1. Microorganisms in pig slurry leach through agricultural soil to field drains Anita Forslund 1, Jesper S. Krog 2, Anna Charlotte Schultz 3, Lars E. Larsen 2, Jeanne Kjaer 4, Preben Olsen 5, Anders Dalsgaard 1 1 Department of Veterinary Disease Biology, University of Copenhagen-HEALTH 2 National Veterinary Institute, Section of Virology, Technical University of Denmark 3 Division of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark 4 Department of Water and Natural Resources, Rambøll 5 Department of Agroecology, Aarhus University

2. 2 Bio Molecular biologist Ph.D. in virology Zoonotic viruses –Influenza (Currently main focus) –Hepatitis E virus The work presented here was part of my Ph.D

3. 3 Overview Introduction to hepatitis E virus –HEV disease and zoonotic –And why it is interesting in the context of water contamination. Study design and results Conclusion and discussion Perspective/future work

4. 4 Background - History and clinical manifistations of HEV Condition was termed non-A non-B hepatitis until isolation of virus 1st HEV isolation in 1983 (Belayan, 1983) HEV causes human epidemics and outbreaks in the eastern world (India, Pakistan, China) and Africa Acute selflimiting infection in most cases –Chronic infection in immunosuppressed patients Symptoms of infected people Asymptomatic Mild (headache, nausea, abdominal pain etc.) (Food poisoning) Fulminant hepatitis (can be fatal) 4

5. 5 Background - HEV in pigs HEV in found pigs worldwide Spain 1985-97: 98% of pig herds were positive for HEV IgG (Casas et al. 2009) 49.5% of 4 – 22 week old Danish pigs excrete HEV (RNA) fecally (Breum et al, 2010) ~92% of Danish pig herds are HEV positive (IgG) Infected pigs are asymptomatic. 5

6. 6 PB. Christensen et al. 2008 Significant difference Background - Zoonosis

7. 7 Background - Zoonotic aspects of HEV Water environment in the western world? Bad hygiene Low sanitary conditions Consumption of undercooked pork products.

8. 8 Background – Aim of study Risk for spread of zoonotic pathogens, e.g. hepatitis E virus, Salmonella and Cryptosporidium to the aquatic environment 1.5 million 12 million 26 million ton slurry 1 kg of slurry pr m 2 of all fields 60% of the total area of Denmark

9. 9 STUDY METHOD AND RESULTS

10. 10 The study Test slurry for microrganisms Apply the slurry to the test field (Silstrup) Wait for rain and monitor drain and wells Collect water and test for microorganisms 1 m 3.5 m Ground water level Sample cont. Study setup

11. 11 Study setup Silstrup test field, with tile drains installed. All drains are connected to a single outlet from where sampling is performed. Sandy clay loam soil, heavily fractured and bioturbated Three types of samples were collected Event samples: Collected when flow in drains is high (Heavy rain) Weekly samples: A pooled sample of water running in drainage system over a week Well samples: From monitoring wells (1,5 – 2 m b.g.s) and horizontal drain wells ( 3.5 m b.g.s)

12. 12 Results – Test of slurry prior to application Somatic coliphages 24 – 200 nm Hepatitis E virus ss RNA virus, 32 nm Enterococcus spp. diameter of 0.5–1 µm Rotavirus group A (RV-A) ds RNA virus (segmented), 70 nm Porcine circovirus type 2 (PCV2) ss DNA virus, 17 nm E. coli 2 μm long and 0.5 μm in diameter HEVPCV2RV-A Somatic coliphages E. colienterococci Real time (RT)-PCRu/mlPFU/mlCFU/ml 1.2×10 4 5.4×10 4 3.8×10 5 2.2×10 5 6.0×10 4 3.6×10 4 Viruses Fecal indicators

13. 13 Results – Weekly samples HEVPCV2RV-A Somatic coliphages E. coli enterococc i Real time (RT)-PCRu/mlPFU/mlCFU/ml 1.2×10 4 5.4×10 4 3.8×10 5 2.2×10 5 6.0×10 4 3.6×10 4

14. 14 Results – Drain, event 14 events

15. 15 Results – Drain, event - bacteria

16. 16 Results – Drain, event – HEV, PCV2

17. 17 Results – Drain, event- Rotavirus and phages Somatic coliphages Rotavirus Somatic coliphages as a model for Rotavirus?

18. 18 Results – Wells

19. 19 Conclusions 1 m 3 m Ground water level All microorganisms were detected in drain water Potential spread to water environment (Shellfish, recreational water) RV-A was found in the ground water Potential contamination of drinking water? Somatic coliphages does not model RV-A migration RV-A HEV PCV2 RV-A E. Coli Enterococcus spp. Somatic coliphages

20. 20 Conclusion and discussion As all three viruses discovered in the pig slurry appeared in the drainage water, it indicates that virus present in pig slurry do spread to the water environment. Interaction with the water environment could be a possible route of transmission of porcine viruses (HEV)? Are private wells in close proximity to fields at risk? Are the viruses infectious?

21. 21 Stream Lake Fjords Perspectives and upcoming work ? Stream Fjord with shellfish production

22. 22 Acknowlegements Thank you for your attention The Danish Ministry for Food, Agriculture and Fisheries (DFFE) for funding. PATHOS project - Leaching of pathogens and estrogens from manure separation products to freshwater Charlotte K Hjulsager, National Veterinary Institute, Denmark, for help with PCV2 analysis.