Biochar filters as an on-farm treatment to reduce pathogens when irrigating with wastewater-polluted sources.

Microbial contamination of vegetables due to irrigation with wastewater-polluted streams is a common problem around most cities in developing countries because wastewater is an available source of water and nutrients but wastewater treatment is often inadequate. On-farm treatment of polluted water is a feasible option to manage microbial risks in a multi-barrier approach. Current evidence indicates good suitability of biochar filters for microbe removal from wastewater using the hydraulic loading rate (HLR) designed for sand filters, but their suitability has not been tested under on-farm conditions. This study evaluated the combined effect of several variables on removal of microbial indicators from diluted wastewater by biochar filtration on-farm and the correlations between removal efficiency and HLR. Columns of biochar with three different effective particle diameters (d10) were fed with diluted wastewater at 1x, 6x, and 12x the design HLR and two levels of water salinity (electrical conductivity, EC). Influent and effluent samples were collected from the columns and analyzed for bacteriophages (ɸX174 and MS2), Escherichia coli, Enterococcus spp., and Saccharomyces cerevisiae. Microbe removal decreased with increasing HLR, from 2 to 4 to 1 log10 for bacteria and from 2 to 0.8 log10 for viruses, while S. cerevisiae removal was unaffected. Effective particle diameter (d10) was the main variable explaining microbe removal at 6x and 12x, while EC had no effect. Correlation analysis showed removal of 2 log10 bacteria and 1 log10 virus at 3x HLR. Thus biochar filters on-farm would not remove significant amounts of bacteria and viruses. However, the design HLR was found to be conservative. These results, and some technical and management considerations identified, can assist in the development of a scientific method for designing biochar filters for on-farm and conventional wastewater treatment.

Pathogens in crop production systems irrigated with low-quality water in Bolivia.

In dry areas, the need for irrigation to ensure agricultural production determines the use of all available water sources. However, the water sources used for irrigation are often contaminated by untreated or minimally treated wastewater. Microbial risks from reusing wastewater for vegetable irrigation can be addressed by installing environmental barriers that pathogens must cross to reach humans in the reuse system. Knowledge of pathogen flows inside the system and pathogen removal potential is the first step towards devising a risk management strategy. This study assessed microbe prevalence in farming systems in the Bolivian highlands that use wastewater-polluted sources for irrigation of lettuce. Samples of soil, lettuce and different water sources used in the farming systems were taken during one crop season and concentrations of coliphages, Escherichia coli and helminth eggs were measured. The results showed high spread of these microorganisms throughout the whole system. There was a significant correlation between microbial quality of water and of the harvested produce for several microorganisms. The microbial prevalence in protected shallow wells was found to be significantly lower than in other water sources. These findings can help formulate feasible risk management strategies in contexts where conventional technologies for microbial removal are not possible.

Fate and survival of Salmonella Typhimurium and Escherichia coli O157:H7 in repacked soil lysimeters after application of cattle slurry and human urine.

BACKGROUND: Use of cattle slurry as a fertiliser is common practice around the world. Human urine use is not as common, but owing to its fertiliser value this might change in the future. It is essential to minimise the transfer of enteric pathogens through fertilisation, with respect to both animal and public health. Therefore the objective of this research was to study the survival and transport of Salmonella Typhimurium and Escherichia coli O157:H7 in two agricultural soils when applied to soil along with either cattle slurry or human urine over a period of 180 days. RESULTS: Both Salmonella and E. coli O157:H7 were more rapidly reduced when applied together with human urine than when applied with cattle slurry. However, both pathogens persisted in low amounts at 20 and 50 cm depth in both soils throughout the whole study period. No Salmonella or E. coli O157:H7 was detected in the leachate over the 180 day study. CONCLUSION: The risk of disease transmission is higher when cattle slurry is used as fertiliser compared with human urine. However, the risk of groundwater infiltration would be low as long as water velocity through the soil is moderate. Increased knowledge of pathogen persistence in soil after fertiliser application is a valuable tool for improving risk evaluations and formulating guidelines for the use of cattle and/or human wastes in cropping soils.

Thermal treatment for pathogen inactivation as a risk mitigation strategy for safe recycling of organic waste in agriculture.

Thermal treatment at temperatures between 46.0°C and 55.0°C was evaluated as a method for sanitization of organic waste, a temperature interval less commonly investigated but important in connection with biological treatment processes. Samples of dairy cow feces inoculated with Salmonella Senftenberg W775, Enterococcus faecalis, bacteriophage ϕX174, and porcine parvovirus (PPV) were thermally treated using block thermostats at set temperatures in order to determine time-temperature regimes to achieve sufficient bacterial and viral reduction, and to model the inactivation rate. Pasteurization at 70°C in saline solution was used as a comparison in terms of bacterial and viral reduction and was proven to be effective in rapidly reducing all organisms with the exception of PPV (decimal reduction time of 1.2 h). The results presented here can be used to construct time-temperature regimes in terms of bacterial inactivation, with D-values ranging from 0.37 h at 55°C to 22.5 h at 46.0°C and 0.45 h at 55.0°C to 14.5 h at 47.5°C for Salmonella Senftenberg W775 and Enterococcus faecalis, respectively and for relevant enteric viruses based on the ϕX174 phage with decimal reduction times ranging from 1.5 h at 55°C to 16.5 h at 46°C. Hence, the study implies that considerably lower treatment temperatures than 70°C can be used to reach a sufficient inactivation of bacterial pathogens and potential process indicator organisms such as the ϕX174 phage and raises the question whether PPV is a valuable process indicator organism considering its extreme thermotolerance.

Design, Methods, and Participation in Riksmaten Young Children-A Swedish National Dietary Survey.

Background: National dietary surveys provide essential data for risk benefit assessments of foods and nutrients, for management and policy development. Physical activity measurement and biomonitoring can provide important complementary data but are less commonly included. Objectives: This study aimed to describe the study design and methods of the cross-sectional Swedish national dietary survey Riksmaten Young Children (Riksmaten småbarn), of children aged 9 mo, 18 mo, and 4 y. Participation/dropout rates for the 2 older age groups are also presented. The impact of different recruitment strategies is discussed. Methods: Children (N = 16,655) were randomly selected from the population register; invitations to guardians were sent by post and where possible, followed up by telephone. Food intake was assessed by a 2-d food diary and/or questionnaire. Height and weight were reported after measurement. Physical activity (accelerometery, 7 d) and stool, blood, and urine samples were assessed in subgroups. Results: Food consumption data were collected in 1828 children (11% of the invited; 18 mo: n = 1078, and 4 y: n = 750). Of participants also in subgroups, 71% provided physical activity data (n = 1307), 60% stool samples (n = 630), and 51% blood and/or urine samples (n = 593). The study population represented all geographic regions and types of municipalities in Sweden, but participating households had both higher education level and higher income than the target population. Only minor differences were seen in participation rates between recruitment via post and telephone compared with those through post only (12% compared with 10%). Repeated contact attempts were needed for the majority of participants (65%). Despite the low-participation rate, 99% of the participants completed the study once started. Conclusions: Although it was a challenge to recruit participants, Riksmaten Young Children provides a substantial amount of information at national level, representative in terms of sex, geography, and family structure. The underrepresentation of households with lower socioeconomic position must be considered when generalizing results.

The Virucidal Effect of the Chlorination of Water at the Initial Phase of Disinfection May Be Underestimated If Contact Time Calculations Are Used.

For the microbiological safety of drinking water, disinfection methods are used to remove or inactivate microorganisms. Chlorine and chlorine dioxide are often used as disinfectants in drinking water treatment plants (DWTPs). We investigated the effectiveness of these chemicals in inactivate echovirus 30 (E30), simian 11 rotavirus (RV SA11), and human adenovirus type 2 (HAdV2) in purified water from a DWTP. Within two minutes of contact, chlorine dioxide inactivated E30 by 4-log10, RV SA11 by 3-log10, and HAdV2 could not be detected, while chlorine reduced E30 by 3-log10, RV SA11 by 2-3log10, and HAdV2 by 3-4log10. However, viral genomes could be detected for up to 2 h using qPCR. The CT method, based on a combination of disinfectant concentration and contact time, during such a short initial phase, is problematic. The high concentrations of disinfectant needed to neutralize organic matter may have a strong immediate effect on virus viability. This may lead to the underestimation of disinfection and overdosing of disinfectants in water with organic contamination. These results are useful for the selection of disinfection systems for reuse of treated wastewater and in the risk assessment of water treatment processes using chlorine and chlorine dioxide.

Microbiological hazards associated with the use of water in the post-harvest handling and processing operations of fresh and frozen fruits, vegetables and herbs (ffFVHs). Part 1 (outbreak data analysis, literature review and stakeholder questionnaire).

The contamination of water used in post-harvest handling and processing operations of fresh and frozen fruit, vegetables and herbs (ffFVHs) is a global concern. The most relevant microbial hazards associated with this water are: Listeria monocytogenes, Salmonella spp., human pathogenic Escherichia coli and enteric viruses, which have been linked to multiple outbreaks associated with ffFVHs in the European Union (EU). Contamination (i.e. the accumulation of microbiological hazards) of the process water during post-harvest handling and processing operations is affected by several factors including: the type and contamination of the FVHs being processed, duration of the operation and transfer of microorganisms from the product to the water and vice versa, etc. For food business operators (FBOp), it is important to maintain the microbiological quality of the process water to assure the safety of ffFVHs. Good manufacturing practices (GMP) and good hygienic practices (GHP) related to a water management plan and the implementation of a water management system are critical to maintain the microbiological quality of the process water. Identified hygienic practices include technical maintenance of infrastructure, training of staff and cooling of post-harvest process water. Intervention strategies (e.g. use of water disinfection treatments and water replenishment) have been suggested to maintain the microbiological quality of process water. Chlorine-based disinfectants and peroxyacetic acid have been reported as common water disinfection treatments. However, given current practices in the EU, evidence of their efficacy under industrial conditions is only available for chlorine-based disinfectants. The use of water disinfection treatments must be undertaken following an appropriate water management strategy including validation, operational monitoring and verification. During operational monitoring, real-time information on process parameters related to the process and product, as well as the water and water disinfection treatment(s) are necessary. More specific guidance for FBOp on the validation, operational monitoring and verification is needed.

Composting for avian influenza virus elimination.

Effective sanitization is important in viral epizootic outbreaks to avoid further spread of the pathogen. This study examined thermal inactivation as a sanitizing treatment for manure inoculated with highly pathogenic avian influenza virus H7N1 and bacteriophages MS2 and 6. Rapid inactivation of highly pathogenic avian influenza virus H7N1 was achieved at both mesophilic (35°C) and thermophilic (45 and 55°C) temperatures. Similar inactivation rates were observed for bacteriophage 6, while bacteriophage MS2 proved too thermoresistant to be considered a valuable indicator organism for avian influenza virus during thermal treatments. Guidelines for treatment of litter in the event of emergency composting can be formulated based on the inactivation rates obtained in the study.

The UV Dose Used for Disinfection of Drinking Water in Sweden Inadequately Inactivates Enteric Virus with Double-Stranded Genomes.

Irradiation with ultraviolet light (UV) at 254 nm is effective in inactivating a wide range of human pathogens. In Sweden, a UV dose of 400 J/m2 is often used for the treatment of drinking water. To investigate its effect on virus inactivation, enteric viruses with different genomic organizations were irradiated with three UV doses (400, 600, and 1000 J/m2), after which their viability on cell cultures was examined. Adenovirus type 2 (double-stranded DNA), simian rotavirus 11 (double-stranded RNA), and echovirus 30 (single-stranded RNA) were suspended in tap water and pumped into a laboratory-scale Aquada 1 UV reactor. Echovirus 30 was reduced by 3.6-log10 by a UV dose of 400 J/m2. Simian rotavirus 11 and adenovirus type 2 were more UV resistant with only 1-log10 reduction at 400 J/m2 and needed 600 J/m2 for 2.9-log10 and 3.1-log10 reductions, respectively. There was no significant increase in the reduction of viral viability at higher UV doses, which may indicate the presence of UV-resistant viruses. These results show that higher UV doses than those usually used in Swedish drinking water treatment plants should be considered in combination with other barriers to disinfect the water when there is a risk of fecal contamination of the water.

Ammonia disinfection of hatchery waste for elimination of single-stranded RNA viruses.

Hatchery waste, an animal by-product of the poultry industry, needs sanitation treatment before further use as fertilizer or as a substrate in biogas or composting plants, owing to the potential presence of opportunistic pathogens, including zoonotic viruses. Effective sanitation is also important in viral epizootic outbreaks and as a routine, ensuring high hygiene standards on farms. This study examined the use of ammonia at different concentrations and temperatures to disinfect hatchery waste. Inactivation kinetics of high-pathogenic avian influenza virus H7N1 and low-pathogenic avian influenza virus H5N3, as representatives of notifiable avian viral diseases, were determined in spiked hatchery waste. Bovine parainfluenza virus type 3, feline coronavirus, and feline calicivirus were used as models for other important avian pathogens, such as Newcastle disease virus, infectious bronchitis virus, and avian hepatitis E virus. Bacteriophage MS2 was also monitored as a stable indicator. Coronavirus was the most sensitive virus, with decimal reduction (D) values of 1.2 and 0.63 h after addition of 0.5% (wt/wt) ammonia at 14 and 25°C, respectively. Under similar conditions, high-pathogenic avian influenza H7N1 was the most resistant, with D values of 3.0 and 1.4 h. MS2 was more resistant than the viruses to all treatments and proved to be a suitable indicator of viral inactivation. The results indicate that ammonia treatment of hatchery waste is efficient in inactivating enveloped and naked single-stranded RNA viruses. Based on the D values and confidence intervals obtained, guidelines for treatment were proposed, and one was successfully validated at full scale at a hatchery, with MS2 added to hatchery waste.

Bifidobacterial survival in surface water and implications for microbial source tracking.

Bifidobacteria are common in the intestines of humans and other animals. Sorbitol-fermenting Bifidobacteria (SFB) are associated with human faecal pollution. The ratio of total Bifidobacteria (TB) to SFB has been proposed to be a means for determining the source (human or animal) of faecal pollution. In this study, SFB were detected in all sewage samples (n = 39) and in 42% of samples of animal origin (n = 24): pig (5/6 positive), poultry (4/8), horse (1/4), and cow (0/6). The TB:SFB ratio was significantly higher in animal samples than in sewage samples (p < 0.01), with the exception of stored poultry manure, which had ratios (1.4-11) similar to the sewage samples (1-17). The lowest ratio in the other animal samples was 40 (pig). The TB:SFB ratio in sewage remained stable over time in surface water, and it may be a useful screening tool to determine the source of faecal pollution in surface water. Bifidobacteria died off in a log linear manner at 4 and 22 degrees C, with rates of disappearance of 0.16 and 0.58 log(days-1), respectively. Faecal coliforms (FC) disappeared more slowly (0.09 and 0.43 log(days-1), respectively). The FC:SFB ratio proved to be an inadequate tool for determining the age of sewage pollution.

Assessing microbial contamination and antibiotic resistant bacteria using zebra mussels (Dreissena polymorpha).

Aquatic pollution with faecal bacteria and subsequent consumption of contaminated water or food is a worldwide issue that causes severe health effects (e.g. meningitis, salmonellosis, dysentery). In addition, the excessive use of antibiotics in animal husbandry and human medicine has enhanced the selective pressure on pathogenic bacteria, further increasing human health risks and detrimental effects on natural microbial communities. This urges the need to monitor faecal contamination using a time-integrated approach, as grab water samples can miss pathogen peaks. We tested the ability of zebra mussels (Dreissena polymorpha) to take up and depurate faecal indicator bacteria such as Escherichia coli and intestinal enterococci. Furthermore, we quantified the frequency of antibiotic resistant bacteria in water and mussels both in controlled laboratory tests and under in situ conditions downstream of a sewage treatment plant (STP). Laboratory results show that bacterial indicators in mussels were 132 times higher than their concentration in water, and that mussels retained bacteria up to 2 days after pulse exposure. Field results show decreasing bacterial concentrations in both water and mussels downstream the STP, with maximum E. coli concentrations ranging 173-9 cfu mL-1 in water and 2970-330 cfu g-1 in mussels. Similarly, enterococci ranged 59-4 cfu mL-1 and 1450-240 cfu g-1 in water and mussels, respectively. High proportions of antibiotic resistant E. coli were found in mussels (72%) and water (65%), and slightly lower proportion of resistant enterococci was found in mussels (47%) and in water (34%). Moreover, 33% of the bacteria isolated from mussels were resistant to multiple antibiotics, which emphasizes that resistance is a common feature in surface waters and highlights the need for safe water management. Our results show that zebra mussels provide an efficient, time-integrating tool for quantifying faecal indicators, including resistant and multidrug resistant bacteria.

Evaluating Dissemination Mechanisms of Antibiotic-Resistant Bacteria in Rural Environments in China by Using CTX-M-Producing Escherichia coli as an Indicator.

It is becoming increasingly recognized that the environment plays an important role both in the emergence and in dissemination of antibiotic-resistant bacteria (ARB), Mechanisms and factors facilitating this development are, however, not yet well understood. The high detection rate of CTX-M genes in environmental sources provides an opportunity to explore this issue. In this study, 88 CTX-M-producing Escherichia coli were isolated from 30 pig feces samples from 30 pig farms and 201 environmental samples. CTX-M-producing E. coli was detected with the following frequencies in the different types of samples: pig feces, 73%; river water, 64%; river sediment, 52%; wastewater, 31%; drinking water, 23%; outlet sediment, 21%; soil, 17%; and vegetables, 4.4%. Dissemination of CTX-M-producing E. coli to different environmental matrices was evaluated by analyzing the genetic relatedness of isolates from different environmental sources, and putative transmission routes through bird feces, pig feces, drinking water, river sediment, river water, and wastewater were hypothesized. Dissemination through these routes is likely facilitated by anthropogenic activities and environmental factors. Wild birds as potential vectors for dissemination of CTX-M-producing E. coli have the capacity to spread ARB across long distances. Regional dissemination between different environmental matrices of CTX-M-producing E. coli increases the exposure risk of humans and animals in the area.

Dissemination of extended-spectrum ß-lactamase-producing Escherichia coli carrying mcr-1 among multiple environmental sources in rural China and associated risk to human health.

Antibiotic resistance among gram-negative bacteria is increasingly becoming a problem of global concern. Particularly problematic is the emergence of resistance to last-resort antibiotics such as carbapenems and colistin. The increasing number of reports on the plasmid-mediated colistin resistance gene mcr-1 in isolates worldwide is raising concerns for the future usefulness of this class of antibiotics. Dissemination of mcr-1 is believed to have originated mainly from animal breeding, however, the role of the environment as a transmission source is not yet fully understood. In the current study, 89 extended-spectrum ß-lactamase-producing Escherichia coli isolated from 231 samples from different environmental sources in 12 villages in a rural area of Shandong, China, were screened for mcr-1. 17 (19.1%) mcr-1-positive isolates were found from different environmental sources, aggregated in 6 villages. Plasmids of three different Inc-groups carrying mcr-1 were confirmed, indicating that the widespread geographical distribution of mcr-1 in the local area is due to a number of different plasmids. Additionally, almost a third (29.4%) of the isolates carried virulence factors associated to intestinal pathogenic E. coli. These results illustrate the high complexity of the transmission patterns of mcr-1 among different environmental matrices on a local scale and the potential for the environment to facilitate dissemination and emergence of antibiotic-resistant and virulent strains of bacteria.

Characterization of Clinically Relevant Strains of Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae Occurring in Environmental Sources in a Rural Area of China by Using Whole-Genome Sequencing.

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, and a common cause of healthcare-associated infections such as pneumonia, septicemia, and urinary tract infection. The purpose of this study was to survey the occurrence of and characterize K. pneumoniae in different environmental sources in a rural area of Shandong province, China. Two hundred and thirty-one samples from different environmental sources in 12 villages were screened for extended-spectrum ß-lactamase-(ESBL)-producing K. pneumoniae, and 14 (6%) samples were positive. All isolates were multidrug-resistant and a few of them belonged to clinically relevant strains which are known to cause hospital outbreaks worldwide. Serotypes, virulence genes, serum survival, and phagocytosis survival were analyzed and the results showed the presence of virulence factors associated with highly virulent clones and a high degree of phagocytosis survivability, indicating the potential virulence of these isolates. These results emphasize the need for further studies designed to elucidate the role of the environment in transmission and dissemination of ESBL-producing K. pneumoniae and the potential risk posed to human and environmental health.

Presence of antibiotic residues in various environmental compartments of Shandong province in eastern China: Its potential for resistance development and ecological and human risk.

OBJECTIVES: To investigate the occurrence of antibiotic residues in different types of environmental samples including water samples in rural Shandong province, China. Further, to characterize the potential ecological risk for development of antibiotic resistance in the environment, and the potential direct human health risk of exposure to antibiotics via drinking water and vegetables. METHODS: Environmental samples (n = 214) (river water, waste water, drinking water, sediments, manure, soil and edible parts of vegetables) were collected in twelve villages in Shandong province in eastern China. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to determine the concentration of antibiotic residues. The ratio of the measured environmental concentrations (MEC) to the predicted no-effect concentrations (PNEC) was used to evaluate the ecological risk (risk quotient, RQ) for development of antibiotic resistance. The potential risks to human health through exposure to antibiotics in drinking water were assessed by comparing measured environmental concentrations (MEC) and predicted no-effect concentration in drinking water (PNECDW), and in vegetables by comparing estimated daily intake (EDI) to ADI. RESULTS: Sulfapyridine, sulfamethoxazole, ciprofloxacin, enrofloxacin, levofloxacin, norfloxacin, chloramphenicol, florfenicol, doxycycline, and metronidazole were detected at concentrations ranging between 0.3 and 3.9 ng/L in river water, 1.3 and 12.5 ng/L in waste water, 0.5 and 21.4 ng/L in drinking water, 0.31 and 1.21 µg/kg in river sediment, 0.82 and 1.91 µg/kg in pig manure, 0.1 and 11.68 µg/kg in outlet sediment, 0.5 and 2.5 µg/kg in soil, and 6.3 and 27.2 µg/kg in vegetables. The RQs for resistance development were >1 for enrofloxacin, levofloxacin, and ranged between 0.1 and 1 for ciprofloxacin. MECs/PNECDW ratios were <1 from exposure to antibiotics through drinking water for both adults and children. EDI/ADI ratios were <0.1 from exposure to antibiotics by vegetable consumption. CONCLUSIONS: Antibiotic pollutants were ubiquitous in various environmental compartments of Shandong province of China. Risk estimates indicated a potential for the measured levels of enrofloxacin, levofloxacin and ciprofloxacin in waste water to pose an ecological risk for resistance selection, and further studies are needed to validate this finding. The investigated antibiotics did not appear to pose an appreciable direct human health risk from environmental exposure through drinking water or vegetables consumption. However, they might still pose a risk for resistance development.

Study protocol for One Health data collections, analyses and intervention of the Sino-Swedish integrated multisectoral partnership for antibiotic resistance containment (IMPACT).

INTRODUCTION: To effectively minimise the emergence and dissemination of antibiotic resistant bacteria, a holistic One Health approach is called for. The Sino-Swedish Integrated Multisectoral Partnership for Antibiotic Resistance Containment is a cross-sectoral and integrated project on antibiotic resistance, conducted in Shandong Province in China. This paper outlines the overall study protocol for the project. To our knowledge, this is the first research programme aiming to take a true holistic approach across multiple sectors simultaneously in China, and the first to incorporate both antibiotic use and infection prevention and control in addition to antibiotic resistance patterns. The project aims to address gaps in current knowledge and seeks to improve the situation through a system-wide intervention. By using a One Health approach we can address important research questions that individual discipline investigations are unable to. The results obtained should thus more closely reflect the world in which human health, animal health and the environment are inextricably and intimately interlinked. METHODS AND ANALYSIS: Both quantitative and qualitative studies are included for households from 12 villages, their surrounding environment and a tertiary care hospital in a nearby town. The studies include analyses of antibiotic consumption for humans and pigs; qualitative and quantitative data on perceptions, knowledge and attitudes; faecal carriage of extended spectrum ß-lactamase and carbapenemase-producing Enterobacteriaceae from pigs and humans, and occurrence in household drinking water, surface water, waste water and clinical bacterial isolates from the hospital. Carriage of methicillin-resistant Staphylococcus aureus in humans, household pigs and clinical bacterial isolates is also investigated. Furthermore, potential inter-relationships between these sources are analysed. A multifaceted One Health intervention is designed and implemented in 6 of the 12 villages. Repeated and continuous data collections take place over 2 years, where the repeated data collection is performed after 1 year of intervention. Comparisons are made between intervention and control villages, before and after the intervention. ETHICS: Ethics approval was obtained from the first Affiliated Hospital, College of Medicine, Zhejiang University, China, reference number 2015#185 and 2015#283.

Inactivation of Viruses and Bacteriophages as Models for Swine Hepatitis E Virus in Food Matrices.

Hepatitis E virus has been recognised as a food-borne virus hazard in pork products, due to its zoonotic properties. This risk can be reduced by adequate treatment of the food to inactivate food-borne viruses. We used a spectrum of viruses and bacteriophages to evaluate the effect of three food treatments: high pressure processing (HPP), lactic acid (LA) and intense light pulse (ILP) treatments. On swine liver at 400 MPa for 10 min, HPP gave log10 reductions of ≥4.2, ≥5.0 and 3.4 for feline calicivirus (FCV) 2280, FCV wildtype (wt) and murine norovirus 1 (MNV 1), respectively. Escherichia coli coliphage ϕX174 displayed a lower reduction of 1.1, while Escherichia coli coliphage MS2 was unaffected. For ham at 600 MPa, the corresponding reductions were 4.1, 4.4, 2.9, 1.7 and 1.3 log10. LA treatment at 2.2 M gave log10 reductions in the viral spectrum of 0.29-2.1 for swine liver and 0.87-3.1 for ham, with ϕX174 and MNV 1, respectively, as the most stable microorganisms. The ILP treatment gave log10 reductions of 1.6-2.8 for swine liver, 0.97-2.2 for ham and 1.3-2.3 for sausage, at 15-60 J cm-2, with MS2 as the most stable microorganism. The HPP treatment gave significantly (p < 0.05) greater virus reduction on swine liver than ham for the viruses at equivalent pressure/time combinations. For ILP treatment, reductions on swine liver were significantly (p < 0.05) greater than on ham for all microorganisms. The results presented here could be used in assessments of different strategies to protect consumers against virus contamination and in advice to food producers. Conservative model indicators for the pathogenic viruses could be suggested.

A theoretical approach to using faecal indicator data to model norovirus concentration in surface water for QMRA: Glomma River, Norway.

Monitoring of faecal indicator organisms, such as Escherichia coli, in environmental and drinking waters is inadequate for the protection public health, primarily due to the poor relationship between E. coli and the occurrence of human pathogens, especially viruses, in environmental samples. Nevertheless, measurements of faecal indicator organisms within the risk based approach, can provide valuable information related to the magnitude and variability of faecal contamination, and hence provide insight into the expected level of potential pathogen contamination. In this study, a modelling approach is presented that estimates the concentration of norovirus in surface water relying on indicator monitoring data, combined with specific assumptions regarding the source of faecal contamination. The model is applied to a case study on drinking water treatment intake from the Glomma River in Norway. Norovirus concentrations were estimated in two sewage sources discharging into the river upstream of the drinking water offtake, and at the source water intake itself. The characteristics of the assumed source of faecal contamination, including the norovirus prevalence in the community, the size of the contributing population and the relative treatment efficacy for indicators and pathogens in the sewage treatment plant, influenced the magnitude and variability in the estimated norovirus concentration in surface waters. The modelling exercise presented is not intended to replace pathogen enumeration from environmental samples, but rather is proposed as a complement to better understand the sources and drivers of viruses in surface waters. The approach has the potential to inform sampling regimes by identifying when the best time would be to collect environmental samples; fill in the gaps between sparse datasets; and potentially extrapolate existing datasets in order to model rarer events such as an outbreak in the contributing population. In addition, and perhaps most universally, in the absence of pathogen data, this approach can be used as a first step to predict the source water pathogen concentration under different contamination scenarios for the purpose of quantifying microbial risks.

Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II.

The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.