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Identification of Toxoplasma Gondii Genotypes in California Sheep

Hoar, Bruce; Li, Xunde; Atwill, Edward
University of California - Davis
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1) Develop and optimize a PCR-RFLP diagnostic test for T. gondii in sheep tongue, heart, and brain samples.

2) Determine specific genotypes present in a random sample of mature (>1 year of age) sheep presented for harvest at a commercial slaughter facility.

3) Use spatial data analysis to detect potential clusters of specific genotypes.

4) Develop a logistic regression model to assess the relationship between infection and purported risk factors.

More information

Millions of consumers become ill from pathogenic organisms in or on food every year in the United States. A recent publication estimates that infection with Toxoplasma gondii is responsible for 24% of human foodborne deaths annually. Some of the risk factors that have been identified for human infection with T. gondii include eating rare lamb, raw ground beef, locally produced cured, dried, or smoked meat, drinking unpasteurized goat's milk and having 3 or more kittens. While only a small percentage of exposed humans develop clinical toxoplasmosis, the disease in immunocompromised patients has a high mortality rate. Recent investigations have determined that genetic variability among T. gondii isolates may be associated with disease severity. Genetic typing of T. gondii, based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), has indicated that 3 lineages predominate in North America and Europe, but isolates with much greater diversity also have been recovered. In addition to its role as a foodborne disease agent, T. gondii is associated with significant production impacts for sheep and goat producers. Infection of susceptible pregnant females can result in placental invasion and transplacental infection of the fetus. Typical clinical signs are abortion and production of stillborn or weak lambs, often accompanied by a small mummified fetus. California has over 600,000 sheep, yet the serologic prevalence for this important pathogen was last surveyed in 1977 with 24% of samples being seropositive. The genetic variability of isolates present in California sheep flocks has never been determined. Given the high seroprevalence found in previous studies of sheep in California, it is logical to conclude that consumption of sheep meat represents a potential risk for human infection with T. gondii. We believe that it is important to improve our understanding of the prevalence of the various genotypes of T. gondii in sheep presented for slaughter. This will allow us to develop a database for comparison to human cases to assist with source identification. We will also determine risk factors associated with infection of sheep and thereby assist producers in developing control programs to reduce the impact of the disease in their flocks.

We will target mature animals (>1 yr) exclusively for inclusion in our study. This will result in primarily old ewes being sampled, the animals at greatest risk of production losses due to toxoplasmosis. To detect a prevalence of 10%, with a standard error of 2.5%, a sample size of 144 animals is required. At the slaughter facility, tongue, brain, heart, and blood will be collected aseptically and placed into appropriate containers, placed on ice, and transported to the laboratory. Owner and animal information will be collected, as available. With consent of the slaughter facility, we will contact the owner and administer a questionnaire that will assess management of the flock. Samples will be processed based on a method described elsewhere with modifications. Briefly, 100 g of tissue sample (free of fat and connective tissue) will be cut into pieces of approximately 1 cm3. The cut tissue will be put into a Stomacher bag with filter and cell lysis buffer containing 100 mM Tris HCl pH 8.0, 5 mM EDTA pH 8.0, 0.2% SDS, 200 mM NaCl, 40 mg/l proteinase K at the ratio of 2.5 ml per g of sample. Samples will be homogenized in a Stomacher for 2 min at high speed followed by incubation overnight at 55C in a water bath. After incubation, the samples will be mixed well and 50 ml homogenized sample will be subjected to DNA extraction using a phenol / chloroform extraction method. The genotypes of T. gondii isolates will be determined by PCR-RFLP and DNA sequencing as previously described. Briefly, for the PCR-RFLP, multi-locus analysis at the B1 and SAG1, SAG2, SAG3 and GRA6 genes will be performed. PCR products will be visualized using ethidium bromide staining on 1-2% agarose gels. PCR product from each reaction will be digested with restriction enzymes to identify diagnostic restriction fragment patterns on subsequent agarose gels. For DNA sequencing, a subset PCR products of T. gondii B1, SAG1 and GRA6 loci will be sequenced at the UC Davis DNA Sequencing Facility. DNA sequences will be analyzed using Vector NTI software (Invitrogen Carlsbad, CA) and compared to published T. gondii sequences using a NCBI (National Center for Biotechnology Information) online BLAST search tool. Based on these results, the distribution of T. gondii archetypal I, II, III and atypical genotypes will be determined. Prevalence data will be analyzed using descriptive statistics. Logistic regression will be used to assess the relationship between risk factors identified from the questionnaire and presence of infection. A geographic information system (ArcGIS v10, ESRI, Redlands, CA) map of California will be combined with the results of our genotyping analysis to facilitate visualization of areas where various genotypes of T. gondii are found. In order to investigate clustering of genotypes, spatial and temporal data analyses will be performed. Purely spatial and purely temporal clustering will be assessed using SaTScan software (version 1.0.3, M. Kulldorff and National Cancer Institute, Bethesda, MD) while the Knox test will be used to evaluate whether observed time-space clustering is present in the data.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens