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Popular Science Monthly/Volume 29/October 1886/The Microbes of Animal Diseases

THE MICROBES OF ANIMAL DISEASES.[1]
By E. L. TROUESSART.

THE first of the virulent and contagious diseases in which the presence of a microbe was positively ascertained was anthrax, or splenic fever, which attacks most of our horned animals, and especially cattle and sheep.

As early as 1850, Davaine had observed the presence of minute rods in the blood of animals which died of splenic fever; but it was only in 1863, after Pasteur's first researches into the part played by microbes in fermentations, that Davaine suspected these rods of being the actual cause of the disease. He inoculated healthy animals with the tainted blood, and thus ascertained that even a very minute dose would produce a fatal attack of the disease, and the rods, to which he gave the name of Bacteridia, could always be discovered in enormous numbers in the blood.

The microbe so named by Davaine must from its characteristics be assigned to the genus Bacillus, and is now termed Bacillus anthracis. This disease, which affects men as well as animals, is characterized by general depression, by redness and congestion of the eyes, by short

PSM V29 D781 Bacillus anthracis.jpg
Fig. 1.Bacillus anthracis of splenic fever in different stages of development; bacilli, spores, and curled filaments (.much enlarged). Fig. 2.Bacillus anthracis, produced in Guinea-pig by inoculation; corpuscles of blood and bacilli.

and irregular respiration, and by the formation of abscesses, which feature, in the case of the human subject, has procured for it the name of malignant pustule. The disease is quickly terminated by death, and an autopsy shows that the blood is black, that intestinal hæmorrhage has occurred, and that the spleen is abnormally large, heavy, and gorged with blood; hence the name of splenic fever. The disease is generally inoculated by the bite of flies which have settled upon carcasses and absorbed the bacteria, or by blood-poisoning through some accidental scratch, and this is especially the case with knackers and butchers who break and handle the bones of animals which have died of anthrax.

The period of incubation is very short. An ox which has been at work may return to the stall apparently healthy. He eats as usual; then lies down on his side and breathes heavily, while the eyes are still clear. Suddenly his head drops, his body grows cold; at the end of an hour the eye becomes glazed; the animal struggles to get up, and falls dead. In this case, the illness has only lasted for an hour and a half (Empis).

In order to prove that the disease is really caused by Bacillus anthracis, Pasteur inserted a very small drop of blood, taken from an animal which had recently died of anthrax, in a glass flask which contained an infusion of yeast, neutralized by potassium and previously sterilized. In twenty-four hours the liquid, which had been clear, was seen to be full of very light flakes, produced by masses of bacilli, readily discernible under the microscope. A drop from the first flask produced the same effect in a second, and from that to a third, and so on. By this means the organism was completely freed from all which was foreign to it in the original blood, since it is calculated that, after from eight to ten of such processes, the drop of blood was diluted in a volume of liquid greater than the volume of the earth. Yet the tenth, twentieth, and even the fiftieth infusion would, when a drop was inserted under the skin of a sheep, procure its death by splenic fever, with the same symptoms as those produced by the original drop of blood. The bacillus is, therefore, the sole cause of the disease.

These cultures have often since been repeated by numerous observers, so that the microbe has been studied in all its forms, and the extent of its polymorphism has been ascertained. At the end of two days the bacterium, which, while still in the blood, is of a short abrupt form, displays excessively long filaments, which are sometimes rolled up like a coil of string. In about a week many of the filaments contain refracting, somewhat elongated nuclei. These nuclei presently form chaplets, in consequence of the rupture of the cell-wall of the rod which gave birth to them; others, again, float in the liquid in the form of isolated globules. These nuclei are the spores or germs of the microbes, which germinate when placed in the infusion, become elongated, and reproduce fresh bacilli.

These spores are much more tenacious of life than the microbes themselves. The latter perish in a temperature of 60°, by desiccation, in a vacuum, in carbonic acid, alcohol, and compressed oxygen. The spores, on the other hand, resist desiccation, so that they can float in the air in the form of dust. They also resist a temperature of from 90° to 95°, and the effects of a vacuum, of carbonic acid, of alcohol, and compressed oxygen.

In 1873 Pasteur, aided by Chamberland and Roux, carried on some experiments on a farm near Chartres, in order to discover why this disease is so common in some districts, in which its spread can not be ascribed to the bite of flies. Grass, on which the germs of bacteridia had been placed, was given to the sheep. A certain number of them died of splenic fever. The glands and tissues of the back of the throat were very much swelled, as if the inoculation had occurred in the upper part of the alimentary canal, and by means of slight wounds on the surface of the mucous membrane of the mouth. In order to verify the fact, the grass given to the sheep was mixed with thistles and bearded ears of wheat and barley, or other prickly matter, and in consequence the mortality was sensibly increased.

In cases of spontaneous disease it was surmised that the germs which were artificially introduced into food in the course of these experiments are found upon the grass, especially in the neighborhood of places in which infected animals had been buried. It was, in fact, ascertained that these germs existed above and around the infected carcasses, and that they were absent at a certain distance from their burial-place. It is true that putrid fermentation destroys most of the bacteria, but before this occurs a certain number of microbes are dispersed by the gas disengaged from the carcass; these dry up and produce germs, which retain their vitality in the soil for a long while.

The mechanism by means of which these germs are brought to the surface of the soil and on to the grass on which the sheep feed is at once simple and remarkable. Earth-worms prefer soils which are rich in humus or decomposing organic substance, and seek their food round the carcass. They swallow the earth containing the germs of which we have spoken, which they deposit on the surface of the soil, after it has traversed their intestinal canals, in the little heaps with which we are all acquainted. The germs do not lose their virulence in their passage through the worms' intestines, and, if the sheep swallow them together with the grass on which they browse, they may contract the disease. The turning-up of the soil by the spade or plow may produce the same effect.

A certain warmth is necessary for the formation of germs; none are produced when it falls below 12°, and the carcasses buried in winter are therefore less dangerous than those buried in the spring and summer. It is, in fact, in hot weather that the disease is most prevalent. Animals may, however, contract it even in their stalls from eating dry fodder on which germs of these bacteria remain.

Pasteur and his pupils performed an experiment in the Jura in 1879, which clearly shows that the presence of germs above the trenches in which carcasses have been buried is the principal cause of inoculation. Twenty oxen or cows had perished, and several of them were buried in trenches in a meadow where the presence of these germs was ascertained. Three of the graves were surrounded by a fence, within which four sheep were placed. Other sheep were folded within a few yards of the former, but in places where no infected animals had been buried. At the end of three days three of the sheep folded above the graves had died of splenic fever, while those excluded from them continued to be healthy. This result speaks for itself.

Malignant pustule, which is simply splenic fever, affects shepherds, butchers, and tanners, who handle the flesh and hide of tainted animals. Inoculation with the bacillus almost always occurs in consequence of a wound or scratch on the hands or face. In Germany, fatal cases of anthrax have been observed, in which the disease has been introduced through the mouth or lungs, as in the case of the sheep observed by Pasteur. The human subject appears, however, to be less apt to contract the disease than herbivora, since the flesh of animals affected by splenic fever, and only killed when the microbe is fully developed in the blood, is often eaten in farm-houses. In this case the custom prevalent among French peasants of eating overcooked meat constitutes the chief safeguard, since the bacteria and their germs are thus destroyed.

The rapidity with which anthrax is propagated by inoculation generally renders all kinds of treatment useless: if, however, the wound through which the microbe is introduced can be discovered, it should be cauterized at once. This method is often successful in man. The pustule is cauterized with red-hot iron, or with bichloride of mercury and thymic acid, two powerful antiseptics, certain to destroy the bacteridium. It is expedient, as a hygienic measure, to burn the tainted carcasses, and, if this is not done, they should be buried at a much greater depth than is usually the case.

But the preservative means on which chief reliance is now placed is vaccination with the virus of anthrax. Pasteur has ascertained that when animals are inoculated with a liquid containing bacteridia of which the virulence has been attenuated by culture carried as far as the tenth generation, or even further, their lives are preserved. They take the disease, but generally in a very mild form, and it is an important result of this treatment that they are henceforward safe from a fresh attack of the disease; in a word, they are vaccinated against anthrax.

In the cultures prepared with the view of attenuating the microbe, it is the action of the oxygen of the air which renders the bacteridium less virulent. It should be subjected to a temperature of from 42° to 43° in the case of Bacillus anthracis, to enable it to multiply, and at the same time to check the production of spores which might make the liquid too powerful. At the end of the week, the culture, which at first killed the whole of ten sheep, killed only four or five out of ten. In ten or twelve days it ceased to kill any; the disease was perfectly mild, as in the case of the human vaccinia. After the bacteridia have been attenuated, they can be cultivated in the lower temperature of from 30° to 35°, and only produce spores of the same attenuated strength as the filaments which form them (Chamberland). The vaccine thus obtained in Pasteur's laboratory is now distributed throughout the world, and has already saved numerous flocks from almost certain destruction. Although this process has only been known for a few years, its results are such that the gain to agriculture already amounts to many thousands of pounds.

Toussaint makes use of a slightly different mode of preparing a vaccine virus, which is, however, analogous to that of Pasteur. He subjects the lymph of the blood of a diseased animal to a temperature of 50°, and thus transforms it into vaccine. Toussaint considers the high temperature to be the principal agent of attenuation, and ascribes little or no importance to the action of the oxygen in the air.

Chamberland and Roux have recently made researches with the object of obtaining a similar vaccine by attenuating the primitive virus by means of antiseptic substances. They have ascertained that a solution of carbolic acid of one part in six hundred destroys the microbes of anthrax, while they can live and flourish in a solution of one part in nine hundred, but without producing spores, and their virulence is attenuated. When a nourishing broth is added to a solution of one in six hundred, the microbe can live and grow in it for months. Since the chief condition of attenuation consists in the absence of spores, this condition seems to be realized by the culture in a solution of carbolic acid, one in nine hundred, and it is probable that a fresh form of attenuated virus may thus be obtained. Diluted sulphuric acid gives analogous results. However this may be, the vaccine prepared by Pasteur's process is the only one which has been largely used, and which has afforded certain results to cattle-breeders.

Public experiments, performed before commissions composed of most competent men, have clearly shown the virtue of the protective action. In the summer of 1881 the initiation was taken by the Melun Society of Agriculture. Twenty-five sheep and eight cows or oxen were vaccinated at Pouilly-le-Fort, and then reinoculated with blood from animals which had recently died of anthrax, together with twenty-five sheep and five cows which had not been previously vaccinated. None of the vaccinated animals suffered, while the twenty-five test sheep died within forty-eight hours, and the five cows were so ill that the veterinary surgeons despaired of them for several days.

This experiment was publicly repeated in September, 1881, by Thuillier, Pasteur's fellow-worker, whose death we have recently had to deplore, before the representatives of the Austro-Hungarian Government; and again near Berlin, in 1882, before the representatives of the German Government, and always with the same success. Up to April, 1882, more than one hundred and thirty thousand sheep and two thousand oxen or cows had been vaccinated; and since that time the demand for vaccine from Pasteur's laboratory has reached him from every quarter.

The sickness of barn-door poultry, which is commonly called cholera, is caused by the presence in the blood of a small micrococus or bacterium in the form of the figure 8, differing, therefore, in form from Bacillus anthracis, but also an aërobie. It may be cultivated in chicken-broth, neutralized by potash, while it soon dies in the extract of yeast, which is so well adapted to Bacillus anthracis. The microbe of this disease may also be attenuated by culture, and it may be done more easily than in the case of anthrax, since it is not necessary to raise the temperature, as the bacterium of fowl-cholera does not produce spores under culture. Pasteur has therefore been able to prepare an attenuated virus well adapted to protect fowls from further attacks of this disease.

The disease affecting swine, which is called rouget, or swine-fever, in the south of France, has been recently studied by Detmers in the United States, where it is also very prevalent, and by Pasteur in the department of Vaucluse. It is a kind of pneumo-enteritis. These observers consider that the disease is caused by a very slender microbe, formed, like that of fowl-cholera, in the shape of the figure 8, but more minute. Others say that there is a bacillus which was observed by Klein as early as 1878 in swine attacked by this disease. In spite of the apparent contradiction, it is probable that we have only two forms of the same microbe, for the bacillus in Klein's culture at first

PSM V29 D786 Swine fever microbes.jpg
Fig. 3.—Swine Fever: section of a lymphatic gland, showing a blood-vessel filled with microbes (much enlarged). (Klein.)

resembles Bacterium termo, in the form of an 8, before it is elongated into rods. Pasteur has succeeded in making cultures of microbes in the figure 8. He has inoculated swine with the attenuated form, after which they have been able to resist the disease, so there is reason to hope that in the near future this new vaccine, containing the attenuated microbe, may become the safeguard of our pig-sties.

An epidemic which raged in Paris in 1881 was called the typhoid fever of horses, and was fatal to more than fifteen hundred animals belonging to the General Omnibus Company in that city. This disease is also produced by a microbe, with which Pasteur was able to inoculate other animals (rabbits); for this purpose he made use of the serous discharge from the horses' nostrils. The inoculated rabbits died with all the symptoms and lesions characteristic of the disease. The attenuation of this microbe by culture is difficult, since at the end of a certain time the action of the air kills it. Pasteur has, however, found an expedient by which to accomplish his purpose. When the culture is shown to be sterile in consequence of the death of the microbe, he takes as the mother-culture of a fresh series of daily cultures the one which was made on the day preceding the death of the first mother-culture. In this way he has obtained an attenuated virus with which to inoculate rabbits, and the same result might undoubtedly be obtained in the case of horses.

There are many other contagious diseases which affect domestic animals, and which are probably due to microbes, such as, for instance, the infectious pneumonia of horned cattle. This w r as probably the first disease in which the protective effects of inoculation were tried, according to Wilhelm's method. This method consisted in making an incision under the animal's tail with a scalpel dipped 1 in the purulent mucus or blood taken from the lung of a beast which had died of pneumonia; sometimes the serous discharge from the swelling under the tail of an inoculated animal was used for others. Fever and loss of appetite ensued, lasting from eight to twenty-five days, but the animal was afterward safe from further attacks of the disease. Cattle-plague, or contagious typhus, is likewise ascribed to the presence of a microbe with which we are as yet imperfectly acquainted.

Experimental septicæmia is entitled to special mention, since it has too often been confounded with anthrax, and has been unskillfully produced with the intention of vaccinating animals in accordance with Pasteur's process. This occurs when too long an interval (twenty-four hours) elapses after the death of an animal, before taking from it the blood intended for vaccine cultures. After this date the blood noPSM V29 D787 Septic vibrio bacillus of malignant oedema.jpgFig. 4.—Septic vibrio, bacillus of malignant ædema (Koch): a, taken from spleen of Guinea-pig; b, from a mouse's lung. longer contains Bacillus anthracis, which is succeeded by another microbe termed Vibrio septicus, differing widely from the anthrax microbe in form, habit, and character (Fig. 4). Bacillus anthracis is straight and immobile, while the Septic Vibrio is sinuous, curled, and mobile. Moreover, it is anaërobic, and does not survive contact with the air, but it thrives in a vacuum or in carbonic acid. Since Bacillus anthracis is, on the other hand, an aërobie, it is clear that the two microbes can not exist simultaneously in the blood or in the same culture-liquid. The inoculation with this fresh microbe is no less fatal; its action is even more rapid than that of Bacillus anthracis, but the lesions are not the same; the spleen remains normal, while the liver is discolored. The septic vibrio is only found in minute quantities in the blood, so that it has escaped the notice of many observers. It is, however, found in immense numbers in the muscles, in the serous fluid of the intestines, and of other organs. It is very common in the intestines, and is probably the beginning of putrefaction.

Rabies is a canine disease which is communicated by a bite, and the inoculation of man and other animals by the saliva. We are not yet precisely acquainted with the microbe which causes the disease, but Pasteur's recent researches have thrown considerable light on its life history, which is still, however, too much involved in obscurity. It must first be observed that the hypothetical microbe of rabies, which no one has yet discovered, should not be confounded with the microbe of human saliva; this is found in the mouths of healthy persons.

The following conclusions are the result of Pasteur's researches into the virus of rabies.

This virus is found in the saliva of animals and men affected by rabies, associated with various microbes. Inoculation with the saliva may produce death in three forms: by the salivary microbe, by the excessive development of pus, and finally by rabies. The brain, and especially the medulla oblongata, of men and animals which have died of rabies, is always virulent until putrefaction has set in. So also is the spinal cord. The virus is, therefore, essentially localized in the nervous system. Rabies is rapidly and certainly developed by trephining the bones of the cranium, and then inoculating the surface of the brain with the blood or saliva of a rabid animal. In this way there is a suppression of the long incubation which ensues from simple inoculation of the blood by a bite or intravenous injection on any part of the body. It is probable that in this case the spinal cord is the first to be affected by the virus introduced into the blood; it then fastens on its tissues and multiplies in them.

As a general rule, a first attack which has not proved fatal is no protection against a fresh attack. In 1881, however, a dog, which had displayed the first symptoms of the disease of which the other animals associated with him had died, not only recovered, but failed to take rabies by trephining, when reinoculated in 1882. Pasteur is now in possession of four dogs which are absolutely secured from infection, whatever be the mode of inoculation and the intensity of the virus. All the other test-dogs which were inoculated at the same time died of rabies. In 1884, Pasteur found the means of attenuating the virus. For this purpose he has inoculated a morsel of the brain of a mad dog into a rabbit's brain, and has passed the virus proceeding from the rabbit through the organism of a monkey, whence it becomes attenuated and a protective vaccine for dogs. This is the first step toward the extinction of this terrible disease.

Glanders, again, is a disease easily transmitted from horses to man. Glanders, or farcy, is caused by the presence of a bacterium, observed as early as 1868 by Christot and Kiener, and more recently studied at Berlin by Schütz and Löfler. This microbe appears in the form of very fine rods (bacillus) in the lungs, liver, spleen, and nasal cavity. Babès and Havas found this bacillus in the human subject in 1881. Experimental cultures have been made simultaneously in France and Germany, and have given identical results.

Bouchard, Capitan and Charrin made their cultures in neutralized solutions of extract of meat, maintained at a temperature of 37°. By means of successive sowings, they have obtained the production of unmixed microbes, presenting no trace of the original liquid, and this was done in vessels protected from air-germs. These cultures may be carried to the eighth generation. Asses and horses inoculated with liquid containing the microbes produced by this culture have died with the lesions characteristic of glanders (glanderous tubercles in the spleen, lungs, etc.). Cats and other animals which have been inoculated in the same way die with glanderous tubercles in the lymphatic glands and other organs.

It follows from these experiments that the microbe which causes this disease is always reproduced in the different culture-liquids with its characteristic form and dimensions; that uni-ungulates can be inoculated with it, as well as man and other animals. In fact, this microbe is the essential cause of the disease.

We have already spoken of muscardine, a silk-worm's disease produced by a microscopic fungus; two other diseases are caused by distinct microbes, of which we must shortly speak. In the silk-worm nurseries, in which this disease prevails, the silk-worms which issue from the eggs, technically called seed, are slowly and irregularly developed, so as to vary greatly in size. Many die young, and those which survive the fourth molt shrink and shrivel away; they can hardly creep on to the heather to spin their cocoon, and produce scarcely any silk.

On an examination of the worms which have died of this disease, De Quatrefages ascertained the presence of minute stains on the skin and in the interior of the body, which he compared to a sprinkling of black pepper; hence the name pebrine. Under the microscope these stains assume the form of small mobile granules like bacteria, which Cornalia termed vibratile corpuscles, on account of their movements. Finally, Osimo and Vittadini ascertained the existence of these corpuscles in the eggs, and consequently showed that the epidemic might be averted by the sole use of healthy eggs, of which the soundness should be established by microscopic examination.

It was at about this date (1865) that Pasteur undertook the exhaustive study of pebrine; but Béchamp was the first to pronounce the disease parasitic, resembling muscardine in this respect, and caused by the attacks of a microbe—or microzyma, to adopt Béchamp's name—of which the germ or spore is derived from the air, at first attacking the silk-worm from without, but multiplying in its interior, and developing with its growth, so that the infected moth is unable to lay its eggs without depositing the spores of the microbe at the same time, and thus exposing the young grub to attack as soon as it is born. Pasteur's own researches soon induced him to adopt the same view. The PSM V29 D790 Nosema bombis pebrine microbe.pngFig. 5.Nosema bombysis, pebrine microbe (x 500 diam.) pebrine microbe was long regarded as a true bacterium, successively described as Bacterium bombycis, Nosema bombycis (Fig. 5), and Panistophyton ovale. Balbiani's recent researches tend to show that it should be assigned to another group, much nearer to animals, and designated Sporozoaria. These protista, still regarded as plants by many naturalists, chiefly differ from bacteria by their mode of growth and reproduction, in which they resemble the parasitic protozoaria, termed Psorospermia, Coccidies, and Gregarinidæ.

In Sporozoaria, growth by fission, the rule in all bacteria, has not been observed; this distinction is fundamental. Sporozoaria multiply by free spore-formation in a mass of sarcode substance (protoplasm), resulting from the encysting of the primitive corpuscles (mother-cells). The formation of numerous spores may be observed within the mother-cells, having the appearance of pseudonavicellæ or spores of gregarinidæ and psorospermia (parasites of vertebrate animals). Balbiani forms these organisms, which are found in many insects, into a small group, which he terms Microsporidia.

The ripe spores are the vibratile corpuscles of Cornalia. They closely resemble the spores of some bacilli (B. amylobacter, for instance), and their germination is likewise effected by perforation of the spore at one end, and issue of the protoplasm from the interior. This, however, does not issue in a rod-like form (Bacillus), but in that of a small protoplasmic mass, with amœboid movements, a characteristic not observed in any bacterium (Balbiani). The other species of silk-worms which have been recently introduced, notably the oak silk-worm from China (Attacus Pernyi), are attacked by microsporidia analogous to those of pebrine.

Pasteur has indicated the mode of averting the ravages of this disease. He has thus addressed the breeders: "If you wish to know whether a lot of cocoons will yield good seed, separate a portion of them and subject them to heat, which will accelerate the escape of the moth by four or five days, and examine them under the microscope to ascertain whether corpuscles of pebrine are present. If they are, send all the cocoons to the silk-factory. If they are not diseased, allow them to breed, and the seed will be good and will hatch out successfully. In a word, start with absolutely healthy seed, produced by absolutely pure parents, and rear them under such conditions of cleanliness and isolation as may insure immunity from infection." When the disease is developed, fumigation with sulphurous acid is recommended, or preferably with creosote or carbolic acid, which do not affect the silk-worms (Béchamp), and which hinder the development of microsporidia. These fumigations likewise keep the litter from becoming corrupt, and in a properly conducted nursery the litter is kept dry.

Wrongly confounded with pebrine, the disease flacherie is still more destructive to silk-worms. The symptoms are remarkable. The rearing of silk-worms often goes on regularly up to the fourth molt, and success seems assured, when the silk-worms suddenly cease to feed, avoid the leaves, become torpid, and perish, while still retaining an appearance of vitality, so that it is necessary to touch them in order to ascertain that they are dead. In this state they are termed morts-flats. A few days, sometimes even a few hours, suffice to transform the most flourishing nursery into a charnel-house. Pasteur examined these morts-flats, and found that the leaves contained in the stomachPSM V29 D791 Micrococcus bombycis flacherie microbe.pngFig. 6.Micrococcus bombycis (Cohn) Flacheriemicrobe (x 500 diam.). and intestine were full of bacteria, resembling those which are developed when the leaves are bruised in a glass of water and left to putrefy (Fig. 6). In a healthy specimen, of good digestion, these bacteria are never found. It is therefore evident that the disease is owing to bad digestion, and becomes rapidly fatal in animals which consume an enormous amount of food, and do nothing but eat from morning to night. The digestive ferments of unhealthy silk-worms do not suffice to destroy the bacteria of the leaves, nor to neutralize their injurious effects. These bacteria are really the cause of the disease, for if even a minute quantity of the leaves taken from the intestine of diseased silk-worms be given to healthy specimens, they soon die of the same disease. It is, therefore, essentially contagious, and, in order to prevent the diseased silk-worms from contaminating the healthy by soiling the leaves on which the latter are about to feed, as much space should be assigned to them as possible.

Good seed should also be selected, since it has been ascertained that some lots of seed are more liable to the disease than others. The affection does not indeed begin in the egg, as in pebrine, but the question of heredity comes in. It is clear that, when a silk-worm has been affected by flacherie without dying of it, its eggs will have little vitality, and the grubs which issue from them will be predisposed by their feeble constitution to contract the disease.

 

  1. From "Microbes, Ferments, and Molds." By E. L. Trouessart. Vol. lvi, "International Scientific Series." New York: D. Appleton & Co. 1886.