Issued JUIIP 12, 1911. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 137. A. D. MELVIN, CHIEF OF BUREAU. ANTHRAX, . SPECIAL REFERENCE TO THE PRODUCTION OF IMMUNITY. BY CHARLES F. DAWSON, M. D., D. V. S., Veterinarian, Delaware College Agricultural Experiment Station, UNDER THE DIRECTION OF JOHN R. MOHLER, V. M. D., Chief of the Pathological Division, Bureau of Animal Industry. 'alifornia ?ional ility WASHINGTON: GOVERNMENT PRINTING OFFICE. 1911. Issued June 12, 1911. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 137. A. D. MELVIN, CHIBF OP BUREAU. ANTHRAX, WITH SPECIAL REFERENCE TO THE PRODUCTION OF IMMUNITY. BY CHARLES F. DAWSON, M. D., D. V. S., Veterinarian, Delaware College Agricultural Experiment Station, UNDER THE DIRECTION OF JOHN R. MOHLER, V. M. D., Chief of the Pathological Division, Bureau of Animal Industry. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1911. THE BUREAU OF ANIMAL INDUSTRY. Chief: A. D. MELVIN. Assistant Chief: A. M. FARRINGTON. Chief Clerk: CHARLES C. CARROLL. Animal Husbandry Division: GEORGE M. ROMMEL, chief. Biochemic Division: M. DORSET, chief. Dairy Division: B. H. RAWL, chief. Inspection Division: RICE P. STEDDOM, chief; MORRIS WOODEN, R. A. RAMSAY, and ALBERT E. BEHNKE, associate chiefs. Pathological Division: JOHN R. MOHLER, chief. Quarantine Division: RICHARD W. HICKMAN, chief. Zoological Division: B. H. RANSOM, chief. Experiment Division: E. C. SCHROEDER, superintendent. Editor: JAMES M. PICKENS. 2 LETTER OF TRANSMITTAL U. S. DEPARTMENT OF AGRICULTURE, BUREAU or ANIMAL INDUSTRY, Washington, D. C., March W, 1911. SIR : I have the honor to transmit for publication the accompanying manuscript on the subject of anthrax, by Dr. Charles F. Dawson, of the Delaware College Agricultural Experiment Station, the experi- mental work having been carried out in cooperation with the Patho- logical Division of this bureau. Anthrax is one of the oldest and most widely distributed diseases of animals, and is particularly destructive to cattle and sheep. The existing method of combating the infection is to endeavor to control it by means of a vaccine devised by Pasteur. Two injections are, however, necessary by this method, and an active immunity is not established for about four weeks. Two important results have been accomplished through the work described in this paper. A single vaccine has been produced which may be substituted for the above-mentioned double vaccine, thus cut- ting down the time one-half. The other result is the production of an antianthrax serum that will confer an immediate passive immu- nity. Thus the two combined are a distinct advantage over the exist- ing method in the case of sudden outbreaks, where there is no time to prepare for the disease in the usual way. This serum can also be used in conjunction with the double vaccine of Pasteur. I recommend that the paper be published in the bulletin series of this bureau. Respectfully, A. D. MELVIN, Chief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. 3 CONTENTS. Page. Introduction 5 Historical 5 The anthrax bacillus 6 Cultural and morphological characters 7 Channels of infection 9 Infection of the soil 10 Methods of diagnosis 11 Preparation of specimens 11 Animal inoculations 12 Different forms of anthrax 13 Symptoms 14 Post-mortem appearances 15 The cause of death 15 Disposal of carcasses lg The official method in Delaware 17 Susceptibility of the various animals 17 Anthrax in man 19 Malignant pustule 19 Pulmonary and intestinal anthrax 20 The anthrax season 20 Prevalence of anthrax in Delaware 22 Methods of producing immunity in animals 24 The existing methods of double vaccination (Pasteur method) 24 Effectiveness of the method 25 A test of Pasteur vaccines 26 Experiments with various substances to test immunizing powers 28 Anthraxin 28 Pyocyanase 30 Anthraxase 35 Anthraxoin 38 A commercial vaccine in pill form 40 Preparation of an effective single vaccine 41 Tests of the single vaccine 42 A serum for producing immediate immunity 43 Experiments with the serum 44 Conclusion ,... 47 ANTHRAX, WITH SPECIAL REFERENCE TO THE PRODUCTION OF IMMUNITY. INTRODUCTION. Anthrax is primarily a disease of herbivorous animals, and occurs as an epizootic in sheep, cattle, horses, and mules, the animals being susceptible in the order named. In experimental work the small ani- mals, such as mice, guinea pigs, and rabbits, are extremely suscep- tible. The French name for the disease is " charbon ; " the German, '' Milzbrand." When it occurs in domestic animals it is also some- times known as splenic fever. In natural outbreaks anthrax usually has the character of a rapidly fatal septicemia, with the presence of large numbers of the characteristic bacilli in the blood. While the disease does not occur as a natural infection in man in the same sense as in animals, man frequently becomes infected from handling infected animals or their products, such as hides and hair. When man becomes infected through abrasions of the skin, the result- ing disease process is carbunculous in nature and is known as " malig- nant pustule." When the infection takes place in the lungs, the dis- ease is known under the name " woolsorters' disease." A third form in man may attack the intestinal tract. The latter two forms are rapidly fatal. In malignant pustule the disease may remain localized and the patient may recover. If, however, the bacilli migrate from the local lesion to the blood stream, death rapidly ensues, the patient dying from septicemia or toxemia. HISTORICAL. Anthrax is one of the oldest diseases. Moses records it in Exodus 9 : 9. Homer, Ovid, Plutarch, Virgil, Pliny, and many others have mentioned it in their writing. It exists in all countries and in all latitudes. It was formerly very destructive to human life as well as to animals. In 1617, near Naples, G0,000 people are reported to have died from anthrax. In San Domingo, in 1770, 15,000 persons perished in the short period of six weeks. This enormous death rate was probably due, in part, to eating the carcasses of animals dead from the disease. 5 6 ANTHRAX. It was not until the middle of the nineteenth century that scientists began the serious study of this very destructive disease. In 1849, Pollender, while studying the blood of animals dead from anthrax, discovered numerous rod-shaped, microscopic bodies, which he claimed were the cause of the disease. This claim remained unsub- stantiated until 1863, when Davaine announced that the bodies dis- covered by Pollender were bacteria, and showed that the blood of an animal could not cause anthrax in another animal when injected into it unless it contained these bodies. To this bacterium Davaine gave the present name, Bacillus anthracis. Davaine's views were not immediately accepted, but others took up the work, and in 1876 Dr. Robert Koch, in making his first contribution to bacteriology, announced that he had been successful in proving the correctness of Davaine's work on anthrax, and the question was then accepted as settled. Koch isolated the bacilli in pure culture and demonstrated their ability to form spores. Pie claimed that natural infection takes place through the intestinal tract, although he was at first unable to demonstrate this by feeding food infected by artificial cultures. Later, Pasteur succeeded in producing anthrax by feeding hay sprayed with cultures of Bacillus anthracis when he at the same time mixed with the hay thistles or any plants that would cause pricking or abrasions of the intestinal tract so that the bacilli could pass into the tissues. THE ANTHRAX BACILLUS. On account of the comparatively large size of the Bacillus anthra- cis it being from 5 to 20 micromillimeters long and from 1 to 1.5 micromillimeters broad, although both longer and shorter forms occur and the great readiness and certainty with which it kills experimental animals, it was the earliest disease to be studied with the microscope and by modern bacteriological methods. We first learned to demonstrate spore formation and spore staining from anthrax cultures. The germ is exceedingly easy to cultivate, grow- ing on almost any medium ajid at a wide range of temperature. It has many of the general morphological characters of the whole group of bacteria, and is, therefore, very useful for teaching pur- poses. The bacillus has a place in the earliest researches on im- munity, Pasteur having shown in 1880-1882 that animals inoculated with strains of the bacilli which had been devitalized to a certain extent by cultivation at high temperatures did not contract anthrax when exposed, while those not so protected did contract the disease. Spores of anthrax are among the most resistant things in nature, and are, therefore, useful in testing the germicidal power of disin- fectants. If a drop of blood is taken immediately after death from an animal dead from anthrax and examined with a microscope, large numbers THE ANTHRAX BACILLUS. 7 of nonmotile bacteria will be seen. A much better view of them can be obtained if a so-called blood-smear preparation is stained with methylene blue or any of the basic anilin dyes. It will be noticed that the ends of all the bacilli are sharply cut across. If viewed with a higher power, it will be seen that the ends bulge at the corners, making the ends appear slightly concave. The cells are also noticed to have a capsule, and when several cells are lying end to end the capsule seems common to the entire chain of cells. If in making preparations of anthrax blood great care is taken not to overheat, but just enough to " set " the specimen, and methy- lene blue is used as the stain, we can, while viewing the slide by reflected light with the unaided eye, notice a violet coloration of the entire specimen. If we now place the slide under the microscope, we can see that the space between the bacillus and its capsule is stained a reddish color. This is known as McFadyeans color test, and if the specimen is properly prepared is to be observed in all anthrax preparations made from dead animals. The test is most successful in those preparations where the bacilli are very numerous, and could be of great importance in field work where a microscope may not be at hand. CULTURAL AND MORPHOLOGICAL CHARACTERS. The bacillus of anthrax has such definite and constant cultural and morphological characters that it can hardly be mistaken for any other bacterium. When growing on the surface of agar-agar plate cultures beautiful colonies occur, appearing as wavy wreaths resem- bling locks of hair or bits of raw cotton thrown on the surface. It is supposed that the whole mass consists of thousands of contiguous cells forming one long chain, wound up in all directions. The groAvth in gelatin tubes is characteristic in that it liquefies the medium and the growth resembles an inverted fir tree. In milk a small amount of acid is formed, so that milk is slightly coagulated and slowly peptonized. In peptone bouillon virulent bacilli make a much more luxuriant growth than do the attenuated bacilli or vaccines. The heaviest growth is near the surface, where it can obtain oxygen, and consists of a dense mass of intertwined bacilli in chains which readily fall to the bottom of the tube if it be even slightly shaken. This heavy growth does not occur when bouillon is inoculated with attenuated bacilli. Growth on all media proceeds best at 35 C. The minimal tempera- ture for growth is 12 C., and the maximal 45 C. The vegetating bacillus is killed by a short exposure (15 minutes) to 60 C. moist heat, and by desiccation in a few days at ordinary temperatures. The spores, on the other hand, will stand almost any disinfecting process ANTHBAX. that any infected material can stand without greatly changing its physical characters. Hence it is impossible to disinfect hides con- taining anthrax spores without spoiling them for leather making. Spores require a boiling temperature in moist heat, and a much higher temperature in dry heat to destroy them. Spores live for a long time probably 10 years in the soil or in water, and it is because of this great resistance that pastures once infected remain so for an indefinite period. The bacillus can grow either in the presence or absence of oxygen, but it grows best when air or oxygen is present. Spores can form only in the presence of oxygen, and this accounts for the fact that the bacillus never sporulates in the living blood nor in cultures grown under anaerobic conditions. When spores begin to form, a tiny bright spot appears in the center of the parent cell, which soon increases to the diameter of the cell and always remains in its center. The cell protoplasm seems to be used up in the production of the spore, which now forms the resting stage of the organism. This spore remains quiescent until it is again placed in a suitable environment for growth, when it sprouts and assumes the bacillary or infective stage. Although the spore, or resting stage, of Bacillus anthracis is very resistant to all natural and artificial disinfectants, the bacillar or infective stage is quite vulnerable. The bacilli locked up in the air- less blood vessels or cavities of the body not only do not spore, but rapidly disintegrate after death. These important facts are of incal- culable value in the suppression of the disease by nature, and furnish the strongest kind of an argument for the prompt burial of animals dead from the disease. If such an animal be opened, however, the above-mentioned natural protection against the spread of the infec- tion is removed, as the bacilli will continue to multiply and sporula- tion will finally ensue. The following experiments were made to demonstrate the foregoing facts: The spleen of a guinea pig just dead from anthrax was minced and placed in a Petri dish exposed to air at room temperature. Microscopic examinations of this material 16 hours after showed that every bacillus had spored, the protoplasmic cellular material staining poorly. Another experiment made to determine the effect of such exclusion of the air as burial can afford showed that 24 hours are required for spore formation. In order to determine the effect of total exclusion of air upon the question of viability of the bacilli, capillary glass tubes were filled with heart's blood from a mouse just dead from anthrax and sealed in a flame. These sterile tubes were filled under precautions which prevented the ingress of extraneous bacteria; hence there were only anthrax bacteria in them, and the disintegrating effects of post- CHANNELS OF INFECTION. 9 mortem bacilli through the septic ferments which they form in a decomposing carcass were not present. Examinations and cultures from these tubes were made at intervals up to the twenty-third day, and while the bacilli were lessened in numbers, they were still viable. In buried carcasses, even though they be not opened, putrefactive bacteria grow very rapidly and their excretions cause a granular degeneration of the anthrax bacilli. If air is admitted to the carcass these excretions have no deleterious effect upon the spores that have formed, and such a carcass infects the soil in which it is buried, while if the carcass had not been opened, and care had been taken to disinfect the soiled exterior and air-containing cavities of the animal, the putrefactive bacteria would kill off the anthrax bacilli. The length of time necessary for this destruction varies with the temperature, being about a week in hot weather. CHANNELS OF INFECTION. There are four routes by which animals may become infected, namely, by ingestion, by inoculation, by- inhalation, and by trans- mission. Ingestion. The first and most frequent, and therefore the most important, is by ingestion. This form is acquired when animals are turned out to pasture. Inoculation. The method of infection next in importance is by inoculation, which is brought about by contact of abrasions and wounds with infected material, or by flies or other insects. A case of spontaneous inoculation arising in the course of my ex- periments the only one which occurred in the work was where a ram, immune to anthrax, was severely bled to furnish blood for experiments. He was kept in a stall where a sheep had died three months previously of experimental anthrax. A considerable wound was made by the operation and the animal was found dead of an- thrax on the second day after having been bled. Inhalation. The form third in importance is by inhalation. Here spores are breathed in with dust arising from infected material or surfaces. Transmission. A fourth form, which is probably rare, is that of transmission from the mother to the fetus in utero.' I have observed this in the case of an experimental cow which had been immunized with a single anthrax vaccine and subsequently tested with virulent bacilli. The mother at no time appeared sick, but on the sixteenth day after receiving the virulent bacilli she dropped a dead calf, which appeared to be a full-term one. Not suspecting the cause of the trouble, the calf and membranes were buried. Fortunately, a loop of the discharge found on the ground was plated and developed a pure cultivation of 60,000 anthrax bacilli, the virulence of which 89049 Bull. 13711 2 10 ANTHRAX. was demonstrated on a guinea pig. The cow was in no way sick, and daily microscopic examinations of the vaginal discharges showed these were free of anthrax bacilli. INFECTION OF THE SOIL. Soil becomes infected with anthrax by discharges from animals suffering with the disease, and by the dead bodies of animals which are allowed to decompose upon the surface. These discharges con- sist of small hemorrhages which occur from the mucous membranes in the nose and bowels, mixed with the nasal secretions 'and with the feces. These sometimes do not occur until just before death. In some cases the discharges are so limited as almost to escape obser- vation, but a microscopic examination in such cases would reveal large numbers of bacilli. Hence it is very important to limit to the smallest area possible the wanderings of an animal so affected. It is doubtful if the feces of the smaller experimental animals contain anthrax bacilli. One observer reports the presence of anthrax spores in the feces of guinea pigs, but I have never found them. Pasteur, in his memorable paper on anthrax before the Academy of Sciences in Paris in 1880, gave his opinion as to another manner in which the surface becomes infected with this disease. He said in part : Earthworms are the bearers of the germs, and they bring this terrible para- site to the surface of the ground from the depths to which it has been buried. It is in the little cylinders of earth, of flue earthy particles, that the worms ex- crete and deposit at the surface of the ground after morning dews, or after rains, that are to be found, besides a crowd of other germs, the germs of charbon (anthrax). While it does not seem impossible that earthworms may swallow the spores of anthrax and cast them out upon the surface, that close observer, Koch, was unable to verify the facts. He held that pas- tures remain infected on the surface from preexisting cases, and that the parasite remains on or near the surface from season to season and infects animals which graze upon such soil. When the grass gets short in dry weather, spores that may have been carried into the soil by previous rains either rise again by capillarity or are brought to the surface by the animals pulling the grass up by the roots. The writer has examined many earthworm casts from an- thrax graves of various ages from 15 years to 1 month and has failed to find anthrax spores in them. Pasteur's work was done be- fore the days of solid media, and hence the plate-culture method of isolation was not used by him. Another disease, malignant edema, is easily produced in guinea pigs and rabbits, and greatly resembles anthrax clinically, as well as in the morphology of the organism METHODS OF DIAGNOSIS. 11 causing it. These two germs would greatly resemble each other when viewed with a microscope made in 1880. In order to test further the question as to whether the surface above an anthrax grave becomes infected by earthworms, or otherwise, the writer has for the past three years pastured sheep and cattle upon soil in which there are many anthrax graves, and no case of spontane- ous anthrax has arisen. The animals were frequently observed graz- ing in the little hollows formed by the sinking in of the graves, and it would seem that this is sufficient proof of the questionableness of Pasteur's early theory regarding this source of infection. METHODS OF DIAGNOSIS. Anthrax is a disease that we absolutely know is caused by the para- site ascribed to it. We can tell just how long an animal will live after it has been inoculated with a given culture, provided the culture is of maximum strength. We know that a virulent culture will, in proper dose kill the laboratory animals, as well as sheep, in 48 hours, and that we can always recover the germ in the blood of such animals and reproduce the same disease in another animal by inoculating it with this blood. Hence, all of Koch's postulates can be fulfilled. Different races of anthrax bacilli vary in virulence. Those of highest virulence will kill guinea pigs and rabbits in 0.00001 c. c. doses of a 24-hour bouillon culture 1 in 48 hours, and the same culture would kill a sheep in 95 hours when given a 0.0001 c. c. dose subcu- taneously. Very young animals are much more susceptible to anthrax than adults, and hence care must be exercised in vaccinating young animals. A sucking rabbit a few days old died in 18 hours from a 0.1 c. c. dose of bouillon culture, and the virulence of the bacillus, which had become somewhat lessened, was greatly enhanced. A diagnosis can be made by the veterinary scientist by two meth- ods; either by a bacterioscopic examination of blood smears made soon after death or by animal inoculations. On account of the nature of the disease it is highly desirable that the first method be used, as no time should be lost in coming to a decision. It is easy to reach a de- cision if the specimen has been properly prepared and if the micro- scopist is familiar with the organism. PREPARATION OF SPECIMENS. A small drop of blood thinly spread on a piece of glass or paper and dried immediately is all that is required. It is best to obtain blood for this purpose by making a needle puncture into the jugular vein, as sometimes the bacilli are not numerous in the peripheral blood vessels. A specimen prepared in this way will arrive at the laboratory just in the condition in which it was collected. There will be no bacteria in it that were not there when it was prepared. 12 ANTHRAX. Sometimes specimens arrive consisting of two smeared surfaces of glass stuck together. These are dangerous, and it is next to impos- sible to separate them. They should be boiled and thrown away, no attempt being made to make an examination. The most dangerous anthrax package, and one which is not allowed in the mails, is the bottle of blood. These have arrived broken, and in one instance a positive diagnosis was made from blood smeared on the outside of the package by the sender's hands. Again, when parts of organs, or blood in volume) are sent in, decomposition will have taken place and a diagnosis may be impossible. The fruit- jar pack- age, containing spleen, liver, heart, etc., sent by express, is another most dangerous package. These have arrived broken, with liquid dripping from them. When not broken their opening is generally attended with dangerous difficulties^ The top is usually screwed down so tight that considerable force must be used to open it, and when this is accomplished the gases which have- formed will spray the liquids upon one's hands and clothing. A diagnosis of a blood-smear preparation is made by first " set- ting " the specimen by heat, passing the glass slide through the flame three times, occupying a second each time. The specimen is now stained with methylene blue or fuchsin, dried between two pieces of filter paper, and mounted for examination. If anthrax bacilli are present they can be easily seen stained blue or red, according to the stain used. They appear as rods with square ends. They are never round or blunt. They will almost always appear in chains, although they frequently appear as single cells when torn apart in making the preparation. Spores are never found in a specimen prepared from a recently dead animal. AVith lenses of 1,000-1,200 amplification the ends appear somewhat concave, from bulging of the corners of the cylinder. When such an organism is found in the blood of an animal recently dead the disease may be called anthrax, as it is the only one in animals that provides such a picture. ANIMAL. INOCULATIONS. When there is reason for doubt, animal inoculation must be re- sorted to. Mice, guinea pigs, and rabbits may be used, and these are susceptible in the order named. The suspected material is ground up in a sterile mortar containing normal salt solution. The solid particles are allowed to settle or are filtered off, and a few drops to 0.5 c. c. of the liquid are injected subcutaneously into guinea pigs. If anthrax be present the animal will die in two or three days, and the bacillus can be recovered from its blood and examined as in the first method. Plate cultures should also be made from the extract DIFFERENT FORMS OF ANTHRAX. 13 of the suspected substance, as it is possible to isolate the anthrax organism and thus come to a decision from a study of the anthrax colonies if any develop, as such colonies have a characteristic appear- ance. Inoculation into experimental animals of cultures made from these colonies, if they be anthrax, will cause the disease, and thus any doubt as to their identity will be removed. DIFFERENT FORMS OF ANTHRAX. Anthrax manifests itself in different forms according to the seat of invasion, kind of animal, virulence of the bacillus, season of the year, and time of the outbreak. The most acute type of the disease is seen oftenest in cattle and sheep and is known as apoplectic or fulminant anthrax. This form appears suddenly, without premonitory symptoms. The animal is suddenly seized with trembling, a haggard expression, swaying, diffi- cult breathing, and cyanosis. Convulsions and death occur in from a few minutes to two or three hours. Blood} 7 discharges usually occur during the last hour from the nose and rectum. These are very infectious, as the bacilli are present in large numbers, and constitute an important way in which pastures become infected. For this- reason such animals should be confined where these dis- charges can be .disinfected. The second form, known as anthrax fever or internal anthrax, differs from the fulminant or apoplectic form only in its duration. The symptoms in the last stages are practically those seen in the most acute type, except that they are more intense and of longer duration. There will be excitability, restlessnass, high fever, oozing of blood from the nose, eyes, ears, rectum, and thinner parts of the skin of the axilla or thigh ; tremors, dullness, prostration, grind- ing of the teeth, colicky pains, difficult breathing, arching of the back, rolling of the eyes, convulsions, and death in sheep in 24 hours, in Battle in from 2 to 5 days, and in horses in from 1 to 5 days. The third form is local or external anthrax. In this form the disease is at first localized in the mouth, throat, or skin in cattle, and the same form appears most often in horses in the tongue, throat, neck, breast, withers, shoulders, flank, or thigh. These swellings are firm or doughy, insensitive at certain parts, and tend to become gangrenous. There is no tendency to suppuration, and they do not crackle on pressure a diagnostic point when there is doubt as to whether the case is one of anthrax or of blackleg. When cut into something which should never be done, as the exudation always contains the anthrax bacillus there escapes either a pale, straw-colored, or bloody liquid. 14 ANTHRAX. SYMPTOMS. The symptoms of anthrax will vary with the species and with the type of the disease, except that in the last stages of any of the three types the most pronounced symptoms are identical. In the most acute type the animal may appear at first to be perfectly well and keep along with its fellows even when its temperature is very high as high as 106 F. Along with such a temperature we shall of course find a rapid pulse and increased respiration. When one is standing close beside such an animal the heart beats may be plainly heard. Soon other symptoms, such as grinding of the teeth, tremors, and standing with head down, appear. Then appear drooping of the head and ears and-. a disposition to lie down. Animals that have been lively will now decline to rise unless- handled roughly. They become stupid and sleepy and very weak in the hind parts. Whereas the temperature has all along been high, it now shows a sharp decline, and before death may become subnormal. The visible mucous mem- branes are a dusky red, especially those of the rectum and vulva. There is a bloody nasal discharge. The feces will be coated with a bloody mucus. Local swellings appear in the mouth, throat, neck, and breast (especially in horses), and there are sharp attacks of colic and convulsions which end the misery of the animal in from 12 to 48 hours after the disease is first noticed. Pregnant animals are liable to abort and thus greatly spread the infection through the copious discharges. An outbreak has its highest mortality at its onset, while later on some animals, especially horses and mules, may recover. In the most acute types, which occur mostly in cattle and sheep, the animal is found dead. A cow which seemed well at night is found dead in the morning, or if death occurs in the daytime, the illness is of short duration, occupying only a few minutes or' one or two hours. In these sudden attacks the symptoms follow each other so rapidly and death is so sudden that sometimes the owner is convinced that the animal has been poisoned. The attack is ushered in w r ith trem- bling, anxious expression, high fever, rolling of the eyes, and con- vulsive movements, soon followed by general convulsions and death. In the local form or cutaneous anthrax in cattle, swellings appear suddenly on different parts of the body at one or many places, and the animal dies with the same symptoms as occur in the most acute type when the bacilli reach the circulating blood from these local lesions. When the infection occurs in the tongue or pharnyx, we have in the first case gloss anthrax, and in the latter pharyngeal anthrax, the symptoms varying somewhat according to the part most affected ; but the general constitutional symptoms will be those already de- scribed. In some cases the most prominent symptoms at first will be enormous swellings of the rectal mucous membrane. POST-MORTEM APPEARANCES. 15 In the local form, or cutaneous anthrax, in the horse, the swellings occur at the points of entrance of the bacilli or spores, where there are abrasions of the skin or mucous membrane, or where biting in- sects have brought the infection from a previous case. These swell- ings appear suddenly at the point of inoculation and are character- ized by a rapidly spreading edema. The general symptoms are not so urgent, the fever is less intense, and the mortality, while not so great as in the more acute form, is still high. POST-MORTEM APPEARANCES. An animal that has died of anthrax will nearly always be found much bloated, with blood oozing from the nose and rectum. There will be evidences on the ground that the animal died a violent death, in convulsions. Local swellings will be present or absent according to the type of the disease. In the rapidly fatal cases little change will be noted either in the blood or internal organs beyond those produced by high blood pressure, indicated by a swollen spleen and engorged liver. The carcass itself the edible portions would show nothing that would make it doubtful as food. In the more prolonged cases a hemorrhagic condition will be noted in all the internal organs. The blood will be tarry in appearance and will not clot. The heart is often light in color, while on the inside it will be found deeply stained and containing dark, uncoagulated blood. The liver may be found enlarged and is easily torn in han- dling, presenting on its surface hemorrhagic areas. The spleen is often specially enlarged and distorted in shape, and ruptures on handling. The bacilli of anthrax can be found in largest numbers in those organs where the lesions are most pronounced, namely, the spleen, liver, and engorged lymphatic glands, but they are commonly found in any part of the vascular system. In the serous cavities, such as the pericardia!, the pleural, and the peritoneal, may be found a san- guineous fluid consisting of serum, red and white cells, and anthrax bacilli. The hyperemia and areas of extra vasated blood may appear at any point of the body where the bacilli have become localized and enormously multiplied, forming capillary embolisms, which consist of broken-down blood corpuscles, and bacilli. Hence they are fre- quent in the tongue, throat, lungs, stomach, and intestinal walls; the mesentery and the omentum; the skin, connective tissue, and the muscles. THE CAUSE OF DEATH. From the manner of death it would seem that the poison of an- thrax acts specifically upon the center of respiration, this in turn allowing a fatal accumulation of carbon dioxid in the blood. That 16 ANTHRAX. it is the replacement of the oxygen of the blood by carbon dioxid that is the immediate cause of death in anthrax is further shown by the loss in the blood of the property of coagulation, it being known that carbon dioxid precipitates and throws out of action the fibrin-form- ing element of the blood fibrinogen. DISPOSAL OF CARCASSES. The disposal of an anthrax carcass should not be left to the owner nor should anyone be allowed to open a carcass of an animal that is supposed to have died from anthrax. The diagnosis can only with certainty be made by a competent bacteriologist. He need only be furnished a drop of blood from the suspected animal. This blood can be drawn from the jugular by means of a syringe, or a small nick sufficient to allow a drop to escape may be made with a suitable in- strument. This blood should be smeared upon a piece of glass or piece of paper, dried immediately, and forwarded to the bacteriolo- gist. Any blood escaping during the above operation should at once be disinfected. The State should appoint a sanitary agent in every election district, whose duty it should be to dispose of anthrax car- casses, according to instructions issued by a competent authority, as it is only by the proper disposal of the carcass and by vaccination that the disease can be controlled. While the majority of our farmers recognize the advisability of promptly burying the carcass, there are those who would allow the animals to lie where they die to decompose or be eaten by dogs and buzzards. Parts of the carcass may be car- ried by dogs to adjoining or distant pastures and spread the infection. Spores forming in the carcass opened in this way would pass through the intestinal canal of buzzards and dogs and permanently infect pastures miles away. The soil upon which such an animal has lain would certainly be infected and remain so for many years. During the last few years deep burial, rather than efforts at crema- tion, has been practiced, with good results so far as can be ascertained. While cremation properly carried out is undoubtedly the safest method of disposing of an anthrax carcass, it is practically impos- sible to destroy absolutely all vestiges of the body and bacilli-laden bodj- fluids, and unless this is done the whole operation so far as it goes is almost a useless waste of time, material, and labor. In the cremation of carcasses in the field even the earth upon which it has lain should be thoroughly and deeply burned over so that the heat will penetrate to a depth sufficient to insure the death of the anthrax bacilli which certainly passed into the soil with the body fluids when rupture occurred from the heat. We know enough of the biology of the anthrax bacillus to take advantage of the fact that if it is kept locked up in the closed cavities and blood vessels of the body where no air can reach it, not only will the bacilli fail to produce the re- DISPOSAL. OF CARCASSES. 17 sistant spore, but they will actually decompose and become noninfec- tious, so that the bacilli which are in the blood vessels of an unopened animal perish. On the other hand, those bacilli which have come away in the pulmonary and rectal discharges during life will, if they are provided with sufficient material to keep them moist, promptly go into the spore or resting stage. These constitute the main source of danger, and in this way a considerable area may become infected. For similar reasons, hemorrhages into air-containing cavities, such as the lungs, trachea, head, sheath, vagina, and rectum, all favor the production of the resistant spore. Hence, if we introduce into the cavities a disinfectant which will surely kill the spores, and also at the same time take care to disinfect the soiled exterior surfaces of the animal, and then bury such an animal without opening it, we may reasonably conclude that such a carcass will not thereafter be a source of infection. THE OFFICIAL METHOD IN DELAWARE. In Delaware the method of procedure as advised and as carried out by the State board of agriculture is as follows: Formalin is injected in both directions into the trachea. Formalin-soaked cotton is pressed up the nostrils and into the mouth, rectum, vagina, or sheath. The grave is dug where the animal dies, when possible. The body of the animal is then either thoroughly wetted down with a 4 per cent solu- tion of formalin or kerosene, or coal oil is poured over the uppermost side of the animal and is set on fire. Only enough kerosene is applied to burn off the hair, as it would defeat the purpose to burn the animal sufficiently to cause rupture of the body. The carcass is then rolled into the grave with the other side uppermost. This side is now sprinkled with the oil and fired, after which the dirt upon which the animal has lain is disinfected "with formalin and is then shoveled into the grave first. The grave is now filled and banked up as is the cus- tom. Such graves should be surrounded by a fence. In fact, there should be a graveyard widely fenced in, and when an animal is known to have anthrax, it should be placed therein and allowed to die where it is to be buried. Such an inclosure should never be used for any other purpose, and should be so located that drainage from it upon other land will not occur. SUSCEPTIBILITY OF THE VARIOUS ANIMALS. While all the farm animals and even man himself will contract anthrax, we know that the degree of susceptibility varies not only in the different species, but also in individuals of the same species. We find that the herbivora are very susceptible and that the carnivora 89049 Bull. 13711 3 18 ANTHRAX. *., are less so, while the omnivora occupy a place between these two extremes. Cattle and sheep, being extremely susceptible, contract the fulminating type in nearly every case, while horses and mules frequently live several days, exhibiting the local or skin form, and some cases recover. It could probably be shown that any ruminant is more susceptible than other herbivora because of the anatomical and physiological characteristics of their respective digestive organs. In ruminants we find the immense rumen, with its moisture, heat, and alkaline reaction, an admirable incubator for bacteria. The out- ward passage of the contents of the rumen is slow, so there is plenty of time for anthrax spores swallowed with the food to develop into bacilli and multiply into prodigious numbers before they pass through the other two stomachs into the true or acid stomach, where some of them may be digested, but where many may pass out unharmed into the alkaline intestine and be absorbed with the food, finally entering the blood stream suspended in the chyle. It would now require only a few hours for them to multiply in the blood into sufficient numbers to produce their fatal poison. It is doubtless in this way that cattle which seem well when last seen and fed at night come down suddenly with the disease and are found dead in the morning. Swine are less susceptible than horses and cattle, yet when swine eat the carcasses of animals dead from anthrax they may contract the disease. As in other species, the young sucking pigs are more susceptible than the adults. As swine usually contract the disease by eating the dead animals, they generally have the disease in the throat or intestine. The symptoms in such cases do not vary materially from like cases in the other species. Dogs and cats contract the disease similiarly to hogs and are classed among the less susceptible. The symptoms are those to be expected, and consist of swelling of the throat, difficult swallowing, vomiting, profuse bloody diarrhea, high fever, and death. Birds of prey are said to be immune to the disease. Chickens are less susceptible than swine, yet Pasteur showed that chickens were quite susceptible when immersed in cold water and then inoculated. Likewise frogs, which are ordinarily immune, can be successfully inoculated after immersion in warm water. Young pigeons of certain breeds are easily infected artificially, and sparrows, finches, canaries, yellow-hammers, and redbreasts have been successfully inoculated. In chickens anthrax runs a very rapid and fatal course in 24 hours. They can contract the disease by eating the carcass or dis- charge of an animal dead from anthrax. As in other animals, a differential diagnosis between this disease and other chicken diseases of like type should be based upon the discovery of the anthrax bacillus in the blood along with the symptoms of sudden debility ANTHRAX IN MAN. 19 and high fever. Anthrax swellings will occur on the comb and wattles, around the eyes, and on the tongue, palate, and feet. The bird is extremely weak, has violent tremors and convulsions, and bloody diarrhea, and dies within 24 hours. ANTHRAX IN MAN. Anthrax is not, strictly speaking, a human disease, yet it is not rare in the practice of physicians who attend the workers in tanneries where imported hides are converted into leather, nor is it rare in per- sons whose occupation brings them in otherwise contact with hides,, wool, hair, furs, hoofs, bones, rags, felt, glue, or any product made from the bodies of animals that die of anthrax. Hence the disease is found in shepherds, cattlemen, horsemen, farmers, drovers, butchers, tanners, brush makers, and veterinarians. The disease is notoriously common in those who handle imported hides, although man can become infected through the bites of flies or other infected insects. The sound skin is sufficient protection against the entrance of the bacillus, but the slightest abrasion is sufficient as a point of inoculation in handling infected material, or even for inocu- lation by the ordinary housefly that has visited the discharges of an animal suffering with anthrax. The process of tanning does not always disinfect an anthrax hide. That hair is a medium of infec- tion is shown by outbreaks of anthrax among brush makers. Per- sons who handle imported wool and rags contract the disease by in- haling the spores in the dust which arises. This spore develops into the bacillus, and a case of pulmonary anthrax is established. This form is known as woolsorters' disease, or ragpickers' disease. Those who handle bones and carcasses in fertilizer, glue, and rendering establishments located in anthrax districts are peculiarly exposed to infection. In man the large majority of cases occur on the face, prob- ably because this part of the body is more liable to be attacked by biting insects. Anthrax in man is a local disease, and we do not find the bacilli in as large numbers in the blood or other organs, such as the liver, spleen, and kidneys, as we do in the lower animals. The bacilli can, however, be found in abundance in the local lesion. MALIGNANT PUSTULE. Malignant pustule, or local anthrax of the skin, usually occurs on the face, hands, arms, or neck of those who handle imported hides. In from one to three days after infection takes place a small red pimple appears. This soon changes to a vesicle, which is very pain- ful. The center of the vesicle rapidly becomes necrotic, forming a black eschar, which soon becomes surrounded by a ring of vesicles. 20 ANTHRAX. The surrounding tissues become congested and edematous, and the lymphatics are involved. The fever is quite high, and the patient is very sick from the absorption of poison from the local disease. It runs a rapid course, and the patient may die from toxemia. Most of these cases are, fortunately, susceptible of successful treat- ment. This consists of early and thorough excision of the pustule and all infected surrounding tissue, followed by the local application of strong disinfectants. At Guy's Hospital, in London, 13 out of 15 cases were cured by excision, even though in 12 of these cases the inflammation had spread to the surrounding parts or had involved the lymphatic glands with more or less severe constitutional disturb- ance. In some cases the disease process goes no further than the formation of the eschar, which then becomes a scab, with subsidence of the inflammatory process, and recovery. In the majority of cases, however, the disease runs a regular course; if not excised early the bacilli enter the blood stream and the case terminates fatally from a modified form of the same disease as occurs in cattle, with the excep- tion that the bacilli are not found in large numbers in the internal organs nor is the spleen greatly enlarged. It may, therefore, be said that in man death is largely due to the absorption of toxins from the local lesions. When the pustule is situated on the extremities the per- centage of recoveries under treatment is large. PULMONARY AND INTESTINAL ANTHRAX. Pulmonary anthrax, or woolsorters' disease, is caused by the inha- lation of spores by those who handle hides or other infected material. The initial lesion is local and is situated in the lower trachea. It consists of a swollen and hemorrhagic condition of the mucous mem- brane, with great enlargement of the mediastinal and bronchial glands and of effusions into the pleural and pericardial cavities and the lungs. Externally may occur also cutaneous edema over the chest and neck and inflamed glands. This form is rapidly fatal, the patient dying from suffocation and toxemia. Intestinal anthrax is also of local origin and has about the same pathology and terminations as the pulmonary form. There is intense inflammation of the intestinal mucous membrane and involvement of the neighboring lymphatic glands. Intestinal infection takes place when anthrax-infected products are eaten. THE ANTHRAX SEASON. Anthrax is peculiarly a disease which may be said to be a seasonal one; that is, the disease makes its appearance with the advent of certain kinds of weather. In March, 1907, in Delaware we were visited with some exceptionally warm, springlike weather of con- THE ANTHBAX SEASON. 21 siderable duration, and on the 22d of that month the first case oc- curred for that year, an exceptionally early start. In 1908 the first case occurred on May 2. In 1909 the first case did not occur until July 23, but the disease persisted until November 6, whereas in 1907 the last case was on September 24, and in 1908 on August 24. So that we may say the season lasts, in Delaware, from March to No- vember, being most prevalent in the summer months. As the disease is, generally speaking, a warm-weather one, and as we know that the causative agent is present throughout the year, there must be some conditions which come with warm weather which predispose to the onset of the disease, either by affecting the receptivity of the animals or by increasing the infective properties of the germ of the disease. Possibly both conditions prevail. There can be little doubt that the resting stage of the Bacillus anthracis the spore will quickly develop into the bacillar or infecting stage under the influ- ence of heat, moisture, and organic matter. These conditions pre- vail on the pasture in warm weather, and the writer has demon- strated that the germ of anthrax will vegetate in a 2 per cent hay infusion. Hence, when spores of anthrax come in contact with hay infusions in pastures, especially in meadows, there is produced a viru- lent culture which will infect the animal eating them. The animals themselves are probably more susceptible to anthrax when turned out upon fresh pastures, as the ingestion of rank grasses, which are acid in reaction, lessens the normal alkalinity of the blood and thereby increases the susceptibility to anthrax. This greater alkalinity of the blood in carnivora may be the cause of their greater resistance to the disease, while the loss of alkalinity of the blood by the herbivora, and especially by ruminants such as cattle and sheep, through the ingestion of such large quantities of acid grass, may account for their increased susceptibility during the grass-growing season. At all events there are certain weather factors which de- termine, in a measure, the onset of the anthrax season, and given such conditions the season can be predicted with tolerable certainty. This season exists when we have had a long, dry period, followed by light rains or infrequent heavy rains, and then by extreme heat. This kind of weather will not only produce bacterial multiplication, but will cause a rapid growth of rank acid grasses, and these Avhen eaten will increase the susceptibility of animals to anthrax by decreasing the alkalinity of their blood. Anthrax, then, may be said to be a pasture disease, which exists mostly in warm weather. If it were a stable disease, why should we not have cases occurring in winter, and why is it that the disease rarely occurs in animals that are not turned out on pasture? We rarely see a case of anthrax in a carriage horse that is fed in the stall, but it is not of infrequent occurrence in horses that are turned 22 ANTHRAX. out to pasture. Cases are rare in city horses, but common in the farm horse. The city horse must eat the same hay and grain, gener- ally speaking, as the farm horse. If it were the hay and grain that produce the disease, we should find anthrax as common in city stalls as on the farm. Anthrax rarely occurs in small towns. It is distinctly a disease of animals that graze on infected fields. A recent case emphasizes this. An expressman delivered some goods to a farmer who lives 1 mile from this town (Newark, Del.). This farm has anthrax every season. The expressman's horse must have grazed upon infected soil while there, as it was sick within 48 hours with high fever and edematous swellings at the root of the neck and down the front legs. A microscopic examination of the exudate showed the presence of anthrax bacilli, and the animal died in a few hours. This animal lived in the stable with others, and was fed the same feed. No case has occurred since, and it was the only animal from that stable that visited the infected farm. The great importance of an early diag- nosis and isolation was demonstrated in this case. Within a few hours after a swelling was noticed anthrax was suspected and some of the bloody subcutaneous exudate was aspirated and brought to the writer by the attending veterinarian for diagnosis. It was thus possible to remove the animal from the stable to a yard where other animals could not become infected long before any infecting dis- charge had taken place. The animal was embalmed, washed down with formalin solution, as previously described in this article, and buried in a six-foot grave as soon as the same could be dug. The stables have been used uninterruptedly ever since (four months), and no fresh case of anthrax has occurred. PREVALENCE OF ANTHRAX IN DELAWARE. Anthrax has been officially recognized as existing in this State (Delaware) since August, 1892. It is highly probable that the disease had its origin in imported hides used in the tanneries at Wilmington for making leather. Large quantities of scraps accumulate during the process of leather making, and these were sold to farmers who com- posted them and later spread the infected material upon the land. Since the spores of anthrax would live and retain their virulence in a compost heap, it is easy to see how the lands first became infected. While it is generally conceded that meadow pastures are most pro- ductive of the disease, the tillable lands were probably infected first, as above stated. The meadow pastures have retained their infective properties because of their constant use for grazing, and particularly because of fresh cases of anthrax occurring upon them. The higher tillable lands have, however, in many caes become disdnfected by the lapse of time, cultivation, and drainage. PREVALENCE OF ANTHRAX IN DELAWARE. 23 Estimates made by veterinarians practicing in the State show that there are from 175 to 200 farms in Delaware that are permanently infected with anthrax. These farms are located, in a general way, in that territory which drains into the Delaware River and the Dela- ware Bay. The infected territory comprises about one-third of the total area of the State. While anthrax has been known to exist in the State only since 1892, it is highly probable that the first cases occurred much earlier and that the disease is as old as the morocco-leather industry. The originally infected territory must have been the cultivated lands upon which infected compost fertilizer was spread by the farmer him- self. Animals dying on such farms before the nature of the disease was known here were either hauled out dead or turned out while yet alive upon the marshes to die. Doubtless they would in some cases be set adrift in the creeks and thus infect any shore or marsh upon which they drifted. As the marshes are always wet and contain decaying vegetation in abundance, the conditions for the development of the microbe of anthrax are perfect, while upon the dry cultivated lands, even though the microbe be present, the conditions are, owing to the comparative absence of water, much more unfavorable to the multiplication of the causative agent. It is because of these condi- tions that marsh pastures are regarded as being more productive of the disease. The wash water from the tanneries at Wilmington can no doubt infect the streams into which it flows and cause the disease in animals either grazing upon them or drinking from them. Those farms and marshes now infected will remain so for many years, even though no new cases occur upon them. This liability to outbreaks of anthrax on a farm has- a depressing effect, not only upon the value of the land, but upon all agricultural operations. While there has been no epidemic of anthrax in Delaware for several years, and the number of deaths from the disease has been exceeded by many ordinary diseases of which little notice is taken, anthrax must always be considered a menace to the agricultural wel- fare of the State because of the ever-present liability of an outbreak which might assume the proportion of a genuine epidemic. This liability should be offset by the annual vaccination of every farm animal in the infected territory. Even though several years may have elapsed since the last case occurred on a farm, that farm is liable at any time to be again visited by the disease. 24 ANTHRAX. METHODS OF PRODUCING IMMUNITY IN ANIMALS. THE EXISTING METHOD OF DOUBLE VACCINATION (PASTEUR METHOD). Since 1892 anthrax has been controlled by vaccination by a method devised by Louis Pasteur. This method consists in the subcutaneous injection of attenuated cultures of the bacillus of anthrax. Two in- jections of varying degrees of strength are made at an interval of 12 to 14 days. The first injection consists of 1 cubic centimeter of a culture that has been incubated at 42 to 43 C. for a sufficient time to decrease its virulence to a point where it will kill white mice, but not guinea pigs or rabbits. This, generally requires a period of 24 days, assuming that the culture was made originally from a moist, virulent race of anthrax bacilli. Such cultures are to be made directly from the heart's blood of an animal that has died of anthrax within 48 hours after inoculation. In such blood we find only non- spore-bearing bacilli, and when these are grown at 42 to 43 C. they do not at any time produce spores while this temperature is main- tained. Contrary to the statements of some, however, the bacilli promptly form spores when this temperature is reduced. These spores do not acquire any more virulence, however, than the parent bacilli which produced them. The second injection consists of a culture similarly made and incubated at 42 to 43 C. for a period of 12 to 18 days, or one whose virulence has been reduced to a point where it will not kill rabbits but will kill white mice and guinea pigs, the latter in 3 or 4 days. The author's experience has shown that there is no hard and fast line in the number of days that a culture of a given race of anthrax bacilli must be attenuated. Much depends upon the re- sistance of the bacillus, the character of the culture medium, the exactness of the temperature of the incubator, and the natural re- sistance of the animals used in testing the vaccines. When a culture of proper strength has been obtained and is properly transferred to fresh media about once a month and kept in a cool place where it will not evaporate, it may be used indefinitely as a stock culture for inoculating a liquid medium, which constitutes the vaccine. When such attenuated cultures are inoculated into animals, a very mild and clinically unnoticeable attack of anthrax is produced, which confers an active immunity which persists throughout the anthrax season. The inoculation must be repeated the following season. Certain precautions are necessary in the application of anthrax vaccine. Assuming that the vaccine has been properly prepared, it is the duty of the veterinarian to ascertain that anthrax is not already existing in the animals he is about to vaccinate. As a pre- caution, when there is reason to suspect that anthrax may already METHODS OF PRODUCING IMMUNITY. 25 be existing, no animal showing a fever should be vaccinated, as the disease may be carried from the already infected animal to others upon the point of the inoculating needle. Again, in order to prevent abscess in horses and mules by introducing under the skin strepto- cocci or staphylococci by the point of the needle, the place of injec- tion should be disinfected of. these microbes. The anthrax bacillus can not produce pus, but the germs usually found upon the skin are to be held responsible when abscess occurs from vaccination. The writer has been in the habit of dipping the point of the needle into strong carbolic acid contained in a small vial which may be conveniently carried in the side pocket at the time of vaccinating. None of this acid can enter the needle and kill the vaccine germs, as it is already filled with the vaccine, and none can be introduced under the skin, as it is all removed from the needle in its passage through the skin, and this in turn disinfects the wound made. While every veterinarian knows that hypodermic injections of medicine are daily made with a minimum of abscess production, it must be recognized that the conditions are not similar, and hence it is deemed highly advisable that these precautions be taken, especially in horses and mules. There is usually little or no swelling at the point operated upon. Where abscess occurs the operator is to blame. It means that pus- producing germs have been carried in upon the needle and that either the needle or the skin was not disinfected. Abscess is more likely to occur in horses and mules than in other animals. It has been the practice in Delaware to continue the vaccinated animals at their usual work. We have no data upon the subject show- ing this is unwise, but it is believed that the animals should be shielded as much as possible from excessive work and from extremes of heat or cold or from chilling rains. We advise our vaccinators, who con- sist of regular, practicing veterinarians designated by the governor upon the recommendation of the board of agriculture, to destroy all opened bottles of vaccine that remain unused at the end of a day's work, as it will certainly become contaminated and be spoiled if kept overnight at ordinary temperatures. The vaccine is dispensed in 50-dose bottles hermetically sealed and distinctively labeled, so there can be no mistake made in using it. EFFECTIVENESS OF THE METHOD. The results of vaccination have been as good, as shown by our sta- tistics, as those obtained by the use of any other biological product. That the method is entirely safe is shown by the fact that only one dangerous swelling has been brought to our notice in three years' experience in Delaware. Every year there are instances where, for 26 ANTHRAX. some reason, animals that have been vaccinated die from anthrax, showing that they were not protected by the vaccine. No doubt some of these failures may be due to the animal being missed during vacci- nating. In other cases the failure may be due to the vaccine needle not properly puncturing the skin, so that the vaccine falls upon the ground. These accidents are readily brought about when the animals are unruly. On the other hand, the writer has observed a case of anthrax that occurred in vaccinated animals that is, where it was positively certain that the vaccine was properly prepared and active and had been properly applied. Some cases of nonprotection by vaccination can be explained by the fact that the vaccine itself was inert, and therefore nonprotec- tive. Cultures made from such vaccine failed to grow. The vaccine would, of course, fail in the usual physiological test for an anthrax vaccine, and, of course, would fail to protect the animal against anthrax. The writer has known of instances where for various rea- pons an animal would escape vaccination, and that animal would be the only one to die on that farm. Hence he feels warranted in highly commending vaccination as the most important means of combating this terrible scourge. In France, where vaccination is most popular, and where statistics are reliable because of governmental control, the death rate before vaccination was adopted was, in cattle 5 per cent, and in sheep 10 per cent. After vaccination was adopted the losses were reduced to 0.34 per cent in cattle and 0.94 per cent in sheep. In Delaware the losses in 1907 in horses and cattle that were vaccinated were 0.32 per cent. Vaccination should be practiced every spring, at least a month be- fore it is time to turn the animals out to pasture, as a month is re- quired for the production of immunity. This vaccinating should not be optional with the owner, as at present, but should be compulsory, the State assuming the risk of loss from the use of the vaccine, but not in those cases where it can be shown the animal died not from vaccination but from a natural infection, owing to failure of being protected by the vaccine, or from other causes. A TEST OF PASTETJB VACCINES. Along with the investigation of anthrax for the purpose of dis- covering new methods of treatment and prevention of the disease, and for studying the biology of the causative organism and the gen- eral sanitary aspects of the subject, it was decided to test the efficacy of freshly prepared Pasteur vaccines. It is believed that some of the bad results that have been reported as following the use of Pasteur vaccine is due to carelessness on the part of those preparing and using it. As there is no visible difference in the appearance of the two METHODS OF PRODUCING IMMUNITY. 27 vaccines, Nos. 1 and 2, it is an easy matter to get the bottles mixed or improperly labeled. If the labels should come off the bottles, the vaccinator would have no guide as to which vaccine he was using. The writer has purchased anthrax vaccines on the open market and has found some that were wholly inert, and others that were too strong when tested in the usual way. For the purpose of gaining such information as is possible from the practical experiences of practicing veterinarians who vaccinate against anthrax in Delaware, the writer undertook to prepare Pasteur vaccines for the State board of agriculture, which were to be used as soon as possible after they had reached destination. The cultures con- stituting the vaccines were grown in 50-dose bottles, each bottle con- taining 50 c. c. of bouillon which had been inoculated 24 hours pre- viously with the vaccinal germs. The official vaccinator was fur- nished first with No. 1 vaccine. In 12 days he was shipped the No. 2 vaccine, for use on the same animals. Under this plan it was impos- sible for the bottles of vaccine to get mixed, even though the labels came off. The plan of using fresh vaccine in which the germ is still in the bacillar stage was continued with good results for two seasons. For the last two years the plan of using vaccines that had been incubated for four or five days, or until the bacilli had spored, has been tried. This latter method represents the condition of vaccine as put on the market by commercial houses, and is a suspension of spores instead of bacilli. According to the experiences of these two seasons, we have reason to believe that the vaccine will remain active for several months, and such cultures may be prepared several months in ad- vance of their use, provided the incubation is carried to a point where all growth ceases, or spores form. These spores inherit just that degree of strength possessed by their progenitors, and do not change except with a considerable lapse of time, possibly a year, if kept under favorable conditions. It is highly important that anae- robic conditions be not established in the bottles, as the bacilli will sink to the bottom and will not spore when grown anaerobically. To pre- vent this, it is important that the culture medium be made to reabsorb the air which has been driven out of it during sterilization before being inoculated with the vaccine germs. In preserving stock cultures of the vaccine it is very important that they be incubated for several days before being taken out of the incubator. If they are removed before spores have formed, the more vulnerable bacilli may succumb to existing unfavorable conditions. To these conditions may be ascribed the death of cultures of anthrax vaccine, and the loss of virulence of former virulent cultures of anthrax bacilli, which some- times occur during the course of laboratory work. 28 ANTHRAX. EXPERIMENTS WITH VARIOUS SUBSTANCES TO TEST IMMUNIZING POWERS. Experiments upon the disease have been carried on uninterruptedly for the past three years. These had for their object the preparation of substances in the laboratory and in living animals which could be used in combating anthrax by acting as antitoxins or bactericides, or as vaccines. Realizing the great importance and economy in producing pro- tective and curative substances in the laboratory over the necessarily expensive methods when employing animals for the same purposes, efforts have been made along the line of producing various culture products to be used in combating anthrax. Pyocyanase, made after the method of Emmerich and Loew, gave some good results when tested upon rabbits, but failed upon sheep. Anthraxase, prepared by the writer after the same general method used in producing pyocyanase, was without protective or curative properties, although it produced high fever when injected into rabbits and sheep subcutaneously. Anthraxoin, consisting of a turbid suspension of dead anthrax bacilli, was apparently useless in protecting sheep against anthrax when used similarly to the Pasteur vaccine. A single vaccine, having for its object the production of immunity in two weeks and thus cutting down the necessary period by one-half, was made by incubating a virulent bacillus for about 18 days at 42 to 43 C. Such a culture will kill guinea pigs in about a week, and in strength it thus holds a position between the two vaccines of Pasteur. With it sheep were vaccinated and after 12 days withstood an otherwise fatal infection with virulent bacilli. In some cases, however, the immunity was not sufficiently strong, as was evidenced by the death, now and then, of a sheep when tested with virulent bacilli. The preparation and use of the various substances, together with the results of the experimental work, are described in the following pages. ANTHRAXIN. Anthraxin was made similarly to tuberculin and mallein. The cultures of anthrax were grown for 10 days with daily shaking in glycerinated, peptonized bouillon, the glycerin being used in 4 per cent strength. The cultures were then sterilized by boiling, filtered through Berkefeld filters, and then evaporated to one-tenth of the original volume. A sirupy liquid, much resembling tuberculin, re- sulted. It was tested on rabbits and sheep for immunizing proper- ties, as shown in Table 1, on the following page. EXPERIMENTS WITH ANTHRAXIN. TABLE 1. Experiments with anthraxin. 29 Injections Animal. Date. Anthraxin. Virulent culture anthrax. Temperature. Result and remarks. Rabbit 1 1907. July 10 Ice subcutane- F. July 17, 10 a. m. . . ous. 105, 3 p. m .. July 18 104, 10 a. m.. . do 103, 3pm July 19 Normal July 24, 11 a. m. . . 1 c. c. subcutane- 104 July 24, 1.30p.m.. ous. 105 July 24,5.30p.m.. 105 July 25, 9 a. m Normal . . . July 31, 9 a. m 0. 05 c. c Aug. 2, 9 a. m Dead frm anthrax. Sheep 1 Aug. 27, 1 p. m 5 c. c. subcutane- Aug. 27, 6 p. m ous. Sheep blowing, head Aug. 28, 9 a. m down. Sheep seems well. Sept 3, 9 a. m . 5 c. c. subcutane- Sept. 10, 9 a. m. . . ous. 0. 25 c. c. Sept 12, 9 a m Dead from aithrax. Sheep 2 Aug. 27, 1 p. m 5 c. c. subcutane- Aug. 27, 6 p. m ous. Sheep blowing, head Aug 28, 9 a m down. Seems well. Sept. 3, 9 a. m 5 c. c. subcutane- Sept. 10, 9 a. m ous. . . .do Sept 23, 9 a m 0. 25 c. c Sept. 25, 9 a. m Dead from anthrax. Sheep 3 Aug. 27, 9 a. m 5 c. c. subcutane- Aug. 27, 6 p. m.. ous. Blowing; no fever. Sept. 3, 9 a. m 5 c. c. subcutane- Sept. 5 ous. Diarrhea. Sept. 10, 9 a. m. . . 5 c. c. subcutane- Well. Sept. 15, 9am ous. 0. 25 c. c Sept. 17, 9 a. m. . . Dead from anthrax. Sheep 4 Aug. 27, 9 a. m. . . 5 c. c. subcutane- Aug. 11, G p. m ous. Blowing; no fever. Sept. 13, 9 a. m 5 c. c. subcutane- Diarrhea for several Sept. 10, 9 a. m ous, .do days. Sept 15, 9am 0. 25 c. c Sept. 17, 9 a. m Dead from anthrax. Sheep 5 and 6. . Sept. 3, 9 a. m 5 c. c. subcutane- Sept. ous. Seemed well. Sept. 10, 9 a in 5 c. c. subcutane- Oct. 7, 9 a. m . . ous. 0. 25 c. c. Oct. 9, 9 a. m Both dead from an thrax. From the foregoing experiments we gather that anthraxin was possessed of no immunizing properties whatever. 30 ANTHRAX. PYOCYANASE. Pyocyanase, the next substance experimented with, was prepared as follows: Large flasks of medium were inoculated with Bacillus pyocyaneus and grown at 37 C. until a ropy condition was produced, which required three weeks, the flasks meanwhile being shaken daily. The composition of the medium was as follows: Peptone, 0.5 per cent; glycerin, 0.1 per cent; dipotassium phosphate, 0.1 per cent; magnesium sulphate, 0.01 per cent; sodium chlorid, 0.3 per cent; sodium bicarbonate, 0.1 per cent ; in distilled water (synthetic medium of Emmerich and Loew). When growth had ceased in this medium the growths from a number of agar-agar cultures of the same organ- ism were added and the whole was thoroughly shaken. The culture medium was then neutralized with dilute hydrochloric acid. Car- bolic acid was then added to 0.2 per cent strength as a preservative. The liquid was then evaporated down to one-tenth of its original volume at ordinary room temperature by being placed in pie plates. Then the liquid was dialyzed for 24 hours in running water, filtered through Berkefeld niters, and 0.2 per cent carbolic acid was again added to replace that which was dialyzed out. The resulting liquid, pyocyanase, has a dark coffee color and a pungent odor. The experi- ments to test its immunizing and curative properties upon guinea pigs, rabbits, and sheep are recorded in Tables 2 and 3. TABLE 2. Experiments with unfiltered pyocyanase. Animal. Date. Injections. Temper- ature. Result and remarks. Pyocyanase. Virulent 24- hour culture anthrax. Rabbit 1 Guinea pig 1 1907. Dec. 9, 9 a. m Dec. 10, 9 a. m Dec. 11, 9 a. m 6 c. c. sub- cutaneous. 4 c. c. sub- 0.2 c.c. sub- cutaneous. F. Rabbit dead. Liver ne- crosed, spleen enlarged; anthrax bacilli at local lesion, but none found in blood or in liver and spleen; probablydiedfrom pyocyanase poisoning. Died during night. No ba- cilli in blood, but few at local lesion; probably died from pyocyanase poison- ing. cutaneous. 3 c. c. sub- - Guinea pig 2 Dec. 17, 10a.m... Dec. 18, 10 a. m cutaneous. 1J c. c. sub- 0.1 c. c. sub- cutaneous Dec. 18, 1.30p.m.. cutaneous. 2 c. c. sub- Dec. 16, 10 a. m... cutaneous. 3 c. c. sub- Dec. 17, 10 a. m... cutaneous 0.1 c.c. sub- cutaneous. EXPERIMENTS WITH UNFILTEBED PYOCYANASE. TABLE 2. Experiments with unfiltered pyocyanase Continued. 31 Animal. Date. Injections. Temper- ature. Result and remarks. Pyocyanase. Virulent 24- hour culture anthrax. Guinea pig 2... Guinea pig 3 Rabbit2 Rabbits Rabbit 4 Rabbit5 Rabbit 6 Rabbit? 1907. Dec. 18, 9 a. m Dec. 19, 9 a. m 1J c. c. sub- cutaneous. F. Sick. Chloroformed. No bacilli in blood, but a few at local lesion; pyocyanase poi- soning. Dead from anthrax. Bacilli in blood; another guinea pig treated similarly died of anthrax on third day after inoculation. Found dead of anthrax. Bacilli in blood. Edema at points of injection of pyocyanase, no fever. Animal apparently well. Animal dies from anthrax on eleventh day. Bacilli in blood. Dead from anthrax. An- other rabbit treated simi- larly died of anthrax on third day also; bacilli in blood. Slight swelling at point where pyocyanase was in- jected. Animal dead from anthrax, having lived four days. Considerable reaction at point of injections. Rabbit dead from anthrax on third day. Small reaction from injec- tions. Animal dead from anthrax, having lived four days. Dec. 19, 9 a. m Dec. 20, 10 a. m... 3 c. c. sub- cutaneous. do Dec. 21, 3 p. m 1J c. c. sub- 1908. Jan. 2, 9 a. m Jan. 4 9 it. in cutaneous. 0. Ic.c. sub- cutaneous. Jan. 13 9 a. m Jan. 14, 9 a. m Jan. 15, 9 a. m. . . . 6 c. c. sub- cutaneous. do... 0.2 c. c. sub- cutaneous. 103 102 Jan. 16, 9 a. m Jan. 13., 9 a. m Jan. 14,9 a. m Jan. 15 9 a. m. 6 c. c. sub- cutaneous. 6 c. c. sub- 0.3 c. c. sub- cutaneous. Normal. Jan. 18, 9 a. m cutaneous. Jan. 25, 9 a. in Jan. 13 9 a. m Jan. 14 9 a. m 6 c. c. sub- cutaneous. . do 0.2 c. c. sub- cutaneous. Normal. do . . Jan. 15, 9 a. m..... do Jan. 16, 9 a. m Feb. 3, 10 a. m Feb. 4, 10 a. m i c. c. sub- cutaneous. do 0.3 c.c. sub- cutaneous. 102.8 Feb. 5, 10 a. m ...do.... Feb. 8, 10 a. m Feb. 3, 10 a. m Feb. 4, 10 a. m Feb. 5 10 a. m 1 c. c. sub- cutaneous. do . do... 0.3 2. c. sub- cutaneous. Feb. 7, 10 a. m... Feb. 3, 10 a. m 2 c. c. sub- cutaneous. Feb. 4, 10 a. m do- Feb. 5, 10 a. m 0.3 c.c. sub- cutaneous. 103 103 Feb. 8, 10 a. m 32 ANTHRAX. TABLE 2. Experiments with unfiltered pyocyanase Continued. Animal. Date. Injections. Temper- ature. Result and remarks. Pyocyanase. Virulent 24- hour culture anthrax. Rabbits 1908. Feb. 3, 10 a. in 4 c. c. sub- cutaneous. 1 c. c. sub- cutaneous. F. Some reaction from injec- tions. Abscess formed at point of injection. Animal dead from anthrax. Severe reaction at point of injection. Abscess forms. Animal dead from anthrax on fourth day. Rabbit 9 Feb. 4, 10 a. in Feb. 7, 10 a. m 0.3 c. c. sub- cutaneous. 103.5 Feb. 3, 10 a. m Feb. 4, 10 a. m Feb. 5, 10 a. m 5 c. c. sub- cutaneous. 2 c. c. sub- cutaneous. 0.2 c.c. sub- cutaneous. 104 105 Feb. 8, 10 a. m. . . The foregoing experiments were made with unfiltered pyocyanase. The dead bacilli produce abscesses at points of injection and fre- quently disease of the liver, which contributed to death from anthrax. TABLE 3. Experiments icith filtered pyocyanase. Animal. Date. Injections. Tem- perature. Result and remarks. Pyocyanase. Virulent 24- hour culture of anthrax. Sheep 1 1909. Jan. 4, 10 a. m. . . . 10 c. c. sub- F. 103.6 105 106 103.6 103 Animal sick. Off feed. Dead from pyocyanase poi- soning. Respirations jerky. Dead from anthrax, having lived 5 days. Animal does not seem sick even though temperature is high. Sheep 2 i Sheep 3 Jan. 4, 1 p. m. ... cutaneous. Jan. 4, 3 p. m Jan. 5, 9 a. m 10 c. c. sub- cutaneous. 0.1 c. c. sub- cutaneous. Jan. 5, 4 p. m. . . Jan. 5, 7 p. m Jan. 6, 9a. m Jan. 6, 12m 3 c. c. sub- cutaneous. 103 105.5 106.9 104 Jan. 6, 4 p. m .... Jan. 7, 10 a. m Jan. 7, 11 a. m 1 c. c. sub- cutaneous. Jan. 21, 3 p. m 5 c. c. sub- Jan. 22, 9 a. m cutaneous. 4 c. c. sub- 103.5 102.5 102.5 103 102 102 Jan. 22, 3 p. m. cutaneous. 2 c. c. sub- Jan. 23, 9 a. m cutaneous. 3 c. c. sub- Jan. 23, 3 p. m Jan. 25 9 a. m cutaneous. 2 c. c. sub- cutaneous. 0.1 c. c. sub- cutaneous. Jan. 28, 12m Jan, 2, 10 a, m Jan. 7, 10 a. m 2 c. c. sub- cutaneous. Jfin. 7, 4 p, m . , , 102 107.2 101 101 Jan. 13, 9 a. m 2 c. c. sub- Jan. 13, 3 p. m cutaneous. 5 c. c. sub- cutaneous. 0.1 c. c. sub- cutaneous. EXPERIMENTS WITH FILTERED PYOCYANASE. 33 TABLE 3. Experiments with filtered pyocyanase Continued. Animal. Date. Injections. Tem- perature. Result and remarks. Pyocyanase. Virulent 24- hour culture of anthrax. Sheep 3 1909. Jan. 14, 9 a. m 2 c. C. sub- F. 102.5 102.5 104.5 Anthrax fever setting in. Dead from anthrax (3 days;. Dead two hours later of an thrax (3 days). Rabbit well. Found dead of anthrax (10 days). Animal sick. Animal much better. Much emaciated and swol- len along belly. Dead from anthrax. Post- mortem e x a m i n a tion showed linear abscess in liver; repeated larger doses of pyocyanasc probably caused animal to succumb to anthrax (lived 5 weeks); check rabbit died In 48 hours after inoculation. Animal has kept well. Animal found dead from anthrax, showing immu- nity was only passive (lived 24 days). Sheep 4 Rabbit 10 Jan. 14 1.30 p. m cutaneous. Jan. 15, 10 a. m 2 c. c. sub- Jan. 16 cutaneous. Jan. 8, 10 a. m Jan 81pm 1 c. c. sub- cutaneous. 101 102 Jan. 8, 3 p. m Jan. 9 10 a. m 1 c. c. sub- cutaneous. ...do .. 0.1 c. c. sub- cutaneous. 103 103.5 105.5 103.3 103 Jan 10 4 30 p. m .do Jan. 11 9 a. in 1908 Mar. 5, 9 a. m . 05 c. c. sub- Rabbit 11 Rabbit 12 Rabbit 13 Mar. 6, 9 a. m cutaneous. 0.5 c. c. sub- Mar. 7, 9 a. m cutaneous, do.. Mar. 8, 9 a. m 0.2 c. c. sub- Mar. 9, 9 a. m Mar. 13, 9 a. m 0.5 c. c. sub- cutaneous. cutaneous. 102.8 Mar. 18, 9 a. m Mar. 5, 9 a. m Mar. 6, 9 a. m 1 c. c. subcu- taneous, do 103 103 101.5 102.6 103.7 103 103 Mar. 7, 9 a. m Mar. 8, 9 a. m do 0.2 c. c. sub- cutaneous. Mar. 10, 9 a. m Mar. 18, 9 a. m Mar. 21, 9 a. m Mar. 23, 9 a. m Mar. 24, 9 a. m 1.5c. c. sub- cutaneous. 3 c. c. sub- cutaneous. 5 c. c. sub- cutaneous. Apr. 12, 9 a. m Apr. 13, 9 a. m Mar. 5, 9 a. m Mar. 6, 9 a. m 1.5 c. c. sub- cutaneous. . do... 102 102 103 102.8 102.8 103 104.3 Mar. 7, 9 a. m Mar. 8, 9 a. m do do . 0.2 c. c. sub- cutaneous. Mar. 18, 9 a. m . do Mar. 20, 9 a. m Mar. 24, 9 a. m Apr. 12, 9 a. m 3 c. c. sub- cutaneous. 5 c. c. sub- cutaneous. 0.1 c. c. sub- Apr. 14, 9 a. m cutaneous. Mar. 18, 9 a. m Mar. 18, 2 p. m 5 c. c. sub- cutaneous. do 0.2 c. c. sub- cutaneous. 34 ANTHRAX. TABLE 3. Experiments with filtered pyocyanase Continued. Animal. Date. Injections. Tem- perature. Result and remarks. Pyocyanase. Virulent 24- hour culture of anthrax. Babbit 13 1908. Mar. 18, 4 p. m.. P. 104 104 101 Rabbit found dead from an- . thrax. Period of incuba- tion lengthened to 10 days by pyocyanase; two more rabbits similarly treated reacted the same, and lived 10 days and 7 days, respectively. Simultaneous Injections of "ase" and bacilli. Found dead from anthrax. Lived 21 days. Some swelling which disap- pears in 2 days. Animal has remained well up to Feb. 24, when it was killed by dog (lived 69 days). Animal remains well. Swollen from "ase" injec- tion. Animal has remained well up to Feb. 4, when it was killed by dog (lived 69 days). No lesions on post- mortem; check rabbit died in 48 hours after inocula- tion. Simultaneous injection. Chloroformed to locate posi- tion and stage of develop- ment of bacilli. No spores or bacilli found by micro- scope or culture in local lesion, liver, spleen, or peritoneal cavity. Check rabbit died in 48 hours after inoculation. Rabbit 14 Mar. 19, 9 a. m Mar. 20, 9 a. m ... 5 c. c. sub- Mar. 28 cutaneous. Nov. 24, 9 a. m 1 c. c. sub- Rabbit 15 Rabbit 16 Nov. 24, 3 p. m ... cutaneous. do 0.2 c. c. sub- cutaneous. 103 Dec. 4, 9 a. m do Dec. 5 Dec. 16 3 c. c. sub- Feb. 4 cutaneous. Nov. 24, 11 a. m. . . Nov. 24, 3. p m ... Dec. 4, 9 a. m 2 c. c. sub- cutaneous. do do 0.2 c. c. sub- cutaneous. 104 Dec. 5 Dec. 16 3 c. c. sub- Feb. 4 cutaneous. Nov. 24, 11 a. m... 3 c. c. sub- Rabbit 17 Nov. 24, 3 p. m cutaneous. .. .do... 0.2 c. c. sub- cutaneous. 104 Dec. 4, 9 a. m do ; Dec. 5, 9 a. m 104.8 Dec. 16, 9 a. m 3 c. c. sub- Feb. 4 cutaneous. Nov. 24, 11 a. m... 4 c. c. sub- Nov. 24, 3 p. m cutaneous, .do.. . 0.2c. c. sub- cutaneous. 104 From the foregoing experiments with pyocyanase it will be seen that when given in proper dose and simultaneously with virulent anthrax bacilli, the period of inoculation is greatly lengthened in rabbits. Sheep, however, seem to be very susceptible to poisoning by pyocyanase, and no immunity is conferred upon them by its use. EXPERIMENTS WITH ANTHRAXASE. 35 ANTHRAXASE. Anthraxase was made in about the same manner as pyocyanase, except that the Bacillus dnthracis was substituted for Bacillus pyo- cyaneus. The tests of this substance include cultures grown in ordi- nary bouillon, cultures grown in Emmerich and Loew's medium, and experiments with precipitated anthraxase. The details are given in Tables 4, 5, and 6. TABLE 4. Experiments with anthraxase Cultures grown in ordinary bouillon. Animal. Date. Injections. Temper- ature. Results and remarks. Anthraxase. Anthrax culture. Rabbit 18 Rabbit 19... ... 1908. Mar. 17, 9 a. m Mar. 18, 9 a. m Mar. 19, 9 a. m 0. 75 c.c. sub- cutaneous. 0.50 c.c. sub- cutaneous, do .. "F. 101 101.5 102 102.4 107 Anthrax fever. Animal very sick. Dead from anthrax (lived 5 days). Swollen lymphatics. Anthrax fever. Dead from anthrax on sec- ond day. Anthrax fever. Dead from anthrax on third day. Animal pregnant. Animal died from shock. Died of anthrax on fourth day. Animal excited. 0. 2 c. c. sub- cutaneous. Mar. 20, 9 a. m Mar. 21, 9 a. m 3 c.c. subcu- taneous. 5c. c. subcu- Mar. 23, 9 a. m . taneous. do... Mar. 25, 9 a. m Mar. 17, 9 a. m Mar. 18, 9 a. m .. 1.5 c.c. sub- cutaneous. . ...do 100.5 102 102. C 104.6 Rabbit 20 Mar. 19, 9 a. m Mar. 20, 9 a. m Mar. 21, 9 a. m. 2 c.c. subcu- taneous. 3 c.c. subcu- taneous. 0.2 c. c. sub- cutaneous. Mar. 17, 9 a. m Ic.c. subcu- 101 101.5 Rabbit 21 Mar. 18, 9 a. m taneous. do Mar. 19, 9 a. m 0.2 c. c. sub- Mar. 20, 9 a. m cutaneous. 104. 1 103 Mar. 21, 9 a. in 3 c.c. subcu- Mar. 22 9 a. m taneous. Mar. 23, 9 a. m 0.5c. c. sub- 103 104 104 Rabbit 22 Rabbit 23 Mar. 24, 9 a. m cutaneous. ...do Mar. 20, 9 a. in. . 5 c. c. intra- do venously. 3 c. c. subcu- 0.2 c. c sub- Mar. 23, 9 a. in Mar. 24, 9 a. m . taneous. 2 c. c. subcu- taneous, do cutaneous. 104. 5 104 103.1 Mar. 20, 9 a. m do 5 c. c. intra- venously. 3 c. c. subcu- 0.2 c. c. sub- cutaneous. Mar. 27, 9 a. m ... taneous. 102.9 102.4 Mar. 28, 9 a. m . 4 c. c. subcu- Mar. 30, 9 a. m . . taneous. Mar. 23, 9 a. m 4 c.c. subcu- 105 100. 5 Mar. 24, 9 a. m .. taneous, do Mar. 25, 9 a. in Mar. 20, 9 a. m 5 c.c. subcu- taneous. 0.2 c. c. sub- cutaneous. 103.4 36 ANTHRAX. TABLE 4. Experiments with anthraxase, etc. Continued. Animal. Date. Injections. Temper- ature. Results and remarks. Anthraxase. Anthrax culture. Rabbit 23 1908. Mar. 28. 9 a. m.... 4c. c. subcu- F. Died of anthrax on third day. Animal pregnant. Dead from anthrax on fourth day. Animal pregnant. Much depressed by the in- jections and aborted at 4 p. m. (Control for this se- ries dies in 48 hours after in- oculation with the same culture.) Dead from anthrax on third day. Rabbit 24 Mar. 30, 9 a. m.... taneous. Mar. 23, 9 a. m.... 6c.c. subcu- 104.1 105 103.9 Rabbit 25 Rabbit 26 Mar. 24, 9 a. m taneous. do Mar. 25, 9 a. m Mar. 26, 9 a. m 5 c. c. intra- do.. venously. 3c.c. subcu- taneous. 4c.c. subcu- 0.2 e. c. sub- cutaneous. Mar. 28, 9 a. m 103. 6 Mar. 30, 9 a. m taneous. Mar. 25, 9 a. m Mar. 26, 9 a. in 3 c. c. intra- venously. 5 c. c. intra- 0.1 c. c. sub- cutaneous. 103 103. 8 do venously. 3 c. c. subcu- taneous. 0.5 c. c. sub- Apr. 7, 9 a. m.. .. Apr. 8, 9 a. m cutaneous. ...do... 102 103 Apr. 9, 9 a. m do 0.1 c. c. sub- cutaneous. Apr. 10, 9 a. m do Apr. 11, 9 a. m 2 c.c. subcu- 105 105. 3 Apr. 11, 4 p. m Apr. 12, 9 a. m taneous. 3 c.c. subcu- taneous. TABI.K 5. Experiments with anthraxase. Cultures groir-n in Emmcrieli and Lne-w's medium. Animal. Date. Injections. Temper- ature. Results and remarks. Anthraxase. Anthrax cul- ture. Rabbit 27 1908. Apr. 7, 10 a. m . 0.5 c. c. sub- F. 102 102 102 102.3 102.5 103 Much swollen. Dead from anthrax on fifth day. Dies of anthrax in 2 days. Rabbit 28 Apr. 9, 10 a. m . cutaneous. ...do... 0.2 c. c. sub- cutaneous. Apr. 10, 10 a. m. . . do Apr. 11, 10 a. m. 1 c. c. sub- Apr. 11, 4 p. m cutaneous. 3 c. c. sub- Apr. 12, 4 p. m cutaneous. Apr. 13, 4 p. m Apr. 15, 10 a. m... Apr. 16, 10 a. m... 0.25 c. c. sub- cutaneous. do 100.8 99.5 99.8 101 102.8 Apr. 17, 10 a. m ...do.... Apr. 18, 10 a. m ...do.... Apr. 25, 10 a. m . . .. .do 0.2 c. c. sub- cutaneous. Apr. 26, 10 a. m... Apr. 27, 10 a. m . 0.50 c.c sub- cutaneous. EXPERIMENTS WITH ANTHRAXASE. 37 The following experiments on rabbits and sheep were made with precipitated anthraxase. Absolute alcohol, 9 volumes to 1 of anthrax- ase solution, was employed as the precipitant. The resulting yellow, gummy precipitate was readily soluble in water, to which 0.2 per cent strength of carbolic acid was added. TABLE 6. Experiments witli precipitated anthraxasc. Animal. Dife. Injections. Temper- ature. Result and remarks. Anthraxase. Anthrax culture. Rabbit 29 Rabbit 30 1908. Apr. 15,9 a. m Apr. 16,9 a. m. . 0.5 c.c. sub- cutaneous. do "F. 101 101 101 101 101.8 102 Check rabbit dies. Animal seems perfectly well. Given to test duration of immunity. Found dead of anthrax. Dead of anthrax at 4 p. m. on fifth day. Anthrax fever. Check dies to-day. Animal turned out in yard. Animal found dead. Post- mortem showed death from anthrax and cocci- diosis (lived 12 days). Died of anthrax at 2 p. in. on fourth day. Apr. 17,9 a. ni do Apr. 18,9 a m . do Apr. 25, 9 a. m Apr. 2t>.9 a. m. .. do ...do.... 0.01 c.c. sub- cutaneous. Apr. 27,9 a. m do Apr. 27, 5 p. m 5 c. c. sub- Apr. 28,9 a. m . cutaneous. 103 103 Apr. 29,9 a. m Apr. 30 0.1 c c sub- cutaneous. May 2, 10 a. m 1 c. c. sub- 103.3 102.5 103 102 104.5 105 104 100.3 102. 3 101 102 104.8 102. 5 101 102 102.5 Rabbit 31 . May 2, 2 p. m cutaneous. do do May 4, 10 a. m 3 c. c. sub- cutaneous. May 4, 4 p. m cutaneous. May 5, 11.30 a. m 2 c. c. sub- May 5, 4 p. in. .. cutaneous. May 6, 10 a. m May 2, 10 a. m May 2, 2 p. m 1 c. c. sub- cutaneous, do Sheep 5. . . May 2, 4 p. m ... .do 0.1 c. e. sub- May 4, 10 a. in cutaneous. May 5, 10 a. m May 5, 4 p. m. .. 2 c. c. sub- cutaneous. May 6, 10 a. m May 7, 10 a. ni . . May 8, 10 a. m May 14, 10 a. m . June 29, 10 a. m 5 c. c. sub- Sheep June 29, 12 m.. cutaneous, do .. June 29, 2 p. in do June 29, 4 p. in. .do . 0.1 c. c. sub- June 30, 10 a. m . .do.... cutaneous. Juno 30,12m do June 30, 2 p. m... . .do.... June 30, 4 p. ni do July 2 9 a in do 105. S 104 3 July 2, 10 a. m do July 2, 12 in. do 105.8 Juno 29, 10 a. in do June 29, 12m ...do.... June 29, 2 p.m... ...do.... 38 ANTHRAX. TABLE 6. Experiments with precipitated anthraxasc Continued. Animal. Date. Injections. Temper- ature. Result and remarks. Anthraxase. Anthrax culture. Sheep 6 1908. June 29, 4 p. m June 30, 10a.m... 5 o. c. sub- cutaneous. do O.lc.c. sub- cutaneous. F. % Check guinea pig dies in 2 days. Animal dead of anthrax on sixth day. Found dead of anthrax on fourth day after inocula- tion of test dose. Sheep 7 June 30, 12m do June 30, 2 p. m . ...do June 30, 4 p. m do July 2, 8 a. m do 102.2 102 102 102.8 103 July 2, 10 a. m ...do.... July2, 12m do July 2, 3 p. m ...do. ... July 2, 7 p. m do July 4, 10 a. m do July 28, 10 a. m... ...do.... July 28, 12m .do July 28, 2 p. m do July 28, 4 p. m July 29, 10a.m... do . .do .. 0.1 c. c. suf- cutaneous. 102.3 102 102.3 102.6 106 106 106 106.2 July 29, 12m do Jnlv29, 2p. m do July 29, 4 p. m .do July 30, 10 a. m... do.. July 30, 12m do.... July 30, 2 p. m . . .do July 30, 4 p. m do July 31, 10 a. m ... ANTHRAXOIN. Anthraxoin was prepared and experimented with as follows: It consists essentially of a suspension, in carbolized normal salt solution, of dead, sporeless, anthrax bacilli. It was produced by inoculating, from a fresh anthrax carcass, bottles that had been filled completely with nutrient bouillon and plugged with hardest paraffin stoppers. In this way none but sporeless bacilli were introduced, and under the existing anaerobic conditions no spores could form. After all growth had ceased, at 35 C., these cultures were completely immersed in a water bath maintained at 55 C. for one hour, which was sufficient to devitalize the bacilli but not to destroy any antibodies they might contain. The dead bacilli were separated by filtration from the liquid in which they had grown, and those adhering to the Berkefeld filter were washed off in the requisite amount of carbolized normal salt solution. The suspension experimented with consisted of the bacilli that grew in 2,500 c. c. of bouillon, suspended in 50 c. c. of carbolized normal sodium chlorid solution. The details are given in Table 7. EXPERIMENTS WITH ANTHRAXOIN. TABLE 7. Experiments with anthraxoin. Animal. Date. Injections. Temper- ature. Result and remarks. Anthraxoin. Anthrax cul- ture. Sheep 8 1908. Apr. 24, 10 a. m... 1 c. c. subcu- "F. Sheep dies of anthrax in 2 days. Remains well. Sheep dies of anthrax in 3 days. Sheep dies of anthrax in 2 days. Sheep dies of anthrax in 3 days. Sheep dead from anthrax in 2 days. Sheep dead from anthrax in 4 days. Sheep dead from anthrax in 3 days. Sheep 9 May 8, 10 a. m... taneous. 2 c. c. subcu- May 20, 10 a. m. taneous. 0.25 c. c. sub- May 22, 10 a. m cutaneous. Apr. 24, 10 a. m. 2 c. c. subcu- Sheep 10 May 8, 10 a. m taneous. 0.25c. c. sub- May 9, 10 a. m cutaneous. 102.2 103 101.5 101.5 May 9, 4 p. m May 10, 10 a. m May 11,10a.m.. Apr. 24, 10 a. m 0.5 c. c. sub- Sheep 11 May8, 10 a. m May 20, 10 a. m cutaneous. 1 c. c. subcu- taneous. 25 c. c. sub- May 11, 10 a. m May 24, 10 a. in 1 c. c. subcu- taneous. cutaneous. Sheep 12 0.25 c. c. sub- May 11, 10 a. m . 1 c. c. subcu- cutaneous. Sheep 13 . . May 20, 10 a. m taneous. 0.25 c. c. sub- May 11, 10 a. m. . 1 c. c. subcu- cutaneous. Sheep 14 . May 23, 10 a. m. . taneous. 5 c. c. subcu- June 8, 10 a. m June 10, 10 a. m. taneous. 0.25 c. c. sub- cutaneous. May 11, 10 a. m May 23, 10 a. m. . 1 c. c. subcu- taneous. 5 c. c. subcu- Sheep 15 Rabbit32 June 8, 10 a. in.. taneous. 0.25 c. o. sub- June 12, 10 a. m . cutaneous. Apr. 20, 9 a. m Apr. 26, 12 m 10 c. c. sub- cutaneous, .do... 103 104 104 104 103 103 103.5 103.5 104 103.5 104 Apr. 26, 2 p. m do.. Apr. 26, 4 p. m... .do 0.25 c. c. sub- cutaneous. Apr. 27, 9 a. m.. 12 c. c. sub- Apr. 27, 12 m . cutaneous. .do Apr. 27, 2 p. m do .. Apr. 27, 4 p. m .do Apr. 28, 9 a. m 10 c. c. sub- Apr. 28, 2 p m cutaneous, do Apr. 28, 4 p. m do Apr. 29, 10 a. m Mar. 8, 10 a. m . . 0.5 c. c. sub- Mar. 8, 1 p m cutaneous. 104.1 105.4 101.8 Mar. 8, 4.30 p. m.. Mar. 9, 9 a. m. . 0.2 c. c. sub- cutaneous. 40 ANTHRAX. TABLE 7. Experiments with anthraxoin Continued. Animal. . Date. Injections. Temper- ature. Result and remarks. Anthraxoin. Anthrax cul- ture Rabbit 32 1908. Mar. 10, 9 a. m F. 105.8 Dead from anthrax in 2 days. Dead from anthrax in 2 days. Animal dead from anthrax in 2 days. Rabbit stupid. Rabbit dead from anthrax in 2 days. Skin swollen at injection. Much swollen from injection. Swelling practically gone. Dead from anthrax in 2 days. Guinea pig Babbit 33 Mar. 11, 9 a. in Mar. 8, 10 a. m Mar. 9, 10 a. m Mar. 11, 10 a. m... 0.5 e. c. sub- cutaneous. 0.2 c. c. sub- cutaneous. Mar. 16, 9 a. m . . 0.1 c. c. sub- 101 102.2 102.5 104.3 101.5 102 103.5 Rabbit 34 Mar. 16, 11 a. m... cutaneous. Mar. 16, 12m Mar. 16, 4.30 p. m Mar. 17, 9 a. m Mar. 26, 9 a. m 0.1 c. c. sub- Mar. 26, 4 p. m cutaneous. Apr. 6, 9 a. m 0.01 c. c. sub- cutaneous. Apr. 8, 9 a. m Mar. 16, 9 a. m... 0.2 c. c. sub- 102 103.6 104.5 105.3 102 102.5 105 Rabbit 35 Mar. 16, 11 a. m... futaneous. Mar. 16, 12m Mar. 16, 1.30p.m.. Mar. 17, 9 a. m. . Mar. 26, 9 a. m Mar. 26, 2 p. m 0.2 c. c. sub- cutaneous. Apr. 6, 9 a. m 0.01 c.c. sub- Apr. 8, 9 a. m cutaneous. Mar. 16, 9 a. m Mar. 16, 11 a. m... 0.3 c. c. sub- cutaneous. 102 103.2 103.5 104 105 102.5 102 104.5 Mar. 16, 12 m Mar. 16, 2 p. m Mar. 16, 4 p. m Mar. 17, 9 a. m . Mar. 26, 9 a. m Mar. 26, 4 p. m 0.3 c. c. sub- cutaneous. Apr. 6, 9 a. m. .. 0.01 c. c. sub- cutaneous. Apr. 6, 9 a. m A COMMERCIAL VACCINE IN PILL FORM. A commercial vaccine which, according to the makers, consists of dead anthrax organisms in pill form was also tested. These small pills are placed under the skin by means of a trocar and are claimed by the makers to produce immunity to anthrax. Microscopic exami- nation, as well as cultural and animal experiments, show that the claims of the makers, in so far as the vaccine being dead and harm- less is concerned, are true. One can easily see with the microscope that these little pills consist of dead anthrax bacilli and their spores EXPERIMENTS WITH COMMERCIAL, VACCINE. 41 held together in pill form by a proper excipient. The writer was unable, however, to verify the claim that they produce any im- munity, as is shown in Table 2. A rabbit succumbed in six days, but as it is very rarely that a rabbit can be immunized by a vaccine, a sheep, which animal is easily protected, was also employed, with negative results. TABLE 8. Experiments with commercial vaccine in pill form. Animal. Date. Injections. Temper- ature. Result and remarks. Vaccine. Anthrax culture. Rabbit 36 1908. Apr. 7, 9 a. m 1 pill, sub- jr. Dead from anthrax in 6 days. Sheep dead from anthrax in 48 hours. Sheep 16 Apr. 8, 9 a. m Apr. 9, 4 p. m cutaneous. 1 pill (dis- solved), subcuta neous. do . . do Apr. 10, 4 p. m 1 pill, sub- 0.01 c. c. sub- cutaneous. Apr. 11, 4 p. m cutaneous. 104 104.3 Apr. 13, 4 p. in Apr. 15, 4 p. m Oct. 17, 9 a. m Nov. 2, 9 a. m Nov. 4, 9 a. in 1 pill with trocar. 0.1 c. c. sub- cutaneous. PREPARATION OF AN EFFECTIVE SINGLE VACCINE. The single vaccine, like Pasteur vaccine, consists of cultures of attenuated anthrax bacilli, the only difference being in the degree of attenuation and that, as its name implies, it is applied only once, thus requiring a shorter time and only one handling of the animals. To prepare such a vaccine or attenuated culture, the virulence of an already virulent culture of anthrax was exalted by passage through very young animals. A strain can in this way be pro- duced which will kill a sucking rabbit in 24 hours. From the heart's blood of such a carcass tubes of bouillon are inoculated immediately after death. These cultures are then incubated at 42 to 43 C. for varying periods of time from 12 to 18 days. On the twelfth day a subculture is made from one of the tubes and cultivated at 35 C. On the thirteenth and succeeding days subcultures are made from the remaining tubes until the series has been completed. These six subcultures of attenuated bacilli are now tested on guinea pigs and rabbits, and on sheep if possible. 42 ANTHRAX. More dependence is to be placed upon the animal test than upon the number of days the attenuation process is carried on. A cul- ture of proper strength is generally obtained from tubes that have been attenuated for about 16 days. The proper culture will be one which will not kill rabbits, but which will kill a majority of guinea pigs in a delayed period, say 5 or 6 days. Such an animal should show no swelling at the point of inoculation, and the bacilli should be found only sparingly in the internal organs. When shaken in cultures such a vaccine shows a homogeneous clouding of the medium ; no flocculi persisting, as occurs in virulent cultures. As rabbits and guinea pigs can not be made immune to anthrax by vaccination of this kind, these animals are only useful in testing the pathogenesis of the cultures. For testing the immunizing prop- erty, sheep, which are easily immunized, were employed. Quite a number of sheep were used in the experiment to produce a safe, efficient single vaccine, and as regards the failures along this line, it is only necessary to say that they were entirely due to improper attenuation of the cultures. When the proper attenuation was reached there was no difficulty in immunizing animals by a single vaccina- tion. This was effective against a subsequent, otherwise mortal dose of virulent anthrax bacilli, as the following experiments will show. TESTS OF THE SINGLE VACCINE. EXPEBIMENT NO. 1. On April 15, 1909, a visibly pregnant cow was given subcuta- neously 1 c. c. of single vaccine. She showed no ill effects whatever from the injection. On May 1 the animal was inoculated with 0.2 c. c. of virulent anthrax bacilli from a 24-hour culture which killed a check rabbit and a check cow in 48 hours. On May 19 the cow dropped a fully developed calf which when found was dead, lying with its head bent under the shoulder. Supposing that the calf had been asphyxiated, it and the membranes were buried. However, a platinum loop full of the discharge was plated and a pure culture of 60,000 anthrax bacilli was obtained. A guinea pig inoculated with a culture made from one of these colonies died of anthrax in 48 hours. The cow remained well, and subsequent daily cultivations from vaginal discharges showed no anthrax, bacilli. This experiment was extremely valuable, not only as showing that the cow had been immunized by a single vaccination, but also for showing not only that anthrax can be communicated not only to the fetus, but that it can be thus communicated by an immune mother. It also showed that anthrax bacilli may persist for at least 18 days in the body of an immune animal. When the cow was destroyed for various reasons on May 25 she was in perfect health, and cultures SERUM FOR PRODUCING IMMEDIATE IMMUNITY. 43 and inoculations made with her blood showed no anthrax bacilli present. It is to be regretted that this animal could not have been kept for further observations and experiments. EXPERIMENT No. 2. On September 14, 1909, two sheep were vaccinated with single vaccine, each receiving 1 c. c., and showed no sickness therefrom. On September 28 each sheep was inoculated subcutaneously with 0.2 c. c. of a culture of anthrax bacilli whose virulence had been proven on a rabbit in the same experiment. The sheep at no time showed any sickness. On November 2 both sheep were again tested with virulent bacilli and showed no sickness. EXPERIMENT No. 3. On November 12, 1909, three sheep were vaccinated with 1 c. c. of single vaccine and showed no sickness therefrom. On November 27 one of the animals was tested with 0.2 c. c. of virulent bacilli, and on December 1 the other two were similarly tested. They at no time showed any sickness. These three sheep, together with the two used in experiment No. 2, were used later in experiments to produce an antibacterial serum. A SEBUM FOB PBODUCING IMMEDIATE IMMUNITY. Although the favorable results from vaccination by the Pasteur system have been known for a long time, and owing to the cheapness of the vaccine it would seem that there is nothing more to be desired, the length of time required to produce immunity is one drawback in its use when one is endeavoring to check an existing outbreak of the disease. When vaccination is practiced a month in advance of the animals being turned out to pasture in the spring, the system of Pasteur vaccination is the proper one to use. In existing outbreaks, however, it is evident that any system that requires a month to be- come protective leaves much to be desired, as many animals could be- come infected and die before protection could be afforded them. With the end in view of devising a method whereby an immediate immunity could be established in existing outbreaks or where an im- munity could be brought about in a much shorter time than has been possible under the old system of vaccination, the writer has devoted a large portion of his time for the past three years. As it had been conclusively demonstrated that animals can be immunized by a single vaccine, thus cutting down one-half the period necessary for immuni- zation by the old system of Pasteur, it was decided to experiment upon the production of an antianthrax serum by endeavoring to 44 ANTHRAX. hyperimmunize sheep after the manner of the production of anti- hog-cholera serum, with the exception that whereas in the latter work virulent blood was used, this was precluded in our experiments owing to the dangers attending the handling of large quantities of virulent anthrax blood. Instead, the blood used was that drawn from an animal immune to anthrax, and not from one sick with anthrax. Briefly stated, the writer has produced an antibacterial serum by highly immunizing sheep through repeated inoculations, first of at- tenuated anthrax bacilli, and following these by inoculations of the most virulent races of the bacilli in increasing doses until the animal would withstand with impunity fifty thousand times the minimal lethal dose. Such a serum will protect a sheep against an otherwise mortal dose of bacilli and produce an immediate immunity. It is, therefore, a very valuable adjunct in working against the spread of the disease in existing outbreaks where the usual vaccination is be- ing practiced. The serum will confer a passive immunity immedi- ately, and thus protect the animal against fatal infection until the vaccine confers an active immunity. And should an animal which has been protected by the serum become infected with a virulent anthrax bacillus, the results of this infection will be the production of a much stronger immunity than the vaccine and serum would otherwise confer. It is evident that if a single vaccine, or even a double vaccine, can be used in conjunction with an antibacterial serum to produce im- mediate passive immunity which will persist until the vaccine has had time to bring about active immunity a long step will have been made in controlling this formidable disease. The experiments with this end in view were carried out upon sheep as follows: EXPERIMENTS WITH THE SERUM. EXPEBIMENT NO. 1. On December 15, 1909, a sheep immunized with single vaccine and a subsequent inoculation with virulent bacilli (see Experiments Nos. 2 and 3 with single vaccine) was bled from the carotid artery the femoral artery being small and deep-seated by means of a glass cannula and rubber tubing. When the blood had clotted, 500 c. c. of serum were decanted and injected into the inguinal regions of another sheep which had been similarly immunized. Microscopic examina- tions and inoculations into guinea pigs of this blood showed it to be free of anthrax bacilli. The injected sheep was lame in both hind legs the next day, but this lameness disappeared when the complete absorption of the injected blood had taken place, and no abscess formed. On January 12 this sheep was bled from the carotid artery, EXPERIMENTS WITH THE SEKUM. 45 and when the serum was collected it was preserved by adding to it one part of a 5 per cent solution of carbolic acid to each nine parts of serum. In making this addition of carbolic acid some coagulation will occur if the preservative and serum be not poured simultaneously into another vessel and stirred by a helper. The above serum was without protective properties. All animals upon which it was used died of anthrax, as follows : On January 13, 1910, 12 guinea pigs received increasing doses of serum from 0.1 up to 3 c. c. and simultaneously 0.1 c. c. of virulent anthrax culture. All of these animals died of anthrax in 48 hours. On January 17, 1910, the amounts of serum were increased to 4 c. c., 5 c. c., 6 c. c., 7 c. c., 8 c. c., 9 c. c., and 10 c. c., with a simultaneous dose of 0.1 c. c. of virulent culture. The 7 animals thus tested also died in 48 hours. On February 5, 1910, a sheep received subcutaneously 24 c. c. of the serum and '0.2 c. c. of virulent culture. A control rabbit died in 48 hours and the sheep died of anthrax in three days. On February 9, 10, and 11 another sheep received daily 12 c. c. of the serum and on the 12th a test dose of virulent bacilli. This sheep lived four days and then died of anthrax. These experiments demonstrated that the modified hog-cholera method of serum production could not be applied to anthrax, and it was then decided to try and bring about hyperimmunization by increasing doses of virulent bacilli repeated over a long period. The two sheep used in experiment No. 2 with single vaccine were em- ployed for this purpose as follows : v EXPERIMENT No. 2. On September 14, 1909, two sheep were vaccinated with 1 c. c. of single vaccine. On September 28 they were given a test dose of 0.2 c. c. of virulent bacilli, and remained well, while a check rabbit died in 48 hours. On November 2 each sheep again received 0.2 c. c. of virulent bacilli. On March 4, 1910, each sheep received 0.3 c. c. of virulent bacilli. On March 18 they received 1 c. c. of virulent bacilli, and on April 2 each sheep received 5 c. c. of virulent bacilli. A period of about seven months was thus consumed. On April 14, 1910, these sheep were bled from the carotid artery, producing 1,900 c. c. of serum, to which was added one part of a 5 per cent solution of carbolic acid to each nine parts of serum as a preservative. Prior to the addition of the preservative tests for the presence of anthrax bacilli were made upon guinea pigs and by plate cultures. None were found. This serum was used upon sheep in connection with virulent an- thrax bacilli as shown in Table 9. 46 ANTHRAX. TABLE 9. Experiments with antianthrax serum. Animal. Date. Injections. Results and remarks. Immune serum. Virulent culture. Sheep 17 1910. Apr. 29 May 16 ..do.. . 5c.c. subcutaneous.. 0. 2 c. c. subcutaneous. . . . 2 c. c. subcutaneous. . . . 1 c. c. subcutaneous. . . . 1 c. c. subcutaneous. . . . 2 c. c. subcutaneous. . . . 5 c. c. subcutaneous . . . . 1 c. c. virulent culture . . 1 c. c. subcutaneous. . . . 1 c. c. subcutaneous. . . . 5 c. c. subcutaneous. . . . 1 c. c. virulent culture. . 5 c. c. single vaccine. . . . 1 c. c. virulent culture. . 5 c. c. virulent culture. . 5 c. c. single vaccine. . . . 1 c. c. virulent culture. .5 c. c. virulent culture. . 1 c. c. virulent culture. 1. c. c. single vaccine . . . . 1 c. c. virulent culture. . 5 c. c. virulent culture. 1 . c. c. single vaccine. . . . 5 c. c. virulent culture. . 5 c. c. virulent culture. 1. c. c. single vaccine. . . . 1 c. c. virulent culture. . 5 c. c. virulent culture. . 1 c. c. virulent culture. . 1 c. c. virulent culture. Sheep remains well. Rabbit dies in 48 hours. Not sick. No bad result. Remained well. Died in 48 hours. Remained well. Died in 48 hours. Remained well. ' Do. Dead in 48 hours. Remained well. Do. Do. Died of anthrax in 48 hours (control for sheep 22, 23, and 24, for inoculations June 18). Died of anthrax in 48 hours (control for sheep 22, 23, and 24, for inoculations on July 6. Rabbit (control) Sheep 18 May 21 June 20 July May 21 May 24 June 20 July 6 May 24 May 27 June 20 July 6 May 27 June 20 July fi June 20 June 1 June 18 July 6 June 1 June 18 July June 1 June 18 July 6 June 18 July G 10 c. c. subcutaneous. . .do Rabbit (control) Sheep 19 do 10 c. c. subcutaneous . do Rabbit(control) Sheep 20 do 10 c. c. subcutaneous . . ..do... . Sheep 21 10 c. c. subcutaneous . .do. Rabbit (control) Sheep 22... 5 c.c. subcutaneous. 10 c. c. subcutaneous. . ..do Sheep 23 . . 5 c. c. subcutaneous . . 10 c. c. subcutaneous. do Sheep 24... 5 c. c. subcutaneous. . 10 c. c. subcutaneous. do Sheep 25 (con- trol). Rabbit (control) No doubt the period of time occupied in hyperimmunizing the sheep which produced this highly protective serum can be very much lessened probably one-half. It will be noted that the time covered was about seven months. This was not intentional, but as the sheep had already been immunized for another object, and not used, it was decided to carry out the idea of greatly increasing their immunity by the method followed. It will be seen by referring to the table that an idle period of about three months elapsed between November, 1909, and February, 1910, which might just as well have been em- ployed for the purpose. It is also possible that by giving larger doses of virulent bacilli the immunizing property of the serum may be considerably increased. That the degree of immunity can be established by the number of inoculations and the quantity of virulent culture used was clearly shown in sheep Nos. 18, 19, 20, 21, 22, 23, and 24. They had received CONCLUSION. 47 three injections of serum and three inoculations with bacilli, in very small doses, and while they themselves were immune (see Table 9) their blood, when tested after slaughter on July 18, did not protect other sheep. It is also highly probable that sheep will produce a more effective antianthrax serum for their own species, and cattle for cattle, horses for horses, etc. ; that is, the sera should be homologous. However, no effort was made to test this principle, as the great expense attending such experiments precluded their being carried out. CONCLUSION. The writer does not advise the abandonment of the old Pasteur system of vaccination against anthrax when it is practiced upon ani- mals before they are turned out on the pastures in the spring of the year. When animals are dying, however, vaccination alone requires too long a period to protect, and it is in these outbreaks that the anti- anthrax serum should be used in conjunction with vaccine. The ex- periments have shown that a single vaccine may be used with good results. Where it is desired, however, the serum may be used simul- taneously with the double vaccine or with a single vaccine. o UC SOUTHERN REGIONAL LIBRARY FACILITY A 001 084 327 4 305 Univer* Sout Libj