<OL> <LI> Characterization of host mechanisms associated with mastitis susceptibility and resistance. <LI> Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses. <LI> Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety.
NON-TECHNICAL SUMMARY: Mastitis is the most costly disease of the U.S. dairy industry. Cows with mastitis have reduced milk production, increased risk of culling, and contribute to decreased milk quality. This project will investigate new management strategies and technologies that will help prevent and more successfully cure mastitis in dairy cattle. <P>APPROACH: Objective 1- Specific Aim - Identify and purify the bacterial proteins that are selectively recognized by IgG2 from hyperimmune serum. We will bind purified anti-J5 IgG2 antibodies, that were isolated from hyperimmunized cattle, to an affinity column matrix. We will then elute E. coli protein preparations similar to those used in earlier Western Blot studies, through the column. After washing of the column, we will disassociate the antibody-antigen binding in the column, and collect the proteins that are released. We will then determine their approximate molecular weight and ability to be recognized by serum IgG2 from hyperimmunized cattle by Western Blot. We expect to focus on the proteins previously identified, in the range of 8-10 and 37 kDa, respectively. We will cut the proteins bands from our Western Blot gels, and utilizing the services of the Macromolecular Structure, Sequencing and Synthesis Facility located in the Department of Biochemsitry, we will attempt N-terminal and C-terminal sequence analysis to help determine the identity of the proteins from the E. coli genome database. Clone the identified proteins to increase yield. By use of the mRNA that codes for our selected proteins, we will induce reverse transcription, and then synthesize complimentary DNA (cDNA) that codes for the proteins. We will then insert these cDNA genes into plasmids of E. coli, and express the proteins. The proteins will then be separated by crude fractionation and agarose gel, purified by affinity chromatography, and identified by Western Blot. Test the ability of the cloned proteins to elicit an antibody response in test animals. (antigen recognition) We will immunize test cattle with our purified protein extract mixed with an adjuvant to determine if the proteins are antigenic. This will be determined by measuring serum antibody responses in immunized versus non-immunized animals. We will perform ELISA to detect the presence of anti-J5 IgG2 antibody responses. <P>Objective 2- Specific Aim- Continue to perform clinical efficacy trials of antimicrobial therapy for the treatment of mastitis We will first determine the prevalence of intramammary infections in heifers at calving, based on the results we will test the use of macrolides to reduce the incidence of intramammary infections evaluating outcomes based on microbiology tests, somatic cell count results and antibiotic residue measurement. A partial budget will be calculated to determine economic benefit to prepartum therapy. Results of experiments will offer dairy farmers guidelines to determining heifers IMI prevalence on dairy farms and a tool to control the incidence of IMI infections. Experiments proposed below will be completed on a large commercial dairy herd, with 3000 milking cows, which including 900 to 1000 heifers in first lactation.