Environmental Research 106 (2008) 27–33 Occurrence of Cryptosporidium and Giardia in sewage sludge and solid waste landfill leachate and quantitative comparative analysis of sanitization treatments on pathogen inactivation Thaddeus K. Graczyk a,b,c,Ã , Malgorzata Kacprzak d , Ewa Neczaj d , Leena Tamang a , Halshka Graczyk e , Frances E. Lucy f , Autumn S. Girouard c a Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA b Johns Hopkins Water and Health Center, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA c Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA d Czestochowa University of Technology, Institute of Environmental Health Engineering, 42-200 Chestochowa, Poland e Johns Hopkins University, Baltimore, MD 21218, USA f School of Science, Institute of Technology, Sligo, Ireland Received 27 January 2007; received in revised form 7 May 2007; accepted 18 May 2007 Available online 21 June 2007 Abstract Circulation of Cryptosporidum and Giardia in the environment can be facilitated by spreading of sewage sludge on agricultural or livestock grazing lands or depositing in landfills. Solid waste landfill leachate and sewage sludge samples were quantitatively tested for C. parvum and C. hominis oocysts, and G. lamblia cysts by the combined multiplexed fluorescence in situ hybridization (FISH) and immunofluorescent antibody (IFA) method. Subsequently, the effects of four sanitization treatments (i.e., ultrasound and microwave energy disintegrations, and quicklime and top-soil stabilization) on inactivation of these pathogens were determined. The landfill leachate samples were positive for Giardia, and sewage sludge samples for both Cryptosporididium and Giardia. The overall concentration of G. lamblia cysts (mean; 24.2/g) was significantly higher (Po0.01) than the concentration of C. parvum and C. hominis oocysts (mean; 14.0/g). Sonication reduced the load of G. lamblia cysts to non-detectable levels in 12 of 21 samples (57.1%), and in 5 of 6 samples (83.3%) for C. parvum and C. hominis. Quicklime stabilization treatment was 100% effective in inactivation of Cryptosporidium and Giardia, and microwave energy disintegration lacked the efficacy. Top-soil stabilization treatment reduced gradually the load of both pathogens which was consistent with the serial dilution of sewage sludge with the soil substrate. This study demonstrated that sewage sludge and landfill leachate contained high numbers of potentially viable, human-virulent species of Cryptosporidium and Giardia, and that sonication and quicklime stabilization were the most effective treatments for sanitization of sewage sludge and solid waste landfill leachates. r 2007 Elsevier Inc. All rights reserved. Keywords: Cryptosporidium; Giardia; Sewage sludge; Landfill; Environmental contamination 1. Introduction Cryptosporidium parvum, C. hominis, and Giardia lamblia are human enteric parasites inflicting considerable morbid- ity on healthy people, and Cryptosporidium can cause mortality in the immunosuppressed population (Wolfe, 1992; Graczyk et al., 1997a). Their transmisive stages, i.e., oocysts and cysts, respectively, are shed in large numbers in the feces of infected people or animals, and due to their resistance to environmental stressors, they are ubiquitous in environment (Wolfe, 1992; Graczyk et al., 1997a). C. parvum is propagated through the anthropozonotic cycles (Graczyk et al., 1997a), and C. hominis cycles among people (Morgan-Ryan et al., 2002). The oocysts can remain viable for almost a year in the environment (Tamburrini ARTICLE IN PRESS www.elsevier.com/locate/envres 0013-9351/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2007.05.005 Ã Corresponding author. Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA. Fax: +1 410 955 0105. E-mail address: tgraczyk@jhsph.edu (T.K. Graczyk).