2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This Project plan was reviewed and replaces terminated Project 1265-32000-066-00D. Toxoplasma gondii and Trichinella spiralis are parasites that can cause serious disease in humans and/or animals. Toxoplasma gondii is a single-celled (protozoan) parasite, whereas T. spiralis is a nematode. The major issue with respect to both T. gondii and T. spiralis is food safety, primarily related to pork products. Consumers have long been aware of the potential presence of worms (T. spiralis) in pork and they are becoming increasingly aware of the fact that T. gondii is found in pork and other types of meat. Both parasites pose a risk to consumers who eat raw, undercooked or otherwise improperly prepared meat. The objectives of the research conducted on these parasites includes reducing or eliminating risk of human exposure from contaminated pork or other meat products. For each of these two parasites, understanding their biology and epidemiology is the major focus of research which will lead to improved control. The project plan has 4 specific goals:. 1)to develop an integrated risk model for Toxoplasma in the U.S. pork industry; . 2)to assess the role of chickens in the risk of human exposure to Toxoplasma; . 3)to develop improved serological detection assays for Toxoplasma in humans and chickens; and. 4)to support on-farm certification and export marketing efforts for Trichinella as requested by USDA regulatory agencies. This program falls within components of the ARS 5-Year Food Safety Action Plan 2006-2010 under Component 1, 1.1: Pathogens, Toxins, and Chemical Contaminants-Preharvest; and 1.2: -Postharvest. The proposed research directly addresses 1.1.1: Methodology, “Highly specific serological tests with the necessary sensitivity to detect animal carriers.”; 1.1.2: Epidemiology, “Epidemiological studies that elucidate chemical or pathogen presence and behavior in particularly important, suspected biological niches, including animal manure and other environmental niches.”; 1.1.3: Ecology, Host Pathogen and Chemical Comtaminants Relationships, “Identification of environmental sites, both physical and biological, which provide pathogen reservoirs and sites of amplification.”; 1.1.4: Intervention Strategies, “Interventions that prevent colonization or exclude pathogens from the gut.”; 1.2.3: Production and Processing Ecology, “Determine correlations between specific genetic types or clonal groups and virulence characteristics; and any relationships between the virulence characteristics and human infections.”; 1.2.4: Processing Intervention Strategies, “Increase fundamental understanding of the mechanisms, modes and sites of action at the cellular level of various intervention (inactivation) processes, and combination(s) thereof.”; 1.2.5: Omics, "Improved science-based risk assessment decisions to control foodborne pathogens. In particular, allowing comparisons (molecular systematics) of strains, providing information on genes that contribute to pathogenicity in human and/or animal hosts”; 1.2.7: Risk Assessment, “Develop data, and use these data for the development of predictive models for growth survival and inactivation of pathogens critical in minimally processed, brined, and fresh-cut produce.” It has recently been shown that meat, and in particular pork, is a source of human infection with the parasite Toxoplasma gondii. The National Retail Meats Survey for Toxoplasma gondii demonstrated the presence of viable T. gondii tissue cysts in fresh pork offered for sale to consumers, and consumer advocacy groups are now pointing to meat as a source of infection causing birth defects in newborns. To address these concerns, the U.S. pork industry needs a proactive approach to reduce the incidence of Toxoplasma in pigs. Trichinella spiralis continues to be a significant consumer concern with respect to the safety of pork products. The U.S. requires extensive processing of all ready-to-eat pork products due to this parasite. Trading partners require testing of pork products prior to shipment, and the image of U.S. pork with respect to Trichinella infection impacts accessibility to foreign markets. Although Trichinella is no longer an important public health issue in the U.S., the lack of any type of testing program, or documentation of the safety of U.S. pork is an impediment to increased domestic consumption of fresh product and to international market access. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006) Determine impact of industry standard post harvest meat processing technologies on viability of T. gondii in meat. Conduct strain typing of T. gondii isolates from free range chickens to determine link to human infections. Validate a serological assay (ELISA) for T. gondii infection in humans and chickens. Validate a serological assay (ELISA) for T. spiralis infection in horses. Year 2 (FY 2007) Conduct serological sampling for T. gondii and producer surveys on swine farms using medium risk management systems. Conduct serological sampling on chicken farms using organic, free range, and standard (indoor) management systems. Transfer chicken and human anti-T. gondii ELISA technology to the private sector for purposes of commercial production. Infect horses with Trichinella spiralis and collect serum samples to track antibody responses by ELISA. Year 3 (FY 2008) Perform ELISA on collected serum to determine the prevalence of T. gondii infection in swine on surveyed farms. Perform ELISA on collected serum to determine the prevalence of T. gondii infection in chickens in different management settings. Begin testing of human serum collections using T. gondii oocyst-specific validated ELISA. Collect blood and tissue samples to validate digestion methods and to assess the persistence of T. spiralis muscle larvae in the musculature of the horse. Year 4 (FY 2009) Develop a risk model to predict the likelihood of infection of pigs with T. gondii in high, medium, and low risk management systems. Audit high risk management systems to identify management practices which lead to T. gondii infection of chickens at the farm level. Create reference standard serum bank for Trichinellosis in horses and develop distribution protocols for international serum requests. Year 5 (FY 2010) Develop and test management standards on T. gondii endemic and T. gondii-free swine farms to validate the risk model; transfer set of best practices to industry. Design and transfer to industry a generic management plan (Good Production Practices) to reduce or eliminate exposure of chickens to T. gondii. Develop comprehensive risk analysis of T. gondii infection to humans from meat consumption based on results of ELISA serum analysis and National Retail Meat Survey data. 4a.List the single most significant research accomplishment during FY 2006. National Retail Meats Survey The APDL National Retail Meat Survey for T. gondii was completed in collaboration with the CDC Parasitic Diseases Laboratory. The National Retail Meats Survey determined that viable T. gondii was present in 0.4% of pork samples, but not in beef or chicken, though 1.5% of chicken samples were seropositive, indicating exposure to T. gondii. The National Retail Meats Survey was the first study where prevalence of a pathogen in animals has been directly linked to consumer risk in the retail meat case and is a model for future risk assessment of pathogens from farm to table. These data have been transferred to commodity groups and stakeholders through invited presentations and industry publications, and to the public through Agency press releases. The research supports Component 1 of the National Program Action Plan for Food Safety (National Program 108); Pathogens, Toxins and Chemical Contaminants- Preharvest (1.1) and Postharvest (1.2), 1.1.2: Epidemiology, and 1.1.3: Ecology, Host Pathogen and Chemical Comtaminants Relationships. 4b.List other significant research accomplishment(s), if any. -Globalization and the population structure of Toxoplasma gondii Focusing on the geographic component of genetic variation among Toxoplasma gondii genotypes, we showed that most genotypes are locale-specific, but some are found across continents and are closely related to each other, indicating a recent radiation of a pandemic genotype. Furthermore, we showed that the geographic structure of T. gondii is extraordinary in having one population that is found in all continents except South America, whereas other populations are generally confined to South America, and yet another population is found worldwide. Our evidence suggests that South American and Eurasian populations have evolved separately until recently, when ships populated by rats, mice, and cats provided T. gondii with unprecedented migration opportunities, probably during the transatlantic slave trade. Our results explain several enigmatic features of the population structure of T. gondii and demonstrate how pervasive, prompt, and elusive the impact of human globalization is on nature. The research supports Component 1 of the National Program Action Plan for Food Safety (National Program 108); Pathogens, Toxins and Chemical Contaminants- Preharvest (1.1) and Postharvest (1.2), 1.2.3: Production and Processing Ecology. 4c.List significant activities that support special target populations. None 4d.Progress report. See subordinate project progress reports associated with this in house project 5.Describe the major accomplishments to date and their predicted or actual impact. Methods have been developed for the accurate detection of Toxoplasma gondii infection in pigs, including the use of recombinant antigens in an ELISA test. These tests allow monitoring of animals on Toxoplasma endemic swine farms after GPP have been put into place to determine the success of the implemented management changes and to reduce the number of infected swine that enter the food chain. The test has been licensed by private industry and commercialized. Impacts NP108 Action Plan components 1.1.1.1; 1.1.1.3; 1.2.1.2; 1.4.1.4; 1.4.1.7 Information on the prevalence of Toxoplasma in pigs raised under various management systems was collected. Risk factors associated with acquisition of T. gondii in pigs raised in these management systems were analyzed; cats and mice were found to be the main source of T. gondii infection in pigs. Studies were initiated to identify point sources of oocyst contamination on farms where high prevalence was found. Results of these studies were transferred to stakeholder and industry groups through invited presentations and through articles published in industry publications. Impacts NP108 Action Plan components 1.1.1.1; 1.1.1.3; 1.2.1.2; 1.4.1.4; 1.4.1.7 Post-harvest technologies that render fresh meats safe with respect to Toxoplasma gondii transmission were identified. Methods (cooking, irradiation) were characterized which kill Toxoplasma parasites in meat and meat products. Storing retail pork cuts containing tissue cysts at or below 0oC for 7 days rendered T. gondii non-viable, and thus showed that management of the meat case storage temperature is an under-utilized method for effective control of T. gondii in retail pork. Injection of pork with specific formulations of salt-based enhancement solutions was shown to kill T. gondii tissue cysts in meat within 8 hours of injection, thus rendering infected meat safe for consumers. Results of these studies were transferred to stakeholder and industry groups through invited presentations and through articles published in industry publications. Impacts NP108 Action Plan components 1.1.1.1; 1.1.1.3; 1.2.1.2; 1.4.1.4; 1.4.1.7 An audit and verification system was developed to allow pigs to be certified free from Trichinella infection on the farm. The audit is based on evaluation of management practices which are known to limit risk of exposure of pigs to infection. Periodic testing of certified pigs is conducted using second generation diagnostic tests developed and commercialized through ARS research. The Trichinella Certification Program now has a full time Coordinator within APHIS; the Certification Program was the first pre-harvest food safety program implemented in the U.S., with over 100 producers enrolled in the Program. Impacts NP108 Action Plan components 1.1.1.1; 1.1.1.3; 1.4.1.4; 1.4.1.7 Methods used for the inspection of pigs and horses for Trichinella at slaughter were evaluated and recommendations were incorporated into existing regulations to improve and standardize internationally accepted detection methods. The ARS/CFIA collaborative study provided the data necessary to fully validate Trichinella detection protocols to meet International Organization for Standardization 17025 import standards proposed for all countries and to establish a bank of international reference serum for use in the harmonization of serological assays for trichinellosis in the horse. Impacts NP108 Action Plan components 1.1.1.1; 1.1.1.3; 1.4.1.4; 1.4.1.7 An export market inspection/training program for Trichinella was established by ARS and is continuously maintained in cooperation with the Agricultural Marketing Service. The Program preserves an export market for U.S. exporters exceeding $150 million dollars annually. The Program satisfies export requirements of the EU, Russia, and Singapore; fresh pork or horsemeat originating in the U.S. cannot be exported unless exporting meat packers participate in the Program Impacts NP108 Action Plan components 1.1.1.1; 1.4.1.7 We developed molecular methods to differentiate all currently recognized genotypes/species of Trichinella. Presently, this is the only unequivocal method to diagnose the source of infection and has clinical applications because of its ability to differentiate at the level of single larvae; this method is used worldwide to identify Trichinella species involved in outbreaks of disease. Impacts NP108 Action Plan components 1.1.1.1; 1.1.1.4; 1.4.1.4 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The finding that some currently used meat processing technologies are effective in eliminating risk to consumers of acquiring T. gondii infection from retail pork products has been transferred to stakeholder and industry groups through invited presentations and through articles published in industry publications. These data are important because 1.0% of all market hogs are infected with this parasite; the pork industry has identified eliminating the risk of T. gondii transmission from pork as one of its top food safety priorities. A serological assay (ELISA) for T. gondii infection in humans were tested and validated. The assay can differentiate between oocyst and tissue cyst infections in humans; an initial invention report has been submitted. Collaborations have been developed with the CDC, the University of Chicago, Case Western Reserve University, and the Research Institute of the Polish Mother's Memorial Hospital for access to human serum banks. The ELISA will be used to survey these serum banks to determine the predominant route of exposure of humans to T. gondii. The ARS/CFIA collaborative study on Trichinella in the horse provided the data necessary to fully validate Trichinella detection protocols to meet ISO 17025 import standards proposed for all countries. A bank of international reference serum for use in the harmonization of serological assays for trichinellosis in the horse has been established and tested following OIE guidlines; the serum bank is currently available to the international scientific community. An export market inspection program was maintained in cooperation with the Agricultural Marketing Service in which ARS (APDL) provides training to approximately 20-25 meat inspectors each year and provides support to an inspection program in U.S. slaughter plants. Training and quality assurance testing activities are ongoing which maintain the integrity of the testing program and preserves the U.S. export market to the EU and Russia for fresh pork and horsemeat. Constraints related to the development and application of tests for diagnosing parasitic diseases of swine are related to end-user acceptance of the technology rather than the effectiveness of the tests already developed or presently under development. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Hill, D.E., and Dubey, J.P. Prevalence of viable Toxoplasma gondii in beef, chicken and pork from retail meat stores in the United States: risk assessment to consumers. National Hog Farmer Magazine, December 15, 2005, p. 24. Hill, D.E. Presentation to Jim Dever of the USDA Foreign Agricultural Service (FAS), and Dr. M. Cagil, the Turkish Agriculture Councilor from the Turkish Embassy, on ARS/CFIA collaboration on harmonization of international testing protocols for Trichinella spiralis, May 11, 2006. Baltimore Sun, July 18, 2006. Disease (Trichinella spiralis) found in 3 pigs in Carroll County, MD. Review Publications Hill, D.E., Chirukandoth, S., Dubey, J.P. 2005. Toxoplasma gondii. Animal Health Research Reviews. 6(1):41-61. Sreekumar, C., Vianna, M., Hill, D.E., Miska, K.B., Lindquist, A., Dubey, J.P. 2005. Differential detection of hammondia hammondi from toxoplasma gondii using polymerase chain reaction. Parasitology International. 54(4):267-269. Dubey, J.P., Chapman, J.L., Rosenthal, B.M., Mense, M., Schueler, R.L. 2006. Clinical sarcocystis neurona, sarcocystis canis, toxoplasma gondii and neospora caninum infections in dogs. Veterinary Parasitology. 137:36-49. Dubey, J.P., Hill, D.E., Zarlenga, D.S. 2006. A trichinella murrelli infection in a domestic dog in the united states. Veterinary Parasitology. 137(3-4): 374-378. Dubey, J.P., Gomez, J.E., Bedoya, A., Lora, F., Vianna, M., Hill, D.E., Kwok, O.C., Shen, S.K., Marcet, P.L., Lehmann, T. 2005. Genetic and biologic characteristics of toxoplasma gondii isolates in free-range chickens from Columbia, South America. Veterinary Parasitology. 134:67-72. Gamble, H.R., Pozio, E., Lichtenfels, J.R., Zarlenga, D.S., Hill, D.E. 2005. Trichinella pseudospiralis from a wild pig in Texas. Veterinary Parasitology. 132:147-150. Hill, D.E., Dubey, J.P. 2005. Prevalence of viable toxoplasma gondii in beef, chicken and pork from retail meat stores in the United States; risk assessment to consumers. National Hog Farmer. p. 24. Dubey, J.P., Lopez, B., Alvarez, M., Mendoza, C., Lehmann, T. 2005. Isolation, tissue distribution, and molecular characterization of toxoplasma gondii from free-range chickens from guatemala. Journal of Parasitology 91:955-957. Dubey, J.P., Marcet, P.L., Lehmann, T. 2005. Characterization of toxoplasma gondii isolates in free-range chickens from Argentina. Journal of Parasitology. 91:1335-1339. Dubey, J.P., Rajapakse, R.J., Ekanayake, D.K., Sreekumar, C., Lehmann, T. 2005. Isolation and molecular characterization of Toxoplasma gondii from chickens from Sri Lanka. Journal of Parasitology. 91:1480-1482. Dubey, J.P., Hill, D.E., Jones, J.L., Hightower, A.W., Kirkland, E., Roberts, J.M., Marcet, P.L., Lehmann, T., Vianna, M., Miska, K.B., Sreekumar, C., Kwok, O.C., Shen, S.K., Gamble, H.R. 2005. Prevalence of viable toxoplasma gondii in beef, chicken, and pork from retail meat stores in the united states and risk assesment to consumers. Journal of Parasitology 91:1082-1093. Dubey, J.P. 2005. Unexpected oocyst shedding by cats fed toxoplasma gondii tachyzoites: in vivo stage conversion and strain variation. Veterinary Parasitology 133:289-298. Dubey, J.P., Edelhofer, R., Marcet, P., Vianna, M., Kwok, O.C., Lehmann, T. 2005. Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria. Veterinary Parasitology 133:299-306. Dubey, J.P., Rosypal, A.C., Pierce, V., Scheinberg, S., Lindsay, D.S. 2005. Leishmaniasis-associated placentitis in a dog from maryland. Journal of the American Veterinary Medical Association 227:1266-1269. Dubey, J.P., Fair, P.A., Bossart, G.D., Hill, D.E., Fayer, R., Sreekumar, C., Kwok, O.C., Thulliez, P. 2005. A comparison of four serologic tests to detect antibodies to toxoplasma gondii in naturally-exposed bottlenose dolphins (tursiops truncatus). Journal of Parasitology. 91:1074-1081. Wetzel, D.M., Schmidt, J., Kuhlenschmidt, M.S., Dubey, J.P., Sibley, D. 2005. Gliding motility leads to active cellular invasion by cryptosporidium sporozoites. Infection and Immunity. 73:5379-5387. Lindsay, D.S., Kaur, T., Mitchell, S.M., Goodwin, D.G., Strobl, J., Dubey, J.P. 2005. Buprenorphine does not affect acute murine toxoplasmosis and is recommended as an analgesic in toxoplasma gondii studies in mice. Journal of Parasitology. 91:1488-1490. Dubey, J.P., Lenhart, A., Castillo, C.E., Alvarez, L., Marcet, P., Sreekumar, C., Lehmann, T. 2005. Toxoplasma gondii infections in chickens from Venezuela: isolation, tissue distribution, and molecular characterization. Journal of Parasitology. 91:1332-1334. De Sousa, S., Ajzenberg, D., Canada, N., Freire, L., Correia Da Costa, J.M., Darde, M.L., Thulliez, P., Dubey, J.P. 2006. Biologic and molecular characterization of toxoplasma gondii isolates from pigs from Portugal. Veterinary Parasitology. 135:133-136. Dubey, J.P., Gennari, S.M., Laabruna, M.B., Camargo, L., Vianna, M., Marcet, P.L., Lehmann, T. 2006. Characterization of toxoplasma gondii isolates in free-range chickens from Amazon, Brazil. Journal of Parasitology. 92:36-40.