Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe.
OBJECTIVE
Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum.


RESULTS
FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h.


CONCLUSIONS
Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity.


CONCLUSIONS
Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.
Smith
J J
JJ
Montana Microbiological Services, IIc., Bozeman, MT, USA. jjsmith@qut.edu.au
Gunasekera
T S
TS
Barardi
C R M
CR
Veal
D
D
Vesey
G
G
D000818:Animals; D016785:Cryptosporidium parvum / Q000502:physiology*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006207:Half-Life; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D033661:Oocysts / Q000502:physiology*; D010539:Permeability; D015347:RNA Probes; D016053:RNA, Protozoan / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics; D012263:Ribonucleosides / Q000378:metabolism; D013045:Species Specificity; D014638:Vanadates / Q000378:metabolism
D015347:RNA Probes; D016053:RNA, Protozoan; D012335:RNA, Ribosomal; D012263:Ribonucleosides; C057724:vanadyl ribonucleoside complex; D014638:Vanadates; D012260:Ribonucleases