Comparison of In Vitro Cell Culture and a Mouse Assay for Measuring Infectivity of Cryptosporidium parvum

Abstract

In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r ‫؍‬ 0.85, n ‫؍‬ 25) and for oocysts exposed to ozone and UV light (r ‫؍‬ 0.89, n ‫؍‬ 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

Bile Acids Enhance Invasiveness of Cryptosporidium spp. into Cultured Cells

“…Proteins in supernatants (lanes 1, 2, and 3) and sporozoite pellets (lanes 4, 5, and 6) were resolved by SDS-PAGE followed by silver staining (A) or immunoblotting (B) using Cryptosporidium-specific rabbit polyclonal antibody. drug therapies against the pathogen and for investigating hostparasite interactions (3,17). Due to the low infectivity of C. parvum to cultured cells, many attempts have been made to improve infectivity, including optimization of conditions for excystation (11,15,16,23,24), improvement of nutrients in culture medium (14,23), and other methods, such as centrifugation (26).…”

“…Although the molecular and biochemical mechanisms involved in excystation are poorly understood, it is believed that host environmental factors, such as temperature, pH, proteases, bile salts, and possibly other unknown factors, trigger the excystation of ingested oocysts (19). The newly excysted sporozoites secrete adhesive molecules and other signaling proteins which have been demonstrated to play a key role in the initial attachment and cellular invasion (4, 6).The in vitro cell culture system, while limited to the asexual phase, has been a useful tool for investigating early parasite attachment and invasion and has been applied to measure Cryptosporidium infectivity and for screening of compounds for inhibitory activity (3,17). Several cell lines, including human ileocecal adenocarcinoma (HCT-8), human colonic adenocarcinoma (Caco-2), and Madin-Darby bovine kidney (MDBK) cell lines, are widely used for Cryptosporidium in vitro studies (12,22).…”

“…The in vitro cell culture system, while limited to the asexual phase, has been a useful tool for investigating early parasite attachment and invasion and has been applied to measure Cryptosporidium infectivity and for screening of compounds for inhibitory activity (3,17). Several cell lines, including human ileocecal adenocarcinoma (HCT-8), human colonic adenocarcinoma (Caco-2), and Madin-Darby bovine kidney (MDBK) cell lines, are widely used for Cryptosporidium in vitro studies (12,22).…”

Inactivation of Cryptosporidium parvum under chlorinated recreational water conditions

“…While mouse infectivity is considered a gold standard (Black et al 1996;Bukhari et al 2000), it is an expensive and cumbersome method. Tissue culture has been compared to mouse infectivity with favorable results (Arrowood 2002;Rochelle et al 2002;Joachim et al 2003). Tissue culture, while demanding special skills, is less expensive than mouse infectivity and far less labor-intensive.…”

Potential molecular tools for assessing the public health risk associated with waterborne Cryptosporidium oocysts

“…Infectivity or viability of Cryptosporidium oocysts has been evaluated by several molecular, culture or infectivity assays such as: (i) evaluation of comparative infectivity in CD1 mice and in cell cultures using RT-PCR detection assay targeting hsp70 gene mRNA (Rochelle et al, 2002(Rochelle et al, , 2004, (ii) evaluation of infectivity in HC8 cells using qPCR targeting the 18S rRNA gene (Garvey et al, 2010), (iii) cell culture (CC)-PCR assay targeting hsp70 DNA (Aboytes et al, 2004), and (iv) CC-IFA assay on oocysts recovered by USEPA method 1623 (Quintero-Betancourt et al, 2003;Gennaccaro et al, 2003).…”