Location: Animal Parasitic Diseases Laboratory
2023 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
Progress was made towards all project objectives, which fall under NP108.
In support of Objective 1, we extended our prior work in two important directions. First, we built on our prior published work with Eimeria acervulina to evaluate differentially expressed genes in each of two strains of Eimeria maxima as well as Eimeria tenella. This clarifies what is unique to each species of Eimeria as it sporulates; more importantly to our food safety agenda, this clarifies what master regulators of physiology and development are conserved among such species. The broad base of conserved and strongly expressed genes, throughout development, provide an empirical basis for hypothesizing viability markers for Cyclospora cayentanensis, a key need for industry and regulatory stakeholders.
We completed a series of evaluations of gene expression in aging and dying oocysts. We did this by extracting Ribonucleic acids (RNA) and performing quantitative reverse transcription polymerace chain reaction (qPCR) assays on cohorts of parasites weeks, months, and years since their last passage (held either at room temperature or 4 degrees Centigrade ever since). Bioassays in chickens determined the viability of these oocysts, enabling us to evaluate gene expression in cohorts of parasites enriched for viable or inviable oocysts. Indeed, RNA quality and quantity provided an interim early signal for the physiological condition of these parasites.
The published work on E. acervulina and our published overview of the need for surrogates attracted attention of stakeholder groups and new research partners, enabling concrete progress in developing aptamer-based detection assays for Cyclospora, enabling the training of a successful Artificial Intelligence-based image processing system for oocyst maturation, and establishing as efficacious disinfection systems for Cyclospora based on Ultraviolet (UV) irradiation and ozonation.
In support of Objective 2, we advanced progress towards viability assays in Eimeria and Cyclospora. Our comparative approach provided us the insight that genes comprising at least 1,000 transcripts per million (TPM) throughout sporulation are more likely to be shared among species than are genes that undergo substantial upregulation in any given species. In this sense, core housekeeping genes provide the most suitable fund for viability markers likely to serve practical purposes in Cyclospora, where regulators and industry are plagued with diagnostic assays lacking information about viability (and therefore, risk).
We advanced progress on two projects supported by the Center for Produce Safety. In the first project (Assessing the Efficacy of Filtration for Cyclospora Control), we defined performance of filters comprised of sand and zero valent iron to reduce water contamination with oocysts of coccidian parasites. In the second project (Viability Assays for Cyclospora), we explored means to evaluate (through vital stains and digital PCR). The quantification of targets will support progress in the field, even if the oocyst and sporocyst walls prove impervious to stains even in dead oocysts (which appears may be the case).
We did not embark on some of the anticipated washing experiments outlined for Objective 3, but we made more progress on produce sanitation than we had planned owing to excellent collaboration. In particular, our partners at the University of Tennessee (via a Material Transfer Research Agreement) developed a high-throughput image analyzer that they succeeded in using to establish, as efficacious, three wavelengths of Ultraviolet (UV) light, as well as an ozonation procedure, to arrest development of Eimieria oocysts.
In support of Objective 4A, ARS scientists in Beltsville, Maryland, previously published (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. Our faster approach simultaneously sequences thousands of genomic fragments from parasites and uses a workflow requiring just a few days.
In support Objective 4B, ARS scientists in Beltsville, Maryland, nearly completed a national survey determining whether PQA+ pigs produced in the United States 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, we accelerated testing and drove towards completion of the project. We estimate 90% completion by the end of FY23, and will use a second source of funding (from the National Pork Producers Council) to complete the work by the end of the first quarter of FY24.
In support of Objective 4C, we commenced a study of Sarcocystis in beef, finding near universal infection with an enzootic species (acquired from dogs and wild canids) and beginning the first evaluation (using needed metagenomic tools) of the possibility of zoonotic species, as has been reported from Europe and elsewhere.
Accomplishments
1. Surrogates speed the scientific fight against a produce parasite. Produce growers, packers, and retailers seek to minimize the public health burden and reputational harm posed by outbreaks of Cyclospora cayetanensis, but they lacked proven means to mitigate risk. This parasite is difficult to find, impossible to grow in the lab, and dangerous to work with, slowing the pace of scientific progress. Therefore, ARS scientists in Beltsville, Maryland, hastened progress by studying closely related organisms that infect chickens, quickly completing seminal studies that identify mitigation opportunities such as water filtration. These insights help producers and regulators prevent infection, benefitting the fresh produce industry, grocers, food safety officials, and the safety of food for all Americans.
2. New insights into the development of a parasite compromising food safety. The foodborne parasite Cyclospora cayetanensis causes gastrointestinal distress, but until recently no one had observed key steps in the parasite’s development in the body because all such information had been derived from just a few biopsy specimens of limited quality. ARS scientists in Beltsville, Maryland, overcame this obstacle by examining, in detail, biopsies containing never-before-seen developmental stages. Their discoveries suggest new avenues to block parasite dissemination and survival, and help food safety professionals, physicians, and pathologists better understand, prevent, and mitigate the consequences of infection.
3. Discriminating among parasites contaminating beef. Cattle, like other livestock, experience infections of parasites in the genus Sarcocystis that result in long-lived tissue cysts. Identifying distinctions among these various species advances control efforts and clarifies any food safety risk, since some parasites pose no threat to human health whereas others can cause enteric disease in people. Therefore, USDA scientists in Beltsville, Maryland, undertook a comprehensive reassessment of such parasites in beef, noting morphological and genetic criteria to identify them. In particular, they cleared up longstanding confusion among parasites presenting thick sarcocyst walls, and deposited reference materials into collections so that future workers will forever be able to associate parasite names with physical voucher material. This clarifies important distinctions between Sarcocystis bovifelis and Sarcocystis hirsuta. This information has helped veterinarians and other diagnosticians understand cattle infections, and serves as a basis to refine ongoing efforts to reconsider human health risks posed by Sarcocystis in beef.
Review Publications
Lopez-Orozco, N., Quiroz-Bucheli, A., Kwok, O.C., Dubey, J.P., Canon-Franco, W., Sepulveda-Arias, J. 2022. First serological and molecular detection of Toxoplasma gondii in guinea pigs (Cavia porcellus) used for human consumption in Nariño, Colombia, South America. Veterinary Parasitology. 36. Article e10081. https://doi.org/10.1016/j.vprsr.2022.100801.
Dubey, J.P., Rosenthal, B.M. 2022. Bovine sarcocystosis: Sarcocystis species, diagnosis, prevalence, economic and public health considerations, and association of Sarcocystis species with eosinophilic myositis in cattle. International Journal of Parasitology. 53(9):463-475. https://doi.org/10.1016/j.ijpara.2022.09.009.
Li, X., Bai, Y., Wu, Y., Zeng, W., Xiang, Z., Zhao, H., Zhao, W., Chen, X., Duan, M., Wang, X., Zhu, W., Sun, K., Wu, Y., Zhang, Y., Qin, Y., Rosenthal, B.M., Cui, L., Yang, Z. 2022. PvMSP-3a and PvMSP-3ß genotyping reveals higher genetic diversity in Plasmodium vivax parasites from migrant workers than residents at the China-Myanmar border. Infection, Genetics and Evolution. 106.Article 105387. https://doi.org/10.1016/j.meegid.2022.105387.
Dubey, J.P. 2022. Redescription, deposition of life cycle stages specimens of Sarcocystis bovifelis Heydorn, Gestrich, Mehlhorn, and Rommel, 1975, and amendment to Sarcocystis hirsuta Moulé, 1888. Parasitology. 149(12):1575-1589. https://doi.org/10.1017/S0031182022001044.
