Research Project: Zoonotic Parasites Affecting Food Animals, Food Safety, and Public Health

Location: Environmental Microbial & Food Safety Laboratory

2020 Annual Report

Objectives
Objective 1: Conduct whole-genome sequencing to characterize the differences between zoonotic/non-zoonotic and pathogenic/non-pathogenic Cryptosporidium, Giardia, Blastocystis, and Enterocytozoon bieneusi. Objective 2: Develop intervention and treatment strategies against zoonotic parasites Cryptosporidium and Giardia. Subobjective 2.A. Evaluate the ability of probiotics to prevent/ameliorate the negative effects of cryptosporidiosis and giardiasis in rodent models of infections. Subobjective 2.B. Evaluate glucagon-like peptide 2 (GLP-2) and feed additives that enhance basal GLP-2 secretion on pre-weaned calves as an intervention and treatment for cryptosporidiosis and giardiasis. Objective 3: Develop a unique and highly sensitive assay to detect the zoonotic protists Cryptosporidium, Giardia, Blastocystis, Encephalitozoon and Enterocytozoon in food and environmental samples by targeting intracellular viral symbionts of these parasites and water-borne pathogens. Subobjective 3.A. Detecting Cryptosporidium parvum and Giardia duodenalis by targeting intracellular viral symbionts of these parasites. Subobjective 3.B. Identifying viruses and recovering viral RNA from Blastocystis, Encephalitozoon and Enterocytozoon, and develop detection assays based on the viral symbionts.

Approach
Cryptosporidium, Giardia, Blastocystis, and Microsporidia are cosmopolitan microscopic parasites that cause severe diarrheal disease in humans and animals, and can be lethal in immunecompromised individuals. These parasites are spread by fecal contamination, are waterborne, and have been identified as contaminants of fresh fruit and vegetables. To identify the genomic basis of host specificity and virulence for Cryptosporidium, Giardia, Blastocystis, and Enterocytozoon bieneusi, we will conduct whole genome sequencing and use comparative genomic analysis between zoonotic/non-zoonotic and pathogenic/non-pathogenic organisms. Furthermore, because current detection methods lack sensitivity that results in potential underreporting of produce contamination, we will develop new highly sensitive assays based on molecular detection targeting intracellular viral symbionts of these parasites. These assays will enable better detection of zoonotic protist parasites in food, and provide for a better understanding of the role of food animals in the epidemiology of these organisms. Because there are no vaccines or preventable medicines for Cryptosporidium and Giardia, we plan to evaluate different products to prevent disease spread and/or symptoms for Cryptosporidium and Giardia. We will assess products with the potential to be incorporated as feed additives for animals and humans using randomized clinical trials to evaluate their efficacy. To evaluate effectiveness for probiotics we will use rodent challenge models (mice and gerbil), and for GLP-2 and/or Sucram a calf challenge model.

Progress Report
In 2020, significant progress was made for objectives included in this project plan, all of which fall under the National Program 108. We completed sample collection 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). Over 4,000 fecal samples from goats, including from goat kids to adults, from 26 states were processed to concentrate parasite to later extract DNA that will be used to conduct molecular characterization of all isolates and whole genome sequencing of selected isolates. Most of the microscopy has already been completed (3,000 samples) and number of oocysts and cysts per gram of feces for Cryptosporidium and Giardia, respectively, have been noted. A method to produce intact whole genomes directly from fecal isolates of Giardia was established using a combination of cleaning/concentration of parasites techniques and short (Oxford Nanopore MinIon) and long read (Illumina MiSeq) sequencing platforms. Both sequencing platforms complement each other, MiSeq produces short but highly accurate sequences and the MinION produces long reads up to 100s of kb in length assisting with alignment and reducing number of contigs necessary to produce whole genomes. We conducted an assessment of completeness of genome assembly for two Giardia isolates using this methodology and compared resulting genomes to Giardia reference genomes that demonstrated that we can successfully produce complete novel whole genomes from Giardia. This method is now ready to be utilized to produce novel Giardia genomes from fecal isolates to allow performing genome wide analysis that is required to determine the genetic basis of Giardia’s pathogenicity and host specificity as well as genes that may be drug or vaccine targets to aid in the treatment and prevention of this common pathogen. ARS researchers in Beltsville, Maryland, have recently developed a method to study intra-host Blastocystis communities using next generation amplicon sequencing (NGS) that has been successfully used with collaborators from Mexico, Spain, and Brazil to better understand role of mixed infections in humans, wild carnivores, and domestic and wild birds. Results from those studies were published in Parasites & Vectors (doi:10.1186/s13071-019-3814-z), Journal of Eukaryotic Microbiology (doi: 10.1111/jeu.12772), and Parasite Epidemiology and Control (doi.org/10.1016/j.parepi.2020.e001380), respectively. Additional studies to further understand epidemiology of this parasite in humans, horses, cattle, chickens, pigs, goats, deer in collaboration with national and international collaborators are in progress. An NGS protocols to detect and identify species and assemblages of Giardia using the beta-giardin gene was developed by ARS researchers in Beltsville, Maryland. This is one of the most commonly used genes for Giardia molecular characterization and captures variability within assemblages. A selected set of samples were processed using Sanger and NGS typing. Results obtained by both methods were compared, and it was clear that NGS can resolve complex DNA mixtures and detect mixed assemblage infections as well as variations within assemblages. This indicates that previous studies that have relied on Sanger sequencing, may not reflect the extent of diversity resulting in incorrect assumptions on Giardia transmission. The importance of detecting diversity in a sample is critical to understand Giardia epidemiology and its transmission dynamics. A manuscript containing this information will be soon submitted as an invited contribution to Food and Waterborne Parasitology. Additionally, in collaboration with scientists from Spain, we are investigating the presence of zoonotic parasite E. bieneusi in pigs, dog, cats, and wild boars to understand if they could be a source of environmental contamination leading to infection of other animals and humans. Results demonstrated that human-pathogenic E. bieneusi genotypes are present in these animals corroborating their potential role as a source of human infection and environmental contamination. Two manuscripts were published, one that covered dogs and cats in Parasitology Research (doi.org/10.1007/s00436-019-06428-1) and the other one on sympatric Iberian pigs and wild boars in Transboundary Emerging Diseases (doi.org/10.1111/tbed.13658).

Accomplishments
1. Method to detect parasite Cryptosporidium in water. Cryptosporidium is responsible for causing 30,000 cases of severe diarrhea annually in the U.S. The parasite is resistant to most standard water disinfectants, can cause clinical infection at very low numbers, and no effective drug or vaccine exists. ARS scientists developed a method to detect the parasite in water using an RNA assay that targets a virus, Cryspovirus, found inside all species of Cryptosporidium in thousands of copies. This method is more sensitive than current gold standard tests for source and finish water and could be used as a water screening method by regulatory and public health agencies to detect and track Cryptosporidium in untreated and drinking water.

Review Publications
Dashti, A., Santin, M., Cano, L., De Lucio, A., Bailo, B., Hernandez De Mingo, M., Koster, P.C., Fernandez-Basterra, J.A., Aramburu-Aguirre, Lopez-Molina, N., Fernandez-Crespo, J.C., Calero-Bernal, R., Carmena, D. 2019. Occurrence and genetic diversity of Enterocytozoon bieneusi (Microsporidia) in owned and sheltered dogs and cats in Northern Spain. Parasitology Research. 118(10):2979-2987. https://doi.org/10.1007/s00436-019-06428-1.
Calero-Bernal, R., Santin, M., Maloney, J.G., Martin-Perez, M., Habela, M., Fernandez-Garcia, J.L., Figueiredo, A., Najera, F., Palacios, M.J., Mateo, M., Balseiro, A., Barral, M., Lima Barbero, J., Koster, P.C., Carmena, D. 2019. Blastocystis sp. subtype diversity in wild mesocarnivores from Spain. Journal of Eukaryotic Microbiology. 67(2):273-278.
Maloney, J.G., Molokin, A., Cury, M.C., Santin, M., Da Cunha, M.J. 2020. Blastocystis subtype distribution in domestic and captive wild birds from Brazil using next generation amplicon sequencing. Parasite Epidemiology and Control. 9:e00138. https://doi.org/10.1016/j.parepi.2020.e00138.
Lindsay, D., Dubey, J.P., Santin, M. 2019. Coccidia and other Protozoa. Book Chapter. 1015-1027. https://doi.org/10.1002/9781119350927.ch66.
Santin, M. 2020. Giardiasis and cryptoporidiosis. Veterinary Clinics of North America. 36(1):223-238. https://doi.org/10.1016/j.cvfa.2019.11.005.
Fink, M.Y., Maloney, J.G., Keselman, A., Li, E., Menegas, S., Jones, C., Singer, S.M. 2019. Proliferation of resident macrophages is dispensable for protection during Giardia duodenalis infections. ImmunoHorizons. 3(8):412-421. https://doi.org/10.4049/immunohorizons.1900041.