Disinfection of housing systems, with special reference to Salmonella

1. Disinfection of housing systems, with special reference to Salmonella Kim O. Gradel Danish Veterinary Institute, Århus, Denmark Nordic Poultry Consultant and Veterinary Conference, 20-22 November 2003, Stockholm, Sweden

2. Background • Today, little Salmonella is introduced into Danish poultry farms; the occurrence of Salmonella is dominated by persistent infections. • Poultry (animal) houses and equipment are not very ”cleanable”. • The scientific literature on the topic is sparse. • Most documentation on disinfection of animal houses can only be obtained from commercial sources.

3. Cleaning and disinfection projects under the Danish Salmonella control programme • Heat disinfection project • Laboratory tests • Field tests • Chemical disinfection project • Resistance to disinfectants • Surface/carrier disinfection tests

4. Micro- organism Type ”State/condition” Disinfectant Surroundings Type Concentration Organic matter Temperature pH Biofilm Factors that influence disinfection, related to the projects Resistance to disinfectants Heat project Surface disinfection tests

5. Heat disinfection laboratory tests • Principles: • Simulation of real-life conditions. • Worst-case scenarios. • Full factorial design. • Aims: • To find a temperature-humidity-time treatment that kills Salmonella. • Investigate correlations between susceptibility of spiked Salmonella and naturally occurring E. coli.

6. Heat disinfection laboratory tests Factors

7. 24-hour samples 48-hour samples 0-hour samples 1 oC per hour Final heating temperature 20 oC 20 oC 72-hour samples 10-day samples Heat disinfection laboratory tests Overview of tests

8. Heat disinfection laboratory tests Samples prior to heating

9. Heat disinfection laboratory tests Heating of high humidity samples

10. Heat disinfection laboratory tests Results

11. E. coli detected? Salmonella spp. detected on Rambach agar? SUM Yes No Pure culture of Salmonella Salmonella- and non-Salmonella Growth of non-Salmonella Sterile Yes 8 90 10 8 116 No 40 8 3 222 273 SUM 48 98 13 230 389 Heat disinfection laboratory tests:Correlations between results for Salmonella and E. coli

12. Heat disinfection laboratory testsConclusions • Humidity, both before and during heating, was an important factor in the bacterial killing. • In general, there was a higher survival in feed than in faeces. • There were high correlations between the survival of spiked Salmonella and naturally occurring E. coli. • No bacteria were detected at 60 oC and 100% RH after 24 hours of heating (i.e. ”gold” standard for field studies).

13. Heat disinfection field tests • Aim: To test if the gold standard (60 oC & 100% RH during 24 hours) was valid in the field. • Principles: • Salmonella samples before and after heating. • Salmonella results per se. • Non-sterile Rambach agar plates. • Challenge samples, placed at sites where temperature was measured.

14. Heat disinfection field tests:Overview of farms, houses and treatments

15. Heat disinfection field testsResults for Salmonella samples

16. Heat disinfection field testsCorrelations between Salmonella and coliforms on house level

17. Heat disinfection field testsSalmonella”hot” sites

18. Heat disinfection field testsCorrelations between Salmonella and coliforms on site level

19. Heat disinfection field testsResults for challenge samples

20. Heat disinfection field testsConclusions • In tight houses, 60 oC and 100% RH was achieved minimum 10 cm above floor level within one hour and was easily maintained during 24 hours. • 60 oC and 100% RH during 24 hours seemed effective in eliminating Salmonella and putative indicator bacteria. • The addition of 30 ppm formaldehyde at the beginning of the process seemed to lower the lethal temperature by 2-5 oC. • The occurrence of coliforms could be a guidance for the efficacy against Salmonella, although no cut-off values could be predicted.

21. Resistance against 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.

22. Resistance against disinfectantsSelection of isolates from a persistently Salmonella-infected broiler house First isolate selected ”Middle” isolate selected Last isolate selected

23. Resistance against disinfectantsSources of bacterial isolates

24. Resistance against disinfectants Disinfectants used in the study • Formaldehyde (24.5%) • Glutaraldehyde (23%) and benzalkonium chloride (5%) compound (Bio Komplet Plus) • Oxidising compound (blend of peroxygen compounds) (Virkon S) • Phenol (30-45% high boiling tar acids) (FFS) • Iodophor (FAM 30) ”Danish” disinfectants ”English” disinfectants

25. Resistance to disinfectantsMIC-tests (method)

26. Resistance to disinfectantsAdaptation and de-adaptation (method) MIC-tests MIC-tests

27. Resistance to disinfectantsMIC-tests (Salmonella results)

28. Resistance to disinfectantsMore results (data not shown) • No associations between increased MICs for the three disinfectants showing • significant differences for some serotypes (formaldehyde, oxidising • compound, iodophor). • No associations between MICs and use of disinfectants in the • preceding download period. • Adaptation or de-adaptation did not alter any MICs significantly. • No significant increases or decreases in MICs during the persistence • periods (67 broiler houses with two isolates and 21 broiler houses with • more than two isolates), either generally (p = 0.30) or for any individual • disinfectants.

29. Resistance to disinfectantsConclusions • Resistance to commonly used disinfectants • does not play a major role in persistence • of Salmonella in Danish broiler houses. • In the laboratory, it was not possible to adapt • selected strains to the actual disinfectants.

30. Surface disinfection tests • Principles: • Worst-case scenario surface disinfection tests simulating conditions and disinfection procedures encountered in badly cleaned poultry houses, especially at low temperatures. • Factors: • Isolates: S. Enteritidis (low MICs), S. Senftenberg (high MICs), Enterococcus faecalis (putative indicator bacterium). • Poultry house materials: Concrete flags, rusty feed chain links, wooden dowels, jute egg belts. • 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.

31. Surface disinfection testsDesign • High bacterial concentrations in organic matter. • Organic matter ”added to” poultry house materials. • 24 hours of drying. • Disinfection for set time periods. • 25 hours of drying. • Traditional bacteriological procedures with tenfold dilutions, i.e. a most probable number (”semi-quantitative”) method.

32. Surface disinfection testsConcrete flag with feed

33. Surface disinfection testsFeed chain links with feed

34. Surface disinfection testsDisinfection of concrete flags

35. Surface disinfection testsResults for concrete flags

36. Surface disinfection testsResults for feed chain links

37. Surface disinfection testsResults for wooden dowels

38. Surface disinfection testsResults for jute egg belts

39. Surface disinfection testsConclusions I • Efficacy (best first): formalin > glutaraldehyde/benzalkonium chloride > oxidising compound > water. • Except: Feed chain links with fats at 30 oC before and after disinfection, where the oxidising compound apparently was the most effective. • For the effective disinfectants (formalin and glutaraldehyde/benzalkonium chloride) there were no differences between the two Salmonella isolates, whereas S. Senftenberg was more susceptible than S. Enteritidis in tests with the oxidising compound and water, in spite of higher MICs for S. Senftenberg (for formalin and the oxidising compound). • In general, Enterococcus faecalis was more recalcitrant than the two Salmonella isolates, i.e. it is a putative indicator bacterium, e.g. in field trials.

40. Surface disinfection testsConclusions II • Fats seem to be the best protectant for the bacteria among the types of organic matter used. • In general, there were few differences between the different poultry house materials. • Formalin is better than glutaraldehyde/benzalkonium chloride at 6 oC, although it is often stated that formalin is only effective above 16 oC, whereas glutaraldehyde is effective down to 5 oC!

41. Acknowledgments • Kirsten Holm, DVI, Århus, Denmark. • Kirsten Christensen, DVI, Århus, Denmark. • Robert H. Davies, Weybridge, UK. • Janet Corry, Bristol University, UK. • Staff at Landskontoret for Fjerkrærådgivning, Århus, Denmark. • Staff at Danish Meat Research Institute, Roskilde, Denmark. • Participating farmers.