Influence of surface characteristics on the stability of Cryptosporidium parvum oocysts

Abstract

Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.

FIG. 1.

(a) Representative electropherogram of CML microspheres and mesityl oxide dopant. Peaks at the start of the electropherogram (from 0 s to ca. 30 s) result from noise caused by application of the 30-kV energy potential following sample injection. (b) Representative electropherogram of C. parvum oocysts, which were analyzed as received. The single peak at 1.9 min corresponds to the oocysts. The peak indicated by the dashed line represents the retention time for both the mesityl oxide dopant and C. parvum oocysts, showing that the two have the same value. (c) Representative electropherogram for oocysts washed in distilled water. The peak at ca. 2 min represents both oocysts and the mesityl oxide dopant.

FIG. 2.

(a) Aggregation of CML microspheres suspended in distilled water and 0.5 M NaCl. (b) Aggregation of C. parvum oocysts suspended in distilled water and 0.5 M NaCl. Error bars represent 1 standard deviation.

FIG. 3.

Photomicrographs of CML microspheres (a) and oocysts (b) in a Petroff-Hauser counting chamber. The microspheres and oocysts were suspended in a 0.5 M NaCl solution.

FIG. 4.

ATR spectra of C. parvum oocysts and chemicals commonly used in the oocyst purification process.

FIG. 5.

ATR spectra of formalin-treated oocysts (spectrum a) and oocysts not exposed to formalin (spectrum b). Note the absorption bands identified in spectrum a by asterisks that correspond to formaldehyde. These bands may arise from either formaldehyde adsorbed onto the surface of the oocyst or formaldehyde absorbed into the oocyst. Also note the absence of these bands in spectrum b.

FIG. 6.

Diffuse reflectance FTIR spectra of C. parvum oocysts from Cornell and Idaho.