Histochemical and Immunohistochemical Evidence of a Bacterium Associated with Lesions of Epizootic Bovine Abortion

Abstract

Abstract. 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.

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

“…Recent development of an immunohistochemical assay to visualize the bacterium in tissue has increased the specificity of diagnosis, 1 and conventional PCR has increased both sensitivity and specificity of diagnosis.…”

“…1 Genomic DNA was extracted from cryopreserved samples according to manufacturer's instructions i with the following modifications: 40 µl of proteinase K was used during the overnight digestion of tissues. Purified DNA was eluted from spin columns using 100 µl of nuclease-free water.…”

“…[6][7][8][9] A loss of cortical thymocytes and extensive infiltration of macrophages into the medulla of the fetal thymus are pathognomonic for EBA. 1,7,9 Fetuses also have elevated levels of immunoglobulins (Ig) that are specific for the bacterium. 2,12 The development of a reliable bovine challenge model in which lesions indistinguishable from those observed in naturally infected animals provided a much needed experimental system with which to identify the etiologic agent and advance EBA pathogenesis studies.…”

“…2,12 The development of a reliable bovine challenge model in which lesions indistinguishable from those observed in naturally infected animals provided a much needed experimental system with which to identify the etiologic agent and advance EBA pathogenesis studies. 1,9,16 Upon identification of a novel deltaproteobacterium as the causative agent of EBA (aoEBA) in 2005, new methodologies were developed to identify the bacterium in fetal and tick tissue 1,4,10,17 ; sensitivity and specificity of a polymerase chain reaction (PCR)-based diagnosis approached 88% and 100%, respectively. 10 While diagnostic capabilities have improved, techniques for quantitative assessment of bacterial load in fetal tissues are needed as this bacterium has yet to be cultivated in vitro.…”

Whole Genome Analysis of Sierra Nevada Virus, a Novel Mononegavirus in the Family Nyamiviridae

“…4 Subsequent experimental studies in heifers have indicated that BA-T virus was not the causative agent of EBA. 5,6 The disease has now been associated with a deltaproteobacterium transmitted by the same tick species, 7,8 but the BA-T virus has remained uncharacterized. Because the original designation, bovine abortion-tick (BA-T) virus, is misleading, we propose instead the name "Sierra Nevada virus" (SNVV) for the new virus.…”