Kim Oren Gradel Department of Infectious Diseases Aalborg Hospital Aarhus University Hospital

1. Salmonella in chickens, their houses, and consumers: excerpts from the Danish farm-to-fork chain Kim Oren Gradel Department of Infectious Diseases Aalborg Hospital Aarhus University Hospital Aalborg, Denmark Koret School of Veterinary Medicine, 8 April 2008

2. Main infection sources in humans Danish Zoonosis Centre: Annual report on Zoonoses in Denmark 2006. Koret School of Veterinary Medicine, 8 April 2008

3. Table of contents • Salmonella control programmes in chickens • Salmonella in chicken houses • Salmonella in humans Koret School of Veterinary Medicine, 8 April 2008

4. Broiler production Import of day-old chicks Central rearing: Zoonosis directive samples as from 1994 Central rearing: Salmonella control programme as from 1996 Central rearing Parent flocks Parent flocks: Zoonosis directive samples as from 1994 Parent flocks: Salmonella control programme as from 1996 Feed mills Feed mills: All feed heat treated to 81 oC as from the late 80’ies Hatchery Hatchery: Salmonella control programme as from 1996 Broiler farms: Sampled at 3 weeks of age as from 1989 Broiler farms Koret School of Veterinary Medicine, 8 April 2008

5. Table egg production Import of day-old chicks Central rearing: Zoonosis directive samples (1994) Central rearing: Salmonella control programme (1996) Central rearing Parent flocks: Zoonosis directive samples (1994) Parent flocks: Salmonella control programme (1996) Parent flocks Feed mills: All feed heat treated to 81 oC as from the late 80’ies Hatchery: Salmonella control programme (1996) Hatchery Feed mills Commercial rearing Commercial rearing: Salmonella control programme (1996) Table egg houses Table egg houses: Salmonella control programme (1996) Table egg houses: Voluntary programme (cloacal swabs before slaughter) as from 1992 Koret School of Veterinary Medicine, 8 April 2008

6. Two DVMs employed at the Danish Veterinary Laboratory in 1996 • Principal tasks: • Overview of Salmonella situation (databases) • Tracking infection sources (databases, enquiries, visits to farms) • Advising Salmonella infected farmers (visits to farms) Koret School of Veterinary Medicine, 8 April 2008

7. Combating Salmonella in poultry • Reduction principles: • Vaccination (not allowed in DK) • Antibiotics (should not be an option) • Competitive exclusion (variable results) • Elimination principles (Scandinavia): • Culling of infected flocks • Cleaning and disinfection of infected premises • Which is why: you’ll hear little about Salmonella in chickens, but more about Salmonella in chicken houses! Koret School of Veterinary Medicine, 8 April 2008

8. Table of contents • Salmonella control programmes in chickens • Salmonella in chicken houses • Salmonella in humans Koret School of Veterinary Medicine, 8 April 2008

9. Persistently Salmonella-infected broiler houses Gradel KO, unpublished results Koret School of Veterinary Medicine, 8 April 2008

10. Questionnaire-based field study Koret School of Veterinary Medicine, 8 April 2008

11. Questionnaire-based field study: overview May-June 1997: First time occurrence of these two Salmonella types in the Danish broiler sector! Parent flock infected with S. Typhimurium, PT66 (ST66) Parent flock infected with S. Enteritidis, PT8 (SE8) Hatchery 51 farms, comprising 84 broiler houses, had SE8 and/or ST66 detected Koret School of Veterinary Medicine, 8 April 2008

12. Questionnaire-based field study: Methods • 78 broiler houses (93%) on 42 farms (82%) were visited • Each visit: • assessment of rodents, beetles, equipment, cleaning standard • the farmer was asked about routine procedures • Broiler houses with Salmonella in ≥2 crops (multiple-infection houses) were compared with broiler houses with Salmonella in 1 crop (single-infection houses) Koret School of Veterinary Medicine, 8 April 2008

13. Main results: factors that decreased the risk of multiple-infection houses • ST66 rather than SE8 • Lower number of positive samples (1-5) in the first crop • Antiseptic soap and water in the ante-room • Equipment for removal of dead birds did not cross the hygiene barriers • Gravel alongside the broiler house • Systematic check of indoor rodent-bait depots • Combined surface and pulse fog disinfection (in contrast to separate use of each of these) Koret School of Veterinary Medicine, 8 April 2008

14. Interpretations and practical implications • Many variables! • Two interpretations: • Significant factors have a specific impact on Salmonella persistence • Significant factors illustrate the advantage of general order and system Koret School of Veterinary Medicine, 8 April 2008

15. Disinfection projects under the Salmonella control programme • 2000 (three years after the implementation of the Salmonella control programme): money allocated to research • Focus: persistently Salmonella infected premises • Two projects: • “Heat disinfection project” • “Chemical disinfection project” Koret School of Veterinary Medicine, 8 April 2008

16. Background for disinfectant studies • Most reported disinfection tests are suspension tests: • Impressive results (best case scenarios) • Therefore widely used by the disinfectant companies! • Extrapolation to realistic conditions is very difficult • Very little research on disinfection of animal buildings Koret School of Veterinary Medicine, 8 April 2008

17. Heat laboratory tests Koret School of Veterinary Medicine, 8 April 2008

18. Heat laboratory tests: aim • Finding a time-temperature-humidity gold standard that eliminates Salmonella and relevant indicator bacteria under worst-case scenario conditions (i.e., poorly cleaned poultry houses). Koret School of Veterinary Medicine, 8 April 2008

19. Heat laboratory tests: factors Koret School of Veterinary Medicine, 8 April 2008

20. Heat laboratory tests: results Koret School of Veterinary Medicine, 8 April 2008

21. Heat disinfection tests: correlations between Salmonella and E. coli Koret School of Veterinary Medicine, 8 April 2008

22. Heat laboratory tests: gold standard for field studies • No bacteria were detected at 60 oC and 100% RH after 24 hours of heating Koret School of Veterinary Medicine, 8 April 2008

23. Heat field tests Koret School of Veterinary Medicine, 8 April 2008

24. Heat field tests: principles I Koret School of Veterinary Medicine, 8 April 2008

25. Heat field tests: principles II Koret School of Veterinary Medicine, 8 April 2008

26. Heat field tests: sampling • 300 Salmonella samples taken before and after heat treatment • Challenge samples in each house at the 12 sites where temperature was logged every 5 minutes: • Feed: Enterococcus faecalis, E. coli • Faeces: Enterococci, E. coli Koret School of Veterinary Medicine, 8 April 2008

27. Heat field tests: overview of farms, houses and treatments Koret School of Veterinary Medicine, 8 April 2008

28. Heat field tests: results for Salmonella samples Koret School of Veterinary Medicine, 8 April 2008

29. Heat field tests: conclusions • In tight houses: 60 oC and 100% RH achieved minimum 10 cm above floor level within one hour and was easily maintained • 60 oC and 100% RH during 24 hours: effective in eliminating Salmonella and putative indicator bacteria • 30 ppm formaldehyde seemed to lower the lethal temperature by 2-5 oC • Today, 5-6 years later, Salmonella has not been detected in any of the heat treated layer houses Koret School of Veterinary Medicine, 8 April 2008

30. Heat field tests: implications • Steam and formaldehyde implemented in other places: • Visit by DVMs from Agricola Tre Valli, Italy (Europes 3rd biggest poultry company) • Building a steam generator in Japan • Enquiries from Spain and Sweden • Economically feasible: • Farmers’ expenditures due to persistent Salmonella infections: $100,000-200,000 or more • The owner of Denmark’s biggest table layer farm went bankrupt after spending $2,200,000 on Salmonella before heat treatment was documented • Cost of heat treating poultry house: $10,000 Koret School of Veterinary Medicine, 8 April 2008

31. Resistance to disinfectants Koret School of Veterinary Medicine, 8 April 2008

32. Resistance to disinfectants • Hypothesis: There is an association between persistence of Salmonella in poultry houses and the common use of a few types of disinfectants in these • Aims: • To see if minimum inhibitory concentrations (MICs) against five commonly used disinfectants could be related to Salmonella persistence or use of disinfectants in Danish broiler houses. • To see if resistance against the five disinfectants could be introduced and maintained in the laboratory Koret School of Veterinary Medicine, 8 April 2008

33. MIC-tests MIC-tests Resistance to disinfectants:adaptation and de-adaptation Koret School of Veterinary Medicine, 8 April 2008

34. MICs for isolates from Danish chicken houses ”Non-persistent” serotypes ”Danish” disinfectants ”English” disinfectants ”Persistent” serotypes Higher MICs Koret School of Veterinary Medicine, 8 April 2008

35. Resistance to disinfectants:conclusions • No associations between MICs and use of disinfectants in the preceding download period • No associations between MICs and Salmonella persistence • Adaptation or de-adaptation did not alter any MICs beyond one doubling dilution, i.e., within normal biological variation Koret School of Veterinary Medicine, 8 April 2008

36. Surface disinfection tests Koret School of Veterinary Medicine, 8 April 2008

37. Surface disinfection tests: general principles • Worst-case scenario surface disinfection tests simulating conditions and disinfection procedures encountered in badly cleaned poultry houses, especially at low temperatures Koret School of Veterinary Medicine, 8 April 2008

38. Surface disinfection tests: factors • Isolates: S. Enteritidis, S. Senftenberg, Enterococcus faecalis • Poultry house materials: Concrete, rusty metal, wood, jute • Organic matter: Feed, fats, egg yolk. • Disinfectants: formalin, glutaraldehyde/benzalkomium chloride, oxidising compound, water (control) • Temperatures before and after disinfection: 6/11/20/30 and 6/11/30 oC, respectively • Disinfection time: 5, 15, 30 minutes Koret School of Veterinary Medicine, 8 April 2008

39. Surface disinfection tests:concrete flag with feed Koret School of Veterinary Medicine, 8 April 2008

40. Surface disinfection tests:feed chain links with feed Koret School of Veterinary Medicine, 8 April 2008

41. Surface disinfection tests: results I From: Gradel, K.O.: Disinfection of empty animal houses - scientific evidence for applied procedures.In Kurladze, G.V. (ed.): Environmental Microbiology Research Trends. New York, USA: Nova Science Publishers, Inc., 2007, pp. 59-98. ISBN 978-1-60021-939-9. Koret School of Veterinary Medicine, 8 April 2008

42. Surface disinfection tests: results II • S. Senftenberg was more susceptible than S. Enteritidis in tests with the oxidising compound and water, in spite of higher MICs for S. Senftenberg • In general, Enterococcus faecalis was more recalcitrant than the two Salmonella isolates. Thus, it is a putative indicator bacterium in field trials Koret School of Veterinary Medicine, 8 April 2008

43. Future recommendations • More worst-case scenario laboratory studies with various conditions (different types of organic matter, temperature, disinfectants) • Field intervention studies • Standardized monitoring programmes (e.g., Hazard Analysis Critical Control Points) • Useful for many other micro-organisms than Salmonella Koret School of Veterinary Medicine, 8 April 2008

44. Salmonella control programmes in chickens • Salmonella in chicken houses • Salmonella in humans Koret School of Veterinary Medicine, 8 April 2008

45. Interest of study group • Zoonotic Salmonella and Campylobacter • Bacteremia, ”all” micro-organisms • Patient-related factors : • age • chronic diseases • medicine • Registry-based research Koret School of Veterinary Medicine, 8 April 2008

46. Salmonella bacteremia Koret School of Veterinary Medicine, 8 April 2008

47. Salmonella bacteremia • Prognostic study for 111 patients • Possible prognostic factors, e.g.: • Age • Chronic diseases • Salmonella serotype • Antibiotic treatment • Leukocytes • C-reactive protein • Albumin • Haemoglobin • Creatinine Koret School of Veterinary Medicine, 8 April 2008

48. Salmonella bacteremia Koret School of Veterinary Medicine, 8 April 2008

49. Seasonal variation of salmonellosis Koret School of Veterinary Medicine, 8 April 2008

50. Non-hospitalized Endogenous factors: The seasonal variation ”minimizes” Exogenous factors: The seasonal variation ”maximizes” Hospitalized Bacteremia Seasonal variation of salmonellosis • Background: the ”typical” seasonal variation was less evident for bacteremia patients • Hypothesis: Koret School of Veterinary Medicine, 8 April 2008