Ayalkibet Hundesa

Molecular detection of pathogens in water - the pros and cons of molecular techniques.

Pollution of water by sewage and run-off from farms produces a serious public health problem in many countries. Viruses, along with bacteria and protozoa in the intestine or in urine are shed and transported through the sewer system. Even in highly industrialized countries, pathogens, including viruses, are prevalent throughout the environment. Molecular methods are used to monitor viral, bacterial, and protozoan pathogens, and to track pathogen- and source-specific markers in the environment. Molecular techniques, specifically polymerase chain reaction-based methods, provide sensitive, rapid, and quantitative analytical tools with which to study such pathogens, including new or emerging strains. These techniques are used to evaluate the microbiological quality of food and water, and to assess the efficiency of virus removal in drinking and wastewater treatment plants. The range of methods available for the application of molecular techniques has increased, and the costs involved have fallen. These developments have allowed the potential standardization and automation of certain techniques. In some cases they facilitate the identification, genotyping, enumeration, viability assessment, and source-tracking of human and animal contamination. Additionally, recent improvements in detection technologies have allowed the simultaneous detection of multiple targets in a single assay. However, the molecular techniques available today and those under development require further refinement in order to be standardized and applicable to a diversity of matrices. Water disinfection treatments may have an effect on the viability of pathogens and the numbers obtained by molecular techniques may overestimate the quantification of infectious microorganisms. The pros and cons of molecular techniques for the detection and quantification of pathogens in water are discussed.

Quantification and Stability of Human Adenoviruses and Polyomavirus JCPyV in Wastewater Matrices

ABSTRACT Human adenoviruses (HAdV) and human polyomavirus JCPyV have been previously proposed as indicators of fecal viral contamination in the environment. Different wastewater matrices have been analyzed by applying real-time quantitative PCR procedures for the presence, quantity, and stability of a wide diversity of excreted HAdV and JCPyV. High quantities of HAdV and JCPyV were detected in sewage, effluent wastewater, sludge, and biosolid samples. Both viruses showed high stability in urban sewage. These results confirm the suitability of both viruses as indicators of human fecal viral pollution.

Raw Sewage Harbors Diverse Viral Populations

ABSTRACT At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity. IMPORTANCE At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected. At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected.

Identification of Human and Animal Adenoviruses and Polyomaviruses for Determination of Sources of Fecal Contamination in the Environment

ABSTRACT The Adenoviridae and Polyomaviridae families comprise a wide diversity of viruses which may be excreted for long periods in feces or urine. In this study, a preliminary analysis of the prevalence in the environment and the potential usefulness as source-tracking tools of human and animal adenoviruses and polyomaviruses has been developed. Molecular assays based on PCR specifically targeting human adenoviruses (HAdV), porcine adenoviruses (PAdV), bovine adenoviruses (BAdV), and bovine polyomaviruses (BPyV) were applied to environmental samples including urban sewage, slaughterhouse, and river water samples. PAdV and BPyV were detected in a very high percentage of samples potentially affected by either porcine or bovine fecal contamination, respectively. However, BAdV were detected in only one sample, showing a lower prevalence than BPyV in the wastewater samples analyzed. The 22 slaughterhouse samples with fecal contamination of animal origin showed negative results for the presence of HAdV. The river water samples analyzed were positive for the presence of both human and animal adenoviruses and polyomaviruses, indicating the existence of diverse sources of contamination. The identities of the viruses detected were confirmed by analyses of the amplified sequences. All BPyV isolates showed a 97% similarity in nucleotide sequences. This is the first time that PAdV5, BAdV6, and BPyV have been reported to occur in environmental samples. Human and porcine adenoviruses and human and bovine polyomaviruses are proposed as tools for evaluating the presence of viral contamination and for tracking the origin of fecal/urine contamination in environmental samples.

Development and application of a one-step low cost procedure to concentrate viruses from seawater samples

A novel and simple procedure for concentrating adenoviruses from seawater samples is described. The technique entails the adsorption of viruses to pre-flocculated skimmed milk proteins, allowing the flocs to sediment by gravity, and dissolving the separated sediment in phosphate buffer. Concentrated virus may be detected by PCR techniques following nucleic acid extraction. The method requires no specialized equipment other than that usually available in routine public health laboratories, and due to its straightforwardness it allows the processing of a larger number of water samples simultaneously. The usefulness of the method was demonstrated in concentration of virus in multiple seawater samples during a survey of adenoviruses in coastal waters.

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

ABSTRACT In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.

Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment

The Adenoviridae family comprises a wide diversity of viruses that may be excreted for long periods in feces or urine. Previous studies have suggested that the detection of human and animal adenoviruses as well as human and animal polyomaviruses by PCR could be used as an index of fecal contamination of human and animal origin. In this study, quantitative PCR assays targeting specifically porcine adenoviruses have been developed and applied to fecal and environmental samples, including pig slurries, urban sewage, slaughterhouse sewage and river water samples. To develop real-time quantitative PCR for the detection and quantitation of porcine adenoviruses, primers and a TaqMan probe targeting a 68-bp region of the porcine adenovirus hexon gene were designed to amplify specifically porcine adenovirus, and the conditions of the reaction were optimized. The assay detected 1-10 genome copies per test tube and was specific in showing no positive results when samples containing human or bovine adenoviruses were analyzed. Fecal samples contained mean concentrations of porcine adenoviruses of 10(5) GC/g while slaughterhouse wastewater samples showed mean values of 10(3) GC/ml. The assay detected porcine fecal pollution in samples that were highly diluted and had been collected at a considerable distance from the input source, such as river water. In general, the data presented here provide a quantitative tool for the analysis of porcine adenoviruses as indicators of the presence of porcine contamination in the environment, and support the detection of porcine adenoviruses by real-time quantitative PCR as a promising and valuable tool for source-tracking studies.

Development of a quantitative PCR assay for the quantitation of bovine polyomavirus as a microbial source-tracking tool

Adenoviruses and polyomaviruses are two distinct DNA viral families that are excreted in high concentrations and distributed in human and animal populations. Targeting specific virus included in these families has proved to be a promising and useful tool for tracing specifically sources of environmental contamination. In this study, a quantitative PCR assay that is specific for bovine polyomaviruses was developed and used to determine the excretion level and concentration of bovine polyomaviruses in urine and environmental samples, including urban sewage, slaughterhouse sewage, and river water. A set of primers and a TaqMan probe were designed to target a 77-bp region of the bovine polyomavirus VP1 gene, and the conditions of the reaction were optimized. A detection limit was established at 1-10 genome copies per test tube. The assay was specific and produced negative results when samples containing human or porcine fecal contamination were analyzed. This is, to our knowledge, the first description of bovine polyomaviruses excreted in bovine urine samples (mean values of 10(4) GC/l). Bovine polyomaviruses were also detected and quantified in slaughterhouse wastewater and river waters, which shows the spread of these viruses in many environmental samples containing contamination of bovine origin. The procedure described in this paper provides a quantitative source-tracking tool for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.

New methods for the concentration of viruses from urban sewage using quantitative PCR

Viruses are among the most important pathogens present in water contaminated with feces or urine and represent a serious risk to human health. Four procedures for concentrating viruses from sewage have been compared in this work, three of which were developed in the present study. Viruses were quantified using PCR techniques. According to statistical analysis and the sensitivity to detect human adenoviruses (HAdV), JC polyomaviruses (JCPyV) and noroviruses genogroup II (NoV GGII): (i) a new procedure (elution and skimmed-milk flocculation procedure (ESMP)) based on the elution of the viruses with glycine-alkaline buffer followed by organic flocculation with skimmed-milk was found to be the most efficient method when compared to (ii) ultrafiltration and glycine-alkaline elution, (iii) a lyophilization-based method and (iv) ultracentrifugation and glycine-alkaline elution. Through the analysis of replicate sewage samples, ESMP showed reproducible results with a coefficient of variation (CV) of 16% for HAdV, 12% for JCPyV and 17% for NoV GGII. Using spiked samples, the viral recoveries were estimated at 30-95% for HAdV, 55-90% for JCPyV and 45-50% for NoV GGII. ESMP was validated in a field study using twelve 24-h composite sewage samples collected in an urban sewage treatment plant in the North of Spain that reported 100% positive samples with mean values of HAdV, JCPyV and NoV GGII similar to those observed in other studies. Although all of the methods compared in this work yield consistently high values of virus detection and recovery in urban sewage, some require expensive laboratory equipment. ESMP is an effective low-cost procedure which allows a large number of samples to be processed simultaneously and is easily standardizable for its performance in a routine laboratory working in water monitoring. Moreover, in the present study, a CV was applied and proposed as a parameter to evaluate and compare the methods for detecting viruses in sewage samples.

Analysis of the evolution in the circulation of HAV and HEV in eastern Spain by testing urban sewage samples

The aim of the study was to analyse the evolution of the prevalence of HAV and HEV in the population of eastern Spain by analysing the viruses excreted in urban sewage. Raw urban sewage samples were collected and analysed during several years using RT-PCR techniques and sequencing analysis. Two limiting regions were analysed, one of them having implemented HAV vaccination programs. Acute symptomatic HEV cases were also examined. Results were compared with those from previous studies in the area using identical methodology. The percentage of positive HAV samples in urban sewage fell from 57.4% to 3.1% in 5-10 years in the two studied areas in Spain. Around 30% of the urban sewage samples were positive for HEV in the absence of agricultural sources of contamination. HEV RNA was also detected in four clinical cases of acute hepatitis. The dramatic reduction in the presence of HAV in raw urban sewage observed in eastern Spain could be most likely related to the general improvement in sanitation. However, these improvements would not have an equivalent effect on the circulation of HEV and this observation could be explained by the presence of animal reservoirs for HEV, which act as external sources of infections.