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United States Department of Agriculture

Agricultural Research Service

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Research Project: Characterization OF...MOLECULES from Es Products of Trichinelia Spiralis/t. Pseudospiralis L1 & Their Use in Immunodiagnosis

Location: Animal Parasitic Diseases

2013 Annual Report


1a.Objectives (from AD-416):
This project will have both pure and applied components. From the viewpoint of basic research, this project will focus on unknown aspects of the zoonotic parasite Trichinella pseudospiralis and its accompanying biology. This is a far less studied species of the genus Trichinella relative to the more common T. spiralis. It is the more ancestral of the genus and biologically most distinct. Research will use tools of functional proteomics and molecular-taxonomy together with molecular-biogeography to compare geographical isolates of Trichinella from several regions of South America. From an applied research perspective, the project will focus on identification of potential T.pseudospiralis/T. spiralis L1 larvae antigens and biomolecules among the excreted and secreted products and characterizing their peptide epitopes with the intent of preparing recombinant molecules that can be tested and evaluated for immunodiagnostic applications.


1b.Approach (from AD-416):
The main objective of the project is to more accurately characterize the protein composition of the excretory-secretory (ES) products of two major Trichinella species with different life-styles; T. spiralis (encysted form) / T. pseudospiralis (non-encysted form), with special attention devoted to cystatin. The approach will involve modern biochemical and molecular mass-spectrometric methods. In addition, based upon recent knowledge of the genome of T. spiralis we also plan to “mine” the genome and transcriptome for putative antigenic determinants. This work will involve a detailed characterization of the proteomes T. spiralis and T. pseudospiralis muscle larvae and in particular the ES products using 2D gel electrophoresis followed by mass-spectrometric methods. We hope to identify species-specific and genus-specific antigens that can be expressed in bacterial or yeast expression systems, purified and characterized. Once characterized, key antigens and antigenic determinants will be identified by peptide mapping. These recombinant molecules and/or synthetically generated peptide epitopes will then be tested according to a standardized ELISA format and/or by immunoblotting for their diagnostic potential. The project will also advance biogeographical studies of individual species of Trichinella isolates from various regions of South America (Argentina and Chile).


3.Progress Report:

The main objective of this project is to accurately characterize the protein composition of the excretory- secretory (ES) products of two major Trichinella species, each with different life-styles; T. spiralis (encysted form) and T. pseudospiralis (non-encysted form). Special attention is being devoted to cystatins, a family of cysteine protease inhibitors that work on peptidases. This is being investigated using modern biochemical and molecular mass-spectrometric methods, and bioinformatics based upon recent knowledge of the genome of T. spiralis which was generated by ARS scientists. Using these data bases, the genome and transcriptome of T. spiralis will be mined for putative antigenic determinants. This work also involves a detailed characterization of the proteomes of T. spiralis and T. pseudospiralis muscle larvae and in particular the ES products using 2D gel electrophoresis followed by mass-spectrometry in the hope of identifying species-specific and genus specific antigens that can be cloned, expressed and characterized. Once characterized, key antigens and antigenic determinants will be identified by peptide mapping. These recombinant molecules and/or synthetically generated peptide epitopes will be tested by ELISA and immunoblotting for their diagnostic potential. This project will advance biogeographical studies of individual species of Trichinella isolates from various regions of South America (Argentina and Chile). Currently, libraries of both T. spiralis and T. pseudospiralis have been generated, data mining of the now completed T. spiralis genome has begun and geographical isolates if both T. spiralis and T. pseudospiralis is ongoing.


Last Modified: 9/2/2014
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