Microorganisms in pig slurryleachthroughagriculturalsoil to fielddrains Anita Forslund1, Jesper S. Krog2, Anna Charlotte Schultz3, Lars E. Larsen2, Jeanne Kjaer4, Preben Olsen5, Anders Dalsgaard1 1Department 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
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
Overview • Introduction to hepatitis E virus • HEV disease and zoonotic • And why it is interesting in the context of watercontamination. • Study design and results • Conclusion and discussion • Perspective/future work
Background - History and clinicalmanifistations of HEV • Conditionwastermed non-A non-B hepatitis until isolation of virus • 1st HEV isolation in 1983 (Belayan, 1983) • HEV causes human epidemics and outbreaksin the easternworld (India, Pakistan, China) and Africa • Acuteselflimitinginfection in most cases • Chronicinfection in immunosuppressed patients • Symptoms of infectedpeople • Asymptomatic • Mild (headache, nausea, abdominalpain etc.) (Food poisoning) • Fulminant hepatitis (canbe fatal)
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 areasymptomatic.
Background - Zoonosis PB. Christensen et al. 2008 Significant difference
Background - Zoonotic aspects of HEV Bad hygiene Low sanitary conditions Consumption of undercooked pork products. Water environment in the western world?
Background – Aim of study 1.5 million 12 million 26 million ton slurry 1 kg of slurry pr m2 of all fields 60% of the total area of Denmark • Risk for spread of zoonotic pathogens, e.g. hepatitis E virus, Salmonella and Cryptosporidium to the aquatic environment
Test slurry for microrganisms Apply the slurry to the test field (Silstrup) Wait for rainand monitor drain and wells Collectwater and test for microorganisms The study Study setup 1 m Sample cont. Groundwaterlevel 3.5 m
Studysetup • 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)
Results – Test of slurry prior to application Viruses Porcine circovirus type 2 (PCV2) ss DNA virus, 17 nm Hepatitis E virus ss RNA virus, 32 nm Rotavirus group A (RV-A) ds RNA virus (segmented), 70 nm Fecal indicators E. coli 2 μm long and 0.5 μm in diameter Somatic coliphages 24 – 200 nm Enterococcus spp. diameter of 0.5–1 µm
Results – Drain, event 14 events
Results – Drain, event- Rotavirus and phages Somatic coliphages as a model for Rotavirus? Rotavirus Somaticcoliphages
Conclusions All microorganismsweredetected in drainwater Potential spread to waterenvironment (Shellfish, recreationalwater) RV-A wasfound in the groundwater Potential contamination of drinkingwater? Somatic coliphages does not model RV-A migration HEV PCV2 RV-A E. Coli Enterococcusspp. Somatic coliphages 1 m Groundwaterlevel 3 m RV-A
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?
Perspectives and upcomingwork • Stream • Lake • Fjords ? Fjord withshellfishproduction Stream
Acknowlegements • 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. Thankyou for your attention