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

Location: Environmental Microbial & Food Safety Laboratory

2022 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 2022, 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, generated high quality G. duodenalis assemblage A and D genomes directly from cysts obtained from feces of naturally infected hosts using a hybrid sequencing (Illumina MiSeq and Oxford Nanopore MinION platforms) and assembly strategy. Findings on these culture free Giardia genomes were published in the journal Current Research in Microbial Sciences (https://doi.org/10.1016/j.crmicr.2022.100114). Additionally, ARS researchers in Beltsville, Maryland, completed the quantification of Cryptosporidium oocysts and Giardia cysts using Immunofluorescence Microscopy (IFA) 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). For isolates with large quantities of Cryptosporidium and Giardia, a multistep cleaning process paired with a hybrid sequencing and assembly strategy is in progress to generate reference quality Giardia and Cryptosporidium genomes. In addition, the genome of an isolate of Cyclospora cayetanensis obtained from a pediatric patient via a collaboration with the Hospital Infantil de Morelia "Eva Sámano de Lopez Mateos" that contained high numbers of oocysts is being produced using the same hybrid sequencing and assembly strategy. For Objective 2, ARS researchers in Beltsville, Maryland, used their recently developed next generation amplicon sequencing (NGS) method to investigate mixed assemblage infections in dairy cattle, sheep, and human populations to understand impact of those mixed infections in epidemiology of Giardia. A total of 2539 pre-weaned dairy calves from a nationwide project conducted in collaboration with NAHMS/APHIS were screened for Giardia using molecular tools. Giardia duodenalis was frequently identified in dairy calves (1013/2539; 39.9%). The ability of NGS and Sanger sequencing to detect mixed assemblage infections was compared using a subset of the positive samples (n=314). NGS detected six-times more mixed infections compared with Sanger sequencing, demonstrating superior sensitivity to detect assemblages present in low abundances and highlighting the advantages of NGS application in molecular epidemiological studies of Giardia. Results from this study were published in the journal Veterinary Parasitology (doi.org/10.1016/j.vetpar.2022.109702). Additionally, all laboratory work and NGS analysis has been completed and manuscripts are in preparation for two other Giardia projects, a sheep study including Giardia-positive lambs as part of a collaboration project with the Complutense University of Madrid as well as a human study including samples from Colombian children as part of a collaboration with El Bosque University. In a joint effort with the Carlos III Health Institute, the most comprehensive study carried out to date in the Iberian Peninsula in domestic and wild ungulates that includes molecular characterization of Blastocystis and Enterocytozoon bieneusi including validation of novel genetic variants is in progress. For Objective 3, ARS researchers in Beltsville, Maryland, are using NGS to study mixed Giardia and Blastocystis species/assemblage/subtype infections to elucidate the epidemiology of those parasites in a longitudinal dairy cattle survey that included 990 fecal specimens collected 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. Blastocystis NGS analysis was completed. Because potentially novel Blastocystis subtypes were detected, a MinION strategy to amplify full-length Blastocystis small subunit rRNA gene sequences is being used for their validation. Additionally, NGS was used to: 1) conduct the first Blastocystis study in white-tailed deer that found that deer were commonly infected with a wide diversity of subtypes, including two novel subtypes, zoonotic subtypes, and subtypes frequently reported in domestic animals (doi.org/10.3390/microorganisms9061343); 2) determine the prevalence of Blastocystis in Turkish immunodeficient and immunocompetent diarrheic patients that demonstrated the advantage of using NGS to understand the role of intra-subtype variation of Blastocystis in pathogenicity (doi.org/10.1371/journal.pntd.0009779); 3) complete the first molecular characterization of Blastocystis in cattle in Spain that demonstrated that mixed subtype infections and zoonotic subtypes are frequent in cattle (doi.org/10.3390/vetsci8090191); and 4) conduct the first study in Colombia to characterize subtypes of Blastocystis in farm animals that showed great diversity of subtypes including subtypes previously identified in humans indicating that farm animals could play a role in transmission to humans (doi.org/10.3389/fvets.2021.732129).

Accomplishments
1. Developed a hybrid assembly strategy to produce Giardia genomes. The parasite Giardia duodenalis is a common cause of diarrhea in humans and is responsible for numerous foodborne and waterborne outbreaks. It also infects a wide range of livestock, wildlife, and companion animals and is considered a species complex consisting of eight assemblages that have different host specificity and pathogenicity. Whole genome sequencing and comparative genomics can assist in identifying traits related to host specificity and disease outcomes. However, genomic data from cysts obtained directly from infected hosts are lacking. ARS scientists in Beltsville, Maryland, developed a protocol to obtain genomes from freshly isolated cysts using a multistep cleaning process combined with a hybrid assembly strategy using long read (Oxford Nanopore MinION) and short read (Illumina MiSeq) sequencing. Using this approach, some of the most contiguous genomes available for G. duodenalis assemblages A and D were generated which will aid the Giardia research community to advance diagnostic, vaccine, and drug development.

2. First report of Blastocystis in white-tailed deer. Blastocystis is one of the most common intestinal parasites of humans and wild and domestic animals worldwide. A high genetic diversity has been observed among morphologically indistinguishable isolates from mammalian and avian hosts. Wildlife populations, including deer, have the potential to serve as reservoirs of parasitic diseases for both humans and domestic animals either through direct contact or through contamination of food or water resources. However, there is not information on the occurrence and subtype distribution of Blastocystis in white-tailed deer in the United States. ARS scientists in Beltsville, Maryland, conducted the first study to assess Blastocystis subtype diversity and occurrence in white-tailed deer worldwide using next generation amplicon sequencing. White-tailed deer were found to be commonly infected/colonized with a wide diversity of subtypes including subtypes frequently reported in humans and domestic animals indicating that deer could play an important role in transmission to humans and domestic animals.

3. Assessment of Giardia duodenalis mixed assemblage infections cattle. Giardia is a common intestinal parasite of humans and animals worldwide. It frequently infects cattle, regardless of the husbandry system, producing a high number of Giardia cysts signifying potential for human exposure from environmental contamination. Cattle can harbor both animal-adapted and human pathogenic assemblages, but conventional molecular methods lack the sensitivity to detect mixed infections leaving their occurrence in cattle underexplored and underestimated. ARS scientists in Beltsville, Maryland, investigated the presence of mixed assemblage infections in dairy calves using next generation sequencing (NGS) and conventional Sanger sequencing. Six-times more mixed infections were detected using NGS (18%) compared to conventional sequencing (3%). These findings highlight the advantages of NGS application in molecular epidemiological studies to uncover mixed assemblage infections which is needed to better understand Giardia epidemiology, establish routes of transmission, and assess the potential role of cattle and other animals as a source of environmental contamination with zoonotic assemblages.

Review Publications
Higuera, A., Herrera, G., Jimenez, P., Garcia, D.J., Pulido, M., Bulla, D.M., Pinilla, J.C., Moreno, A., Maloney, J.G., Santin, M., Ramirez, J.D. 2021. Identification of multiple Blastocystis subtypes in domestic animals from Colombia using amplicon-based next generation sequencing. Frontiers in Veterinary Science. https://doi.org/10.3389/fvets.2021.732129.
Maloney, J.G., Jang, Y., Molokin, A., George, N.S., Santin, M. 2021. Wide genetic diversity of Blastocystis in white-tailed deer (Odocoileus virginianus) from Maryland, USA. Microorganisms. https://doi.org/10.3390/microorganisms9061343.
Sarzhanov, F., Dogruman-All, F., Santin, M., Maloney, J.G., Gureser, A.S., Karasartova, D., Taylan-Ozkan, A. 2021. Investigation of neglected protists Blastocystis and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods. PLOS Neglected Tropical Diseases. 15(10):e0009779. https://doi.org/10.1371/journal.pntd.0009779.
Dashti, A., Rivero-Juarez, A., Santin, M., George, N.S., Köster, P.C., Lopez-Lopez, P., Rosalde, M.A., Garcia-Bocanegra, I., Gomez-Villamandos, J., Caballero-Gomez, J., Frias, M., Bailo, B., Ortega, S., Maudica, A.A., Calero-Bernal, R., Gonzalez-Barrio, D., Rivero, A., Briz, V., Carmena, D. 2021. Diarrhoea-causing enteric protist species in intensively and extensively raised pigs (Sus scrofa domesticus) in Southern Spain. Part I: Prevalence, genetic diversity, and zoonotic potential. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14388.
Rivero-Juarez, A., Dashti, A., Santin, M., Köster, P.C., Lopez-Lopez, P., Risalde, M.A., Garcia-Bocanegra, I., Gomez-Villamandos, J., Caballero-Gomez, J., Frias, M., Ballo, B., Ortega, S., Muadica, A.S., Calero-Bernal, R., Gonzalez-Barrio, D., Rivero, A., Briz, V., Carmena, D. 2021. Diarrhoea-causing enteric protist species in intensively and extensively raised pigs (Sus scrofa domesticus) in Southern Spain. Part II: Association with Hepatitis E virus susceptibility. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.14408.
Abarca, N., Santin, M., Maloney, J.G., George, N.S., Molokin, A., Cardona, G., Sashti, A., Köster, P., Bailo, B., Hernández-De-Mingo, M., Muadica, A.S., Calero-Bernal, R., Carmena, D. 2021. Molecular detection and characterization of Blastocystis sp. and Enterocytozoon bieneusi in cattle in Northern Spain. Veterinary Sciences. https://doi.org/10.3390/vetsci8090191.
Maloney, J.G., Molokin, A., Solano Aguilar, G., Dubey, J.P., Santin, M. 2022. A hybrid sequencing and assembly strategy for generating culture free Giardia genomes. Current Research in Microbial Science. https://doi.org/10.1016/j.crmicr.2022.100114.
Hublin, J.S., Maloney, J.G., George, N.S., Molokin, A., Lombard, J.E., Urie, N.J., Shivley, C., Santin, M. 2022. Enhanced detection of Giardia duodenalis mixed assemblage infections in pre-weaned dairy calves using next generation sequencing. Veterinary Parasitology. 304:109702. https://doi.org/10.1016/j.vetpar.2022.109702.
Maloney, J.G., George, N.S., Molokin, A., Santin, M. 2021. An Illumina MiSeq based amplicon sequencing method for the detection of mixed parasite infections using the Blastocystis SSU rRNA gene as an example. Methods in Molecular Biology. https://doi.org/10.1007/978-1-0716-1681-9_5.