Development of methods and confirmation of human enteropathogenic microsporidia in water

Abstract

Not too long ago, microsporidia were looked upon as a small group of pathogens of interest in agriculture and to a few scientists devoted to basic biology. With the discovery that a number of these obligate intracellular protozoa are pathogenic to humans, a need for the elucidation of transmission routes of these human pathogens has arisen. Unfortunately, no methods are currently available for recovery, detection, or species determination of microsporidia that may be present in the environment This dissertation begins with an introduction into the basic biology of microsporidia, describes the development of complete methods for recovery, detection, and species determination of microsporidia in water, provides validation of these methods, and finally provides the first environmental screening of various water sources. Results show that traditional methods for recovery of microsporidia display very low recovery percentages (less than 5%), and do not allow for confirmative analyses. Methods are described that provide average spore recovery of 38% from finished waters and new PCR approach is also described which specifically amplifies HEM DNA extracted from environmental samples. This molecular method demonstrates the ability to detect less than 10 spores in concentrated water samples and the ability to accurately determine the species of the microsporidia, in these samples. Using these newly developed methods, human enteropathogenic microsporidia (HEM) were shown to be present in a wide variety of water samples including surface water, groundwater, effluents, and drinking water. Thus, this dissertation takes the first step in answering the hypothesis, that HEM are indeed waterborne pathogens.