Location: Environmental Microbial & Food Safety Laboratory
2019 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 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.
Accomplishments
Review 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. https://doi.org/10.1007/s00436-018-6149-3.
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. https://doi.org/10.1016/j.meegid.2019.04.013.
Li, W., Santin, M., Feng, Y. 2019. Host specificity in Enterocytozoon bieneusi and public health implications. Trends in Parasitology. 35(6):436-451. https://doi.org/10.1016/j.pt.2019.04.004.
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. https://doi.org/10.1007/s12639-018-1060-5
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. https://doi.org/10.1016/j.mimet.2018.11.022.
