PCR-mediated recombination between Cryptosporidium spp. of lizards and snakes

The presence or absence of genetic recombination has often been used as one of the criteria for Cryptosporidium species designation and population structure delineation. During a recent study of cryptosporidiosis in reptiles that were housed in the same room, 4 lizards were found to have concurrent infections of C. serpentis (a gastric parasite) and C. saurophilum (an intestinal parasite), and 6 snakes were concurrently infected with C. serpentis, C. saurophilum and a new Cryptosporidium as indicated by PCR-RFLP analysis of the SSU rRNA gene. DNA sequence analysis of cloned PCR products confirmed the diagnosis of mixed infections. Surprisingly, it appeared that 11 of the 22 clones (8 and 14 clones from a lizard and a snake, respectively) had chimeric sequences of two Cryptosporidium spp. BootScan analysis indicated the existence of recombinants among the cloned sequences and detection of the informative sites confirmed the BootScan results. Because the probability for genetic recombination between gastric and intestinal parasites is small, these hybrid sequences were likely results of PCR artifacts due to the presence of multiple templates. This was confirmed by PCR-sequencing analysis of single-copy templates using diluted DNA samples. Direct sequencing of 69 PCR products from 100- to 1,000-fold diluted DNAs from the same snake and lizard produced only sequences of C. serpentis, C. saurophilum and the unnamed Cryptosporidium sp. Thus, care should be taken to eliminate PCR artifacts when determining the presence of genetic recombination or interpreting results of population genetic studies.