Occurrence of Cryptosporidium and Giardia in sewage sludge and solid waste landfill leachate and quantitative comparative analysis of sanitization treatments on pathogen inactivation
Introduction
Cryptosporidium parvum, C. hominis, and Giardia lamblia are human enteric parasites inflicting considerable morbidity 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 and Pozio, 1999), e.g., animal liquid waste (Hutchison et al., 2005a, Hutchison et al., 2005b), and Giardia cysts can retain their viability outside the host for at least 2 months (deRegnier et al., 1989). An infected person can shed up to 3×109 oocysts (Okhuysen et al., 1999) or up to 5×108 cysts over the course of infection (Medema and Schijven, 2001). Extrapolation from human volunteer dose–response experiments (Rendtorff, 1954; Dupont et al., 1995) indicates that a dose of a single oocyst or cyst results in 0.4%, and 2.0% probability of cryptosporidiosis and giardiasis, respectively (Medema and Schijven, 2001).
As municipal solid waste contains fecal material from a variety of sources, when water from rainfall contacts the waste in a landfill, the liquor that leaches from the decomposing refuse is highly contaminated with human pathogens (Sobsey, 1978; Yangin et al., 2002; Szostakowska et al., 2004). Studies on human infectious agents in sewage showed that the number of Cryptosporidium oocysts and Giardia cysts can be very high (Madore et al., 1987; Straub et al., 1993; Bukhari et al., 1997; Chauret et al., 1999; Robertson et al., 2000). Such high concentrations are due to the high input of oocysts and cysts (Rimhanen-Finne et al., 2001, Rimhanen-Finne et al., 2004). Land applications of sewage sludge-end products is an ecologically important means of utilization of nitrogen and phosphorus. However, spreading sludge on agricultural lands, which has increased during the last years due to economic and environmental reasons (Rimhanen-Finne et al., 2004), facilitates circulation of Cryptosporidium and Giardia in the environment. It also contaminates shallow aquifers and potable waters (Straub et al., 1993), thus poses a human and animal health threat. Also, their very low ID50 values (Wolfe, 1992; Okhuysen et al., 1999) and very high D-values (Hutchison et al., 2005a, Hutchison et al., 2005b) indicate a health risk for workers exposed to sewage and landfill leachates (Rimhanen-Finne et al., 2004; Westrell et al., 2004; Hutchinson et al., 2005a). Therefore, sanitization treatments applied to landfill leachates and sewage sludge are of high public health importance. Ultrasonic or microwave energy disintegrations, and quicklime or soil stabilization can be used for sanitization of landfill leachate and sewage sludge; however, information on effectiveness of these treatments on inactivation of Cryptosporidium and Giardia is incomplete (Ashokkumar et al., 2003; Rimhanen-Finne et al., 2001, Rimhanen-Finne et al., 2004; Collins et al., 2005).
The effectiveness of sanitization treatments applied to solid waste landfill leachate and sewage sludge measured by inactivation efficacy of Cryptosporidium and Giardia requires a reliable, quantitative method for the enumeration of potentially viable pathogens. Microscopy has low sensitivity and requires a skilled microscopist (Rimhanen-Finne et al., 2001). Direct immunofluorescent antibody (IFA) (Santos et al., 2004; Rimhanen-Finne et al., 2004) usually overestimates the Cryptosporidium and Giardia load because the antibodies cross-react with other species of Cryptosporidium not virulent for humans (Graczyk et al., 1996), small unicellular algae (Clancy et al., 1994), and nonviable oocysts (Graczyk et al., 1997b). PCR-based methods, although very sensitive and specific, do not allow for pathogen viability assessment, and are highly sensitive for massive amounts of microorganisms and compounds present in sludge acting as PCR inhibitors (Rimhanen-Finne et al., 2001). Vital dyes, e.g., DAPI stain, in conjunction with IFA can be used for viability assessment of oocysts (Hutchison et al., 2005a, Hutchison et al., 2005b). Recovery of Cryptosporidium oocysts and Giardia cysts from environmental samples remains a technologically complex process, but even more challenging is subsequent species-specific identification and viability assessment of these pathogens. The fluorescence in situ hybridization (FISH) technique meets both of these challenges (Graczyk et al., 2003; Smith et al., 2004). The multiplex FISH method uses fluorescently labeled oligonucleotide probes designed to hybridize with sequences of 18S rRNA of either C. parvum or C. hominis, and G. lamblia, respectively (Deere et al., 1989; Dorsch and Veal, 2001; Smith et al., 2004; Lemos et al., 2005). Because rRNA is only present in large copy numbers in viable organisms (Dorsch and Veal, 2001; Smith et al., 2004), FISH allows species-specific identification by providing visualization of potentially viable oocysts and cysts, and facilitates their enumeration (Graczyk et al., 2003, Graczyk et al., 2004). However, extended rRNA half-life may result in viability overestimation using FISH (Smith et al., 2004). Furthermore, multiplexed FISH has been combined with a direct IFA against the wall antigens of Cryptosporidium and Giardia, and this approach has been successful for simultaneous detection of C. parvum, C. hominis, and G. lamblia in environmental and clinical samples (Graczyk et al., 2003, Graczyk et al., 2004, Graczyk et al., 2006; Lemos et al., 2005). In order to support FISH results related to potential viability of C. parvum and C. hominis oocysts, we used in vitro excystation assay (Graczyk et al., 1997b).
The purposes of the present study were to quantitatively determine the intensity of natural contamination of sewage sludge and solid waste landfill leachate with C. parvum and C. hominis oocysts and G. lamblia cysts, and to assess the effectiveness of four sanitization treatments, i.e., ultrasound and microwave energy disintegrations, and quicklime and top soil stabilization on inactivation of these oocysts and cysts.
Section snippets
Materials and methods
The sewage sludge originated from two urban wastewater treatment plants, Pajeczno (51°09′N; 19°00′E) and Myszkow (50°35′N; 19°19′E), Poland. Both plants received human and industrial waste while none of them received animal manure. Sewage sludge from Pajeczno was biologically stabilized and de-watered. A sewage sludge sample (10 kg) was transported to the laboratory in a cooler, vigorously mixed upon arrival and agitated with top-soil in closed plastic, 15 dm3-capacity, containers at the three
Results
C. parvum and C. hominis oocysts, and G. lamblia cysts were identified in control sewage sludge samples from both Pajeczno and Myszkow (Table 1). FISH and IFA labeling clearly differentiated potentially viable vs. non-viable oocysts. Potentially viable oocysts identified by FISH were intact, revealed a small gap between the oocyst wall and internal structures, and the sporozoites were visible. The excystation index for the oocysts from Pajeczno and Myszkow was 85% and 93%, respectively, and the
Discussion
The present study demonstrated that the overwhelming majority of transmissive stages of human-virulent Cryptosporidium and Giardia species were potentially viable as per FISH and in vitro excystation assays while in the sewage sludge. Thus, given: (a) the massive amounts of sewage sludge utilized by agriculture or deposited in landfills (Straub et al., 1993; Szostakowska et al., 2004; Westrell et al., 2004; Gale 2005); (b) environmental robustness of these pathogens (deRegnier et al., 1989;
Acknowledgments
The study was supported by the Organization for Economic Co-Operation and Development (OECD) (Grant No. AGR/PR20061), Paris, France; Fulbright Specialist Fellowship (Grant 2225 to Graczyk), Washington, DC, USA; Johns Hopkins Center in Urban Environmental Health, Baltimore, MD, USA (Grant No. P30 ES03819); Alternatives Research & Development Foundation; NOAA Chesapeake Bay Office, Annapolis, MD, USA (Grant No. NA04NMF4570426); and the Johns Hopkins Center for a Livable Future, Baltimore, MD, USA.
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