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.

Analysis of gene gain and loss in the evolution of predatory bacteria.

Predatory bacteria are ubiquitously distributed in nature in including in aquatic environments, sewage, intestinal tracts of animals and humans, rhizophere and, soils. However, our understanding of their evolutionary history is limited. Results of recent studies have shown that acquiring novel genes is a major force driving bacterial evolution. Therefore, to gain a better understanding of the impact of gene gain and loss in the evolution of bacterial predators, this study employed comparative genomic approaches to identify core-set gene families and species-specific gene families, and model gene gain and loss events among 11 genomes that represented diverse lineages. In total, 1977 gene families were classified. Of these 509 (pattern 11111111111) were present all of the 11 species. Among the non-core set gene families, 52 were present only in saltwater bacteria predators and had no ortholog in the other genomes. Similarly 109 and 44 were present only in the genomes of Micavibrio spp. and Bdellovibrio spp., respectively. In this study, the gain loss mapping engine GLOOME was selected to analyze and estimate the expectations and probabilities of both gain and loss events in the predatory bacteria. In total, 354 gene families were involved in significant gene gain events, and 407 gene families were classified into gene loss events with high supported value. Moreover, 18 families from the core set gene family were identified as putative genes under positive selection. The results of this study suggest that acquisition of particular genes that encode functional proteins in metabolism and cellular processes and signaling, especially ABC systems, may help bacterial predators adapt to surrounding environmental changes and present different predation strategies for survival in their habitats.

Purification and Host Specificity of Predatory Halobacteriovorax Isolates from Seawater.

Halobacteriovorax (formerly Bacteriovorax) is a small predatory bacterium found in the marine environment and modulates bacterial pathogens in shellfish. Four strains of Halobacteriovorax originally isolated in Vibrio parahaemolyticus O3:K6 host cells were separated from their prey by an enrichment-filtration-dilution technique for specificity testing in other bacteria. This technique was essential, since 0.45-µm filtration alone was unable to remove infectious Vibrio minicells, as determined by scanning electron microscopy and cultural methods. Purified Halobacteriovorax strains were screened for predation against other V. parahaemolyticus strains and against Vibrio vulnificus, Vibrio alginolyticus, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium DT104, all potential threats to seafood safety. They showed high host specificity and were predatory only against strains of V. parahaemolyticus. In addition, strains of Halobacteriovorax that were predatory for E. coli O157:H7 and S. Typhimurium DT104 were isolated from a tidal river at 5 ppt salinity. In a modified plaque assay agar, they killed their respective prey over a broad range of salinities (5 to 30 ppt). Plaques became smaller as the salinity levels rose, suggesting that the lower salinities were optimal for the predators' replication. These species also showed broader host specificity, infectious against each other's original hosts as well as against V. parahaemolyticus strains. In summary, this study characterized strains of Halobacteriovorax which may be considered for use in the development of broad-based biocontrol technologies to enhance the safety of commercially marketed shellfish and other foods.

Hematologic and IgG responses of heifers experimentally infected with the agent of epizootic bovine abortion.

BACKGROUND: Epizootic bovine abortion (EBA) is a tick-transmitted abortive disease of beef cattle in the western United States. Infected cattle do not have clinical signs until abortion occurs, usually within the last trimester of gestation. There is little information on the hematologic response of the dam following infection. OBJECTIVE: The purpose of this study was to determine if changes in blood leukocytes and serum IgG concentrations could be detected following experimental infection of pregnant heifers with the etiologic agent of EBA (aoEBA). METHODS: Twelve Angus heifers were infected during gestation with the aoEBA using an inoculum prepared from the thymus of an infected fetus. Five pregnant heifer controls were given an inoculum prepared from the thymus of an aoEBA-negative calf. PCVs, total and differential leukocyte counts, and serum IgG concentrations were measured weekly following administration of the inocula until abortion or calving. Gross and microscopic examinations were performed on all aborted fetuses to confirm infection. RESULTS: Eleven of 12 heifers in the treatment group aborted, and significant findings were decreased lymphocyte counts at weeks 1 and 14 postinoculation and increased monocyte counts at week 4 compared with control animals. Serum IgG concentrations were significantly increased at weeks 6-8 and 11 in the treatment group. CONCLUSIONS: Leukogram changes are subtle in infected cattle. Future research efforts should be aimed at development of an antibody test specific for detection of previously infected animals, which could graze safely on EBA-endemic pastures.

Selective predation and rapid evolution can jointly dampen effects of virulent parasites on Daphnia populations.

Parasites are ubiquitous and often highly virulent, yet clear examples of parasite-driven changes in host density in natural populations are surprisingly scarce. Here, we illustrate an example of this phenomenon and offer a theoretically reasonable resolution. We document the effects of two parasites, the bacterium Spirobacillus cienkowskii and the yeast Metschnikowia bicuspidata, on a common freshwater invertebrate, Daphnia dentifera. We show that while both parasites were quite virulent to individual hosts, only bacterial epidemics were associated with significant changes in host population dynamics and density. Our theoretical results may help explain why yeast epidemics did not significantly affect population dynamics. Using a model parameterized with data we collected, we argue that two prominent features of this system, rapid evolution of host resistance to the parasite and selective predation on infected hosts, both decrease peak infection prevalence and can minimize decline in host density during epidemics. Taken together, our results show that understanding the outcomes of host-parasite interactions in this Daphnia-microparasite system may require consideration of ecological context and evolutionary processes and their interaction.

Analyses of the vrl gene cluster in Desulfococcus multivorans: homologous to the virulence-associated locus of the ovine footrot pathogen Dichelobacter nodosus strain A198.

Major parts of the virulence-associated vrl locus known from the gammaproteobacterium Dichelobacter nodosus, the causative agent of ovine footrot, were analyzed in the genome of the sulfate-reducing deltaproteobacterium Desulfococcus multivorans. In the genome of D. multivorans 13 of the 19 vrl genes described for D. nodosus are present and highly conserved with respect to gene sequence and order. The vrl locus and its flanking regions suggest a bacteriophage-mediated transfer into the genome of D. multivorans. Comparative analysis of the deduced Vrl proteins reveals a wide distribution of parts of the virulence-associated vrl locus in distantly related bacteria. Horizontal transfer is suggested as driving mechanism for the circulation of the vrl genes in bacteria. Except for the vrlBMN genes D. multivorans and Desulfovibrio desulfuricans G20 together contain all vrl genes displaying a high degree of similarity. For D. multivorans it could be shown that guanine plus cytosine (GC) content, GC skew, di-, tri- or tetranucleotide distribution did not differ between the vrl locus and its flanking sequences. This could be a hint that the vrl locus originated from a related organism or at least a genome with similar characteristics. The conspicuous high degree of conservation of the analyzed vrl genes may result from a recent transfer event or reflect a function of the vrl genes, which is still unknown and not necessarily disease associated. The latter is supported by the evidence for expression of the vrl genes in D. multivorans, which has not been described as pathogen or to be associated to any disease pattern before.

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.

Coral disease diagnostics: what's between a plague and a band?

Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.