Research Project: Detection and Characterization of Zoonotic and Emerging Parasites Affecting Food Safety and Public Health

Location: Environmental Microbial & Food Safety Laboratory

2023 Annual Report

Objectives
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.

Approach
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.

Progress Report
In 2023, significant progress was made for all objectives included in this project plan, which fall under the National Program 108. For Objective 1, ARS researchers in Beltsville, Maryland, continued generating high quality G. duodenalis genomes directly from cysts obtained from naturally infected hosts using a hybrid sequencing (Illumina and Nanopore Technology) and assembly strategy. Specifically, genomes from G. duodenalis assemblages B, C, D, and E were obtained. Additionally, the genome of a Cyclospora cayetanensis isolate obtained via a collaboration with the Hospital Infantil de Morelia "Eva Sámano de Lopez Mateos" was generated. The bioinformatic analysis of this genome has been completed and a manuscript is in preparation. Additionally, ARS researchers in Beltsville, Maryland, are conducting molecular detection of protist parasites from a nationwide project that included over 4,000 fecal samples from goats conducted in collaboration with the National Animal Health Monitoring Service (NAHMS) of Animal and Plant Health Inspection Service (APHIS) using Sanger and next generation amplicon sequencing (NGS). Isolates of Cryptosporidium and Giardia are being selected to continue to conduct a multistep cleaning process using immunomagnetic beads to undertake a hybrid sequencing and assembly strategy to generate reference quality G. duodenalis assemblages and Cryptosporidium species genomes. In addition, use of non-fat dry milk to assist with DNA extraction for purified Cryptosporidium oocysts and Giardia cysts is currently being assessed. Preliminary data suggest that DNA recovery increases in those samples in which non-fat dry milk was included. This is a critical finding that could decrease the minimum number of parasite forms necessary to extract enough DNA to undertake the generation of hybrid sequencing to produce genomes from naturally infected hosts for which low numbers of parasites are available. For Objective 2, ARS researchers in Beltsville, Maryland, are testing an NGS approach using Nanopore Technology to detect mixed infections of Cryptosporidium species and genotypes using primers that amplify the 800 bp region of the small subunit rRNA gene (SSU RNA) that is currently the gold standard for genotyping Cryptosporidium. Additionally, they are using their NGS methods to investigate in goats in the United States mixed assemblage and subtype infections of G. duodenalis and Blastocystis, respectively, to understand impact of mixed infections in the epidemiology of those parasites. Specifically, a subset representing 20% of sampled goats randomly selected from the over 4,000 goats obtained from a nationwide project conducted in collaboration with NAHMS/APHIS are being screened. Additionally, NGS analysis has been completed for two other Giardia projects using NGS in sheep and humans in collaboration with the Complutense University of Madrid and El Bosque University, respectively and two manuscripts are in preparation. A MinION strategy developed by ARS researchers in Beltsville, Maryland, to amplify full-length Blastocystis small subunit rRNA gene sequences is being used for validation of novel subtypes in collaboration with other international institutions including: 1) ST33 and ST34 in Colombian horses (https://doi.org/10.3390/microorganisms10091693); 2) ST35 in a Brazilian child, ST36 in a Mexican wild bat, ST37 Mexican wild rodent, and ST38 in a captive European water vole in UK (https://doi.org/10.3390/microorganisms11010046); 3) ST40 in muskoxen in Greenland (https://doi.org/10.1016/j.ijpara.2023.05.005); and 4) ST41 in a Colombian patient undergoing colorectal cancer screening (https://doi.org/10.1111/jeu.12978). For Objective 3, ARS researchers in Beltsville, Maryland, are using their NGS method to detect the presence of Giardia mixtures in baby romaine that was spiked with different numbers of cysts and washed according to the FDA’s BAM Chapter 19b. Additionally, they used their Blastocystis NGS protocol to: 1) study mixed Blastocystis subtype infections in the first and most comprehensive longitudinal study conducted in dairy cattle that included fecal collections from 30 calves from birth to 24 months of age at a dairy farm in Maryland to determine the prevalence, mixed infections, and age distribution of genetic variants (https://doi.org/10.1186/s13071-023-05795-0); 2) conduct the first Blastocystis study in wild rabbits and hares as part of an extensive study meant to establish the potential public health significance of intestinal parasites in those animals that found that they were commonly infected with subtypes frequently reported in humans as well as with other zoonotic pathogens of public health importance (G. duodenalis assemblage B and Encephalitozoon intestinalis) (https://doi.org/10.1111/zph.13018); and 3) investigate Blastocystis genetic diversity and potential risk factors for Blastocystis infection in rural schoolchildren from Colombia that demonstrated an association between presence of Blastocystis and sociodemographic variables (gender, age, number of people per household, toilet location, companion animals or chickens in the house, handwashing habits, and nausea within 15 days prior sampling) (https://doi.org/10.1007/s00436-023-07841-3). In a joint effort with the Carlos III Health Institute and University of Aveiro, the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal and Spain to investigate E. bieneusi and Blastocystis occurrence and their genetic diversity in wild carnivores, wild ungulates, and domestic animals was completed. The study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals (https://doi.org/10.1093/mmy/myad018 and https://doi.org/10.1093/mmy/myac070). The Blastocystis analysis has been completed and two manuscripts are in preparation.

Accomplishments
1. MinION sequencing strategy to generate full-length Entamoeba small subunit rRNA (SSU rRNA) gene sequences. Characterization of gut pathogens causing diarrhea shared by humans and animals is crucial to understand their genetic diversity, host specificity, and impact on host health and disease. Entamoeba, one of the most common gut protozoans of humans and animals, is transmitted by contaminated food and water. The current validation system for novel Entamoeba genetic variants requires generation of full-length sequences of the small subunit ribosomal RNA gene. However, no method is available to obtain full-length sequences from the complex sample matrixes in which Entamoeba is usually identified. A protocol developed by ARS scientists in Beltsville, Maryland, using a Nanopore sequencing strategy was successfully used to generate the first full-length sequences of five Entamoeba sequence types isolated from sheep and muskox samples. This protocol provides an essential tool to properly identify Entamoeba genetic variants and to get an insight into their distribution to comprehend transmission pathways, host specificity, zoonotic potential, and pathogenicity.

2. First large-scale longitudinal study of Blastocystis. Blastocystis is a common intestinal protist in humans and animals worldwide. Transmission occurs via the fecal-oral route with infections acquired via contact with infected hosts or ingestion of contaminated food or water. Epidemiology studies using molecular tools have generated data on subtypes of Blastocystis from human and animal hosts, however, no large-scale longitudinal studies have ever been conducted for Blastocystis. Longitudinal studies are needed to understand patterns of infection prevalence and subtype diversity and their relationship with host age. ARS scientists in Beltsville, Maryland, conducted the most comprehensive longitudinal study of Blastocystis ever performed. Next generation amplicon sequencing was used to simultaneously define Blastocystis prevalence and subtype diversity in 30 dairy calves that were followed from birth through 24 months. Blastocystis presence increased with time and a remarkable mixture of Blastocystis subtypes were identified. Subtype diversity increased with age, and distinct patterns in subtype prevalence and chronicity were observed, supporting the importance of subtype discrimination to understand host infection. This study represents a significant advance in understanding Blastocystis infection dynamics in a host population over time.

3. Next generation sequencing to evaluate Blastocystis genetic diversity in horses. Blastocystis is a common enteric parasite found in humans and animals worldwide. Extensive genetic diversity has been described for Blastocystis subtypes, which do not exhibit strict host specificity. Thus, zoonotic transmission may occur through either direct contact or through fecal contamination of food or water. Horses play an important role within human culture and can harbor human pathogens such as Blastocystis. However, data on Blastocystis in horses is scarce. ARS scientists in Beltsville, Maryland, in collaboration with the Universidad Nacional de Colombia-Sede Bogotá conducted the first study to assess Blastocystis subtype diversity and occurrence in horses worldwide using next generation amplicon sequencing. Horses were found to be Blastocystis-positive (44%) and mixed subtype infections were common (56%). Twelve subtypes were identified including ten known subtypes (ST1, ST3–ST6, ST10, ST14, ST25, ST26) and 2 novel subtypes (ST33 and ST34). Seven of those subtypes have been previously reported in humans and most Blastocystis-positive horses harbored one of those subtypes (89%). This comprehensive study indicates that horses frequently harbor potentially zoonotic Blastocystis subtypes that could contribute to environmental contamination and the transmission of Blastocystis to humans.

Review Publications
Wang, T., Guo, Y., Roelling, D.M., Li, N., Santin, M., Lombard, J., Kvac, M., Naguib, D., Zhang, Z., Feng, Y., Xiao, L. 2022. Sympatric recombination in zoonotic Cryptosporidium leads to emergence of populations with modified host preference. Nature Microbiology. 39(7). Article e150msac150. https://doi.org/10.1093/molbev/msac150.
Vioque, F., Dashti, A., Santin, M., Ruiz-Fons, F., Koster, P.C., Hernandez-Castro, C., Garcia, J.T., Bailo, B., Ortega, S., Olea, P., Arce, F., Chicharro, C., Nieto, J., Gonzalez, F., Vinuela, J., Carmena, D., Barrios, D. 2022. Wild micromammal host spectrum of zoonotic eukaryotic parasites in Spain. Occurrence and genetic characterization. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14643.
Dashti, A., Santin, M., Koster, P.C., Ballo, B., Ortega, S., Imana, E., Habela, M.A., Rivero-Juarez, A., Vicente, J., We&H, G., Arnal, M.C., Morrondo, P., Armenteros, J.A., Balseiro, A., Cardona, G.A., Martinez, C., Ortiz, J.A., Calero-Bernal, R., Gonzalez-Barrio, D., Carmena, D. 2022. Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain. Medical Mycology. 60(9). Article myac070. https://doi.org/10.1093/mmy/myac070.
Baek, S., Maloney, J.G., Molokin, A., George, N.S., Cortes Vecino, J.A., Santin, M. 2022. Diversity of Blastocystis subtypes in horses in Colombia and identification of two new subtypes. Microorganisms. 10:1693. https://doi.org/10.3390/microorganisms10091693.
Hernández-Castro, C., Martínez-Rosado, L.L., Dashti, A., Köster, P.C., Bailo, B., Orozco, M.C., Santin, M., González-Barrio, D., Carmena, D. 2023. Co-infection by Cryptosporidium meleagridis and Enterocytozoon bieneusi in an HIV+ Colombian patient. Parasitologia. 3:48-52. https://doi.org/10.3390/parasitologia3010006.
Rego, L., Castro-Scholten, S., Cano, C., Jimenez-Martin, D., Koster, P.C., Caballero-Gomez, J., Bailo, B., Dashti, A., Hernanadez-Castro, C., Cano-Terriza, D., Vioque, F., Maloney, J.G., Santin, M. 2023. Iberian wild leporidae as hosts of zoonotic enteroparasites in Mediterranean ecosystems of Southern Spain. Zoonoses and Public Health. 70(3):223-237. https://doi.org/10.1111/zph.13018.
Hernández, P.C., Maloney, J.G., Molokin, A., George, N.S., Morales, L., Chaparro-Olaya, J., Santin, M. 2023. Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals signifcant associations between contact with animals and infection risk. Parasitology Research. 122(7):1451-1462. https://doi.org/10.1007/s00436-023-07841-3.
Maloney, J.G., Molokin, A., Seguí, R., Maravilla, P., Martínez-Hernández, F., Villalobos, G., Tsaousis, A.D., Gentekaki, E., Carmena, D., Santin, M., Muñoz-Antolií, C., Klisiowicz, D.R., Oishi, C.Y., Toledo, R. 2023. Identification and molecular characterization of four new Blastocystis subtypes designated ST35-ST38. Microorganisms. 11:43. https://doi.org/10.3390/microorganisms11010046.
Figueiredo, A.M., Dahsti, A., Santin, M., Koster, P.C., Torres, R.T., Fonseca, C., Mysterud, A., Carvalho, J., Sarmento, P., Neves, N., Hipólito, D., Palmeira, J.D., Teixeira, D., Lima, C. 2023. Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal. Medical Mycology. 6(12). Article myad018. https://doi.org/10.1093/mmy/myad018.
Chacin-Bonilla, L., Santin, M. 2023. Cyclospora cayetanensis infection in developed countries: Potential endemic foci. Microorganisms. 11(3):540. https://doi.org/10.3390/microorganisms11030540.
Santin, M., Molokin, A., Maloney, J.G. 2023. A longitudinal study of Blastocystis in dairy calves from birth through 24 months demonstrates dynamic shifts in infection rates and subtype prevalence and diversity by age. Parasites & Vectors. 16(1):177. https://doi.org/10.1186/s13071-023-05795-0.
Stensvold, C.R., Berg, R.P., Maloney, J.G., Molokin, A., Santin, M. 2023. Molecular characterization of Blastocystis and Entamoeba of muskoxen and sheep in Greenland. International Journal for Parasitology. 53(11-12):673-685. https://doi.org/10.1016/j.ijpara.2023.05.005 .
Tapia-Veloz, E., Gozalbo, M., Guillen, M., Dashti, A., Bailo, B., Koster, P.C., Santin, M., Carmena, D., Trelis, M. 2023. Prevalence and associated risk factors of intestinal parasites among schoolchildren in rural Ecuador, with emphasis on the molecular diversity of Giardia duodenalis, Blastocystis sp. and Enterocytozoon bieneusi. PLOS Neglected Tropical Diseases. 17(5). Article e0011339. https://doi.org/10.1371/journal.pntd.0011339.
Hernández-Castro, C., Maloney, J.G., Agudelo-López, S.P., Toro-Londoño, M.A., Botero-Garcés, J.H., Orozco, M.C., Quintero-Quinchia, Y.C., Correa-Cote, J.C., Múnera-Duque, A., Ricaurte-Ciro, J.C., Londoño-Álvarez, L.I., Escobar, R.M., Köster, P.C., Carmena, D., Santin, M. 2023. Identification and validation of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening. Journal of Eukaryotic Microbiology. 70. Aricle e12978. https://doi.org/10.1111/jeu.12978.