The premature discharge of the products of conception from the uterus is a not infrequent occurrence among domestic animals, and doubtless various factors may from time to time operate in its causation. For a long time, however, practical husbandmen have recognized an epizoötic or contagious kind of abortion, a definite transmissible disease in which the loss of the fetus is the most prominent characteristic. The transmissibility of contagious abortion of cows appears to have been demonstrated experimentally for the first time by Brauer. Experimental transmission has been performed by a number of investigators subsequently, the work of Nocard (1886) furnishing conclusive evidence upon this point.
It is certain that a disease, or possibly more than one disease, of this nature is a source of serious loss to the live stock industry in the United States, and there can no longer be any doubt that a considerable part of this loss is due to the definite specific disease prevalent on the continent of Europe and in England, and known as Contagious, Infectious, or Epizoötic Abortion. The purpose of this bulletin is the brief presentation of some of the facts concerning the cause, prevention and restriction of this disease, which have been established by modern investigation, for the information of men engaged in live stock production.
BACTERIOLOGICAL INVESTIGATIONS ELSEWHERE
Nocard carried out the first extensive bacteriological investigation of contagious abortion. In microscopic preparations of the diseased placenta he was able to recognize numerous short bacilli and micrococci. These were also found in the amniotic fluid. He obtained pure cultures of these two organisms, but failed to induce abortion upon inoculating these cultures into other animals. Neither of the germs obtained in culture could therefore be regarded as the causative agent in the disease.
In 1895 Bang and Stribolt undertook the investigation of this disease, and their results are now generally regarded as the most important of all the contributions to the study of this subject. A cow showing all the symptoms of impending abortion was purchased and slaughtered. The unopened uterus was removed to the laboratory where it was opened with special precautions to avoid all contamination. An abundant, grayish yellow, odorless exudate was found between the ovum and the inner wall of the uterus. Upon standing this exudate separated into two layers, a reddish yellow serum above, and a grayish yellow partly solid layer below. In microscopic preparations of this exudate, stained with Loeffler's methylene blue, numerous very small bacilli were found, apparently in pure culture, some of them lying free, but large numbers of them crowded together inside cells. These latter appeared at first to be micrococci, but more careful examination proved them to be really short rods. Bang and Stribolt were able to cultivate this organism in tubes of a gelatin-agar-serum medium, the germ developing only in a particular zone beginning about 5 mm. beneath the surface of the medium and extending downward 10 to 15 mm. After considerable work with cultures, they concluded that the bacillus is neither an aerobe nor an anaerobe, in the ordinary sense, but exhibits a very peculiar behavior in respect to oxygen, requiring for its development a partial pressure of oxygen somewhat less than that present in the atmosphere. They were unable to obtain growth of the germ in the presence of the ordinary atmosphere, nor in the absence of oxygen (Pyrogallol method). Curiously enough, by placing their tube cultures in an atmosphere of pure oxygen, they obtained cultures developing in two zones, one near the top and the other near the bottom of the tube, indicating that there are two optima in the oxygen requirement of the organism. This very interesting character of the organism received great attention at their hands, but nevertheless Bang points out that typical development such as he has pictured was not always obtained, a number of factors seeming to cause variation in the position and extent of the developmental zones in these tube cultures. By exhausting the air above the medium in the tube, the growth was made to extend to the surface. In this way they were able to obtain growth of the bacillus on plates, but they did not work out a reliable plate method, preferring to employ the dilution tube cultures for separation in all their work. Bang and Stribolt subsequently examined pieces of placenta from a large number of cases of contagious abortion, and found the bacillus microscopically in practically all cases. Sometimes they were abundant, in other instances very scarce. Most of this material was badly contaminated, yet, from that sent in during the colder season they successfully isolated the bacillus in pure culture in a majority of the cases. In three fetuses the bacillus was found in the intestinal contents in pure culture; in one fetus it was isolated from the blood. Two cows with mummified fetus in utero were examined post mortem. These fetuses had been dead 9 months and 5 months respectively but the surrounding exudate still contained the abortion bacillus and pure cultures of it were obtained from each case. Uterine exudate kept in the refrigerator still contained living abortion bacilli after seven months.
Having found the same bacillus microscopically in a series of cases of abortion, and having obtained it in pure culture from a number of them, it now remained for Bang and Stribolt to produce the disease by inoculation of these cultures into healthy animals. Four pregnant cows were obtained without knowledge of their previous history. Two of them were inoculated by intravaginal application of pure cultures, and two by intravaginal application of pieces of afterbirth from aborting cows. No abortion resulted in any of the cows and at slaughter 19 to 29 days after inoculation, there was no evidence of the disease. This result was surprising, as Brauer had induced abortion by the second of the above-mentioned procedures in from 9 to 21 days, Lehnert in from 12 to 20 days, and Trinchera in 9 to 13 days. The authors thought that the animals may have been immune on account of a previous attack of the disease, or that possibly the interval between innoculation and slaughter (19 to 29 days) may have been too brief for the disease to have developed. For the next experiment two cows were purchased from a region where abortion was unknown. Pregnancy began January 14 and January 16, 1896. On April 14, a rich culture of the abortion bacillus was injected well up into the anterior end of the vaginal canal of each of these cows. The inoculation was repeated in the same way on May 23, and again on June 4. One cow aborted June 24, the fetus evidently having been dead some days. The abortion bacillus was isolated from the afterbirth. The other cow showed the signs of impending abortion on June 23, and was slaughtered on June 24. The condition inside the uterus resembled in every respect that observed in the cow from which the original culture had been isolated, and the bacillus was present in pure culture. In these cows the disease had appeared 10 weeks after the first inoculation. A third cow was inoculated by intravaginal application January 19, 1897, and subcutaneously March 6, in both instances with pure cultures of the bacillus. Premature delivery of a living calf occurred April 9, 80 days after the first inoculation. Abortion was also caused in sheep by intravaginal application and by intravenous injection of pure cultures. Inoculation by the latter method proved to be more certain in these animals, and the incubation period after intravenous injection was only 7 days in one case and 12 days in another. Intravenous inoculation of a mare resulted in a premature delivery after 28 days. In all these cases the bacillus was recovered from the afterbirth.
In 1902, Preisz at Budapest isolated the same bacillus from two cases of contagious abortion in cows. He confirmed the findings of Bang in respect to the oxygen requirements of the organism, and was able to obtain cultures by a variety of methods on ordinary media. Apparently his cultures were less vigorous than those of Bang, for they soon died out, their resistance to germicides was slight, and all his inoculations into animals, including two pregnant cows, two pregnant guinea pigs, and one pregnant rabbit, as well as a number of other small animals, were without positive result. Preisz named the organism "Corynebacterium abortus endemici (s. infectiosi)."
In 1908, Nowak at the University of Krakau in Austria made a very important contribution to the study of this disease. He found the culture method of Bang and Stribolt very useful for the identification of the organism when obtainable in pure culture, and when the contaminating bacteria were few in number. When other bacteria were numerous, as is frequently the case in material obtained for examination, he found this method difficult. The pyrogallol method of Preisz also proved to be unreliable in his hands. Eventually he devised a method of plate culture which proved to be very useful. Ordinary agar was melted and cooled to 50° C. then mixed with about one fourth its volume of naturally sterile blood serum, and poured into sterile Petri dishes where it was allowed to solidify. The piece of placenta or other material to be examined was then streaked over several of these plates in succession, and the plates were incubated for 24 hours at 37° C., to allow contaminating bacteria to develop. The plates were next placed in a glass jar together with a culture of Bacillus subtilis, one square centimeter of culture surface of the latter organism being provided for each 15 cc. capacity of the jar. The jar was sealed and placed at 37° C. for three days, at the end of which time excellent surface colonies of Bang's bacillus were obtained. By the application of this method Nowak has succeeded in isolating the bacillus from the blood and intestinal contents of a number of fetuses, and from uterine discharge, when other methods failed. He has also observed that one could gradually decrease the amount of culture surface of B. subtilis employed in succeeding cultures and eventually get the bacillus of Bang to grow in the presence of atmospheric air, altho the cultures were relatively poor ones. Nowak also confirmed Bang by obtaining cultures in an atmosphere of nearly pure oxygen, as well as in ordinary air under a pressure of three atmospheres. His cultures were evidently vigorous for some of them were successfully transplanted after two years. Nowak used ordinary agar as a medium with considerable success, and found glucose agar to be almost as favorable to the growth of the bacillus. For the detection of the germ in pathological material, however, these media proved to be inferior to the serum-agar mixture in several cases. Cultures were obtained in broth and in milk without coagulation, contrary to the statement of Preisz. No gas was produced in sugar broth. Nowak inoculated a number of pregnant laboratory animals, and produced abortion with great regularity in guinea pigs and rabbits by subcutaneous, intravenous and intraperitoneal injection. He did not succeed in producing abortion by intravaginal application nor by feeding. No tests were made upon larger animals much to his regret, as in his opinion the experiments of Bang upon cows still left something to be desired in the way of experimental evidence.
McFadyean and Stockman (1909) have investigated the contagious abortion of cattle in Great Britain, and have found it to be identical with that studied by Bang in Denmark. They were able to produce the disease in cows by intravenous injection of natural virus and of active pure cultures, without a failure in eight experiments. By intravaginal application they caused the disease twice with cultures and three times with natural virus, but also failed to obtain any result in three trials with the natural virus. Subcutaneous inoculation was successful three times in five trials. By feeding they produced the disease three times in four trials. These authors consider ingestion to be an important mode of contracting contagious abortion in nature.
Zwick (1910) has made a preliminary report of the bacteriological investigation of contagious abortion at the German Imperial Health Office. By a comparative study of cultures, the unity of the disease in Denmark, Germany, England, and Holland has been established. Certain individual differences were detected in the various culture strains examined, and it was found that the bacillus could be readily cultivated upon various ordinary laboratory media, and that it could also adapt itself to an aerobic existence, thus confirming the work of Nowak. In one instance the bacillus grew aerobically immediately upon isolation from the animal body. Abortion was induced in sheep, goats, and rabbits by intravenous injection, intravaginal application, and also by feeding. Work upon the use of abortin (analogous to tuberculin) for diagnosis, and upon the agglutination and complement fixation tests, was in progress at the time the report was made.