Pathogen Research

       In 1993, a now infamous outbreak of the opportunistic protist Cryptosporidium parvum affected over 400,000 individuals in Milwaukee, Wisconsin (MacKenzie et al., 1994). While public outcry over this incident has led to extensive monitoring for C. parvum in drinking water, other microbial pathogens may represent a similar threat to the public supply. The U.S. Environmental Protection Agency recently listed the protozoal parasite microsporidia as one such pathogen of emerging concern.

        Microsporidia are obligate intracellular parasites that are of increasing concern as a source of infection in the immunocompromised. A number of clinical studies exist that detail the manifestations of microsporidia infections in human beings, however, to date, little research exists to document the detection and distribution of the organism in drinking water, a potentially significant source of human infection. The spore stage of the protist is resistant to traditional chlorination techniques and may pass through water filtration equipment because of its small size, increasing its significance as a drinking water contaminant.

        Traditional microbial assays including light and electron microscopy and immunological methods fail to detect all infectious species of concern in humans. Our laboratory has adapted a PCR-based assay originally described by Kock et al. (1997) that allows the specific identification of all species of microsporidia that infect humans. The method relies on the PCR-based amplification of the small-subunit-rRNA gene of the organism; species identification is made by size comparisons of the resulting DNA fragment. Figure 1 below shows results from the amplification of a sample contaminated with two infectious species of microsporidia: Encephalitozoon intestinalis and Encephalitozoon hellem. The peaks (in black) are easily distinguished from each other at 417 bp and 426bp, respectively.

Figure 1: Peaks (in black) resulting from the amplification of the SSU-rRNA gene of two infectious species of microsporidia: Encephalitozoon intestinalis and Encephalitozoon hellem (red peaks represent a 500 bp DNA ladder).

The method outlined is highly sensitive, specific to individual species of the organism and is widely available at a relatively low cost. Currently, the laboratory is refining filtration techniques for separating nicrosporidia spores from large volume water samples and will proceed to field application and testing of the described method within the year.

References:

Kock, N.P., Petersen, H., Fenner, T., Sobottka, I., Schmetz, C., Deplazes, P., et al. (1997) "Species-specific identification of microsporidia in stool and intestinal biopsy specimens by the polymerase chain reaction," Eur J Microbiol Infect Dis 16(5):369-376.

MacKenzie, W.R., Hoxie, N.J., Proctor, M.E., Gradus, M.S., Blair, K.A., Peterson, D.P., et al. (1994) "A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply," N Engl J Med 331(3):161-167.

Parasitic Microsporidia