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

Epizootic bovine abortion (EBA), first identified in the 1950s, is a major contributor of economic loss to western U.S. beef producers. The causative agent proved elusive for over fifty years until a novel Deltaproteobacteria was identified as the etiologic agent in 2005. The microbe, which has yet to be successfully cultured in vitro, has proven difficult to purify from necropsy tissues. Thus, phylogenetic characterization has been limited to analysis of the 16S ribosomal RNA (rRNA) gene (AF503916), which placed this bacterium in the order Myxococcales, suborder Sorangiineae, family Polyangiaceae and most closely related to Sorangium cellulosum. The focus of the current study was to further expand the morphologic characterization and taxonomic placement of this bacteria, named here as Pajaroellobacter abortibovis. Modified Gram staining, combined with transmission electron microscopy, provide strong evidence that the bacterium is gram negative. Flow cytometric analysis identified the presence of P. abortibovis in murine leukocytes. While attempts to sequence ten universally conserved protein-coding genes using previously published degenerative primers failed, redesigned primers based solely upon Deltaproteobacteria facilitated the partial sequencing of two genes; fusA (JQ173112) and pyrG (JQ173111). Primers designed in a similar fashion generated a partial sequence of the 23S rRNA gene (JQ173113) These sequences, combined with a revised 16S rRNA phylogenic analysis, support the placement of this bacteria as a unique genus separate from Sorangium.

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

The current study was directed at developing and validating an indirect fluorescent antibody test (IFAT) capable of detecting antibodies specific for the agent of epizootic bovine abortion (aoEBA). Sensitivity and specificity was determined by comparing antibody titers from 114 fetuses infected with aoEBA with 68 fetuses diagnosed with alternate infectious etiologies. Data established specificity at 100% and sensitivity at 94.7% when cutoff criteria for a positive test were assigned at a titer of ≥1,000. Potential cross-reactivity was noted in samples from 3 fetuses with antibody titers of 10 or100; all were infected with Gram-positive organisms. The remaining 65 fetuses infected with microbes other than aoEBA, and an additional 12 negative reference sera, did not have detectable titers. The IFAT-based serology assay is rapid, reproducible, and unaffected by fluid color or opacity. Total fetal immunoglobulin (Ig)G was also evaluated as an aid for diagnosing EBA. Significantly higher concentrations of IgG were identified in fetuses infected with aoEBA as compared to those with alternate infectious etiologies. The presence of IgG is a sensitive indicator of EBA and increases the specificity of FAT-based serologic diagnosis when titers are 10 or 100. Taken together, serology and IgG analyses suggest that the incidence of EBA may be underestimated.

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

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 ß-actin gene. Reaction efficiencies were 100.9% for the 16S aoEBA gene and 93.1% for the bovine ß-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 host-tissue 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.

Age-prevalence of Otarine Herpesvirus-1, a tumor-associated virus, and possibility of its sexual transmission in California sea lions.

Otarine Herpesvirus-1 (OtHV-1) is a gammaherpesvirus routinely detected in urogenital tumor tissues of adult sea lions dying during rehabilitation, To investigate the epidemiology of this virus and guide the development of a mathematical model of its role in the multifactorial etiology of cancer in California sea lions, polymerase chain reaction (PCR) amplification of an OtHV-1 specific fragment of the DNA polymerase gene was used to look for evidence of OtHV-1 infection in urogenital and pharyngeal swabs and peripheral blood mononuclear cells (PBMC) of sea lions of different ages. Samples were also examined from pregnant females and their late term in utero or aborted fetuses to investigate potential for vertical transmission. Prevalence of infection in 72 adult females was 22%, whereas it was 46% in 52 adult males, and was significantly lower in 120 juvenile animals (6%). OtHV-1 DNA was most often detected in the lower reproductive tract of the adult animals, especially the males, and rarely in the pharynx or urogenital tract of juvenile animals. These data suggest sexual transmission may an important route of transmission. Additional studies are required to confirm this mode of transmission. Additionally, the virus was detected in a single prematurely born pup, suggesting the possibility of perinatal transmission. No indication of a PBMC associated viremia was evident in adults using standard PCR or in juveniles using standard and real time PCR.

Antimicrobial properties of the chelating agent EDTA on Streptococcal bovine mastitis isolates.

To determine the efficacy of the chelating agent EDTA on microbial growth, separate cultures of two streptococcal bovine mastitis isolates, Streptococcus agalactiae and Streptococcus uberis, were exposed to known concentrations of EDTA. Bacterial cultures of 10(8) CFU/ml were exposed to concentrations of EDTA ranging from 30 to 100 mM in an in-vitro-milk environment. Multiple replications of cultures exposed to EDTA were plated during a two-hour time course. A concentration of 100 mM EDTA resulted in a 90% reduction of S. agalactiae and a 99% reduction of S. uberis. Under these experimental conditions, EDTA treatments in cultures of both isolates exhibited from 1 to 2 log reductions suggesting that EDTA is a potentially effective antimicrobial against streptococcal isolates implicated in causing bovine mastitis.

Infectious disease and the decline of Steller sea lions (Eumetopias jubatus) in Alaska, USA: insights from serologic data.

Serologic data were examined to determine whether infectious disease may have played a role in the decline of Steller sea lions (Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands, USA. Available published data, unpublished data, and recent collections (1997-2000) were compared and reviewed. Data were stratified by geography to compare the declining western Alaskan population in the Aleutian Islands through eastern Prince William Sound to the increasing population in southeastern Alaska. Prevalences of antibodies from the 1970s to the early 1990s were noted for Leptospira interrogans, Chlamydophila psittaci, Brucella spp., phocid herpesvirus-1, and calciviruses. Serum samples collected from 1997-2000 were tested for antibodies to these agents as well as to marine mammal morbilliviruses, canine parvovirus, and canine adenovirus-1 and -2. Conclusions could not be drawn about changes in antibody prevalence to these agents during the decline of Steller sea lions, however, because data were incomplete or not comparable as a result of inconsistencies in testing techniques. Despite these shortcomings, results provided no convincing evidence of significant exposure of Steller sea lions to morbilliviruses, Brucella spp., canine parvovirus, or L. interrogans. Steller sea lions have been exposed to phocid herpesviruses, caliciviruses, canine adenovirus, and C. psittaci or to cross-reactive organisms in regions of both increasing and decreasing sea lion abundance. Based on similar antibody prevalence estimates from the increasing and decreasing populations, these agents are unlikely to have been the primary cause of the population decline. They may have contributed to the decline or impeded population recovery, however, because of undetected mortality and morbidity or reductions of fecundity and body condition in animals under other stresses. Systematic monitoring for disease agents and their effects is needed to determine whether infectious disease currently plays a role in the decline and lack of recovery of Steller sea lions.

Coccidioidomycosis in Przewalski's horses (Equus przewalskii).

Coccidioidomycosis is a rare, often subclinical infection in domestic animals caused by the fungus Coccidioides immitis. Because of an apparent high incidence of coccidioidomycosis in Przewalski's horses (Equus przewalskii) housed at a single facility, necropsy records and biomaterials from animals that died between 1984 and 2000 were reviewed (n = 30, 15 males, 15 females). Coccidioidomycosis was the leading cause of death (33%) in this population with lesions in the lungs and tracheobronchial lymph nodes of all animals and variable involvement of the skeletal muscle, heart, kidney, liver, skin, brain, spinal cord, spleen, as well as other regional lymph nodes. At the time of death, affected horses tended to be younger than unaffected animals, were from multiple lineages, and males were over represented. During the same time period, no other exotic equids (n = 76) housed at the same facility were diagnosed with coccidioidomycosis, suggesting that environmental factors are not the sole cause of the high incidence in E. przewalskii. Numbers of the lymphocyte subsets (CD3, CD4, CD5, CD8, CD21+ cells) quantified by flow cytometry were similar between Przewalski's horses and domestic horses (Equus caballus). Although responses of lymphocyte blastogenesis assays were similar between Przewalski's (n = 5) and domestic horses (n = 5) in response to the T cell mitogen concanavalin A, lymphocytes from two of the Przewalski's horses failed to proliferate in response to Coccidioides. One of these horses had systemic disease and the second developed coccidioidomycosis 2 yr later. These results suggest that the immune system of some Przewalski's horses fails to respond appropriately to Coccidioides.