UNIVERSITY OF CALIFORNIA PUBLICATIONS COLLEGE OF AGRICULTURE AGRICULTURAL EXPERIMENT STATION BERKELEY, CALIFORNIA BOVINE INFECTIOUS ABORTION AND ASSOCIATED DISEASES OF CATTLE AND NEW-BORN CALVES I. METHODS OF CONTROLLING ABORTION GEOEGE H. HART II. THE IMPORTANCE OF BACTERIUM ABORTUM OF BANG AND OTHER MICRO- ORGANISMS IN BOVINE INFECTIOUS ABORTION JACOB TRAUM III. MANIFESTATIONS OF ABORTION DISEASE WITHOUT DEMONSTRABLE ETIOLOGICAL FACTOR JACOB TRAUM and GEORGE H. HART IV. DISEASES OF THE GENITAL TRACT OF CATTLE FREOUENTLY ASSOCIATED WITH ABORTION FRED M. HAYES V. THE DIAGNOSIS OF PREGNANCY IN CATTLE GEORGE H. HART VI. SCOURS AND PNEUMONIA IN CALVES GEORGE H. HART and JACOB TRAUM BULLETIN No. 353 January, 1923 UNIVERSITY OF CALIFORNIA PRESS BERKELEY 1923 David P. Barrows, President of the University. EXPERIMENT STATION STAFF HEADS OF DIVISIONS Thomas Forsyth Hunt, Dean. Edward J. Wickson, Horticulture (Emeritus). , Director of Eesident Instruction. C. M. Haring, Veterinary Science, Director of Agricultural Experiment Station. B. H. Crocheron, Director of Agricultural Extension. C. B. Hutchison, Plant Breeding, Director of the Branch of the College of Agriculture, Davis. H. J. Webber, Subtropical Horticulture, Director Citrus Experiment Station. William A. Setchell, Botany. Myer E. Jaffa, Nutrition. Ralph E. Smith, Plant Pathology. John W. Gilmore, Agronomy. Charles F. Shaw, Soil Technology. John W. Gregg, Landscape Gardening and Floriculture. Frederic T. Bioletti, Viticulture and Fruit Products. Warren T. Clarke, Agricultural Extension. Ernest B. Babcock, Genetics. Gordon H. True, Animal Husbandry. Walter Mulford, Forestry. James T. Barrett, Plant Pathology. W. P. Kelley, Agricultural Chemistry. H. J. Quayle, Entomology. Elwood Mead, Rural Institutions. H. S. Reed, Plant Physiology. L. D. Batchelor, Orchard Management. W. L. Howard, Pomology. ^Frank Adams, Irrigation Investigations. C. L. Roadhouse, Dairy Industry. R. L. Adams, Farm Management. W. B. Herms, Entomology and Parasitology. John E. Dougherty, Poultry Husbandry. D. R. Hoagland, Plant Nutrition. G. H. Hart, Veterinary Science. L. J. Fletcher, Agricultural Engineering. Edwin C. Voorhies, Assistant to the Dean. DIVISION OF VETERINARY SCIENCE G. H. Hart. J. Traum. F. M. Hayes. J. R. Beach. * In cooperation with Division of Agricultural Engineering, Bureau of Public Roads, U. S. Department of Agriculture. CONTENTS PAGE I. Methods of Controlling Abortion: 271 The Location of Bacterium abortum Organisms in the Body of Infected Animals 271 Escape and Life of the Organisms outside of the Body of Animals 274 Method of Drawing Blood for the Agglutination and Complement Fixation Tests 277 Discussion of this Method of Diagnosis 279 Procedure in Uninfected Herds 280 Procedure in Infected Herds 282 Outline of Vaccination Experiments Now Being Conducted by this Station 288 Abortion, a Self-Limiting Disease 290 Summary and Conclusions 291 II. The Importance of Bacterium abortum of Bang and Other Microorganisms in Bovine Infectious Abortion 294 Introduction 294 Review of the Investigations bearing on the Cause of Infectious Abor- tion of Cattle 296 Pathogenicity Tests of Various Strains of Bacterium abortum 308 Observations upon Forty Aborted Fetuses Received at Berkeley 309 Discussion 320 The Power of Bacterium abortum to Produce Manifestations of Abortion Disease 321 The Failures of Bacterium abortum to Produce Manifestations of Abortion Disease 321 The Extent of Bacterium abortum Infection 322 Other Organisms Connected with Manifestations of Abortion Disease 323 Conclusions 327 III. Manifestations of Abortion Disease Without Demonstrable Etiological Factor History of the Herd 327 Cases of Abortion 327 Summary of the Cases 330 Other Cases Showing Difficulties of Breeding 340 Feeding of the Herd 341 Condition of Animals to Date 342 Other Factors Associated with Breeding Difficulties as Possible Con- tributing Causes of Abortion 342 Febrile Diseases and Injury 342 Poisonous Plants, Spoiled Feed, and Alfalfa 343 Impaired Breeding Ability on the Part of the Bull 345 The Effect of Diet on Reproduction 345 PAGES IV. Associated Diseases of the Genital Tract of Cattle 347 Retained Placenta 348 Metritis and Endometritis 354 Pyometra 356 Cervicitis 358 Salpingitis 359 Cystic Degeneration of the Ovaries 362 Discussion 366 V. The Diagnosis of Pregnancy in Cattle 367 The Estrus Cycle 367 Changes taking Place in the Genital Tract during the Estrus Cycle Phases , 368 Irregularities in the Estrus Cycle 370 Changes in the Genital Organs occuring in Pregnancy... 373 External Manifestations of Pregnancy 377 Economic Value of the Knowledge of the Presence and Stage of Preg- nancy : 377 Summary and Conclusions 379 VI. Scours and Pneumonia in Calves 380 Introduction 380 Bacterial Causes 381 Avenues of Infection 384 Incidence of the Disea c e 387 Factors aside from Bacterial Agents in the Causation of the Disease.... 388 Prevention 390 Treatment 391 VII. References : 393 VIII. Glossary 397 ILLUSTRATIONS PAGE Non-pregnant Genital Tract of the Cow 293 Illustrating Method Used in Growing Bacterium abortum under C0 2 310 Gravid Uterus of Cow at Full Term, Laid Open to Show Fetal Sac with Faint Outline of Fetus 326 A Cotyledon from a Pregnant Uterus 349 Enlarged Cystic Ovary on Left, with Normal Ovary on Right 363 Cross-Section of the Cystic Ovary (left) shown in Figure V 363 Cow with Long-standing Cystic Ovaries 364 Cow with Cystic Ovaries of Two Years' Standing 365 Microscopic Section of Ovary of Cow, Showing a Graafian Follicle with Ovum 369 Figure 10. Three Pairs of Ovaries from Cows, each Pair Showing an Ovary Containing a Corpus Luteum 371 Figure 11. Same as Figure 10, with the Ovaries Containing the Corpus Lutei Cut in Cross-Section 371 Figure 12. Microphotograph of Spermatozoa from a Bull, magnified x800 375 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. I METHODS OF CONTROLLING ABORTION By GEOEGE H. HAET THE LOCATION OF BACTEEIUM ABOETUM OEGANISMS IN THE BODY OF INFECTED ANIMALS The location in the animal body of the causative organism of abortion has been a very important fundamental problem in out- lining methods for the control of the disease and one which has been the subject of much study and experimentation. W. L. Williams 89 in 1916, and again in 1921 90 carefully outlined the theory that calves in infected herds or calves fed raw milk containing Bacterium dbor- tum or other organisms became infected, continued to harbor the organisms until they reached maturity, and were very liable to abort with their first pregnancies. This theory has been rather definitely refuted by experiments of Dick and Duebler 18 in Pennsylvania, by Simms and Miller 71 in Oregon, and by observations in various parts of this and other countries. Prior to and coincident with the refuta- tion of this theory, experimental work designed to ascertain the sites in the body of the infected animal which constituted the seat of temporary and permanent location of the abortion organisms was carried on extensively. The result, of this work has greatly increased our knowledge regarding this important phase of the disease. Today the work of Schroeder and Cotton 65 - 66 > 67 , Mohler and Traum 49 , Buck, Creech and Ladson 8 in the U. S. Bureau of Animal Industry, and of other investigators has established the fact there are four definite locations of Bacterium abortum in the body of infected animals. It is important for owners of live stock to know something of these seats of localization and to understand how the organisms leave the body of one animal and gain access to the bodies of other animals. Udder and Supramammary Lymph Glands. — The udder and the supramammary lymph glands are probably of first importance in this regard because in mature cows in which the udder has been active they become the more or less permanent seats of the organisms in a considerable percentage of infected animals. The organisms will remain in these glands and be given off very regularly in the milk for a period of years — in one cow, under the observation of 272 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION Schroeder 03 , for a period of seven years. On the other hand, except in one case of apparently natural infection, reported by Carpenter 10 , and one case in an adult virgin four-year-old animal following intra- venous inoculation by Schroeder 65 , the organism has not been found in the undeveloped udder or supramammary lymph glands of unbred heifers. The general opinion supported by field results, is that in such animals, these glands are very rarely seats of the infection even though the animals may be exposed to it during the early period of their lives. Infection is at most so unusual as to be of little practical importance in formulating measures for the control of the disease. Pregnant Uterus and Discharges From Genital Tract. — The sec- ond important place in which the organism is found is in the pregnant uterus and in discharges from the genital tract following abortion or normal parturition. The premature expulsion of the fetus is the final act in the chain of events following infection with the Bacterium abortum Bang. The organism may produce this event at any stage of gestation, from shortly after conception to the normal time for the birth of the calf. A certain period of time, which varies in individual animals, is required from the entrance of the organisms into the animal's body until abortion occurs, and the end of gestation may intervene before this has taken place. The offspring in such a case will be born apparently normal and alive, although the uterus, membranes, and discharges are teeming with the organisms. For this reason all animals in infected herds must at this time be looked upon as possible spreaders of the organism even though they have not and may never abort. According to Theobald Smith 73 , who has made a very careful and enlightening histo-pathological study of the mode of entrance and changes produced by Bacterium abortum in the pregnant uterus of cattle, the bacteria are deposited in the maternal cotyledons by the blood stream. From here they pass through to the fetal cotyledons and gradually spread out over the epithelium of the chorion or external fetal membrane, and in this process they destroy the cells and cause them to be exfoliated. They then pass through into the amniotic fluid, and, in the physiological process of the swallowing of this fluid by the fetus, they are carried into the stomach, or they may reach the interior of the body of the fetus direct from the placenta through the umbilical vein. The cotyledons and membranes, therefore, become infected first in the progress of the bacteria and may be the only parts containing the organisms when gestation ends and parturition occurs. Experiments by Schroeder and Cotton 65 have rather definitely shown, on the other hand, that the non-pregnant uterus does not harbor the organisms Bulletin 353] BOVINE INFECTIOUS ABORTION 273 and that they leave an infected womb in a few weeks after parturi- tion or abortion. The longest time it was found by them to remain in the womb was fifty-one days following the emptying of the uterus. Organisms in great numbers experimentally placed in the empty uterus remained there only a few days. Bulls experimentally infected by placing the organisms in the sheath and smearing them over the penis just prior to copulation did not transmit infection to the female. Thus it would appear that the empty uterus is not a favor- able seat for the multiplication of the organisms even though they do gain access to it. This condition changes, however, after preg- nancy, and McFadyean and Stockman 42 and others have shown that experimentally cattle may become infected by the vaginal route after pregnancy has been established for some weeks or months. Genital Tract of Bulls. — The third definite location of the organ- isms is in the genital tract or the accessory glands of a certain per- centage of bulls. Here it may set up small foci of inflammation and abscess formation from which the organisms may constantly escape from the animal through the discharges from the genital tract. Even though the experiments of Schroeder and Cotton 07 tend to show that organisms excreted from such an animal into the vagina of the female at the time of copulation would not infect her, the animal would nevertheless be passing the organisms on to litter and other material which came in contact with his discharges. The lesions of the genital tract of the bull may be recognized on physical examina- tion in certain rare cases, but failure to find such a condition is not definite evidence that the animal is free from infection. This condi- tion may be determined by the agglutination test applied to the animal's blood. G astro -Intestinal Tract of Calves. — The fourth seat of the organ- ism is the gastro-intestinal tract of calves during the periods when they are fed on infected milk. It is not generally believed that the organisms remain permanently in the bodies of these animals after they are weaned, because of the fact that the udder, the permanent seat of infection, is not subject to such infection until it becomes active prior to and following the first parturition. Definite experi- mental evidence that such animals excrete abortion bacilli with the intestinal discharges has not been established. As they are frequently ingesting the organisms in large numbers with the milk, however, this should be considered a possible means of spreading the infection until it is definitely proved otherwise. Outside of these four locations, the organism is not found with any regularity in other parts of the animal body. Schroeder and 274 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION Cotton 65 have injected them in suspensions into the circulating blood and could not recover them from the blood after a period of two hours. In unborn infected fetuses and aborts, it is found with great regularity in the lungs and stomach contents, having reached the inside of the body through the blood stream from the cotyledons or in the physiological process of swallowing the amniotic fluid which becomes infected with the organisms after they have passed through the cotyledons and chorion. ESCAPE AND LIFE OF THE OEGANISMS OUTSIDE OF THE BODIES OF ANIMALS Since abortion is a chronic disease, its manifestations are not recognized for weeks or months after infection occurs, a fact that in some cases makes it difficult to trace the source of the infection. Not only is a knowledge of the location of the organisms in the animal body necessary, but also their method of escape and existence outside of the body are factors of equal importance in controlling the disease. Present knowledge leads us to believe that the organism is strictly parasitic and cannot multiply outside the body of a susceptible animal. It is found in nature only as the result of contamination with dis- charges from infected animals in which it is present. Under favorable conditions of moisture and protection from sunlight, however, it will live for a period of weeks or even months outside of the body of the host animal. An abortion occuring in pasture, it can readily be seen, offers an ideal means of spreading the organisms over the feed supply of the remainder of the herd. The same condition arises when abortion occurs around farm buildings or when the animal is allowed to mix indiscriminately with the herd in pasture or corral for several weeks following an abortion, while she may be still discharging the organisms from her vulva. Under such conditions animals have been seen to smell and actually lick up visible amounts of vaginal discharge from the ground. The throwing of infected litter and bedding from the stable into a corral is also a source of infection because of the very common habit of cattle to eat straw and other bedding soiled with urine and feces, from the ground of a corral even when good hay is available in feed racks. As the after- birth and uterine discharge may be teeming with the organisms even when parturition is normal, this is the period of time when all animals in infected herds must be looked upon as possible spreaders of the infection. Bulletin 353] BOVINE INFECTIOUS ABORTION 275 Bulls gaining access to the premises from outside sources may spread the organisms with discharges from their urethra. Although from our present knowledge we believe only a small percentage of bulls are so infected, their discharges are definitely known to be a source of such infection and therefore should be guarded against. Calves during the milk drinking period may be passing the organ- isms with their feces and thus contaminating their surroundings. Bacterium abortum is very frequently present in large numbers in milk, and care should therefore be exercised not to allow such milk to become a source of infection. Possible opportunity for such a means of infecting herds to occur under ordinary farm conditions is present when unpasteurized skim-milk from creameries .is taken to uninfected premises; also when excess skim-milk and overflow milk from a creamery or skimming station are allowed to run into a drain pipe which opens into a flowing stream. Organisms from such a stream passing through pasture lands and furnishing drinking water may be taken into the bodies of susceptible animals even at a distance of several miles from the plant. It is possible for infection to be carried from a cow giving off the organisms with her milk, to a susceptible cow, on the hands of the milker or the teat cups of a milking machine, in which event the infection would enter the uninfected animal by way of the teat duct and establish itself first in the udder. It should therefore be recognized that there are many ways by which the organism, after leaving the body of an infected animal, by one of the comparatively few and well understood channels, may gain access to the surroundings of other even remotely situated animals and set up a new focus of the disease. The direct route, however, by which animals become infected with the organism under practical ranch conditions in the vast majority of cases is by way of the digestive tract through the ingestion of contaminated food. The original contamination on the uninfected ranch occurs through the purchase and addition to the clean herd of one or more animals infected with the organisms and giving them off from their bodies without necessarily having themselves any observable breeding difficulties. Experimental evidence shows that animals may become infected in other ways, such as by way of the vagina after pregnancy has become established; by way of the teat duct and udder, (Schroeder and Cotton 65 ), by way of the con- junctiva (Seddon 68 ), in laboratory animals; but these sources of infection probably do not exist or are of only secondary importance under ordinary conditions of handling cattle on the farm. 276 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION The direct transmission of the infection from the male to the female, or vice versa in the act of copulation is no longer held to be an important means of spreading the disease, if it is a factor at all. This addition to our knowledge regarding the disease minimizes the importance of the one-time widespread recommendation and frequent application of the routine practice of douching the sheath of the bull before and after each service. Such treatment at best only disinfects the sheath and exterior of the penis, while in infe2ted bulls the organ- isms have been found in the seminal vesicles and epididymis where they cannot be reached by disinfectants and from which they are discharged into the vagina with the semen at the time of copulation. The important factor to recognize, therefore, is that the infection is spread from herd to herd largely by the addition to the uninfected herd of infected animals from outside sources. The great amount of movement of live stock in the ordinary channels of trade has been the means of spreading this disease, as well as the other great chronic scourge, tuberculosis, among the live stock throughout the whole state and country. Frequently an unscrupulous owner of live stock, despite a knowledge of the existence of the disease in his herd, has advertised it for sale and through the medium of the public auction has dis- tributed the animals in small groups or individually to many new premises, so that a high percentage of the dairy herds of any size, and many of the range herds, have become infected with the disease. Live stock men must recognize the importance of this means of spreading disease and help to develop proper regulations to prevent it. Owners have too often in the past looked upon regulations for disease control as unnecessary hindrances to the operation of their business. The success of the Federal Accredited Herd Plan in tuberculosis eradication is evidence that cooperation of cattle men in solving disease problems leads to success and the consequent financial stabilizing of the industry. When purchases are to be made — and it is fully recognized that this is and always will be very frequently necessary for the continuation of the live stock business — certain efforts should be made to see that the animals have a good breeding history and that they are free from infection. The agglutination and the complement fixation tests are means at our disposal of helping to ascertain whether or not infection exists in animals to be purchased. Bulletin 353] BOVINE INFECTIOUS ABORTION 277 METHOD OF DRAWING BLOOD FOR THE AGGLUTINATION AND COMPLEMENT FIXATION TESTS Drawing blood is an operation that is being more and more com- monly carried out and at times must be done in the absence of veterin- ary assistance. The apparatus required depends on the point from which the blood is to be taken. It may be collected from the ear, the tail, or the jugular vein. From dairy cows confined in stanchions, the jugular vein offers the most convenient source from which to draw it, by means of a sharp hypodermic needle such as is used in a blackleg or hog cholera syringe. These needles should be boiled before use. In going from cow to cow, however, it is only necessary to wash them out with disinfectant solution, followed by clear tap water. Care must be exercised that the lumen is open before insert- ing them, as otherwise no blood will flow when the vein is tapped. Wire stilets for cleaning particles of blood clots from the lumen should be at hand. The most desirable needle for the purpose is one with the stem about two inches long and from fourteen to sixteen gauge. The animal's head is restrained by an assistant with the fingers of one hand in the nostrils, or with a halter, or nose tongs, pulling the nose around to the right side and, if necessary, tying it to the upper cross-piece supporting the stanchion. If long hair is present on the left side of the neck, clip it with scissors or clippers and sponge the area with 2 per cent compound solution of cresol or other similar disinfectant. With the thumb of the left hand press upon the left jugular groove, which will cause the jugular vein to enlarge by retarding the flow of blood to the heart so that it can be distinctly seen. With the needle held firmly in the right hand, it is inserted directly over the center of the swollen vein. A strong push is required to insert even a sharp needle through the thick skin of cattle as compared to the much thinner hide of the horse. It is rare for the needle to be successfully pushed through the skin and into the vein at the same time. In case it has been inserted directly over the center and in the same general direction of the vein, a second sharp thrust, with the left hand keeping the vein well distended, will usually carry it in. Blood will then flow freely from the needle and should be collected in clean dry glass vials or bottles. It is very important that the containers be dry as a very small amount of moisture will cause the blood to hemolyze/ thereby coloring the serum, which may form a precipitate or otherwise render interpretation of the test 278 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION difficult or impossible. About 15 mils (one tablespoon) of blood is a satisfactory amount; when difficulty is experienced in getting it to flow readily, a less amount can be made to suffice, but in no case should this be less than one teaspoonful. A list for identification of the cows may be kept on paper and the bottles simply numbered on the corks, or, if a label is available, the cow's description and number may be placed on it and the label pasted on the bottle. After the blood has clotted in the bottles, they should be shaken or sharply struck against the palm of the hand to loosen the clot from the sides, allowing it to contract and the serum to separate. In animals which have to be handled in chutes — thick-necked bulls and the beef breeds — it is difficult to make the jugular vein stand out with sufficient prominence and also difficult for the operator to be in a position to get the needle into it. Under these conditions blood must be drawn from the ear or tail. In valuable animals where it is not desirable to slit the cartilage of the ear, the hair may be clipped short on the upper surface of the ear and a vein cut without cutting through the cartilage. The principal objections to ear bleed- ing is the small amount of blood which usually flows from the incision and the time required to get a number of samples. The tail therefore offers a more favorable source under these con- ditions. In drawing blood from this appendage, the incision is made on the under surface, as the blood vessels of the tail pass along the under surface and to each side of the coccygeal vertebrae. The incision should be made with a sharp knife and preferably over the center of the vertebrae, rather than at the end, so as to avoid the possibility of opening a joint. Any point on the tail above the brush may be selected as the point of incision. When the artery is com- pletely severed, blood flows very freely and, after sufficient is collected the hand should be held over the incision to stop excessive bleeding. Sometimes it is necessary to wrap the incision with cotton and a bandage for a day or two. With experience, the incision can be made deep enough in most cases to get sufficient blood without pro- ducing an excessive flow. There is no danger of an animal bleeding to death from such an incision; the wound in all cases under our observation has healed readily, leaving practically no scar. The blood samples should be sent by express at once to the labora- tory. In warm weather or when long shipments are necessary, it is preferable to let the clot contract over night in a cool place and then decant the clear serum off into clean dry bottles and ship. Only the clear serum is used in making the test ; when this cannot be obtained, Bulletin 353] BOVINE INFECTIOUS ABORTION 279 the reading of the test is interfered with. When decanting cannot be carried out, the whole blood, during shipment in warm weather should be iced. DISCUSSION OF THIS METHOD OF DIAGNOSIS While the agglutination and complement fixation tests have a definite place in the control of abortion and will undoubtedly come into much wider use in the future than they have in the past, live stock owners should realize the limitations of the procedure. The agglutina- tion test, which is the one usually employed, consists in bringing small amounts of the serum of the animal to be tested in contact with a suspension of abortion organisms in physiological salt solution in test tubes and allowing it to stand for twenty-four to forty-eight hours. In case the serum shows a positive reaction, the blood of the animal will contain what are termed agglutinins, and these will agglu- tinate the abortion organisms so that they will .clump together in the bottom of the tube, leaving the supernatant physiological salt solution clear. If the cow is not a reactor, her serum will contain no agglutin- ins, the abortion organisms will not become clumped, and the fluid will remain cloudy, the condition it is in when the serum is added. The agglutinins are specific for the infecting organisms and will agglutinate no others. A positive reaction to the agglutination test indicates that the animal from which the blood was drawn is, or recently has been infected with the disease. It is no proof that she will abort, because some animals become infected with Bacterium abortum and never abort. These animals may, however, spread the disease to other animals, as already mentioned. In testing blood from whole herds of animals, the agglutination test gives an idea of the extent of the infection in the herd. Animals giving a positive reaction are the ones most likely to abort in the future. According to Schroeder, about 60 per cent of the positive reactors will expel the organisms with their milk. Some animals shortly after abortion will give a negative agglutination test. In two first-calf cows under our control which had probably never aborted, a suspicious reaction to the agglutination test was obtained. They were sent to be slaughtered, and blood taken at time of killing was nega- tive. Despite this finding, Bacterium abortum was present in the supramammary lymph glands of both animals. Single negative reac- itons are, therefore, not positive proof of absence of infection. Nega- tive reactions of whole herds are much more valuable. 280 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION Isolation or removal of positive reactors, where they are so few in number as to make it practical, with repeated agglutination tests on the remainder, may successfully remove infection in certain selected cases. The test is rendered temporarily or permanently valueless by the previous administration of killed abortion organisms, in the form of bacterins, or of live abortion organisms in the form of vaccine. PEOCEDUEE IN UNINFECTED HEEDS The recommendations to owners of live stock in regard to this disease vary more or less in each individual case. In dealing with a disease of such widespread nature, the conditions met with vary exceedingly. Thus, in one part of the state we may see a herd of several hundred head of range cattle to which no additions are made except bulls from time to time. These animals are constantly kept on patented land and do not mix with other animals. The range may be divided into several isolated pastures or be practically all in one. Owners of other similar herds take their cattle to the Forest Reserve during the summer season, at which time they are in more or less intimate association with other cattle. These are very different situations from that confronting the owner of a dairy herd kept con- stantly corralled on a comparatively small piece of high-priced land with no opportunity to segregate groups of animals or to raise young stock. Pure-bred herds offer problems entirely different from grade herds. It is therefore very difficult to cover these extremes and the gradations between them in such a way that the recommended pro- cedures will not appear impractical to some owners. The fact remains, however, that the condition desired in all cases depends on the principles deduced from the careful study and experimentation of many investigators in various parts of the world and on the results gained from field experience in applying these principles in the actual handling of herds. In many cases the ideal procedure cannot be carried out, but, if the ideal is recognized, it can be approximated as nearly as the individual circumstances render possible, and satis- factory, partial, or poor results expected accordingly. The disease is very widespread — statements have been made by some that practically no large dairy herds are free from it. But when we consider that if new infection is excluded, the disease may run its course in infected herds, then it is perfectly proper for an owner to assume that his herd is no longer infected and to proceed accordingly. As the greatest source of spreading the abortion infec- tion is the adding of new stock from infected herds, the fundamental Bulletin 353] BOVINE INFECTIOUS ABORTION 281 recommendation is to raise sufficient heifers to replace the adult animals and thus reduce the purchase of animals to a minimum. When this cannot be done, the safest class of females to add are those between weaning age and breeding age. Animals during this period of their lives are least likely to carry the organism in their bodies. It is generally believed and has been proved in practical experience, that these animals so rarely carry infection that under all ordinary conditions this is a safe procedure. However, Bacterium abortum has been isolated in rare cases from the udder of heifers which have never lactated (Carpenter 10 and Schroeder 65 ). Agglutination tests recently reported by Barnes 5 showed 13.4 per cent positive reactions in eighty-two unbred heifers over one year of age from seven herds, and 19 per cent of twenty-six between six months and one year of age from five herds. Therefore, where all precautions are being taken, such heifers should have their blood tested. This knowledge that females rarely carry the organisms permanently in their bodies until it has found lodgment in the udder and supramammary lymph glands — and this occurs only after the gland has been actively engaged in the secretion of milk — does not apply to the male. Our knowledge is at present less definite with regard to this sex. It has been conclusively demonstrated that the organisms may secure a permanent foothold and set up pathological processes in the male genital tract without in any observable way interfering with the general health or breeding ability of the animal. At just what age this may occur is not definitely known and while in those cases in which this condition has been reported the animals were sexually mature, it is known that bulls become sexually mature at an early age. Therefore, on adding bulls past the weaning age to clean herds, they should first have the agglutination test applied. In both pure-bred and grade herds it is necessary from time to time to add adult females from outside sources. It is with such animals that infection is most liable to be introduced. While adult females from close-by herds, with whose breeding history the pur- chaser is acquainted, may be added with comparative safety, it is a good general rule of procedure to have the blood of such animals tested. Too much dependence cannot be placed on single negative tests in such animals, and when little is known of the breeding history and conditions render it possible, a second examination of their blood should be made after thirty to sixty days. This is recommended even though the animals cannot be kept isolated in the meantime. Owners of clean herds are frequently faced with two other situa- tions which may become sources of infection. One of these is sending 282 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION females away to be bred, and the other is admitting outside females to the premises for breeding to the herd sire. As already stated, there is a considerable amount of evidence at hand today to show that the act of copulation in itself is not a means of spreading infection from female to male, or vice versa. The danger is rather that outside cows will spread infection on the premises, either from vaginal discharge containing the organism, when the cows are brought to be bred too soon after abortion or parturition, or by contamination of the food of the home animals; by contact with milk of the outside cows. Females going away to infected ranches are more liable to become infected by ingesting food contaminated with the organism than by service from the bull. Cows brought on premises to be bred should therefore remain on neutral territory, i.e., territory not occupied by the home animals, and at least two months should have elapsed since parturition. Animals sent away to be bred in herds, the abortion history of which is unknown, should be sent only to places where proper precautions are taken against infection. Finally, owners of uninfected herds should watch the water supply and drainage conditions as a possible source of contamination. Under range conditions, where cattle are taken into Forest Reserve range and there mingle with other herds, the opportunities for keep- ing out infection are difficult. Even under such conditions, it is common for a group of a comparatively few cattlemen to largely control the range in particular valleys and meadows, and these men should consider as a group, the abortion problem, at least during the period of the year when their cattle are running together. In small herds which are members of bull clubs, it is also necessary to consider all the animals in such a club as a unit in dealing with this disease. PROCEDURE IN INFECTED HEEDS The degree by which the presence of infectious abortion in a herd manifests itself in actual abortion varies widely. In some cases it spreads through the herd until the incidence of premature expulsion of the fetus reaches 50 to 75 per cent of the pregnant animals; in a few rare instances, as high as 90 per cent have been known to abort. In other cases the disease seems to spread slowly, never becoming a serious factor in the calf crop or dairy production, and, in a few rare instances in small herds, a single case of abortion definitely due to Bacterium abortum Bang is all that has been observed to occur. Mild infections sometimes develop sudden virulence and cause great losses. This phenomenon has been termed by Williams "abortion Bulletin 353] BOVINE INFECTIOUS ABORTION 283 storms," and the potential danger of such an infection should not be underestimated. On account of the fact that abortion is a self -limiting disease, many substances have received widespread credit as curative agents without having any practical value as such. Among these may be mentioned the carbolic acid and methylene blue treatments and there is a host of proprietary preparations. Any method outlined for controlling this infection in the herd must be carried out on a fairly large scale before definite conclusions can be deduced as to its merit and credit given for good results. Each infected herd offers problems peculiar to itself, and considerable thought should be given by the owner and his veterin- arian to the method to be followed in limiting losses. They should familiarize themselves with an early diagnosis of abortion due to Bacterium abortum Bang, and all cases of abortion should be con- sidered to be of this nature unless they are definitely proved other- wise. If a laboratory is close at hand, the fetus and membranes can be examined for the presence of the organisms. The more practical method of ascertaining definite knowledge of the presence of the infection consists in having the agglutination test applied to the blood of the suspected animals. Three distinct methods of procedure for controlling the disease are outlined below, either one of which may be used alone in certain selected cases, or they may be combined to advantage when it appears that better results are to be obtained thereby. These methods are: 1. The repeated testing of the entire herd by the agglutination test, and the removal of reactors. 2. The isolation method. 3. The vaccination method. In average-sized herds in which the infection appears, blood should be taken from all the animals of breeding age, male and female. In case this shows but a very small percentage of infected animals and other conditions are favorable, it is feasible to expect that good results will follow the use of the first method for controlling the disease. Repeated testing of the entire herd by the Agglutination Test, and removal of reactors.— Actual details of handling abortion under this method are rare in published rep'orts. The work of E. M. Robinson 61 in South Africa is therefore of great interest in this regard. He experimentally demonstrated the feasibility of eradicating the infec- tion from a herd by this method. The experimental herd, consisting of sixty-three cattle — cows, heifers, and one bull, taken from a herd of one hundred head — were taken to a clean farm. The remainder 284 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION of the herd consisted of aborters and reactors, except for one positive cow which, at the time they were moved in 1915, gave a positive agglutination test and was accidentally included. In January, 1916, a test was applied to all the animals and this cow and four others reacted positively. These animals were isolated, and two aborted, one was barren, and two calved normally but the presence of abortion organisms were demonstrated in their milk. Another agglutination test in March, 1916, showed three more reactors. These were removed ; one aborted, one calved normally, and one did not breed. After this test, the reactors were no longer isolated on the farm, but removed to different premises. A third test of the remaining cattle was made in May, 1916, and showed only one positive reactor. This cow was removed and aborted in July of the same year. In July, 1916, a fourth test was applied to the herd and showed two reactors. Both of these cows were removed. One calved normally and one gave birth to a premature living calf. Bacterium abortum was isolated from the milk of both animals. In August, 1916, a fifth test was made and there were no positive reactors. Two years later there had still been no positive reactors in this herd or among the clean herd into which they were introduced. This experiment shows that the animals reacting positively are the most likely ones to abort and rather frequently are giving off the organisms from their bodies. By the application of five agglutination tests and the removal of eleven head from the herd of sixty-three animals, over a period of about eight months, the infection was eradicated from the herd. An effort to apply this method would be practical for owners with two or more herds on different ranches, and for those with a small percentage of reactors where opportunity exists to raise young stock to replace the adult cows, thus rendering outside purchases unneces- sary. With the more general application of the agglutination test to entire herds of cattle, opportunities for demonstrating the value of this plan will undoubtedly present themselves. The Isolation Method. — It is under this plan that we believe the disease can be handled to the best advantage in the great majority of herds. It is based on our knowledge of the means of escape of the organisms from the bodies of infected animals and consists in so handling the animals that the escaping organisms will have least opportunity of gaining access to the bodies of uninfected animals. To do this, isolated maternity quarters must be provided in which animals about to abort or calve normally are kept. They should Bulletin 353] BOVINE INFECTIOUS ABORTION 285 preferably be provided with entirely separate stalls, opening to the outside only, for individual animals, and should be disinfected or fumigated after each animal- is removed from them. Bedding, after- birth, and drainage from such quarters should be burned, buried, or so handled that cattle cannot gain access to them. Such animals should be kept isolated from the herd until all discharge from the genital tract has ceased and, preferably until an examination by a veterinarian shows the genital organs to be in a normal condition. This has the double advantage of keeping the animals from spreading infection and making possible the recognition of abnormal conditions, which may thus be treated at once. The earlier such conditions are properly treated, the less is the danger of the animal becoming per- manently sterile. Even though it is practically possible to keep such animals isolated only during the comparatively short period when their milk is unfit for commercial use, it will be advantageous to do so. Calves, during the milk-drinking period, should be kept isolated from the remainder of the herd, and manure from their quarters should be so handled that cattle cannot come in contact with it or their food and water become contaminated by it. Another source of contamination is carelessly-handled milk. Milk house drainage, contaminated with spilled milk, opening into irri- gation ditches, or on to green alfalfa pasture, or alfalfa cut and fed green is a possible source of infection to well animals. During the general application of this method, the buildings and surrounding premises should be disinfected weekly, monthly, or quarterly, depend- ing on the amount of infection present and the degree of completeness of the isolation methods which practical conditions render possible of maintenance. Unbred heifers past the weaning age should be kept isolated, and, particularly, bred heifers should be separated from adult cows during the period of pregnancy. If only two groups can be maintained, unbred and bred heifers should be kept together, and separated from the cows. The herd sire, if a non-reactor, may be used to breed such animals with comparative safety if they are taken to neutral clean ground for breeding. Under these conditions, bulls giving a positive agglutination test should be eliminated from the herd. If positively reacting high-priced bulls must be maintained for breeding the adult cows, they should be kept corralled and given the necessary exercise in the corral or in such a way that possibly infected discharges from their genital tract will not contaminate the feed of other animals in the herd. Too great stress cannot be laid on the importance of this method of handling abortion. It is the means of 286 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION obtaining successful results, as has been demonstrated by field trials in various parts of the world and particularly reported upon in this country by Simms and Miller 71 in 'Oregon, and by Dick 18 and Duebler 17 in Pennsylvania. The Vaccination Method. — Three bacteriological products have been prepared for use in herds infected with this disease. These are dead abortion germs known under the name of abortion bacterins, live abortion germs known as abortion vaccine, and abortion serum made from the blood of cattle or horses immunized against Bacterium abortum Bang. One other bacteriological preparation known as abortin should be mentioned here. This was first prepared by McFad- yean and Stockman in England by a method very similar to the method of preparing tuberculin and mallein. It was further studied and its method of preparation refined in this country by Meyer and Hardenbergh 47 in 1913. At the present time no definite field of usefulness for this product in the diagnosis or handling of abortion disease can be recognized. Abortion Bacterin. — The first of these products to be widely placed on the market was abortion bacterin. It contained a suspension in salt solution or other fluid of abortion organisms grown on cultures in the laboratory and killed by means of heat. This being the first bacteriological product put out for the disease, it was exploited by commercial laboratories and was very widely used. As was to be expected, losses in some herds stopped after it was administered, but this occurs in a certain percentage of outbreaks when no treatment is given. Carefully checked experiments to ascertain its value have been carried out in England and Germany, and by the Wisconsin and Michigan agricultural experiment stations in this country. The conclusion reached in all cases was that no value can be attributed to the administration of this product. Abortion Vaccine. — Abortion vaccine, consisting of live abortion germs, was originally introduced and used experimentally in sheep", goats, and cattle by Bang 4 who obtained definite protective results in his carefully controlled experiments. Later it was studied extensively and used in the field by McFadyean and Stockman of the British Ministry of Agriculture. It is at present being distributed by this governmental agency to owners of live stock in England. It is pro- duced commercially and distributed by a number of firms manufactur- ing biological products in the United States. Carefully checked experiments by Hadley 25 of Wisconsin, Huddle- son 33 of Michigan, and the U. S. Bureau of Animal Industry, as reported by Schroeder 63 , seem to definitely show that its adminis- Bulletin 353] BOVINE INFECTIOUS ABORTION 287 tration is followed by a lower incidence of abortion in the treated than in unvaccinated control animals. Thus, in Hadley 's experiments of 127 unbred heifers vaccinated, 77.9 per cent calved normally while only 66.7 per cent of the controls did so. The best results were obtained in open cows which had never aborted. In this group the vaccine was 91.8 per cent effective, which was in marked contrast to 44.4 per cent of normal carvings for the controls. In open cows which had aborted, the vaccine was of little value, and, as was to be expected, in cows which were pregnant at the time of vaccination but had never aborted, there was a higher percentage of abortions in the vaccinated than in the controls. The total vaccinated cattle from which his data were obtained numbered 439 head, of which 14.1 per cent aborted, while in the 101 controls 31.2 per cent aborted. The results varied in different groups; the highly favorable ones were obtained in the group consisting of open adult cows which had never aborted. The vaccinated cattle also showed a lower sterility rate and an increased breeding efficiency over the controls. Despite the compara- tively large number of animals in the experiment, the writer warns against the danger of passing premature judgment on the value of any therapeutic agent for contagious abortion. Huddleson's experiments cover a much smaller number of animals and the animals were not under such close observation, but he con- cludes in regard to Herd A: "These data apparently indicate a decrease in the abortion and sterility rate of the treated animals and a marked increase in the breeding efficiency of the treated over the untreated animals." In regard to his work on both herds A and B he states : ' ' These data while very suggestive are too few to warrant final conclusions as to the value of vaccine treatment. ' ' Schroeder 's report covers twenty-three cattle which were part of a drove of sixty-six, none of which, according to the tests that could be made, was infected with bovine infectious abortion. Eleven received subcutaneous injection of pure living cultures of Bacterium abortum about two months before they were served by the bull. Four received repeated injections of killed cultures of the abortion organism after they had become pregnant, and eight were retained untreated as controls. The twenty-three cattle were equally and similarly exposed to abortion infection. The exposure was via the digestive tract and the material used was obtained from actual cases of infectious abortion. Of the eleven cows treated with live organisms before conception, ten calved normally and one aborted; of the four that received dead organisms, two calved normally and two aborted; seven of the eight controls aborted. 288 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION Despite the general concensus of opinion of investigators that the vaccine does reduce the incidence of actual abortion, and despite its continued distribution by the British Government over a number of years, the situation with regard to each individual infected herd should be considered thoroughly before this procedure is resorted to. This method is diametrically opposite to that outlined in the first plan. While there are conditions in which it is to be recommended, they certainly do not include any of those herds which might offer an opportunity to try out the first plan. Neither is it to be recom- mended for herds from which animals are constantly being sold for dairy or breeding purposes to go into possibly uninfected herds. In our opinion, owners are justified in using this method in heavily infected herds with a high rate of actual abortions, when the animals are confined in corrals on small pieces of land with no oppor- tunity to segregate groups of animals or raise young stock. Additions to the herd from outside sources are therefore constantly necessary, and sales of animals from such premises are usually for immediate slaughter. Vaccine treatment is the simplest method of handling the disease and therefore the one which owners and veterinarians are liable to use without full consideration of the ultimate results. It is at best a means of reducing the manifestations of the disease, but does not eliminate infection from the herd. Reported results from herds so handled do not give information as to the number of vaccinated animals which become permanent carriers of this infection, nor mention subsequent history over a period of years following the treatment, which is essential before this can be recommended, on a wholesale scale. OUTLINE OF VACCINATION EXPERIMENTS NOW BEING CONDUCTED BY THIS STATION In order to obtain more information in regard to this method of treatment, a portion of the special abortion investigation appropria- tion made at the last session of the legislature is being used in the following outlined experiments which are now under way. This investigation is designed to furnish information on the im- portant and still unsettled question of the actual value of live abortion organisms in producing immunity. It is expected that it will throw additional light on the localization of the injected bacteria and deter- mine whether or not it is necessary, in the production of immunity in Bacterium abortum infection, to have actual multiplication and activ- ity of the organism in the animal body, or whether it is an immunity Bulletin 353] BOVINE INFECTIOUS ABORTION 289 that is conferred upon an animal simply as the result of having been infected with cultures of the causative agent. It is expected that this investigation will also show the extent to which the infection, resulting from both the inoculation to produce immunity and the ingestion to produce infection, may be injurious to the animals infected and also to animals associated with them. The question as to whether transmission of infection by the bulls, from infected groups to non-infected check groups, is likely to occur will be of great interest. There have been purchased fifty-five heifers of breeding age and two young bulls known to be free from abortion infection as determined by history and laboratory tests. These will be divided into four groups: Group 1. — Twenty heifers to be inoculated subcutaneously with live Bacterium abortum organisms prepared after the method used in the laboratory of the British Ministry of Agriculture. After two months, these animals are to be bred by placing the two bulls in the pasture with them. These bulls will have already served animals in Group 2. After pregnancy has been established, they are to be subjected to infection by the ingestion of Bacterium abortum organ- isms. This is the principal experimental group to determine the efficiency of live abortion organisms in the prevention of abortion. Group 2. — Fifteen animals to be bred without any preliminary treatment by the two bulls before they are placed in Group 1. Ten of these animals, after pregnancy has become established, are to be subjected to infection with Bacterium abortum organisms in the same manner as Group 1. Five are to be kept as association animals with Groups 1 and 2. This is to be a check on the ability of our infective methods to produce abortion and on the control of the experiment in Group 1 ; also, an experiment with the five head on the extent to which pregnant cattle are in danger of becoming infected when exposed to aborting cows. Group 3. — Ten animals are to be vaccinated and bred at the same time and in the same manner as Group 1, but not to receive any further treatment until after parturition. This group is to be examined after parturition for the presence of Bacterium abortum in the milk as a result of the vaccination. Some or all of these animals may be slaughtered after the milk examination is completed and attempts made to isolate Bacterium abortum from the internal organs. Group 4. — Ten animals, five of which are to be bred, without previous treatment, by the bulls after they have served the animals in Groups 1, 2 and 3. We intend to have the other five animals left open so that in case opportunity offers, the bulls can serve them shortly after having served an aborting cow. This group, therefore, is to be a check on the bulls which have served vaccinated or recently aborting cows acting as carriers to non-infected females through the medium of copulation. 290 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION As an aid to the field observations on these four groups, laboratory tests of the blood of the fifty-five animals will be made at frequent intervals. The germs used for infecting the thirty animals are to be obtained from such sources as will constitute, as nearly as possible, the means of spreading the organism from the carrier of the infection, such as the milk of several cows infected with Bacterium abortum and aborted fetuses containing the organism. Bacterium abortum of known viru- lence from laboratory cultures will also be given. During this experiment, bacteriological examination of aborted fetuses from various ranches will be made. It is the intention in this work to examine approximately one hundred fetuses in order to ascertain the frequency in this section of the country with which Bacterium abortum Bang or other organisms are associated with the premature expulsion of the young. In this way, it will be possible to keep in constant touch with a source of supply of infective material of various kinds. Abortion Serum. — If this material has any immunizing value it is of a passive nature and will not last for a longer period than a few weeks. Recently a theoretical field of usefulness for it has been brought forward in combination with dead abortion germs for safely carrying animals in infected herds through the latter months of pregnancy. No checked evidence is at hand to show that it has any value in this connection. Its field of usefulness under our present knowledge is restricted to very narrow limits. ABOKTION A SELF-LIMITING DISEASE Despite the excessive losses it causes to the live stock industry and the widespread distribution of this infection, the fact should always be kept in mind that from the standpoint of the individual herd it is a self -limiting disease. This is due to the gradual acquiring of a tolerance or immunity to the particular strain of the organism, and, provided no new infection is added from the outside, it will cease to produce its manifestations over a varying period of time even though no special precautions are taken to limit its ravages. The period of time required for this varies greatly and is apparently longer in dairy herds than in beef herds. The latter are the class of herds where isolation methods are the most difficult to carry out. In such cases advantage should be taken of the natural production of immunity, and it is believed that this may be hastened by selection and breeding in such a way that animals which breed normally in Bulletin 353] BOVINE INFECTIOUS ABORTION 291 spite of the presence of the infection and those which develop immunity after one abortion can be kept for breeding. Those that are seriously affected by the disease and have retained afterbirth or abort a second time, or are difficult to get with calf and so on, may be culled out. Observations made by Potter 57 , in Kansas, on range cattle are particularly interesting in this regard. Herd immunity has been observed by all students of this disease in the field and its development is the reason that so many worthless substances have acquired a reputation as curative agents. In a large number of herds it is prevented from manifesting itself by the con- stant bringing in of fresh infection with the purchase of outside animals. In the certified dairies around the San Francisco Bay region there are at present about 1800 head of dairy cattle. In order to keep out tuberculosis, it has been the practice for the past seven or eight years to raise sufficient young stock in these dairies to replace the old cows, which has reduced the purchase of outside cattle to a minimum. This has automatically reduced losses from abortion to a point where it is of little financial significance. Therefore the presence of the disease to a severe extent in any particular herd where practical conditions render it impossible to introduce the recognized control measures should not necessarily cause the owner to hastily consign all the animals to slaughter. In some instances where the disease appeared in very virulent and widespread form, it has been known to clear up to a remarkable extent over a period of a single year. In the progress of the disease in a herd a certain percentage of animals are left temporarily or permanently sterile, which greatly adds to the financial loss it causes. SUMMARY AND CONCLUSIONS The localization of Bacterium dbortum Bang in the bodies of infected bovine animals and the means by which it leaves the body are fairly well understood. A knowledge of these fundamental facts is essential on the part of live stock owners to prevent infection from getting into the herd and to control it after it has gained access. The agglutination and complement fixation tests, although they have their limitations, are important factors in furnishing informa- tion in regard to the status of individual animals and particularly individual herds in regard to the presence or absence of infection and the amount of same. 292 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION The greatest source of infection with the disease and the means by which it is introduced into herds in the vast majority of cases is the addition of infected cattle. Other more remote means exist but they always involve the contamination of surroundings with discharges from infected cattle and the transportation of the con- taminated material to a point where cattle gain access to it without in the meantime exposing it to conditions that will destroy the organisms. Food and water supply are the great vehicles for so transporting the infectious agent. Animals purchased from outside sources and community pasture or ranges are always a potential clanger of the introduction of the disease. Depending on the practical conditions involved, danger from this source may be more or less completely eliminated. When infection gains access to the herd definite information is at hand on the means to be taken to limit the losses from its presence. Isolation methods of dealing with the disease are first to be recommended. While under certain conditions the use of live abortion germ vaccine can be recommended after due consideration of all the factors involved, this course of procedure should not be adopted by owners without full knowledge of the facts and possible consequences. The use of dead abortion germs is very generally believed to be worthless. Less is known regarding the possible value of abortion serum, but it is not probable that it will ever take an important place as a means of controlling or preventing this disease. The disease in any herd is self -limiting provided new infection is not constantly added and in a high percentage of herds will in time be reduced to a minimum even though very meager precautions are taken against it. This important fact renders it unwise to quickly dispose of whole herds of cattle because the infection is present in them. In range cattle the self-limiting aspect of the disease seems to be more noticeable or manifests itself more quickly than in high-pro- ducing, closely-housed dairy herds. Bulls containing Bacterium abortum in their discharges, while they may not infect cows during the act of copulation, are neverthe- less spreaders of the bacteria contained in their discharges. Bulletin 353 BOVINE INFECTIOUS ABORTION 293 Fig. 1. — Non-pregnant genital tract of the cow. 1. Left ovary and broad ligament. 2. Eight ovary. 3. Fallopian tube. 4. Horn of the uterus. 5. Corpus luteum protruding from ovary. 6. Vagina. 7. Horn of uterus opened. 8. Body of uterus and internal os. 9. External os. Cervical canal between 8 and 9. 294 UNIVERSITY OF CALIFORNIA — EXPERIMENT STATION II THE IMPORTANCE OF BACTERIUM ABORTUM OF BANG AND OTHER MICROORGANISMS IN BOVINE INFECTIOUS ABORTION By JACOB TKAUM The abortion disease of cattle is rather universally accepted as being caused by an infection. Other agents, nevertheless, have been and are being held responsible for the production of abortion in these animals. Principal among these are mechanical injuries and con- sumption of moldy, ergotized or otherwise spoiled food. Recently the lack of certain vitamins and failure to maintain the proper mineral balance especially in high-producing cows have been suggested as contributing causes to abortion and other reproductive disturbances. Part II of this bulletin discusses the causes of the infectious type of bovine abortion; the other forms of abortion will be discussed in Part III. In this, as in other infections, more than one manifestation of disease is produced, and one or more of these may be present; and while in the infection under consideration, the premature expulsion of the fetus is the most outstanding manifestation of its presence, the abortion, nevertheless, does not always occur. This is fully recog- nized, and practically every definition of bovine infectious abortion,* as the disease is now termed, includes other evidences of the existence of the infection. The report of the committee on abortion of the American Veterinary Medical Association 59 contains the following definition: "Bovine infectious abortion is characterized as a rule by an interference with the development of the fetus, frequently resulting in its premature expulsion, either dead or alive, viable or unviable. There is also a frequent inflammation of the fetal mem- branes and of the maternal cotyledons together with frequent reten- tion of the afterbirth. A cow may be the carrier and disseminator of the germs of the disease without showing symptoms of her dangerous condition. ' ' *This disease has been known as contagious, infectious, or epizootic abortion, abortion disease, and slinking of the calves; the name bovine infectious abortion has been suggested by the committee on abortion of the American Veterinary Medical Association. Bulletin 353] BOVINE INFECTIOUS ABORTION 295 This, in substance, is the definition of the disease given by most writers on the subject and with few exceptions they also consider that the infectious type of abortion is caused principally by the Bacterium abortum of Bang. The above mentioned report states that "The essential cause of bovine infectious abortion is Bacterium abortus (Bang)." E. C. Schroeder 63 , of the U. S. Bureau of Animal Industry, in a paper presented at the Twenty-fifth Annual Meeting of the U. S. Livestock Sanitary Association, states: "The prime etiological factor of bovine infectious abortion was discovered by Bang and Stribolt." Simms and Miller, 71 of the Oregon Experi- ment Station, in a report of experiments with this disease, state: "The conclusion has been reached that Bacillus abortus (Bang) is the principal cause of abortion among Oregon cattle." The term 'infec- tious abortion' as used in this paper will refer to infection with this organism. Practically all textbooks, such as Hutyra and Marek's "Special Pathology and Therapeutics of Diseases of Domestic Ani- mals," Hoare's "System of Veterinary Medicine," Ostertag and Zwick in Kolle and Wassermann's "Handuch der Pathogenen Microorganismen, " accept the Bang organism as the essential cause in infectious abortion of cattle. On the other hand, Moussu 52 while admitting that the Bacterium abortum of Bang may be responsible for contagious abortion, never- theless insists that an entirely different bacillus belonging to the paracoli group causes a large proportion of abortions ; especially did he find this so in France. W. L. Williams, of the New York State Veterinary College at Cornell University, and some of his colleagues, even though firm believers in the infectious nature of abortion, have persistently refused to accept Bacterium abortum as the proved principal cause of infectious abortion in cattle, and a considerable part of Williams' writings are given up to a discussion of the experi- ments presented as evidence to prove that Bacterium abortum of Bang is the principal cause of the infection; and in his latest writings 84 states: "It has not been determined what, if any, effect upon the reproductive efficiency of a herd the elimination of Bacterium abortus infection would cause." 296 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION REVIEW OF THE INVESTIGATIONS BEARING ON THE CAUSE OF INFECTIOUS ABORTION OF CATTLE To properly discuss and evaluate the results of investigational work which have a bearing on the causative agent of bovine infectious abortion requires the presentation of details. This will be done to the extent that space and importance of the work warrant. The first report of well-organized attempts to isolate a definite bacterial causative agent of abortion is written by No card 53 , who at the instigation of the French Government investigated this disease in 1885. Nocard gave a very thorough description of the pathological findings, and incriminated as the possible causes a coccus and a short bacillus, which he found in the exudate between the uterus and the chorion, the outer covering of the fetus. With neither of these organisms, however, did he produce the disease. In 1897, Bang 3 reported the results of investigations by himself and Striboldt, in which they discovered the organism now known as Bacterium abortum. A cow showing evidence of approaching abor- tion was purchased and slaughtered. An elastic ligature was tied around the vagina, and the specimen* reached the laboratory six hours after slaughter. Using every precaution to eliminate extraneous organisms, they found in the exudate between the uterus and the chorion a very small organism which appeared to be in pure culture. These organisms were found as individuals, also crowded together in masses, and frequently enclosed in cells. The investigators at first thought the organism was a coccus, but on more careful examination with a higher power glass, the bacillary nature of the organism was definitely demonstrated. In shake cultures, consisting of gelatin, agar, and serum, they found a growth developing in a definite zone, beginning about 5 mm. beneath the surface of the medium and extend- ing downward from 10 to 15 mm. They further found that the organism grew also in an atmosphere of very high oxygen tension. In the course of time they examined twenty-one specimens includ- ing pieces of afterbirth, uterine exudate, and aborted fetuses. In almost all cases of uterine exudate, microscopic examination demon- strated the presence of the organism, but always contaminated with other organisms. They succeeded, however, in obtaining pure colonies of the organism by subculturing during the cold time of the year in the specimens received. In three cases where fetuses were sent in, they obtained pure culture of the germ from the small intestines without any evidence of other bacteria. In one case they were able Bulletin 353] BOVINE INFECTIOUS ABORTION 297 to obtain the organism also from the blood and in another from the blood, stomach, and medulla oblongata. In two cases of mummified fetuses of five and nine months' standing, respectively, they obtained pure cultures of Bacterium abortum in each case. Having repeatedly found Bacterium abortum, and having been able to obtain it in pure culture, they next set out to produce the disease in order to definitely demonstrate that it was the cause of infectious abortion in cattle. Four cows bought in the stockyards of Copenhagen were used for this purpose. Two of them received intravaginal inoculations of cultures. After nineteen and twenty- nine days, respectively, these animals showed no evidence of the disease. They were slaughtered and the autopsies were negative. Into the vaginas of the other two cows were placed pieces of retained afterbirth. Thirty-three and thirty-five days afterward, respectively, these animals were slaughtered and autopsy findings again were negative. Bang explained the cause of his failures as being possibly due to the fact that the animals used in the experiment were of unknown history and might have previously aborted and therefore may have been immune. Further, the reason these animals were killed so soon after injection was because the work of Brauer, Len- hert, and Trinchera indicated that the period of incubation for intra- vaginal inoculation of abortion material is from nine to twenty-one days. About this time the work of the Committee of the Highland Agri- cultural Society of Scotland was brought to the notice of Bang through the article on "Epizootic Abortion" by Pemberthy; in this the report of the Committee indicated that the period of incubation with sheep and cattle was from five and one-half to ten weeks. Bang and his co-workers purchased two cows from a territory where abor- tion was unknown. When these cows were three months pregnant, they were inoculated intravaginally with a culture of the Bang organ- ism. Thirty-nine days later they were again given an intravaginal injection, and twelve days from the second injection they were given a third intravaginal injection of culture. Seventy-one days after the first injection, one of the cows aborted. Six hours after the abortion, the afterbirth was examined and cultured, and from this Bacterium abortum was isolated. Cultures and smears also showed the presence of micrococci. The second cow was slaughtered on the same day while showing symptoms of approaching abortion. Abor- tion bacilli were obtained in pure culture from the exudate. Another cow suffering from pyelonephritis was given an intravaginal inocula- tion of culture of Bacterium abortum on January 19, 1897, and on 298 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION March 6 was given a subcutaneous inoculation of culture. Eighty days after the first inoculation a premature but living calf was dropped. The calf died from dysentery at fourteen days. Bacterium dbortum was obtained in the uterine exudate. Bang concluded that this was a case of uterine catarrh produced by the inoculated organism. On June 12, 1897, Bang 4 inoculated 36 c.c. of a bouillon serum culture of the germ intravenously into a four-year-old cow in her fourth month of pregnancy. Three months later this animal aborted and the exudate contained abortion bacilli. This animal, Bang states, came from a farm where abortion did not exist. An eight-year-old cow from an uninfected environment received on June 12, 1897 a culture of Bacterium dbortum and on September 7, abortion exudate by mouth. On November 26, she gave birth to a small living calf, terminating a pregnancy which started March 14, 1897. The after- birth was covered with abortion exudate, rich in bacilli. Bang states that he was able by intravenous inoculation to produce abortion in cows, sheep, goats, and rabbits. In 1902, Preisz 58 of Budapest, Hungary, reported the finding of Bacterium dbortum in vaginal discharge forwarded to him for examin- ation. In one case the discharge was collected eleven days and in another, fourteen days after abortion. His photograph of the shake culture, the coagulation of the milk, and the successful growing of the organism by the sodium hydroxide-pyrogallic acid method makes the germ a doubtful Bacterium dbortum. This may account for his inability to produce abortion in two pregnant cattle by intravenous and intravaginal injection. He was also unable to produce abortion in guinea pigs inoculated intravenously, intraperitoneally, subcutane- ously, and intravaginally. In 1908, Nowak 56 of Austria, reported results of a bacteriological study of the Bacterium dbortum of Bang, in which he made a valuable contribution, especially in the method of isolating this organism from bacteriological material. In brief, his method consisted of smearing the specimen on agar plate to which had been added one quarter of its volume of sterile serum. This plate was incubated for twenty-four hours; at the end of that time all the colonies were marked and the plates placed in a jar which contained a culture of Bacillus subtilis. One square centimeter was provided for every 15 c.c. capacity of the jar. The jar was sealed and placed in the incubator for three days. Material which yielded negative results by the Bang method frequently gave positive results with Nowak 's method. By sub- cutaneous, intravenous, and intraperitoneal injection, Nowak was Bulletin 353] BOVINE INFECTIOUS ABORTION 299 able to produce abortion in guinea pigs and in rabbits. By feeding and by intravaginal inoculation, his results were negative. In 1909, McFadyean and Stockman 42 , reporting for the committee appointed by the Department of Agriculture and Fisheries to inquire into epizootic abortion in Great Britain, gave the results of work started in December, 1905. In the course of their investigations they isolated an organism which satisfied the morphologic and biologic characteristics of Bacterium abortum, differing, however, with respect to the optimum growth requirements set forth by Bang. McFadyean and Stockman at times succeeded in growing the organism directly on sloped medium, and in shake cultures the growth was always limited to the zone described by Bang but might appear on the surface and in the water of condensation. Comparisons with cultures furn- ished them by Bang convinced the English investigators that they had isolated the Bang organism. In their experimental work they inoculated twenty-eight heifers, as follows: Intravaginally : Six heifers with natural material and three with cultures. Of the former, three were positive and three were negative ; of the latter, two were positive and one non-pregnant. Per os: Four heifers, inoculated with natural material. Two were positive, one negative, and one non-pregnant. One heifer, inoculated with culture per os, was positive. Subcutaneously : Four, inoculated with natural material. Two were positive and two negative. One animal was inoculated with culture and proved to be positive. Intra- venously : Two animals were inoculated with natural material and seven with cultures. Of the former, one was positive and one non- pregnant; of the latter, all were positive. Summarizing the results of these experiments, it is noted that out of sixteen animals inoculated with natural materials, eight were posi- tive, six negative, and two non-pregnant. Of the twelve inoculated with cultures, eleven were positive and one non-pregnant. Of the nineteen positives, seven actually aborted, one gave birth to a weak calf three weeks ahead of time, two were killed while showing signs of approaching abortion, nine were killed at various intervals after inoculation and showed the abortion exudate. In all cases they claimed to have demonstrated the presence of Bacterium abortum. McFadyean and Stockman desired to determine the extent in which this infection was present in England, and in the course of a survey they examined the stomach contents of fifty-one fetuses and found twenty-two positive and twenty-nine negative for Bacterium abortum. Thirty-five fetal membranes were examined of which thirty- three were found positive and two negative; the latter two were 300 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION reported too putrid for examination. Forty-two swabs of vaginal discharge were examined; twenty-nine were found positive and thirteen negative. The diagnosis in all cases was based entirely upon the microscopic examination of the material. In 1913, Zwick and Zeller 91 reported the work undertaken by the German Imperial Board of Health on a study of the infectious abortion of cattle in Germany. The report includes work done from December 1908 to December 1911. Their first problem was to deter- mine whether or not the organism described by Bang was responsible for infectious abortion present in Germany. To that end they exam- ined fifty-two aborted fetuses, nine prematurely-born calves, eighteen afterbirths, two uteri, and one vaginal discharge. Of the fifty-two fetuses, thirty-five proved positive for the Bang organism. In thirty out of the thirty-five cases Bacterium abortum was obtained in pure culture, but the five other cases were contaminated, two with Bac- terium coli, two with streptococci, and one with Bacterium proteus. Regarding the seventeen negative cases, the investigators report that five had histories which indicated a doubtful presence of infectious abortion. Examination of these five yielded three sterile cases and two which contained Bacterium coli in pure and mixed cultures. Cultures from seven other negative fetuses remained sterile although the histories and serum tests from the dams indicated Bacterium abortum infection. One fetus yielded sterile cultures although its history indicated infection. Two negative fetuses showed on micro- scopic examination Bacterium abortum-like organisms but the cultures were sterile. One negative case yielded Bacillus pyogenes* Of the nine prematurely-born calves, two yielded sterile cultures and seven coli organisms, of which six were in pure culture and one with a coccus. In the eighteen afterbirths examined, the results show five positive for Bacterium abortum and thirteen negative for that organism. Four of the positive ones were of mixed cultures and one proved positive after the intravaginal inoculation of sheep. Of the thirteen negative ones, six were badly decomposed and seven showed mixed cultures mostly of Bacterium coli and cocci. One uterus from a cow slaughtered before abortion took place yielded organisms of Bacterium abortum in pure cultures. The other uterus was from a cow that had aborted four weeks previously and was negative. The one vaginal discharge examined yielded negative results ; it was from a cow which had aborted twenty days previously. * According to Brown and Orcutt 7 this organism belongs to the corynebacteria, but since this change has not as yet been adopted by the Society of American Bacteriologists, it has not been made here. Bulletin 353] BOVINE INFECTIOUS ABORTION 301 Zwick and Zeller confined their animal inoculations to sheep, goats, guinea pigs, and rabbits, and obtained positive results in a very large percentage by introducing the organism into the vagina, subcutis, peritoneum, and mouth. In twenty-four aborted fetuses, Holth 32 obtained cultures of Bac- terium abortum from the gastro-intestinal contents of nineteen cases. In sixteen of the nineteen, no other organisms were found. Two of the nineteen fetuses yielded cultures of Bacterium abortum from the blood. From one of the five cases in which the Bang organism was not found, a pure culture of Bacillus pyogenes was recovered. In the last decade much literature on infectious abortion of cattle has been published both in America and elsewhere, and some of it contains findings that have an important bearing on the point in question. Prior to that time very little work had been done on the etiology of this disease. Chester 14 of the Delaware Experiment Sta- tion reports a study of a herd in which eight cows aborted. From that herd he was able to obtain a piece of placenta, removed by hand, from which he made cultures on agar slants. He obtained one rapidly- growing organism and a second slower-growing one which he identi- fied as the colon organism, and which at that time he thought the possible cause of the disease. A pregnant cow was given 5 c.c. of a bouillon culture of the organism through the vagina and no ill effects resulted. In 1904, Moore 50 reported work done by Law and himself in 1897-98, in which they found bacteria belonging to the colon group, in fetal membranes, cotyledons, and uterine discharge in a fresh condition from seven cases of infectious abortion. Other bacteria present to a greater or less extent were bacilli, micrococci, and strep- tococci. He states, "Its power to produce abortion was tested in a few cases on pregnant cows, all of which delivered their calves at full term. We have no experimental evidence, therefore, that this organism stands in a causal relation to the trouble." The first investigators to isolate and identify Bacterium abortum as being responsible for abortion in cattle in this country were McNeal and Kerr 43 ' 44 in 1910. They examined afterbirths and discharges from seventeen cows, ten of which were term deliveries, and in these the abortion organism was not found. Seven were premature deliver- ies, and of these one was not clinically an abortion case. Two of the six abortion cases were examined before they adopted the Nowak plate method and negative results were obtained. Of the remaining four, two gave positive cultures of Bacterium abortum. In the third case only a small bit of the afterbirth was available for examination, 302 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION and in the fourth a small quantity of vaginal discharge which had been picked up from the ground. They inoculated four pregnant guinea pigs subcutaneously with culture isolated from these cases; premature evacuation of the uterus resulted, with death of the fetuses after three and a half, eight, six, and seven days, respectively. In the first pig two fetuses were practically fully developed and covered with hair. In this instance Bacterium abortum was isolated only from the subcutaneous tissue of the mother at the point of inocula- tion. In the other three cases, the fetuses were undeveloped and the condition was that of true abortion. In these instances the abor- tion organism was demonstrated at the point of inoculation, and in the interior of the uterus in pure culture in all three. McNeal and Kerr stated, "From this rather limited series of examinatijons we may conclude that the bacillus of Bang is the microbic cause of at least some of the contagious abortion of cattle in this country." In 1912, Good 24 of the Kentucky Experiment Station, reported having obtained pure cultures of Bacterium abortum from discharges and fetal membranes of aborting cows. Surface 80 , in 1913, reported the following experimental infection : A cow which had never aborted came from a herd in which abortion did not exist. She had been tested several times by the agglutination and complement fixation tests, always with negative results. When about five months pregnant, she received 20 c.c. of Bacterium abortum culture, on November 25, 1912, and aborted on January 6, 1913, fifty-two days after the inoculation. The cultures used were obtained from Denmark and grown aerobically. Cultures were recovered from the afterbirth and fetal stomach in twenty-four hours, both in plates according to the Nowak method, and also in plates kept in the incu- bator under ordinary atmospheric condition. This very rapid growth under ordinary atmospheric condition is given by Surface as addi- tional proof that he produced abortion with the culture with which he inoculated. Moore and Fitch 51 in 1911 inoculated five pregnant cows intra- venously with cultures of Bacterium abortum. Abortion took place in all five at the end of seven, twenty-six, sixty-five, eighty-five, and eighty-five days, respectively, after inoculation. Characteristic lesions of abortion were present in every instance except in the first animal. This experiment is of doubtful value since two of these five cows had aborted, two had been sterile, and but one had calved the pre- ceding year. In 1912, four more cows, which so far as known had never aborted, were fed cultures of Bacterium abortum. Three of these animals aborted and one had retained afterbirth. Serum tests Bulletin 353] BOVINE INFECTIOUS ABORTION 303 were not made before feeding these animals. All of them, however, showed an increase in agglutinins after feeding, and two of the aborters, which were definitely negative on the first two tests, after feeding, developed an agglutination of 1 to 600. In 1913, four more cows, were fed abortion organisms. There was a rise of agglutinins following the feeding in all cases. Three were still pregnant at the time of the writing of the report. One dropped a weak calf at 263 days of pregnancy. In 1913 Mohler and Traum 49 reported the finding of Bacterium ab or turn in eleven out of fifteen aborted fetuses. W. L. Williams 85 , in 1914, reported experiments with six heifers raised carefully under his direction. These he infected by intra- venous inoculation of a culture of the same strain as used by Moore and Fitch. One of the six died of tuberculosis. Five others were killed from three and a half to six months after inoculation, respec- tively. Four were normal and no abortion organisms were found; the fifth showed in the utero-chorionic space abortion exudate from which Bacterium abort um was isolated. Another lot of three animals 86 were inoculated intravenously. One of these aborted, one calved at the end of 272 days but had retained afterbirth and metritis ; the third calved at the end of 284 days with prompt expulsion of the fetus but metritis developed nine days after parturition. In another group of three heifers, two were given cultures by the mouth. One of them which reacted strongly before the culture was given aborted. The other calved normally. Following the recording of the above experiments of Moore and Fitch and of his own, Williams states : ' ' With full regard for the defects in the evidence, it is nevertheless extensive and important, and justifies amply the general conclusion that the Bang organism is the essential cause of contagious abortion, a conclusion freely open to revision whenever new evidence may demand." In the reports of the New York State Veterinary College for years 1917-18 87 and 1918-19, 88 published in 1919 and 1920, respec- tively, Williams reviews the work of Nocard, Bang, McFadyean and Stockman, Moore and Fitch, his own experiments just mentioned, and the report of Bland, in which the latter gives a record of inoculating five pregnant cows and nine pregnant heifers by intravenous inocu- lation for the purpose of immunization (inoculation by mistake since Bland thought these animals were not pregnant). Of these fourteen animals, only two cows and one heifer aborted. Williams also refers to the cultures made by Day of the United States Bureau of Animal Industry at Chicago. From the uterine cavity of twenty-five preg- 304 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION nant cows, seventeen showed no growth; two, streptococci and short bacilli ; three, staphylococci and short bacilli ; one, streptococci and staphylococci. In a similar work done by Hagan at the Buffalo stock yards, in which he examined twenty-six uteri, sixteen of which were pregnant, he found two sterile, seven which yielded coli-like organisms, one micrococci, three streptococci, three streptococci and staphylococci, and in one of these there was also found paracolon. Two out of the sixteen showed exudation in the utero-chorionic space ; in one a micrococcus was found and in the other a streptococcus. It appears that Williams lays too much stress upon the findings by Day and Hagan of the various organisms in pregnant and non-pregnant uteri and attributes to these organisms an unproved importance in cattle abortion. In 1918 Theobald Smith 72 reported the finding of spirilla or vibrios in pure culture from the fetuses of fourteen cases of abortion. He states that the condition of the fetus is much the same whether spirilla or bacteria of abortion are present, and that changes in the chorion due to vibrio cannot be distinguished with the naked eye from those due to the organism of Bang 73 . In 1919, Smith 74 reported the .finding of twelve more cases of spirilla infection in aborted fetuses. One of these twelve was associated with Bacterium abortum. In the grouping of cases of abortion according to the associated bacteria, in one herd Smith 75 recorded sixty-two cases from which Bacterium abortum was isolated, twenty-six cases of pure spirilla infection (one of the spirilla cases was associated with Bacterium abortum infection) ; another group of twenty-one abortions from which neither Bacterium abortum nor Vibrio fetus* was isolated — Smith considers that one or two of the twenty-one cases may possibly belong to the group of Bacterium abortum infections and several may come within the group of Vibrio fetus. Two others were cases of infection with Bacillus pyogenes, in which the preceding pregnancy had been cut short by Bacterium abortum infection. In eight cases miscellane- ous rapidly-growing bacteria appeared in the cultures. Of these several may be regarded as cases in which bacteria gained entrance after expulsion, the fetus being large enough to live a short time after birth. One was clearly a case of asphyxiation at birth and one was regarded by the attendant as traceable to fighting several days before expulsion of the fetus. Seven were considered bacteria-free. Smith states, "How many of these seven are due to injury or toxic substance and food factors cannot be stated." *Name suggested by Smith for the spirillum isolated from abortion cases and accepted by the Commitee of the Society of American Bacteriologists on Charac- terization and Classification of Bacterial Types. Bulletin 353] BOVINE INFECTIOUS ABORTION 305 Smith 74 succeeded in producing a disease of the fetal membrane and finding the vibrios in two out of four pregnant cows, injected intravenously with cultures of his Vibrio fetus. Bacterium abortum infection was ruled out in these cases. Among the twenty-six cases associated with Vibrio fetus of which one also contained Bacterium abortum, there were no heifers involved. All were second or later pregnancies. Recently, however, Smith, Little, and Taylor 77 found three cases of abortion in native heifers in the same herd associated with Vibrio fetus. Vibrios have been recovered from abortions in ewes in the British Isles by McFadyean and Stockman 41 and in this country by C. M. Carpenter 9 . The former also report the finding of vibrios in cattle abortion in Ireland and in Wales. Bacterium abortum was absent in these cases. In the report of the New York State Veterinary College for the year 1920-21, C. M. Carpenter 10 recorded the bacteriological studies of cattle genital tracts and fetuses. Among these are included the findings in eleven pregnant apparently normal uteri. In these he found six uteri sterile; Bacterium abortum was found in fetal fluids in one case ; three uteri showed streptococci, one Pseudomonas pyocy- aneus, and one showed an unidentified rod. Cultures from eighteen normal non-pregnant uteri yielded no growth in seven uteri, four showed staphylococci, and three streptococci in the cultures. Three developed streptococci and staphylococci, and one Pseudomonas pyocyaneus in the cultures. In twenty-three fetuses from apparently normal pregnant uteri, Carpenter obtained ten negatives, three showed streptococci alone, three staphylococci alone, and three streptococci and staphylococci together. Three showed Bacterium abortum in pure culture in the stomach ; one of the three showed no other organism ; in another strep- tococci were found in the meconium of the rectum. Bacteriological examination of twenty-five aborted fetuses yielded the following results: Bacterium abortum was found in ten cases. In nine it was found in pure culture, eight times in the stomach alone or also in other organs, and once in the genital tract. In five of these nine cases, no other organism was found in the fetus. In the tenth case staphylococci and Bacterium abortum were found in the heart's blood. Streptococci alone were found in two cases. In two other fetuses streptococci associated with other organisms were ob- tained. In four fetuses staphylococci alone or associated with other organisms were obtained. In one case a Gram positive rod, and in another a Gram positive spore-bearing rod was found. 306 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION Carpenter also made a bacteriological examination of three mummi- fied fetuses. In one he found Streptococcus viridans. In the other, two organisms from the colon-aerogenes group were obtained. One of the latter cases was associated with Bacterium abortum. Rettger, White, and Chapman 00 in a recent publication reported results of their attempt to produce abortion by artificial inoculation of cultures of Bacterium abortum. In their first trials where fourteen feedings of Bacterium abortum in capsules were given to eight cattle between the ages of two to twelve years, they obtained uniformly negative results. In the second attempt ten calves were fed cultures in pasteurized milk and after weaning were given cultures in cap- sules. Here again they obtained negative results. In both experi- ments the feeding of the organism in capsules did not incite the production of agglutinins or complement fixing bodies. Ten calves were used as controls. One of these became a confirmed reactor to the serologic tests and aborted, another aborted later. In another experiment three heifers were injected subcutaneously with culture of Bacterium abortum. One aborted and the others gave positive reactions. The inner surface of the vulvas of three other heifers was scarified and coated with cultures of Bacterium abortum. One of these gave a positive reaction and aborted five months after infection. The other two remained negative to the test, one calving normally and the other found to be non-pregnant. Eight heifers received superficial intra-urethral injection of cultures of Bacterium abortum. Six became reactors to the serological test. One gave only partial reactions and one was negative. Two of the reactors dropped premature small but live calves. In a further experiment, seven animals received deep intravaginal injection of Bacterium abortum. Three of the seven became confirmed reactors, two aborted, another gave birth to a small calf and a third calved normally. The other four resisted injection and remained negative to the serological tests. Huddelson 33 , in his studies on artificial immunization against bovine infectious abortion used fifteen heifers, none of which had ever given a positive blood test, nor had they ever so far as known been subjected to abortion infection before this experiment. Six were immunized, four were kept as normal controls, five were controls on the mode of infection after immunization and conception. Three of these five received intravenous injection of 1 c.c. of Bacterium abortum and two were fed living cultures of Bacterium abortum. Three of the normal controls calved normally and one failed to con- ceive. Each of the five infected controls, however, aborted about ten weeks subsequent to the appearance of antibodies in the blood serum. Bulletin 353] bovine infectious abortion 307 Seddon 68 in his studies on abortion diseases records the infec- tion of a cow by drenching on the sixty-second day of her pregnancy, which resulted in premature calving on the 262nd day and the finding of Bacterium abortum in the uterine exudate. Another animal which was injected subcutaneously with Bacterium abortum aborted eighty- two days after infection, on the 249th day of pregnancy. The uterine exudate contained abortion bacilli (cultures and guinea pig inocula- tions). A third heifer on the sixty-third day of gestation received intravenously, living Bacterium abortum, and 108 days after the inoculation the animal aborted. Organisms were found in the fetus and in the exudate. A fourth heifer when pregnant 225 days received abortion organisms in the vagina. She calved the 268th day of pregnancy, which was sixty-seven days after the infection. The uterine exudate contained Bacterium abortum. He does not state in his article the number of animals, if any, which were negative to the inoculation. Sven Wall's 83 extensive studies on alterations in the uteri in abortion and chronic metritis are pertinent to our discussion. His findings were as follows: In two gravid uteri showing metritis, chorionitis, and placentitis, Bacterium abortum alone was found in the exudate. In another gravid uterus containing odorless thick pus in the utero-chorionic space, Bacillus pyogenes alone was found ; this case showed chorionitis and metritis, but no placentitis. In a fourth gravid uterus, between the unaltered fetal membranes and the red- colored, partly denuded uterine mucosa, about fifteen cubic centi- meters of yellow, odorless, slimy pus were observed lying at the internal opening of the cervical canal. Smears showed streptococci and probably Bacterium coli. This case was two days old when examined. In two uteri, containing mummified fetuses, Bacterium abortum alone was found. From four other cases of uteri containing mummi- fied fetuses Wall obtained from one, Bacillus pyogenes, from another Bacillus pyogenes and a streptococcus, from the third case a strepto- coccus, and from the fourth a streptococcus, Bacterium coli and a diphtheroid. From the uterine cavity of two cattle that came to autopsy some days after abortion, he isolated Bacterium abortum. In one case this organism was associated with streptococci and in the other with Bacillus pyogenes and Bacterium coli. In three cases of retained afterbirth and in fifteen cases of chronic metritis he found, as a rule, either streptococci or Bacillus pyogenes or both, and in some instances these were complicated with Bacterium coli or Bacterium proteus putrefaction. 308 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION PATHOGENICITY TESTS OF VARIOUS STRAINS OF BACTERIUM ABORTUM Since the work of Smith and Fabyan 79 and that of Schroeder and Cotton 66 upon production of the disease in guinea pigs by inoculation of Bacterium abortum, these experimental animals have been used a great deal for determining the presence and the isolation of Bac- terium abortum from suspected materials. They have also been used for the study of comparative pathogenicity of various strains of Bacterium abortum. Schroeder 64 reports that, together with W. E. Cotton, he studied three porcine strains, one from Indiana, the second from Illinois, and the third from California. They found these strains to differ in their pathogenicity for guinea pigs. They were not wholly like any of the numerous other bovine strains studied by them. The lesions produced by porcine strains in guinea pigs were grosser in character, bone lesions were commoner, and post-orbital abscesses developed. One of the porcine strains produced abortion in cattle and sows. From a comparative study of ten porcine strains from three out- breaks in California and of five bovine strains of Bacterium abortum, the writer 82 concluded: "Porcine strains, on the whole, proved to be more virulent for guinea pigs, causing, with few exceptions involv- ment of the testes and one or both radio-carpal regions, also general adenitis. Thirteen of the twenty-two guinea pigs inoculated with porcine strains succumbed within two months, while none of the twelve inoculated with bovine strains died of infection. The dose for each guinea pig in the above series was similar. ' ' Meyer, Fleischner, and Shaw 46 , in their study on the pathogen- icity of Bacterium melitensis and Bacterium abortum for guinea pigs, review the work done and results obtained in guinea pig inoculation with Bacterium abortum by Smith and Fadj^an, Schroeder and Cot- ton, T. C. Evans, Krage, Thomsen, Holth, Robinson and Zeller. From their review, it can readily be seen that the lesions in guinea pigs have not been found by many of the above mentioned workers. Meyer, Fleischner and Shaw studied the pathogenicity of four bovine strains upon twenty-two guinea pigs ; eighteen were inoculated intratesticularly and four subcutaneously. Two strains were patho- genic for guinea pigs in fifteen out of sixteen cases, and the other two strains produced lesions and infection in one each of three inocu- lated intratesticularly with each strain. One of their pathogenic strains which had been isolated from milk, was two years old. The history of the other strain is unknown. The dose used in the above inoculations was from 1000 to 2000 million viable organisms. They Bulletin 353] BOVINE INFECTIOUS ABORTION 309 conclude that until knowledge relative to individual susceptibility of the guinea pig has been accumulated, it is impossible to make con- clusive statements, but the few, carefully-controlled observations rather forcibly suggest that old stock cultures are not only less toxic but less invasive than recently isolated strains or infected material derived from bovine, porcine, or cavian sources. These investigators also found Bacterium abortum from sows highly virulent for guinea pigs. Six out of twenty-four animals succumbed in from seventeen to forty days after intraperitoneal inoculation. The strain exhibited pyogenic properties. The testicles, epididymes, and seminal vesicles of all the animals used in the tests were destroyed by suppurative processes. The spleen was usually enlarged. Nine guinea pigs showed involvment of the ribs and long bones, while eleven showed lesions only in the radio-carpal and talo- tibial regions. They also report in tabulated form the lesions found in 114 guinea pigs inoculated with stock cultures, milk and guinea pig material at various periods after inoculations. Huddleson 34 , in his recent study on comparative pathogenicity of several strains of Bacterium abortum, used twenty-one strains on one hundred and twelve guinea pigs. From his work, one can conclude in a general way that older cultures lose their virulence for guinea pigs. In fact, he states: "A majority of strains isolated became non-virulent within one year after isolation. ' ' A study of his results indicates that there appears to be a difference of the individual strain irrespective of time of its isolation. For instance, his strains 20 and 805 were both isolated in May, 1919 ; the former uniformly produced lesions while the latter was uniformly negative. His strain 16 yielded no-lesion guinea pigs in January, 1920 and August, 1921, but was positive April, 1921. One swine culture failed to produce lesions in guinea pigs, while another strain from Illinois yielded positive and negative guinea pigs. OBSERVATIONS UPON FOETY ABORTED FETUSES RECEIVED AT BERKELEY Agglutination tests of blood sera from cattle in a number of herds in various parts of California indicated that abortion was in a large percentage of cases due to Bacterium abortum infection ; never- theless it was deemed advisable to make a bacteriological study of aborted fetuses from dairies within a radius of one hundred miles of Berkeley. In general, the procedure consisted in making three cultures from the stomach contents on cooked blood agar, fetus liver agar, 2 per cent glycerol and 1 per cent glucose agar, respectively, three on the same kind of media from the lungs and one from the meconium, 310 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION and placing these under C0 2 conditions, as suggested by Huddleson. 35 Plates were streaked with stomach contents and meconium from the rectum and kept under ordinary atmospheric conditions in the incu- bator. Figure 2* illustrates the method used in our laboratory to obtain the recommended C0 2 conditions. Fifteen per cent of the air is drawn off by suction and replaced by 10 per cent of carbon dioxide from the tank. It was found necessary to remove 5 per cent more air than was replaced by gas to allow for expansion when placed in the incubator. 9 ~1 -rSfr '■ iff i m i '■■■■... % HI I r lr* HL laws. Fig. 2. — Illustrating Method Used in Growing Bacterium abortum Under C0 2 . The Pressure in the desiccator is reduced by suction until manom- eter reads 144 mm. (marked 15%). C0 2 is allowed to flow in from cylinder until pressure reads 38 mm. (marked 5%). Upon heating to 37° C. in incubator, the pressure will again become approximately atmospheric. Besides inoculating the cultures, one guinea pig was inocu- lated intraperitoneally with stomach contents and another with lung extract. Hanging drop preparations were made at the time of autopsy, with a special view to determining the presence or absence of spirilla. Smears were made from the stomach contents and lung in duplicate and were stained with Gram and other methods as it was thought desirable. Table 1 contains the result of such examina- tion upon forty fetuses made by Hart and the writer. *The modification of the Huddleson method, as illustrated in Figure 2, was suggested by H. Goss of the Nutrition Division. The manometer was also made by him. Bulletin 353] BOVINE INFECTIOUS ABORTION 311 05 •Is t_l o ~ * 1 m © Q 1« 7 -o .2 S r Z .9 ■a-8 + o o 33 - 1 a) '.11 ©"-^ © § ^ § £523 3^ to _ C ■S.2| ■■§•§§; i ill 1 I 03 -, si -9 ! 0(3 _,0Q<5 s © + Evidence and history in- dicate cervix open several days before expulsion + T3 3 3 O + + o o3 If 02 i-l 1 1 o c3 If GQ h3 + 1 o o3 a g> GO h^ 1 1 o o3 a g 5 o S + + 03 03 a g> 8 o la O o o3 a o 03 s •+3 o 03 73 03 a o 03 o3 a o 03 o3 a o 525 t-J + + 1 § T3 1 s i i i + + + 1 1 1 1 1 1 o I o + — Contaminated — Gram negative acid producing rod + 1 1 a o o 1 1 1 I 1 1 © "8 S •a © a a 6 i-O 6 6 o W o t-l 03 bC CI 0) OQ OQ 0) 4d p 03 bfi d 03 03 2 M o u H T3 © > "8 © © ea Q o OS 03 Q CO si 03 Q 03 Q os d o3 1-5 6 T— 1 * i + .a>a 1 1 o o3 a g> + + O o3 o 5 GO h-5 1 1 o o3 If 1 1 o If + + C3 o3 1 1 o a -^ "5 SB Ig o o 2 03 o3 a O 5 03 a o 03 o3 a o 03 o3 a O + M c i i i 1 1 O 1 1 1 1 1 1 Pyogenes colonies 1 + + ,0 o 03 S O — Few Staphy- lococci colonies — Colon, Diphther- oid, and _ coccus 1 1 1 Pyogenes colonies + + + 03 S 11 a a < 6 g -t-5 c3 2 6 00 6 6 o W Si 03 bfi CI 03 03 M O S-l U Date Received d o3 »-5 CO "3 o '3 5 O O + 1 + 1 1 1 a t-1 + + + + + + + + 1 + and unidentified _ organism + 1 1 1 1 1 ,4 o a o + + + + 1 1 + + 1 1 1 1 + 1 1 1 1 1 i •a cp a <1 6 6 6 00 6 d 00 3 * 1 s-l H 02 02 F-| fa o u S-i o Pi m o Pi H -a a; o Q OS OS i—l fa OS CO fa Oi SO fa CI fa 05 oT fa . 6 i-H o a 1 1 O 03 || to O 3 S •S = o w O CD 1 + Staphyloco ecus aureus + 1 CO CO IS "^ 1 1 1 -f- and con- taminating colony + and con- taminating colony — mold — con- taminated — con- taminated + + + 1 1 1 ^3 o 03 £ O 1 1 1 + and 4 con- taminating colonies 1 1 1 + + + 1 1 1 0> ~o3 £ a o3 . ^ O a ^ < T3 03 ~o3 pq 6 6 6 CO o ■P o w M o a CO co o> o a o a u H CO CO e X > o 0) 03 Q OS o3 3 OS © OS 3 OS i— 1 03 3 OS of o3 6 53 i> 00 OS O (N Bulletin 353] BOVINE INFECTIOUS ABORTION 315 • | CD t o '43 o CP CP a & bC , ft ' Ph 1 1 + 1 a a 1 1 o 03 o S 1 1 o 03 3 g> o S CO !-} Stomach — Lung — Placenta — + + o 03 3 g> CO H 1 1 o 03 3 g> CO h-} en O O o CD a; 1 1 1 1 1 (3 — B. pyogenes — B. pyogenes 1 1 1 1 1 1 + + + 1 1 1 o s o QQ — B. pyogenes — B. pyogenes 1 1 1 — unidentified — unidentified — unidentified + + 1 — Unidentified organism — Unidentified organism — Unidentified organism a; s a a 73 03 ft JO >> > £h CD =3 73 6 GO 6 6 3 GO CP Fh H-3 03 03 £5 o CO co CP Fh ft o H CO co CP Fh ft o Fh co CO CP Fh ft X m T5 CD > '3 o CD d Q CM CM OS CM Fh 03 3 CM CM OS CM Fh 03 CM (M o" CO Fh 03 CM CM C3i co" a > 73 ca pq a 1 O N ft 03 a * O 0) o 2 03 03 ^ ft £ a PQ M Cultures made from lung. Abdominal viscera not available. i 1 .5? 8 3 o 1^ ' o3 r~j 3 C b£ Sac 3 << 3 B h5 cm | be 2 ft 1 + a -p 3 || 3 ^ ^ Ph i i r3 o o3 If c72 i-Q 73 CM 1 03 C35 73 «H a * 3 CO (-< o Is ■a S O o 1 1 1 1 1 bfi a a Hi a a a 2 2 2 bC bJO bO a a a 3 £ £ 'o "o *o QUO 1 1 1 "S > 3 cq - O K ph ■*-" b£ 3 Sh C3 ffi bJD > U o o "3 03 2 1 13 o 3 O CD > o 1 1 1 1 1 1 CD A » ft ~ 5 -a I o a || o a O QQ ft ft ft 3 3 3 . O O O H H H bD bC bX) 3 3 3 'o 13 'o o a o 1 1 1 •CD 73 o3 CD 3 O 1 1 1 73 3 c3 A. 3 ^ ,2 o o J3 1 -H ft . s m § 3 1 osj 1 1 s ■a * a p. < H d o -p 6 6 00 S is o CO 02 03 ft 03 03 2 ft 03 03 03 pH ft X pH 03 bfl 3 03 03 03 CD 03 03 03 PH ft Date Received CM CM "C ft < CM > 6 00 CM 9 o CO CO Bulletin 353] BOVINE INFECTIOUS ABORTION 317 DO a Portion of lung filled with air + 1 + Lungs showed expansion with air Normal except there was no union of bones or joints in any portion of body Guinea Pig Inoculations 1 + o 03 + + o o3 o 9 Stomach — Lung — Placenta — 1 + o o3 02 ^ 1 1 ■si 2 2 s a 00 Sg •a J 1 + 1 1 + + + + — Contami- nated 1 1 1 S3 O bfl CP > O + 1 1 — Staphylo- cocci and colon like J3 w cj a O CO — Colon and mold — Colon and mold — Mold -a o3 1 03 d o O + 1 1 1 1 — Staphylococci — albus B. coli and other irrident — Staphylo- cocci and colon like 0) -^> c3 s •a ® a a 6 d 6 o 6 00 d oo o 44 o 9 ■ Eh H 0) bfi 9 co co a; CO E o 9 H H CO T3 > '5 o Q CN CN Oi CO*" rH § CN CN OS © CN o? 3 CN CN <35 CN o3 3 CN <35 lO CN >> c3 CN a> d 9 •-5 6 CN CO CO CO CO CO CO 318 UNIVERSITY OF CALIFORNIA EXPERIMENT STATION GO s Lungs showed evidence of having been filled with air + + 1 + 3 O + + o c3 I! CO r-3 + + o C3 S bC 8 § CO h-3 1 1 o c3 s a CO.h-3 + + o c3 CO t-3 09 "9 u Is ■a J o o + 1 1 1 bfi C S3 + + 1 + and con- taminating organism + — and 3 con- taminating organisms 1 1 1 + + 1 q a O — Staphylococci and colon + Staphylococci and colon + and 2 con- taminating organisms + *d co * g a o O o 1 1 1 + + + 4) 03 s 11 a a S d 00 6 00 6 How H CO Oh CO co a o Date Received