Quantification of Enteric Viruses,Pathogen Indicators,and Salmonella Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods

The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of Salmonella enterica in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 10⁴ MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for Salmonella enterica, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.Over the last decade, thousands of people in the United States have been infected with waterborne diseases, a large number of whom were hospitalized. Most of the waterborne disease outbreaks in the United States that occurred between 1991 and 2004 were related to microbial agents, i.e., viruses, bacteria, and parasites (5, 30). The majority of the outbreaks involved unidentified agents, and the Environmental Protection Agency (EPA) suspects that many of the outbreaks due to unidentified sources were caused by enteric viruses (46). Indeed, viruses have a high potential for groundwater pollution due to their small size and low die-off rates. The occurrence of enteric viruses in groundwater has been reported (1, 7, 12, 17). In the United States, approximately 5.6 million dry tons of biosolids are generated annually and 60% of the biosolids are applied to land (36).Several studies have reported the occurrence of enteric viruses in biosolids (18, 35, 47); however, information on the quantity and infectivity of enteric viruses in biosolids is still limited, and most studies focused solely on enteroviruses (41). Few studies have reported the levels of human adenoviruses (HAdV) in biosolids (6, 47), and no quantitative results have yet been reported on some of the emerging viruses, such as hepatitis A virus (HAV) and noroviruses (NV). Also, only one or two types of enteric viruses were quantified in the previous studies; therefore, it is hard to determine and compare the prevalence of different types of enteric virus in biosolids, since the samples and sample processing methods varied from study to study. A few studies focused on the viral infectivity of biosolids, and the results showed that infectious astrovirus and enteroviruses were still present in the treated biosolids (9, 18, 42). However, no results on the occurrence of adenoviruses in biosolids have been reported.PCR techniques have been used in most of the recent environmental virology studies. In comparing these techniques to cell culture, the main advantages of PCR methods for virus detection are fast results, less labor intensiveness, high specificity and sensitivity, and the capability of detecting difficult-to-culture or nonculturable viruses (for examples, human noroviruses and adenovirus 40/41). Quantitative real-time PCR (qPCR), which is considered the latest advancement in PCR technology, can provide both qualitative and quantitative results. However, PCR results may not reflect the infectivity of the samples since PCR only detects the genes of the pathogens; therefore, integrated cell culture-PCR (ICC-PCR) was developed to identify the specific infectious enteric viruses. ICC-PCR has been used to detect infectious enteric viruses in river water, tap water, beach water, and wastewater effluent samples (28, 29, 39, 50). However, Buffalo green monkey (BGM) cell culture, currently recommended by the EPA, has been compared with other cell lines, such as A549 and PLC/PRC/5 (28, 39), and the results showed that enteric viruses were propagated better with these cell lines than with BGM cells.The main objective of this work was to investigate the occurrence and the quantitative levels of the enteric viruses in class B mesophilic anaerobically digested (MAD) biosolid samples by molecular and cell culture methods. These results can be used for risk assessment at biosolid application sites. Also, enteric viruses have been suggested as fecal source tracking indicators (21, 32), and the levels of enteric viruses in biosolids reported in this study would be useful for the determination of which enteric virus is a better fecal source tracking indicator at biosolid application sites. MAD biosolids were chosen since they are the most common type of class B biosolid produced in the United States (47). Three dewatered biosolid samples were also included for comparison purposes. The levels of human adenovirus (HAdV), adenovirus type 40/41 (HAdV 40/41), enterovirus (EV), norovirus GI (NV GI) and NV GII, human polyomavirus (HPyV), and hepatitis A virus (HAV) were quantified by qPCR methods. BGM and A549 cell lines were used to quantify the infectious viruses in the biosolids, and the effectiveness of these two cell lines'' ability to propagate infectious viruses was compared. The occurrence of infectious EV and HAdV in biosolids was determined by ICC-PCR, and the serotypes of the infectious adenoviruses propagated on A549 were further determined. The levels of pathogen indicators and Salmonella enterica are also presented in this study.