C.Frazar, P.A.Orlandi, DVA, OARSA, FDA, Laurel, MD
Cyclospora cayetanensis is a human parasitic pathogen that has been associated with outbreaks of food-borne illness attributed to contaminated fresh produce (raspberries, basil, mesculin lettuce). As with other pathogenic microorganisms associated with food- and waterborne contamination, this parasite has a direct impact on regulatory, economic and public health issues. Unlike most food-borne bacterial pathogens however, there is a considerable lack of information concerning its environmental biology, its host reservoirs, and the basic concepts of its pathophysiology. On a molecular level, only the small subunit rRNA gene and the first internal transcribed spacer region have been described. Beyond their usefulness in rapid molecular detection methods and phylogenetic analysis, however, such information has been of limited value. In contrast, the use of RT-PCR to detect differentially expressed molecular targets rather than genomic housekeeping genes may provide a means to assess parasite viability. From a regulatory perspective discrimination between an infectious oocyst and a non-infectious or a dead oocyst in a food sample is essential, as the latter two do not necessarily present a serious human health risk. As heat shock proteins (HSPs) are thought to play an important role in oocyst sporulation, mRNA expression may correlate oocyst detection with viability and infectivity. In the present study we identified a putative HSP70 homologue from C. cayetanensis. By aligning HSP70 gene sequences from closely related organisms such as Eimeria spp. and Cryptosporidium spp. and comparing highly conserved regions, we designed a series of DNA primers. PCR amplification using these primers yielded a 970 bp amplicon. BLAST analysis of this sequence revealed considerable homology to the HSP70 of C. parvum. Based on the information obtained thus far, we are currently developing a refined strategy to obtain and sequence the remainder of the C. cayetanensis HSP homologue. RT-PCR analysis will then be used to examine any correlation between the expression of this protein and oocyst sporulation and infectivity.