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Title:   Author  item
                                            KNIEL, K - 1254-40-00item
                                            Higgins, Jamesitem
                                            Trout, Jamesitem
                                            Fayer, Ronalditem
                                            Jenkins, Mark
  Submitted to: Journal of Microbiological Methods
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 5/15/2004
                                    Publication  Date: 6/1/2004
                                    Citation: Kniel, K.E., Higgins, J.A., Trout, J.M., Fayer, R., Jenkins, M.C. (2004) Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts.  Journal of Microbiological Methods 58: 189-195.Interpretive Summary: Cryptosporidiosis is an intestinal disease of humans and animals caused by the protozoan parasite Cryptosporidium parvum.  The disease is prevalent in young calves, which suffer weight loss associated with diarrhea and inappetance.  There are no approved drugs or disinfectants available to treat infected individuals or destroy the parasite.  One method to prevent infection is to reduce exposure of humans and animals to the parasite by a combination of land management and water filtration.  These management techniques rely on sensitive methods to detect the parasite in water.  All of the techniques that are presently used to detect C. parvum oocysts are neither sensitive nor specific enough to identify low numbers of organisms in water.  The present study describes a dot-blot method is directed at an obligate viral symbiont of the parasite.  This virus, termed CPV, is present at 2000 particles per oocysts, and as such increases the sensitivity of detection by at least 1000-fold.  The technique should be useful for detecting C. parvum in water.
                                Technical Abstract: The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite.   Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein.  Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite.  Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen.  Water samples were seeded with C. parvum oocysts and incubated at 4 C, 20 C, or 25 C for greater than three months to determine if CPV levels were correlated with oocyst infectivity.  Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence.  While sporozoite infectivity declined by more than 75% after one month at 25 C, the CPV signal was similar to that of control samples at 4 C.  By 3 months at 20 C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal.  This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.


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