ARS | Publication request: DEVELOPMENT AND APPLICATION OF AN IMPROVED SEMI-QUANTITATIVE TECHNIQUE FOR DETECTING LOW LEVEL CRYTOSPORIDIUM PARVUM INFECTIONS IN MOUSE TISSUE USING POLYMERASE CHAIN REACTION

Submitted to: Journal of Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 1997
Publication Date: 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 to treat this disease; moreover, only a 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 now reflects an infectious parasite dose that is more relevant to normal infections. The PCR technique was then applied to test the effect of irradiation treatment on the development of C. parvum in mice. It appears that radiation doses greater than 20 kRad are necessary to prevent infection in mice. By employing this PCR method, laboratories can now evaluate anti-cryptosporidial drugs and reagents using parasite doses that are relevant to human cryptosporidial infections, and more reliably quantitate the effect of drugs on parasite survival.

Technical Abstract: An improved semi-quantitative technique was developed for measuring low infectious doses of Cryptosporidium parvum in neonatal mice using polymerase chain reaction (PCR). Separate litters of neonatal mice were inoculated with 0, 100, 1000, or 10,000 C. parvum oocysts and killed 96 hr post-infection. A segment of the ileum or the entire whole intestine was then removed from subgroups of mice in each litter and total DNA was extracted using standard procedures. By employing a CP15/60-based semi-quantitative PCR technique, C parvum DNA was detected in mice infected with as few as 100 oocysts. DNA isolated from the ileum of infected mice produced a more intense PCR signal than DNA isolated from the whole intestine. This technique was used to study the intracellular development of C. parvum sporozoites that had been exposed in the oocyst stage to either 0, 15, 20, 25, or 30 kRad gamma irradiation. A CP15/60 PCR signal was observed in ileum tissue from mice infected with 0 kRad- or 15 kRad-irradiated C. parvum oocysts. A very slight PCR signal was generated by PCR on ileum tissue DNA from mice infected with 20 kRad-irradiated oocysts, while no signal was observed in PCR on intestinal DNA from mice infected with oocysts exposed to higher radiation doses.