New method for the concentration and detection of Cyclospora cayetanensis in environmental waters

Cyclospora cayetanensis is a sporulating parasitic protozoan that infects the epithelium of the upper small intestinal tract. It has been identified as both a food and waterborne pathogen endemic in many developing countries. It is an important agent of Traveler's Diarrhea in developed countries and in 1996–1999 was responsible for numerous foodborne outbreaks in the United States and Canada.; There has been much debate as to where to place C. cayetanensis taxonomically due to its homology with Eimeria species. To date, the only nucleic acid sequences available are for the ribosomal DNA of C. cayetanensis and three other Cyclospora species; within these a high degree of sequence conservation has been observed. This homology has hindered identification of C. cayetanensis in environmental samples using the only published polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methodology.; Due to this difficulty, an alternative nested PCR method was developed which can detect <1 oocyst seeded into 10 liters of surface water. A theoretical analysis of the primers revealed that they would amplify all known Cyclospora, some Eimeria species as well as Isospora robini, RFLP with Alu I was used to distinguish C. cayetanensis from the other species.; To determine whether or not C. cayetanensis was present in southern California environmental waters, five sites along the Santa Ana River were selected for a three-season survey (July 1998–March 1999). Four samples were identified as positive using the traditional PCR and RFLP protocol. The new PCR and RFLP method was used to re-examine 121 of the surface water samples. While none, including the earlier presumptive positive samples, were positive for C. cayetanensis, however, two appear to be Cyclospora species. This research therefore, shows that genetically similar organisms will result in false positive samples using published nested primers and RFLP unless they are microscopically confirmed. The nested primers and RFLP method presented in this dissertation, however, are able to distinguish between Cyclospora species without the need for microscopic confirmation.