Research Project: Foodborne Parasites and their Impact on Food Safety

Location: Animal Parasitic Diseases Laboratory

2022 Annual Report

Objectives
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.

Approach
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
Good progress was made towards all project Objectives with notably accelerated progress in defining genes whose expression typifies mature, infectious parasite oocysts (Objective 1) and systems for tracing outbreaks of Trichinella (Objective 4). For the Objective 1, we completed and published (a year ahead of schedule) a comprehensive assessment of gene expression through a 24-hour interval of sporulation in the chicken parasite Eimeria acervulina, a surrogate for Cyclospora cayetanensis, an emerging human parasite threatening produce safety. That study identified coordinated expression patterns in many genes, identifying those especially strongly expressed in mature, sporulated oocysts. These serve as biomarkers for infectious oocysts and have engendered considerable interest in the research community. Their discovery supported two successful applications for extramural funding, and new material transfer research agreements, because they provide the means to further develop viability assays capable of supporting public health surveillance and evaluate mitigation strategies. Although we had no Milestones for Objective 2 in FY22, the progress described above established momentum for developing viability assays for Eimeria and (by extension) Cyclospora. Specifically, we designed assays to quantify expression of genes upregulated in mature, sporulated oocysts and developed a new grant proposal to leverage two additional technologies. We submitted a successful 2-year proposal to: Test vital stains for their ability to selectively abrogate PCR amplification from dead parasites, allowing means to quantify living parasites whose membranes repel such stains. Use droplet digital PCR to absolutely quantify such targets, without reference to a standard curve. We had no milestones for Objective 3, whose purpose is to determine the efficacy of washing procedures to limit contamination of produce with oocysts. Nonetheless, we advanced progress towards that goal in two ways. First, we developed a new Material Transfer Research Agreement (MTRA) with the University of Tennessee to aid their development of high-throughput screens for treatments that abrogate parasite maturation. Second, we were invited to join the CycloCore Consortium and submitted a proposal to USDA-NIFA that would substantially leverage ARS investments in this area. Other CycloCore partners (including a team in Norway) have good experience in evaluating washing procedures, and one purpose of this working group is to develop and share resources and materials. In FY23, as planned, we will develop standard operating procedures for introducing non-zoonotic Eimeria surrogates to the produce facility on our campus. For Objective 4A, we completed and published (two years ahead of schedule) a paper demonstrating Rad-Seq as a powerful, rapid tool to individuate outbreak lineages of Trichinella spiralis. Prior work seeking to trace outbreaks of this foodborne parasite faced steep hurdles owing to the inbred nature of this parasite in Europe and the Americas. The best prior attempts employed a “DNA fingerprinting” method based on characterizing variation in a dozen or more genes harboring repetitive sequences of varying length. Although our team contributed to those prior successes, we found the process too demanding and slow to provide much practical use to those attempting to manage ongoing outbreaks. We achieved much faster success by harvesting, and sequencing, thousands of genomic fragments from parasites originating from either of several swine farms (in Poland, where the parasite remains endemic) or from surrounding wildlife. Using a workflow requiring just a few days, this method readily identifies kinship among the parasites from a given farm. It showed that rats and pigs on one farm were being infected by the same parasites; and it ruled out several suspected wildlife sources of infection, whose parasites were genetically variable and distinct from those on the swine farms. As pleased as we were with this achievement, we have sought additional time-savings improvements, resulting in another (submitted) manuscript that uses draft whole genome sequencing to generate equivalent epidemiological insights in half the time. Ultimately, these efforts will have impact if the tools are fast enough, and cheap enough, for public health authorities and veterinarians to use them. Having substantially met our 5 year goals for this part of the project, we will seek refinements that drive towards this user-friendly goal. We advanced Objective 4B through renewed energy towards establishing whether U.S. Swine constitute a “Negligible Risk” Production environment for Trichinella. This effort, augmented by the Animal Plant Health Inspection Service (APHIS) funding, was slowed by social distancing imperatives imposed by the COVID pandemic; however, the pace of testing has accelerated since Spring, 2022 to drive towards completion of the project in July 2023. During maximized telework, the team of student interns worked with the Lead Scientist to publish a review of diagnostic and surveillance methods. We devoted no energy to advancing serological diagnostic methods. For Objective 4C, we prepared the ground for a large-scale evaluation of Sarcocystis in beef by preparing the laboratory (deemed in need of a new biosafety cabinet), recruiting a research team, and conducting extensive literature reviews and species redescriptions of parasites in this genus that infect cattle. Three manuscripts were written that address the attributes, and confusing taxonomic nomenclature, for species that infect cows consuming water or food contaminated with feline, canine, or human feces. Light microscopy provides very limited means of differentiating among these parasites; for some decades, Transmission Electron Microscopy (coupled with experimental infections in carnivores) provided means to establish the identity of each parasite type- necessary in order to establish which parasites threaten human health. We began building on literature pairing such tools with molecular diagnostics, preparing a manuscript demonstrating the means to characterize the species composition of all such parasites in a given sample of beef. To build a stable foundation for the survey upon which we will embark in FY23, we also redescribed certain species of uncertain provenance, leveraging unparalleled USDA expertise and archival histological specimens.

Accomplishments
1. Defining a produce safety parasite. Produce recalls incur financial harms to growers and grocers when parasites of Cyclospora contaminate herbs, leafy greens, or berries. Confronting this food safety threat requires better definition of the pathogen and the disease it causes. Here, USDA scientists discovered entirely new stages of parasite development in human infections, revising the fundamental understanding of how the parasite establishes infection and causes disease. This discovery may influence the detection and diagnosis of human disease and may suggest new avenues for treating sickened individuals or reducing their likelihood of spreading disease to others.

2. Discerning which parasites are infectious. Increasingly sensitive assays allow food producers and regulators to detect the presence of parasite oocysts, but such tests typically cannot say whether the parasites are mature and infectious. USDA scientists therefore monitored maturing parasites for changes in gene expression. They studied a model coccidian parasite (Eimeria acervulina, which contributes to billions of dollars in annual losses to poultry producers), looking for genes that likely also typify infectious stages of Cyclospora cayetanensis. This discovery has attracted widespread attention, leading to two grants, two new research partnerships with academic food safety researchers, and participation in a major consortium proposal to accelerate progress in defining and mitigating this consequential threat to produce safety.

3. New tools to trace a pork parasite. Trichinella spiralis once posed a major threat to pork safety. Since improvements in farm biosecurity, wild game consumption accounts for virtually all the outbreaks in America; abroad, pork infections remain more common. Tracing the origins of foodborne outbreaks benefits international trade and public health but attempts to do so with Trichinella have traditionally proven difficult, time-consuming, and inconclusive. Therefore, USDA researchers worked with Polish colleagues to devise rapid means to sample genomic variation in Trichinella spiralis. These laboratory procedures and analysis pipeline delivered actionable tracing data in just two days. This achievement enables veterinary and public health authorities better understand and manage the health risks, and financial risks, posed by this foodborne parasite.

Review Publications
Tucker, M.S., Obrien, C.N., Jenkins, M.C., Rosenthal, B.M. 2021. Dynamically expressed genes provide candidate viability biomarkers in a model coccidian. PLoS ONE. 16(10):e0258157. https://doi.org/10.1371/journal.pone.0258157.
Wang, S., Zeng, W., Zhao, W., Xiang, Z., Zhao, H., Yank, Q., Li, X., Duan, M., Li, Z., Wang, X., Rosenthal, B.M., Yang, Z. 2022. Comparison of in vitro transformation efficiency methods for Plasmodium falciparum. Molecular and Biochemical Parasitology. https://doi.org/10.1016/j.molbiopara.2021.111432.
Bilska-Zajak, E., Rosenthal, B.M., Thompson, P.C. 2021. Trich-tracker - a practical tool to trace T. spiralis transmission based on rapid, cost-effective sampling of genome-wide genetic variation. International Journal for Parasitology. https://doi.org/10.1016/j.ijpara.2021.08.002.
Dubey, J.P., Charlesworth, J., Pritt, B. 2022. Transmission electron microscopy on a case of Cyclospora cayetanensis infection from an immune-competent case confirms and extends prior detailed descriptions of its notably small endogenous stage. Parasitology. 149:1397-1405. https://doi.org/10.1017/S0031182022000786.
Dubey, J.P., Murata, F., Cerqueira-Cezar, C., Kwok, O.C., Su, C. 2021. Bears as reservoirs of zoonotic parasites: a 50 year retrospective review based on serological and genetic evidence. Journal of Parasitology. 107(3):519-528. https://doi.org/10.1645/21-16.
Delgado De Las Cueva, G., Prakas, P., Rudaityte-Lukosiene, E., Garcia-Gil, M., Martinez-Gonzalez, M., Butkauskas, D., Mowery, J.D., Dubey, J.P., Habela, M., Calero-Bernal, R. 2021. First description of Sarcocystis species infecting Barbary sheep (Ammotragus lervia). Parasitology Research. 2021. https://doi.org/10.1007/s00436-021-07239-z.
Barburas, D., Cozma, V., Ionica, A., Abbas, I., Mircean, V., D'Amico, G., Dubey, J.P., Gyorke, A. 2022. Intestinal parasites of buffalo calves from Romania: molecular characterization of Cryptoporidium spp. and Giardia duodenalis, and the first report of Eimeria bareillyi. Folia Parasitologica. 69(15):Article e2022. https://doi.org/10.14411/fp.2022.015.
Chen, X., Zhang, J., Pan, M., Zhao, H., Qin, P., Wang, S., Si, Y., Yang, Q., Li, X., Zeng, W., Zheng, X., Wu, Y., Duan, M., Li, X., Wang, X., Maxier, D., Zhang, Y., Zhao, W., Rosenthal, B.M., Huang, Y., Yang, Z. 2021. Novel diagnostic methods using loop-mediated isothermal amplification (LAMP) for diagnosing malarial infections and preventing clinical relapse. Parasites & Vectors. https://doi.org/10.1186/s13071-021-04764-9.
Feng, S., Wang, S., Zeng, W., Zhong, D., Hu, Y., Bai, Y., Ruan, Y., Si, Y., Zhao, H., Yang, Q., Li, X., Chen, X., Zhang, Y., Li, C., Xiang, Z., Wu, Y., Cheng, F., Su, P., Rosenthal, B.M., Yang, Z. 2021. Polymorphism of antifolate drug resistance in Plasmodium vivax from local residents and migrant workers returned from the China-Myanmar Border. PLOS Neglected Tropical Diseases. 11:683423. https://doi.org/10.3389/fcimb.2021.683423.
Barlow, A., Roy, K., Hawkins, K., Ankarah, A.A., Rosenthal, B.M. 2021. Evaluating testing and assurance methods for Trichinella surveillance programs. Food and Waterborne Parasitology. https://doi.org/10.1016/j.fawpar.2021.e00129.