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Recent studies on epizootic bovine abortion
THERIOGENOLOGY RECENT STUDIES ON EPIZOOTIC BOVINE ABORTION D.G. McKercher, J.H. Theis, E.M. Wada, E.C. Loomis, V. Bolton, E H. Ito
After many years of intensive study, concepts concerning epizootic bovine abortion (EBA) are undergoing fundamental changes. The demonstration that the Ornithodoros coriaceus tick is a natural vector of the infection led to the isolation of a virus which, although not an abortifacient agent per se, appears to be associated in 0. cori-Y aceus ticks with an agent which causes what is believed to be EBA. The nature of this agent is as yet unknown. However, such findings provide increasing evidence that an agent, or agents, in addition to the chlamydia which was isolated during the early studies of the disease, are involved etiologically in EBA. Indications are, therefore, that EBA is a disease syndrome, rather than a specific disease entity caused by a single etiologic agent.
From the Department of Veterinary Microbiology, School of Veterinary Medicine (McKercher, Wada and Ito): the Department of Medical Microbiology, School of Medicine (Theis and Bolton); and the Department of Entomology, College of Agriculture and Environmental Sciences (Loomis), University of California, Davis, California 95616. Supported by U.S. Department of Agriculture, Agricultural Research Services Contract 12-14-100-9084 (45), and LDR Epizootic Bovine Abortion Fund 3-447256-19900. The authors gratefully acknowledge the kind cooperation of Dr. B. Osburn, Department of Pathology, School of Veterinary Medicine, for providing the histopathologic data. Introduction There is evidence that epizootic bovine abortion (EBA) has existed in California for many years (11, but it was not until the early 1950's that serious attempts were made to characterize the condition and to discover its cause. During this latter period, field observations indicated that the disease might be vector transmitted, with ticks being the most likely suspects. Subsequently, a chlamydial agent was isolated from aborted bovine fetuses having lesions characteristic of EBA (2). Since the agent produced abortion on inoculation it was believed,on this basis, to be the cause of the disease. However, since chlamydiae were not known at that time to be vector transmitted or associated with ticks, the vector concept was largely discounted. Later, a chlamydial agent was isolated from several species of tick and from assorted rodents indigenous to foothill areas (3). These findings revived the vector concept and gave rise to the existence of a
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hypothetical vector-reservoir cycle in foothill areas in which the pregnant cow became infected as an interloper host. Evidence for such a biologic interplay was strengthened by the demonstration that the bovine chlamydial isolate from an aborted fetus, when inoculated intradermally to simulate tick exposure, caused cattle to abort (4). In view of this evidence, a study was undertaken with the object of verifying involvement of the chlamydial agent as the cause of EBA and identifying additional agents that might be implicated etiologitally. The study was carried out on a herd of beef cattle on a ranch in a typical Sierra foothill area. The procedure entailed the collection of a representative cross section of the arthropod and rodent populations of the ranch, from which attempts were made to isolate chlamydiae or other agents by culture in chicken embryos. Relatively large numbers of rodents (Neotoma, Peromyscus, Microtus, Citellus, Perognathus), and arthropods (Dermacentor, Culicoides,Chrysops, Trombiculi, Simulids, fleas, deer keels,lice, and some ectoparasites from rabbits) were cultured in addition to tissues from several Scleroporus lizards and two deer. The findings were negative throughout. It was then decided to abandon this approach as being too unselective. Attempts to recover agents from fetuses aborted as a result of infection diagnosed clinically as EBA had been continued from the time the disease was first studied on an intensive basis. However, with the exception of two chlamydial isolates recovered in the late 1950's (2) no further isolations of any agent were made. The consistent failure to do so was attributed to the long postinfection interval, during which the agent disappeared from the fetal tissues, or remained in such low concentration as to be essentially impossible to isolate. To overcome this difficulty, it was decided to remove the fetuses from pregnant heifers after they returned from grazing in EBA enzootic areas in the hope of obtaining some in which active infection was in progress. The following three experiments were based largely on this approach A local beef herd which ranged in an EBA-enzootic area, and in which EBA was an annual problem, was selected for the initial study. On return of the herd from the range pastures in late June or early July, which almost always preceded the abortion outbreak, 10 pregnant heifers were obtained. Since the abortion rate varied from 10% to 30% or more each year, this number was considered minimal in order to ensure that at least one or two of the heifers would be carrying an infected fetus. One heifer was killed at each of 10 successive weekly intervals and the fetus removed. Tissues were selected for culture in cell monolayers, in explant culture, and in chicken embryos. The results were disappointing in that no gross evidence of fetal pathology was observed in any fetus, with the exception of one which was aborted several days before it was scheduled for study. While it had gross lesions considered indicative of EBA infection, no agent was
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isolated from its tissues, or from any of the other nine fetuses whose tissues were also cultured. Histopathologic findings were inconclusive in the aborted fetus, and several of the grossly normal fetuses, selected at random for detailed histopathologic study, were negative for tissue changes. In view of the possibility that infection occurred shortly after range exposure, the approach was modified the following year in that individual heifers were removed at intervals and the fetuses were processed. From 15 to 20 heifers, determined by palpation to be pregnant, were eartagged and bled prior to their transfer to the range. After 3 to 4 weeks, by which time tick exposure was assumed to have occurred, one animal was removed each week, and the fetus was cultured as in the previous year. In addition, after the specimens had been passaged three times in cell culture and chicken embryos, and grown out in explant cultures, these cultures were inoculated into pregnant heifers to determine whether an agent which might have escaped detection was present. Again the results were disappointing. One fetus had lesions indicative of EBA infection. However, no agent was recovered from this fetus, or from the others, by serial blind passage of their tissues in the culture systems used. Also, the pregnant heifers into which 3rd passage chicken embryo and cell-cultured materials were inoculated, delivered normal calves at term. Shortly after completion of this study, it was demonstrated that Ornithodoros coriaceus ticks transmitted an agent,while feeding, which caused pregnant heifers to abort (5). The fetuses had lesions much like those described for EBA. It was, therefore, now possible to identify infected ticks by feeding them on pregnant heifers, and to use such ticks as a natural means of infecting experimental cattle. A study was then made using this method of infection. Although it did not resolve the question of the etiology of EBA, it provided some rather interesting information concerning an additional agent and, for that reason, the study is described in some detail in the present work. Materials and Methods Experimental Cattle - Beef breed heifers which had been reared in a tick-free area were used in this study. Tick Exposure - After breeding, and just prior to transfer of the heifer herd to the spring grazing range in an EBA-enzootic area, a number of heifers in excess of that needed for the experiment, were pregnancy tested and separated from the remainder of the herd. They were maintained in a tick-free area until used in the experiment. From 1 to 3-112 months after the estimated day of conception, 12 of the heifers were exposed experimentally to 0. coriaceus ticks. The ticks were collected from four geographic lo&ions in California and had been demonstrated to be capable of inducing abortion in pregnant heifers by prior feediwexposure. Each geographically
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representative group of ticks was so divided among the total number of heifers to be exposed that each of the latter was fed upon by many ticks from each of the four geographic areas in which they were collected. For exposure, a special harness was placed over a shaved area of the back of each heifer while she was restrained in a squeeze chute (5). The ticks were added to cups in four separate batches and observed while they fed to ensure that exposure had occurred. The number applied per heifer ranged from 24 to 113. When the engorged ticks detached, they were returned to the appropriate container, thus making it possible to keep those fed on any one heifer separate from the others, and to preserve their geographic identity for further reference. At intervals following tick exposure, seven of the heifers were slaughtered, and their fetuses removed and studied. Chicken Embryo Culture Techniques - Tissues obtained for culture from each fetus were placenta, pooled lung and spleen, pooled liver and kidney, and a miscellaneous pool containing brain, lymph node, muscle, and thymus gland. The supernatant (s/n) portions of 50% suspensions (a) of the four preparations in a kanamycin-vancomycin-streptomycin (KVS) solution, were diluted 10-l, and 10m2 in the same diluent for inoculation. Two-tenth ml amounts of each diluted preparation were inoculated via the yolk sac route into a series of not less than 8 to 10, 6- or 'I-day-oldchicken embryos. Embryos which died within 48 hours after inoculation were discarded. Those which died subsequently, and those surviving by the 8th day, were harvested and portions of yolk sac and body tissues of each were pooled. For the initial subpassage, the placenta and pooled lung and spleen passed materials, were combined, as were the remaining two, i.e. the pooled liver and kidney, and the miscellaneous tissue pool, and diluted as for the original inoculums. Subsequent tissue harvestings were made as described. For the 2nd subpassage a single pool was prepared, diluted 10d3 and passaged as indicated above. If no evidence of infection appeared in this passage, the original fetal tissues were considered to have been negative for an agent, and further culture was discontinued. Cell Culture Techniques - Individual 50% suspensions of liver, kidney, spleen, lung, brain, bone marrow, lymph node, thymus gland, and placenta from each fetus were prepared in minimal essential medium (MEM). The s/n fraction of each, obtained by centrifugation at 1500 rpm for 15 minutes, was diluted 1:lO and 1:50. Each preparation was inoculated, in a volume of 0.05 ml, to bovine embryonic lung (BEL) cell~~?nolayers, at passage levels from five to nine, in microplate wells. From 8 to 12 microcultures were inoculated titer (MT) per specimen. After holding for 60(;fn at room temperature (23 C) for containing 10% by volume of adsorption to occur, 0.05 ml of MEM fetal calf serum (FCS), was added to each inoculated monolayer. (a)
Consisting of 1 gm of each, streptomycin, vancomycin and kanamycin per liter of phosphate buffered saline (Dulbecco formula)
(b) Cooke Engineering Company, Alexandria, VA (c) Grand Island Biological Company, Santa Clara, CA
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The latter were incubated at 37 C in an atmosphere containing 5% by volume of carbon dioxide, and were observed daily for evidence of cytopathic (CP) changes through the 7th to 10th day postinoculation. For subpassage,the monolayer cultures of each specimen were frozen and thawed once and pooled. After light centrifugation,the s/n fractionswere diluted and inoculatedas described for the original passage. White blood cell inoculumswere obtained by centrifugingwhole blood, drawn in an anticoagulant,at 1500 rpm for 15 minutes, and removing the buffy coat layer. The latter was alternatelyfrozen and thawed several times, and prepared for inoculationas for the above tissue specimens. (d) from Explant cultures were grown out in 30 ml plastic bottles fragments of the organs listed above, in MEM containing 10% by volume of FCS. The cultures were incubated 7 to 10 days as already described, by which time complete monolayers had formed. Subpassagewas made in plastic bottles at 10 to 12 day intervals by trypsinizingthe monolayers and suspending the cells in MEM containing 5% FCS. Results The most significant finding was that although two of the five tick-exposedcontrol heifers gave birth to normal calves at term, two delivered premature calves and one aborted. Lesions were present in all three, although they were considered to be relatively nonspecific. Unfortunately,only one term and one prematurely delivered calf were cultured, in addition to the aborted fetus (No. 4). Both calves were negative for an agent. However, changes of a suspiciousnature were observed in cell monolayers inoculatedwith tissues of the aborted fetus. As these disappearedby the 3rd passage, they were considered to be artifacts. Nonetheless,the fact that three of the five heifers either delivered prematurely or aborted, supports findings made in an earlier study (6) to the effect that 0. coriaceus ticks harbor an abortifacientagent or agents for catFle. Whether any of the heifers from which the fetuses were removed at slaughter would have aborted or delivered a premature calf is not known. Only one of these fetuses (No. 8) had evidence of infection and a virus which had been isolated from ticks in earlier studies, but thus far found to be nonabortifacient (61, was recovered from it. The other heifers contained apparently normal fetuses at slaughter in which no pathologic changes were evident, and from which no isolationswere made. The fact that control heifer (No. 4) which aborted was estimated to have conceived only 25 days prior to tick exposure might be of considerablesignificance. This exposure was at least 63 days earlier in gestation than were any of the other exposures of the pregnant heifers in the study. The possible reason for the absence of lesions, or for failure to recover an agent from the heifers whose pregnancies
(d) Corning Glass Works, Corning, NY AUGUST-SEPTEMBER
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were interrupted, is that exposure occurred too late in gestation to infect all but this one fetus. However, this hypothesis is not compatible with findings in the other two pregnant heifers (Nos. 89 and 91) in the control group which, although they were estimated to be 122 and 140 days, respectively, into gestation when tick exposed, they delivered weak, premature calves which were, presumably, infected earlier. The results are summarized in Table 1. Discussion Contradictory findings such as the above have characterized studies of EBA from the beginning. Despite the long years devoted to investigations of this difficult disease problem, it still remains largely an enigma. Although progress has been slow, it must be recognized that, because of its very nature, abortion is an extremely difficult problem to study. The act of abortion -per se is only the final in a series of events which takes place over a prolonged period under conditions which make monitoring all but impossible. During this lengthy interval, a number of extraneous factors may intervene, resulting in misinterpretations of findings and faulty conclusions. To control a disease on a rational basis, the cause must be known. Thus far, the only agent isolated from fetuses aborted as a result of infection diagnosed clinically as EBA is a chlamydial agent. Initially it was believed to be the sole cause of the condition, as it caused abortion on experimental inoculation, consistent with the fact that chlamydiae cause abortion in other ruminant species. In view of the urgency of the EBA problem and the economic benefits which would be derived by early control of the disease, an inactivated vaccine, prepared according to the method used for the ovine chlamydial abortion vaccine (7), was tested in heifers by experimental challenge. Several attempts failed to demonstrate any protective effect by the vaccine (8). Moreover, virgin heifers which had been inoculated with the viable chlamydial agent aborted when challenge inoculated during the ensuing pregnancy. The same results were obtained when the immunity of cows which had aborted from the naturally occurring disease was challenged during the subsequent gestation. This apparent lack of immunity to experimental challenge contrasted sharply with field reports to the effect that an immunity to the natural infection develops as a result of a single abortion. It was felt that this discrepancy might be due to the method of experimental immunity challenge used, rather than to the apparent inability of the agent to immunize under experimental conditions. It was recognized that if natural exposure could be simulated and used as immunity challenge under experimental conditions, the discrepancy might be resolved. Therefore, since the agent was believed to be vector transmitted, the immunity of vaccinated cattle was challenged by intradermal inoculation to simulate vector exposure.
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TJSERIOGENOLOGY This approach proved highly successful in that in several experiments none of the vaccinated cattle aborted. In contrast, practically 100% of control animals either did, or delivered calves prematurely (9). These results appeared to reconcile the discrepancy between failure to demonstrate, by experimental challenge, immunity to EBA in vaccinated cattle, and evidence for immunity from field exposure. The explanation appears to lie in the fact that, as is the case with other chlamydial infections, the immunity associated with chlamydial abortion is of the cell-mediated type. Briefly, this type of immune response functions as follows.* Lymphocytes, sensitized to an antigen as a consequence of either natural infection or vaccination, become activated on subsequent re-exposure to the same antigen and respond by elaborating lymphokinases which possess various activities. One lymphokinase (chemotaxin) attracts phagocytic cells to the location of the antigen. Another enhances their phagocytic capabilities, while a 3rd (migration-inhibiting factor - MIF) retains them in intimate contact with the antigen. As a result, the latter is phagocytized and disposed of. In view of its cell-mediated nature, immunity to chlamydial abortion is believed to ensue when cattle become sensitized to the agent as a result of natural infection acquired, apparently, by vector transmission. On re-exposure the following year, the agent is again deposited into the skin by the vector, where it is retained temporarily because of the density of the surrounding tissues. Before the agent can replicate and escape from these foci into which it is implanted, the sensitized lymphocytes have elaborated the various lymphokinases which mobilize and retain phagocytic cells at the sites where the antigen is present, and endow them with marked ingestive properties. Thus, the agent is disposed of before it can become dispersed in the tissues and reach the blood stream. Essentially, the same response was elicited in the vaccinated heifers when the challenge inoculum was deposited in the skin by intradermal inoculation. However, in early studies of immunity to EBA, the challenge agent was inoculated through the skin of the vaccinated cattle, either by subcutaneous or intramuscular inoculation (8). As a result, it spread quickly from the inoculation sites before the phagocytic cells had become fully mobilized. Once it was widely dispersed in the body, the phagocytes were incapable of coping with it, and blood stream invasion with ensuing abortion, occurred as a consequence. It has been observed that when cattle are maintained in an EBAenzootic area, they abort only once from the infection and then become resistant to such abortions in subsequent years. However, if transferred to a non-enzootic area they apparently lose this immunity, as they again abort on return to the enzootic area. Such behavior is compatible with that of a cellular-type immunity, which is characteristic of chlamydial infection, in that constant antigenic restimulation is required to maintain resistance at a protective level. Therefore, further evidence for involvement of a chlamydial agent in
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the etiology of EBA is provided. Having demonstrated that the vaccine was protective under experimental conditions (4,9), it was tested in cattle in an area where EBA was reported to occur. Unfortunately, only several herds with a limited number of cattle in each were available, and conditions were such that it was not possible to monitor the animals closely. In any event, it was reported that, insofar as could be determined, there was no evidence of a reduced abortion rate among the vaccinees as compared with the control animals. Assuming that this were a valid observation, and that exposure to the chlamydial abortion agent had occurred, two possible explanations could account for these findings. As chlamydiae have been isolated from both 0. coriaceus and Dermacentor occidentalis ticks, exposure to these tic?& could have resulted -either of two outcomes. Dermacentor occidentalis ticks are shallow feeders, the penetration not exceeding that of intradermal injection. Experimentally, it was found that vaccinated heifers were refractory to challenge exposure given via this route (4,9). Therefore, had these ticks been responsible for the field exposure, which obviously took place, abortion would not have been expected to occur. However, if exposure were due to 0. coriaceus ticks, abortion most likely would have ensued, as these-ticks are deep feeders and the agent would have been deposited in the subcutaneous tissues of the exposed animals. As already indicated, in earlier studies, when cattle were challenge inoculated by either subcutaneous or intramuscular injection, abortion ensued on a regular basis (8). The reason for this appears to be that the skin, in which the cellmediated immune response functions most effectively, particularly in the case of vector transmitted infections, was circumvented. Therefore, the possibility that the abortions which occurred in the field trial herds in the present study were due to chlamydiae cannot be ruled out. This is particularly relevant, in view of the fact that chlamydiae have been demonstrated to cause abortion in other ruminants. Also, it has been demonstrated by simulated vector exposure to the bovine chlamydial agent that EBA is most likely an arthropod-transmitted infection (4). Moreover, the fact that ticks collected from foothill areas harbored chlamydiae indicates not only that such agents exist in areas inhabited by cattle, but that a means of transporting them to cattle is available (3). Finally, the nature of the immune response of cattle to infection with the chlamydial abortion agent is compatible with that of vector-type exposure to an agent which evokes a cellmediated immune response. On the other hand, it is highly unrealistic to believe that undiscovered causes of bovine abortion still do not exist, and particularly in foothill areas which abound with arthropods and rodents, but where few epizootiologic studies have been carried out. In fact, evidence to this effect became apparent during studies of a virus which was isolated recently from members of the O.coriaceus species collected in an EBA epizootic area (6). This virus, as yet unidentified, appears to be unrelated
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etiologically to abortion. However, pregnant heifers exposed to 0. coriaceus ticks which did not harbor this virus delivered weak calves from 21 to 30 days prematurely which, at necropsy, were found to possess lesions suggestive of EBA infection. In view of these findings, it appears that the ticks must have harbored a still unknown abortifacient agent. It is entirely possible that this agent is a virus. However, in attempts to isolate agents from ticks and aborted fetuses, the inoculums were heavily treated with antibiotics to control nonviral contaminants, and culture systems designed for viral isolation were utilized. Under such circumstances rickettsial or chlamydial agents would not be recovered. It is possible, therefore, that the suspected agent might well be either of these. Nonetheless, regardless of its identity, these findings provide growing evidence that a number of agents are involved etiologically in the EBA syndrome, of which one is a chlamydia. The extent to which the latter is involved remains uncertain. In the case of most infectious agents this would be relatively easy to determine by serologic means. Unfortunately, because of a common complement fixing group antigen, which is shared essentially by all mammalian chlamydiae, it is not possible to identify the specific chlamydia responsible in each case for the diverse infections which these agents cause. Cattle are prone to several including conjunctivitis, arthritis, and pneumonitis, in addition to abortion. Consequently, most, if not all cattle, possess complement-fixing chlamydial antibody. While the persistence of such antibody is generally of short duration, it prevails in cattle because of an asymptomatic enteric chlamydial infection which maintains a constant antigenic stimulation (10). Unfortunately, the only practical procedure for detecting chlamydial antibody is the complement-fixation test. Therefore, until such time as additional procedures are devised to identify specific chlamydiae, serology will play little or no role in either epidemiologic or diagnostic studies of infections caused by these agents. Isolation of the chlamydial agent from aborted bovine fetuses as a means of diagnosis and for monitoring vaccine field trials is unsatisfactory. The disease is contracted on foothill pastures where many of the abortions take place. The inaccessibility of the animals in these extensive primitive areas renders it largely impossible to obtain the fetuses. However, this is probably of little practical importance because, under even the most favorable experimental conditions, it is extremely difficult to recover the agent from aborted fetuses. The reason appears to be that the agent fails to survive the long period between infection of the dam and expulsion of the fetus. In fact, lesions present in aborted fetuses indicate that the fetal infection usually occurs long in advance of the abortion. In the case of such chlamydial infections as pneumonitis, conjunctivitis, or even arthritis, in which clinical evidence of disease is apparent in the host, and access to materials for culture is available, isolation can be accomplished fairly consistently. In the case of abortion, however, replication of the agent occurs mainly in the placenta and in the fetus itself, without any clinical evidence being displayed
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THERIOGENOLOGY by the infected cow. It appears, too, that the agent is not shed into the general circulation of the latter; hence no opportunity for its isolation from this source is provided. The fact the chlamydiae are recovered much more readily from aborted ovine and caprine fetuses might reflect the shorter gestation period characteristic of these species, which enables the agent to survive until abortion occurs. The unavailability of aborted fetuses in a satisfactorycondition is a severe handicap insofar as histopathologic studies are concerned. While several gross lesions have been associated with naturally occurring EBA, most are of a nonspecific nature. Generally speaking, in our experience at least , gross lesions are absent in most fetuses diagnosed as having been aborted as a result of EBA. Thus, lack of serologic procedures, the circumstances which mitigate against isolation of the agent from fetuses, and the unreliability of either clinical or pathological diagnoses represent major handicaps in dealing with chlamydial abortion. There are two possible approaches to defining more precisely the extent to which the chlamydial agent is involved etiologically in the EBA syndrome. One is to conduct field trials in EBA enzootic areas, on at least 5000 vaccinees and an equal number of control cattle, and to make a statistical analysis of the numbers of calves produced by each group. This would indicate the total percentage of abortions in the herds under study and the proportion due to chlamydial abortion. However, such an undertaking would be costly and would involve a highly coordinated team effort on the part of various specialist groups over a period of months. A much more limited approach, both cost-wise and administratively, would entail the exposure of equal numbers of vaccinated and unvaccinated (control) pregnant heifers maintained under experimental conditions, to O.coriaceus ticks collected in EBA enzootic areas and determined to be capable of inducing abortion in cattle. Either study would also indicate whether agents in addition to the chlamydia are involved etiologically in the EBA syndrome, and whether the proportion of abortions due to the latter is sufficiently high to warrant the adoption of preventive measures. References 1.
Traum, J., and Hart, G.H. Bovine Infectious Abortion. III. Manifestation of Abortion Disease without Demonstrable Etiological Factor. Univ. Calif. Agr. Expt. Sta. Bull., -353:327-346 (1923).
2.
Storz, J., and McKercher, D.G. Etiological Studies on Epizootic Bovine Abortion. Zentbl. f. Vet-Med., -9: Heft 4, 411-427; Heft 5, 520-541 (1962).
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3.
Eddie, B., Radovsky, F.J., Stiller, D., and Kumada, N. Psittacosis-L~phogranuloma-Venereum (PL) Agents (Bedsonia, Chlamydia) in Ticks, Fleas, and Native Animals in California. Am. J. Epidemiol., 90:449-460 (1969).
4.
McKercher, D.G., Crenshaw, G.L., Theis, J.H., Wada, E.M., and Mauris, C.M. Experimentally Induced Immunity to Chlamydial Abortion of Cattle. J. Inf. Dis., 128:231-234 (1973).
5.
Schmidtmann, E.T., Bushnell, R.B., Loomis, E.C., Oliver, N.M., and Theis, J.H. Experimental and Epizootiologic Evidence Associating Ornithodoros coriaceus Koch (Atari-Argasidae) with Exposure of Cattle to Epizootic Bovine Abortion in California. J. Med. Ent. In Press (1976).
6.
Wada, E.M., McKercher, D.G., Castrucci, G., and Theis, J.H. Preliminary Characterization and Pathogenicity Studies of a Virus Isolated from Ticks (Ornithodoros coriaceus) and from Tick-Exposed Cattle. Am. J. Vet. Res. In Press, (1976).
7.
McEwen, A.D., Dow, J.B., and Anderson, R.D. Enzootic Abortion in Ewes. An Adjuvant Vaccine Prepared from Eggs. Vet. Rec., -67: 393-394 (1955).
8.
McKercher, D.G., Robinson, E.A., Wada, E.M., Saito,J.K., and Franti, C.E. Vaccination of Cattle Against Epizootic Bovine Abortion. Cornell Vet. -59:211-226 (1969).
9.
McKercher, D.G., Crenshaw, G.L., Franti, C.E. Vaccination Against Abortion. J. Am. Vet. Med. Ass.,
10.
Wada, E.M., Mauris, C.M., and Epizootic Bovine ~~hlamydia~) 163:889-891 (1973). -
York, C.J., and Baker, J.A. A New Member of the PsittacosisL~phogranu~oma Group of Viruses that Causes Infection in Calves. J. Exp. Med, -93:587-604 (1951).
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109
109
94
88
19
26
43
15
25
122
140
4
ES
91
97
-__
107
48
Normal Control
112
41
Tick Exposure Controls
88
109
31
--..
24
___
---
81
-__
---
89
82
37
68
111
..--
61
113
---
54
83
84
47
102
40
87
112
Term Delivery (274 ddysf
Weak Calf (257 days)
Neg Neg
__-
Weak Calf (249 days)
nd
Questionable Myocarditis, thymic atrophy, lytnphoid hyperplasia, focal hepatic necrosis
-__
Abortion (130 days)
Petechiaein ocular and nasal mucosa
Term Delivery(269 days) Neg
nd Edema, liver lesions
-...-
Neg
___
r-_--
Term Delivery(266 days)
__-
nd
_--
Neg
Neg
Neg
*eg
Neg
__-
Neg
_--
Neg
Neg
Neg
-_-
Neg
Neg
Neg
Neg
_--
Neg
Neg
---
Virus
Lymphoid hyperplasia Neg
Neg
Neg
Agent PregnancyTermindtrd Isolated By
Histopathologic Fetal Lesions
Agent f‘~wi Findingsmade in Attemptsto Isolatean Abortif,icirnt the Fetusesof Tick (Ornithodoroscoriaceus)I::<[.~::,w*l Hriit:rs
Tick Exposure No. of Killed E Fetus Gross Fetal (Days PostTicks Removed (Day Lesions Conception) Applied Post-Tick Exposure Neg 93 34 109
14
a
Heifer No.
TABLE I
After many years of intensive study, concepts concerning epizootic bovine abortion (EBA) are undergoing fundamental changes. The demonstration that the Ornithodoros coriaceus tick is a natural vector of the infection led to the isolation of a virus which, although not an abortifacient agent per se, appears to be associated in 0. cori-Y aceus ticks with an agent which causes what is believed to be EBA. The nature of this agent is as yet unknown. However, such findings provide increasing evidence that an agent, or agents, in addition to the chlamydia which was isolated during the early studies of the disease, are involved etiologically in EBA. Indications are, therefore, that EBA is a disease syndrome, rather than a specific disease entity caused by a single etiologic agent.
From the Department of Veterinary Microbiology, School of Veterinary Medicine (McKercher, Wada and Ito): the Department of Medical Microbiology, School of Medicine (Theis and Bolton); and the Department of Entomology, College of Agriculture and Environmental Sciences (Loomis), University of California, Davis, California 95616. Supported by U.S. Department of Agriculture, Agricultural Research Services Contract 12-14-100-9084 (45), and LDR Epizootic Bovine Abortion Fund 3-447256-19900. The authors gratefully acknowledge the kind cooperation of Dr. B. Osburn, Department of Pathology, School of Veterinary Medicine, for providing the histopathologic data. Introduction There is evidence that epizootic bovine abortion (EBA) has existed in California for many years (11, but it was not until the early 1950's that serious attempts were made to characterize the condition and to discover its cause. During this latter period, field observations indicated that the disease might be vector transmitted, with ticks being the most likely suspects. Subsequently, a chlamydial agent was isolated from aborted bovine fetuses having lesions characteristic of EBA (2). Since the agent produced abortion on inoculation it was believed,on this basis, to be the cause of the disease. However, since chlamydiae were not known at that time to be vector transmitted or associated with ticks, the vector concept was largely discounted. Later, a chlamydial agent was isolated from several species of tick and from assorted rodents indigenous to foothill areas (3). These findings revived the vector concept and gave rise to the existence of a
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hypothetical vector-reservoir cycle in foothill areas in which the pregnant cow became infected as an interloper host. Evidence for such a biologic interplay was strengthened by the demonstration that the bovine chlamydial isolate from an aborted fetus, when inoculated intradermally to simulate tick exposure, caused cattle to abort (4). In view of this evidence, a study was undertaken with the object of verifying involvement of the chlamydial agent as the cause of EBA and identifying additional agents that might be implicated etiologitally. The study was carried out on a herd of beef cattle on a ranch in a typical Sierra foothill area. The procedure entailed the collection of a representative cross section of the arthropod and rodent populations of the ranch, from which attempts were made to isolate chlamydiae or other agents by culture in chicken embryos. Relatively large numbers of rodents (Neotoma, Peromyscus, Microtus, Citellus, Perognathus), and arthropods (Dermacentor, Culicoides,Chrysops, Trombiculi, Simulids, fleas, deer keels,lice, and some ectoparasites from rabbits) were cultured in addition to tissues from several Scleroporus lizards and two deer. The findings were negative throughout. It was then decided to abandon this approach as being too unselective. Attempts to recover agents from fetuses aborted as a result of infection diagnosed clinically as EBA had been continued from the time the disease was first studied on an intensive basis. However, with the exception of two chlamydial isolates recovered in the late 1950's (2) no further isolations of any agent were made. The consistent failure to do so was attributed to the long postinfection interval, during which the agent disappeared from the fetal tissues, or remained in such low concentration as to be essentially impossible to isolate. To overcome this difficulty, it was decided to remove the fetuses from pregnant heifers after they returned from grazing in EBA enzootic areas in the hope of obtaining some in which active infection was in progress. The following three experiments were based largely on this approach A local beef herd which ranged in an EBA-enzootic area, and in which EBA was an annual problem, was selected for the initial study. On return of the herd from the range pastures in late June or early July, which almost always preceded the abortion outbreak, 10 pregnant heifers were obtained. Since the abortion rate varied from 10% to 30% or more each year, this number was considered minimal in order to ensure that at least one or two of the heifers would be carrying an infected fetus. One heifer was killed at each of 10 successive weekly intervals and the fetus removed. Tissues were selected for culture in cell monolayers, in explant culture, and in chicken embryos. The results were disappointing in that no gross evidence of fetal pathology was observed in any fetus, with the exception of one which was aborted several days before it was scheduled for study. While it had gross lesions considered indicative of EBA infection, no agent was
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isolated from its tissues, or from any of the other nine fetuses whose tissues were also cultured. Histopathologic findings were inconclusive in the aborted fetus, and several of the grossly normal fetuses, selected at random for detailed histopathologic study, were negative for tissue changes. In view of the possibility that infection occurred shortly after range exposure, the approach was modified the following year in that individual heifers were removed at intervals and the fetuses were processed. From 15 to 20 heifers, determined by palpation to be pregnant, were eartagged and bled prior to their transfer to the range. After 3 to 4 weeks, by which time tick exposure was assumed to have occurred, one animal was removed each week, and the fetus was cultured as in the previous year. In addition, after the specimens had been passaged three times in cell culture and chicken embryos, and grown out in explant cultures, these cultures were inoculated into pregnant heifers to determine whether an agent which might have escaped detection was present. Again the results were disappointing. One fetus had lesions indicative of EBA infection. However, no agent was recovered from this fetus, or from the others, by serial blind passage of their tissues in the culture systems used. Also, the pregnant heifers into which 3rd passage chicken embryo and cell-cultured materials were inoculated, delivered normal calves at term. Shortly after completion of this study, it was demonstrated that Ornithodoros coriaceus ticks transmitted an agent,while feeding, which caused pregnant heifers to abort (5). The fetuses had lesions much like those described for EBA. It was, therefore, now possible to identify infected ticks by feeding them on pregnant heifers, and to use such ticks as a natural means of infecting experimental cattle. A study was then made using this method of infection. Although it did not resolve the question of the etiology of EBA, it provided some rather interesting information concerning an additional agent and, for that reason, the study is described in some detail in the present work. Materials and Methods Experimental Cattle - Beef breed heifers which had been reared in a tick-free area were used in this study. Tick Exposure - After breeding, and just prior to transfer of the heifer herd to the spring grazing range in an EBA-enzootic area, a number of heifers in excess of that needed for the experiment, were pregnancy tested and separated from the remainder of the herd. They were maintained in a tick-free area until used in the experiment. From 1 to 3-112 months after the estimated day of conception, 12 of the heifers were exposed experimentally to 0. coriaceus ticks. The ticks were collected from four geographic lo&ions in California and had been demonstrated to be capable of inducing abortion in pregnant heifers by prior feediwexposure. Each geographically
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representative group of ticks was so divided among the total number of heifers to be exposed that each of the latter was fed upon by many ticks from each of the four geographic areas in which they were collected. For exposure, a special harness was placed over a shaved area of the back of each heifer while she was restrained in a squeeze chute (5). The ticks were added to cups in four separate batches and observed while they fed to ensure that exposure had occurred. The number applied per heifer ranged from 24 to 113. When the engorged ticks detached, they were returned to the appropriate container, thus making it possible to keep those fed on any one heifer separate from the others, and to preserve their geographic identity for further reference. At intervals following tick exposure, seven of the heifers were slaughtered, and their fetuses removed and studied. Chicken Embryo Culture Techniques - Tissues obtained for culture from each fetus were placenta, pooled lung and spleen, pooled liver and kidney, and a miscellaneous pool containing brain, lymph node, muscle, and thymus gland. The supernatant (s/n) portions of 50% suspensions (a) of the four preparations in a kanamycin-vancomycin-streptomycin (KVS) solution, were diluted 10-l, and 10m2 in the same diluent for inoculation. Two-tenth ml amounts of each diluted preparation were inoculated via the yolk sac route into a series of not less than 8 to 10, 6- or 'I-day-oldchicken embryos. Embryos which died within 48 hours after inoculation were discarded. Those which died subsequently, and those surviving by the 8th day, were harvested and portions of yolk sac and body tissues of each were pooled. For the initial subpassage, the placenta and pooled lung and spleen passed materials, were combined, as were the remaining two, i.e. the pooled liver and kidney, and the miscellaneous tissue pool, and diluted as for the original inoculums. Subsequent tissue harvestings were made as described. For the 2nd subpassage a single pool was prepared, diluted 10d3 and passaged as indicated above. If no evidence of infection appeared in this passage, the original fetal tissues were considered to have been negative for an agent, and further culture was discontinued. Cell Culture Techniques - Individual 50% suspensions of liver, kidney, spleen, lung, brain, bone marrow, lymph node, thymus gland, and placenta from each fetus were prepared in minimal essential medium (MEM). The s/n fraction of each, obtained by centrifugation at 1500 rpm for 15 minutes, was diluted 1:lO and 1:50. Each preparation was inoculated, in a volume of 0.05 ml, to bovine embryonic lung (BEL) cell~~?nolayers, at passage levels from five to nine, in microplate wells. From 8 to 12 microcultures were inoculated titer (MT) per specimen. After holding for 60(;fn at room temperature (23 C) for containing 10% by volume of adsorption to occur, 0.05 ml of MEM fetal calf serum (FCS), was added to each inoculated monolayer. (a)
Consisting of 1 gm of each, streptomycin, vancomycin and kanamycin per liter of phosphate buffered saline (Dulbecco formula)
(b) Cooke Engineering Company, Alexandria, VA (c) Grand Island Biological Company, Santa Clara, CA
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The latter were incubated at 37 C in an atmosphere containing 5% by volume of carbon dioxide, and were observed daily for evidence of cytopathic (CP) changes through the 7th to 10th day postinoculation. For subpassage,the monolayer cultures of each specimen were frozen and thawed once and pooled. After light centrifugation,the s/n fractionswere diluted and inoculatedas described for the original passage. White blood cell inoculumswere obtained by centrifugingwhole blood, drawn in an anticoagulant,at 1500 rpm for 15 minutes, and removing the buffy coat layer. The latter was alternatelyfrozen and thawed several times, and prepared for inoculationas for the above tissue specimens. (d) from Explant cultures were grown out in 30 ml plastic bottles fragments of the organs listed above, in MEM containing 10% by volume of FCS. The cultures were incubated 7 to 10 days as already described, by which time complete monolayers had formed. Subpassagewas made in plastic bottles at 10 to 12 day intervals by trypsinizingthe monolayers and suspending the cells in MEM containing 5% FCS. Results The most significant finding was that although two of the five tick-exposedcontrol heifers gave birth to normal calves at term, two delivered premature calves and one aborted. Lesions were present in all three, although they were considered to be relatively nonspecific. Unfortunately,only one term and one prematurely delivered calf were cultured, in addition to the aborted fetus (No. 4). Both calves were negative for an agent. However, changes of a suspiciousnature were observed in cell monolayers inoculatedwith tissues of the aborted fetus. As these disappearedby the 3rd passage, they were considered to be artifacts. Nonetheless,the fact that three of the five heifers either delivered prematurely or aborted, supports findings made in an earlier study (6) to the effect that 0. coriaceus ticks harbor an abortifacientagent or agents for catFle. Whether any of the heifers from which the fetuses were removed at slaughter would have aborted or delivered a premature calf is not known. Only one of these fetuses (No. 8) had evidence of infection and a virus which had been isolated from ticks in earlier studies, but thus far found to be nonabortifacient (61, was recovered from it. The other heifers contained apparently normal fetuses at slaughter in which no pathologic changes were evident, and from which no isolationswere made. The fact that control heifer (No. 4) which aborted was estimated to have conceived only 25 days prior to tick exposure might be of considerablesignificance. This exposure was at least 63 days earlier in gestation than were any of the other exposures of the pregnant heifers in the study. The possible reason for the absence of lesions, or for failure to recover an agent from the heifers whose pregnancies
(d) Corning Glass Works, Corning, NY AUGUST-SEPTEMBER
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were interrupted, is that exposure occurred too late in gestation to infect all but this one fetus. However, this hypothesis is not compatible with findings in the other two pregnant heifers (Nos. 89 and 91) in the control group which, although they were estimated to be 122 and 140 days, respectively, into gestation when tick exposed, they delivered weak, premature calves which were, presumably, infected earlier. The results are summarized in Table 1. Discussion Contradictory findings such as the above have characterized studies of EBA from the beginning. Despite the long years devoted to investigations of this difficult disease problem, it still remains largely an enigma. Although progress has been slow, it must be recognized that, because of its very nature, abortion is an extremely difficult problem to study. The act of abortion -per se is only the final in a series of events which takes place over a prolonged period under conditions which make monitoring all but impossible. During this lengthy interval, a number of extraneous factors may intervene, resulting in misinterpretations of findings and faulty conclusions. To control a disease on a rational basis, the cause must be known. Thus far, the only agent isolated from fetuses aborted as a result of infection diagnosed clinically as EBA is a chlamydial agent. Initially it was believed to be the sole cause of the condition, as it caused abortion on experimental inoculation, consistent with the fact that chlamydiae cause abortion in other ruminant species. In view of the urgency of the EBA problem and the economic benefits which would be derived by early control of the disease, an inactivated vaccine, prepared according to the method used for the ovine chlamydial abortion vaccine (7), was tested in heifers by experimental challenge. Several attempts failed to demonstrate any protective effect by the vaccine (8). Moreover, virgin heifers which had been inoculated with the viable chlamydial agent aborted when challenge inoculated during the ensuing pregnancy. The same results were obtained when the immunity of cows which had aborted from the naturally occurring disease was challenged during the subsequent gestation. This apparent lack of immunity to experimental challenge contrasted sharply with field reports to the effect that an immunity to the natural infection develops as a result of a single abortion. It was felt that this discrepancy might be due to the method of experimental immunity challenge used, rather than to the apparent inability of the agent to immunize under experimental conditions. It was recognized that if natural exposure could be simulated and used as immunity challenge under experimental conditions, the discrepancy might be resolved. Therefore, since the agent was believed to be vector transmitted, the immunity of vaccinated cattle was challenged by intradermal inoculation to simulate vector exposure.
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TJSERIOGENOLOGY This approach proved highly successful in that in several experiments none of the vaccinated cattle aborted. In contrast, practically 100% of control animals either did, or delivered calves prematurely (9). These results appeared to reconcile the discrepancy between failure to demonstrate, by experimental challenge, immunity to EBA in vaccinated cattle, and evidence for immunity from field exposure. The explanation appears to lie in the fact that, as is the case with other chlamydial infections, the immunity associated with chlamydial abortion is of the cell-mediated type. Briefly, this type of immune response functions as follows.* Lymphocytes, sensitized to an antigen as a consequence of either natural infection or vaccination, become activated on subsequent re-exposure to the same antigen and respond by elaborating lymphokinases which possess various activities. One lymphokinase (chemotaxin) attracts phagocytic cells to the location of the antigen. Another enhances their phagocytic capabilities, while a 3rd (migration-inhibiting factor - MIF) retains them in intimate contact with the antigen. As a result, the latter is phagocytized and disposed of. In view of its cell-mediated nature, immunity to chlamydial abortion is believed to ensue when cattle become sensitized to the agent as a result of natural infection acquired, apparently, by vector transmission. On re-exposure the following year, the agent is again deposited into the skin by the vector, where it is retained temporarily because of the density of the surrounding tissues. Before the agent can replicate and escape from these foci into which it is implanted, the sensitized lymphocytes have elaborated the various lymphokinases which mobilize and retain phagocytic cells at the sites where the antigen is present, and endow them with marked ingestive properties. Thus, the agent is disposed of before it can become dispersed in the tissues and reach the blood stream. Essentially, the same response was elicited in the vaccinated heifers when the challenge inoculum was deposited in the skin by intradermal inoculation. However, in early studies of immunity to EBA, the challenge agent was inoculated through the skin of the vaccinated cattle, either by subcutaneous or intramuscular inoculation (8). As a result, it spread quickly from the inoculation sites before the phagocytic cells had become fully mobilized. Once it was widely dispersed in the body, the phagocytes were incapable of coping with it, and blood stream invasion with ensuing abortion, occurred as a consequence. It has been observed that when cattle are maintained in an EBAenzootic area, they abort only once from the infection and then become resistant to such abortions in subsequent years. However, if transferred to a non-enzootic area they apparently lose this immunity, as they again abort on return to the enzootic area. Such behavior is compatible with that of a cellular-type immunity, which is characteristic of chlamydial infection, in that constant antigenic restimulation is required to maintain resistance at a protective level. Therefore, further evidence for involvement of a chlamydial agent in
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the etiology of EBA is provided. Having demonstrated that the vaccine was protective under experimental conditions (4,9), it was tested in cattle in an area where EBA was reported to occur. Unfortunately, only several herds with a limited number of cattle in each were available, and conditions were such that it was not possible to monitor the animals closely. In any event, it was reported that, insofar as could be determined, there was no evidence of a reduced abortion rate among the vaccinees as compared with the control animals. Assuming that this were a valid observation, and that exposure to the chlamydial abortion agent had occurred, two possible explanations could account for these findings. As chlamydiae have been isolated from both 0. coriaceus and Dermacentor occidentalis ticks, exposure to these tic?& could have resulted -either of two outcomes. Dermacentor occidentalis ticks are shallow feeders, the penetration not exceeding that of intradermal injection. Experimentally, it was found that vaccinated heifers were refractory to challenge exposure given via this route (4,9). Therefore, had these ticks been responsible for the field exposure, which obviously took place, abortion would not have been expected to occur. However, if exposure were due to 0. coriaceus ticks, abortion most likely would have ensued, as these-ticks are deep feeders and the agent would have been deposited in the subcutaneous tissues of the exposed animals. As already indicated, in earlier studies, when cattle were challenge inoculated by either subcutaneous or intramuscular injection, abortion ensued on a regular basis (8). The reason for this appears to be that the skin, in which the cellmediated immune response functions most effectively, particularly in the case of vector transmitted infections, was circumvented. Therefore, the possibility that the abortions which occurred in the field trial herds in the present study were due to chlamydiae cannot be ruled out. This is particularly relevant, in view of the fact that chlamydiae have been demonstrated to cause abortion in other ruminants. Also, it has been demonstrated by simulated vector exposure to the bovine chlamydial agent that EBA is most likely an arthropod-transmitted infection (4). Moreover, the fact that ticks collected from foothill areas harbored chlamydiae indicates not only that such agents exist in areas inhabited by cattle, but that a means of transporting them to cattle is available (3). Finally, the nature of the immune response of cattle to infection with the chlamydial abortion agent is compatible with that of vector-type exposure to an agent which evokes a cellmediated immune response. On the other hand, it is highly unrealistic to believe that undiscovered causes of bovine abortion still do not exist, and particularly in foothill areas which abound with arthropods and rodents, but where few epizootiologic studies have been carried out. In fact, evidence to this effect became apparent during studies of a virus which was isolated recently from members of the O.coriaceus species collected in an EBA epizootic area (6). This virus, as yet unidentified, appears to be unrelated
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etiologically to abortion. However, pregnant heifers exposed to 0. coriaceus ticks which did not harbor this virus delivered weak calves from 21 to 30 days prematurely which, at necropsy, were found to possess lesions suggestive of EBA infection. In view of these findings, it appears that the ticks must have harbored a still unknown abortifacient agent. It is entirely possible that this agent is a virus. However, in attempts to isolate agents from ticks and aborted fetuses, the inoculums were heavily treated with antibiotics to control nonviral contaminants, and culture systems designed for viral isolation were utilized. Under such circumstances rickettsial or chlamydial agents would not be recovered. It is possible, therefore, that the suspected agent might well be either of these. Nonetheless, regardless of its identity, these findings provide growing evidence that a number of agents are involved etiologically in the EBA syndrome, of which one is a chlamydia. The extent to which the latter is involved remains uncertain. In the case of most infectious agents this would be relatively easy to determine by serologic means. Unfortunately, because of a common complement fixing group antigen, which is shared essentially by all mammalian chlamydiae, it is not possible to identify the specific chlamydia responsible in each case for the diverse infections which these agents cause. Cattle are prone to several including conjunctivitis, arthritis, and pneumonitis, in addition to abortion. Consequently, most, if not all cattle, possess complement-fixing chlamydial antibody. While the persistence of such antibody is generally of short duration, it prevails in cattle because of an asymptomatic enteric chlamydial infection which maintains a constant antigenic stimulation (10). Unfortunately, the only practical procedure for detecting chlamydial antibody is the complement-fixation test. Therefore, until such time as additional procedures are devised to identify specific chlamydiae, serology will play little or no role in either epidemiologic or diagnostic studies of infections caused by these agents. Isolation of the chlamydial agent from aborted bovine fetuses as a means of diagnosis and for monitoring vaccine field trials is unsatisfactory. The disease is contracted on foothill pastures where many of the abortions take place. The inaccessibility of the animals in these extensive primitive areas renders it largely impossible to obtain the fetuses. However, this is probably of little practical importance because, under even the most favorable experimental conditions, it is extremely difficult to recover the agent from aborted fetuses. The reason appears to be that the agent fails to survive the long period between infection of the dam and expulsion of the fetus. In fact, lesions present in aborted fetuses indicate that the fetal infection usually occurs long in advance of the abortion. In the case of such chlamydial infections as pneumonitis, conjunctivitis, or even arthritis, in which clinical evidence of disease is apparent in the host, and access to materials for culture is available, isolation can be accomplished fairly consistently. In the case of abortion, however, replication of the agent occurs mainly in the placenta and in the fetus itself, without any clinical evidence being displayed
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THERIOGENOLOGY by the infected cow. It appears, too, that the agent is not shed into the general circulation of the latter; hence no opportunity for its isolation from this source is provided. The fact the chlamydiae are recovered much more readily from aborted ovine and caprine fetuses might reflect the shorter gestation period characteristic of these species, which enables the agent to survive until abortion occurs. The unavailability of aborted fetuses in a satisfactorycondition is a severe handicap insofar as histopathologic studies are concerned. While several gross lesions have been associated with naturally occurring EBA, most are of a nonspecific nature. Generally speaking, in our experience at least , gross lesions are absent in most fetuses diagnosed as having been aborted as a result of EBA. Thus, lack of serologic procedures, the circumstances which mitigate against isolation of the agent from fetuses, and the unreliability of either clinical or pathological diagnoses represent major handicaps in dealing with chlamydial abortion. There are two possible approaches to defining more precisely the extent to which the chlamydial agent is involved etiologically in the EBA syndrome. One is to conduct field trials in EBA enzootic areas, on at least 5000 vaccinees and an equal number of control cattle, and to make a statistical analysis of the numbers of calves produced by each group. This would indicate the total percentage of abortions in the herds under study and the proportion due to chlamydial abortion. However, such an undertaking would be costly and would involve a highly coordinated team effort on the part of various specialist groups over a period of months. A much more limited approach, both cost-wise and administratively, would entail the exposure of equal numbers of vaccinated and unvaccinated (control) pregnant heifers maintained under experimental conditions, to O.coriaceus ticks collected in EBA enzootic areas and determined to be capable of inducing abortion in cattle. Either study would also indicate whether agents in addition to the chlamydia are involved etiologically in the EBA syndrome, and whether the proportion of abortions due to the latter is sufficiently high to warrant the adoption of preventive measures. References 1.
Traum, J., and Hart, G.H. Bovine Infectious Abortion. III. Manifestation of Abortion Disease without Demonstrable Etiological Factor. Univ. Calif. Agr. Expt. Sta. Bull., -353:327-346 (1923).
2.
Storz, J., and McKercher, D.G. Etiological Studies on Epizootic Bovine Abortion. Zentbl. f. Vet-Med., -9: Heft 4, 411-427; Heft 5, 520-541 (1962).
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3.
Eddie, B., Radovsky, F.J., Stiller, D., and Kumada, N. Psittacosis-L~phogranuloma-Venereum (PL) Agents (Bedsonia, Chlamydia) in Ticks, Fleas, and Native Animals in California. Am. J. Epidemiol., 90:449-460 (1969).
4.
McKercher, D.G., Crenshaw, G.L., Theis, J.H., Wada, E.M., and Mauris, C.M. Experimentally Induced Immunity to Chlamydial Abortion of Cattle. J. Inf. Dis., 128:231-234 (1973).
5.
Schmidtmann, E.T., Bushnell, R.B., Loomis, E.C., Oliver, N.M., and Theis, J.H. Experimental and Epizootiologic Evidence Associating Ornithodoros coriaceus Koch (Atari-Argasidae) with Exposure of Cattle to Epizootic Bovine Abortion in California. J. Med. Ent. In Press (1976).
6.
Wada, E.M., McKercher, D.G., Castrucci, G., and Theis, J.H. Preliminary Characterization and Pathogenicity Studies of a Virus Isolated from Ticks (Ornithodoros coriaceus) and from Tick-Exposed Cattle. Am. J. Vet. Res. In Press, (1976).
7.
McEwen, A.D., Dow, J.B., and Anderson, R.D. Enzootic Abortion in Ewes. An Adjuvant Vaccine Prepared from Eggs. Vet. Rec., -67: 393-394 (1955).
8.
McKercher, D.G., Robinson, E.A., Wada, E.M., Saito,J.K., and Franti, C.E. Vaccination of Cattle Against Epizootic Bovine Abortion. Cornell Vet. -59:211-226 (1969).
9.
McKercher, D.G., Crenshaw, G.L., Franti, C.E. Vaccination Against Abortion. J. Am. Vet. Med. Ass.,
10.
Wada, E.M., Mauris, C.M., and Epizootic Bovine ~~hlamydia~) 163:889-891 (1973). -
York, C.J., and Baker, J.A. A New Member of the PsittacosisL~phogranu~oma Group of Viruses that Causes Infection in Calves. J. Exp. Med, -93:587-604 (1951).
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109
109
94
88
19
26
43
15
25
122
140
4
ES
91
97
-__
107
48
Normal Control
112
41
Tick Exposure Controls
88
109
31
--..
24
___
---
81
-__
---
89
82
37
68
111
..--
61
113
---
54
83
84
47
102
40
87
112
Term Delivery (274 ddysf
Weak Calf (257 days)
Neg Neg
__-
Weak Calf (249 days)
nd
Questionable Myocarditis, thymic atrophy, lytnphoid hyperplasia, focal hepatic necrosis
-__
Abortion (130 days)
Petechiaein ocular and nasal mucosa
Term Delivery(269 days) Neg
nd Edema, liver lesions
-...-
Neg
___
r-_--
Term Delivery(266 days)
__-
nd
_--
Neg
Neg
Neg
*eg
Neg
__-
Neg
_--
Neg
Neg
Neg
-_-
Neg
Neg
Neg
Neg
_--
Neg
Neg
---
Virus
Lymphoid hyperplasia Neg
Neg
Neg
Agent PregnancyTermindtrd Isolated By
Histopathologic Fetal Lesions
Agent f‘~wi Findingsmade in Attemptsto Isolatean Abortif,icirnt the Fetusesof Tick (Ornithodoroscoriaceus)I::<[.~::,w*l Hriit:rs
Tick Exposure No. of Killed E Fetus Gross Fetal (Days PostTicks Removed (Day Lesions Conception) Applied Post-Tick Exposure Neg 93 34 109
14
a
Heifer No.
TABLE I