxin, so-called because they can combine with toxin and hence neutralize it. Antitoxins are specific—that is, an antitoxin which will combine with the toxin of Clostridium tetani will not combine with that of Corynebacterium diphtheriÆ or of Clostridium botulinum, or of any other toxin, vegetable or animal. When a toxin is kept in solution for some time or when it is heated above a certain temperature (different for each toxin) it loses its poisonous character. It may be shown, however, that it is still capable of uniting with antitoxin, and preventing the latter from uniting with a fresh toxin. This confirms the hypothesis that a toxin molecule has at least two groups: a combining or haptophore, and a poisoning or toxophore group. A toxin which has lost its poisonous property, its toxophore group, is spoken of as a toxoid. The theory of antitoxin formation is further supported by the fact that the proper introduction of toxoid, the haptophore group, and hence the real stimulus, can cause the production of antitoxin to a certain extent at least.

The close relationship between toxins and enzymes has already been pointed out. This is still further illustrated by the fact that when enzymes are properly introduced into the tissues of an animal there is formed in the animal an antienzyme specific for the enzyme in question which can prevent its action. The structure of enzymes, as composed of a haptophore, or uniting, and a zymophore or digesting (or other activity) group, is similar to that of toxins, and enzymoids or enzymes which can combine with the substance acted on but not affect it further, have been demonstrated.

These free cell receptors, antitoxins or antienzymes, which are produced in the body by the proper introduction of toxins or enzymes, respectively, have the function of combining with these bodies but no other action. As was pointed out above, this is sufficient to neutralize the toxin or enzyme and prevent any injurious effect since they can unite with nothing else. Since these receptors are the simplest type which has been studied as yet, they are spoken of by Ehrlich as receptors of the first order. Other antibodies which are likewise free receptors of the first, order and have the function of combining only have been prepared and will be referred to in their proper connection. They are mainly of theoretical interest.

Ehrlich did a large part of his work on toxins and antitoxins with ricin, the toxin of the castor-oil bean, abrin, from the jequirity bean, robin from the locust tree, and with the toxins and antitoxins for diphtheria and tetanus. Antitoxins have been prepared experimentally for a large number of both animal and vegetable poisons, including a number for bacterial toxins. The only ones which, as yet, are of much practical importance are antivenin for snake poison, (not a true toxin, however, see p.275), antipollenin (supposed to be for the toxin of hay fever) and the antitoxins for the true bacterial toxins of Corynebacterium diphtheriÆ and Clostridium tetani.

The method of preparing antitoxins is essentially the same in all cases, though differing in minor details. For commercial purposes large animals are selected, usually horses, so that the yield of serum may be large. The animals must, of course, be vigorous, free from all infectious disease. The first injection given is either a relatively small amount of a solution of toxin or of a mixture of toxin and antitoxin. The animal shows more or less reaction, increased temperature, pulse and respiration and frequently an edema at the point of injection, unless this is made intravenously. After several days to a week or more, when the animal has recovered from the first injection, a second stronger dose is given, usually with less reaction. Increasingly large doses are given at proper intervals until the animal may take several hundred times the amount which would have been fatal if given at first. The process of immunizing a horse for diphtheria or tetanus toxin usually takes several months. Variations in time and in yield of antitoxin are individual and not predictable in any given case.

After several injections a few hundred cubic centimeters of blood are withdrawn from the jugular vein and serum from this is tested for the amount of antitoxin it contains. When the amount is found sufficiently large (250 “units” at least for diphtheria per cc.)24 then the maximum amount of blood is collected from the jugular with sterile trocar and cannula. The serum from this blood with the addition of an antiseptic (0.5 per cent. phenol, tricresol, etc.) constitutes “antidiphtheritic serum” or “antitetanic serum,” etc. All sera which are put on the market must conform to definite standards of strength expressed in “units” as determined by the U.S. Hygienic Laboratory. In reality a “unit” of diphtheria antitoxin in the United States is an amount equivalent to 1cc. of a given solution of a standard diphtheria antitoxin which is kept at the above-mentioned laboratory. This statement, of course, gives no definite idea as to the amount of antitoxin actually in a “unit.” Specifically stated, a “unit” of antitoxin contains approximately the amount which would protect a 250 gram guinea-pig from 100 minimum lethal doses of diphtheria toxin, or protect 100 guinea-pigs weighing 250 grams each from one minimum lethal dose each. The minimum lethal dose (M.L.D.) of diphtheria toxin is the least amount that will kill a guinea-pig of the size mentioned within four days. Since toxins on standing change into toxoids to a great extent, the amount, of antitoxin in a “unit,” though protecting against 100 M.L.D., in reality would protect against about 200 M.L.D. of toxin containing no toxoid.

The official unit for tetanus antitoxin is somewhat different, since it is standardized against a standard toxin which is likewise kept at the Hygienic Laboratory. The unit is defined as “ten times the amount of antitoxin necessary to protect a 350 g. guinea-pig for 96 hours against the standard test dose” of the standard toxin. The standard test dose is 100 M.L.D. of toxin for a 350 g. guinea-pig. To express it another way, one could say that a “unit” of tetanus antitoxin would protect one thousand 350 g. guinea-pigs from 1 M.L.D. each of standard tetanus toxin.

Various methods have been devised for increasing the amount of antitoxin in 1cc. of solution by precipitating out portions of the blood-serum proteins and at the same time concentrating the antitoxin in smaller volume. It is not considered necessary in a work of this character to enter into these details nor to discuss the process of standardizing antitoxin so that the exact amount of “units” per cc. may be known.

Charrin and Rogers appear to have been the first (1889) to observe the clumping together of bacteria (Pseudomonas pyocyanea) when mixed with the blood serum of an animal immunized against them. Gruber and Durham (1896) first used the term “agglutination” in this connection and called the substance in the blood-serum “agglutinin.” Widal (1896) showed the importance of the reaction for diagnosis by testing the blood serum of an infected person against a known culture (typhoid fever).

It is now a well-known phenomenon that the proper injection of cells of any kind foreign to a given animal will lead to the accumulation in the animal’s blood of substances which will cause a clumping together of the cells used when suspended in a suitable liquid. The cells settle out of such suspension much more rapidly than they would otherwise do. This clumping is spoken of as “agglutination” and the substances produced in the animal are called “agglutinins.” If blood cells are injected then “hemagglutinins” result: if bacterial cells “bacterial agglutinins” for the particular organism used as “glanders agglutinin” for Pfeifferella mallei, “abortion agglutinin” for Bacterium abortus, “typhoid agglutinin” for Bacterium typhosum, etc.

The phenomenon may be observed either under the microscope or in small test-tubes, that is, either microscopically or macroscopically.

In this case the cells introduced, or more properly, some substances within the cells, act as stimuli to the body cells of the animal injected to cause them to produce more of the specific cell receptors which respond to the stimulus. The substance within the introduced cell which acts as a stimulus (antigen) to the body cells is called an “agglutinogen.” That “agglutinogen” is present in the cell has been shown by injecting animals experimentally with extracts of cells (bacterial and other cells) and the blood serum of the animal injected showed the presence of agglutinin for the given cell. It will be noticed that the receptors which become the free agglutinins have at least two functions, hence at least two chemical groups. They must combine with the foreign cells and also bring about their clumping together, their agglutination. Hence it can be stated technically that an agglutinin possesses a haptophore group and an agglutinating group.

It is probable that the agglutination, the clumping, is a secondary phenomenon depending on the presence of certain salts and that the agglutinin acts on its antigen as an enzyme, possibly a “splitting” enzyme. This is analogous to what occurs in the curdling of milk by rennet and in the coagulation of blood. This probability is substantiated by the fact that suspensions of bacteria may be “agglutinated” by appropriate strengths of various acids.

The formation of agglutinin in the body for different bacteria does not as yet appear to be of any special significance in protecting the animal from the organism, since the bacteria are not killed, even though they are rendered non-motile, if of the class provided with flagella, and are clumped together. The fact that such bodies are formed, however, is of decided value in the diagnosis of disease, and also in the identification of unknown bacteria.

In many bacterial diseases, agglutinins for the particular organism are present in the blood serum of the affected animal. Consequently if the blood serum of the animal be mixed with a suspension of the organism supposed to be the cause of the disease and the latter be agglutinated, one is justified in considering it the causative agent, provided certain necessary conditions are fulfilled. In the first place it must be remembered that the blood of normal animals frequently contains agglutinins (“normal agglutinins”) for many different bacteria when mixed with them in full strength. Hence the serum must always be diluted with physiological salt solution (0.85 per cent.). Further, closely related bacteria may be agglutinated to some extent by the same serum. It is evident that if they are closely related, their protoplasm must contain some substances of the same kind to account for this relationship. Since some of these substances may be agglutinogens, their introduction into the animal body will give rise to agglutinins for the related cells, as well as for the cell introduced. The agglutinins for the cell introduced “chief agglutinins,” will be formed in larger quantity, since a given bacterial cell must contain more of its own agglutinogen than that of any other cell. By diluting the blood serum from the animal to be tested the agglutinins for the related organisms (so-called “coagglutinins” or “partial agglutinins”) will become so much diminished as to show no action, while the agglutinin for the specific organism is still present in an amount sufficient to cause its clumping. Agglutinins are specific for their particular agglutinogens, but since a given blood serum may contain many agglutinins, the serum’s specificity for a given bacterium can be determined only by diluting it until this bacterium alone is agglutinated. Hence the necessity of diluting the unknown serum in varying amounts when testing against several known bacteria to determine for which it is specific, i.e., which is the cause of the disease in the animal.

The agglutinins in the serum may be removed from it by treating it with a suspension of the cells for which agglutinins are present. If the “chief” cell is used all the agglutinins will be absorbed. If related cells are used, only the agglutinins for this particular kind are removed. These “absorption tests” furnish another means of determining specificity of serum, or rather of determining the “chief agglutinin” present.

Just as an unidentified disease in an animal may be determined by testing its serum as above described against known kinds of bacteria, so unknown bacteria isolated from an animal, from water, etc., may be identified by testing them against the blood sera of different animals, each of which has been properly inoculated with a different kind of known bacteria. If the unknown organism is agglutinated by the blood of one of the animals in high dilution, and not by the others, evidently the bacterium is the same as that with which the animal has been inoculated, or immunized, as is usually stated. This method of identifying cultures of bacteria is of wide application, but is used practically only in those cases where other methods of identification are not readily applied, and especially where other methods are not sufficient as in the “intestinal group” of organisms in human practice.

The diagnosis of disease in an animal by testing its serum is also a valuable and much used procedure. This is the method of the “Widal” or “Gruber-Widal” test for typhoid fever in man and is used in veterinary practice in testing for glanders, contagious abortion, etc. In some cases a dilution of the serum of from 20 to 50 times is sufficient for diagnosis (Malta fever), in most cases, however, 50 times is the lowest limit. Evidently the greater the dilution, that is, the higher the “titer,” the more specific is the reaction.

PRECIPITINS.

Since agglutinins act on bacteria, probably through the presence of substances within the bacterial cell, it is reasonable to expect that if these substances be dissolved out of the cell, there would be some reaction between their (colloidal) solution and the same serum. As a matter of fact Kraus (1897) showed that broth cultures freed from bacteria by porcelain filters do show a precipitate when mixed with the serum of an animal immunized against the particular bacterium and that the reaction is specific under proper conditions of dilution. It was not long after Kraus’s work until the experiments were tried of “immunizing” an animal not against a bacterium or its filtered culture, but against (colloidal) solutions of proteins, such as white of egg, casein of milk, proteins of meat and of blood serum, vegetable proteins, etc. It was ascertained that in all these cases the animal’s serum contains a substance which causes a precipitate with solutions of the protein used for immunization. The number of such precipitating serums that have been made experimentally is very large and it appears that protein from any source when properly introduced into the blood or tissues of an animal will cause the formation of a precipitating substance for its solutions. This substance is known, technically as a “precipitin.” The protein used as antigen to stimulate its formation, or some part of the protein molecule (haptophore group), which acts as stimulus to the cell is spoken of as a “precipitinogen,” both terms after the analogy of “agglutinin” and “agglutinogen.” In fact the specific precipitation and agglutination are strictly analogous phenomena. Precipitins act on proteins in (colloidal) solution and cause them to settle out, agglutinins act on substances within cells which cells are in suspension in a fluid and cause the cells to settle out. Ehrlich’s theory of the formation of precipitins is similar to that of agglutinins, and need not be repeated. Substitute the corresponding words in the theory of formation of agglutinins as above given and the theory applies.

The precipitin reaction has not found much practical use in bacteriology largely because the “agglutination test” takes its place as simpler of performance and just as accurate. The reaction is, however, generally applicable to filtrates of bacterial cultures and could be used if needed. The so-called “mallease” reaction in glanders is an instance.

Precipitins find their greatest usefulness in legal medicine and in food adulteration work. As was noted above, if animals, rabbits for example, are immunized with the blood of another animal (human beings) precipitins are developed which are specific for the injected blood with proper dilution. This forms an extremely valuable means of determining the kind of blood present in a given spot shown by chemical and spectroscopic tests to be blood and has been adopted as a legal test in countries where such rules of procedure are applied. Similarly the test has been used to identify the different kinds of meat in sausage, and different kinds of milk in a mixture. An extract of the sausage is made and tested against the serum of an animal previously treated with extract of horse meat, or hog meat, or beef, etc., the specific precipitate occurring with the specific serum. Such reactions have been obtained where the protein to be tested was diluted 100,000 times and more. Biological relationships and differences have been detected by the reaction. Human immune serum shows no reaction with the blood of any animals except to a slight extent with that of various monkeys, most with the higher, very slight with the lower Old World and scarcely any with New World monkeys.

It is a fact of theoretical interest mainly that if agglutinins and precipitins themselves be injected into an animal they will act as antigens and cause the formation of antiagglutinins or antiprecipitins, which are therefore receptors of the first order since they simply combine with these immune bodies to neutralize their action, have only a combining or haptophore group. Also if agglutinins or precipitins be heated to the proper temperature they may retain their combining power but cause no agglutination or precipitation, i.e., they are converted into agglutinoid or precipitinoid respectively after the analogy of toxin and toxoid.

Precipitins like agglutinins possess at least two groups—a combining or haptophore group and a precipitating (sometimes called zymophore) group. Hence they are somewhat more complex than antitoxins or antienzymes which have a combining group only. For this reason Ehrlich classes agglutinins and precipitins as receptors of the second order.

Before Koch definitely proved bacteria capable of causing disease several physiologists had noted that the red corpuscles of certain animals were destroyed by the blood of other animals (Creite, 1869, Landois, 1875), and Traube and Gescheidel had shown that freshly drawn blood destroys bacteria (1874). It was not until about ten years afterward that this action of the blood began to be investigated in connection with the subject of immunity. Von Fodor (1885) showed that saprophytic bacteria injected into the blood are rapidly destroyed. FlÜgge and his pupils, especially Nuttall in combating Metchnikoff’s theory of phagocytosis, announced in 1883, studied the action of the blood on bacteria and showed its destructive effect (1885–57). Nuttall also showed that the blood lost this power if heated to 56°. Buchner (1889) gave the name “alexin” (from the Greek “to ward off”) to the destroying substance and showed that the substance was present in the blood serum as well as in the whole blood, and that when the serum lost its power to dissolve, this could be restored by adding fresh blood. Pfeiffer (1894) showed that the destructive power of the blood of animals immunized against bacteria (cholera and typhoid) was markedly specific for the bacteria used. He introduced a mixture of the blood and the bacteria into the abdominal cavity of the immunized animal or of a normal one of the same species and noted the rapid solution of the bacteria by withdrawing portions of the peritoneal fluid and examining them (“Pfeiffer’s phenomenon”). Belfanti and Carbone and especially Bordet (1898) showed the specific dissolving action of the serum of one animal on the blood corpuscles of another animal with which it had been injected. Since this time the phenomenon has been observed with a great variety of cells other than red blood corpuscles and bacteria—leukocytes, spermatozoa, cells from liver, kidney, brain, epithelia, etc., protozoa, and many vegetable cells.

It is therefore a well-established fact that the proper injection of an animal with almost any cell foreign to it will lead to the blood of the animal injected acquiring the power to injure or destroy cells of the same kind as those introduced. The destroying power of the blood has been variously called its “cytotoxic” or “cytolytic” power, though the terms are not strictly synonymous since “cytotoxic” means “cell poisoning” or “injuring,” while “cytolytic” means “cell dissolving.” The latter term is the one generally used and there is said to be present in the blood a specific “cytolysin.” The term is a general one and a given cytolysin is named from the cell which is dissolved, as a bacteriolysin, a hemolysin (red-corpuscle-lysin), epitheliolysin, nephrolysin (for kidney cells), etc. If the cell is killed but not dissolved the suffix “cidin” or “toxin” is frequently used as “bacteriocidin,” “spermotoxin,” “neurotoxin,” etc.

The use of the term “cytolysin” is also not strictly correct, though convenient, for the process is more complex than if one substance only were employed. As was stated above, the immune serum loses its power to dissolve the cell if it is heated to 55° to 56° for half an hour, it is inactivated. But if there be added to the heated or inactivated serum a small amount of normal serum (which contains only a very little cytolytic substance, so that it has no dissolving power when so diluted) the mixture again becomes cytolytic. It is evident then that in cytolysis there are two distinct substances involved, one which is present in all serum, normal or immune, and the other present only in the immune cytolytic serum. This may be more apparent if the facts are arranged in the following form:

1. I. Immune serum dissolves cells in high dilution.
2. II. Heated immune serum does not dissolve cells.
3. III. Normal serum in high dilution does not dissolve cells.
4. II. + III., i.e., Heated immune serum plus diluted normal serum dissolves cells.

Therefore, there is something in heated immune serum necessary for cell dissolving and something different in diluted normal serum which is necessary. This latter something is present in unheated immune serum also, and is destroyed by heat. Experiment has shown that it is the substance present in all serum both normal and immune that is the true dissolving body, while the immune substance serves to unite this body to the cell to be destroyed, i.e., to the antigen. Since the immune body has therefore two uniting groups, one for the dissolving substance and one for the cell to be dissolved, Ehrlich calls it the “amboceptor.” He also uses the word “complement” to denote the dissolving substance, giving the idea that it completes the action of dissolving after it has been united to the cell by the amboceptor, thus replacing Buchner’s older term “alexin” for the same dissolving body.

AMBOCEPTORS.

The theory of formation of amboceptors is similar to that for the formation of the other types of antibodies. The cell introduced contains some substance, which acts as a chemical stimulus to some of the body cells provided with proper receptors so that more of these special receptors are produced, and eventually in excess so that they become free in the blood and constitute the free amboceptors. It will be noticed that these free receptors differ from either of the two kinds already described in that they have two uniting groups, one for the antigen (cell introduced) named cytophil-haptophore, the other for the complement, complementophil haptophore. Hence amboceptors are spoken of as receptors of the third order. They have no other function than that of this double combining power. The action which results is due to the third body—the complement. It will be readily seen that complement must possess at least two groups, a combining or haptophore group which unites with the amboceptor, and an active group which is usually called the zymophore or toxophore group. Complements thus resemble either toxins, where the specific cell (antigen) is injured or killed, or enzymes, in case the cell is likewise dissolved. This action again shows the close relation between toxins and enzymes. Complement may lose its active group in the same way that toxin does and becomes then complementoid. Complement is readily destroyed in blood or serum by heating it to 55° to 56° for half an hour, and is also destroyed spontaneously when serum stands for a day or two, less rapidly at low temperature than at room temperature.

Amboceptors appear to be specific in the same sense that agglutinins are. That is, if a given cell is used to immunize an animal, the animal’s blood will contain amboceptors for the cell used and also for others closely related to it. Immunization with spermatozoa or with epithelial or liver cells gives rise to amboceptors for these cells and also for red blood corpuscles and other body cells. A typhoid bactericidal serum has also some dissolving effect on colon bacilli, etc. Hence a given serum may contain a chief amboceptor and a variety of “coamboceptors,” or one amboceptor made up of a number of “partial amboceptors” each specific for its own antigen (“amboceptorogen”). Amboceptors may combine with other substances than antigen and complement, as is shown by their union with lecithin and other “lipoids,” though these substances seem capable of acting as complement in causing solution of blood corpuscles.

COMPLEMENTS.

As to whether complements are numerous, as Ehrlich claims, or there is only one complement, according to Buchner and others, need not be discussed here. In the practical applications given later as means of diagnosis it is apparent that all the complement or complements are capable of uniting with at least two kinds of amboceptors.

If complement be injected into an animal it may act as an antigen and give rise to the formation of anticomplement which may combine with it and prevent its action and is consequently analogous to antitoxin. If amboceptors as antigen are injected into an animal there will be formed by the animal’s cells antiamboceptors. As one would expect, there are two kinds of antiamboceptors, one for each of its combining groups, since it has been stated that it is always the combining group of any given antigen that acts as the cell stimulus. Hence we may have an “anticytophil amboceptor” or an “anticomplementophil amboceptor.” These antiamboceptors and the anticomplements are analogous to antitoxin, antiagglutinin, etc., and hence are receptors of the first order.

ANTISNAKE VENOMS.

A practical use of antiamboceptors is in antisnake venoms. Snake poisons appear to contain only amboceptors for different cells of the body. In the most deadly the amboceptor is specific for nerve cells (cobra), in others for red corpuscles, or for endothelial cells of the bloodvessels (rattlesnake). The complement is furnished by the blood of the individual bitten, that is, in a sense the individual poisons himself, since he furnishes the destroying element. The antisera contain antiamboceptors which unite with the amboceptor introduced and prevent it joining to cells and thus binding the complement to the cells and destroying them. With this exception these antibodies are chiefly of theoretical interest.

FAILURE OF CYTOLYTIC SERUMS.

The discovery of the possibility of producing a strongly bactericidal serum in the manner above described aroused the hope that such sera would prove of great value in passive immunization and serum treatment of bacterial diseases. Unfortunately such expectations have not been realized and no serum of this character of much practical importance has been developed as yet (with the possible exception of Flexner’s antimeningococcus serum in human practice. What hog cholera serum is remains to be discovered).

The reasons for the failure of such sera are not entirely clear. The following are some that have been offered: (1) Amboceptors do not appear to be present in very large amount so that relatively large injections of blood are necessary, which is not without risk in itself. (2) Since the complement is furnished by the blood of the animal to be treated, there may not be enough of this to unite with a sufficient quantity of amboceptor to destroy all the bacteria present, hence the disease is continued by those that escape. (3) Or the complement may not be of the right kind to unite with the amboceptor introduced, since this is derived from the blood of a heterologous (“other kind”) species. In hog-cholera serum, if it is bactericidal, this difficulty is removed by using blood of a homologous (“same kind”) animal. Hence Ehrlich suggested the use of apes for preparing bactericidal sera for human beings. The good results which have been reported in the treatment of human beings with the serum of persons convalescing from the same disease indicate that this lack of proper complement for the given amboceptor is probably a chief factor in the failure of sera from lower animals. (4) The bacteria may be localized in tissues (lymph glands), within cavities (cranial, peritoneal), in hollow organs (alimentary tract), etc., so that it is not possible to get at them with sufficient serum to destroy all. This difficulty is obviated by injecting directly into the spinal canal when Flexner’s antimeningococcus serum is used. (5) Even if the bacteria are dissolved it does not necessarily follow that their endotoxins are destroyed. These may be merely liberated and add to the danger of the patient, though this does not appear to be a valid objection. (6) Complement which is present in such a large excess of amboceptor may just as well unite with amboceptor which is not united to the bacteria to be destroyed as with that which is, and hence be actually prevented from dissolving the bacteria. Therefore it is difficult to standardize the serum to get a proper amount of amboceptor for the complement present.

COMPLEMENT-FIXATION TEST.

Although little practical use has been made of bactericidal sera, the discovery of receptors of this class and the peculiar relations between the antigen, amboceptor and complement have resulted in developing one of the most delicate and accurate methods for the diagnosis of disease and for the recognition of small amounts of specific protein that is in use today.

This method is usually spoken of as the “complement-fixation” or the “complement-deviation test” (“Wassermann test” in syphilis) and is applicable in a great variety of microbial diseases, but it is of practical importance in those diseases only where other methods are uncertain—syphilis in man, concealed glanders in horses, contagious abortion in cattle, etc. A better name would be the “Unknown Amboceptor Test” since it is the amboceptor that is searched for in the test by making use of its power to “fix” complement.

The principle is the same in all cases. The method depends, as indicated above, on the ability of complement to combine with at least two amboceptor-antigen systems, and on the further fact that if the combination with one amboceptor-antigen system is once formed, it does not dissociate so as to liberate the complement for union with the second amboceptor-antigen system. If an animal is infected with a microÖrganism and a part of its defensive action consists in destroying the organisms in its blood or lymph, then it follows from the above discussion of cytolysins that there will be developed in the blood of the animal amboceptor specific for the organism in question. If the presence of this specific amboceptor can be detected, the conclusion is warranted that the organism for which it is specific is the cause of the disease. Consequently what is sought in the “complement-fixation test” is a specific amboceptor. In carrying out the test, blood serum from the suspected animal is collected, heated at 56° for half an hour to destroy any complement it contains and mixed in definite proportions with the specific antigen and with complement. The antigen is an extract of a diseased organ (syphilitic fetal liver, in syphilis), a suspension of the known bacteria, or an extract of these bacteria. Complement is usually derived from a guinea-pig, since the serum of this animal is higher in complement than that of most animals. The blood of the gray rat contains practically as much. If the specific amboceptor is present, that is, if the animal is infected with the suspected disease, the complement will unite with the antigen-amboceptor system and be “fixed,” that is, be no longer capable of uniting with any other amboceptor-antigen system. No chemical or physical means of telling whether this union has occurred or not, except as given below, has been discovered as yet, though doubtless will be by physico-chemical tests, nor can the combination be seen. Hence an “indicator,” as is so frequently used in chemistry, is put into the mixture of antigen-amboceptor-complement after it has been allowed to stand in the incubator for one-half to one hour to permit the union to become complete. The “indicator” used is a mixture of sheep’s corpuscles and the heated (“inactivated”) blood serum of a rabbit which has been injected with sheep’s blood corpuscles and therefore contains a hemolytic amboceptor specific for the corpuscles which is capable also of uniting with complement. The indicator is put into the first mixture and the whole is again incubated and examined. If the mixture is clear and colorless with a deposit of red corpuscles at the bottom, that would mean that the complement had been bound to the first complex, since it was not free to unite with the second sheep’s corpuscles (antigen)—rabbit serum (hemolytic amboceptor) complex—and destroy the corpuscles. Hence if the complement is bound in the first instance, the specific amboceptor for the first antigen must have been present in the blood, that is, the animal was infected with the organism in question. Such a reaction is called a “positive” test.

On the other hand, if the final solution is clear but of a red color, that would mean that complement must have united with the corpuscles—hemolytic amboceptor system—and destroyed the corpuscles in order to cause the clear red solution of hemoglobin. If complement united with this system it could not have united with the first system, hence there was no specific amboceptor there to bind it; no specific amboceptor in the animal’s blood, means no infection. Hence a red solution is a “negative test.” The scheme for the test may be outlined as follows:

Incubate one-half hour in a water bath or one hour in an incubator.

Then add the indicator which is

Incubate as above.

In practice all the different ingredients must be accurately tested, standardized and used in exact quantities, and tests must also be run as controls with a known normal blood of an animal of the same species as the one examined and with a known positive blood.

It should be stated that in one variety of the Complement Fixation Test, namely, the “Wassermann Test for Syphilis” in human beings, an antigen is used which is not derived from the specific organism (Treponema pallidum) which causes the disease nor even from syphilitic tissue. It has been determined that alcohol will extract from certain tissues, human or animal, substances which act specifically in combining with the syphilitic amboceptor present in the blood. Alcoholic extracts of beef heart are most commonly used. Details of this test may be learned in the advanced course in Immunity and Serum Therapy.

The complement-fixation test might be applied to the determination of unknown bacteria, using the unknown culture as antigen and trying it with the sera of different animals immunized against a variety of organisms, some one of which might prove to furnish specific amboceptor for the unknown organism and hence indicate what it is. The test used in this way has not been shown to be a practical necessity and hence is rarely employed. It has been used, however, to detect traces of unknown proteins, particularly blood-serum proteins, in medico-legal cases in exactly the above outlined manner and is very delicate and accurate.

It has been mentioned that Metchnikoff, in a publication in 1883, attempted to explain immunity on a purely cellular basis. It has been known since Haeckel’s first observation in 1858 that certain of the white corpuscles do engulf solid particles that may get into the body, and among them bacteria. Metchnikoff at first thought that this engulfing and subsequent intracellular digestion of the microÖrganisms were sufficient to protect the body from infection. The later discoveries (discussed in considering Ehrlich’s theory of immunity) of substances present in the blood serum and even in the blood plasma which either destroy the bacteria or neutralize their action have caused Metchnikoff to modify his theory to a great extent. He admitted the presence of these substances, though giving them other names, but ascribed their formation to the phagocytes or to the same organs which form the leukocytes—lymphoid tissue generally, bone marrow. It is not within the province of this work to attempt to reconcile these theories, but it may be well to point out that Ehrlich’s theory is one of chemical substances and that the origin of these substances is not an essential part of the theory, so that the two theories, except in some minor details, are not necessarily mutually exclusive.

Sir A.E. Wright and Douglas, in 1903, showed that even in those instances where immunity depends on phagocytosis, as it certainly does in many cases, the phagocytes are more or less inactive unless they are aided by chemical substances present in the blood. These substances act on the bacteria, not on the leukocytes, and change them in such a way that they are more readily taken up by the phagocytes. Wright proposed for these bodies the name opsonin, derived from a Greek word signifying “to prepare a meal for.” Neufeld and Rimpau at about the same time (1904), in studying immune sera, observed substances of similar action in these sera and proposed the name bacteriotropins, or bacteriotropic substances. There is scarcely a doubt that the two names are applied to identical substances and that Wright’s name opsonin should have preference.

The chemical nature of opsonins is not certainly determined, but they appear to be a distinct class of antibodies and to possess two groups, a combining or haptophore and a preparing or opsonic group and hence are similar to antibodies of Ehrlich’s second order—agglutinins and precipitins. Wright also showed that opsonins are just as specific as agglutinins are—that is, a micrococcus opsonin prepares micrococci only for phagocytosis and not streptococci or any other bacteria.

Wright showed that opsonins for many bacteria are present in normal serum and that in the serum of an animal which has been immunized against such bacteria the opsonins are increased in amount. Also that in a person infected with certain bacteria the opsonins are either increased or diminished, depending on whether the progress of the infection is favorable or unfavorable. The opsonic power of a serum normal or otherwise is determined by mixing an emulsion of fresh leukocytes in normal saline solution with a suspension of the bacteria and with the serum to be tested. The leukocytes must first be washed in several changes of normal salt solution to free them from any adherent plasma or serum. The mixture is incubated for about fifteen minutes and then slides are made, stained with a good differential blood stain, Wright’s or other, and the average number of bacteria taken up by at least fifty phagocytes taken in order in a field is determined by counting under the microscope. The number so obtained Wright calls the phagocytic index of the serum tested. The phagocytic index of a given serum divided by the phagocytic index of a normal serum gives the opsonic index of the serum tested. Assuming the normal opsonic index to be 1, Wright asserts that in healthy individuals the range should be not more than from 0.8 to 1.2, and that an index below 0.8 may show a great susceptibility for the organism tested, infection with the given organism if derived from the individual, or improper dosage in case attempts have been made to immunize by using killed cultures, vaccines, of the organism.

On the occasion of the author’s visit to Wright’s clinic (1911) he stated that he used the determination of the opsonic index chiefly as a guide to the dosage in the use of vaccines.

Most workers outside the Wright school have failed to recognize any essential value of determinations of the opsonic index in the use of vaccines. Some of the reasons for this are as follows: The limit of error in phagocytic counts may be as great as 50 per cent. in different series of fifty, hence several hundred must be counted, which adds greatly to the tediousness and time involved; the variation in apparently healthy individuals is frequently great, hence the “normal” is too uncertain; finally the opsonic index and the clinical course of the disease do not by any means run parallel. Undoubtedly the method has decided value in the hands of an individual who makes opsonic determinations his chief work, as Wright’s assistants do, but it can scarcely be maintained at the present time that such determinations are necessary in vaccine therapy. Nevertheless that opsonins actually exist and that they play an essential part in phagocytosis, and hence in immunity, is now generally recognized.

BACTERIAL VACCINES.

Whether determinations of opsonic index are useful or not is largely a matter of individual opinion, but there is scarcely room to doubt that Wright has conferred a lasting benefit by his revival of the use of dead cultures of bacteria, bacterial vaccines, both for protective inoculation and for treatment. It is perhaps better to use the older terms “vaccination” and “vaccine” (though the cow, vacca, is not concerned) than to use Wright’s term “opsonic method” in this connection, bearing in mind that the idea of a vaccine is that it contains the causative organism of the infection as indicated on p.253.

As early as 1880 Touissant proposed the use of dead cultures of bacteria to produce immunity. But because injections of such cultures were so frequently followed by abscess formation, doubtless due to the high temperatures used to kill the bacteria, the method was abandoned. Further, Pasteur and the French school persistently denied the possibility of success with such a procedure, and some of them even maintain this attitude at the present time. The successes of Wright and the English school which are being repeated so generally wherever properly attempted, leave no doubt in the unprejudiced of the very great value of the method and have unquestionably opened a most promising field both for preventive inoculation and for treatment in many infectious diseases. That the practice is no more universally applicable than are immune serums and that it has been and is still being grossly overexploited is undoubted.

The use of a vaccine is based on two fundamental principles. The first of these is that the cell introduced must not be in a condition to cause serious injury to the animal by its multiplication and consequent elaboration of injurious substances. The second is that, on the other hand, it must contain antigens in such condition that they will act as stimuli to the body cells to produce the necessary antibodies, whether these be opsonins, bactericidal substances, or anti-endotoxins. In the introduction of living organisms there is always more or less risk of the organism not being sufficiently attenuated and hence of the possibility of its producing too severe an infection. In using killed cultures, great care must be exercised in destroying the organisms, so that the antigens are not at the same time rendered inactive. Hence in the preparation of bacterial vaccines by Wright’s method the temperature and the length of time used to kill the bacteria are most important factors. This method is in general to grow the organisms on an agar medium, rub off the culture and emulsify in sterile normal salt solution (0.85 per cent. NaCl). The number of bacteria per cc. is determined by staining a slide made from a small volume of the emulsion mixed with an equal volume of human blood drawn from the finger and counting the relative number of bacteria and of red blood corpuscles. Since the corpuscles are normally 5,000,000 per c.mm., a simple calculation gives the number of bacteria. The emulsion of bacteria is then diluted so that a certain number of millions shall be contained in each cc., “standardized” as it is called, then heated to the proper temperature for the necessary time and it is ready for use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added unless the vaccine is to be used up at once. The amounts of culture, salt solution, etc., vary with the purpose for which the vaccine is to be used, from one or two agar slant cultures and a few cc. of solution, when a single animal is to be treated, to bulk agar cultures and liters of solution as in preparing antityphoid vaccine on a large scale.

Agar surface cultures are used so that there will be as little admixture of foreign protein as possible (see Anaphylaxis, p.289 et seq.). Normal saline solution is isotonic with the body cells and hence is employed as the vehicle.

Lipovaccines.—The suspension of bacteria in neutral oil was first used by Le Moignac and Pinoy who gave the name “lipovaccines” (??p??=fat) to them. It was claimed that the reaction following injection of these vaccines was less severe than with saline vaccines in many instances; also, that the bacteria were much more slowly absorbed. For these two reasons it was hoped that much larger numbers of bacteria could be injected at one dose and one injection would suffice instead of three or more as ordinarily used. The technique of preparation, standardization and killing of the organisms has not as yet been sufficiently well established to warrant the general substitution of lipovaccines for ordinary saline suspensions.

Vaccines are either “autogenous” or “stock.” An “autogenous” vaccine is a vaccine that is made from bacteria derived from the individual or animal which it is desired to vaccinate and contains not only the particular organism but the particular strain of that organism which is responsible for the lesion. Stock vaccines are made up from organisms like the infective agent in a given case but derived from some other person or animal or from laboratory cultures. Commercial vaccines are “stock” vaccines and are usually “polyvalent” or even “mixed.” A “polyvalent” vaccine contains several strains of the infective agent and a “mixed” contains several different organisms.

Stock vaccines have shown their value when used as preventive inoculations, notably so in typhoid fever in man, anthrax and black-leg in cattle. The author is strongly of the opinion, not only from the extended literature on the subject, but also from his own experience in animal, and especially in human cases, that stock vaccines are much inferior and much more uncertain in their action when used in the treatment of an infection, than are autogenous vaccines. This applies particularly to those instances in which pneumococci, streptococci, micrococci, and colon bacilli are the causative agents but to others as well. The following are some of the reasons for this opinion: The above organisms are notoriously extremely variable in their virulence. While there is no necessarily close connection between virulence and antigenic property, yet since virulence is so variable, it is rational to assume that antigenic property is also extremely variable. Individuals vary just as much in susceptibility and hence in reactive power, and generally speaking, an individual will react better in the production of antibodies to a stimulus to which he has been more or less subjected, i.e., to organisms derived from his own body.

In the preparation of a vaccine great care must be used in heating so that the organisms are killed, but the antigens are not destroyed. Many of the enzymes present in bacteria, especially the proteolytic ones, are not any more sensitive to heat than are the antigens, hence are not destroyed entirely. Therefore a vaccine kept in stock for a long time gradually has some of its antigens destroyed by the uninjured enzymes present with them, and so loses in potency. Therefore in treating a given infection it is well to make up a vaccine from the lesion, use three or four doses and if more are necessary make up a new vaccine. If the above statements are borne in mind and vaccines are made and administered accordingly, the author is well satisfied that much better results will be secured.

In accordance with the theory on which the use of vaccines is based, i.e., that they stimulate the body cells to produce immunizing antibodies, it is clear that they are especially suitable in those infections in which the process is localized and should not be of much value in general infections. In the latter case the cells of the body are stimulated to produce antibodies by the circulating organisms, probably nearly to their limit, hence the introduction of more of the same organisms, capable of stimulating though dead, is apt to overtax the cells and do more harm than good. It is not possible to tell accurately when this limit is reached, but the clinical symptoms are a guide. If vaccines are used at all in general infections they should be given in the early stages and in small doses at first with close watch as to the effect. In localized infections only the cells in the immediate neighborhood are much stimulated, hence the introduction of a vaccine calls to their aid cells in the body generally, and much more of the resulting antibodies are carried to the lesion in question. Manifestly surgical procedures such as incision, drainage, washing away of dead and necrotic tissue with normal saline solution, not necessarily antiseptics, will aid the antibodies in their action and are to be recommended where indicated.

In the practical application of any remedy the dosage is most important. Unfortunately there is no accurate method of determining this with a vaccine. Wright recommended determining the number of the organisms per cc. as before mentioned, and his method or some modification of it is still in general use. From what was said with regard to variation, both in organisms and in individuals, it can be seen that the number of organisms is at least only a very rough guide. This is further illustrated by the doses of micrococcus (staphylococcus) vaccines recommended by different writers, which vary from 50,000,000 to 2,000,000,000 per cc. The author is decidedly of the opinion that there is no way of determining the dosage of a vaccine in the treatment of any given case except by the result of the first dose. Hence it is his practice to make vaccines of a particular organism of the same approximate strength, and to give a dose of a measured portion of a cubic centimeter, judging the amount by what the individual or animal can apparently withstand, without too violent a reaction. If there is no local or general reaction or if it is very slight and there is no effect on the lesion, the dose is too small. If there is a violent local reaction with severe constitutional symptoms clinically, and the lesion appears worse, the dose is too large. There should be some local reaction and some general, but not enough to cause more than a slight disturbance, easy to judge in human subjects, more difficult in animals. In cases suitable for vaccine treatment no serious results should follow from a properly prepared vaccine, though the process of healing may be delayed temporarily. Wright claimed, and many have substantiated him, that always following a vaccination there is a period when the resistance of the animal is diminished. This is called the “negative phase,” and Wright considered this to last as long as the opsonic index remained low, and when this latter began to increase the stage of the “positive” or favorable phase was reached. As has been stated the opsonic index is pretty generally regarded as of doubtful value, though the existence of a period of lowered resistance is theoretically probable from the fact that antibodies already present in the blood will be partially used up in uniting with the vaccine introduced and that the body cells are called upon suddenly to do an extra amount of work and it takes them some time to adapt themselves. This time, the “negative phase,” is much better determined by the clinical symptoms, general and especially local. It is good practice to begin with a dose relatively small. The result of this is an indication of the proper dosage and also prepares the patient for a larger one. The second dose should follow the first not sooner than three or four days, and should be five to seven days if the first reaction is severe. These directions are not very definite, but clinical experience to date justifies them. It is worth the time and money to one who wishes to use vaccines to learn from one who has had experience both in making and administering them, and then to remember that each patient is an individual case, for the use of vaccines as well as for any other kind of treatment.

AGGRESSIN.

Opsonins have been shown to be specific substances which act on bacteria in such a way as to render them more readily taken up by the leukocytes. By analogy one might expect to find bacteria secreting specific substances which would tend to counteract the destructive action of the phagocytes and bactericidal substances. Bail and his co-workers claim to have demonstrated such substances in exudates in certain diseases and have given the distinctive name “aggressins” to them. By injecting an animal with “aggressins,” antiaggressins are produced which counteract their effects and thus enable the bacteria to be destroyed. The existence of such specific bodies is not generally accepted as proved. The prevailing idea is that bacteria protect themselves in any given case by the various toxic substances that they produce, and that “aggressins” as a special class of substances are not formed.

Dallera, in 1874, and a number of physiologists of that period, observed peculiar skin eruptions following the transfusion of blood, that is, the introduction of foreign proteins. In the years subsequent to the introduction of diphtheria antitoxin (1890) characteristic “serum rashes” were not infrequently reported, sometimes accompanied by more or less severe general symptoms and occasionally death—a train of phenomena to which the name “serum sickness” was later applied, since it was shown that it was the horse serum (foreign protein) that was the cause, and not the antitoxin itself. In 1898 Richet and Hericourt noticed that some of the dogs which they were attempting to immunize against toxic eel serum not only were not immunized but suffered even more severely after the second injection. They obtained similar results with an extract of mussels which contain a toxin. Richet gave the name “anaphylaxis” (“no protection”) to this phenomenon to distinguish it from immunity or prophylaxis (protection).

All the above-mentioned observations led to no special investigations as to their cause. In 1903, Arthus noticed abscess formation, necrosis and sloughing following several injections of horse serum in immediately adjacent parts of the skin in rabbits (“Arthus’ phenomenon”). Theobald Smith, in 1904, observed the death of guinea-pigs following properly spaced injections of horse serum. This subject was investigated by Otto and by Rosenau and Anderson in this country and about the same time von Pirquet and Schick were making a study of serum rashes mentioned above. The publications of these men led to a widespread study of the subject of injections of foreign proteins. It is now a well-established fact that the injection into an animal of a foreign protein—vegetable, animal or bacterial, simple or complex—followed by a second injection after a proper length of time leads to a series of symptoms indicating poisoning, which may be so severe as to cause the death of the animal. Richet’s term “anaphylaxis” has been applied to the condition of the animal following the first injection and indicates that it is in a condition of supersensitiveness for the protein in question. The animal is said to be “sensitized” for that protein.25 The sensitization is specific since an animal injected with white of chicken’s egg reacts to a second injection of chicken’s egg only and not pigeon’s egg or blood serum or any other protein. The specific poisonous substance causing the symptoms has been called “anaphylotoxin” though what it is, is still a matter of investigation. It is evident that some sort of an antibody results from the first protein injected and that it is specific for its own antigen.

A period of ten days is usually the minimum time that must elapse between the first and second injections in guinea-pigs in order that a reaction may result, though a large primary dose requires much longer. If the second injection is made within less time no effect follows, and after three or more injections at intervals of about one week the animal fails to react at all, it has become “immune” to the protein. Furthermore, after an animal has been sensitized by one injection and has reacted to a second, then, if it does not die from the reaction, it fails to react to subsequent injections. In this latter case it is said to be “antianaphylactic.”

It must be remembered that proteins do not normally get into the circulation except by way of the alimentary tract. Here all proteins that are absorbed are first broken down to their constituent amino-acids, absorbed as such and these are built up into the proteins characteristic of the animal’s blood. Hence when protein as such gets into the blood it is a foreign substance to be disposed of. The blood contains proteolytic enzymes for certain proteins normally. It is also true that the body cells possess the property of digesting the proteins of the blood and building them up again into those which are characteristic of the cell. Hence it appears rational to assume that the foreign proteins act as stimuli to certain cells to produce more of the enzymes necessary to decompose them, so that they may be either built up into cell structure or eliminated as waste. If in this process of splitting up of protein a poison were produced, then the phenomena of “anaphylaxis” could be better understood. As a matter of fact Vaughan and his co-workers have shown that by artificially splitting up proteins from many different sources—animal, vegetable, pathogenic and saprophytic bacteria—a poison is produced which appears to be the same in all cases and which causes the symptoms characteristic of anaphylaxis. On the basis of these facts it is seen that anaphylaxis is simply another variety of immunity. The specific antibody in this case is an enzyme which decomposes the protein instead of precipitating it. The enzyme must be specific for the protein since these differ in constitution. Vaughan even goes so far as to say that the poison is really the central ring common to all proteins and that they differ only in the lateral groups or side chains attached to this central nucleus. The action of the enzyme in this connection would be to split off the side chains, and since these are the specific parts of the protein, the enzyme must be specific for each protein. The pepsin of the gastric juice and the trypsin of the pancreas split the native proteins only to peptones. As is well known, these when injected in sufficient quantity give rise to poisonous symptoms, and will also give rise to anaphylaxis under properly spaced injections. They do not poison normally because they are split by the intestinal erepsin to amino-acids and absorbed as such. Whether Vaughan’s theory of protein structure is the true one or not remains to be demonstrated. It is not essential to the theory of anaphylaxis above outlined, i.e., a phenomenon due to the action of specific antibodies which are enzymes. On physiological grounds this appears the most rational of the few explanations of anaphylaxis that have been offered and was taught by the author before he had read Vaughan’s theory along the same lines.

On the basis of the author’s theory the phenomena of protein immunity and antianaphylaxis may be explained in the following way which the author has not seen presented. The enzymes necessary to decompose the injected protein are present in certain cells and are formed in larger amount by those cells to meet the increased demand due to injection of an excess of protein. They are retained in the cell for a time at least. If a second dose of protein is given before the enzymes are excreted from the cells as waste, this is digested within the cells in the normal manner. If a third dose is given, the cells adapt themselves to this increased intracellular digestion and it thus becomes normal to them. Hence the immunity is due to this increased intracellular digestion.

On the other hand, if the second injection is delayed long enough, then the excess enzyme, but not all, is excreted from the cells and meets the second dose of protein in the blood stream and rapidly decomposes it there, so that more or less intoxication from the split products results. This uses up excess enzyme, hence subsequent injections are not digested in the blood stream but within the cells as before. So that “antianaphylaxis” is dependent on the exhaustion of the excess enzyme in the blood, and the condition is fundamentally the same as protein immunity, i.e., due to intracellular digestion in each case.

As has been indicated “serum sickness” and sudden death following serum injections are probably due to a sensitization of the individual to the proteins of the horse in some unknown way. Probably hay fever urticarial rashes and idiosyncrasies following the ingestion of certain foods—strawberries, eggs, oysters, etc., are anaphylactic phenomena.

In medical practice the reaction is used as a means of diagnosis in certain diseases, such as the tuberculin test in tuberculosis, the mallein test in glanders. The individual or animal with tuberculosis becomes sensitized to certain proteins of the tubercle bacillus and when these proteins in the form of tuberculin are introduced into the body a reaction results, local or general, according to the method of introduction. The practical facts in connection with the tuberculin test are also in harmony with the author’s theory of anaphylaxis as above outlined. Milder cases of tuberculosis give more vigorous reactions because the intracellular enzymes are not used up rapidly enough since the products of the bacillus are secreted slowly in such cases. Hence excess of enzyme is free in the blood and the injection of the tuberculin meets it there and a vigorous reaction results. In old, far-advanced cases, no reaction occurs, because the enzymes are all used in decomposing the large amount of tuberculous protein constantly present in the blood. The fact that an animal which has once reacted fails to do so until several months afterward likewise depends on the fact that the excess enzyme is used in the reaction and time must elapse for a further excess to accumulate.

The anaphylactic reaction has been made use of in the identification of various types of proteins and is of very great value since the reaction is so delicate, particularly when guinea-pigs are used as test animals. Wells has detected the 0.000,001 g. of protein by this test. It is evident that the test is applicable in medico-legal cases and in food examination and has been so used.