Other vaccine attempts NVP-BKM120 cell line have included a variety of subunit vaccines, none of which provided complete protection against heterologous challenge [3] and [4]. In addition, while infection with one strain of A. marginale sensu stricto typically precludes infection with another, multiple cases of superinfection have been described [5], [6] and [7]. Vaccine failures are due to expression of variants of the major surface proteins

MSP2 and MSP3. A. marginale creates a wide array of antigenic variants by substitution of whole or partial pseudogene cassettes into a single genomic expression site by segmental gene conversion [8], [9], [10] and [11], with increasing complexity of the expressed mosaic proteins [12]. Following persistent infection, the immune system has

Ku-0059436 order been exposed to a majority of the simple variants, which prevents another strain with similar variants from establishing concurrent infection. However, if the second strain has a unique pseudogene, novel variants generated by segmental gene conversion allow superinfection to take place [13]. In addition to MSP2 and MSP3, a variety of other variable surface antigens have been found in A. marginale; these have been called the msp2 superfamily [14]. Generally, these are all members of the pfam01617 (Surface Ag 2), which has related members in several other bacterial genera. Several of these have been found in cross-linked surface antigen complexes, and have been suggested as vaccine candidates [15]. A recent study by Agnes et al. used sera from cattle infected with A. marginale subspecies centrale to determine antigens that are cross-protective from sensu stricto challenge [16]. Several other studies have implicated components of the bacterial type 4 secretion system as vaccine candidates [17], [18] and [19]. In this paper, we examine multiple strains of A. marginale sensu

stricto, using high-throughput sequencing techniques to examine the members of unless the pfam01617 family and the other previously suggested vaccine components to determine their degree of conservation. Proteins that are widely conserved between all strains are candidates for inclusion in cross-protective vaccines. Further, the techniques described can be used to examine other organisms with significant numbers of repeats, allowing rapid determination of conserved proteins for diagnosis and vaccine development. A. marginale genomic DNA was isolated from highly infected bovine blood taken at the acute stage of infection. Organisms were purified from uninfected erythrocytes and white cells by passage through a cellulose column (C-6288, Sigma, St. Louis, MO) and frozen [20]. Genomic DNA was isolated from organisms using Qiagen genomic DNA kits according to manufacturer protocols.