DEVELOPMENT AND APPLICATION OF AN IMPROVED SEMIQUANTITATIVE TECHNIQUEFOR DETECTING LOW-LEVEL CRYPTOSPORIDIUM-PARVUM INFECTIONS IN MOUSE-TISSUE USING POLYMERASE CHAIN-REACTION

An improved semiquantitative technique was developed for measuring low infectious doses of Cryptosporidium parvum in neonatal mice using pol ymerase chain reaction (PCR). Separate litters of neonatal mice were i noculated with 0, 10(2), 10(3), or 10(4) C. parvum oocysts and killed 96 hr postinfection. A segment of the ileum or the entire whole intest ine was then removed from subgroups of mice in each litter and total D NA was extracted using standard procedures. By employing a CP15/60-bas ed semiquantitative PCR technique, C. parvum DNA was detected in mice infected with as few as 10(2) oocysts. DNA isolated from the ileum of infected mice produced a more intense PCR signal than DNA isolated fro m the whole intestine. This technique was used to study the intracellu lar 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 wit h O-kRad- or 15-kRad-irradiated C. parvum oocysts. A very slight PCR s ignal was generated by PCR on ileum tissue DNA from mice infected with 20-kRad-irradiated oocysts, whereas no signal was observed in PCR on intestinal DNA from mice infected with oocysts exposed to higher radia tion doses.