2013 Annual Report
1a.Objectives (from AD-416):
1. Develop serological assays that differentiate swine chronically infected with zoonotic T. spiralis from swine only transiently exposed to T. murrelli.
2. Estimate how frequently pastured pigs and feral swine are exposed to infection with enzootic and zoonotic species of trichinella.
1b.Approach (from AD-416):
Screen expression libraries of T. spiralis and T. murrelli with hyper-immune sera from swine to identify clones encoding species-specific diagnostic antigens. Develop antibodies that discriminate among the two types of infection. Test in experimentally infected swine. Apply to panels of serum collected from feral swine and pastured pigs. Validate against genotyped parasite specimens obtained from tissue samples matched with serum.
Established and validated procedures to strip antigenic proteins of sugars which would otherwise dominate the immune response. In order to identify proteins that may uniquely provoke immune responses to either T. spiralis or T. murrelli, it was necessary to first remove shared, irritating sugars that would otherwise dominate the immune response. Methods were established to do so.
Established conditions for individuating proteins by two dimensional gel electrophoresis. Secreted protein preparations were separated in two dimensions (size and charge), in order to compare each parasite according to the proteins that it expresses at various stages. In addition, protein preparations from each parasite were probed with serum from pigs that had been exposed to one of these parasites or the other. Ongoing efforts will verify, through replication, those proteins exclusively recognized by pigs exposed to T. spiralis, on the one hand, or T. murrelli, on the other.
Established the identity of candidate antigens. Peptide sequencing of particular proteins identified as being differentially recognized by the two types of serum represented a recent, marked step forward. That is, spots recognized as having migrated to unique positions were excised from the gel, broken into constituent peptide fragments, and identified according to their amino acid sequence.