Microorganisms in pig slurry leach through agricultural soil to field drainsAnita Forslund1, Jesper S. Krog2, Anna Charlotte Schultz3, Lars E. Larsen2, Jeanne Kjaer4, Preben Olsen5, Anders Dalsgaard1

1 Department of Veterinary Disease Biology, University of Copenhagen-HEALTH2 National Veterinary Institute, Section of Virology, Technical University of Denmark3 Division of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark4 Department of Water and Natural Resources, Rambøll5 Department of Agroecology, Aarhus University

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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

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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

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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)

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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.

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PB. Christensen et al. 2008

Significant difference

Background - Zoonosis

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Background - Zoonotic aspects of HEV

Water environment in the western world?

Bad hygiene

Low sanitary conditions

Consumption of undercooked

pork products.

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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 m2 of all fields

60% of the total area of Denmark

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STUDY METHOD AND RESULTS

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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

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Study setupSilstrup 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)

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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

HEV PCV2 RV-A Somatic

coliphages

E. coli enterococci

Real time (RT)-PCRu/ml PFU/ml CFU/ml

1.2×104 5.4×104 3.8×105 2.2×105 6.0×104 3.6×104

Viruses

Fecal

indicators

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Results – Weekly samplesHEV PCV2 RV-A Somatic

coliphages

E. coli enterococc

i

Real time (RT)-PCRu/ml PFU/ml CFU/ml

1.2×104 5.4×104 3.8×105 2.2×105 6.0×104 3.6×104

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Results – Drain, event

14 events

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Results – Drain, event - bacteria

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Results – Drain, event – HEV, PCV2

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Results – Drain, event- Rotavirus and phages

Somatic coliphages

Rotavirus

Somatic coliphages as a model for Rotavirus?

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Results – Wells

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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

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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?

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• Stream

• Lake

• Fjords

Perspectives and upcoming work

?

Stream

Fjord with shellfish production

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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.