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.     

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.     

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.     

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.     

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.     

Salmonella enterica subsp. arizonae-associated abortion in a mare

Abortion in mares can be a financially devastating event to horse owners and the equine industry. In this case report, we describe the gross, histopathological and microbiological findings associated with an abortion caused by Salmonella enterica subsp. arizonae, which should be considered in cases of abortion and metritis. Gross and histopathological evaluation of fetal and placental tissue were consistent with ascending septic placentitis resulting in fetal septicaemia. S. enterica subsp. arizonae was isolated from the placenta, lung and fetal gastric contents. Diagnosis was confirmed by MALDI-TOF MS. To our knowledge, this subspecies of S. enterica has not been previously reported as an isolate in a case of late-term equine abortion. A retrospective search of abortion cases at our institution was done, and no similar cases were identified for comparison. This case demonstrates S. enterica subsp. arizonae, although not previously reported as a cause of abortion in pregnant mares and uncommonly isolated from equids in the literature, may cause late-term abortions in susceptible animals. This pathogen should be considered in cases of abortion outbreaks on a farm and merits investigation of the carrier status of mares within a herd.     

Evidence for Parachlamydia in bovine abortion

Bovine abortion of unknown infectious aetiology still remains a major economic problem. In this study, we focused on a new possible abortigenic agent called Parachlamydia acanthamoebae. Retrospective samples (n=235) taken from late-term abortions in cattle were investigated by real-time diagnostic PCR for Chlamydiaceae and Parachlamydia spp., respectively. Histological sections of cases positive by real-time PCR for any Chlamydia-related agent were further examined by immunohistochemistry using specific antibodies. Chlamydophila abortus was detected only in three cases (1.3%) by real-time PCR and ArrayTube Microarray playing a less important role in bovine abortion compared to the situation in small ruminants in Switzerland. By real-time PCR as many as 43 of 235 (18.3%) cases turned out to be positive for Parachlamydia. The presence of Parachlamydia within placental lesions was confirmed in 35 cases (81.4%) by immunohistochemistry. The main histopathological feature in parachlamydial abortion was purulent to necrotizing placentitis (25/43). Parachlamydia should be considered as a new abortigenic agent in Swiss cattle. Since Parachlamydia may be involved in lower respiratory tract infections in humans, bovine abortion material should be handled with care given the possible zoonotic risk.     

Leptospira abortion in horses

Leptospira infection was diagnosed as the cause of 4 late-term equine abortions/stillbirths and 1 neonatal death in Louisiana. The most consistent gross and microscopic lesions were icterus and interstitial nephritis, respectively. Diagnoses were based on visualization of compatible spirochetes in Warthin-Starry-stained sections of kidney, liver, and placenta. Confirmation by immunofluorescence was made in 2 cases.     

Descriptive epidemiology of late-term abortions associated with the mare reproductive loss syndrome in central Kentucky.

Epidemiological and pathological findings of 433 late-term abortions associated with the mare reproductive loss syndrome (MRLS) in central Kentucky were identified by reviewing the records of the University of Kentucky Livestock Diseases Diagnostic Center. The distribution of dates of abortion was clustered during a brief period of time, presumably from a simultaneous environmental exposure. The most common pathological findings were microscopic pulmonary lesions consisting of squamous epithelial cells present in alveoli with or without concurrent infiltration of inflammatory cells (neutrophils, macrophages, or monocytes) in the interstitium or within alveoli. Isolation of a non-beta-hemolytic Streptococcus (52% of fetuses) or an Actinobacillus sp. (19% of fetuses) was common. Placentitis or funisitis was identified in 44% of fetuses. No single pathological finding, however, was pathognomonic for MRLS-associated late-term abortion. This report describes the pathological findings characterizing the MRLS-associated abortion. A cause of MRLS could not be determined from necropsy findings.     

Fetal and placental lesions in bovine abortion due to Tritrichomonas foetus

Laboratory records and histologic material from 13 cases of naturally occurring, early- to late-term bovine abortion due to Tritrichomonas foetus were examined. Placentitis was present in all placentas. Pyogranulomatous bronchopneumonia was in six of the 11 lungs which were examined. Trichomonad organisms were demonstrated within the placental stroma in all cases and free or phagocytized in pulmonary airways in seven cases.     

Tularemia in range sheep: an overlooked syndrome?

Abortion and death caused by Francisella tularensis were well recognized in range flocks of domestic sheep in Idaho, Montana, and Wyoming in the first 6 decades of the 20th century. The current report describes 4 episodes of tularemia in 3 range flocks in Wyoming and South Dakota in 1997 and 2007 (1 flock was affected twice). Flock owners reported that ticks were unusually numerous and commonly present on sheep during outbreaks. Tularemia presented as late-term abortions (3 episodes) or listlessness and death in lambs and, to a lesser extent, ewes (1 episode). Lesions were multifocal pinpoint necrotic foci in tissues, particularly spleen, liver, and lung. An immunohistochemical procedure demonstrated F. tularensis, particularly in necrotic foci. The diagnosis was corroborated by bacterial isolation and, in individual cases, by serology, fluorescent antibody assay, and/or polymerase chain reaction detection of F. tularensis. Diagnosticians in endemic areas should include tularemia as a differential diagnosis when investigating late-term abortions or outbreaks of fatal illness in young lambs, particularly in years of high tick activity and when characteristic necrotic foci occur in spleen, liver, and lung.     

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.     

Etiologic agents detected in a 10-year study of bovine abortions and stillbirths.

During a 10-year period, specimens from 8,995 bovine abortions were submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory; 8,962 of these specimens were suitable for examination. A supportable diagnosis was made in 2,942 (32.82%) of the abortions examined. An infectious cause was determined for 2,723 (30.38%) and a noninfectious cause for 219 (2.44%). Bacteria caused 1,299 (14.49%), viruses caused 948 (10.57%), and fungi caused 476 (5.31%). The infectious bovine rhinotracheitis virus was associated with 485 (5.41%) and the bovine viral diarrhea virus with 407 (4.54%). The most common noninfectious causes of abortion or stillbirth were anomalous development, 112 (1.25%); dystocia, 56 (0.62%); and twinning, 37 (0.41%). The cause of 6,020 (67.17%) abortions was not determined. Lesions, many suggesting the presence of infection, were present in 1,554 (17.34%) fetuses or placentas in which no infectious agent could be demonstrated.     

Chlamydia-related abortions in cattle from Graubunden, Switzerland

In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.     

Bovine abortions attributable to Listeria ivanovii: four cases (1988-1990).

During a 3-year period, 4 cases of bovine abortion attributable to Listeria ivanovii were diagnosed from 243 bovine fetuses submitted for diagnostic evaluation. Listeria monocytogenes was isolated only once from a bovine fetus during this same time period. Pathologic findings were similar to those seen in abortions attributable to L monocytogenes. Consistent management factors were not recognized and breed susceptibility was not apparent. Listeria ivanovii is most often associated with abortions from sheep and is rarely reported from cattle. On the basis of findings in this study, L ivanovii must be included as a potential cause of bovine abortions.     

Diagnostic studies of the fetus, placenta and maternal blood from 265 bovine abortions

During 1981, the fetus, placenta and maternal serum were received from each of 265 bovine abortions. These specimens were examined using histopathological, histochemical, bacteriological, mycological, endocrinological, immunological, serological and virological techniques. The cause of abortion was identified in 98 (37%) cases. Of these diagnosed abortions 27 (28%) were due to infection with fungi, 17 (17%) to Salmonella spp, 11 (11%) to Campylobacter fetus and 10 (10%) to Corynebacterium pyogenes infection. Of the remaining 33 (34%) diagnosed abortions 8 were due to Leptospira sp, 5 due to protozoan (probably Sarcocystis) encephalitis and 20 due to miscellaneous bacterial infections (including Brucella abortus) and fetal deformities. Bovine virus diarrhoea virus was isolated in 5 (2%) abortions, however the significance of the isolations was not known. Infectious bovine rhinotracheitis virus and chlamydia were not identified as causal agents in any abortion. In 18 (7%) abortions no definite etiologic agent was identified despite pathological findings suggestive of infection. Fetal heart blood serology and immunoglobulin values were not reliable indicators of infectious abortion. Of the remaining 149 (56%) abortions, 17% had high maternal serum cortisol levels and twin fetuses occurred in 9%.     

Diagnostic des avortements infectieux chez les bovins laitiers.

During a two year period, March 1977 to April 1979, a total of 92 bovine abortions were studied. The cause of abortion was determined in 34.8% of the cases examined. Opportunistic bacteria, the most commonly diagnosed cause of abortion, accounted for 31.2% of the cases. Leptospirosis was associated with 28.1% of the abortions, infectious bovine rhinotracheitis and fungi in respectively 15.7%, bovine viral diarrhea in 6.2%. A congenital abnormality accounted for one case (3.1%). In 23 cases (25%), there was no definitive diagnosis, in spite of evidence of experience with pathogen or suggestive findings of pathology, but insufficient evidence to warrant diagnosis. No findings were recorded in 35.8% of the (possibly noninfectious) cases and in only four cases (4.4%), specimens were unsatisfactory for examination.     

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.     

An epidemiologic study of late-term abortions in dairy cattle in Denmark, July 2000-August 2003

Abortion in dairy cows in well-managed dairies is not common but differences have been reported probably due to variation in animal health, nutrition and management, as well as difficulties in observing the aborted material. A 38-month study of 507 large Danish dairy herds revealed 3354 late-term abortions and 224,419 calvings were recorded. During the study period, a total of 3717 submissions were made to the Danish Institute for Food and Veterinary Research (DFVF). A broad spectrum of abortive agents was isolated but none were found to be statistically associated with abortions. The number of abortions in a month on a dairy was significantly (P<0.001) associated with the number of cows in the third trimester but explained only 11% (R(adj)(2)=0.114) of the variability of the reported abortions. A total of 23 herds (4.5%) reported 531 abortions (15.8%). Although a marginally significant (P=0.11) risk of increased abortions was found to exist in bovine viral diarrhea virus- (BVDV) infected herds, it could be at least partially explained by additional calvings in those herds. Temporal correlation between inseminations and abortions was statistically significant (P<0.001) with the highest correlation (r=0.47-0.51) after lagging abortions on insemination by 6-8 months. No indication of spatial clustering was detected for either specific-abortogenic pathogens or high aborting dairies using either Cuzick-Edwards' (P>0.17) or spatial scan tests (P>0.23). Ederer-Myers-Mantel test was applied to 3 years of data on the highest aborting dairies and showed that July had nearly double the expected number of maximum monthly abortions (P<0.001). These findings provide further insight into the reported abortion pattern in Danish dairies and may facilitate planning future control programs.