Quantitative duplex TaqMan real-time polymerase chain reaction for the assessment of the etiologic agent of epizootic bovine abortion

Abstract

Abstract. Epizootic bovine abortion (EBA), also commonly known as "foothill abortion," is a late-term abortion primarily in beef cattle with significant economic impacts in California, Nevada, and Oregon. The causative agent is a novel deltaproteobacterium (aoEBA) closely related to the order Myxococcales and vectored by the soft-shelled tick Ornithodoros coriaceus. Historically, diagnosis has relied upon the pathologic examination of the fetus and the presence of elevated fetal serum immunoglobulins. Identification of the etiologic agent, a unique deltaproteobacterium, permitted the development of a quantitative duplex real-time polymerase chain reaction (qPCR) using a unique 90-bp sequence of aoEBA 16S ribosomal RNA gene in conjunction with an 88-bp sequence of the bovine β-actin gene. Reaction efficiencies were 100.9% for the 16S aoEBA gene and 93.1% for the bovine β-actin gene. Application of the duplex TaqMan to a set of aoEBA-infected fetal bovine necropsy tissues demonstrated the assay to be robust in quantitatively identifying the aoEBA bacteria and establishing hosttissue pathogen load. Consistent with previously reported immunohistochemical data, organized lymphoid tissue generally carried the heaviest bacterial load as compared to non-lymphoid tissue. The newly developed duplex TaqMan assay will facilitate diagnosis in difficult cases and provide an invaluable tool for delineating the pathogenesis of EBA.

Assessment of a fluorescent antibody test for the detection of antibodies against epizootic bovine abortion

“…This is consistent with previous demonstrations that an infected fetus can mount a robust immune response that is likely responsible for bacterial clearance prior to abortion. 4 Data from the current study suggests EBA is underdiagnosed. Three aoEBA-infected fetuses with minimal lesions and 5 fetuses diagnosed with undetermined etiologies not suggestive of EBA were determined to have antibody titers ≥1,000.…”

“…Tissues from fetuses with lesions consistent with or suspected of EBA and fetuses with IFAT antibody titers ≥1,000 but undetermined etiologies were examined using IHC as previously described. 1 A quantitative PCR assay specific for aoEBA 4 was conducted on frozen necropsy tissues in cases where IHC results were equivocal or negative and frozen tissues were available. Thymus, spleen, prescapular lymph node, and mesenteric lymph node were the preferred tissues for PCR analysis.…”

“…Thymus, spleen, prescapular lymph node, and mesenteric lymph node were the preferred tissues for PCR analysis. 1,4 Fetuses were assigned a definitive diagnosis of EBA if aoEBA bacteria were detected in 1 or more tissues (aoEBAinfected). The remainder was categorized as either those diagnosed with infectious etiologies from microorganisms other than aoEBA or those with undetermined etiologies (Table 1).…”

“…10 Diagnosis has been historically based on histology, elevated fluid immunoglobulin (Ig)G, 13 and dam history. The etiologic agent of EBA (aoEBA) is now known to be a novel organism in the class Deltaproteobacteria within the order Myxococcales , 11 and diagnosis is currently being supplemented with immunohistochemical staining (IHC) 1 and polymerase chain reaction (PCR) 4,11 assays to detect the microbe.…”

Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology

“…Identification of this Deltaproteobacterium, Pajaroellobacter abortibovis , allowed for the development of new diagnostic methodologies ( Blanchard et al 2010 , 2014 ; Brooks et al 2011 ). The visualization of P. abortibovis through immunohistochemistry and DNA-based diagnostic techniques using PCR have increased diagnostic sensitivity ( Anderson et al 2006 ; Brooks et al 2016 ).…”