Development and application of an improved semiquantitative technique for detecting low-level Cryptosporidium parvum...

An improved semiquantitative 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, 10(2), 10(3), or 10(4) C. parvum oocysts and killed 96 hr postinfection. 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 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 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, whereas no signal was observed in PCR on intestinal DNA from mice infected with oocysts exposed to higher radiation doses.