Detection of Expression Alteration of Cytokines in the Intestine of Balb/c Mice Infected with Cryptosporidium parvum using Relative Fluorescence Quantitative PCR Method

Cryptosporidium parvum (C. parvum) is a parasitic protozoan that causes cryptosporidiosis in mammalian intestinal tract. In this study, C. parvum infection model was established in Balb/c mice, followed by extraction of total RNA from small intestine of infected mice at 1, 3, 7 and 14 dpi, respectively. The relative expression of IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-6 in the small intestine of Balb/cmice infected with C. parvum, were then detected using Ct value comparison method (RT-QPCR 2dct). The primers were designed according to the sequence of mouse cytokines in GenBank. The expression levels of the cytokines were normalized to GAPDH gene expression. Fluorescence threshold (Ct value) obtained from RT-QPCR(2-Delta Delta Ct) method with GAPDH standard curve (linearity R-2=1), followed by sequencing analysis showed that all tested cytokines were amplified. The PCR products exhibited consistent melting curves, suggesting reliable specificity of the cytokine primers. Compared with the control group, the expression levels of IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6 of infected group were all up-regulated, demonstrating an important role of cytokines in controlling C. parvum infection. Hence, these findings suggest that expression of IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-6cytokines could be used as reference for diagnosis of C. parvum infection.