Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. This work seeks to develop serological assays to differentially diagnose swine exposure to Trichinella spiralis or Trichinella murrelli. The reason for doing so is that swine exposed to T. spiralis are likely to harbor abundant, life-long larval burdens posing significant risk to food safety, whereas swine exposed to T. murrelli generally mount successful immune responses. Current methods to screen serum, however, make no distinction between the two conditions. Experimental infections needed to procure larvae of each parasite type were completed; crude extracts and preparations of Excretory-Secretory products (containing immunodominant antigens) were obtained. Peptides from each were fluorescently labeled and run on 2D gels, transferred to membranes, and probed with hyperimmune serum from pigs harboring infections with either T. spiralis or T. murrelli. By refining conditions of electrophoresis, candidate diagnostic epitopes (differentially or exclusively expressed in one parasite or the other, as well as epitopes exclusively recognized by one type of immune serum or the other) are being evaluated. This year, assays were also performed on protein preparations from which sugar residues (glycans) were first removed, because some of these sugars, though highly immunogenic, are common to each parasite (and therefore unsuited for use in discriminating one type of infection from the other). Finally, a substantial portion of the T. spiralis genome was tested for its suitability as a scaffold for short sequence reads derived from the T. murrelli genome. Parameters derived from this 'proof of principle' will be used in ongoing efforts to assemble and compare the T. murrelli genome. Preliminary data derived from this project were also presented at the 13th International Congress on Trichinellosis.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. This work was initiated in April in order to develop serological assays to differentially diagnose swine exposure to Trichinella spiralis or Trichinella murrelli. The reason for doing so is that swine exposed to T. spiralis are likely to harbor abundant, life-long larval burdens posing significant risk to food safety, whereas swine exposed to T. murrelli generally mount successful immune responses; current methods to screen serum, however, make no distinction between the two conditions. In the few months since initiating this project, much progress can already be reported. A visiting scientist was recruited to commence experimental infections intended to procure needed numbers of larvae of each parasite type. Crude extracts, and preparations of Excretory-Secratory products (containing immunodominant antigens) were obtained. Peptides from each were fluorescently labeled and run on 2D gels, transferred to membranes, and probed with hyperimmune serum from pigs harboring infections with one or the other infection. In addition to a large suite of shared epitopes, these experiments have identified candidate constituents which may prove to be specific to one of the two parasites, and recognized by the immune repertoire of swine. In the future, we intend to sequence, clone, and express candidate loci in order to test their suitability as the basis for specific immunoscreening reagents. Meanwhile, working with our APHIS partners and using summer students hired for this purpose, we have commenced screening of swine sera in order to identify farmed and feral animals harboring antibodies to either of these zoonotic parasites. Pending completion of our reagent development, these sera (and their matched tissue samples) will provide a means to validate our diagnostic test and help characterize the regional epidemiology of each agent. This collaboration was coordinated via in-person meetings among all three coordinators and regular interaction with staff, via phone meetings with our APHIS collaborators, and by written communication via emails; a progress report was delivered at the Project Director�s Meeting, July 29th, 2011, accompanied by a poster presentation.
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