Research Project: Zoonotic Parasites Affecting Food Animals, Food Safety, and Public Health

Location: Environmental Microbial & Food Safety Laboratory

2017 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
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
1. 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 Uberlândia 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.

2. 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.

Review Publications
Da Cunha, M.J., Cury, M.C., Santin, M. 2017. Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in brazilian captive birds. Parasitology Research. 116:487-493. https://doi.org/10.1007/s00436-016-5309-6.
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.