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 amyloglucosidase (CPAG) could serve as a indicator for C. parvum oocyst viability. 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 these C. parvum oocysts was inoculated into neonatal mice which were necropsied 4 days 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 months, unlike oocysts stored for 9 or 11 months, contained CPAG mRNA that was detectable by RT-PCR. A gradual decrease in the RT-PCR signal 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 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 may be useful for differentiating viable from non-viable C. parvum oocysts and for studying the expression of the gene for amyloglucosidase in vitro.
Jenkins
M C
MC
Immunology and Disease Resistance Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA.
Trout
J
J
Abrahamsen
M S
MS
Lancto
C A
CA
Higgins
J
J
Fayer
R
R
D000818:Animals; D002470:Cell Survival; D016785:Cryptosporidium parvum / Q000166:cytology*; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000302:isolation & purification; D016053:RNA, Protozoan / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D016053:RNA, Protozoan; D005087:Glucan 1,4-alpha-Glucosidase