ORIGINAL ARTICLE Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts R. Reinoso 1,2 , E. Becares 3 and H.V. Smith 2 1 Environmental Research Institute, University of Leo ´ n, Leo ´ n, Spain 2 Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, Scotland, United Kingdom 3 Department of Ecology, Faculty of Biological and Environmental Sciences, University of Leo ´ n, Leo ´ n, Spain Introduction The increased requirement for wastewater reuse has resulted in attention being focussed on wastewater-treat- ment processes and their ability to inactivate or reduce the numbers of pathogens to acceptable levels (Cooper 1991). The apicomplexan parasite, Cryptosporidium, is responsible for numerous outbreaks of human disease (Caccio ´ et al. 2005; Karanis et al. 2006). Cryptosporidiosis is also of veterinary importance as livestock can be infected with various Cryptosporidium species that cause morbidity, with subsequent economic consequences. Cryptosporidium infects a large range of host species, and the infected hosts can excrete large numbers of viable, environmentally robust oocysts that contaminate waste- water, watersheds, surface and ground waters. Cryptosporidium parvum is the major species responsi- ble for clinical disease in humans and other neonatal mammals. The broad host range of C. parvum, together with the high output of oocysts, ensures a high level of contamination in the environment and both infected human beings and livestock can contribute to the num- bers of waterborne oocysts both through sewage effluent discharges, including those containing contributions from abbatoirs, and the disposal of sewage sludge to land. Cryptosporidium spp. oocysts are common contaminants of water, and have been responsible for numerous outbreaks of waterborne disease (Fayer et al. 2000; Gold and Smith 2001), yet oocysts can be detected in drinking water in the absence of disease in the community (Smith and Grimason 2003; Savioli et al. 2006). Robertson et al. (2000) demonstrated that, while conventional sewage Keywords ammonia, Cryptosporidium parvum, pH, temperature, viability, water. Correspondence Roberto Reinoso, Environmental Research Institute, University of Leo ´ n, La Serna 58, 24007 Leo ´ n, Spain. E-mail: rreit@unileon.es 2007 ⁄ 1769: received 15 December 2006, revised 26 June 2007 and accepted 12 September 2007 doi:10.1111/j.1365-2672.2007.03620.x Abstract Aims: To evaluate individual and combined effects of temperature (4, 18 and 25°C), pH (7 and 10), ammonia (5 and 50 mg l )1 ) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. Methods and Results: The viability of oocysts was evaluated using the fluoro- genic vital dyes assay (4¢,6-diamidino-2-phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P <0Æ005). Exposure of oocysts to pH 10 for 6 days at 25°C reduced oocyst viability from 80% to 51%. Simi- larly, the exposure of C. parvum oocysts to 5 mg NH 3 l )1 and 50 mg NH 3 l )1 for 4 days reduced their viability from between 80% to 41Æ5% and 14Æ8%, respectively. Conclusions: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low con- centrations of ammonia, as commonly found in alga-based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. Significance and Impact of the Study: This study provides relevant data on the inactivation of C. parvum oocysts in alga-based wastewater-treatment systems in the northwest of Spain. Journal of Applied Microbiology ISSN 1364-5072 980 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 980–986 ª 2007 The Authors