1. Chemical disinfectants commonly used in the poultry sector and their impact on Salmonella – an outline of methods and results Kim O. Gradel Danish Veterinary Institute, Å rhus, Denmark SYMPOSIUM: DISINFECTION IN ANIMAL PRODUCTION Tuesday 18 November, 2003, Aarhus, Denmark

2. Disposition Resistance against disinfectants Minimum inhibitory concentration (MIC) tests Adaptation and de-adaptation tests Conclusions Surface disinfection tests Materials and methods Results Conclusions General conclusions

3. Chemical tests related to general disinfection factors Micro- organism DisinfectantSurroundings Type State/condition Organic matter Temperature pH Biofilm Type Concentration Resistance to disinfectants Surface disinfection tests

4. 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.

5. Resistance against disinfectants: epidemiological tools Since 1992, for all Danish broiler flocks/crop cycles: samples for Salmonella have been submitted. The use of disinfectants has been registered by a veterinarian visiting the flock. All data have been registered in a central database.

6. Resistance against disinfectants: persistently Salmonella-infected broiler houses

7. Salmonella serotype Number of crops with the same Salmonella type 1 2 3 4 5 6-10 11-20 21-30 > 30 Enteritidis 5 1 6 4 2 Typhimurium 2 7 4 3 1 2 2 Tennessee 4 4 1 1 2 4.12:b:- 1 3 2 2 4 6 4 5 1 Infantis 9 2 3 3 6 4 Indiana 1 1 2 1 1 3 Resistance against disinfectants: persistence in broiler houses In this study, 67 and 21 broiler houses were represented with two or more than two isolates, respectively.

8. Resistance against disinfectants: persistently Salmonella-infected broiler houses First isolate selected Middle isolate selected Last isolate selected

9. Resistance against disinfectants: sources of bacterial isolates

10. 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

11. Resistance against disinfectants: MIC-tests

12. Resistance against disinfectants: adaptation and de-adaptation MIC-tests

13. Resistance to disinfectants: MIC-tests (Salmonella results)

14. Resistance to disinfectnats: Changes in MICs during the persistence period 67 broiler houses represented with two isolates. 21 broiler houses represented with more than two isolates. Generally, no significant changes (p = 0.30). Oxidising compound has relatively most increases, but p = 0.10.

15. Resistance to disinfectants: more results (data not shown) No associations between MICs and use of disinfectants in the preceding download period. Adaptation or de-adaptation did not alter any MICs beyond one doubling dilution, i.e. within normal biological variation.

16. Resistance to disinfectants: conclusions 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.

17. 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 o C, respectively. Disinfection time: 5, 15, 30 minutes.

18. Surface disinfection tests: design 1. High bacterial concentrations in organic matter. 2. Organic matter added to poultry house materials. 3. 24 hours of drying. 4. Disinfection for set time periods. 5. 25 hours of drying. 6. Traditional bacteriological procedures with tenfold dilutions, i.e. a most probable number ( semi- quantitative ) method.

19. Surface disinfection tests: concrete flag with feed

20. Surface disinfection tests. feed chain links with feed

21. Surface disinfection tests: disinfection of concrete flags

22. esults for concrete flags Surface disinfection tests: results for concrete flags CfuGpfTBTADT S. EnteritidisS. Senftenberg ForG/BOksWaForG/BOksWa L1020113000010002 L20 1130004ND0002 L2011 3000330022 L20663001350011 L20661500240021 H206615000144550022 H2066502450125

23. Surface disinfection tests: results for feed chain links OMTBTADT S. EnteritidisS. SenftenbergEnterococcus faecalis ForG/BOksWaForG/BOksWaForG/BOksWa FA 30 4332005512110032 663055 35 44 FE 11 300055 23001055 6630001055450055 663000415355001555 6615001155 00115552 6650044555401044554

24. Surface disinfection tests: results for wooden dowels OMTBTADT S. EnteritidisS. SenftenbergEnterococcus faecalis ForG/BOksWaForG/BOksWaForG/BOksWa FA 30 210322020433000100000201233122100322 66305334335555455423333>2 2 455442413332345 FE 11 30000110111 000 100122000111232545 6630000 111443000 211122100112453 6615001010111232000 111012001112545 665210010222333000100212133112111334555

25. Surface disinfection tests: results for jute egg belts cfuTBTADT S. EnteritidisS. SenftenbergEnterococcus faecalis ForG/BOksWaForG/BOksWaForG/BOksWa L11 300001222444550114 L663000450113 L661501250003 H11 3002550245214355 H661503550225 H665355502451055

26. Surface disinfection tests: conclusions I Efficacy (best first): formalin > glutaraldehyde/benzalkonium chloride > oxidising compound > water. Except: Feed chain links with fats at 30 o C before and after disinfection, where the oxidising compound apparently was the most effective. Formalin is better than glutaraldehyde/benzalkonium chloride at 6 o C, although it is often stated that formalin is only effective above 16 o C, whereas glutaraldehyde is effective down to 5 o C!

27. Surface disinfection tests: conclusions II 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. 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.

28. General conclusions For Salmonella, resistance to commonly used disinfectants does not seem to be an important aspect of persistence. Results from unrealistic tests are difficult to extrapolate to realistic tests. For Salmonella and other vegetative bacteria, results indicate that the surroundings are more important for the efficacy of disinfection than the type of bacteria.

29. Acknowledgments Kirsten Holm, DVI, Å rhus, Denmark. Luke Randall, Weybridge, UK. Ian McLaren, Weybridge, UK.