Development of a method for the detection of Cryptosporidium in water and selected studies on hepatitis A virus

Abstract

Cryptosporidium is a protozoan parasite that causes gastroenteritis in man and animals. One mode of transmission of cryptosporidiosis is by the fecal-oral route. A method was developed for the concentration and detection of Cryptosporidium oocysts in water to study this organism's occurrence in the environment and its potential for waterborne disease transmission. Oocysts from an infected calf were used in experiments and monoclonal antibody labeled with fluorescein isothiocyanate was used for oocyst detection. A concentration method was developed using spun polypropylene cartridge filters. Optimal conditions for concentration, filter elution, filter porosity, and detection were determined by passage of 20-L volumes of tapwater seeded with 10⁵-10⁶ oocysts through the filters. The best method incorporated a 1-μm filter; 2 L eluent containing 0.1% Tween 80; backflush of eluent through filter; cutting the filter; mechanically shaking filter and eluate; and for 378-L volumes, three successive washings of filter material. Modifications were made when attempting to recover 10²-10³ oocysts in 378-L volurnes. These included addition of 1% Tween 80 and 1% sodium dodecyl sulfate (both made in distilled water) to the pellet, followed by homogenization, sonication, Sheather's flotation, and examination of the entire final preparation using a slide antibody test. To distinguish oocysts from other organisms such as yeast which may cross-react with the antibody, crystal violet and acid-fast stains were used. Cryptosporidium oocysts were isolated from secondarily treated sewage and identified on the basis of size, shape, reaction with antibody, acid-fastness, and inability to take up crystal violet. Limited studies with hepatitis A virus (HAV), a cause of waterborne disease, were performed using the HAS-15 strain of HAV and the FRhk-4 cell line. Four procedures used for concentration and detection of HAV in water were developed. They were radioimmunoassay for detection of viral antigen, production of HAV stocks, radioimmunofocus assay for quantitation of infective virus, and inhibition of certain strains of enteroviruses other than HAV, by guanidine.