This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Species of microsporidia that infect humans and nonhuman primates (NHPs) have been detected in surface and drinking water sources. As a result, Encephalitozoon intestinalis and Enterocytozoon bieneusi were placed on USEPA's drinking water Contaminant Candidate List and Occurrence Priorities List and on the NIH Biodefense Category B list pathogens of concern for waterborne transmission. Of the over 4000 outdoor-housed nonhuman primates at TNPRC, 35-40% are shedding Enterocytozoon bieneusi (phylum Microsporidia) in feces that drain into the surrounding discharge water system. This provides a natural case study site to apply and validate methods to monitor Ent. bieneusi (and other species) in surface water at pre- and post-treatment sites and prior to discharge into the constructed wetland system. The initial objectives of this project were to develop and apply a quantitative real time PCR (qRT-PCR) method for detecting microsporidia in environmental water samples at TNPRC. E. bieneusi spores obtained from bile of infected NHPs at necropsy were used to PCR amplify rDNA that was cloned into a plasmid for calibrating gene copy numbers. Intact total spores were also utilized for generating a spore number calibration curve. Both gene copy and spore number calibration curves generated linear results with R2 values of 0.9978 and 0.9807 respectively and with Ct values of 35 and 34 for one copy and one spore, respectively. The qRT-PCR method was successfully applied to detecting E. bieneusi in rhesus macaque fecal specimens and the results support the application of qRT-PCR for detection of E. bieneusi in discharge water samples.
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