A STUDY OF THE EFFECTS OF SOURCE, CLEANING PROTOCOLS AND
AGING OF COMMERCIALLY AVAILABLE STRAINS OF
Cryptosporidium parvum is a recognised cause of waterborne gastro-enteritis. A number of outbreaks have been reported and documented. Water treatment should provide an effective barrier against the contamination of drinking water but disinfection with chlorine is ineffective. In an attempt to find an alternative to chlorine, a number of disinfection studies have been conducted using a wide range of disinfectants and commercially available isolates of oocysts. Such commercial isolates are either of human or animal origin and have been passaged a number of times through an animal and cleaned to produce stocks for disinfection studies. Evidence suggests that the way in which oocysts are cleaned to remove faecal material can affect their electrophoretic mobility and hence their surface chemistry(1).
Commercial isolates are usually cleaned from faecal material by a variety of different processes including continuous centrifugation, sucrose, sodium chloride or caesium chloride centrifugation and ether or ethyl acetate floatation, the latter to remove fat. Some of these processes include the addition of sulphuric acid to help remove particulate material. Such cleaning processes may alter the antigenic structure of the oocyst, its susceptibility to disinfectants and both factors when the oocyst becomes aged. Time has been shown to affect the susceptibility of oocysts to ozone(2) and may certainly affect the detection of surface epitopes by immunofluorescent staining.
Disinfection studies require the assessment of viability before, during and after exposure. In the UK dye exclusion using 4’,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) has been used as a simple measure of viability(3). An alternative method involves excystation of sporozoites(4). These two tests give a measure of viability but do not provide information about infectivity. In addition, they both have their failings. Disinfection procedures that kill the sporozoites but do not disrupt the integrity of the oocyst may not permit the penetration of PI and therefore underestimate inactivation. Sporozoites exposed to certain chemicals may spontaneously lyse in an excystation procedure.
Inoculation of animals, in particular mice, gives a measure of infectivity but the procedure requires that mice are kept and proper assessment includes histology of the gastrointestinal system to demonstrate that replication is taking place(5). The mouse model has the disadvantage that more than one oocyst may be required to initiate infection. Fluorescent in-situ hybridisation (FISH) now provides an alternative for simple viability assessment(6) whereas inoculation of tissue culture and propagation of sporozoites(7) can be used to demonstrate infectivity. These two techniques are of greater interest in that they work with small numbers of oocysts and hence their sensitivity is much lower. They could be used to assess the viability of oocysts in raw and treated water concentrates.
Cleaning techniques as well as affecting antigenic structure may alter the ability to assess viability using one or more of the above techniques. It therefore becomes important to understand the effect that cleaning techniques have on the oocyst and on the ability to assess viability. In addition commercially available suspensions of oocysts may be contain a mixture of different types of oocysts. Under such circumstances, proper assessment of commercially available strains as to their suitability for disinfection studies needs to be assessed. To add a further confusing factor, the use of 2.5% potassium dichromate is a popular way of preserving the viability of oocysts. If possible any assessment should take into account the source of the oocysts, the means of propagation, the cleaning protocols used, whether potassium dichromate or other preservatives have any influence, and the type of measure used to assess viability.
Assessment of oocyst viability by dye permeability requires the penetration of DAPI but not PI into oocysts that are viable. Sporozoite nuclei stain blue-white with DAPI and are designated DAPI+ PI-. When oocysts are non-viable, the sporozoite nuclei stain with both DAPI and PI and these are designated DAPI+ PI+ and when the contents fail to stain, these are designated DAPI- PI-. This third state can be because the oocysts contain no sporozoites, ie. they are empty. Alternatively they may become non-permeable to the dyes, still retain sporozoites and may be viable. The difference between DAPI- PI- oocysts containing sporozoites and empty oocysts can be confirmed by phase contrast or differential interference contrast microscopy. Examples of permeable and non-permeable oocysts are given at the end of this report in the form of photomicrographs.
Non-permeable oocysts have been reported in faecal material(3). Conversion of permeable (DAPI+ PI-) to non-permeable (DAPI- PI-) oocysts has also been reported(8). Non-permeable oocysts have been found in fresh calf faeces and cleaned oocysts were found to show the same dye exclusion(9). Dye exclusion was shown to be a function of the oocyst wall and not of the metabolic activity of the sporozoites. Non-permeable oocysts converted to permeable oocysts as they aged and then they became non-viable. Conversion was found to be faster at room temperature. In addition, this conversion was found to be faster in oocysts incubated in ammonia(10). Non-permeability makes assessment of viability by dye exclusion more difficult.