Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/1996
Publication Date: N/A
Citation: N/A Interpretive Summary: Cryptosporidiosis is a serious intestinal disease of humans and calves caused by the protozoan parasite Cryptosporidium parvum. In the last decade there have been several outbreaks of cryptosporidiosis in the human population due to contaminated water supplies. At present, no drugs have been approved and few disinfectants have proven effective against this parasite. Developing therapies against Cryptosporidium will require a reliable method of assessing anti-cryptosporidial compounds in a mouse model. The present study describes a molecular-based approach to measure Cryptosporidium infection in mice. The technique utilizes polymerase chain reaction (PCR) to amplify very minute amounts of parasite DNA from mouse intestinal tissue. This PCR technique is more sensitive and reliable than traditional enumeration methods. Also, the molecular-based technique is more rapid and less expensive than conventional assays. By employing this PCR method, laboratories can now evaluate anti-cryptosporidial drugs and reagents using parasite doses that are relevant to human cryptosporidial infections.
Technical Abstract: 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, 102, 103, or 104 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 103 and 104 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 102 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.