1a. 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.
1b. 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.
3. Progress Report:
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.
1. 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.
2. 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.
5. Significant Activities that Support Special Target Populations:
Responsible for organizing the Leadership Experience during the summers of 2016 that provides students from Hispanic Serving Institutions (HSI) with internships at multiple laboratories in Beltsville, Maryland (Beltsville Agricultural Research Center and Beltsville Human Nutrition Research Center) through a partnership with USDA National Institute of Food and Agriculture (NIFA) Hispanic-Serving Institutions Education Grants Program. The program has benefited 38 students from various Hispanic Serving Institutions located in Puerto Rico, Florida, New Mexico, California and Texas. Additionally, responsible for organizing the NIFA-HIS New Project Directors training in 2015 in Beltsville, Maryland with the purpose of encouraging collaborations between faculty and students from HSI and NEA scientists. Hosted a Hispanic summer internship student from University of Puerto Rico at Mayaguez Campus, a Hispanic-Serving Institution. The student learned about detection and molecular characterization of zoonotic protists relevant to food safety.
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. https://doi.org/10.1007/s00436-016-5087-1.
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.