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Research Project: Detection and Control of Foodborne Parasites for Food Safety

Location: Animal Parasitic Diseases Laboratory

2017 Annual Report

Objectives
Objective 1: Refine current immunological assays to investigate rates of human exposure to oocysts of Toxoplasma gondii. Subobjective 1.A (Hill): Refine and validate the TgERP ELISA (Toxoplasma gondii Embryogenesis Related Protein) and a Luminex bead-based immunoassay for use in human and veterinary models. Subobjective 1.B (Hill): Evaluate other candidate antigens to enhance the ability to detect exposure to oocysts in individuals with either low (recent infection) or high avidity (chronic infection) antibodies. Subobjective 1.C (Hill): Using sera collected from Americans via NHANES, determine what proportion of those infected with Toxoplasma harbor antibodies to oocysts. Objective 2: Identify mitigation strategies that reduce Toxoplasma oocysts contamination on fruits and leafy greens. Subobjective 2A (Hill): Evaluate the effectiveness of bioassay, tissue culture, and PCR using apoptosis-specific targets for determination of viability of Toxoplasma oocysts after treatment with cold plasma, monochromatic blue light, pulsed light, laser enhanced acoustic waves, gaseous chlorine dioxide, and ozone to inactivate T. gondii oocysts from the surface of fruits, vegetables, and low moisture foods (LMF). Objective 3: Elucidate the molecular epidemiology and molecular genetics of environmental Toxoplasma oocyst contamination and define virulence and persistence of particular genotypes in food animals. Subobjective 3.A (Dubey): Evaluate whether there are genetically distinct subsets of T. gondii in swine and deer. Subobjective 3.B (Dubey, Rosenthal): Evaluate whether the T. gondii oocysts that account for most infections are derived from local, distinct, and genetically homozygous populations. Objective 4: Determine and validate methods for improved inactivation and surveillance of meat-borne exposure to Toxoplasma gondii and Trichinella sprialis. Subobjective 4.A (Hill, Dubey): Develop a model for pork dry curing processes, taking into account five common measurements monitored during curing – salt/brine concentration, water activity (aw), pH, temperature, and time, for inactivation of Trichinella spiralis, Toxoplasma gondii, and Salmonella. The study will be performed in two phases – an initial multi-factorial modeling phase using ARS’s Pathogen Modeling Program and low, internal, and high endpoints for common curing treatments, and a final validation phase. Subobjective 4.B (Hill): Support the technical aspects of the new National Surveillance Program for Trichinella by 1) assisting in the development a sampling framework; 2) development of a high throughput serological assay for Trichinella and Toxoplasma capable of providing the means to document prevalence to less than 1 infection per million pigs; 3) by evaluating more selective diagnostic antigens to improve sensitivity and specificity; and 4) by assisting in the investigation of any positive findings (tracebacks, genotyping).

Approach
Our project combines translational and applied research to improve monitoring and surveillance for zoonotic parasites, and develops models for their control. Fundamental research proposes to refine new immunological assays to detect human exposure to the oocyst stage of Toxoplasma, and to develop in vitro assays for Toxoplasma oocyst viability after curative treatment of fruits and vegetables. Applied research will develop methods to monitor and inactivate pathogens associated with pork products. Our overall goal is to mitigate the impact of these potentially harmful parasites, thereby protecting consumers and maintaining the vitality of the U.S. pork industry.

Progress Report
In collaboration with scientists at the USDA-ARS Eastern Regional Research Center (ERRC), we completed the validation of endpoint defining curing processes necessary for developing a multi-factorial model for pork curing for inactivation of Trichinella and Toxoplasma. The endpoint processes established the survival limits of the 2 parasites with respect to salt/brine concentration, aw (water activity), pH, temperature, and time. Prediction of inactivation of Trichinella and Toxoplasma in pork meat can be accomplished within the defined endpoints which circumscribe most commonly used parameters for curing of fresh pork (Sub-objective 4a). Conducted quarterly training program for packer analysts for approved direct detection methods for T. spiralis in pork and horsemeat. Conducted testing of analysts and evaluated test results in consultation with the APHIS and the Agricultural Marketing Service (AMS) to maintain integrity of the analyst training program. These efforts support export marketing efforts as requested by USDA regulatory agencies (Sub-objective 4b). Completed the National Retail Meat Survey for Toxoplasma in organic pork and American lamb. The survey identified the risk of Toxoplasma infection to consumers from these meat products using samples collected from 25 MSAs nationwide. These data will be used to discern consumer risk from meat products from animals raised in uncontrolled management systems (Sub-objective 4b).

Accomplishments
1. Prevalence and genetic characterization of Toxoplasma gondii in free-range chickens from grocery stores and farms. Toxoplasmosis continues to be a major public health concern in the U.S. The consumption of undercooked meat is suspected as an important mode of this parasite’s transmission, and free range chickens have been found to be infected. In order to determine whether chickens available for purchase pose a significant risk, a large sample of chicken hearts were obtained from local markets and tested for T. gondii infection. Viable T. gondii was not isolated from any hearts by bioassays in mice and cats. These results indicate that unlike free range back yard chickens, chickens from grocery stores do not pose an appreciable risk for consumers. These reassuring data will be helpful to producers, retailers, food safety authorities, and epidemiologists.

2. Curing parameters to inactivate Trichinella spiralis in ready-to-eat meats. Ready-to-eat meats are protected from spoilage and foodborne pathogens through curing processes that use salt, seasonings, and other additives. Past regulations required costly testing of each pork curing process to demonstrate its ability to kill larvae of the foodborne parasite Trichinella spiralis and it would be preferable to predict how well a proposed new method would eliminate risk, especially given growing consumer interest in low-salt meat products. ARS scientists in Beltsville, Maryland, found that salt concentrations above 1.3%, in combination with fermentation to pH 5.2 or below, kills greater than 96% of Trichinella larvae in stuffed sausages within 24-28 hrs and inactivates them all within 10 days. These curing parameters reliably predict inactivation of Trichinella spiralis, eliminating the need for individual product testing. These results will protect public health and will be of great value to food producers seeking protocols and formulas that safeguard consumer health.

U.S. DEPARTMENT OF AGRICULTURE

Animal Parasitic Diseases Laboratory: Beltsville, MD