Viable Cryptosporidium parvum oocysts exposed to chlorine or other oxidising conditions may lack identifying epitopes
Introduction
The enteric protozoan parasite Cryptosporidium parvum is a well-documented cause of water-borne disease in humans. Low oocyst concentrations are a potential risk, and the organism can cause serious illness when ingested by immunosuppressed individuals 1, 2. Infection occurs when oocysts, shed in the faeces of infected individuals, are ingested by new hosts. Cryptosporidium oocysts are a particular problem to the water industry, as they are present in many surface water supplies 3, 4, 5and are not inactivated by standard disinfection procedures 6, 7. Water-borne outbreaks of cryptosporidiosis have been traced to water treatment plants that have met all current microbiological and chemical standards [1]. Many water utilities now monitor both treated and raw waters for the presence of Cryptosporidium oocysts independently of other tests.
The most commonly used method for detecting Cryptosporidium oocysts from environmental samples involves the concentration of large volumes of water, followed by the elution of particles and purification of oocysts by density gradient centrifugation. Prepared slides of the concentrated material are stained with fluorochrome coupled monoclonal or polyclonal antibodies and examined by epifluorescence microscopy 8, 9. This method is time consuming and tedious, recoveries are low, and it is unsuitable for water samples containing significant amounts of particulate matter 10, 11.Recently a more rapid, sensitive and reliable method has been reported. It is based on flocculating concentration followed by staining the concentrate with a fluorochrome coupled mAb. Oocyst purification is accomplished by flow cytometry and the material is examined by epifluorescence microscopy 11, 12, 13. Both techniques are limited by the specificity and reliability of antibodies that bind to epitopes on the oocyst wall. There are several commercially available antibodies used for the detection of C. parvum. Although specific to Cryptosporidium species, none are specific to the causative agent of human cryptosporidiosis, C. parvum [14].
The aim of this study was to investigate the ability of four commercially available antibodies to detect C. parvum oocysts from water samples that have been exposed to chlorine and oxidising conditions that may be experienced in the environment. The viability of C. parvum oocysts which were exposed to sodium hypochlorite and sodium meta-periodate was also examined.
Section snippets
Oocysts
Cryptosporidium parvum oocysts (from cervine faeces) were obtained from Moredun Animal Health Institute, U.K. Oocysts were stored live in PBS [0.9% (w\v) NaCl, 0.02% (w\v) KH2PO4, 0.29% (w\v) NaHPO4, 0.2% (w\v) KCl, pH 7.4] at 4°C. For experiments not requiring excystation, oocysts were heat inactivated by incubation at 60°C for 30 min.
Excystation studies
In-vitro excystation was performed as described by Vesey et al. [15]. Aliquots of live oocyst suspensions (100 μl) were mixed with 1 ml of acidified (pH 2.75)
Western blotting
Western blotting was used to access the range and nature of epitopes bound by four commercially available antibodies (Table 1) used to detect C. parvum oocysts. Fig. 1Fig. 2 (lanes a) indicate that all four antibodies recognised an apparent complex of very high Mr proteins, of approximately 120 kDa, 116 kDa and 112 kDa, with the 112-kDa band being the most prominent. All antibodies except MM recognised additional bands in the same group at approximately 105 kDa and 108 kDa, and BHM and BHP
Discussion
The detection of C. parvum in environmental waters is challenging, because very low numbers of the organism must be detected in heterogeneous samples 2, 11, 14. Immunofluorescence is currently the most widely used method for detecting C. parvum oocysts in water samples 8, 11.Nevertheless, caution needs to be exercised when using currently available antibodies. It has been demonstrated previously that all four antisera used in this study recognise the same or a similar epitope using competition
Acknowledgements
Anthony G. Moore was supported by a post-graduate scholarship award from the School of Biological Sciences, University of Western Sydney, Nepean and CSIRO Australia, and an Australian Research Council small grant to School of Biological Sciences, University of Western Sydney, Nepean. Graham Vesey was supported by a post-graduate award from AWT, Ensight, NSW, Australia. The authors would like to thank Mr Marc Ramond for his technical assistance in some of the experiments. Keith Williams
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