An update on challenges and threats in Salmonella control programs and their solutions

1. An update on challenges and threats in Salmonella control programs and their solutions Prof. Dr. Filip Van Immerseel Ghent University Faculty of Veterinary Medicine, Department of Pathology, Bacteriology and Avian Diseases filip.vanimmerseel@UGent.be Budapest, 25-01-2012

2. What to talk about? • Epidemiology / prevalence of Salmonella in humans and chickens • EU legislation • Pathogenesis of Salmonella infections • Control methods, focussing on vaccination

3. History of human Salmonella infections and regulatory consequences (EU level) Emergence of human cases of Salmonella Enteritidis starting from 1989

4. Directive 92/117 ‘for the protection against specified zoonoses and zoonotic agents in animals and products of animal origin in order to prevent outbreaks of food-borne infections and intoxications’ Directive 92/117 Rules for monitoring and control of Salmonella in breeding flocks • Rules for monitoring and detection • Focus on breeding flocks • Top-down approach for control • eradication! • No measures in layer and broiler flocks

5. History of human Salmonella infections and regulatory consequences (EU level) • EC92/117 not effective • Breeding flocks • Layers + broilers • Humans VERTICAL HORIZONTAL HORIZONTAL

6. History of human Salmonella infections and regulatory consequences (EU level) • EC92/117 not effective • Blocking vertical transmission not sufficient • Still high contamination level in layer and broiler flocks • Still increase in human cases

7. Statements Scientific committee on veterinary measures relating to public health (12 april 2000): • ‘Measures in place to control food-borne zoonoses are insufficient’ • ‘The monitoring and collection of epidemiological data need to be improved’

8. ‘Measures in place to control food-borne zoonoses are insufficient’ ‘The monitoring and collection of epidemiological data need to be improved’ Regulation 2160/2003 To ensure detection and control of Salmonella and other zoonotic agents at all production stages Directive 2003/99 Detailed rules on monitoring and reporting of zoonoses

9. Directive 2003/99‘to ensure that zoonoses, zoonotic agents and related antimicrobial resistance are properly monitored, and that food-borne outbreaks receive proper epidemiological investigation, to enable the collection in the Community of the information necessary to evaluate relevant trends and sources’ Establishment of national reference laboratories Assessments of trends and sources of zoonotic agents and antimicrobial resistance Submission of an annual report to the EC

10. Directive 2003/99‘to ensure that zoonoses, zoonotic agents and related antimicrobial resistance are properly monitored, and that food-borne outbreaks receive proper epidemiological investigation, to enable the collection in the Community of the information necessary to evaluate relevant trends and sources’

12. Regulation 2160/2003‘to ensure that proper and effective measures are taken to detect and control Salmonella and other zoonotic agents at all relevant stages of production, processing and distribution, particularly at the level of primary production, including in feed, in order to reduce their prevalence and the risk they pose to public health’ Establishment of national monitoring and control programs, to be approved by the EC National control programs should: • provide for the detection of zoonoses and zoonotic agents in accordance with specified requirements and minimum sampling rules • specify the control measures to be taken following the detection of zoonoses and zoonotic agents, in particular to protect public health, including implementation of the specific measures

13. Regulation 2160/2003 Specific measures: slaughter of breeding flocks positive for Salmonella Enteritidis and Typhimurium, destruction of eggs eggs must not be used for direct human consumption (as table eggs) unless they originate from a commercial flock of laying hens subject to a national programme established (from 12/2009) fresh poultry meat may not be placed on the market for human consumption unless Salmonella is absent in 25g of meat (from 12/2010)

14. Community targets • Targets established for the reduction fo Salmonella serotypes of public health significance • To reach the targets, control methods can be made obligatory, while others can be prohibited

15. How to define community targets? Decision 2004/665 to study Salmonella prevalence in layers EFSA baseline studies to estimate across the EU the prevalence of Salmonella spp. in flocks of laying hens and broilers Decision 2005/636 to study Salmonella prevalence in broilers COMMUNITY TARGETS for layers and broilers Regulation 1003/2005 defining targets for breeding flocks Regulation 646/2007 defining targets for broiler flocks Regulation 1168/2006 defining targets for laying hen flocks

16. EFSA baseline study data (layers) + target Salmonella Enteritidis + Typhimurium

17. EFSA baseline study data (broilers) + target Salmonella Enteritidis + Typhimurium

18. Regulation 1177/2006‘regarding requirements for the use of specific control methods in the framework of the national programmes for the control of salmonella in poultry’ • No antimicrobials shall be used to control Salmonella • Vaccination is obligatory in MS when S. Enteritidis flock contamination > 10% • Live vaccines are prohibited • if they cannot be distinguished from wild type strains • during production unless the safety of the use has been demonstrated

19. History of human Salmonella infections and regulatory consequences (EU level) Voluntary measures Awareness Public and governmental pressure

20. History of human Salmonella infections and regulatory consequences (EU level) Mandatory measures

21. Trends and sources reports

22. Serotype distribution – human cases Enteritidis + Typhimurium cause 80% of cases !

23. Similar situation worldwide (data WHO 2005)

24. Emerging human problem: monophasic 4,5,12:i,- Slide provided by W. Rabsch

25. Percentage of human monophasic 4,5,12:i,- isolates to total Typhimurium isolates Slide provided by W. Rabsch

26. Emerging human problem: monophasic 4,5,12:i,- Slide provided by W. Rabsch

27. Is the monophasic strain a Typhimurium strain? Based on molecular typing : YES ‘Public health risk posed by the emerging monophasic Typhimurium strains is comparable to that of other epidemic S. Typhimurium strains’ Important regulatory consequences: include in community targets!

28. Salmonella in layers: pathogenesis and control

29. Egg contamination routes • External egg contamination • Internal egg contamination • After eggshell penetration • After reproductive tract colonization

30. External (outer shell) egg contamination • Any contaminated environment after oviposition is possible source : any bacterial species can contaminate eggshell • Most likely not the most important route for transmission to humans

31. Internal egg contamination • After eggshell penetration No evidence that egg-contaminating serotypes would be more capable to cross the eggshell and membranes

32. Internal egg contamination • After reproductive tract colonization (during egg formation) Systemic spread from the intestine

34. Are there serotype differences in oviduct colonization? • 6 groups of 24 chickens; iv infection using 6 strains • Chickens were intravenously inoculated at the age of 22 weeks • Inoculation dosis: 1 x 108 cfu/ml • Euthanasia at 14 days post-infection • Bacteriological analysis of spleen and oviduct SV Virchow SE 147 Enteritidis SE S1400 Enteritidis ST Typhimurium SHa Hadar SHe Heidelberg

35. Data: spleen colonization All serotypes can spread sytemically to internal organs

36. Data: oviduct colonization Salmonella Enteritidis (and Typhimurium) strains seem to colonize the reproductive organs more efficient

37. What does it mean? • Salmonella can reach reproductive tract but is ‘eliminated or not’ depending on serotype/strain • Oviduct (not ovary) is a harmful environment for some serotypes/strains because of antibacterial factors (e.g. egg white) • Most likely intracellular persistence and incorporation in eggs at egg formation • Salmonella Enteritidis (and Typhimurium) is superior in oviduct colonization compared to other serotypes/strains

38. What happens in the egg? • Egg formation takes 24 hrs (20hrs in uterus for shell development): long incubation time of bacteria with egg white/yolk • Can Salmonella cope with antibacterial activity of egg white at 42°C for 24hrs?

39. Keller et al. (1995). Infect. Immun. 63 : 2443 - 2449. Points to antibacterial activity of egg white during egg formation

40. Kang et al. (2006). Epidemiol. Infect. 134 : 967-976. Very strong antibacterial activity of egg white at chicken body temperature

41. Difference between serotypes? Enteritidis (and to a lesser extent Typhimurium) can cope much better with antibacterial activity of egg white It has protective mechanisms that are yet not defined

42. Prevention/control in layers • 1 or 2 serotypes to focus on • Reduce shedding • Decrease systemic spread VACCINATION !!!

43. Vaccination active immunisation live vaccines inactivated vaccines passive immunisation maternal transferred antibodies antibody containing egg powder Vaccination of laying hens LIVE vs INACTIVATED: cell-mediated/humoral vs humoral immune response

44. What is the ideal live vaccine strain ? • Should yield a high degree of protection against intestinal and systemic infection, including oviduct colonization • Should yield a high degree of protection against egg contamination • Should have a good efficacy against S. Enteritidis and if possible Typhimurium, including the monophasic variant

45. What is the ideal vaccine strain ? • Other characteristics: • easy to administer (drinking water) • contain markers facilitating the differentiation from Salmonella wild-type strains • application of vaccines should not interfere with Salmonella detection methods • humoral antibody response after vaccination should be distinguishably from a Salmonella wild-type response to allow the use of serological detection methods • adequately attenuated for poultry, other animal species, humans and the environment • the vaccines should not affect performance of the animal

46. Result of laying hen vaccination

47. Efficacy of AviPro vaccines on oviduct colonization and egg contamination by Salmonella Enteritidis • 4 groups of animals : • Non-vaccinated • AviPro Salmonella VacE day 1, week 4 and 16 • AviPro Salmonella VacT day 1, week 4 and 16 • AviPro Salmonella Duo day 1, week 4 and 16 • Challenge infection intravenous with 10exp7 cfu of a Salmonella Enteritidis field strain • Sampling eggs for 3 weeks • Euthanasia 3 weeks post-infection : bacteriological analysis of internal organs

48. Efficacy of AviPro vaccines on oviduct colonization and egg contamination by Salmonella Enteritidis Reduction of internal organ colonization, including reproductive tissues, even after iv infection !

49. Efficacy of AviPro vaccines on oviduct colonization and egg contamination by Salmonella Enteritidis Reduced egg contamination !

50. Efficacy of AviPro vaccines on systemic infection by Salmonella monophasic 4,5,12:i,- • 3 groups of animals : • Non-vaccinated • AviPro Salmonella VacT day 1 • AviPro Salmonella Duo day 1 • Challenge infection oral with 10exp8 cfu of a Salmonella Typhimurium and 4,5,12:i,- strain at day 14 • Bacteriological analysis at week 1 and 2 post-infection (day 21 and 28)