Application of human and animal viral microbial source tracking tools in fresh and marine waters from five different geographical areas
Graphical abstract
Introduction
The use of integrated river basin management planning is necessary to mitigate the impacts of climate change and to protect water resources. In Europe, this is being implemented through the ‘Programs of Measures’ as outlined in Article 11 of Directive 2000/60/EC, the Water Framework Directive. Climate change will undoubtedly influence water quality in rivers, lakes and marine waters used for drinking water abstraction, recreational activities, shellfish harvesting and assimilation of point and diffuse fluxes of human and livestock effluents. Thus, the risk profile and treatment interventions necessary for sewage and potable waters must be changed. The fourth assessment report of the Intergovernmental Panel on Climate Change (IPCC) noted that higher temperatures, changes in precipitation regimes and more frequent weather-related disasters are primary changes that represent risks for agriculture, food, and particularly water supplies. Moreover, increasing urban expansion and associated intensification of farm production are increasing microbial loads, which are then discharged into receiving waters worldwide. Achieving legal water quality criteria will, therefore, require intelligent and integrated application of sustainable treatment and urban drainage technologies (SUDs), upgrade of wastewater treatment plants (WWTP), enhanced storm water retention facilities, and application of agricultural best management practices (BMPs) designed to reduce diffuse pollution from livestock farming.
There is increasing concern regarding the levels of fecal pollution in surface waters due to point-source discharges from community sewage treatment plants and livestock concentrations derived from slaughterhouse discharges (Collins et al., 2005, Jamieson et al., 2004). Also, non-point diffuse microbial pollution may originate from direct fecal voiding by grazing livestock, manure spreading or urban surface water runoff, which can derive from roof and road surfaces contaminated with avian, domestic and wild animal feces and also from cross-connections to urban sewerage system, all of which can exhibit high microbial loads (Llopart-Mascaró et al., 2010, Brownell et al., 2007, Bercu et al., 2011). The identification of fecal contamination sources in water using specific markers is a key step for good management and remediation protocols.
Microbial Source Tracking (MST) encompasses a group of methodologies that aim to identify and, in some cases, quantify the dominant sources of fecal contamination in the environment, particularly in water resources (Field et al., 2004, Fong and Lipp, 2005). MST plays a very important role in informing remediation strategies directed against specific pollutant sources. Molecular markers used for MST are target sequences in host-associated microorganisms or sequences derived directly from the host. These can come from prokaryotes, eukaryotes and viruses. Widely-used human and animal-associated markers have been identified in the order Bacteroidales (Bernhard and Field, 2000a, Bernhard and Field, 2000b) and other bacteria. Polymerase chain reaction (PCR) assays have been developed and validated for markers of fecal contamination from humans and a diversity of animals (reviewed in Roslev and Bukh, 2011). Although these molecular markers represent interesting methods for MST, they do have significant limitations. For example, (i) there is a lack of absolute host specificity among human- and animal-associated microbial markers; (ii) there is a lack of temporal stability of some host-associated microbial markers in different host groups and (iii) non-fecal sources of markers potentially exist (Roslev and Bukh, 2011, Stapleton et al., 2009). The use of highly host specific, ubiquitous and stable MST markers that produce persistent excretions in their human or animal hosts could overcome these limitations.
In recent years, many studies have examined human adenovirus (HAdV) and JC polyomavirus (JCPyV) as human fecal indicators, as they are persistently excreted by infected humans both with and without clinical symptoms in the feces or urine (Bofill-Mas et al., 2001). Thus, they are commonly detected in urban wastewater in all geographical areas throughout the annual cycle (Koralnik et al., 1999, Bofill-Mas et al., 2001, Bofill-Mas et al., 2006, Schlindwein et al., 2010, Kokkinos et al., 2011, Rodriguez-Manzano et al., 2012, Bofill-Mas et al., 2013). Traditionally, standard fecal indicator bacteria (FIB) are used to indicate the presence of human or animal fecal contamination. However, FIB counts cannot discriminate between animal or human contamination, whereas AdV and PyV are host-specific and derive from the gastrointestinal and urinary tracts (Bofill-Mas et al., 2000, Maluquer de Motes et al., 2004). In a study using PCR, Harwood et al. (2009) suggested that human polyomaviruses were the most specific human marker for MST among various tools analyzed. Importantly, candidate viral MST indicators do not multiply in the environment and are more resistant to environmental stressors, such as UV irradiance from sunlight and water treatment processes, than FIBs (Bofill-Mas et al., 2013). They may, therefore, represent a better index for viral pathogens such as Hepatitis A and E viruses and noroviruses (NoV) than do the common FIBs used as regulatory parameters world-wide, e.g. Escherichia coli and intestinal enterococci. A large diversity of concentration protocols for viruses in water have been described (Albinana-Gimenez et al., 2009). Viral detection consists of several steps: concentration of viruses from the environmental water sample into a suitable volume, extraction of the DNA or RNA and detection or quantification of the viral segment with molecular techniques. Low viral concentration and viral viability are the main handicaps of these PCR techniques.
Efficient and cost-effective techniques for virus pre-concentration in water have been developed using skimmed milk direct flocculation procedures (Calgua et al., 2008, Calgua et al., 2013a), which have potential for the routine analysis of viruses in water samples. Moreover, sensitive and reliable molecular detection techniques based on real-time PCR designed for specific DNA viruses, such as HAdV and JCPyV, porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV), have been suggested in previous studies for the quantification of these specific markers, offering the potential to delineate human and/or animal fecal contributions in environmental water matrices (Bofill-Mas et al., 2006, Bofill-Mas et al., 2013, Hundesa et al., 2006, Hundesa et al., 2009 and 2010).
This study aimed to test the applicability of human and animal adenoviruses and polyomaviruses in widely diverse river catchments by applying skimmed milk flocculation and qPCR to define sources of fecal contamination in all areas and scenarios.
Section snippets
Control viruses and plasmids
Human adenovirus type 35 (ATCC, LGC Standards AB, Borås, Sweden) stocks were produced by infecting A549 cells cultured in Earl's minimum essential medium (EMEM) supplemented with 1% glutamine, 20 μg of streptomycin and 20U of penicillin per mL and 10% (growth medium) or 2% (maintenance medium) of heat-inactivated fetal bovine serum (FBS). Viruses were released from cells by freezing and thawing the cultures 3 times. Then, a centrifugation step at 3000 g for 20 min was applied to eliminate cell
Quantification of viral markers in five case studies
In this study, samples collected over eighteen months in five river catchments with different land uses and climatological conditions were analyzed for four well-known viral fecal markers (HAdV, JCPyV, PAdV and BPyV). Common SOPs were used in all laboratories for the analysis of the samples. Positive and negative control process produced the expected positive and negative results in all the assays. Inhibition was observed when high levels of organic matter or sand occurred in water samples. In
Discussion
In this study, novel parameters were investigated as microbial source-tracking tools designed to map the origins of fecal contamination to human (HAdV and JCPyV), porcine or bovine (PAdV and BPyV) viruses. DNA-based viral tools were applied in five different river scenarios in five countries: Greece, Spain, Sweden, Hungary and Brazil.
The protocols used in the study have been shown to be easily applicable in routine analysis of a wide diversity of water matrices. The estimated recovery
Conclusions
- 1.
The novel MST tools described in this paper have been shown to be specific, sensitive and provide quantitative data describing source-specific fecal impact in river catchments in different geographical areas.
- 2.
The protocols and viral markers applied in this multi-laboratory study have proven to be robust, cost-effective and applicable for routine MST analysis in all types of water matrices and geographical areas.
- 3.
The human (HAdV and JCPyV) and animal (PAdV and BPyV) viruses analyzed in this study
Acknowledgments
The VIROCLIME Project is funded under the EU Seventh Framework Program, Contract No. 243923. The described study was supported by a collaborative European project coordinated by David Kay and Peter Wyn-Jones as vice-coordinator from the University of Aberystwyth, United Kingdom (VIROCLIME, contract no. 243923). Marta Rusiñol was a fellow of the Catalan Government “AGAUR” (FI-DGR), and Xavier Fernández was a fellow of the University of Barcelona (APIF). Finally, we would like to thank to the
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