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Title:   Author  item
                                            Jenkins, Markitem
                                            Obrien, Celiaitem
                                            Santin-Duran, Monica
  Submitted to: Food and Waterborne Parasitology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 11/26/2015
                                    Publication  Date: 12/15/2015
                                    Citation: Jenkins, M.C., Obrien, C.N., Santin, M. 2015. A sensitive method for detecting and genotyping Cryptosporidium parvum oocysts. Food and Waterborne Parasitology. doi: 10.1016/j.fawpar.2015.11.001.Interpretive Summary: Cryptosporidiosis is an intestinal diarrhea disease of humans and animals caused by the protozoan Cryptosporidium.  A unique feature of Cryptosporidium is that the parasite can cause infections in humans at extremely low doses (~ 130 oocysts), which makes it necessary to develop sensitive methods for detecting the parasite in environmental samples so that precautions, such as boiled water alerts, can be instituted.  In the present study, a sensitive molecular method for the parasite was developed that detects the virus that is harbored by Cryptosporidium oocysts.  This method could detect as little as 5 Cryptosporidium oocysts in a sample, and avoids the need for more laborious molecular techniques that are subject to contamination.  This study also provided a means of utilizing the same nucleic acid material for detecting the parasite and characterizing the strain of Cryptosporidium present in a sample.  This assay should be useful to public health officials who are attempting to identify environmental samples containing Cryptosporidium oocysts.
                                Technical Abstract: Cryptosporidium parvum oocysts represent a considerable health risk to humans and animals because the parasite has a low infectious dose and usually exists in low numbers in environmental samples, which makes detection problematic.  The purpose of this study was to evaluate Cryspovirus as a target for sensitive detection of C. parvum in various sample matrices.  RT-PCR directed to the viral RNA sequence coding for the 40 kDa capsid protein could detect as little as 5 oocysts in a sample of calf feces.  Analysis of DNA extracted from a similar number of oocysts using PCR directed to the Cryptosporidium 18S rDNA sequence revealed that nested PCR was necessary to obtain a detectable PCR signal.  Although less sensitive than Cryspovirus RT-PCR, nested 18S PCR provided amplification products for DNA sequencing for Cryptosporidium species analysis.  DNA from samples containing C. parvum oocysts was also useful for gp60 sequence-based genotyping.  The CPV RT-PCR and CP18S nested PCR assays were applied to RNA and DNA extracted from finish and source water that had been spiked with various numbers of C. parvum oocysts.  The RT-PCR assay could detect as few as 10 C. parvum oocysts in both source and finish water, while nested 18S PCR was required to obtain the same level of sensitivity.  Thus, an two-step assay for detecting Cryptosporidium oocysts by Cryspovirus RT-PCR followed by species and genotype determination through sequencing nested 18S PCR amplicons was developed for analysis of calf feces and both source and finish water samples.   By using DNA that contains viral RNA, the assay avoids nested PCR for the initial detection of Cryptosporidium oocysts, and if positive, allows to subsequent evaluation of DNA for Cryptosporidium species and genotype analysis.


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