Progress 12/20/15 to 12/19/20
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: The Laboratory⿿s maximized telework has clearly impacted our research project considering that we are not able to conduct research in our usual way. Those projects that require sample processing in the lab were all put on hold or conducted at a slower pace to allow for the 25% capacity limit. There will be delays on producing data, submitting data to collaborators, and some deadlines will just have to be shifted to accommodate to the adjustments made on our research schedule due to Covid- 19 pandemic. We have, however, tried to reduce the long-term impact by shifting as much as possible our efforts to focus in processing and analyzing data that could be achieved while teleworking. Both scientists in this project have been focusing their efforts in analyzing data and writing manuscripts. In addition, scientists have made a significant effort to direct their team that includes, 2 support scientists and 1 post doc to ensure that all were being productive while teleworking by assigning specific projects and tasks that were doable while teleworking and by making themselves available via e-mail or zoom meetings to resolve any issues that could occur while at a maximum telework schedule. ACCOMPLISHMENTS 02 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.
Impacts
(N/A)
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.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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.
Impacts
(N/A)
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.
- 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.
- Lindsay, D., Dubey, J.P., Santin, M. 2019. Coccidia and other Protozoa. Book Chapter. 1015-1027.
- Santin, M. 2020. Giardiasis and cryptoporidiosis. Veterinary Clinics of North America. 36(1):223-238.
- 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.
- 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.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. In 2019, significant progress was made for all three objectives and their sub-objectives, all of which fall under the National Program 108. For Objective 1, progress was made in elucidating the molecular epidemiology of zoonotic parasites, Cryptosporidium, Giardia, Enterocytozoon bieneusi, and Blastocystis. We completed and published the largest and most comprehensive Blastocystis study carried out in food animals worldwide in collaboration with the National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS) (Parasitology Research, https://doi.org/10.1007/s00436-018-6149-3). The molecular investigation included 2,539 fecal samples from dairy heifer calves from more than 100 farms in 13 states. Blastocystis was detected in 73 (2.9%) fecal samples, and molecular characterization showed a wide diversity of subtypes (STs) with eleven identified, seven previously reported (ST3, ST4, ST5, ST10, ST14, ST17, and ST21) and four novel subtypes (named ST23 to ST26). Zoonotic subtypes 3, 4, and 5 were frequently found and represented 67% (49) of the positive specimens in this population. Our results suggest that cattle could serve as important reservoirs of infection for humans and other domestic animals highlighting the potential risk of zoonotic transmission for Blastocystis. Because mixed infections with several Blastocystis subtypes are frequent observed, ARS researchers in Beltsville, Maryland, developed a method to study intra-host Blastocystis communities using next generation amplicon sequencing (NGS) that was compared using a set of 75 positive samples to Sanger sequencing. It was demonstrated that NGS has greater sensitivity to study mixed infections and capability to revealed subtype diversity. In addition, nine more infections with potentially zoonotic STs were detected by NGS than Sanger. Indeed, subtype 3, the most common subtype found in humans, was found in 37% (28) of specimens tested by NGS but in only four specimens using Sanger. Our findings clearly indicated that mixed Blastocystis infections may be far more common than previously thought due to the limitations of current detection methods that will help to better understand transmission dynamics and answer many unresolved epidemiological questions for this parasite. This method was recently published in the journal Infect Genetic and Evolution (doi: 10.1016/j.meegid.2019.04.013). Using this technology, we conducted further studies to better understand role of mixed infections in the epidemiology of this parasite in humans, horses, chickens, and wild carnivores in collaboration with scientists from Mexico, Colombia, Brazil, and Spain. ARS researchers in Beltsville, Maryland, successfully obtained satisfactory coverage and depth with whole genome sequencing of Giardia duodenalis cysts purified using immunomagnetic separation directly from animal feces. Two different technologies, short reads using an Illumina MiSeq Plaform and long-reads using Oxford Nanopore MinIon platform, were used to obtain the assembly of whole genomes from G. duodenalis zoonotic assemblage A and host-specific Assemblage D. Currently we are conducting comparisons of protein coding regions and potential virulence factors from these genomes that may reveal differences related to their pathogenicity and host specificity. Genome wide analysis is required to add context to our understanding of Giardia pathobiology through comparison among assemblages and this data will increase our ability to more finely discriminate and characterize the differences between zoonotic and non-zoonotic, and highly pathogenic and less pathogenic isolates. Information will be presented at the International Giardia and Cryptosporidium Conference in Rouen (France) in June 2019. ARS researchers in Beltsville, Maryland, used NGS and Sanger protocols to detect and identify subtypes of Cryptosporidium parvum in dairy cattle. The same fragment of the glycoprotein 60 (gp60) gene, the most commonly used for subtyping, was amplified by both methods. Results obtained by both methods were compared and it was clear that NGS identified much higher levels of within-host diversity than Sanger analysis. Sanger sequencing identified only one gp60 subtype in each sample while NGS identified the same subtype, but also identified additional within-host subtypes. This result demonstrated that NGS can resolve complex DNA mixtures and detect low-abundance intra-isolate variants. This indicates that previous studies that have relied on Sanger sequencing, may not reflect the extent of within-host diversity resulting in incorrect assumptions on Cryptosporidium transmission. The importance of within- host genetic diversity it critical to understand cryptosporidiosis epidemiology and transmission dynamics of Cryptosporidium. More extensive studies using NGS on a wider range of samples are in progress to determine the extent of Cryptosporidium within-host genetic diversity. Additionally, in collaboration with scientists from Spain we are investigating the presence of zoonotic parasite E. bieneusi in domestic and wild animals as they that might 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 corroborating their potential role as a source of human infection and environmental contamination. One manuscript has been submitted to Parasitology Research and another one is about to be submitted. For Objective 2, because current literature indicates the importance of studying the effect in gut microbiome as it is first line of defense against enteric pathogens such as Cryptosporidium and Giardia, we have improved current methods used in the lab to compare the microbial communities using 16S sequencing of DNA extracted from mice fecal samples. We will be using that methodology to investigate the effect of Cryptosporidium and Giardia infections in gut microbiome in untreated mice infected and uninfected to later test the effect of probiotics on mice infected with those parasites and their gut microbiomes. For Objective 3, an improved PCR method was developed for detecting Encephalitozoon and Enterocytozoon in calf feces was recently published in the Journal of Microbiological Methods (doi: 10.1007/s12639-018-1060-5) . The assay incorporates an internal standard to control for false negative reactions due to PCR inhibitors and a nested PCR step to increase sensitivity.
Impacts
(N/A)
Publications
- Maloney, J., Lombard, J., Urie, N., Shivley, C., Santin, M. 2018. Zoonotic and genetically diverse subtypes of Blastocystis in U.S. dairy calves. Parasitology Research. 118(2):575-582.
- Maloney, J.G., Molokin, A., Santin, M. 2019. Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections. Infection, Genetics and Evolution. 73:119-125.
- Li, W., Santin, M., Feng, Y. 2019. Host specificity in Enterocytozoon bieneusi and public health implications. Trends in Parasitology. 35(6):436- 451.
- Jenkins, M.C., O'Brien, C.N., Parker, C.C. 2018. An optimized procedure for detecting enterocytozoon intestinalis and encephalitozoon bieneusi using polymerase chain reaction technology. Journal of Parasitic Diseases. 43(1):75-82.
- Sato de Souza, M., O'Brien, C., Santin, M., Jenkins, M.C. 2018. A highly sensitive method for detecting Cryptosporidium parvum oocysts recovered from source and finish water using RT-PCR directed to Cryspovirus RNA. Journal of Microbiological Methods. 156:77-80.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. In 2018, significant progress was made for all three objectives and their sub-objectives, all of which fall under the National Program 108. For Objective 1, progress was made in elucidating the molecular epidemiology of zoonotic parasites, Cryptosporidium, Giardia, and Blastocystis. We completed molecular testing for presence of Blastocystis of 2,539 fecal samples collected from calves on more than 100 farms in 13 of the top dairy states in collaboration with the National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS). This is the largest and most comprehensive Blastocystis study carried out in food animals worldwide. Blastocystis was detected in 73 fecal samples, and molecular characterization showed a wide diversity of subtypes with eleven subtypes identified, seven previously reported (ST-3, ST-4, ST-5, ST-10, ST-14, ST-17, and ST-21) and four novel subtypes (named ST-23 to ST-26). Zoonotic subtypes 3, 4, and 5 were found in 67% (49) of the positive specimens suggesting that cattle could serve as a reservoir of infection for humans and other domestic animals highlighting the potential risk of zoonotic transmission for Blastocystis. Because mixed infections with several Blastocystis subtypes were frequent observed we developed a method to study intra-host Blastocystis communities using next generation amplicon sequencing (NGS) that will help to better understand transmission dynamics and answer many unresolved epidemiological questions for this parasite. ARS researchers in Beltsville, Maryland, are currently comparing this new method that uses next generation amplicon sequencing on the Illumina MiSeq platform to cloning and Sanger sequencing using primers that amplify a fragment of the SSU rDNA gene for the detection of Blastocystis mixed subtype infections. ARS researchers in Beltsville, Maryland, successfully obtained satisfactory coverage and depth with whole genome sequencing of Giardia cysts purified directly from animal feces. Genome-wide analysis of Giardia isolates is required to add context to our understanding of its pathobiology through comparison among assemblages and this data will increase our ability to more finely discriminate and characterize the differences between zoonotic and non-zoonotic, and highly pathogenic and less pathogenic isolates. In addition, in collaboration with CDC scientists, 90 Cryptosporidium parvum isolates obtained from calves that were subjected to whole genome sequence using next generation sequencing on the Illumina HiSeq platform are being used to conduct comparative genomic analyses that include C. parvum samples obtained from humans. Additionally, in collaboration with scientists from Spain, the presence of zoonotic parasites, E. bieneusi and Blastocystis, in wild carnivores that might be a source of environmental contamination leading to infection of other animals and humans was studied. Results demonstrated that human-pathogenic E. bieneusi genotypes and Blastocystis subtypes are present in wild carnivores corroborating their potential role as a source of human infection and environmental contamination. For Objective 2, because current literature indicates the importance of studying the effect in gut microbiome as it is first line of defense against enteric pathogens such as Cryptosporidium and Giardia, we have refined methodology to compare the microbial communities using 16S sequencing of DNA extracted from mice fecal samples. We are about to initiate a study that will use that methodology to investigate the effect of Cryptosporidium and Giardia infections in gut microbiome in untreated mice infected and uninfected to later test the effect of probiotics on mice infected with those parasites and their gut microbiomes. For Objective 3, an improved PCR method was developed for detecting Encephalitozoon and Enterocytozoon in calf feces. The assay incorporates an internal standard to control for false negative reactions due to PCR inhibitors and a nested PCR step to increase sensitivity. The assay showed good agreement with commercial immunofluorescence assays and is more objective than identifying the extremely small (~ 1 micron) microsporidian spores in complex samples such as calf feces. A method for purifying spores from feces was also developed that assists in lowering the amount of organic material prior to DNA extraction. Accomplishments 01 Enterocytozoon (E.) bieneusi in wild carnivores. The parasite E. bieneusi is the most frequently diagnosed Microsporidia species in humans worldwide; also, it is mainly associated with chronic diarrhea and wasting syndrome, E. bieneusi has emerged as an important public health concern because it impacts humans as well as domestic and wild animals. Although wildlife may play an important role in the transmission of E. bieneusi, limited data exists about the impact of this parasite in wild carnivores. ARS scientists in Beltsville, Maryland, in collaboration with scientists at the Spanish Parasitology Reference and Research Laboratory in Majadahonda, Spain, conducted the most comprehensive study to date for E. bieneusi in wild carnivores. Out of 190 wild carnivore fecal samples collected from five Spanish regions, twenty-five (13.2%) specimens from three host species (European badger, beech marten, and red fox) were positive for E. bieneusi. The study provides an expansion in our understanding of the epidemiology (distribution) of this parasite in wildlife by expanding the host range of this parasite with two new hosts (European badger, beech marten) and documented the presence of human-pathogenic genotypes that corroborates the potential role of wild carnivores as a source of human infection and environmental contamination. 02 Molecular characterization of Cryptosporidium in poultry. The zoonotic disease Cryptospoidiosis, recognized as a significant cause of diarrhea worldwide, is caused by the microscopic parasite Cryptosporidium. Information regarding Cryptosporidium on poultry species that includes molecular characterization is scarce, even though it is an important poultry parasite. To fill this gap, a study to determine the occurrence and genetic diversity of Cryptosporidium was conducted in several Brazilian poultry species by a joint effort between scientists at ARS in Beltsville, Maryland, and at Universidade Federal de Uberl�ndia, Brazil. Out of the 155 fecal specimens collected from different poultry species in the state of Minas Gerais (Brazil), 23 (14.8%) were positive, establishing that Cryptosporidium is a common poultry parasite in Brazil and this study also provided a better understanding of the worldwide distribution of this parasite. This was the first report of Cryptosporidium in Brazilian turkeys and quails. For the third most common species of Cryptosporium involved in human cryptosporidiosis, we discovered two novel genotypes as well three subtypes previously identified in humans in Brazil, indicating that they can be potentially zoonotic.
Impacts
(N/A)
Publications
- Rodrigues Da Cunha, M.J., Cury, M.C., Santin-Duran, M. 2018. Molecular characterization of Cryptosporidium spp. in poultry from Brazil. Veterinary Parasitology. 118:331-335.
- Santin, M., Calero-Bernal, R., Carmena, D., Mateo, M., Balseiro, A., Barral, M., Limabarbero, J.F., Habela, M.A. 2017. Molecular characterization of Enterocytozoon bieneusi in wild carnivores in Spain. Journal of Eukaryotic Microbiology.
- Shivley, C., Lombard, J., Urie, N., Kopral, C., Santin, M., Earleywine, T., Olson, J., Garry, F. 2018. Preweaned heifer management on US dairy operations: Part VI. Factors associated with average daily gain in preweaned dairy heifer calves. Journal of Dairy Science.
- Urie, N., Lombard, J., Shivley, C., Ashley, A., Kopral, C., Santin, M. 2018. Preweaned heifer management on US dairy operations: Part III. Factors associated with Cryptosporidium and Giardia in preweaned dairy heifer calves. Journal of Dairy Science.
- Murphy, H.R., Cinar, H., Gopinath, G., Im, A., Noe, K.E., Chatman, L.D., Miranda, N.E., Wetherington, J.H., Neal-Mckinney, J., Piresc, G.S., Sachs, E., Stanya, K.J., Jognson, C.L., Nascimiento, F., Santin, M., Molokin, A., Samadpour, M., Janagama, H., Kahler, A., Miller, C., Da Silva, A.J. 2017. Interlaboratory validation of an improved U.S. Food and Drug Administration method for detection of Cyclospora cayetanensis in produce using TaqMan real-time PCR. International Journal of Food Microbiology. 69:170-178.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. In 2017, significant progress was made for all three objectives and their sub-objectives, all of which fall under the National Program 108. For Objective 1, progress was made in elucidating the molecular epidemiology of zoonotic parasites, Cryptosporidium, Giardia, and Blastocystis. All 2, 539 samples collected from calves on more than 100 farms in 13 of the top dairy states in collaboration with the National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS) were tested molecularly for presence of Giardia duodenalis. This is the largest study carried out in dairy calves using a multilocus analysis approach. Giardia duodenalis in calves was common (40%) and positive calves were found in all 13 states studied with a prevalence ranging from 25-61%. Molecular characterization identified Assemblage A (11%), D (01%), E (86.9%), and mixed A/E (2%). At present, in collaboration with CDC scientists, 90 Cryptosporidium isolates that were subjected to whole genome sequence using Hi Seq and being used to conduct comparative genomic analyses. ARS researchers in Beltsville, Maryland, are conducting the molecular characterization to identify prevalence and subtypes of Blastocystis and to select samples to prepare libraries for next generation sequencing analysis. So far, 36 (9.9%) samples of 363 calves tested were Blastocystis-positive with positives from 7 of the 13 states tested. Two Blastocystis subtypes, ST-4 and ST-5, were identified both of which have also been reported in humans. Given the prevalence, broad geographic distribution, and presence of zoonotic subtypes in Blastocystis infected calves, our results highlight the potential risk of zoonotic transmission and suggests that cattle could serve as important reservoirs of infection for humans and other domestic animals. Additionally, in collaboration with scientists from Brazil, the presence of zoonotic parasites, E. bieneusi, Cryptosporidium, and Giardia, in captive birds that might also be a source of environmental contamination leading to infection of other animals and humans was studied. Our results demonstrate that human-pathogenic E. bieneusi genotypes, D and Peru6, and G. duodenalis assemblage A are present in captive birds, corroborating their potential role as a source of human infection and environmental contamination. For Objective 2, Real Time PCRs to detect and quantify Cryptosporidium and Giardia in feces and tissues were optimized. The ability to quantify is critical to evaluate the effect of probiotics on the development of cryptosporidiosis and Giardiasis in rodent models. Animal Protocol and Biosafety Protocol (SOP) were approved by the Institutional Animal Care and Use Committee and Biosafey Committee, respectively. Currently, we are testing different infection doses of Cryptosporidium and Giardia to determine best infection dose to proceed to evaluate the effect of probiotics on animals infected with those parasites. For Objective 3, RT-PCR to detect the viral symbiont Cryspovirus was used in surface (source) water and drinking water spiked with Cryptosporidium that was processed using standard EPA 1623 method. RT-PCR was able to detect very low numbers of oocysts, undetectable by immunofluorescence assay (IFA), indicating a potential application to routine testing of environmental water. Accomplishments 01 Microscopic parasites found in Brazilian captive birds. Enterocytozoon bieneusi, Cryptosporidium, and Giardia are environmentally ubiquitous parasitic pathogens that cause serious human and animal intestinal diseases. Although it has been indicated that captive birds could play an important role in the transmission of these zoonotic parasites to humans and animals, there is limited data on the presence or impact of these parasites. To fill this knowledge gap, ARS and Universidade Federal de Uberl�ndia scientists collected fecal examined from captive birds in the state of Minas Gerais, Brazil, and examine the prevalence of the three parasites in feces. Enterocytozoon bieneusi, Cryptosporidium, and Giardia, were detected in captive bird�s fecal material. Molecular characterization revealed that human-pathogenic E. bieneusi and Giardia duodenalis were present in captive Brazilian birds in Brazil corroborating their potential role as a source of human infection and environmental contamination. 02 Sensitive molecular assay to detect the parasite Cryptosporidium parvum in source and finish water. Cryptosporidium, a microscopic parasite responsible for the largest waterborne outbreak in the history of the U. S., is estimated to cause 30,000 cases of acute, severe diarrhea (Cryptosporidiosis) annually in the U.S. The disease is most prominent in young children or immune-compromised individuals, such as those infected with the AIDS virus; further, Cryptosporidiosis usually stems from the ingestion of water contaminated with the parasite. The parasite is resistant to most standard water disinfectants, can cause clinical infection at very low numbers, and no effective prophylactic or therapeutic regimen exists that can prevent or ameliorate the disease. ARS scientists developed a RNA assay that targets a virus, Cryspovirus, found inside of all species of Cryptosporidium, and its efficacy to detect the parasite in water inoculated with various numbers of C. parvum oocysts. The RNA assay was more the sensitive and capable of detecting lower numbers of the parasite than current gold standard test for source and finish water suggesting that detection of Cryptosporidium oocysts may require molecular assays to prevent outbreaks caused by the ingestion of water containing "low numbers of oocysts (the parasite's infect stage)." This method could be used as a water screening method by regulatory and public health agencies to detect and track Cryptosporidium in untreated and drinking water.
Impacts
(N/A)
Publications
- da Cunha, M.J.R., Cury, M.C., Santin, M. 2016. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in brazilian captive birds. Parasitology Research. 116:487-493.
- Durso, L.M., Miller, D.N., Snow, D.D., Santin, M., Henry, C.G., Woodbury, B.L. 2016. Evaluation of fecal indicators and pathogens in a beef cattle feedlot vegetative treatment system. Journal of Environmental Quality. 46(1):169-176.
- Connor, E.E., Wall, E.H., Bravo, D.M., Clover, C.M., Elsasser, T.H., Baldwin, R.L., Santin, M., Kahl, S., Vinyard, B.T., Walker, M.P. 2017. Reducing gut effects from Cryptosporidium parvum infection in dairy calves through prophylactic glucagon-like peptide 2 therapy or feeding of an artificial sweetener. Journal of Dairy Science. 100(4):3004-3018.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. In 2016, significant progress was made for all three objectives. For Objective 1, progress was made in elucidating the epidemiology of zoonotic parasites, Cryptosporidium and Giardia. In collaboration with the National Animal Health Monitoring Service of Animal and Plant Health Inspection Service (APHIS), 2,539 samples were collected from calves on more than 100 farms in 13 of the top dairy states and were examined by immunofluorescence microscopy (IFA) for Cryptosporidium and Giardia. Overall, presence of both parasites was very common with 42.2% of the calves infected with Cryptosporidium and 31% infected with Giardia. At present, ARS researchers in Beltsville, Maryland are conducting the molecular characterization to identify species and genotypes critical to estimate zoonotic potential. Findings will aid with understanding sources and routes of transmission necessary to improve strategies for the control and prevention of cryptosporidiosis and giardiasis in bovines. Additionally, ARS researchers in Beltsville, Maryland performed PCRs to detect the presence, in those calves, of zoonotic parasites E. bieneusi and to later molecularly characterize the genotypes to determine also the public heath impact of E. bieneusi genotypes in dairy calves. In collaboration with scientists from Brazil, the first molecular characterization of E. bieneusi in chickens and sheep from Brazil was conducted. Findings suggest that E. bieneusi is widespread in Brazilian chickens and sheep. Because zoonotic genotypes were identified, these food animals could play an important role in the transmission of this parasite to humans in Brazil. For Objective 2, in collaboration with a scientist from Argentina, ARS scientists in Beltsville, Maryland investigated the effects of administration of probiotic Enterococcus faecalis CECT 7121 on C. parvum infection in immunosuppressed mice. The ARS scientists in Beltsville, Maryland demonstrated that this probiotic interfered with C. parvum infection when both probiotic and parasite were present in the same intestinal location suggesting that probiotic supplementation can alleviate the negative effects of C. parvum infection. For Objective 3, an RT-PCR method to detect the viral symbiont Cryspovirus was developed. This assay was able to detect less than 5 C. parvum oocysts from C. parvum-containing calf feces demonstrating that it could provide a sensitive primary screening tool for detecting Cryptosporidium in samples. Accomplishments 01 Enterocytozoon bieneusi genotypes from cattle. Microsporidia are widely recognized as important human pathogens with E. bieneusi as the most common species infecting humans and animals, including cattle. Although Brazil has the second largest cattle herd in the world and it is the largest exporter of beef there are no data on the presence or impact of E. bieneusi on this important population. To fill this knowledge gap, fecal specimens were collected from 452 cattle from pre- weaned calves to adult cattle in the state of Rio de Janeiro. In this first report of E. bieneusi in Brazilian cattle, a significantly higher prevalence was found in dairy cattle than in beef cattle, and calves less than 2 months of age and those 3-8 months of age versus heifers and adults. Molecular characterization of the internal transcribed spacer (ITS) revealed 12 genotypes; five previously reported in cattle (BEB4, BEB8, D, EbpA and I), and seven novel genotypes (BEB11 to BEB17). The finding of zoonotic genotypes highlights the risk of human contamination with E. bieneusi spores through direct contact, especially with young dairy cattle, and environmental contamination affecting water and plants. 02 Development of a novel highly sensitive method for detecting Cryspovirus in Cryptosporidium parvum. Cryptosporidium, a protozoan parasite responsible for the largest waterborne outbreak of infectious disease in the history of the U.S., is estimated to cause 30,000 cases of severe, acute diarrhea annually in the U.S. It can result in a chronic devastating gastrointestinal infection and death in immune compromised persons. There is also increasing evidence of fresh produce contaminated with Cryptosporidium resulting in sporadic cases as well as outbreaks of foodborne infection. Waterborne and foodborne parasites are definitely underreported and there is a clear need to improve detection by developing more sensitive and cheaper assays to detect the often-low numbers of parasites present in contaminated food and water. A variety of protozoa, including Cryptosporidium, Giardia, harbor viral symbionts. Assays directed to viral symbionts of the target protozoa should be considerably more sensitive than methods directed to the parasite itself because the viruses exist in thousands of copies/ parasite. ARS scientists in Beltsville, Maryland developed an RT-PCR method to detect the viral symbiont Cryspovirus. This RT-PCR directed to Cryspovirus could detect less than 5 C. parvum oocysts from C. parvum-containing calf feces demonstrating that it could provide a sensitive primary screening tool for detecting Cryptosporidium in water, food and clinical samples.
Impacts
(N/A)
Publications
- Da Cunha, M.J., Cury, M.C, Santin, M. 2016. Widespread presence of human- pathogenic Enterocytozoon bieneusi genotypes in chickens. Veterinary Parasitology. 217:108-112.
- Fiuza, V., Lopes, C., Oliviera, F., Fayer, R., Santin, M. 2016. New findings of Enterocytozoon bieneusi in beef and dairy cattle in Brazil. Veterinary Parasitology. 216:46-51.
- Del Coco, V.F., Sparo, M.D., Sidoti, A., Santin, M., Basualdo, J.A., Codorba, M.A. 2016. Effects of Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in mice. Parasitology Research. 115(8) :3239-3244.
- Fiuza, V., Lopes, C., Cosendey, R., Oliviera, F., Fayer, R., Santin, M. 2016. Zoonotic Enterocytozoon bieneusi Genotypes found in Brazilian sheep. Research in Veterinary Science. 107:196-201.
- Jenkins, M.C., Obrien, C.N., Santin, M. 2015. A sensitive method for detecting and genotyping Cryptosporidium parvum oocysts. Food and Waterborne Parasitology. doi: 10.1016/j.fawpar.2015.11.001.
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