Simple method for long-term copro-preservation of Cryptosporidium oocysts for morphometric and molecular analysis

Preservation of Cryptosporidium oocysts in faecal specimens containing 75% ethanol is suitable for subsequent morphometric and molecular analysis. No significant morphologic alteration occurred after storage at ambient temperatures, ranging from 22 to 38 degreesC, for more than 2 years. After washing, sugar floatation and DNA extraction, a nested polymerase chain reaction targeting the small subunit ribosomal RNA gene successfully amplified Cryptosporidium DNA in all 15 isolates examined. The sensitivity of detection by polymerase chain reaction (PCR) was found to be as high as 1.25 oocysts per reaction (mean = 3.01, SD = 1.14). Importantly, a 2.2-kb of the complete DNA sequence of a gene encoding Cryptosporidium thrombospondin-related adhesive protein (TRAP-C1) was also consistently amplified by PCR in all isolates. The PCR-amplified product can be used as a good template for sequencing. Therefore, this simple procedure should be useful for epidemiological analysis of clinical samples from outbreaks, endemic or sporadic cases of cryptosporidiosis when long-term storage of oocysts is required.