Characterization of Pajaroellobacter abortibovis, the etiologic agent of epizootic bovine abortion. Brooks RS(1), Blanchard MT(1), Clothier. J Vet Diagn Invest. May;14(3) Diagnosis of epizootic bovine abortion in Nevada and identification of the vector. Hall MR(1), Hanks D, Kvasnicka W. Kennedy PC, Casaro AP, Kimsey PB, Bon Durant RH, Bushnell RB, Mitchell GM. The development of the fetal lesions of epizootic bovine abortion (EBA) was.

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Investigation of these losses has defined a specific fetal syndrome characterized by late-term abortion or birth of weak or dead calves. Although the unusual clinical presentation and unique fetal pathology associated with EBA have been recognized since the s, the identity of the etiologic agent is unknown.

In this study, suppression-hybridization PCR was used to identify a fragment of the 16S rRNA gene of a previously undescribed bacterium in thymus tissue derived from affected fetuses.

Phylogenetic analysis revealed that this pathogen was a deltaproteobacterium closely related to members of the order Myxococcales.

A specific PCR was subsequently developed to detect the presence of this bacterium in DNA extracted from fetal thymuses. The bacterium was also detected in the argasid tick Ornithodoros coriaceuswhich is the recognized vector of EBA. These data imply a close association between this novel agent and the etiology of EBA.

Epizootic bovine abortion EBAsometimes also called foothill abortion, is endemic in California’s coastal range and in the foothill regions of the Sierra Nevada. EBA continues to be the primary diagnosed cause of abortion in beef cattle in California and is estimated to be responsible for the loss of 45, to 90, beef calves annually.

EBA is transmitted to susceptible heifers by the bite of the soft argasid tick, Ornithodoros coriaceus referred to as the pajaroello tick 14 The tick lives in tree and brush litter in deer and cattle beds, feeds rapidly in less than an hour under experimental conditionsand then departs the host. Identification of this vector was facilitated by the demonstration that the geographic distribution of the tick largely parallels that of EBA 14 EBA is defined as a specific fetal syndrome characterized by late-term abortion or birth of weak or dead calves 8.

The unique fetal pathology of EBA has classically served as the only definitive basis of diagnosis 9 EBA-associated lesions are chronic, developing progressively over a period of 3 months or more. Gross lesions include mucosal and thymic hemorrhages, lymphadenopathy, splenomegaly, hepatomegaly, and ascites. Histologic examination of fetal tissues, particularly the lymphoid organs, is required to confirm a diagnosis 9 Thymic lesions unique to EBA include a loss of cortical thymocytes and extensive infiltration of macrophages into the medulla.

Widespread inflammatory lesions with a vascular orientation are evident in most tissues. Affected fetuses also have markedly elevated immunoglobulin levels, suggestive of a vigorous fetal immune response.

A number of detailed studies have incriminated a variety of microbes as potential causative agents of EBA, including a member of the chlamydial group originally called psittacosis-lymphogranuloma-venereumuncharacterized viruses, Borrelia coriaceaeand an uncharacterized spirochete 311121516171819202728bovin However, upon more detailed examination, all have been excluded as the probable EBA etiologic xbortion.

Characterization of Pajaroellobacter abortibovis, the etiologic agent of epizootic bovine abortion.

The recent development of a challenge system in which inoculation of EBA-diseased fetal thymus into pregnant 90 to days of gestation heifers could result in consistent and predictable transmission of the EBA agent has facilitated efforts to define the causative organism Abortin treatment with antibiotics at the time of EBA challenge abolished infection of the fetus epiaootic therefore implicated a prokaryotic organism as the likely causative agent Although all bacterial culture attempts have been unsuccessful, this breakthrough provided sufficient direction to develop a molecular approach for the identification of the etiologic agent.

Similar approaches have been successfully used boovine identify microbial pathogens, including those bovije for cat scratch fever, Whipple’s disease, human ehrlichiosis, hepatitis C, and hantavirus pulmonary syndrome 5 Fetal necropsy tissues were obtained from a combination of field-infected and experimentally infected heifers, as previously described The heifers were exposed to the agent of EBA by i natural exposure fetuses were submitted by ranchersii tick feeding under experimental conditions, or iii inoculation with tissue homogenate derived from fetuses previously diagnosed as being EBA positive.

Inoculation of heifers with tissue homogenates derived from apparently healthy fetuses was used as a control in these experiments. Fetuses from experimentally exposed heifers abortkon obtained either by surgical removal following dam euthanasia captive bolt in the third trimester to days postchallenge or as dead, weak, or healthy calves following natural parturition.

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In an additional experiment, six heifers were challenged with aliquots of a single thymus tissue pool previously demonstrated to be capable of transmitting EBA Three of these heifers were simultaneously treated with penicillin for 1 week and tetracycline for 3 epizoootic, while the remaining three were left epkzootic to serve as challenge controls Two of the three healthy calves born from antibiotic-treated dams were euthanized immediately prior to consumption of colostrumand tissues were collected for PCR analysis.

Necropsy tissues were collected using a sterile technique. Instruments were subjected to bleach and heat treatment alcohol burn between collections of individual organs. Tissues collected for histopathology formalin included brain, lung, heart, liver, kidney, thymus, lymph nodes, spleen, adrenal, muscle, and digestive tract samples.

A diagnosis of EBA was established on the basis of histopathology and epizoitic serum immunoglobulin G, as previously defined 910 Two different fetal bovine thymic pools containing tissue homogenates from two EBA-positive tester or three EBA-negative driver individual cases were prepared by phenol-chloroform extraction It has been hypothesized that driver-tester hybridization suppresses the amplification of ubiquitous bacterial 16S rRNA sequences, obvine unique bacterial sequences present only in the EBA-positive tissue samples to be amplified.

Amplicons identified on 1. In the tick studies, ticks were collected from locations where EBA was considered to be endemic, including the eastern slope of the Sierra Nevada mountain range in California and southeast Oregon, using dry abortiion as an attractant 614 The ticks were halved longitudinally with a disposable razor prior to DNA extraction.

These primer regions showed the most variation compared with other bacterial sequences in multiple-nucleotide alignments.

epizootlc Since nucleic acid samples extracted from ticks can often aborfion activity inhibitory to PCR, it was vital to establish an internal amplification control to discriminate true- and false-negative PCR results.

The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the following accession numbers: The present study employed an shPCR technique in an eppizootic to identify a bacterial agent bovinee in fetal thymic tissues collected from EBA cases.

This approach was used because preliminary cloning experiments using universal bacterial primers yielded sequences identical to those of Pseudomonas sp. Since subsequent PCR showed that these agents were present in both EBA-positive and -negative tissues, their presence was assumed to be the result of postmortem contamination of the samples. The use of shPCR allowed prokaryotic sequences derived from bovine tissues to be amplified in preference to contaminating bacteria.

Furthermore, since this technique utilizes gene-specific primers directed toward the commonly sequenced 16S rRNA gene, phylogenetic comparisons with other bacteria were possible. From the first experiment, sequences corresponding to a novel betaproteobacterium and Ochrobactrum anthropi were identified.

Two specific PCRs were subsequently developed to detect these agents separately in a panel of individual bovine thymus tissues derived from five EBA-positive and four EBA-negative animals.

Although these agents were detected in some of the DNA samples, the pattern for neither bacterium paralleled the clinical and histopathological diagnosis of EBA data not shown.

Second and third repetitions of the shPCR experiments were performed. In these experiments, an additional 13 clones were sequenced. Epizootic specific PCR was developed described in Materials and Methods to detect this deltaproteobacterium in bovine tissues. Epiziotic results using the small panel of nine bovine tissues see above showed complete concordance with the EBA status of the animals.

In light of these data, this bacterium was chosen as a eoizootic candidate for the putative agent of EBA. Consensus phylogram of 2, neighbor-joining trees generated for a 1,bp fragment of the 16S rRNA eppizootic of the putative agent causing EBA abortioon with sequences from 39 representative genera 7 of the class Deltaproteobacteria.

GenBank accession numbers are shown. Escherichia colia member of the class Gammaproteobacteriawas included as an outgroup in this analysis. The scale represents percent nucleotide substitutions per site. A combination of direct sequencing bp from 25 EBA-positive fetuses representing 16 field cases, four experimental tick transmissions, and five experimental tissue transmissions, the last using thymus homogenate derived from an EBA-positive fetus and Eepizootic blotting of these amplicons was used to confirm PCR specificity.

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Furthermore, the PCR failed to amplify DNA extracted from pure cultures of a selection of bacterial pathogens typically encountered in veterinary medicine or even P. Three heifers experimentally challenged with thymus tissue and simultaneously treated with antibiotics demonstrated the EBA agent to be prokaryotic These three heifers gave birth to healthy calves; two were euthanized immediately prior to consumption of colostrum for PCR analysis; all tissues were PCR negative.

In contrast, the three challenge control fetuses whose dams did not receive antibiotics all presented with classic EBA-associated lesions, and their tissues were PCR positive. Thymuses from all seven abortikn were PCR positive. Six of seven lymph nodes both prescapular and mesenteric and six of the seven spleens were also positive.

Characterization of Pajaroellobacter abortibovis, the etiologic agent of epizootic bovine abortion.

These preliminary results suggest that the agent is preferentially associated with lymphoid tissues. In addition to identifying this novel agent in diseased fetal bovine tissues, PCR has also demonstrated the presence of the deltaproteobacterium in the tick vector, O.

Preliminary data demonstrated the EBA agent to be present in 4 of 11 nymphs tested and 2 of 8 female ticks tested; The nucleic acid sequences were the same five were directly sequenced to confirm the identity of the PCR product as that aboftion in EBA fetal bovine tissues.

Epizoofic study demonstrates a strong association between the presence of a novel deltaproteobacterium and the development of EBA. The agent was detected in thymic tissues from affected fetuses and in the argasid tick vector of EBA. In the absence of a cultivable agent, it is proposed that these molecular data are sufficient evidence to classify this bacterium as the etiologic agent of EBA.

The majority of guidelines that have been suggested for sequence-based determination and incriminating episootic a aborton microorganism in disease 5 have been satisfied.

The phylogenetic location of the putative EBA etiologic agent was surprising, since none of the closest relatives have been demonstrated to be mammalian pathogens. The novel bacterium described in this paper represents only the second member of the delta subdivision of the class Proteobacteria to be a mammalian pathogen the first was Lawsonia intracellularis [ 413 ], the causative agent of porcine proliferative enteropathy.

The PCR developed in novine study proved to be a useful tool, allowing the detection of the novel bacterium in Epizoottic extracted from bovine tissues and ticks.

The inability to identify the agent in all ticks would be consistent with the low-level infection achieved by experimental transmission of EBA to cattle by tick bite. In summary, this work demonstrates a close relationship between a novel bacterium and EBA.

Efforts are under way to define additional gene sequences of this unique pathogen, to visualize the agent in affected tissues, and to visually link the rRNA sequence to the bacterium using in situ hybridization techniques on diseased fetal necropsy tissue.

In addition, with the development of a diagnostic PCR, efforts can be initiated to better define the ecology of EBA, including the identification of additional reservoirs of the bacterium and defining the association between the Abrtion agent and the tick vector. We acknowledge the assistance of Mike Oliver for tick collection and Richard Gniewek and Svetlana Khaiboullina for assistance in identifying the agent in ticks and bovine tissues, respectively.

National Center for Biotechnology InformationU. Journal List J Clin Epizootid v.

Blanchardbogine Brian M. Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC.

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Diagnosis of epizootic bovine abortion in Nevada and identification of the vector.

Nucleotide sequence accession numbers. Heifer exposure n Diagnosis a No. Non-EBA diagnoses included bovine viral diarrhea, neosporosis, Haemophilus somnusand unknown etiologies.

Tissue Presence of agent in fetus a: Denaturing gradient gel electrophoresis: Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Characterization of Borrelia coriaceae antigens with monoclonal antibodies. Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: Sequence-based identification of microbial pathogens:

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