Protective immunity induced through two calving seasons following administration of live epizootic bovine abortion agent (EBAA) vaccine.

A live, infectious vaccine candidate for epizootic bovine abortion, designated EBAA Vaccine, USDA-APHIS Product code #1544.00, has been reported to be both safe and effective. Previous studies established that a single dose of EBAA vaccine administered to cows at potencies of either 2000 or 500 live P. abortibovis-infected murine spleen cells (P.a.-LIC) induced protective immunity for a minimum of 5 months. The current study employed 19 pregnant cows that were challenged with P. abortibovis in their 2nd trimester of gestation; 9 were vaccinated 17.2-months earlier as 1-year-olds with 2000 P.a.-LIC and 10 served as negative controls. Eighty-nine percent of the vaccinates gave birth to healthy calves as compared to 10% of challenge controls. Vaccine efficacy was significant when analyzed by prevented fractions (87.7%; 95% CI=0.4945-0.9781). Serologic data supports previous findings that pregnant cows with detectable P. abortibovis antibodies are immune to P. abortibovis challenge as demonstrated by the birth of healthy calves.

Protection of Cattle against Epizootic Bovine Abortion (EBA) Using a Live Pajaroellobacter abortibovis Vaccine.

Epizootic bovine abortion (EBA) is an arthropod-borne bacterial disease that causes significant economic loss for cattle producers in the western United States. The etiologic agent, Pajaroellobacter abortibovis, is an intracellular pathogen that has yet to be cultivated in vitro, thereby requiring novel methodologies for vaccine development. A vaccine candidate, using live P. abortibovis-infected cells (P.a-LIC) harvested from mouse spleens, was tested in beef cattle. Over the course of two safety studies and four efficacy trials, safety risks were evaluated, and dosage and potencies refined. No incidence of anaphylaxis, recognized health issues or significant impact upon conception rates were noted. Vaccination did result in subclinical skin reactions. Early fetal losses were noted in two trials and were significant when the vaccine was administered within 21 days prior to conception. Administration of the EBA agent (EBAA) vaccine as a single dose, at a potency of 500 P.a-LIC, 56 days prior to breeding, provided 100% protection with no early fetal losses. Seroconversion occurred in all animals following EBAA vaccination and corresponded well with protection of the fetus from epizootic bovine abortion.

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

Epizootic bovine abortion (EBA), or "foothill abortion," is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus, or "pajaroello tick," was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice.

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.

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca).

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify "insults" and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic knowledge derived from longitudinal immunologic studies in killer whales, as was the target of the present study, has the potential to improve diagnostics and health related decision making for this and other domestic and free-ranging cetacean species.

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.

The immune response of foals to natural infection with equid herpesvirus-2 and its association with febrile illness.

Equid herpesvirus-2 (EHV-2) infection is ubiquitous in horses. Although EHV-2 infection has been associated with several disease syndromes, its true pathogenic significance in horses remains uncertain. Epstein-Barr virus (EBV), another gammaherpesvirus, has been shown to cause febrile illness in humans related to its immunopathologic effects. Thus, the purpose of this study was to describe the ontogeny of the immune response of a cohort of 9 foals to natural infection with EHV-2 by evaluating serial complete blood counts, lymphocyte morphology, cytokine gene expression in peripheral blood mononuclear cells (PBMC), viral load in nasal swabs and blood, and antigen-specific cellular immune responses of PBMC, in conjunction with clinical evaluation of the foals. The occurrence of fever in foals was not related to lymphocytosis or specific changes in lymphocyte morphology, cytokine gene expression, or viral load, but tended to be associated (P

Differential levels of mRNA transcripts encoding immunologic mediators in mammary gland secretions from dairy cows with subclinical environmental Streptococci infections.

Dry-off, and the period around parturition, are associated with increased susceptibility to intramammary infections in dairy cows. The immunological profiles of mammary gland secretions during these periods are not well described. The objective of the present study was to better characterize association(s) between chronic subclinical Environmental Streptococci infections at dry-off and relative levels of mRNA transcripts encoding multiple immunologic mediators present in cells derived from mammary gland secretions at dry-off and continuing through parturition. The chronic subclinical bacterial infections in the present study were characterized by multiple isolations of Streptococcus species and elevated SSC for a minimum of three weeks prior to dry-off. The majority of differences between principal and control quarters were identified at dry-off. Transcript levels of IL-17, IL2Rα and iNOS were increased while pro-inflammatory cytokine IL-6, and the regulatory cytokine IL-10, were reduced. Following antibiotic treatment of mammary glands, IL-17 transcripts remained elevated over the course of the study, indicative of a persistent insult. IL-4 transcript levels were modestly elevated at 7 days following dry-off and significantly elevated at 14 days, consistent with activated T(H)1 and T(H)2 lymphocytes in the principal quarters, respectively. From a temporal perspective, transcript levels of IL-8 decreased in all animals through the dry-off period animals and returned to pre-dry-off levels at parturition; levels of iNOS peaked at parturition. Five of the six principal cows experienced recurrent bacterial mastitis during the subsequent lactation; four were in the same quarter as was initially infected with Streptococcus and three of these four were due to coliforms. Taken together, this apparent chronic susceptibility of select mammary glands to bacterial infection would suggest a physiologic and/or immunologic dysfunction. Identification of factor(s) that contribute to the predisposition of mammary glands to developing mastitis should facilitate development of new control strategies.

Cellular immune responses in cetaceans immunized with a porcine erysipelas vaccine.

Clinical erysipelas represents a significant health problem in managed cetacean species. Vaccination was suspended in many oceanariums in the past due to losses associated with vaccine-induced hypersensitivities which were deemed to be a greater threat than clinical erysipelas. A perceived shift in clinical presentation of erysipelas from a chronic dermatologic form to an acute systemic form in dolphins sparked interest in re-initiating vaccination with improved subunit vaccines of Erysipelothrix rhusiopathiae. This manuscript describes the development and application of in vitro correlates of immunity (T(H)1, T(H)2 and T(REG)) in Tursiops truncatus induced by immunization with a commercial porcine 65 kDa subunit E. rhusiopathiae vaccine. Variable degrees of pre-existing T cell memory were identified prior to vaccination. Vaccine-induced IFN gamma responses were consistent with a T(H)1 response and associated with elimination of erysipelas in all vaccinated animals. Comparative analysis between six-month and 12-month vaccination booster regimes demonstrated maintenance of superior memory in the six-month group; however, anamnestic responses induced by booster were only identified in the 12-month group. To our knowledge, this is the first study to develop and apply advanced immunologic analyses for assessing vaccine efficacy in captive or free-ranging wildlife.

Serial passage of the etiologic agent of epizootic bovine abortion in immunodeficient mice.

Molecular studies have provided convincing evidence that a unique deltaproteobacterium is the causative agent of epizootic bovine abortion (EBA). Bovine fetuses, infected following dam exposure, are the only identified susceptible mammalian host. The inability to cultivate the bacterial agent of EBA (aoEBA) in vitro, associated with the substantial cost of bovine experimentation, drove efforts to identify an alternative laboratory animal host. Mice with severe combined immunodeficiency (SCID) were chosen as a potential host after immunocompetent mice proved resistant to infection. SCID mice inoculated with aoEBA-infected bovine fetal thymus homogenates began to show clinical signs at 2 months and became increasingly cachectic over the next 1-2 months. Following a 2nd passage (P2) through SCID mice, three susceptible pregnant heifers were inoculated with P2 murine tissue homogenates. All three fetuses presented with lesions indistinguishable from naturally occurring EBA, confirming successful passage of the bacterial pathogen in SCID mice. All murine (P1 and P2) and bovine fetal tissues contained aoEBA as determined by PCR; 16S bacterial ribosomal nucleotide sequences were identical in all murine and fetal bovine tissues examined. Bacteria in fetal bovine tissues were determined to be heavily opsonized, based upon microscopic evaluation of tissues stained with either FITC-conjugated anti-bovine IgG or biotin-conjugated anti-bovine IgG in conjunction with avidin-FITC. Unlike the near-term bovine fetus, the absence of an antibody response in infected SCID mice permits harvest of unopsonized bacteria for development of serologic assays.

Quantitation of leukocyte gene expression in cetaceans.

Real-time quantitation of cytokine mRNA is a routine immunologic technique, especially fitting for use in those species for which monoclonal antibodies are not available. Quantitative gene expression assays were developed to assist in the immunologic assessment of three cetacean species including bottlenosed dolphins, Pacific white-sided dolphins and beluga whales. Nine cytokine genes (IL-2, -4, -10, -12, -13, -18, TNFalpha, TGFbeta and IFNgamma) and Cox-2 were selected for analysis. Most mitogen-induced mononuclear leukocyte responses were similar between the three cetacean species with either up- or down-regulation of cytokine genes. IL-10 expression was highly variable between species. No TH/1TH2 polarization was evident. Cytokine gene analysis has the potential to identify immune system perturbations induced by environmental insult as well as providing diagnostic tools for characterizing immune responses to environmental antigens and vaccines.

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.

Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion).

Epizootic bovine abortion (EBA) is endemic in California's coastal range and the foothill regions of the Sierra Nevada, where it has been the primary diagnosed cause of abortion in beef cattle for >50 years. 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 1950s, 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. Using histopathology as the definitive diagnosis for EBA, this PCR demonstrated 100% specificity and 88% sensitivity. The bacterium was also detected in the argasid tick Ornithodoros coriaceus, which is the recognized vector of EBA. These data imply a close association between this novel agent and the etiology of EBA.

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.

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

In the 43 years since the first description in California, epizootic bovine abortion (EBA) has been considered but not definitively diagnosed as a cause of late-term abortions on Nevada ranches. Examination of aborted full-term bovine fetuses obtained from Nevada ranches revealed gross abnormalities consistent with EBA (enlarged lymph nodes, petechial hemorrhages of the oral mucosa and conjunctiva, ascites, and splenohepatomegaly), and EBA was confirmed by histologic examination of fetal tissues. The histologic thymic changes were characteristic of EBA and included severe histocytic thymusitis with depletion of thymocytes, interlobular hemorrhage, and fibrinocellular exudation. The gross enlargement of lymph nodes was the result of cortical follicular hyperplasia and histiocytic lymphadenitis. In addition, widespread, predominately nonsuppurative histologic lesions typical of EBA were observed in most organs, including the brain, lung, heart, liver, and spleen. Furthermore, the presence of Ornithodorus coriaceus, the argasid tick vector of EBA, was established by tick collection using CO2 traps. The tick was identified on ranches and in geographic areas (northern and northwestern counties of Nevada) coincident with diagnosis of multiple cases of EBA. This study establishes the presence of EBA as a cause of late-term abortion in Nevada. Additionally, identification of the EBA tick vector, O. coriaceus, in the same areas as the abortions provides strong evidence that the disease is endemic.