CHARACTERIZATION OF A CRYPTOSPORIDIUM PARVUM-SPECIFIC CDNA CLONE AND DETECTION OF PARASITE DNA IN MUCOSAL SCRAPINGS OF INFECTED MICE

A cDNA library was constructed using total RNA extracted from oocysts and sporozoites of the protozoan parasite Cryptosporidium parvum. The expression library was screened with an anti-C. parvum antiserum and a clone, Cp3.4, with a 2043 bp insert, was extracted. Southern blot ana lysis demonstrated a single copy gene that was located on a 1.6 Mb chr omosome. The gene was found to be C. parvum specific as Cp3.4 did not cross-hybridise with chromosomal DNA from three other apicomplexan par asites. The cDNA encodes a polypeptide with a predicted membrane helix at its C-terminal end which is flanked by stretches of acidic amino a cids. Overall, the polypeptide has a low isoelectric point (pI) of 3.9 4. A total of 21 glycine/proline-rich octapeptides were identified whi ch represented variations of a consensus sequence. The function of thi s protein is yet unknown. Using Cp3.4-specific PCR primers, this C. pa rvum gene could be amplified from as little as 0.8 pg of purified para site DNA in a single polymerase chain reaction. Less than 0.1 ng of DN A from the ileum mucosa of immunosuppressed adult mice that had been i nfected with C. parvum oocysts was required to detect the parasites. I n non-immunosuppressed mice that were infected and which did not shed oocysts in numbers detectable by acid-fast staining, parasite developm ent could be detected in 25 ng of total mucosa DNA, This PCR approach may be a valuable technique for the detection of parasite infections i n situations where conventional staining methods fail, such as chronic , low-grade infections or the detection of parasites in potential rese rvoir hosts. (C) 1998 Elsevier Science B.V. All rights reserved.