Objective 1: Characterize genomes of zoonotic parasites to conduct comparative genomics to determine the genetic basis of host specificity, pathogenicity, and virulence. Objective 2: Develop improved molecular assays for detection of zoonotic parasites in food and environmental samples. Objective 3: Elucidate the epidemiology of water and food borne parasites Cyclospora, Cryptosporidium, Giardia, Blastocystis and Microsporidia by identifying unique and emerging genetic variants using molecular tools.
Protist parasites, including Cyclospora, Cryptosporidium, Giardia, Blastocystis and Microsporidia, are some of the most common causes of food and waterborne intestinal illness in the United States and around the world. Yet, our knowledge of the basic biology, epidemiology, and transmission sources of these organisms remains incomplete. This knowledge gap is due in part to the need for better tools to study the prevalence, persistence, and genomic composition of protist parasites in their natural environments. This project plan will address these research needs using a multi-faceted approach. We will develop culture free methods of parasite collection and concentration that will be used to produce novel genomes for zoonotic protist parasites and use those genomes to conduct comparative genomic studies to determine the genes responsible for host specificity, pathogenicity, and virulence. We will develop next generation amplicon sequencing assays and bioinformatic pipelines to improve both detection and resolution of mixed infections of zoonotic protist parasites. The more sensitive newly developed collection and sequencing tools will aide in the determination of the extent and sources of contamination of fresh produce and water to better characterize the movement and population structure of zoonotic parasites across the food continuum. We will characterize the genetics of zoonotic protist parasites in various food animals, fresh produce, and water to detect emerging zoonotic parasites. This information is key to understanding zoonotic transmission risk and the complex associations between humans, food animals, and wildlife. The data and new molecular tools generated by this project plan can be used by scientists, regulators, and industry to better understand the risk zoonotic protist parasites pose to human health and to develop intervention strategies to improve food and water safety.
In 2021, significant progress was made for Objectives 1 and 2 included in this project plan, which fall under the National Program 108. For Objective 1, ARS researchers in Beltsville, Maryland, completed DNA extractions and Immunofluorescence Microscopy (IFA) for Cryptosporidium and Giardia to determine prevalence of infection for the largest and most comprehensive study to identify zoonotic protists (Cryptosporidium, Giardia, E. bieneusi and Blastocystis) carried out in goats in the United States in collaboration with the National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS). The study included over 4,000 fecal samples from goats, including from goat kids to adults, from 26 states. Details of testing for Cryptosporidium and Giardia for each sample were submitted to APHIS to produce reports that were sent to stakeholders (farmers) that participated in the study. Using extracted DNA, we have initiated molecular characterization of isolates to identify species and genotypes for Cryptosporidium, Giardia, E. bieneusi and Blastocystis and investigate public health impact of the species/genotypes found in goats in this study. For Objective 2, ARS researchers in Beltsville, Maryland, have recently developed a method to study intra-host/sample Giardia communities using next generation amplicon sequencing (NGS) that has been successfully used to detect species and assemblages of Giardia to better understand mixed infections. This assay can resolve complex DNA mixtures and detect mixed assemblage infections as well as variations within assemblages. Detecting diversity in a sample is critical to understand Giardia epidemiology and transmission dynamics. Results from validation of the assay were published in the journal Food and Waterborne Parasitology (doi: 10.1016/j.fawpar.2020.e00098). Additional studies of mixed infection in dairy cattle are in progress and are using this assay to further understand epidemiology of Giardia.
1. Developed a next generation amplicon sequencing method to evaluate Giardia genetic diversity. The protozoan Giardia duodenalis is extremely common and responsible for ~280 million human cases of diarrhea every year. It also infects a wide range of animals and is considered a species complex consisting of eight assemblages with differing host specificity and pathogenicity. Molecular characterization to identify Giardia variants present in samples is critical to understand transmission, public health significance, and pathogenicity of this parasite as the network of potential infection sources of is complex. Furthermore, mixed infections are often missed using current detection technology, but better characterization of mixed infections is needed to fully understand Giardia’s complex epidemiology. A next generation amplicon sequencing protocol developed by ARS scientists in Beltsville, Maryland, was capable to detect mixed genetic variants of Giardia and detect low abundance Giardia variants. This method provides a powerful tool for an accurate determination of the population structure of Giardia in clinical samples, environmental samples, and fresh produce. It will assist in identifying potential sources of environmental contamination to help establish effective parasite control measures.
Maloney, J.G., Molokin, A., Santin, M. 2020. Assessment of next generation amplicon sequencing of the beta-giardin gene for the detection of Giardia duodenalis assemblages and mixed infections. Food and Waterborne Parasitology. 21:e00098. https://doi.org/10.1016/j.fawpar.2020.e00098.