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 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.
1. 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.
2. 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 Uberlândia, 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.
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. https://doi.org/10.1016/j.rvsc.2018.03.010.
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. 101(10):9245-9258. https://doi.org/10.3168/jds.2017-14022.
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. 101(10):9199-9213. https://doi.org/10.3168/jds.2017-14060.
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.