Molecular analysis of the 18S rRNA gene of Cryptosporidium serpentis in a wild-caught corn snake (Elaphe guttata guttata) and a five-species restriction fragment length polymorphism- based assay that can additionally discern C. parvum from C. wrairi

Abstract

An adult wild-caught corn snake (Elaphe guttata guttata) was presented for humane euthanasia and necropsy because of severe cryptosporidiosis. The animal was lethargic and >5% dehydrated but in good flesh. Gastric lavage was performed prior to euthanasia. Histopathologic findings included gastric mucosal hypertrophy and a hemorrhagic erosive gastritis. Numerous 5- to 7-microm-diameter round extracellular organisms were associated with the mucosal hypertrophy. A PCR, acid-fast stains, Giemsa stains, and an enzyme immunoassay were all positive for Cryptosporidium spp. PCR and restriction fragment length polymorphism (RFLP) analysis on gastric lavage and gastric mucosal specimens, and subsequent sequencing of the 18S rRNA gene, enabled a distinct molecular characterization of the infecting organism as Cryptosporidium serpentis. Until recently, studies on snake Cryptosporidium have relied on host specificity and gross and histopathologic observations to identify the infecting species. A multiple alignment of our sequence against recently published sequences of the 18S rRNA gene of C. serpentis (GenBank accession no. AF093499, AF093500, and AF093501 [L. Xiao et al., unpublished data, 1998]) revealed 100% homology with the C. serpentis (Snake) sequence (AF093499) previously described by Xiao et al. An RFLP method to differentiate the five presently sequenced strains of Cryptosporidium at this locus was developed. This assay, which uses SpeI and SspI, complements a previously reported assay by additionally distinguishing the bovine strain of Cryptosporidium from Cryptosporidium wrairi.

FIG. 1

Electrophoretic profile of the RFLP analysis from VspI digestion of the 18S rRNA gene of Cryptosporidium spp. Lane 1, C. parvum; lane 3, C. serpentis from gastric mucosa; lane 5, C. serpentis from gastric lavage; lane 7, 100-bp DNA ladder (Gibco BRL). Lanes 2, 4, and 6 are empty. Arrows indicate previously reported digestion of C. muris 18S rRNA (15).

FIG. 2

C. serpentis (guttata) (AF151376) 18S rRNA gene (1,743 bp).

FIG. 3

Schematic representation showing the resulting fragments of an SspI-SpeI digestion of the 18S rRNA gene in Cryptosporidium spp.