Identification and characterization of a novel pathogen causing bovine abortion

Epizootic bovine abortion (EBA), commonly known as foothill abortion, is the leading cause of beef cattle abortion in California, responsible for the loss of an estimated 45,000 to 90,000 calves per year. In 2005, a novel deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA). Thus far, it is not possible to grow this organism in culture using traditional microbiological techniques; rather, it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. This led to the development of a live bacterial vaccine consisting of a quantifiable number of aoEBA-infected mouse spleen cells. Difficulties and costs associated with production of this live bacterial vaccine motivated our investigation into the development of a recombinant vaccine as an alternative approach to help prevent EBA. The objectives of this study were to perform a de novo genome assembly for the novel aoEBA deltaproteobacterium, and subsequently identify and validate potential antigenic proteins as candidates for the future development of a recombinant vaccine. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following their exposure to the aoEBA deltaproteobacterium. 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 50 × coverage into a 1.82 Mbp complete closed circular deltaproteobacterial genome, containing 2250 putative protein coding sequences. The phylogenetic analysis of aoEBA 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 candidate proteins that were likely to be antigenic. Proteins translated from the top nine candidate antigenic genes were run on Western blots and tested against serum from mice and cattle that had been infected by the bacterium. Six of the top nine candidates were bound by antibodies in the serum from infected mice, and the top candidate was strongly bound by serum from infected mice, cows and calves, but not serum from controls. This study provides the basic background information required to further the development of a recombinant vaccine for this orphan disease, which is California's leading cause of abortio