CULTIVATION OF THE HOG CHOLERA VIRUS

The work of Hecke on the cultivation, of hog cholera virus was confirmed with ease. Virus was grown in the presence of fresh minced swine testicle in flasks containing Tyrode solution, on the chorioallantoic membrane of embryonated eggs, and on the surface of swine serum agar. In flasks it was grown for 14 transfers; while on eggs it was grown for 13 transfers, followed by an equal number of transfers on agar, making 26 transfers in all. Only one strain of virus was used and we do not know whether all strains can be cultivated so readily or whether we were particularly fortunate in the selection of the strain used. Neither do we know whether swine testicle is better than other tissues for growth. The cultured virus produces characteristic hog cholera when injected into swine, and its effect can be neutralized with commercial anti-hog cholera serum. No evidence of attenuation of the virus was obtained, the last culture being highly virulent when small amounts were injected. No evidence for the adaptation to the egg could be secured, since passages without swine testicle on the membrane or intravenously for 2 transfers resulted in a loss of the virus. No contaminating virus that might favor the cultivation could be detected by animal or egg inoculation. Not only has the virus been cultivated but it has been demonstrated in large amounts in the culture. Four suspensions containing slightly over 0.5 mg. of protein nitrogen produced typical hog cholera when 1 x 10–6 cc. was injected, and one suspension made in the same way was active in one-tenth this amount. Few titrations on what is commonly known as hog cholera virus, i.e. the serum from acutely ill pigs, are available. We made one such titration and produced a delayed disease with 1 x 10–5 cc. of infectious serum. It seems probable that the culture virus is more active than the commonly used virus and that its practical use in hog cholera vaccination and hyperimmunization would result in a considerable saving. All of the methods used yielded active cultures, but the serum agar method is the one of choice since larger amounts of suspension can be obtained with less labor

A STUDY OF PARATYPHOID BACILLI ISOLATED FROM CASES OF HOG-CHOLERA

During the course of some experimental work on hog-cholera, paratyphoid bacilli were isolated from 16 per cent of the pigs. Culturally these organisms are the same as paratyphoid 18 isolated from man, while they show several differences from hog-cholera bacilli. In their slight pathogenic effect on rabbits they also differ from the hog-cholera bacillus. In their agglutination in sera produced by the injection of living cultures, one of the cultures, isolated from a chronic case, corresponds to Bacillus enteritidis, while the other five are apparently in a class by themselves. They resemble paratyphoid β more closely than hog-cholera bacilli, but the type of clumps formed and absorption experiments show that they are different from either. Whether these differences are enough to make it necessary to put them into a class by themselves is questionable, but the fact that when injected into rabbits they produce an immunity to the hog-cholera bacillus, while paratyphoid β does not, is additional evidence in favor of such a classification. Complement fixation experiments have been of little value in differentiating the members of this group, but on the contrary show their close relationship. It seems probable that some of the cultures that are described in the literature as hog-cholera bacilli really belong to this group, which would account for much of the confusion that exists in the classification of the interesting, truly pathogenic bacillus that at one time was thought to be the cause of hog-cholera and in the series of animals with which we have worked has not appeared once. Whether the ingestion of pork containing these bacilli would cause disease in man is a question that can only be decided by a more careful bacteriological study of the organisms causing food poisonings and paratyphoid fever

INCREASED VIRULENCE OF THE HOG-CHOLERA BACILLUS PRODUCED BY PASSAGE THROUGH RABBITS

By passage through a series of eleven rabbits a culture of the hogcholera bacillus has increased its virulence a thousand times. A subcutaneous injection of twenty organisms, or 0.00000001 cc. of a 24 hour bouillon culture, or a drop of a bouillon culture rubbed lightly into the shaven skin, produces, in the rabbit, a characteristic disease resulting in death on or about the 6th day

STUDIES ON BACILLUS MURISEPTICUS, OR THE ROTLAUF BACILLUS, ISOLATED FROM SWINE IN THE UNITED STATES

In the United States organisms, which culturally are mouse septicemia or swine erysipelas bacilli, have been isolated from the tonsils of five of sixteen pigs examined. These pigs all had hog-cholera, but it is probable that the bacilli were in the tonsils before they were infected with hog-cholera, and there is no evidence that they played any part in the disease. The distribution of the infection seemed to be restricted as most of the pigs from which the bacilli were obtained came from one litter. As we do not have clinical rotlauf, or swine erysipelas, in this country, as these organisms, in Europe, have been found in a large percentage of apparently normal swine, and as the disease is produced with difficulty by the injection of cultures, the question may be raised whether they are not secondary invaders rather than the primary cause of the disease with which they have been associated, or else whether the resistance of swine on the European continent does not differ from that of our breeds as a result of differences in foods. It is possible that the mouse septicemia bacilli found in this country may differ culturally from those present in animals with swine erysipelas. With this in mind, the carbohydrate reactions, as well as other cultural characters not necessary for the identification of the bacilli isolated, have been studied. The disease produced by the injection of these bacilli into mice and pigeons has been studied and shown to be largely an intracellular process. The organisms are taken up by the endothelial cells lining the veins and capillaries; there they multiply and soon kill the cells. It has also been shown that the only type of cell in the blood stream which contains bacteria is the endothelial leucocyte, and the probabilities are that the free phagocytes have been detached from the lining of the vessels. The disease is acute, and the indications are that in the cells the bacilli find a favorable medium for their growth. While phagocytosis may in general be an immune reaction, in this case it appears to favor the parasite rather than the host

EFFECTS OF ENZYMES IN SERUM ON CARBOHYDRATES AND THEIR RELATION TO BACTERIOLOGICAL TECHNIQUE

It has been shown that enzymes in serum will change maltose, dextrin, and starch so that they will react as dextrose in media. These enzymes are destroyed by heating to 60°C. for 15 minutes, but they are present in sera that have been refrigerated for as long as 18 months. The practice of using carbohydrate media containing unheated serum should be discouraged, and if it is used the possibility that the carbohydrate may be changed by the enzymes present must be considered

THE SIGNIFICANCE OF AGGLUTININS IN THE IMMUNITY OF THE RABBIT TO THE HOG-CHOLERA BACILLUS

Rabbits may show a high agglutination titer to the hog-cholera bacillus and have no immunity and on the other hand immune animals may have a comparatively low agglutination titer. In other words, with this organism the height of the agglutination titer does not indicate the degree of immunity. As this bacillus so closely resembles the typhoid bacillus biologically and pathologically, it seems safe to conclude, until evidence is brought forth to the contrary, that in man the height of the agglutination titer does not indicate the actual degree of immunity to the latter organism. The same would apply to other members of the typhoid-colon group. It would not be wise to draw a more general conclusion until other organisms have been tested. This does not mean that agglutinins are not related to immunity but it brings up the question of the wisdom of using them as a guide in immunization with the colon-typhoid group. When injected into the normal, vaccinated, or immune rabbit, the virulent hog-cholera bacillus is rapidly clumped and disappears from the circulation. 40 minutes after injection these organisms can be found in phagocytes in the liver. The fact that the normal rabbit gives this intra vitam agglutination is an exception to the findings of Bull that virulent organisms remain in the circulation for some time after injection

BACILLI OF THE HOG-CHOLERA GROUP (BACILLUS CHOLERÆ SUIS) IN MAN

1. The organisms isolated by Hirschfeld from febrile cases resembling paratyphoid fever and named Paratyphoid C can be placed in the hog-cholera bacillus group by their agglutination absorption properties though they are not typical culturally. 2. When fed to a pig a febrile disease resulted from which the animal recovered. After injection of hog-cholera virus the organisms fed were found generally, distributed and some of them had lost cultural characters so that they are brought into the class of typical hog-cholera bacilli except for their low virulence for rabbits. 3. While hog-cholera bacilli have many opportunities to infect man. they either are not able to grow in the human body or, what is less likely, they do grow and lose the characters that distinguish them

A NON-GAS-PRODUCING STRAIN OF THE HOG-CHOLERA BACILLUS ISOLATED FROM AN OLD LABORATORY CULTURE

In a stock culture of the hog-cholera bacillus, which was passed through a series of rabbits 14 years ago, an organism was found that differs from the original culture in that it fails to form gas from the carbohydrates that are usually attacked by this organism, while acid formation persists. This new strain is agglutinated by an anti-hog-cholera bacillus serum and produces in rabbits and mice a disease similar to that caused by the typical cultures. The failure to form gas has persisted over a period of 18 months and all attempts to cause the strain to revert to the original condition have failed. It resembles in many respects Bacillus typhosus and it may be that some of the so called typhoid cultures that are not agglutinated by antityphoid serum are non-gas-producing paratyphoids. Attempts to produce a similar change in a more recently isolated culture of the hog-cholera bacillus by means of animal passages and changes in the environment have been negative

THE IMMUNITY PRODUCED BY THE GROWTH OF TETANUS BACILLI IN THE DIGESTIVE TRACT

1. A method for the production of tetanus by the injection of a fixed number of spores is described together with the tests made in selecting animals for experimental work. 2. Guinea pigs fed a single serological type of tetanus bacilli will, after 6 months, show considerable amounts of antitoxin in their sera and will manifest immunity to the type fed. To other types they are just as susceptible as are controls. 3. Animals fed several types are immunized to each of these types. It is pointed out in the discussion that the digestive tract of man may carry several types and that he probably reacts in a manner resembling the guinea pig carriers. 4. Guinea pigs that carry tetanus bacilli and have antitoxin in their sera show little if any resistance to tetanus toxin. 5. As there is no relation between the amount of antitoxin in the blood and immunity to tetanus we believe that other bodies, specific for type, must occur and make for the immunity observed

THE TRANSMISSION OF EQUINE ENCEPHALOMYELITIS VIRUS BY AËDES AEGYPTI

In confirming Kelser's work on the transmission of equine encephalomyelitis of the western type by Aëdes aegypti it has been learned that the mosquitoes must be fed virus of high titer if positive results are to be secured. A period of from 4 to 5 days after feeding either on infected guinea pigs or on brain containing virus must elapse before the disease is transmitted by biting, but after this time transmission regularly results for a period of about 2 months. By inoculation, virus can be demonstrated in the bodies of infected mosquitoes for the duration of life. Although virus multiplies in the mosquitoes and is generally distributed in their bodies, repeated attempts to demonstrate it in the eggs from females known to be infected as well as in larvae, pupae, and adults from such eggs have been uniformly negative. Larvae have not taken up virus added to the water in which they were living. Male mosquitoes have been infected with virus by feeding but they have not transmitted the virus to normal females, nor have males transmitted the virus from infected to normal females. When virus of the eastern instead of the western type is used transmission experiments with Aëdes aegypti are negative. Apparently this virus is incapable of penetrating the intestinal mucosa of the mosquito. If, however, it is inoculated into the body cavity by needle puncture it persists and transmission experiments are positive