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

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.