1a.Objectives (from AD-416)
To develop improved serological detection assays for Salmonella and Toxoplasma in humans, pigs, and chickens. Currently available serological tests for Salmonella in swine contain antigens from serovars that do not predominate in U.S. Midwestern swine. As a result, serological tests perform poorly using sera from these animals. We will use serovar specific antigen isolates from Salmonella to develop a serological test which is more sensitive and specific for detection of Salmonella serovars which predominate in Midwestern swine. Until recently, differentiation of foodborne versus oocyst transmission of oxoplasma to humans was impossible. Identification of stage specific antigens from Toxoplasma oocysts have made the development of a validated serological assay possible. We will use these antigens to develop diagnostic methods to determine the most common transmission route of Toxoplasma in humans, and validate a serological test for detection of Toxoplasma infection in chickens.

1b.Approach (from AD-416)
Though previous studies have identified characteristics of high risk management systems, medium and low risk systems have not been completely characterized. We will identify those management systems and strategies that reduce or eliminate Toxoplasma from swine herds on the farm, and develop a comprehensive risk model for the swine industry. Though pork has been identified as a potential source of human infection for Toxoplasma, recent studies suggest that chicken may also be a risk for human transmission. We will analyze Toxoplasma prevalence in chickens raised in different management systems and develop practical interventions for reducing risk of exposure.

Until recently, differentiation of foodborne versus oocyst transmission of Toxoplasma to humans was impossible. Identification of stage specific antigens from oocysts have made the development of a validated serological assay possible. We will use these antigens to develop diagnostic methods to determine the most common transmission route of Toxoplasma in humans.

The U.S. export market for pork and horsemeat is dependent upon industry compliance with the testing requirements of importing countries. The need for international validation, standardization, and other quality control requirements for digestion-based Trichinella testing is critical as countries compete for export markets. We will collaborate with trading partners and international food safety organizations to harmonize testing procedures for Trichinella in meat products destined for export.

3.Progress Report
Coordinated with APHIS and Maryland Department of Agriculture (MDAg) to conduct an epidemiological investigation of a Trichinella and Toxoplasma infections on a swine farm. Worked with MDAg vets and local animal control officers to capture and sample potentially infected sylvatic carnivores near the infected swine farm. Isolated T. gondii and T. spiralis from euthanized animals, and conducted genotyping studies on recovered organisms. Characterization of the transmission cycle indicates that sylvatic carnivores do not maintain infection with Trichinella spiralis in the absence of infected pigs. This work relates to NP 108 Action Plan Component 1a, Pathogens, Toxins, and Chemical Contaminants Preharvest, ii, Epidemiology, to determine the origin and routes of transmission of epizootic pathogens. Surveyed organically raised Midwestern pigs for infection with Trichinella and Toxoplasma gondii; found no Trichinella, but >80% prevalence of Toxoplasma infection. This work relates to NP 108 Action Plan Component 1a, Pathogens, Toxins, and Chemical Contaminants Preharvest, ii, Epidemiology, to determine the origin and routes of transmission of epizootic pathogens. Isolated O-group somatic antigens from 9 Salmonella serovars for development of a mixed antigen ELISA for Midwestern swine. This work relates to NP 108 Action Plan Component 1a, Pathogens, Toxins, and Chemical Contaminants Preharvest, iv, Intervention Strategies, to reduce colonization and shedding of epizootic pathogens by food producing animals. Conducted quarterly testing of analysts and evaluated test results in consultation with the Animal and Plant Health Inspection Service (APHIS) and the Agricultural Marketing Service (AMS) to maintain integrity of the analyst training program. This work relates to NP 108 Action Plan Component 1b, Pathogens, Toxins, and Chemical Contaminants Postharvest, iv, Processing Intervention Strategies, to inactivate microorganisms to varying degrees.

4.Accomplishments
1. Determination of predominate transmission route of Toxoplasma gondii. Currently, the predominant route of infection in human toxoplasmosis is unknown, making the development of risk reduction strategies difficult. Using a newly developed ELISA assay containing a recombinant T. gondii oocyst protein, surveys were conducted of food animals, rodents, and human populations from highly endemic regions in Chile to determine the predominant route of exposure to T. gondii. Results indicate that >80% of infections in food animals and humans tested result from exposure to infectious oocysts, identifying this infection route as the target for intervention strategies aimed at reducing Toxoplasma infection in humans. Rodents do not appear to be a significant reservoir of infection. This accomplishment addresses NP 108 Component 1a, Pathogens, Toxins, and Chemical Contaminants-Preharvest; i) Methodology and ii) Epidemiology.

2. Identification of endemic Toxoplasmosis in pigs on a farm in Maryland and genetic characterization of T. gondii. The prevalence of Toxoplasma gondii was investigated on a poorly managed pig farm in Maryland. Serum and tissue samples from 48 of the 100 pigs on the farm were available for T. gondii evaluation. Serological testing was performed using both ELISA and the modified agglutination test (MAT). Antibodies to T. gondii were detected by ELISA in 12 of 48 animals, while antibodies were detected in 34 of 48 pigs by MAT with titers of 1:10 in 1, 1:20 in 4, 1:40 in 7, 1:80 in 3, 1:160 in 8, 1:320 in 3, 1:640 in 4, and 1:1,280 in 4. Hearts of 16 pigs with MAT titers of 1:10 or higher were bioassayed for T. gondii in cats; 11 cats shed T. gondii oocysts. Hearts of 22 pigs were autolysed and bioassayed only in mice; T. gondii was isolated from 3 of these 22 pigs. Genetic typing of the 14 T. gondii isolates using the SAG1, SAG2, SAG3, BTUB, GRA,6 c22-8, c29-2, L358, PK1 and Apico loci revealed 4 genotypes; 10 isolates belonged to Type II lineage (genotypes #1 and #2), 3 belonged to genotype #3 and 1 belonged to genotype #4. Genotype #1 and #2 have Type II alleles at all genetic loci except the former has Type II allele and the latter has a Type I allele at locus Apico. Both genotypes #1 and #2 are considered to belong to the clonal Type II lineages. Genotype #3 and #4 are non-clonal isolates. Results support low genetic diversity among T. gondii isolates from pigs in the U.S. This accomplishment addresses NP 108 Component 1a, Pathogens, Toxins, and Chemical Contaminants-Preharvest; i) Methodology and ii) Epidemiology.

3. Characterization of high prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption. The prevalence of T. gondii was determined in 383 lambs (< 1 year old) from Maryland, Virginia, and West Virginia. Hearts of 383 lambs were obtained from a slaughter house on the day of killing. Blood was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Sera were first screened using 1:25, 1:50, 1: 100, and 1:200 serum dilutions and hearts selected for bioassay for T. gondii. Antibodies (MAT, 1:25 or higher) to T. gondii were found in 104 (27.1%) of 383 lambs. Hearts of 68 seropositive lambs were used for isolation of viable T. gondii by bioassay in cats, mice or both. In total, 55 isolates of T. gondii were obtained from 68 seropositive lambs. Genotyping of the 55 T. gondii isolates using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB,GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed 14 genotypes. Twenty-six (47%) isolates belong to the clonal Type II lineage (these isolates can be further divided into two groups based on alleles at locus Apico). Eight (15%) isolates belong to the Type III lineage. The remaining 21 isolates were divided into 11 atypical genotypes. Four lambs had infections with two T. gondii genotypes. These results indicate high parasite prevalence, and high genetic diversity of T. gondii in lambs. This is the first in depth genetic analysis of T. gondii isolates from sheep. This accomplishment addresses NP 108 Component 1a, Pathogens, Toxins, and Chemical Contaminants-Preharvest; ii) Epidemiology and iii) Ecology, Host Pathogen and Chemical Comtaminants Relationships.

4. Complete validation of a unique digestion assay to detect Trichinella larvae in horse meat demonstrates its reliability for meeting food safety and trade requirements. A tissue digestion assay using a double separatory funnel (DSF) procedure for the detection of Trichinella larvae in horse meat was validated for application in food safety programs and trade. It consisted of a pepsin-HCl digestion step to release larvae from muscle tissue, followed by two sequential sedimentation steps in separatory funnels to recover and concentrate larvae for detection using a stereomicroscope. The assay, with defined critical control points, was conducted within a quality assurance system compliant with ISO 17025 guidelines. Samples used in the validation were obtained from horses experimentally infected with Trichinella spiralis to obtain a range of muscle larvae densities. Using a 5-g sample size, all samples containing 1.3 – 2 lpg were detected. Similarly, 60 – 100% of samples with infected horse meat containing 0.1 - 0.7 lpg were detected. The data demonstrate that the DSF digestion assay is reliable, efficient and fit for its intended use in food safety and trade. The DSF procedure is the only validated digestion assay for trichinellosis demonstrating consistency and effectiveness at critical levels of sensitivity. This accomplishment addresses NP 108 Component 1a, Pathogens, Toxins, and Chemical Contaminants-Preharvest; i) Methodology, and iii), Ecology, Host Pathogen and Chemical Comtaminants Relationships.

5. Characterization of transplacental Toxoplasmosis in naturally-infected white-tailed deer: isolation and genetic characterization of Toxoplasma gondii from foetuses of different gestational ages. In humans, transmission of Toxoplasma gondii from the mother to the foetus is most efficient during the last trimester of pregnancy but clinical congenital toxoplasmosis is more severe if transmission occurs during the first trimester. Attempts were made to isolate T. gondii by bioassay in mice inoculated with parasites isolated from foetuses of 88 naturally-exposed white-tailed deer from Iowa and Minnesota. Viable T. gondii was found in 6 of 61 deer foetuses in early pregnancy (45-85 days of gestation) from Iowa and in 9 of 27 deer foetuses from Minnesota in mid-gestation (130 -150 days) of a gestational period of 7 months. The 15 T. gondii isolates obtained from fetal deer were PCR-restriction fragment length polymorphism genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and an apicoplast marker, Apico. Five genotypes were revealed, including the clonal Type II and III lineages, and 3 non-clonal genotypes. DNA sequencing analysis of representative isolates at loci SAG2, c22-8, L358 and PK1 revealed that the3 non-clonal genotypes are closely related to the clonal Type I, II and III lineages. It is very likely that these non-clonal genotypes were derived from genetic crosses among the 3 clonal Type I, II and III lineages. The most common genotype was Type II, commonly found in humans in North America and Europe, suggesting a link of transmission from game animals to humans. This accomplishment addresses NP 108 Component 1a, Pathogens, Toxins, and Chemical Contaminants-Preharvest; iii), Ecology, Host Pathogen and Chemical Comtaminants Relationships.

6. Charaterization of genetic diversity among sea otter isolates of Toxoplasma gondii. Sea otters (Enhydra lutris) become infected with Toxoplasma gondii and at times succumb to clinical disease. Here, we determined genotypes of 39 T. gondii isolates from 37 sea otters in 2 geographically distinct locations (25 from California and 12 from Washington State). Six genotypes were identified using 10 PCR-RFLP genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and by DNA sequencing of loci SAG1 and GRA6 in 13 isolates. Of these 39 isolates, 13 (33%) were clonal Type II which can be further divided into 2 groups at the locus Apico. Two of the 39 isolates had type II alleles at all loci except a type I allele at locus L358. One isolate had type II alleles at all loci except the type I alleles at loci L358 and Apico. One isolate had type III alleles at all loci except type II alleles at SAG2 and Apico. Two sea otter isolates had a mixed infection. Twenty-one (54%) isolates had a unique allele at SAG1 locus. Genotyping and DNA sequence analysis for 18 of these 21 isolates at loci SAG1 and GRA6 revealed that there were 2 different genotypes, including the previously identified Type X (four isolates) and a new genotype named Type A (14 isolates). The results from this study suggest that the sea otter isolates are genetically diverse. This accomplishment addresses NP 108 Component 1, a, Pathogens, Toxins, and Chemical Contaminants-Preharvest; iii) Ecology, Host Pathogen and Chemical Comtaminants Relationships.

5.Significant Activities that Support Special Target Populations
None.

6.Technology Transfer

Number of Other Technology Transfer

1

Review Publications
Moura, L., Kelley, P., Krecek, R.C., Dubey, J.P. 2007. Seroprevalence of toxoplasma gondii in cats from Sr. Kitts, West Indies. Journal of Parasitology. 93:952-953.