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.     

Histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion.

Epizootic bovine abortion (EBA), a tick-transmitted disease of pregnant cattle grazing foothill pastures, is a major cause of reproductive failure in California and adjacent states. Affected fetuses develop a chronic disease, resulting in late-term abortion or premature calving. Despite investigations spanning 50 years, to the authors' knowledge, the etiologic agent of EBA has not yet been isolated from affected fetuses or the tick vector. The diagnosis of EBA is based on gross and microscopic lesions. Recently, documentation that the etiologic agent is susceptible to antibiotics and identification of a unique 16S deltaproteobacterial rDNA gene sequence in 90% of thymus tissues from aborted fetuses have supported the role of a bacterial infection as the cause of EBA. To determine whether bacteria could be detected in the tissues, histochemical staining and immunohistochemical procedures were used on formalin-fixed, paraffin-embedded tissues. Use of a modified Steiner silver stain revealed small numbers of intracytoplasmic bacterial rods in 37 of 42 thymic samples from EBA-affected fetuses. Improved detection was achieved by use of immunohistochemical staining with serum from EBA-affected fetuses that resulted in detection of numerous bacterial rods in the cytoplasm of histiocytic cells in the thymus from all 42 EBA-affected fetuses. Immunohistochemical examination of additional tissues from 21 field and experimental EBA cases revealed positively stained intracytoplasmic bacterial rods in many organs with inflammatory lesions. Use of the modified Steiner stain and immunohistochemical staining of tissues from negative-control fetuses failed to reveal organisms. To the authors' knowledge, this is the first report to document morphologic evidence of a bacterium associated with the lesions of EBA.     

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.     

Congenital spirochetosis in calves: association with epizootic bovine abortion

Congenital spirochetosis was encountered as a newly recognized infection of cattle. The spirochete was seen in blood of fetuses with lesions of epizootic bovine abortion. A spirochete with morphologic features similar to those found in the fetuses was detected in Ornithodoros coriaceus ticks. Ticks collected from rangelands were allowed to feed on cows that then produced epizootic bovine abortion-affected fetuses, and the fetuses had spirochetosis. Inapparent spirochetosis also was found in fetuses in clinically normal cattle sent to slaughter. Only a few lesions were seen in abattoir-collected fetuses. Fetal spirochetosis was common in the bovine population studied, and it appeared that infection may be limited only by the availability of the tick vector.     

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.     

Identification of the etiologic agent of epizootic bovine abortion in field-collected Ornithodoros coriaceus Koch ticks

Epizootic bovine abortion (EBA), or foothill abortion as it has often been termed, is a tick-borne disease of pregnant cattle recognized in California, Nevada and Oregon. The primary objective of this study was to better define the relationship of a novel deltaproteobacterium, the putative etiological agent of EBA (aoEBA), with the Pajaroello tick (Ornithodoros coriaceus Koch), the recognized vector of EBA. Three developmental stages of O. coriaceus (larva, nymph, and adult) were collected from five locations in California, Nevada and Oregon. A polymerase chain reaction (PCR), developed for detection of aoEBA, was applied to DNA extracted from ticks. Southern blotting of the PCR products increased the number of ticks determined to be carrying the bacteria by seven-fold, suggesting the majority of infected ticks carry relatively low numbers of the pathogen. An effort was made to determine if an artificial blood meal would stimulate replication of the bacterial pathogen, thereby increasing the frequency in which aoEBA could be identified; no statistically significant effect was evident. The number of ticks determined to be carrying aoEBA varied with geographic location and ranged from 5 to 20%. aoEBA was found in both adults (12% of the males and 12% of the females) and nymphs (13%) but not larvae. Comparative analysis of dissected ticks provided strong evidence that the salivary gland was the most common location of aoEBA in field-collected ticks. No significant correlations were identified between the frequency of infection and tick weight, suggesting that increasing tick age and increased number of blood meals did not increase infectivity.     

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.     

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.     

Failure of dogs to shed oocysts after being fed bovine fetuses naturally infected by Neospora caninum

Neospora caninum is a protozoan that causes abortion in cattle. The dog has recently been identified as a definitive host for N. caninum. To verify if bovine fetuses can infect dogs, nine 2-4-month-old dogs were fed bovine fetuses naturally infected by N. caninum. None of the dogs excreted oocysts, seroconverted, had clinical signs or lesions compatible with N. caninum infection. Additional studies will be necessary to determine the natural mode of infection of dogs by N. caninum.     

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.     

Protective effects against abortion and fetal infection following exposure to bovine viral diarrhea virus and bovine herpesvirus 1 during pregnancy in beef heifers that received two doses of a multivalent modified-live virus vaccine prior to breeding

Objective-To determine whether administration of 2 doses of a multivalent, modified-live virus vaccine prior to breeding of heifers would provide protection against abortion and fetal infection following exposure of pregnant heifers to cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) and cattle with acute bovine herpesvirus 1 (BHV1) infection. Design-Randomized controlled clinical trial. Animals-33 crossbred beef heifers, 3 steers, 6 bulls, and 25 calves. Procedures-20 of 22 vaccinated and 10 of 11 unvaccinated heifers became pregnant and were commingled with 3 steers PI with BVDV type 1a, 1b, or 2 for 56 days beginning 102 days after the second vaccination (administered 30 days after the first vaccination). Eighty days following removal of BVDV-PI steers, heifers were commingled with 3 bulls with acute BHV1 infection for 14 days. Results-After BVDV exposure, 1 fetus (not evaluated) was aborted by a vaccinated heifer; BVDV was detected in 0 of 19 calves from vaccinated heifers and in all 4 fetuses (aborted after BHV1 exposure) and 6 calves from unvaccinated heifers. Bovine herpesvirus 1 was not detected in any fetus or calf and associated fetal membranes in either treatment group. Vaccinated heifers had longer gestation periods and calves with greater birth weights, weaning weights, average daily gains, and market value at weaning, compared with those for calves born to unvaccinated heifers. Conclusions and Clinical Relevance-Prebreeding administration of a modified-live virus vaccine to heifers resulted in fewer abortions and BVDV-PI offspring and improved growth and increased market value of weaned calves.     

An oculo-cerebellar syndrome caused by congenital bovine viral diarrhoea virus-infection

An epizootic characterized by birth of calves severly ataxic and blind were encountered in 3 herds 7–8 months after outbreaks of bovine virus diarrhoea. Serological and virological investigations indicated introduction of bovine viral diarrhoea virus (BVDV) into previously virus-free herds, followed by transplacental virus infection of the fetuses of cows in the first trimester. Clinical, pathological, serological, and microbiological examinations were performed on 10 calves. Pathological findings included microcephaly and cerebellar hypoplasia, ocular malformations, and thymic hypoplasia. BVDV was isolated from tissue and blood of 7 calves, and 4 calves, 1 of which had not received colostrum, had virus-specific neutralizing antibodies.This is the first report on natural occurrence of congenital bovine infection with BVDV among Danish cattle herds resulting in abortion and birth of calves with severe debilitating congenital anomalies. It draws attention to the importance of this virus for bovines of all age groups.     

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.     

Production of cattle immunotolerant to bovine viral diarrhea virus.

Inoculation of bovine virus diarrhea virus into 58 to 125 day old fetuses of bovine virus diarrhea virus seropositive pregnant cows, or inoculation of bovine virus diarrhea virus into seronegative cows 42 to 114 days pregnant, may produce clinically normal calves which are persistently infected with the specific isolate of bovine virus diarrhea virus yet seronegative to the homologous and heterologous isolates. Reinoculation of these persistently infected cattle with their homologous isolate produced no neutralizing antibody response to bovine virus diarrhea virus. These persistently infected cattle were immunocompetent as they developed neutralizing serotiters to infectious bovine rhinotracheitis, parainfluenza-3 viruses and agglutinating serotiters to Pasteurella hemolytica .     

Association of bovine viral diarrhea virus with multiple viral infections in bovine respiratory disease outbreaks

We investigated eleven outbreaks of naturally occurring bovine respiratory diseases in calves and adult animals in the St-Hyacinthe area of Quebec. Specific antibodies to bovine herpesvirus-1, bovine viral diarrhea virus, respiratory syncytial virus, parainfluenza type 3 virus, reovirus type 3, and serotypes 1 to 7 of bovine adenovirus were found in paired sera from diseased animals. Several bovine viruses with respiratory tropism were involved concomitantly in herds during an outbreak of bovine respiratory disease. In addition, concomitant fourfold rises of antibody titers were frequently observed to two or more viral agents in seroconverted calves (61%) or adult animals (38%). Bovine viral diarrhea virus was found to be the most frequent viral agent associated with multiple viral infection in calves only (92%).     

Evaluation of severe disease induced by aerosol inoculation of calves with bovine respiratory syncytial virus

OBJECTIVE: To develop a model of bovine respiratory syncytial virus (BRSV) infection that induces severe disease similar to that seen in some cattle with naturally acquired BRSV infection. ANIMALS: 25 male Holstein calves, 8 to 16 weeks old. PROCEDURE: 17 calves were given a low-passage field isolate of BRSV by aerosolization; 8 control calves were given supernatant from noninfected cell culture. Disease was characterized by evaluating clinical signs, virus isolation and pulmonary function tests, and results of blood gas analysis, gross and histologic postmortem examination, and microbiologic testing. RESULTS: Cumulative incidence of cough, harsh lung sounds, adventitious sounds, and dyspnea and increases in rectal temperature and respiratory rate were significantly greater in infected calves. Three infected calves developed extreme respiratory distress and were euthanatized 7 days after inoculation. Virus was isolated from nasal swab specimens from all infected calves but not from mock infected calves. On day 7 after inoculation, mean PaO2 and PaCO2 were significantly lower, and pulmonary resistance was significantly higher, in infected calves. During necropsy, infected calves had varying degrees of necrotizing and proliferative bronchiolitis and alveolitis with syncytial formation. The 3 calves euthanatized on day 7 had emphysematous bullae in the caudal lung lobes; 1 had unilateral pneumothorax. CONCLUSION AND CLINICAL RELEVANCE: Severe disease similar to that seen in some cattle with naturally acquired BRSV infection can be induced in calves with a single aerosol exposure of a low-passage clinical isolate of BRSV. Our model will be useful for studying the pathogenesis of BRSV infection and for evaluating vaccines and therapeutics.     

Some lesions observed in calves born to cows exposed to the virus of infectious bovine rhinotracheitis in the last trimester of gestation.

Sixteen pregnant cows were challenged with infectious bovine rhinotracheitis virus intranasally. One had a mummified fetus, four aborted, one calf was stillborn, two live fetuses were taken at the abattoir and eight calves were born alive. Of the eight born alive, five were dead by 12 days of age. Four of these had the usual lesions of infectious bovine rhinotracheitis as well as lesions in the intestine and peritoneum and two of the four had a fibrinous pneumonia thought to be caused by aspiration of milk. The lesions, results of virus isolation and fluorescent antibody testing are recorded in these four calves. Attention is drawn to the intestinal lesions, the peritonitis and fibrinous pneumonia and the ease with which the underlying infectious bovine rhinotracheitis infection may be overlooked.     

Results of a new serologic test suggest an association of Waddlia chondrophila with bovine abortion.

Waddlia chondrophila is a little-known intracellular organism belonging to the order Chlamydiales that has twice been isolated from aborted bovine fetuses. To initiate an investigation of the possibility that W. chondrophila may be an abortifacient pathogen, a serologic test was developed and used to screen bovine sera that were submitted to the Washington Animal Disease Diagnostic Laboratory (Pullman, WA). A highly significant statistical association (P < 0.00001) was observed when comparing antibody titers in cows that had aborted with those in other classes of cattle. Although this result is consistent with the possibility that infection with W. chondrophila may be a cause of bovine abortion, it is also possible that seroprevalence simply increases with age or that exposure rates differ between cows and other classes of cattle. Future serologic studies should specifically compare antibody titers between aborting cows and carefully matched nonaborting cohorts.     

Mastitis, polyarthritis and abortion caused by Mycoplasma species bovine group 7 in dairy cattle

OBJECTIVE: To report an outbreak of mastitis, polyarthritis and abortion caused by Mycoplasma sp bovine group 7 in three large, centrally-managed dairies and to review the relevant literature. DESIGN: Epidemiological, clinical and laboratory data were analysed, collated and reported. Multidisciplinary procedures were employed. These included clinical assessment and comprehensive laboratory investigations of affected calves, aborted foetuses and milk samples. Mycoplasma cultures and genetic analyses of isolates were undertaken to identify the aetiological agent. RESULTS: About 30% of 240 calves usually kept in a calf rearing facility developed severe polyarthritis as a result of Mycoplasma sp bovine group 7 infection between 2 and 3 weeks of age. Multiple abortions occurred on these farms. Mycoplasma sp bovine group 7 was recovered from the fibrinopurulent synovial exudates of four 14-day-old calves, from the stomach contents and lungs of two aborted foetuses, from 14 of 21 bulk milk and four of 10 mastitic quarters. Three bulk colostrum samples cultured during the outbreak were negative for mycoplasma. CONCLUSION: Mycoplasma sp bovine group 7 caused significant economic losses as a result of polyarthritis, abortion and mastitis. The disease probably originated from udder infections with spread being facilitated by the decreased use of tetracycline in the treatment of mastitis. Neonatal calves were most likely infected by the consumption of milk contaminated with the organism. Abortions presumably resulted from mycoplasmaemia. This appears to be the first report in Australia of bovine abortion resulting from Mycoplasma sp infection.