Quantification of in vitro and in vivo Cryptosporidium parvum infection by using real-time PCR

Abstract

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.

FIG.1.

Melting-curve analysis of gp40 primers for qPCR performed using SYBR green.

FIG. 2.

(a) IFA used to quantify C. parvum infection in Caco_2A and m-IC_cl2 cells. (b and c) Linear regression analysis comparing qPCR result (y axis) and parasite inoculum (x axis) after a 24-hour infection in m-IC_cl2 (b) and Caco_2A (c) cells. The parasite inoculum ranged from 103 to 106/ml, and these data are representative of two independent experiments performed in triplicate (for each experiment, n = 15).

FIG. 3.

Linear regression analysis comparing qPCR result (y axis) and IFA (x axis) to quantify parasites after a 24-hour infection in m-ICcl2 (a) and Caco_2A (b) cells. The parasite inoculum ranged from 103 to 106/ml, and these data are representative of two independent experiments performed in triplicate (panel a; n = 15) or in duplicate (panel b; n = 10). HPF, high-powered field.

FIG. 4.

Linear regression analysis comparing qPCR result (y axis) and histological measurement (x axis) to quantify parasites after a 24-hour infection in mice. The data are representative of two independent experiments (n = 10). HPF, high-powered field.