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Objectives: Spread of pig slurry as an organic fertilizer is commonly used in Danish agriculture. The slurry is spread untreated so pathogens able to survive in slurry tanks will be widely distributed in the environment. The objective of this study was to examine if hepatitis E virus (HEV), which is known to be excreted in faeces from pigs, will be transported through the soil and into the drainage system of a field due to precipitation or will be retained in the soil matrix. Water from the drainage system is not treated before it is discharged into larger water reservoirs (lake, fjords, streams), and hence could present a risk for virus transmission to wildlife and shellfish. We tested the presence of HEV in water drained from a test field where slurry from a Danish pig farm had been applied and in mussels from different regions in Denmark with fields in close proximity.Methods: Slurry from a Danish pig farm was spread on a tile-drained field of loamy soil. Water that arrived at the drainage system located 1 m below surface was collected over a time period of 4 month. Samples were collected on a weekly basis and when water flow in the drainage system exceeded a certain threshold (an event). In addition, samples of water collected from wells located along the field and groundwater. Archived mussels from different regions in Denmark were included in the study. Virus was concentrated from water using Poly Ethylene Glycol precipitation and virus from the digestive tissue of the mussels was extracted sing proteinase K treatment. Subsequently, viral genomic RNA, from both water and mussels, was purified using the NucliSENS miniMAG system and detection and quantification of HEV and mengovirus (used as process control) were performed by real time RT-PCR.Results: Water samples representing a total of 14 events were tested. HEV was detected in the first event following spread of pig slurry. In agreement with this, the weekly sample of this period also tested positive. HEV was not found in any of the subsequent water samples. Of the 70 blue mussel samples, that mainly originated from fjords, none tested positive.Conclusion: HEV is regarded as a zoonotic virus with pigs as the primary reservoir. The pathway to humans and other mammals is unclear. Here we show that under Danish conditions, spread of pig slurry can cause viral contamination of water reservoirs, making HEV accessible to the population and wildlife. This indicates a possible route of HEV transmission from pigs to other reservoirs. We also show that retention in soil matrix is at a minimum as HEV was detected at first rainfall after application of pig slurry. The viability of the viruses found in this study is still unclear since HEV cannot be cultivated in cells. We did not find any HEV positive mussel samples indicating that the release of HEV from fields is not a concern for shellfish production.

At least 11 linked outbreaks of gastroenteritis with a total of 260 cases have occurred in Denmark in mid January 2010. Investigations showed that the outbreaks were caused by norovirus of several genotypes and by enterotoxigenic Escherichia coli. Lettuce of the lollo bionda type grown in France was found to be the vehicle.

The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low-molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the utilization of purines as nitrogen source in B. subtilis . Based on growth studies of strains with knockout mutations in genes, complemented with enzyme analysis, we could ascribe functions to 14 genes encoding enzymes or proteins of the purine degradation pathway. A functional xanthine dehydrogenase requires expression of five genes ( pucA, pucB, pucC, pucD , and pucE ). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK . Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded by pucF . In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285° on the chromosome and are contained in six transcription units, which are expressed when cells are grown with glutamate as the nitrogen source (limiting conditions), but not when grown on glutamate plus ammonia (excess conditions). Our data suggest that the 14 genes and the gde gene, encoding guanine deaminase, constitute a regulon controlled by the pucR gene product. Allantoic acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the pucABCDE unit was decreased 16-fold, while expression of pucR was decreased 4-fold in the presence of allantoin. We have identified genes of the purine degradation pathway in B. subtilis and showed that their expression is subject to both general nitrogen catabolite control and pathway-specific control.

Viral pathogens in irrigation water are a major threat to public health due to their possibility to cause disease in humans. When using reclaimed water for irrigation it is therefore important to make sure that the water is free from pathogens which can contaminate the crops. In this study we are therefore using metagenomics sequencing with the aim to map the viriome in different water sources. In addition we investigate the possibility to use Human Adenovirus (HAdV) or JC Polyomavirus (JCPyV) as indicator for human fecal contamination. Water has been sampled monthly throughout the treatment process from two urban waste water treatment plants in Copenhagen. All samples are investigated for their viral content and the presence of pathogens by metagenomic sequencing and analyzed specifically for HAdV, JCPyV, norovirus GI and GII (NoV GI and GII) using quantitative (q)PCR. Preliminary qPCR results showed that the average concentration for HAdV within a sample is higher than the average concentration of NoV GI and GII. HAdV could therefore be a good indicator for human fecal contamination in water. The initial analysis of the metagenomic data identifies viruses in all water sources. However, the number of identified pathogenic viral species decreases with treatment of the waste water. Further bioinformatic analyses will investigate the seasonal variations of viral composition within a sample as well as the effect of the treatment system. Updated qPCR and metagenomics data will be presented.

Five methods that detect human enteric virus contamination in lettuce were compared. To mimic multiple contaminations as observed after sewage contamination, artificial contamination was with human calicivirus and poliovirus and animal calici-virus strains at different concentrations. Nucleic acid extractions were done at the same time in the same laboratory to reduce assay-to-assay variability. Results showed that the two critical steps are the washing step and removal of inhibitors. The more reliable methods (sensitivity, simplicity, low cost) included an elution/concentration step and a commercial kit. Such development of sensitive methods for viral detection in foods other than shellfish is important to improve food safety.

Urban cloudburst management may include intentional temporary storage of flood water in parks or other recreational areas. In cities with combined sewers this will expose the population to wastewater and increase the risk of diarrhoeal disease. We present an approach to estimate the risk of diarrhoeal disease after urban flooding of a green area by combining hydraulic modelling, an experimental flood simulation and quantitative microbial risk assessment (QMRA). The method allows the cloudburst manager to reduce unacceptable disease risks during the planning of cloudburst management or on existing facilities.

The amount of animal manure used in modern agriculture is increasing due to the increase in global animal production. Pig slurry is known to contain zoonotic bacteria such as E. coli, Salmonella spp. and Campylobacter spp., and viruses such as hepatitis E virus and group A rotavirus. Coliform bacteria, present in manure, have previously been shown to leach into tile drains. This poses a potential threat to aquatic environments and may also influence the quality of drinking water. As knowledge is especially scarce about the fate of viruses when applied to fields in natural settings, this project sets out to investigate the leaching potential of six different microorganisms: E. coli and Enterococcus spp. (detected by colony assay), somatic coliphages (using plaque assays), and hepatitis E virus, porcine circovirus type 2, and group A rotavirus (by real-time polymerase chain reaction). All six microorganisms leached through the soil entering the tile drains situated at 1-m depth the first day following pig slurry application. The leaching pattern of group A rotavirus differed substantially from the pattern for somatic coliphages, which are otherwise used as indicators for virus contamination. Furthermore, group A rotavirus was detected in monitoring wells at 3.5-m depth up to 2 months after pig slurry application. The detection of viral genomic material in drainage water and shallow groundwater signifies a potential hazard to human health that needs to be investigated further, as water reservoirs used for recreational use and drinking water are potentially contaminated with zoonotic pathogens.