Location: Environmental Microbial & Food Safety Laboratory
2021 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
This is the final report for the project 8042-32000-100-00D terminating in December 2020 that was replaced with 8042-32000-112-00D.
Significant progress was made for objectives included in this project plan, all of which fall under the National Program 108. We performed whole-genome sequencing of selected isolates, with a high number of Giardia cysts and Cryptosporidium oocysts, obtained from goats as part of a collaboration with National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS) to study zoonotic protists in goats in the United States.
A Nanopore sequencing strategy to generate full-length Blastocystis small subunit rRNA (SSU rRNA) gene sequences was developed. This protocol provides a powerful tool for subtype validation and discovery and has already been used to validate seven previously proposed subtypes (ST11, ST23-ST27). Reference sequences were unavailable, and to name three novel subtypes (ST29-ST31). Results from validation of the assay were published in the journal Parasites & Vectors (doi: 10.1186/s13071-020-04484-6. 2020), and studies using this strategy to validate novel subtypes were published in Microorganisms (doi: 10.3390/microorganisms9050997) and Parasitology Research (doi: 10.1007/s00436-021-07170-3). This assay is currently being used to assist collaborators from Spain, Colombia, and Mexico to validate novel Blastocystis subtypes identified in humans and livestock and wild animals.
In collaboration with scientists from Brazil, we investigated the presence of Blastocysts in chickens using NGS to detect and identify subtypes of Blastocystis to understand if chickens could be a source of environmental contamination leading to infection of other animals and humans. The prevalence of Blastocystis in chickens was high, and all positive samples contained one or both potentially zoonotic subtypes ST6 and ST7. Results demonstrated that human-pathogenic Blastocystis subtypes are present in chickens corroborating their potential role as a source of human infection and environmental contamination. One manuscript was published in Parasitology Research (doi: 10.1007/s00436-021-07170-3).
Developed a novel strategy using MinION long-read sequencing to generate full-length Blastocystis SSU rRNA gene sequences. This protocol provides an essential tool for subtype validation and discovery. It has already been used to validate seven previously proposed subtypes for which reference sequences were unavailable and to name three novel subtypes. Moving forward, this method will be crucial for validating novel subtypes to prevent designation of invalid subtypes that could undermine Blastocystis subtype terminology. Furthermore, full-length sequences allow for the robust analyses needed to establish relationships between Blastocystis subtypes and transmission pathways, host specificity, zoonotic potential, and pathogenicity .
Key findings of the project were: 1) developed a next generation amplicon sequencing (NGS) protocol to detect and characterize Blastocystis subtypes as well as to investigate intra-host Blastocystis diversity; 2) conceived and established a method using short and long read sequencing platforms for generating intact whole genomes directly from fecal isolates of Giardia; 3) developed a sensitive molecular assay to detect the parasite Cryptosporidium in source and finish water that targets virus Cryspovirus found inside Cryptosporidium in thousands of copies per parasite; 4) conducted the largest and most comprehensive Blastocystis study carried out in dairy cattle that provided an advance in understanding the epidemiology of these parasites in food animals; 5) conducted a study to determine the occurrence and genetic diversity of Cryptosporidium species in several poultry species that led to the identification of zoonotic species and subtypes of Cryptosporidium in poultry; 5) conducted multiple studies to identify and molecularly characterize of E. bieneusi in cattle, pigs, sheep, birds, dogs, cats, and wildlife that demonstrated that presence of human-pathogenic E. bieneusi genotypes is widespread in wild and domestic animals; 6) established a Nanopore sequencing strategy to generate full-length Blastocystis small subunit rRNA (SSU rRNA) gene sequences that are required to validate novel subtypes; and 7) conducted a study using NGS to detect and identify subtypes of Blastocystis in chickens that revealed a high prevalence of Blastocystis in chickens and all positive samples contained zoonotic subtypes corroborating their potential role as a source of human infection and environmental contamination.
Accomplishments
2. Use of next generation sequencing to evaluate Blastocystis genetic diversity in domestic and wild animals. Blastocystis sp. is one of the most common enteric parasites found in humans and animals worldwide. Extensive genetic diversity has been described for Blastocystis demonstrating that subtypes do not exhibit strict host specificity which raises the possibility of zoonotic transmission through either direct contact or through fecal contamination of food or water. Reports detailing the subtypes and prevalence of Blastocystis in poultry are limited. ARS scientists in Beltsville, Maryland, in collaboration with scientists at the Universidade Federal de Uberlândia (Brazil) assessed Blastocystis prevalence, subtype distribution, and intra-host subtype diversity in samples obtained from chickens using next generation amplicon sequencing. Blastocystis was very prevalent in chickens and molecular characterization revealed that all positive samples contained zoonotic subtypes corroborating their potential role as a source of human infection and environmental contamination.
Review Publications
Maloney, J.G., Molokin, A., Santin, M. 2020. Use of Oxford Nanopore MinION to generate full-length sequences of the Blastocystis small subunit (SSU) rRNA gene. Parasites & Vectors. 13(1):595. https://doi.org/10.1186/s13071-020-04484-6.
Salimo Muadica, A., Messa, A., Dashti, A., Santin, M., Manjate, F., Chirinda, P., Garrine, M., Vubil, D., Acacio, S., Koster, P., Bailo, B., Nhampossa, T., Calero-Bernal, R., Mwenda, J., Mandomando, I., Carmena, D. 2020. First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique. PLOS Neglected Tropical Diseases. 14(6):e0008419. https://doi.org/10.1371/journal.pntd.0008419.
Hublin, J., Maloney, J.G., Santin, M. 2020. Blastocystis in domesticated and wild mammals and birds. Research in Veterinary Science. https://doi.org/10.1016/j.rvsc.2020.09.031.
Vega, L., Herrera, G., Muñoz, M., Patarroyo, M.A., Maloney, J.G., Santin, M., Ramirez, J.D. 2021. How to live in a dysbiosed environment: gut microbiota profiling in patients with co-occurrence of Blastocystis and Clostridioides difficile. Scientific Reports. https://doi.org/10.1371/journal.pone.0248185.
Koster, P., Dashti, A., Bailo, B., Muadica, A.S., Maloney, J.G., Santin, M., Chicharro, C., Miguelanez, S., Nieto, F.J., Cano-Terriza, D., Garcia-Bocanegra, I., Guerra, R., Ponce-Gordo, F., Calero-Bernal, R. 2021. Occurrence and genetic diversity of protist parasites in captive non-human primates, zookeepers, and free-living sympatric rats in the Córdoba Zoo Conservation Centre, southern Spain. Animals. 11(3):700. https://doi.org/10.3390/ani11030700.
Maloney, J.G., Santin, M. 2021. Mind the gap: new full-length sequences of Blastocystis subtypes generated via Oxford Nanopore Minion sequencing allow for comparisons between full-length and partial sequences of the small subunit of the ribosomal RNA gene. Microorganisms. https://doi.org/10.3390/microorganisms9050997.
Maloney, J.G., Da Cunha, M., Molokin, A., Cury, M.C., Santin, M. 2021. Next generation sequencing reveals widespread presence of human-pathogenic Blastocystis subtypes in chickens. Veterinary Parasitology. 12:2219-2231. https://doi.org/10.1007/s00436-021-07170-3.
