A semi-quantitative method for measuring Cryptosporidium parvum infection using polymerase chain reaction

A semi-quantitative method for measuring Cryptosporidium parvum infection in mice using polymerase chain reaction (PCR) was developed for evaluating therapeutic reagents against cryptosporidiosis. A competitor molecule composed of CP15/60 primer-binding sequences flanking an unrelated sequence was synthesized and used in PCR to amplify competitor and target CP15/60 sequences. For estimating relative intestinal parasite numbers, seven-day-old BALB/C mice were infected with 0, 10(2), 10(3), or 10(4) C. parvum oocysts. At 24, 48, 72, and 96 h post-infection, three mice per group were killed and the intestine from each mouse was processed for DNA. Semi-quantitative PCR using serial dilutions of competitor molecule and a constant amount of mouse tissue DNA showed that challenge doses of 10(3) and 10(4) oocysts could be easily detected at both 72 and 96 h post-infection. CP15/60-specific PCR products were not observed at earlier timepoints (24 and 48 h) nor at any timepoint with the 0 or 10(2) challenge doses. The semi-quantitative PCR technique proved to be more sensitive, rapid, and cost-effective compared to conventional histological scoring of cryptosporidial stages in tissue sections from C. parvum-infected mice. A rapid method for extracting DNA from infected mouse intestine was developed and, when used in the semi-quantitative PCR, proved to be as reproducible as conventional DNA extraction methods. (C) 1997 Elsevier Science B.V.