In Vitro Evaluation of Anticryptosporidial Agents Using MDCK Cell Culture and Chemiluminescence Immunoassay

5 4 7 i 3 0 2 3 6 i 5 2 0 2 i 7 2 0 6 i 10 1 5 8 i 6 7 To date, no effective therapeutic compounds are available for clinical use against Ctyptosporidium parvum infections [3]. In vitro drug evaluation systems should facilitate the development and identification of effective therapies. Recently, a highly sensitive chemiluminescence immunoassay (CLIA) has been developed for quantifying C. parvum growth in vitro. Initial data showed C. pawum growth was correlated with the chemiluminescence signal produced in an MDCK cultivation system and that the assay appeared well suited for in vitro anticryptosporidial drug evaluation [4]. To further characterize this system, comparison studies were conducted with different C. parvum isolates and cell lines. Additionally, this system has been used to evaluate more than 60 compounds for anticryptosporidial activities. MATERIALS AND METHODS. C. pawurn isolates including Iowa (calf origin) and Maine (human origin) were generated and purified as previously described [l]. MDCK (ATCC CCL 34), and HCT-8 (ATCC CCL 244) cells were passaged in serum free UltracultureTM medium (BioWhittaker) and seeded into 96-well plates (ViewPlateTM, Packard) for CLTA or 2-well culture slides (Lab-Tek Chamber SlideTM, Nunc) for immunofluorescence assays (IFA). Test compounds were dissolved in 100% DMSO (final concentration in cultures was less than 0.01%) or culture medium. Ultraculture medium with 0.01% DMSO was used as control. Purified oocysts were excysted as previously described [2], diluted with medium to the desired concentrations and promptly dispensed in plates or slides containing 4 day host cells preparations. The inoculum was incubated with the cells for 3 hr, removed by washing with PBS, and replaced with fresh UItraculture medium with or without test compounds. The cultures were incubated at 37°C for 48 hr, washed with PBS, and fixed with Bouin's solution. The CLIA procedure was performed as previously described [4]. Briefly, fixed plates were washed with a Tris-HCI, NaCI, Tween 80 buffer (TBST) and then blocked with 1% BSA-TBST for 30 min at room temperature. Rabbit anti-C. parvum sera was applied to the plates and incubated for I hr. After washing with TBST, the wells were sequentially incubated with biotin-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled streptavidin (KPL). Enhanced luminol (Luminol, 4-iodophenol, and hydrogen peroxide, Aldrich) was used as substrate. The plates were read with a ML3000 Lurninometer (Dynatech) and the relative light units (RLU) were determined. Mean RLUs were calculated from 4 replicate wells and all experiment were repeated at least twice. Monoclonal antibody C3C3, an IgG, antibody directed against C. parvum cytoplasmic antigens found in meront and gamont life cycle stages, was used in the iFA as described previously [2]. Mean parasite numbers were calculated from 3 replicate chambers and all experiment were repeated at least twice. Toxicity was measured using the CellTiter 96TMAQ,,,, assay (Promega). RESULTS AND DISCUSSION. As demonstrated in Table 1, no significant difference was observed in parasite growth (RLU signal) when two different C. parvum isolates were used to infect MDCK cells. The Iowa isolate yielded essentially identical results in both MDCK and HCT-8 cell lines (data not shown). The anticryptosporidial activity of paromomycin in MDCK and HCT-8 cells was both dose-dependent and highly correlated (r2 = 0.94) (Table 2). More than 60 compounds of different structural classes were evaluated using MDCK culture and CLIA (a partial list is shown in Table 3). Anticryptosporidial activities MDCK