Viability of Cryptosporidium parvum Oocysts: Assessment by the Dye Permeability Assay

The paper by Jenkins et al. (6) provides interesting data on Cryptosporidium parvum oocyst permeability and survival. The vital dye assay (4) relies upon oocyst permeability and exhibits limitations, especially in assessment of disinfectant efficacy (3). However, it is rewarding to note that it is considered to have utility and can provide pertinent data.

Although we agree with many points addressed by Jenkins et al. (6), we feel we should make the following three comments which, in part, reiterate conclusions from our previous work (4, 5, 7, 8). These points were perhaps overlooked by Jenkins et al. (6).

(i) In our paper describing the vital dye assay (4), impermeable oocysts (DAPI− PI−) are not described as dead (not viable), as suggested by Jenkins et al. (6). Rather, we concluded (4, 8) that a further “trigger” was required to increase oocyst permeability and thus excystation capability (see Table 1 in reference 4). Intriguingly, in an earlier paper by this research group (1) it appears that they did appreciate that such oocysts (DAPI− PI−) were capable of becoming viable. In this paper (1) they write, with reference to our original paper (4), that “PI-negative, DAPI-negative oocysts are also considered viable, but with the caveat that some treatment, e.g., acidification, is required before excystation will occur.”

We consider oocyst permeability to be a dynamic situation (up until death or excystation), which is reduced by incubation with saliva (8) and storage in cow feces (7) and increased by acidic incubation (8). Rather than simply describing oocysts as alive (viable) and dead (nonviable), our data revealed an additional oocyst state in which the oocysts were impermeable to both dyes and became viable (able to excyst under defined conditions) after a further trigger. Such “quiescent” oocysts could enter either stage, becoming viable or nonviable depending upon environmental factors.

(ii) During correlation of in vitro excystation and the dye permeability assay results, we ensured that any pretreatment was performed on both oocysts to be excysted and oocysts to be subjected to the assay (4, 8). Jenkins et al. (6) used our recommended pretreatment only for oocysts to be excysted and apparently not for oocysts to be subjected to the dye assay; we are therefore not surprised that their results differ from ours. Although we demonstrated a strong positive correlation between in vitro excystation and DAPI+ PI− oocysts, Jenkins et al. (6) did not observe this correlation. Indeed, they report a correlation between DAPI− PI− oocysts and excystation. If the pretreatment used for in vitro excystation by Jenkins et al. had also been used for the dye permeability assay, we would predict that they would have observed a correlation similar to that noted by us (4) and others (2).

Furthermore, if both DAPI+ PI− and DAPI− PI− oocysts are considered to be viable, reductive arithmetic argument shows that PI alone is being used as the indicator of viability. Here DAPI provides no information on oocyst viability, although it may provide some information on alteration of permeability of oocysts to this dye.

(iii) Addition of FITC-conjugated antibody to the assay to assist in oocyst detection during survival studies is pertinent and has been used by us (7) and others (1) as well as by Jenkins et al. (6). However, due to the additional manipulative steps required when DAPI and PI are used, it should be noted that, when viability assessment is conducted simultaneously with detection, oocyst recovery may be reduced. Simultaneous detection and viability assessment should therefore be treated cautiously for environmental monitoring in which detection of oocysts is of primary importance.