Investigations of the relationship between use of in vitro cell culture-quantitative PCR and a mouse-based bioassay for evaluating critical factors affecting the disinfection performance of pulsed UV light for treating Cryptosporidium parvum oocysts in sa

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• 作者：Mary Garvey ; Hugh Farrell ; Martin Cormican ; Neil Rowan
• 关键词：Cryptosporidium parvum ; Pulsed light ; In vitro HCT-8 cell culture-qPCR assay ; Mouse-infectivity bioassay ; Humic acid
• 刊名：Journal of Microbiological Methods
• 出版年：2010
• 出版时间：March 2010
• 年：2010
• 卷：80
• 期：3
• 页码：267-273
• 全文大小：210 K

文摘

Cryptosporidium parvum is an enteric coccidian parasite that is recognised as a frequent cause of water-borne disease in humans. We report for the first time on use of the in vitro HCT-8 cell culture-quantitative PCR (qPCR) assay and the in vivo SCID-mouse bioassay for evaluating critical factors that reduce or eliminate infectivity of C. parvum after irradiating oocysts in saline solution under varying operational conditions with pulsed UV light. Infections post UV treatments were detected by immunofluorescence (IF) microscopy and by quantitative PCR in cell culture, and by IF staining of faeces and by hematoxylin and eosin staining of intestinal villi in mice. There was a good agreement between using cell culture-qPCR and the mouse assay for determining reduction or elimination of C. parvum infectivity as a consequence of varying UV operating conditions. Reduction in infectivity depended on the intensity of lamp discharge energy applied, amount of pulsing and population size of oocysts (P ≤ 0.05). Conventional radiometer was unable to measure fluence or UV dose in saline samples due to the ultra-short non-continuous nature of the high-energy light pulses. Incorporation of humic acid at a concentration above that found in surface water (i.e., ≤ 10 ppm) did not significantly affect PUV disinfection capability irrespective of parameters tested (P ≤ 0.05). These observations show that use of this HCT-8 cell culture assay is equivalent to using the ‘gold standard’ mouse-based infectivity assay for determining disinfection performances of PUV for treating C. parvum in saline solution.