VOL. I.

THE STUDY OF IMMUNITY.

BY M. METCHNIKOFF.

V. THE PROPERTY OF THE MICROBE OF HOG CHOLERA IN THE ORGANISM OF A RABBIT.

WE could never believe that the coccobacillus of hog cholera, with its thin envelope and its poor aspect, should be in a condition to live long in a body absolutely refractory to this microbe. And yet, inoculated in the skin of these rabbits, it provokes a protective suppuration, and maintains its life about three weeks. The pus withdrawn after a few days or more from the subcutaneous abscesses of the rabbit, presents, under the microscope, quite a mass of living and dead leucocytes, in which there are no microbes to be found. But it suffices to sow a little of this pus in broth to obtain an abundant culture of Coccobacillus suinum, -culture always very virulent.

The microbes end, however, by perishing in the pus, and, if it is withdrawn in about three weeks after the inoculation of the virus, the inoculated broth remains perfectly clear and sterile.

Often enough, the subcutaneous abscesses end by opening at the exterior, by which the animal is relieved of a quantity of pus; in other cases the abscesses remain closed and absorb slowly. Two months after the inoculation, the abscess is still voluminous enough, and contains a very thick white pus. Later,

it diminishes and is transformed into a brownish mass diminished in thickness. In all these cases, the pus is sterile, and we find in it no remains of destroyed microbes. The bacteria capable of resisting so long in the refractory organism finally end in death their death occurs, not in the liquid, but in the interior of the phagocytes. If the pus is withdrawn

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NO. 12.

forty-eight hours after the subcutaneous inoculation of the rabbit, the microscope already fails to reveal the microbes. But if a little of this pus is placed in the incubator at 38° C., it is easy to observe that the leucocytes contain masses of bacteria of hog cholera which take on coloring in a most normal manner. These bacteria appear in the form of small oval bacilli, diplobacteria, and also of beads. They develop in the interior of the pus globules and invade the liquid, in which they produce a perfect culture.

These facts demonstrate that the microbes are englobed alive by the pus leucocytes, but that some of the bacteria resist, and when transported to conditions unfavorable to the phagocytes, invade the pus.

The long resistance of the englobed bacteria, gives us to understand that in a few unfavorable circumstances for the organism of the rabbit, the microbe succeeds in developing and killing its host. It is thus that we may explain more easily, the case in which a vaccinated rabbit, tested with vital virus, succumbed at last to pyemia. This disease was provoked by the microbe of hog cholera, and occurred nearly a month after the last inoculation of the virus.

The observation that I have just cited, renders it probable that the microbe, able to resist a long time in the vaccinated rabbit, may end by adapting itself to the medium, and in a favorable moment invade the organism. This should demonstrate that the englobed coccobacilli preserve, not only their vitality, but their virulence in the interior of the phagocytes. The last proposition may be easily studied, because very minute doses suffice to provoke a deadly form of the disease in rabbits.

The pus of the refractory rabbits in which hog cholera bacteria exist in the interior of the leucocytes, is virulent. I

have observed this important fact many times. The pus withdrawn forty-eight hours after inoculation of the vaccinated and refractory rabbit, killed the rabbit (in which it had been injected in the auricular vein) in forty hours. In other experiments the virulence was still more marked. A drop of pus from an inoculated rabbit, withdrawn four days after the inoculation of the virus, and injected in the vein of another rabbit, produced hog cholera with fatal termination in less than twenty-one hours. On preparation of this pus, no microbes were to be observed.

From another vaccinated rabbit, which had resisted several virulent inoculations, the pus injected in the auricular vein of a new rabbit caused death in twenty-nine hours and twenty minutes. This pus had been withdrawn seventeen days after inoculation of the vaccinated rabbit with toxic blood. While the pus formed at the point of inoculation proved so virulent, the serum of the same vaccinated rabbit manifested a very marked preventive property. One cubic centimeter of a very rich culture in this serum, injected in the vein of a rabbit, produced its death. only after seventy-eight hours, notwithstanding the enormous difference between the immense quantity of microbes contained in an abundant culture and the few that this pus contained, so few in number that they were not revealed with the microscope.

We see, according to these experiments, that the pus which has been formed at the spot where the animal was attacked by the microbe, does not at all possess the preventive property which is so remarkable in the serum of the same vaccinated animals.

In cases where the organism contains microbes which during a long time remain alive and preserve their virulence, and where, notwithstanding this, the organism is not invaded, we are always tempted to admit the existence of the antitoxic power. The microbe, though virulent, does not hinder, because its toxines are destroyed at the moment of their production. The absence of an antitoxic property from the blood serum, demonstrated in the second chapter, could not furnish sufficient evidence, because we know that often the phenomena which occur in the living organism are very different from those observed in the serum obtained outside of the organism.

Let us examine the behavior of vaccinated rabbits with regard to the toxines. This question has already been approached by Mr. Selander, who arrived at the conclusion that "immunity against the microbe may be acquired without immunity against the toxines being established." This result has been established by the fact that the rabbits, while vaccinated against the very virulent virus, die when they receive minimum doses of toxic blood. Of the three vaccinated rabbits poisoned by Mr. Selander, one suffered. with toxic injection eighteen days after testing with the mortal virus; a second, nineteen, and a third, twenty-seven days after the test. These rabbits had regained their natural weight, appeared in good health, and yet they all died under the same conditions, and at the same time as the witnesses. Mr. Selander was kind enough to let me witness this experiment, the exactitude of which I can confirm.

Though the fact of the sensitiveness of the vaccinated rabbits to the toxines had already been observed by Mr. Selander I have sought to ascertain them by my own experiments. To that end I injected minimum mortal doses of toxic blood in the veins of three vaccinated rabbits. Intoxication was practiced twenty-one, sixty-two, and one hundred and nine days, after the date of the last test with the virulent virus. The first rabbit, tested four times with the living virus, had become visibly impoverished; but the other. two rabbits were completely recovered from the inoculation and considerably increased in weight. Well, the three rabbits manifested a great deal of susceptibility to the toxic blood, and died before their non-vaccinated witnesses. The toxines injected in the blood of vaccinated rabbits had consequently not been neutralized, nor had they destroyed their

structure.

The vaccinated rabbits are equally sensitive to the doses of the non-fatal toxines. Injected in the auricular veins, these doses provoke a general uneasiness and an elevation of temperature, as in the witnesses not vaccinated. Even the toxic blood heated to 60° C. produces the same effect on the rabbits refractory to the living virus and on the witnesses.

The toxines injected under the skin act in the same manner on vaccinated rabbits and other witnesses.

This accumulation of facts demonstrated that the acquired immunity against hog cholera is not at all due to an antitoxic property of the vaccinated organism.

The resistance of the vaccinated rabbits which have some virulent microbes in their phagocytes, is not truly due to the antitoxic power of their bodies.

As there is no destruction of toxines in the organism of vaccinated rabbits, they rid themselves of the toxic substances by the aid of exaggerated diuresis. The difficulties that we encounter in securing pure urines in sufficient quantities to study in precise manner their toxic power, are such that I have contented myself at this stage with examining the quantity of urine emitted.

In the course of a slow or acute disease, there is no retention of urine, and it is only in exceptional cases that we find the bladder full at autopsies. The quantity of urine emitted demonstrates, rather, the augmentation of diuresis during hog cholera in the rabbits.

I have made a comparative observation on two rabbits, inoculated under the skin with virulent virus, one of which was vaccinated and the other was utilized as a witness. It is in the last that the greatest quantity of urine was passed, both before the inoculation of the virus and during the disease.

There is only one conclusion to draw from these experiments, and that is that the inoculation is not connected with the retention of urine in the rabbits.

Of all the differences which may have been found between the vaccinated rabbits and the witnesses, the most considerable is undoubtedly that which concerns the system of phagocytary defense.

When we inoculate a very virulent mortal virus in the rabbit not vaccinated in the subcutaneous tissue, a hyperemia of all the neighboring vessels is produced, the diapedesis is feeble, and phagocytosis

a progressive tumor is produced in the non-vaccinated rabbits, which also contains a quantity of leucocytes. In case of cure, there is developed at the point of inoculation, a thick pus, formed by the masses of leucocytes.

Mr. Massart has observed that virulent virus of hog cholera contained in glass tubes, and introduced into the abdominal cavity of the rabbits, produces only a feeble attraction for leucocytes, but in the vaccinated rabbits, on the contrary, attracts them very strongly.

There exists then, a parallelism manifest between the resistance of the animal and the activity of the phagocytes.

The subcutaneous injection of toxic blood heated to 58° or 60° C., provokes equally a leucocytory reaction, very different in the vaccinated rabbits and witnesses. While in the former the tumor contains masses of migrated leucocytes produced from the beginning, in the latter the tumor is soft, and contains only a few leucocytes. It is only later, when the witness rabbit enters the stage of cure, that the number of leucocytes augment, and the tumor becomes firmer.

These facts are in perfect accord with the phenomena of the leucocytes in rabbits infected by the microbe, but intoxicated by the toxine of hog cholera. In vaccinated rabbits, the number of leucocytes is materially augmented, while in the witnesses it diminishes in a notable way. Mr. Werigo observed the latter fact for the first time in investigations executed in my laboratory, and he will speak on the subject in his writings.

We see, then, from all that precedes in this chapter, that in the resistance of vaccinated rabbits against hog cholera, the phagocytes which direct themselves. toward the microbes, which englobe them in a living and virulent state, and which end in destroying them in their protoplasma, exert a function of the highest importance. This role is the more considerable because the phagocytes are not seconded by any property whatever capable of destroying the toxic products. of the microbes of hog cholera.

(To be continued.)

THE NEW CHEMISTRY OF THE STOMACH.

BY J. H. KELLOGG, M. D.

(Continued.)

As the work upon which this paper is based includes, so far as the writer knows, the largest number of cases which have been studied by so exact methods of investigation, I think it important to present a brief summary of the results obtained, which will show at a glance the relative frequency of the pathological conditions which are recognized by this mode of investigation, and as will appear, indicate that some of the current notions upon this subject are quite erroneous. With reference to the three general classes, hyperpepsia, hypopepsia, and simple dyspepsia, the cases were divided as follows:

In considering these figures, it should be remembered that hyperpepsia and hyperacidity are by no means coincident conditions; and that each of the three groups included in hyperpepsia has a sub-group in which the acidity is below normal. The old method of analysis would place all of these cases in hypopepsia; but as the cases given show, and as will appear still more clearly from the summary of the particular conditions observed for each group, many cases of hypoacidity are really cases in which there is an excess of stomach work rather than a deficiency, and hence belong to the class of hyperpepsia rather than hypopepsia.

The accompanying tables present at a glance the particular facts observed as regards the relative frequency of excess, deficiency, and equality, in the figures found by analysis in relation to the total acidity (T), the coefficient of digestive work (a), the total chlorine (T), the free HCI (H), the combined chlorine (C), and the sum of free HCl and combined chlorine (H+C), representing the amount of chlorine set free from the bases and prepared to enter into the work of digestion.

The tables also show the relative frequency of the different forms of hyperpepsia, hypopesia, and simple dyspepsia,

Hyperpepsia. The 170 cases found in this class constitute 52.9% of the total number of cases studied.

1. Hyperpepsia with Hyperhydrochlorie (H+). The first group of this class, hyperpepsia, with hyperhydrochlorie, or free HC in excess, presents sixty-three cases, which is 19.6% of the entire number of cases studied, or 37 % of all the cases of hyperpepsia. This is by far the largest single group found. In this group, A is of course nearly always +, yet we find A in six cases, although, as will be noticed, free HCl (H), and combined chlorine (C), are in all the cases of this group. It is evident, then, that these six cases could not be properly classed as cases of hypopepsia, although in a single one of them the total chlorine (T) was slightly deficient. The total chlorine is found in excess in 50 of the 54 cases.

An interesting fact especially worthy of note in relation to this group is the frequency with which a- occurs. Since the figures represented by a are an index to the quality of the digestive work done, or, at any rate, to the chemical quality of C, indicating, when deficient, the presence of neutral chloro-organic compounds, which are as much greater in proportion as a is less than normal, it is evident that in this group of hyperpepsia, in which we find both H+ and C+ and with rare exceptions A+ and T+, or hyperacidity and excessive secretion of chlorine, the digestive product is, if not in the majority of cases, in at least a large minority (42.8%), inferior in quality, a fact which accounts for the remark often made by this class of patients, 66 Doctor, I have a ravenous appetite; I eat more than I ought to eat, and I seem to digest my food without difficulty; nevertheless, I lose in weight continually, and cannot gain an ounce of flesh."

2. Hyperpepsia with Hypohydrochlorie (H-).- In this group, characterized by a deficiency of free HCl, we find seventyfour cases, 23% of the total number of cases studied, or 43.5% of the cases of hyperpepsia. This group is only exceeded by the preceding in the number of cases which it presents.