Histopathologic changes in bovine fetuses after repeated reintroduction of a spirochete-like agent into pregnant heifers: association with epizootic bovine abortion

A spirochete-like organism was found in the plasma of bovine fetuses affected with epizootic bovine abortion (EBA). The spirochete-like organism was frequently found in abattoir-collected fetuses as an inapparent infection, and EBA was found in cattle on foothill rangeland where the vector tick Ornithodorus coriaceus could repeatedly reintroduce the infectious agent into pregnant cattle (superinfection). Epizootic bovine abortion resembled a naturally acquired superinfection in circumstances where the agent was frequently present in the environment under conditions favoring transmission. Therefore, to determine whether fetal lesions could be experimentally induced in utero, spirochete-like organisms collected from clinically normal fetuses at an abattoir were inoculated IV and subcutaneously into 2 pregnant heifers 5 times over a 4-month period to mimic repeated tick transmission in the field. Macroscopic and microscopic examinations of tissues from 2 cesarean-collected fetuses and from 3 calves born at term with the naturally acquired spirochete infection indicated that the calves had evidence of an infection that caused morphologic changes compatible with immunologic stimulation and mild reticuloendothelial hyperplasia. Compared with findings in the calves, lesions in the superinfected fetuses were more severe, and the lesion distribution in various organs was more extensive. The spirochete-like organism appeared to be a mild pathogen because of its persistence in the host. Clinical disease from the infection may only develop with repeated superinfections. Therefore, a relationship between this microorganism and EBA is probable.     

Encephalitis in aborted bovine fetuses associated with Bovine herpesvirus 1 infection.

Brain tissue from 12 aborted bovine fetuses submitted to the Wisconsin Veterinary Diagnostic Laboratory revealed histologic lesions that consisted of glial nodules and variable degrees of mononuclear inflammation, microhemorrhage, neuronal necrosis, and cerebral cortical cavitation. A diagnosis of Bovine herpesvirus 1 (BHV-1) abortion had been made in all of these cases through multiple testing modalities. Brain tissue from 8 of the 12 fetuses was immunohistochemically stained with a monoclonal antibody specific to BHV-1, and, in 5 fetuses, there was positive intralesional staining of neurons, glial cells, and endothelial cells. This preliminary data suggested that herpesviral infection of brain tissue led to the described neurologic lesions. BHV-1 was then amplified from brain tissue in all 12 of the fetuses and was confirmed by partial sequencing of the thymidine kinase and glycoprotein C genes. To the authors' knowledge, neurologic lesions have not previously been described in BHV-1-infected fetuses, nor has BHV-1 previously been identified in bovine fetal brain tissue. The neurologic histopathology attributed to BHV-1 infection in these cases overlaps with the neurologic lesions produced by Neospora caninum, a common etiologic agent of bovine abortion. Therefore, when bovine fetal neurologic lesions are found, both etiologies should be considered and then distinguished by using additional diagnostic tools.     

Experimental transmission of epizootic bovine abortion (foothill abortion)

Advances in defining the biology of epizootic bovine abortion (EBA), including identification of the etiologic agent, have been hampered by the inability to reproduce the disease with confidence. Experimental reproduction of EBA, by feeding the tick vector Ornithodoros coriaceus on susceptible pregnant heifers, is not reliable. The primary objectives of this study were to identify specific tissue(s) obtained from EBA-infected fetuses that could transmit the disease, and then utilize such an infectious challenge system to better define the pathogen, host immunity and geographic distribution of the agent. Described here is the ability to routinely reproduce EBA following inoculation of cryopreserved suspensions of homogenized thymus into susceptible pregnant heifers. This challenge system permitted experiments demonstrating the agent was non-filterable, inactivated upon sonication and susceptible to antibiotics. These findings suggest a prokaryotic microbe and represent a major advance in EBA research. Additional experiments demonstrated that inoculation of the cryopreserved EBA-infectious tissue into heifers, prior to breeding, conferred immunity. Furthermore, such immunized heifers were resistant to challenge with heterologous sources of infectious tissue, suggesting monovalent vaccine development might be feasible. Lastly, challenge studies employing animals from Central Nevada, an area considered free of EBA, demonstrated partial immunity, suggesting the pathogen, and possibly the disease, enjoy a broader distribution than previously thought.     

Comparison of antigenic structure and pathogenicity of bovine intestinal Chlamydia isolate with an agent of epizootic bovine abortion.

A bovine fecal Chlamydia, isolated from a clinically normal cow, had pathogenic capabilities and antigenic structure similar to those of a chlamydial agent of epizootic bovine abortion (EBA). Intravenous inoculation of the fecal chlamydia into a pregnant heifer caused abortion and lesions in the fetus indistinguishable from those of experimentally induced EBA. In serotests with species-specific complement fixation antigens, the intestinal agent was similar to the EBA agent.     

Additional evaluation of undiagnosed bovine abortion cases may reveal fetal neosporosis

Diagnosis of Neospora caninum associated bovine abortion is often made by histological examination of fetal tissues, although this procedure is not very sensitive. One hundred and forty-four undiagnosed bovine abortion cases were evaluated for N. caninum associated bovine abortion using a revised diagnostic protocol. As a result, 12 (8.3%) of these previously undiagnosed bovine abortion cases were definitively diagnosed as N. caninum positive. The 12 new positive cases included 7 that had exhibited histological lesions, and 5 that had not exhibited histological lesions when examined prior to this study. None of the 12 cases had been immunohistochemically (IHC) stained prior to this study. Also, IHC staining implemented during this research revealed tachyzoites without associated histological lesions in at least 1 tissue from 69 (47.9%) of 144 aborted bovine fetuses, and positive IHC staining of cytoplasmic N. caninum antigen in macrophages in at least 1 tissue from 44 (30.6%) of 144 aborted bovine fetuses. These results demonstrate the necessity for more aggressive evaluation of bovine fetuses for neosporosis.     

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.     

The geographic distribution of the putative agent of epizootic bovine abortion in the tick vector, Ornithodoros coriaceus

Epizootic bovine abortion (EBA), also known as “foothill abortion”, is a vector borne disease of beef cattle that graze in the mountainous regions of California, southern Oregon and western Nevada transmitted by the argasid tick Ornithodoros coriaceus. Recently, the putative agent of EBA was identified as a novel Deltaproteobacter in the order Myxococcales. In this study, a TaqMan real-time PCR (TM-PCR) protocol specific to the putative EBA agent was developed. The new real-time TM-PCR assay functioned sensitively and specifically to detect pathogen DNA in field-collected O. coriaceus ticks. The assay had an analytical sensitivity of a single plasmid copy and, when evaluated with a collection of tick-borne pathogens, yielded a positive PCR-result only for the agent of EBA. Use of the TM-PCR represents an effective tool for rapid and highly sensitive assessment of environmental risk and spatial and statistical analysis to highlight areas where there may be increased risk for EBA in susceptible cattle.     

A survey of equine abortion and perinatal foal losses in Hungary during a three-year period (1998-2000)

Cases of equine abortion and perinatal foal losses were investigated in Hungary during a three-year period (1998-2000). Samples from aborted equine fetuses and newborn foals (total n = 96) were examined using bacteriological, virological, pathological, immunohistochemical (IHC), molecular biological and serological methods. The cause of abortion and perinatal foal loss was identified in 67/96 cases (70%); viral infection was found in 22 (23%), viral and bacterial coinfection in 1 (1%), bacterial infection in 23 (24%), protozoan infection in 1 (1%) and fungal infection in 2 cases (2%). Morphological lesions suggestive of infection were recorded in 2 (2%) and non-infectious causes in 16 cases (17%).     

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.     

Studies on the pathogenesis of epizootic bovine abortion

Ornithodoros coriaceus Koch ticks were fed on 37 pregnant cows. The fetuses were obtained from the cows at 23 to 126 days after maternal tick exposure. Characteristic lesions of epizootic bovine abortion were observed only in those fetuses recovered 100 days more or after maternal tick exposure. Fetuses collected between 50 and 100 days showed mild-to-moderate lymphoid and mononuclear cell hyperplasia. Reaction in fetuses studied less than 50 days after maternal tick exposure were mild. Lesions could not be seen in 2 of the youngest fetuses. Increases in serum immunoglobulin concentrations were present only in those fetuses examined 80 days or more after their dams had been exposed to ticks. The specificity of the immunoglobulins could not be determined. Sera from 12 fetuses tested failed to fix complement in tests for group-specific chlamydial antibodies. A wide variety of microbiological cultivation attempts were made to recover the causative agent of epizootic bovine abortion from these fetuses; however, no agent was recovered regularly, and chlamydial organisms were not recovered from any. The significance of 2 recovered agents, apparently viral, is still to be determined. Fetal tissues, both frozen and fresh, collected from fetuses of dams exposed to a feeding of ticks were capable of reproducing the disease after inoculation into pregnant cows or directly into fetuses.     

A survey of causes of bovine abortion occurring in the San Joaquin Valley, California

The causes of abortion in cattle in the San Joaquin Valley of California were surveyed from submissions to the California Veterinary Diagnostic Laboratory at Tulare. Four hundred sixty-eight abortion cases were examined. Most submissions (89%) were from large drylot dairies, milking an average of 814 cows. Abortion evaluations included necropsy, histopathology, bacteriology, virology, and other immunologic and serologic tests. A specific cause was identified in 29.5% of the abortions. Bacterial infections, most of which were sporadic, accounted for 16% of all abortions. Viral causes and protozoal infections were diagnosed in 5.6% and 3.2% of the abortions, respectively. Fetuses with protozoal infection had histologic lesions of focal nonsuppurative necrotizing encephalitis, and protozoa were detected. Similar histologic lesions were seen in 80 additional fetuses (17.1%), and although an etiologic agent was not identified for these cases, a protozoal infection was suspected.     

Bovine abortion caused by Bacillus cereus

Bacillus cereus was identified as an infrequent abortigenic agent in cattle. Necrotizing placentitis with no or sporadic lesions in fetal tissues was seen. Bacillus cereus was isolated in pure culture from fetal tissue and/or placenta. The recent identification of a bovine abortion caused by B cereus, prompted a retrospective survey of the pathology files. Eight of 947 bovine abortions were attributed to B cereus. Bacillus cereus is often mistaken as a contaminant in bovine abortion because of the failure to identify lesions in fetal tissues compatible with bacterial invasion. A necrotizing toxin may be responsible for the placentitis, with expulsion of the fetus before bacterial colonization.     

Lymphocyte blastogenesis and cellular cytotoxicity in a congenital infection of bovine fetuses related to epizootic bovine abortion

The disease referred to as epizootic bovine abortion (EBA) was experimentally induced in bovine fetuses. Dark-field microscopy was used to detect congenital infection with an unclassified spirochaete-like organism. Some of the fetuses collected at abattoirs were also found to be naturally infected with a morphologically similar microorganism. Blood counts and organ weights were correlated with the presence of the microorganism. Lymphocyte blastogenesis increased, the result of in vivo stimulation among the infected fetuses. Phytomitogens (phytohaemagglutinin, concanavalin A and lipopolysaccharide) also stimulated greater responses in infected fetuses when compared to results in normal fetuses. Cellular cytotoxicity was examined by the single cell assay and results indicated that there were fewer cytotoxic lymphocytes among the diseased fetuses. The infected abattoir-collected specimens were obtained from clinically normal adult cattle, and the immunological changes in these fetuses were closely characterised with those of the EBA diseased fetuses. These naturally infected fetuses showed signs of a mild infectious disease.     

Porcine abortion caused by Bacillus sp

Bacillus sp is known to cause abortion in cattle and sheep, but it is rarely reported to cause abortion in swine. Bacillus sp was isolated from aborted fetuses, and lesions compatible with a bacterial infection were detected in 4 of 962 aborted porcine fetuses examined. The infection apparently results in sporadic abortions and is not likely to cause economically significant reproductive failures within a herd.     

Immunohistochemical demonstration of Francisella tularensis in lesions of cats with tularemia.

An immunohistochemical test was developed and validated for detection of Francisella tularensis antigen in tissues of cats with fatal tularemia. Ten cases of naturally occurring tularemia in cats were positive both by isolation of F. tularensis and immunohistochemical identification of F. tularensis antigen. Nine additional cases with lesions typical of tularemia were positive for F. tularensis antigen, although bacterial cultures were not performed. Immunohistochemical identification of F. tularensis in formalin-fixed tissue is valuable for establishing a rapid etiologic diagnosis under circumstances where fresh tissues may not be available for isolation and identification of the organism.     

Pathology of maternal genital tract, placenta, and fetus in equine viral arteritis

Six pregnant mares were given equine viral arteritis virus intravenously. Tissues from genital tracts, placentae, and fetuses were examined by light and electron microscopy to study the mechanism of abortion. Four mares which died with acute disease had diffuse vacuolation of endometrial epithelium and systemic necrotizing vasculitis. Two of these mares had dead fetuses and two had live fetuses; virus was isolated from tissues of one live fetus. Placentae of mares dying from acute disease did not have lesions attributable to infection; virus was isolated from two of these placentae. One of the two mares which recovered from clinical disease aborted a dead fetus eight days after inoculation. The mare had severe necrotizing myometritis and virus was isolated from maternal ovaries and from fetal tissues. The fetus did not have lesions attributable to arteritis virus. These results suggest that although fetal death may occur in utero during acute equine viral arteritis, abortion probably is due to lesions in the uterus of the mare.     

Surface markers on bovine fetal lymphocytes and immunoglobulin synthesis in a congenital infection related to epizootic bovine abortion

Immunological parameters were studied among 23 late-term bovine fetuses. Epizootic bovine abortion (EBA) disease was induced in fetuses by feeding Ornithodoros coriaceus ticks on pregnant heifers. A spirochaete-like microorganism was detected in the blood of diseased fetuses and in inapparent natural infections in some abattoir-collected fetuses. Fetuses were classified according to stages of disease: EBA diseased (n = 10), EBA infected (n = 7) and normal (n = 6). Using flow cytometry, the presence of surface immunoglobulins (sIg) and peanut agglutinin (PNA) receptors were used to detect B and T lymphocytes, respectively. In peripheral blood of normal fetuses, most lymphocytes were identified as T or B cells, whereas about 20 per cent of lymphocytes in EBA diseased fetuses did not reveal the sIg or PNA receptor markers (null cells). Size and shape analyses by flow cytometry detected a population of enlarged lymphocytes in the EBA diseased fetuses. The numbers of cells bearing determinants reactive with monoclonal antibodies specific for bovine T cells (B26A and B29A) and B cells (TH21A) were considerably less than those expressing the PNA receptor and sIg. These results suggested that the monoclonal antibodies were binding to differentiation antigens which were not consistently expressed on the fetal cells. Radio-immunodiffusion was used to measure bovine IgM, IgG1 and IgG2 in fetal serum. The quantities of immunoglobulins were markedly increased in animals infected with the spirochaete-like organism (groups 1 and 2) and were assumed to result from fetal antibody synthesis.     

Induced transplacental transmission of Neospora caninum in cattle.

Three Jersey cows were inoculated SC and IM with 26 million Neospora caninum tachyzoites at 129 (cow 1), 126 (cow 2), and 81 (cow 3) days after mating. Cows remained clinically normal for at least 1 month after inoculation of N caninum. Cow 1 was euthanatized 32 days after inoculation because of gangrenous mastitis. Cow 1 had a live fetus with no gross lesions; however, microscopic lesions were seen in the fetus and consisted of severe nonsuppurative necrotizing encephalitis of the cerebral white matter. Neospora caninum was identified in lesions by staining with anti-N caninum serum in an immunohistochemical test, by bioassays in mice, and by inoculation of bovine monocyte cultures with fetal tissue homogenate. Neither N caninum nor lesions were associated with infection with the protozoon identified in tissues of cow 1. Cows 2 and 3 aborted small autolysed fetuses 101 and 74 days, respectively, after inoculation with N caninum; the fetuses and attached placenta were unsuitable for laboratory investigations. Cows 2 and 3 remained clinically normal 4 months after abortion. Results of this study indicated that N caninum can be transmitted transplacentally in cattle.