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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #440184

Research Project: Foodborne Parasites and their Impact on Food Safety

Location: Animal Parasitic Diseases Laboratory

2021 Annual Report

Objective 1: Describe natural sporulation in coccidian oocysts, including temporal changes in the expression of genes over the course of sporulation and during the degradation of ‘old oocysts’ and morphological changes over the course of sporulation. Sub-objective 1.A. Characterize changes in gene expression as oocysts of E. acervulina mature to their sporulated and infectious state using RNA-Seq to elucidate maturation and identify biomarkers for assaying oocyst viability and infectivity. Sub-objective 1.B: Determine the markers of oocyst senescence by tracking the waning of gene expression and/or the advent of apoptotic signals in E. acervulina. Sub-objective 1.C: Determine whether developmental gene expression patterns established for E. acervulina hold for other Eimeria species infecting poultry. Sub-objective 1.D: Characterize morphological changes of E. acervulina oocysts during sporulation and senescence. Objective 2: Develop assays for coccidian oocyst viability; and using the information from Objective 1, establish gene products strongly up-regulated in infectious, sporulated oocysts of Eimeria. Using comparative genomics, determine homologues in the Cyclospora genome. Sub-objective 2.A: Establish quantitative assays targeting gene products established (above) to undergo the strongest and most consistent up-regulation in mature oocysts of E. acervulina. Sub-objective 2.B: Determine the extent to which these genes’ expression levels predict the infectiousness of oocyst cohorts, using cell culture assays and/or in vivo challenge experiments. Sub-objective 2.C. Identify homologues, in the genome of Cyclospora cayetanensis, of genes consistently and significantly up-regulated in various species of Eimeria infecting poultry. Sub-objective 2.D: Develop quantitative assays designed to measure expression levels of genes deemed most likely over-expressed in mature, infectious oocysts of C. cayetanensis. Objective 3: Evaluate the efficacy of interventions for produce safety using the surrogate. Evaluate known interventions on viability, and on the biomarkers to validate assays for coccidian viability. Sub-objective 3.A: Determine the efficacy of various washing procedures on limiting contamination of produce with oocysts of E. acervulina. Sub-objective 3.B. Improved detection of E. acervulina and C. cayetanensis DNA in contaminating matrices using genome probe capture arrays. Objective 4: Continue to advance the molecular epidemiology of other foodborne zoonotic parasites in livestock and wildlife in the U.S. such as Trichinella in feral swine, pastured swine, and wild carnivores, and Sarcocystis zoonotic species in cattle. Sub-objective 4.A: Determine the efficacy of whole genome and reduced representational sequencing methods to individuate outbreak lineages of Trichinella spiralis. Sub-objective 4.B: Document prevalence of Trichinella in defined compartments of food production and in wild game populations, and the prevalence of anti-Trichinella antibodies in those animals. Sub-objective 4.C: Survey U.S. beef for the presence of Sarcocystis species, including the zoonotic species.

Foodborne parasites exact a serious toll on public health, undermine public confidence in the safety of food, interfere with agricultural marketing and trade, and impose liabilities and exact costs on farmers and food producers. Adopting a “One Health” approach that recognizes commonalities in protecting human, veterinary, and environmental health, we will pursue scientific goals capable of ameliorating burdens imposed by longstanding and emerging problems imposed by parasites contaminating meats and fresh produce. We will first establish a safe and tractable model for Cyclospora cayetanensis, the agent of human cyclosporiasis, using a closely related poultry parasite, Eimeria acervulina. We will use this model to evaluate practical ways to minimize people’s exposure to infection with coccidian oocysts, and will endeavor to supply our regulatory partners with molecular assays to assess parasite viability and infectivity. We will also advance the molecular epidemiology of other foodborne zoonotic parasites in livestock and wildlife in the U.S. such as Trichinella spp. and Sarcocystis zoonotic species. Studies will determine the efficacy of sequencing methods to individuate outbreak lineages of Trichinella spiralis and document prevalence of Trichinella in compartments of food production and in wild game populations. Further studies will analyze the prevalence of Trichinella spp. antibodies in those animals and characterize the presence of Sarcocystis species (including an important zoonotic species) in the U.S. beef supply. Taken together, these studies will address important research gaps and provide powerful tools to producers and food safety regulators for monitoring and ameliorating food safety risks imposed by parasitic infection.

Progress Report
This project was just approved and implemented in May, 2021, but already we are making significant progress. To advance the first objective, we have completed a study of changing gene expression over a 24-hour period during which Eimeria acervulina sporulates (Subobjective 1A). We have identified genes which undergo: • Constitutive expression throughout the interval of sporulation. • Decreased expression in sporulated oocysts. • Increased expression in sporulated oocysts. We have also begun studying how natural aging and heat stress influence gene expression in E. acervulina (Subobjective 1B). We have also designed qPCR and Ampliseq panels designed to measure the expression of candidate biomarkers (Subobjective 2A). We have completed a manuscript describing this early progress and engaged the Office of Technology Transfer in order to establish whether scientific impact will be maximized by publishing these early studies now or by protecting intellectual property (via invention disclosure and patent application) to promote future licensing of technologies based on these discoveries. Meanwhile, we submitted (with ARS and University collaborators) a proposal to the Center for Produce Safety to evaluate the efficacy of filtration to remove coccidian oocyst from irrigation waters. No announcement has been made about the grant, but CPS contacted us for budget details and seem quite convinced that poultry models for Cyclosporosis (Objectives 1-3 of the parent project) indeed merit exploitation to advance efforts to control such foodborne parasites. We have also made striking progress towards Subobjective 4A, completing two manuscripts which open vistas on tracing outbreaks of Trichinella spiralis. In the United States, and in Europe, we have previously documented that this zoonotic parasite (of meat) is highly inbred, impeding efforts to identify or exclude sources of contamination. Working with European collaborators, including a visiting scientist from Poland’s Veterinary Medical Research Institute, we demonstrated that each of two genotyping approaches work. The first approach (using genetic ‘fingerprints’ from markers with variable numbers of repeats, called microsatellites) builds upon earlier ARS discoveries but takes weeks or months to complete. The second approach (simultaneously sampling genome-wide variation from many isolates, using brand-new techniques) solves the problem far more efficiently. Manuscripts for each of these achievements are now in review. Progress on Subobjective 4B was slowed by the extended telework period necessitated by the pandemic (when we suspended testing operations for Trichinella in US Pork). However, a team of young scientists on that project authored a manuscript reviewing actual and possible means to survey swine, pork products, and wild game for evidence of infection (manuscript in revision). Future progress on the project was secured by setting up the lab of our newly-hired scientist and submitting, for review, new Biosafety and Animal Use and Care protocols that will undergird a series of studies on food and water-borne parasitic infections.