The first goal of these experiments was to determine if immunization altered the magnitude or epitope specificity buy INCB28060 of the anti-Msp2 responses as compared to infection; specifically whether immunization as compared to infection shifted the antibody response, in terms of the breadth or magnitude, toward the conserved regions of Msp2. This immunity against conserved region epitopes could prevent immune escape of new variants and result in the clearance observed following challenge of immunized animals but not during natural or experimental infection. The second goal of these experiments was to determine if the breadth or

magnitude of the anti-Msp2 antibody response correlated with control of bacteremia in infected animals or prevention or control of bacteremia in immunized Bortezomib datasheet animals. To address these questions, animals were immunized with purified outer membranes or cross-linked surface proteins from the St. Maries strain of A. marginale, and the resulting specific antibody responses to the hypervariable (HVR) and conserved (CR) regions of Msp2 were mapped and titered. Vaccinees were then challenged with the homologous strain of A. marginale. Importantly, the St. Maries strain, for which the complete genome sequence is available,

was used in these experiments, thus allowing mapping of the Msp2 expressed variants to their original donor pseudogene alleles, analysis of all possible combinations of the HVR, and comprehensive testing of the epitope specificity induced much by immunization versus infection. The immunization and challenge have been previously reported in detail [11]. Briefly, two groups of five calves each were immunized 5 times at 3-week intervals with approximately 35 μg of either A. marginale outer membranes or protein complexes suspended in 1 mg of saponin in a total volume of 1 ml administered subcutaneously. The third group

of five calves was similarly immunized on the same schedule using only adjuvant. Four months after the last immunization, all calves were challenged intravenously with approximately 1 × 104A. marginale (St. Maries strain) in 1 ml Hank’s balanced salt solution. Starting 10 days post-challenge, the packed cell volume and bacteremia, as defined by the percent of infected erythrocytes, were determined daily for all the animals. PCR was used to confirm the lack of infection in the four challenged vaccinees that did not develop microscopically detectable bacteremia based on daily blood smear examination. DNA was isolated from whole blood using a Puregene DNA isolation kit (Qiagen, Valencia CA). Primers that specifically amplify msp5, a single copy gene, were used to detect A. marginale, as previously described in detail [12] and [13]. Amplification was performed in 50 μl volume with 35 cycles of melting at 94 °C for 15 s, annealing at 65 °C for 58 s, and extension for 71 s at 72 °C.