Project : USDA ARS

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

Location: Animal Parasitic Diseases Laboratory

2021 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
We made extensive progress completing important work related to the food safety dangers posed by zoonotic parasites. ARS redirected all its research focused on toxoplasmosis in April 2019, necessitating cessation of activities on many objectives established in May 2016. Some studies relating to experimental and fieldwork concluded before the redirection were published in FY21. These include a series of review articles prepared to enrich a forthcoming revision of Dr. Dubey’s seminal textbook on this parasite and a serological survey of the U.S. swine herd; nonetheless, our team’s primary focus was on developing and pursuing new food safety priorities. These new priorities shine a focus on poultry coccidiosis as a model to make needed headway in managing the foodborne risk posed by Cyclospora cayetanensis, an enteric parasite imposing increasing burdens on human health and increasingly responsible for multi-state outbreaks attributed to fresh produce. In the last year, our team engaged in significant outreach efforts to new stakeholders (the Center for Produce Safety, United Fresh Incorporated, and individual growers that have experienced product recalls) and partnered with other ARS researchers in the food safety program as well as academic leaders in the field (including the President of the International Association for Food Protection) to define and articulate new program priorities; we wrote a new project organized around goals thereby identified, received a ‘minor revision’ review, and implemented the new project in May 2021. Even prior to the new plan’s implementation, the Office National Programs (ONP) endorsed our decision to build stakeholder engagement and seek extramural support for new objectives related to parasitic threats to produce safety; our pre-proposal (to the Center for Produce Safety) delineated sand/zero valent iron filters as a means to remove coccidian oocysts from irrigation waters. We proposed using poultry coccidia (with which our group has ample experience) as a surrogate for Cyclospora, the agent of human disease. The pre-proposal was well received; we were then invited to submit a full proposal, which also appears to have found favor (no formal announcement has yet been made). If filters succeed in removing or harming such parasites, we will deliver a practical pre-harvest control for growers, alleviating concern over a growing food safety threat. We have already made significant progress toward new objectives defined for our successor project (8042-32000-113). In particular, we concluded: • A study of changing gene expression as coccidian oocysts mature (sporulate), identifying biomarkers for parasite viability. Such assays may provide practical means to assess risk and may suggest new avenues for intervention and hygiene. • Two studies describing the first-ever means to trace outbreaks and identify sources of contamination with Trichinella spiralis (mentoring a visitor from the National Veterinary Research Institute of Poland, where endemic parasite transmission on low-resource farms continues to threaten human health and impose costs on pork production). Finally, we solidified our team’s future by successfully recruiting an outstanding new scientist to fill the vacancy created by Dr. Dolores Hill’s retirement. Dr. Asis Khan comes to us from the National Institute of Allergy and Infectious Disease, having previously completed postdoctoral training in one of the Nation’s foremost departments of Medical Microbiology, focusing on parasite genetics, genomics, molecular epidemiology, and virulence. He brings extramural support (from the National Institute of Health (NIH), via a collaboration with the University of Georgia) to make new headway on Cryptosporidium epidemiology and prevention.

Accomplishments
1. A game of cat and mouse. To save human lives, researchers often study animals. The USDA seeks every opportunity to minimize that need. Therefore, USDA researchers and their partners sought to discover a way to grow a cat parasite in mice. This parasite causes toxoplasmosis, sickening pregnant women and their babies. Astonishingly, mice bred and raised to mimic cat digestion supported parasite growth. To hasten the discovery of drugs and vaccines, researchers have sought to build on this landmark achievement.

2. How to prevent foodborne trichinellosis. Many Americans used to become ill by eating pork infected with Trichinella parasites. No longer. Successful measures championed by the USDA reassure consumers, limit producer liability, and promote trade. ARS researchers contributed to new international guidelines for safeguarding meat. ARS partnered with three other USDA agencies (the Agricultural Marketing Service, the Animal and Plant Health Inspection Service, and the Food Safety Inspection Service).

3. A foodborne parasite in meat survives prolonged refrigeration. Refrigeration preserves and protects meat. But USDA researchers suspected that refrigeration does not kill Toxoplasma gondii. The parasite survived in vacuum-packed meat refrigerated for two weeks. Because this parasite causes birth defects and other illnesses, meat should be thoroughly cooked.

4. Dry salt curing kills parasites in meat. Salt curing produces pepperoni and ready-to-eat hams. USDA researchers determined that this procedure kills the parasite causing toxoplasmosis. Salt curing therefore, reduces the risk of birth defects and other illnesses. The findings reassured consumers and helped food producers and regulators manage risk in a cost-effective manner.

5. Dangerous parasites in deer. Many Americans hunt deer and eat venison. Researchers in Beltsville, Maryland, discovered in deer unusual strains of the foodborne parasite causing toxoplasmosis. An outbreak of the severe disease occurred in a group that consumed undercooked venison. These studies remind us that proper cooking ensures venison’s safety.

6. Evolution of SARS-CoV-2. COVID-19 upended life and caused great harm. An international team of infectious disease scholars sought ARS expertise to understand how the virus was spreading and changing. These insights helped epidemiologists understand and respond to the emerging threat.

7. A parasite of livestock and dogs has taken the world by storm. Neosporosis is the most important cause of abortion in cattle. USDA researchers and their partners determined that one parasite lineage has expanded globally. This finding gives hope that the same management tools may succeed wherever the parasite causes harm.

8. Against parasites, the best offense is a good defense. Toxoplasma infects many kinds of birds and mammals. It infects millions of people, some of whom suffer birth defects, mental retardation, and blindness. To understand why, USDA researchers worked with scientists at the University of Pittsburgh to compare harmful and harmless parasites. They discovered differences in how these parasites avoid host defenses. These insights suggested new ways to prevent disease.

9. A comprehensive view of an emerging threat to produce safety. Cyclosporiasis is a poorly understood foodborne illness. USDA and FDA scientists reviewed this parasite’s spread in the USA and outlined research needs that ARS is now pursuing. Success will reduce harm to consumers, growers and grocers.

10. Better detection for food and water-borne parasites. It is difficult to detect parasites that contaminate food and water. USDA researchers and their partners devised a rapid, low-cost and sensitive method to detect three such parasites. These findings aid public health workers, biologists, and parasitologists seeking to reduce risk.

11. Blue mussels scan for parasites in waterways. Ingesting food and water contaminated with parasites causes illness. Detecting scarce parasites is difficult. To overcome this, USDA scientists used blue mussels. These filter feeders concentrate on such parasites. These findings provided a new means to monitor water quality and safeguard health.

U.S. DEPARTMENT OF AGRICULTURE

Animal Parasitic Diseases Laboratory: Beltsville, MD