Detection of animal and human enteric viruses in water from the Assomption River and its tributaries

Animal enteroviruses, reoviruses, and human enteric viruses were detected in water samples (20 L) from a major river system, the Assomption River in the province of Quebec. Animal enteroviruses, probably of porcine origin (this region is a major producer of pork), were isolated on porcine cell cultures and were found in 29 to 60% of water samples from the different sites on the river and in 19 to 48% of the water samples from the tributaries. The average concentration of these animal enteroviruses in water from the Assomption River was 2 to 7 mpniu/L (most probable number of infectious units per litre), and that from the tributaries varied from 3 to 24 mpniu/L. Reoviruses were detected in infected cell cultures by an enzyme-linked immunosorbent assay. Their origin is probably avian (broiler chicken farms) or human (untreated domestic waste waters) and they were detected in 19 to 52% of the water samples from the Assomption River at an average concentration of 3 to 12 mpniu/L. In water samples from the tributaries, 5 to 71% of the samples were positive at an average concentration of 5 to 24 mpniu/L. Human enteric viruses were detected in MA-104 cells by an immunoperoxidase assay using human immune serum globulin. They were detected in 13 to 72% of water samples from the Assomption River and 14 to 71% of the water samples from the tributaries. The average concentration of these human enteric viruses in Assomption River water varied from 1 to 12 and from 2 to 145 mpniu/L in water samples from the tributaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between environmental factors, bacterial indicators, and the occurrence of enteric viruses in estuarine sediments

Current standards for evaluation of the public health safety of recreational and shellfish-harvesting waters are based upon bacteriological analysis, but do not include an evaluation of the number of viruses. The objective of this study was to determine the occurrence of enteric viruses in estuarine sediments and to find a relationship, if any, between the presence of viruses in seawater or sediment or both and various biological and physicochemical characteristics of the environment. Viruses were found in greater numbers in sediment than in overlying seawater on a volume basis. Several types of enteroviruses were isolated: coxsackievirus types A16, B1, and B5, echovirus type 1, and poliovirus type 2. On several occasions, viruses were isolated from sediments when overlying seawaters met bacteriological water quality standards for recreational use. Statistical analysis of the relationship between viruses in seawater or in sediment and other variables measured yielded only one significant association: the number of viruses in sediment was found to be positively correlated with the number of fecal coliforms in sediment. No other physical, chemical, or biological characteristic of seawater or sediment that was measured showed statistically significant association with viral numbers. No correlation was found between bacterial indicators and virus in the overlying waters. The data indicated that evaluation of the presence of bacteria and viruses in sediment may provide additional insight into long-term water quality conditions and that indicator bacteria in water are not reflective of the concentration of enteric viruses in marine waters.     

Occurrence of bacterial indicators and bacteriophages infecting enteric bacteria in groundwater in different geographical areas

AIMS: The aim of this research was to determine the suitability of coliphages (bacteriophages) for assessing the microbial quality of groundwater. METHODS AND RESULTS: The number of several bacterial indicators (faecal coliforms, Escherichia coli, enterococci and spores of sulfite-reducing clostridia) and bacteriophages (somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis) were determined in groundwater of aquifers in various geographical areas. Results show that the relative abundance, determined as percentages of positive detections, of the bacterial indicators and bacteriophages varies depending on the aquifer. CONCLUSIONS: A single bacterial indicator may not be enough to assess microbiological quality in certain aquifers. One bacterial indicator and a bacteriophage parameter provide more information than two bacterial indicators. SIGNIFICANCE AND IMPACT OF THE STUDY: Coliphages (CPH) provide different information from that provided by bacterial indicators on the microbial quality of groundwater in different geographical areas. Easy, fast and inexpensive methods for the detection of CPH are feasible in both industrialized and developing countries.     

Disinfection of human enteric viruses in water by copper and silver in combination with low levels of chlorine.

The efficacy of copper and silver ions, in combination with low levels of free chlorine (FC), was evaluated for the disinfection of hepatitis A virus (HAV), human rotavirus (HRV), human adenovirus, and poliovirus (PV) in water. HAV and HRV showed little inactivation in all conditions. PV showed more than a 4 log10 titer reduction in the presence of copper and silver combined with 0.5 mg of FC per liter or in the presence of 1 mg of FC per liter alone. Human adenovirus persisted longer than PV with the same treatments, although it persisted significantly less than HRV or HAV. The addition of 700 micrograms of copper and 70 micrograms of silver per liter did not enhance the inactivation rates after the exposure to 0.5 or 0.2 mg of FC per liter, although on some occasions it produced a level of inactivation similar to that induced by a higher dose of FC alone. Virus aggregates were observed in the presence of copper and silver ions, although not in the presence of FC alone. Our data indicate that the use of copper and silver ions in water systems may not provide a reliable alternative to high levels of FC for the disinfection of viral pathogens. Gene probe-based procedures were not adequate to monitor the presence of infectious HAV after disinfection. PV does not appear to be an adequate model viral strain to be used in disinfection studies. Bacteroides fragilis bacteriophages were consistently more resistant to disinfection than PV, suggesting that they would be more suitable indicators, although they survived significantly less than HAV or HRV.     

Fate of bacterial indicators,viruses and protozoan parasites in a wastewater multi-component treatment system

The extent of reduction in selected microrganisms was tested at a multi-component wastewater treatment plant that treats sewage for a potential re-use in agriculture. The aim of the investigation was to evaluate possible reciprocal correlation among the different microrganisms and to compare the removal of two encysted pathogenic protozoa with that of microbial indicators, Clostridium perfringens spores, enteroviruses and bacteriophages. Samples collected included the raw wastewater, the chlorinated effluent and the effluent after an ultraviolet light treatment. All of the raw sewage samples were positive for Cryptosporidium oocysts and Giardia cysts, as well as for the other microorganisms tested but the bacteriophage B40-8. The data obtained confirm the removal efficiency of the entire process for indicator bacteria but also show the low and variable removal efficiency for the other microbial parameters, such as Giardia and Cryptosporidium, enteroviruses and Clostridium perfringens spores. Reciprocal correlation between Cryptosporidium and Giardia (oo)cysts and the other microbial groups was not demonstrated. The results confirm the resistance of Clostridium perfringens spores, enteroviruses and protozoa to chlorination and demonstrate the relative persistence of these organisms in the effluents even during the ultraviolet light treatment. The yields also emphasise the influence of the analytical method for the determination of protozoan parasites.     

Detection of noroviruses in tap water in Japan by means of a new method for concentrating enteric viruses in large volumes of freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H(2)SO(4) (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Assessment of the stability of human viruses and coliphage in groundwater by PCR and infectivity methods

Aim: To investigate the potential health hazard from infectious viruses where coliphages, or viruses by polymerase chain reaction (PCR), have been detected in groundwater. Two aspects were investigated: the relationship between infectivity and detection by PCR and the stability of coliphage compared to human viruses. Methods and Results: Virus decay (1 year) and detection (2 years) studies were undertaken on groundwater at 12°C. The order of virus stability from most to least stable in groundwater, based on first‐order inactivation, was: coliphage ΦX174 (0·5 d^?1) > adenovirus 2 > coliphage PRD1 > poliovirus 3 > coxsackie virus B1 (0·13 d^?1). The order for PCR results was: norovirus genotype II > adenovirus > norovirus genotype I > enterovirus. Conclusions: Enterovirus and adenovirus detection by PCR and the duration of infectivity in groundwater followed similar trends over the time period studied. Adenovirus might be a better method for assessing groundwater contamination than using enterovirus; norovirus detection would provide information on a significant human health hazard. Bacteriophage is a good alternative indicator. Significance and Impact of the Study: PCR is a useful tool for identifying the health hazard from faecal contamination in groundwater where conditions are conducive to the survival of viruses and their nucleic acid.     

Correlation of quantitative PCR for a poultry-specific brevibacterium marker gene with bacterial and chemical indicators of water pollution in a watershed impacted by land application of poultry litter

The impact of fecal contamination from human and agricultural animal waste on water quality is a major public health concern. Identification of the dominant source(s) of fecal pollution in a watershed is necessary for assessing the safety of recreational water and protecting water resources. A field study was conducted using quantitative PCR (qPCR) for the 16S rRNA gene of Brevibacterium sp. LA35 to track feces-contaminated poultry litter in environmental samples. Based on sensitivity and specificity characteristics of the qPCR method, the Bayesian conditional probability that detection of the LA35 marker gene in a water sample represented a true-positive result was 93%. The marker's covariance with fecal indicator bacteria (FIB) and metals associated with poultry litter was also assessed in litter, runoff, surface water, and groundwater samples. LA35 was detected in water and soil samples collected throughout the watershed, and its concentration covaried with concentrations of Escherichia coli, enterococci, As, Cu, P, and Zn. Significantly greater concentrations of FIB, As, Cu, P, and Zn were observed in edge-of-field runoff samples in which LA35 was detected, compared to samples in which it was not detected. Furthermore, As, Cu, P, and Zn concentrations covaried in environmental samples in which LA35 was detected and typically did not in samples in which the marker gene was not detected. The covariance of the poultry-specific LA35 marker gene with these known contaminants from poultry feces provides further evidence that it is a useful tool for assessing the impact of poultry-derived fecal pollution in environmental waters.     

Detection and molecular characterization of enteric viruses in environmental samples in Monastir,Tunisia between January 2003 and April 2007

Aims: A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. Methods and Results: A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). Conclusion: Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. Significance and Impact of the Study: Our results address the potential health risks associated with transmission of human enteric viruses through water‐related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.     

Detection and quantitation of human enteric viruses in waste waters: increased sensitivity using a human immune serum globulin--immunoperoxidase assay on MA-104 cells

This study demonstrates that the most sensitive method for the detection and quantitation of cultivable human enteric viruses in water samples after repassage in the MA-104 cell line is the detection of infected cells by the human immune serum globulin--immunoperoxidase (HISG-IP) method recently described by the authors. This immunoperoxidase method is up to 50 times more sensitive than a liquid overlay assay by cytopathic effect in BGM cells. The viral content of waste waters was evaluated with this new methodology. By this method the average viral content of raw sewage (RS) was 900 mpniu/L (most probable number of infectious units per litre), 1056 mpniu/L in primary effluent (PE), and 106 mpniu/L in secondary effluent (SE). With a cytopathic effect assay on BGM cells, values of 85 (RS), 56 (PE), and 2 (SE) mpniu/L were observed, a striking underestimation of the viral content of secondary effluents.     

Evaluation of Adenovirus and E. coli as indicators for human enteric viruses presence in mussels produced in La Spezia Gulf (Italy)

Aims: The purpose of this work was to verify whether E. coli is a good indicator of viral contamination in mussels and Adenovirus could represent a better alternative as indicator organism of viral presence to guarantee consumer health protection. Methods and Results: Eighty samples of mussels from La Spezia Gulf were analysed for E. coli, Salmonella, Adenovirus, Norovirus and hepatitis A virus with cultural and biomolecular tests. The results of bacterial parameters showed E. coli within the law’s limits and the absence of Salmonella. Twelve samples were positive for Adenovirus presence, one for Norovirus genogroup II and two for hepatitis A virus. None of these positive mussels was found to be contaminated with more than one virus at the same time. Conclusion: This study showed that there was not a direct correlation between the presence of human pathogenic viruses and bacterial indicators. Significance and Impact of the Study: Both E. coli and Adenovirus cannot be considered valid substitutes for the direct research of human pathogenic viruses in mussels. To improve consumer health protection, the European Commission will provide standardized methods for Norovirus and hepatitis A virus detection as soon as possible.     

Concentration of Giardia lamblia cysts, Legionella pneumophila, Clostridium perfringens, human enteric viruses, and coliphages from large volumes of drinking water, using a single filtration

Poliovirus, coliphages, Giardia lamblia cysts, Clostridium perfringens spores, and Legionella pneumophila were concentrated simultaneously in a single pass by sequential filtration of large volumes of drinking water through 3- and 1-micron wound electronegative fiberglass cartridge filters (25.4 cm). Filtration was performed under acidic conditions (pH 3.5) in the presence of 0.001 M aluminum chloride to enhance adsorption. Elution of all the microorganisms entrapped or adsorbed to the filters was obtained by a slow backwash elution with a 1.5% beef extract solution, pH 9.75, containing 0.5% Tween 80. Tween 80 was shown to enhance recovery of the bacteriophages, bacteria, and parasites. Giardia cysts were efficiently eluted (71%) and could be reconcentrated by low-speed centrifugation and purified by sucrose density gradient flotation at a final recovery of 52%. Legionella pneumophila cells were eluted at 64% and were further concentrated by low-speed centrifugation at an overall recovery of 55%. C. perfringens spores and coliphages were eluted at efficiencies of 82 and 86%, respectively, and reconcentrated with minimal loss by a detergent - protein flotation method. Poliovirus was eluted at 93% and reconcentrated at 78% efficiency by organic flocculation.     

环境水体中肠道感染菌群与几种典型致病菌的QPCR检测及相关性分析

利用定量PCR(QPCR)方法,对环境水体中的肠道感染菌群及几种致病菌进行4个月的连续检测。结果表明:在污染严重的水体中,埃希氏大肠菌(E.coli)是水中肠道感染菌群的主体,其强度接近于用通用引物检测出的肠道感染菌群强度。当肠道感染菌群拷贝数超过10⁴copies·100 mL^-1时,沙门氏菌(S.typhrmurium)和志贺氏痢疾杆菌(Shigella flexneri)也持续被检出,可以认为是水体病原污染的标志之一;在轻度污染和清洁水体中,当肠道感染菌群拷贝数低于10⁴copies·100 mL^-1时,通过通用引物检测到的肠道感染菌群主要是上述3种病原细菌以外的细菌,且3种病原细菌的出现频度和强度与肠道感染菌群之间没有明显的相关性。     

运用基因芯片技术检测牛、山羊、猪和鸡源性成分

本研究通过对脊椎动物分子标记基因进行序列分析,最终选择线粒体DNA(mtDNA)16S rRNA基因为目标基因,利用一对通用引物,在该引物扩增区间设计了4条特异性基因芯片检测探针及2条质控探针用于对牛、山羊、猪、鸡等4种动物源性成分进行检测。通过对PCR扩增体系及杂交体系的优化,该检测方法能实现对上述4种动物源性成分同时进行快速、准确地检测,具有很好的特异性,灵敏度均达到1pg,最终建立了这4种动物源性基因芯片检测方法。该基因芯片检测技术将为我国进出口饲料中的动物源性成分的鉴别提供新的检测方法和技术支持。     

The roles of enteric bacterial sialidase,sialateO-acetyl esterase and glycosulfatase in the degradation of human colonic mucin

Sialidase activity in normal faecal extracts showed a preference for mucin-related glycoprotein and oligosaccharide substrates, but the presence of two or moreO-acetyl esters at positions C₇–C₉ on the sialic acids retarded the rate of hydrolysis. A specific sialateO-acetyl esterase was detected with a lower total activity relative to sialidase with mucin substrates and having a pH optimum of 7.8 and aK _M of approximately 1mm sialateO-acetyl ester. A specific glycosulfatase activity was found in faecal extracts using the substrate lactit-[³H]ol 6-O-sulfate with a pH optimum of pH 5.0 and aK _M of approximately 1mm.Faecal extracts from ulcerative colitis (UC) patients had higher sialateO-acetyl esterase and glycosulfatase activity, while mucin sialidase activity was unchanged.Metabolically labelled mucin isolated from UC patients contained less sulfate and had lower sialic acidO-acetylation compared with normal mucin. Colonic mucin was degraded more efficiently by faecal extracts from UC patients compared with normal extracts. The UC mucin was degraded more rapidly than the normal mucin by faecal enzyme extracts from both normal and UC subjects. Abbreviations: UC, ulcerative colitis; BSM, bovine submandibular gland mucin; PMSF, phenylmethylsulfonyl-fluoride. Sialic acids are abbreviated according to Schauer [37].