Validation of a Serological Assay using and 18KD Oocyst-specific Protein from Toxoplasma Gondii for Differentiation of Oocyst vs Tissue Cyst

APPROACH: Expression of selected cDNA and validation of serological assay using cloned protein and human sera to determine oocyst vs tissue cyst exposure.

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PROGRESS: 2004/07 TO 2006/09<BR>
Progress Report Objectives (from AD-416) To select the cDNA cloneof the 18.3 kDa protein (oocyst specific) from a library constructed from T. gondii oocysts and validate a serological assay using the recombinant protein. Approach (from AD-416) Expression of selected cDNA and validation of serological assay using cloned protein and human sera to determine oocyst vs tissue cyst exposure. Significant Activities that Support Special Target Populations This report documents research conducted under a Trust agreement between ARS and the National Pork Board. Additional details of the research can be found in the report for the parent project 1265-32000-076-00D, Development of an Integrated Risk Model for Foodborne Zoonotic Parasites in Swine. Reduction of risk of human and food animal infection with the zoonotic parasite, Toxoplasma gondii, is hampered by the lack of data documenting the predominant routes of infection (oocyst vs tissue cyst). Existing serological assays can determine previous exposure to the parasite, but not the infection route. We have identified an oocyst specific 18.3kDa protein that can differentiate between oocyst and tissue cyst-induced T. gondii infection in pigs and humans. An oocyst-specific protein was identified from T. gondii using 2-dimensional Western blots screened with sera from known oocyst induced infection. Matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF- MS) and collision induced dissociation (CID) fragmentation analysis was performed on the identified protein. Polymerase chain reaction (PCR) primers were constructed from the amino acid sequence derived from the mass spectrometry data and PCR was performed using the primers and a sporozoite cDNA library. The sequenced PCR amplicons for the protein were labeled using a non-radioactive method (DIG DNA labeling) for use in library screening to pull out the full length gene for protein expression. Approximately 20 positives were identified. Secondary screening resulted in selection of over 50 positive plaques, and 12 were sequenced and further characterized to confirm the gene. Initial expression studies and immunological screening of the recombinant antigen using anti- T. gondii oocyst infection sera confirmed that the positive clones were reactive with the infection sera. The identified gene was subcloned into the Xmn I/Hind III site of pMal-c2 plasmid vector for protein expression and was expressed as an N-terminal maltose binding fusion protein under the control of the lac repressor. Constitutive expression following subcloning into the pMal-c2 vector was accomplished. Human serum of known infection routes has been used to validate an ELISA assay using the expressed recombinant protein in collaboration with scientists from the CDC and researchers from the University of Chicago. Determination of the predominant route of infection of Toxoplasma gondii in humans and food animals will help to identify strategies for reducing transmission and will increase public perception of pork as a safe food product. Project plans, goals and accomplishments were discussed via email and through written reports.

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PROGRESS: 2005/10/01 TO 2006/09/30<BR>
Progress Report 4d Progress report. This report serves as the final report to document research conducted under a Trust agreement between ARS and the National Pork Board. Additional details of the research can be found in the report for the parent project 1265-32000-076-00D, Development of an Integrated Risk Model for Foodborne Zoonotic Parasites in Swine. Reduction of risk of human and food animal infection with the zoonotic parasite, Toxoplasma gondii, is hampered by the lack of data documenting the predominant routes of infection (oocyst vs tissue cyst). Existing serological assays can determine previous exposure to the parasite, but not the infection route. We have identified an oocyst specific 18.3kDa protein that can differentiate between oocyst vs tissue cyst induced T. gondii infection in pigs and humans. An oocyst-specific protein was identified from T. gondii using 2-dimensional Western blots screened with sera from known oocyst induced infection. Matrix assisted laser desorption/ionization- time of flight mass spectrometry (MALDI-TOF-MS) and collision induced dissociation (CID) fragmentation analysis was performed on the identified protein. Polymerase chain reaction (PCR) primers were constructed from the amino acid sequence derived from the mass spectrometry data and PCR was performed using the primers and a sporozoite cDNA library. The sequenced PCR amplicons for the protein were labeled using a non- radioactive method (DIG DNA labeling) for use in library screening to pull out the full length gene for protein expression. Approximately 20 positives were identified. Secondary screening resulted in selection of over 50 positive plaques, and 12 have been sequenced and further characterized to confirm the gene. Initial expression studies and immunological screening of the recombinant using anti-T. gondii oocyst infection sera confirmed that the positive clones were reactive with the infection sera. The identified gene was subcloned into the Xmn I/Hind III site of pMal-c2 plasmid vector for protein expression and will be expressed as an N-terminal maltose binding fusion protein under the control of the lac repressor. Constitutive expression following subcloning into the pMal-c2 vector was accomplished. Human serum of known infection routes has been used to validate an ELISA assay using the expressed recombinant protein in collaboration with scientists from the CDC and researchers from the University of Chicago. Determination of the predominant route of infection of Toxoplasma gondii in humans and food animals will help to identify strategies for reducing transmission and will increase public perception of pork as a safe food product.