Estimating viability of Cryptosporidium parvum oocysts using reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding amyloglucosidase

The purpose of the present study was to determine if reverse transcriptase- polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amy loglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viabil ity. Oocysts were stored for 1-11 months in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot. of thes e C. panvum oocysts was inoculated into neonatal mice which were necropsied 4 days later for ileal tissue that was analyzed by semi-quantitative PCR t o determine the level of parasite replication. The CPAG RT-PCR assay detect ed RNA from as few as 10(3) C. parvum oocysts. An effect of storage time on both RT-PCR signal and mouse infectivity was observed. RNA from oocysts st ored for 1-7 months, unlike oocysts stored for 9 or 11 months, contained CP AG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR sig nal intensity was observed between 5 and 7 months storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of i leal tissue DNA from mice infected with these same aged oocysts were compar able. The RT-PCR assay of CPAG mRNA in cultured cells infected with viable C. parvum oocysts first detected expression at 12 h with highest expression levels observed at 48 h post-infection. These results indicate that CPAG R T-PCR may be useful for differentiating viable from non-viable C. parvum oo cysts and fur studying the expression of the gene for amyloglucosidase in v itro. (C) 2000 Elsevier Science B,V. All rights reserved.