Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2000
Publication Date: 12/1/2000
Interpretive Summary: Cryptosporidiosis is an intestinal parasitic disease of humans and animals caused by the protozoan Cryptosporidium parvum. The disease is prevalent in the young and in persons with impaired immune systems, such as AIDS patients. There are no effective vaccines or drugs available for treating individuals afflicted with cryptosporidiosis. Therefore, preventing ingestion of C. parvum is of utmost importance. One approach to preventio is to test water destined for human consumption for the presence of the parasite. The present study describes a molecular biology-based approach to not only detect the C. parvum, but also determine whether the parasite is viable. Determining viability is crucial in providing health officials data whether the organism is capable of causing disease and if appropriate disinfection methods, such as boiling water, are necessary. This study showed that as C. parvum ages, the viability of the parasite decreases. This viability is associated with the loss of a signal inside the parasite that codes for an enzyme involved in the survival of the parasite in the environment. This procedure may eventually replace more tedious and time-consuming methods for estimating viability of the parasite.
Technical Abstract: The purpose of the present study was to determine if reverse transcriptase-polymerase chain reaction (RT-PCR) directed at mRNA encoding the enzyme amyloglucosidase (CPAG) could serve as a marker for C. parvum oocyst viability. Oocysts were stored for 1-11 mos. in the refrigerator and at monthly intervals extracted for total RNA for RT-PCR analysis. An aliquot of these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 d later for ileal tissue that was analyzed by semi-quantitative PCR to determine the level of parasite replication. The CPAG RT-PCR assay detected 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 stored for 1-7 mos., unlike oocysts stored for 9 or 11 mos., contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal intensity was observed between 5-7 mos. storage. The intensity of RT-PCR product from oocysts and the signal from semi-quantitative PCR of ileal tissue DNA from mice infected with these same aged oocysts were comparable. 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 RT-PCR provides and accurate and precise method for evaluating viability of C. parvum oocysts.