Characterisation 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 analysis demonstrated a single copy gene that was located on a 1.6 Mb chromosome. The gene was found to be C. parvum specific as Cp3.4 did not cross-hybridise with chromosomal DNA from three other apicomplexan parasites. The cDNA encodes a polypeptide with a predicted membrane helix at its C-terminal end which is flanked by stretches of acidic amino acids. Overall, the polypeptide has a low isoelectric point (pI) of 3.94. A total of 21 glycine/proline-rich octapeptides were identified which represented variations of a consensus sequence. The function of this protein is yet unknown. Using Cp3.4-specific PCR primers, this C. parvum gene could be amplified from as little as 0.8 pg of purified parasite DNA in a single polymerase chain reaction. Less than 0.1 ng of DNA from the ileum mucosa of immunosuppressed adult mice that had been infected with C. parvum oocysts was required to detect the parasites. In non-immunosuppressed mice that were infected and which did not shed oocysts in numbers detectable by acid-fast staining, parasite development could be detected in 25 ng of total mucosa DNA. This PCR approach may be a valuable technique for the detection of parasite infections in situations where conventional staining methods fail, such as chronic, low-grade infections or the detection of parasites in potential reservoir hosts.
Petry
F
F
Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University Mainz, Augustusplatz/Hochhaus, Germany. fpetry@mail.uni-mainz.de
Shirley
M W
MW
Miles
M A
MA
McDonald
V
V
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003457:Cryptosporidiosis / Q000175:diagnosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D018076:DNA, Complementary; D016054:DNA, Protozoan / Q000032:analysis*; D016521:Electrophoresis, Gel, Pulsed-Field; D017125:Genes, Protozoan; D007082:Ileum / Q000469:parasitology; D007413:Intestinal Mucosa / Q000469:parasitology*; D051379:Mice; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
C115997:Cp3.4 protein, Cryptosporidium parvum; D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan