2. “Whats new in Campylobacter infection” Andrew Fox Health Protection /Agency NorthWest

4. Laboratory reporting of selected GI pathogens in England & Wales - 1977 to 2002.

5. Campylobacter Statistics 50,000+ confirmed cases in England and Wales (CDR) Estimated 400,000+ infections annually (IID study) 0.1% cases develop GBS (US estimate) 0-5% cases develop reactive arthritis (Scandinavia) 0.3-5.9% case develop bacteraemia (UK) 10% cases hospitalised, 5 days Meningitis, Cholecystitis, Pancreatitis, Hepatitis, Peritonitis, Myocarditis, Abcess ……

6. BUT… Common source outbreaks are rarely identified and the source of the majority of cases reported in the UK is unknown. In one case control study, epidemiology of infection for over 60% of cases remained unknown. There’s a lot of it about…

8. Transmission pathways for Campylobacter INFECTION

9. Campylobacter world

10. Selective culture Enrichment culture Serotyping Serotype specific phage typing Plasmid profiling PFGE Antibiograms S.enteritidis PT4 S.typhimurium DT104 S.virchow S.ealing S.goldcoast S.typhi The world of Salmonella Enterica

11. The world of E.coli 0157 All E.coli VTEC 0157 Selective culture Enrichment culture 0157 confirmation kits 0157 phage typing 0157 PCR 0157 PFGE

12. Selective culture Enrichment culture (foods) Typing methods Campylobacter spp. Campylobacter world

13. Phenotyping SerotypingSerotyping –Penner serotyping (HS) 65 serotypes65 serotypes –Lior serotyping (HL) >100 serotypes>100 serotypes –Colindale (modified HS) PhagetypingPhagetyping –Preston/Krakhria-Lior/Grajewski/Colindale BiotypingBiotyping –Preston

14. Problems with phenotyping Isolates which fail to reactIsolates which fail to react Need to constantly expand reagent panelNeed to constantly expand reagent panel Limited availability of reagentsLimited availability of reagents Lack of standardisationLack of standardisation Variable expressionVariable expression

15. Molecular sub-typing for C.jejuni Restriction endonuclease analysisRestriction endonuclease analysis RFLPsRFLPs RibotypingRibotyping PFGEPFGE fla typingfla typing PCR RFLPPCR RFLP RAPDRAPD ERICsERICs But problems remain, Ambiguities need combinations Standardisation Roll out

16. Genotyping Still ambiguitiesStill ambiguities –COMBINATORIAL TECHNOLOGIES Fragment analysis methods-significance of small changesFragment analysis methods-significance of small changes Limited phylogenetic anaylsisLimited phylogenetic anaylsis

17. Multilocus Enzyme Electrophoresis (MEE) Aeschbacher and PiffarettiAeschbacher and Piffaretti –Extensive variation –No interspecies recombination

18. Crossroads control and prevention population genetics Ambiguous typing epidemiology sporadic infection Campylobacter infection

19. Campylobacter isolate characterisation Is central to disease surveillance and epidemiology, requiring methods that areIs central to disease surveillance and epidemiology, requiring methods that are –accurate –comprehensive –reproducible

21. Multilocus sequence typing (MLST) Chromosomal DNA Amplify  450-bp fragments of seven house-keeping genes Compare sequences of each gene fragment with the known alleles at the locusAssign alleles at the seven loci to give the allelic profile Compare the allelic profile with those of isolates within a central database via the internet and assign a sequence type (ST) Sequence the seven gene fragments on both strands

22. Camyplobacter jejuni MLST scheme: housekeeping gene targets glt A: citrate synthase unc A: ATP synthase a subunit asp A: aspartase gly A: serine hydroxy methyl transferase gln A: glutamine synthetase pgm: phospho glucomutase tkt: transketolase fla SVR region.

23. C. jejuni sequence types NameaspAglnAgltAglyApgmtktuncA ST-21 2 1 1 3 2 1 5 ST-45 4 7 10 4 1 7 1 ST-206 2 21 5 37 2 1 5 ST-61 1 4 2 2 6 3 17 ST-48 2 4 1 2 7 1 5 ST-257 9 2 4 62 4 5 6 ST-353 7 17 5 2 10 3 6 ST-42 1 2 3 4 5 9 3 ST-403 10 27 16 19 10 5 7 ST-52 9 25 2 10 22 3 6 ST-177 17 2 8 5 8 2 4 ST-354 8 10 2 2 1112 6 ST-22 1 3 6 4 3 3 3 ST-433 2 59 4 38 1712 35 ST-362 1 2 49 4 1166 8 ST-179 1 6 7 2 4032 3 ST-49 3 1 5 17 1111 6

24. Genetic diversity within Penner serotypes

25. Population diversity of C. jejuni Diversity Index Serotype and phagetype 0.989 Serotype, phage and biotype 0.997 Combined genotype 0.930 MLST0.99

26. Do we need yet another typing scheme for Campylobacter? A nucleotide sequence based approach capitalises on technology which is largely automated and increasingly applied to the characterisation of pathogens

27. Advantages of MLST The technique is portable, reproducible and relatively quick, easy and cheap to perform Unlike antigen and antibiotic resistance genes, housekeeping genes are selectively neutral Freedom from reliance on serological typing reagents which are becoming more difficult to produce (recent changes in Animal Procedures Act)

29. Frequency of clonal complexes in different sample sources (n=160) SourceST-21ST-45ST-61 Ruminant35 (41%)11 (13%)17 (20%) Avian3 (11%)10 (35%)_ Wild mammal5 (21%)9 (37%)2 (9%) Environment1 (5%)10 (48%)1 (5%)

30. Comparison of the frequency of ST- complexes from animal and environmental sources with human infections

31. Preston 2000 isolates Number of Clonal Complexes 17 Number of Sequence Types 58 Number of HS serotypes 28

32. Preston 2000 isolates ST clusters versus HS serotype STNo isolatesHS serotype ST-1045HS 5(1); HS 16(1); HS 50(2);HS NT(1) ST-454HS 12(3);HS NT(1) ST-2622HS NT ST-192HS NT

33. Clonal Complexes for C.jejuni isolated from GI infections in Preston area NW England

34. Comparison of clonal complexes for UK human infections: 1991-2000

35. Glastonbury outbreak

36. OutbreakaspAglnAgltAglyApgmtktuncA STFlaSVR Kettering 93 Kettering 93 2 21 5 37 2 1 820611 Kettering 93 Kettering 93 2 21 3 37 2 1 520611 Kettering 93 Kettering 93 2 21 5 37 2 1 8206 11 Kettering 93 Kettering 93 1 7 3 4 8 9 5 42 9 Kettering 93 Kettering 93 2 21 5 37 2 1 820611 Kettering 93 Kettering 93 2 21 3 37 2 1 520611 Kettering 93 Kettering 93 2 21 5 37 2 1 820611 Kettering 93 Kettering 93 1 7 3 4 8 9 5 42 9 Kettering 93 Kettering 93 2 21 5 37 2 1 820611 Kettering 93 Kettering 93 2 21 3 37 2 1 520611 Kettering 93 Kettering 93 2 21 5 37 2 1 820611 Kettering 93 Kettering 93 2 21 3 37 2 1 520611 France France 3 1 5 17 1111 6 4911 Glastonbury 93 Glastonbury 93 1 4 2 2 6 3 17 6114 Investigation of outbreak isolates with MLST

37. HS1 PT76 HS2 PT52 HS4 PT121 HS4 PT55 HS11 PT90 HS19 PT90 ST 21 Complex ST-61 ST 17 ST 22 Multilocus sequence typing of C.jejuni

39. Detection and report of Camp spp. by EIA or PCR DNA Purification and typing Further report to GP, EHO, CCDC, RE 24 - 48 hours 24 hours 3 - 5 days Cluster analysis and investigation Campylobacter detection and typing from faeces or foods - future prospects

40. C. jejuni sequence types NameaspAglnAgltAglyApgmtktuncA ST-21 2 1 1 3 2 1 5 ST-45 4 7 10 4 1 7 1 ST-206 2 21 5 37 2 1 5 ST-61 1 4 2 2 6 3 17 ST-48 2 4 1 2 7 1 5 ST-257 9 2 4 62 4 5 6 ST-353 7 17 5 2 10 3 6 ST-42 1 2 3 4 5 9 3 ST-403 10 27 16 19 10 5 7 ST-52 9 25 2 10 22 3 6 ST-177 17 2 8 5 8 2 4 ST-354 8 10 2 2 1112 6 ST-22 1 3 6 4 3 3 3 ST-433 2 59 4 38 1712 35 ST-362 1 2 49 4 1166 8 ST-179 1 6 7 2 4032 3 ST-49 3 1 5 17 1111 6

42. Place and Time and Type 7 cases in 20 days 21 cases all year in NW and S Lancs HA C.jejuni HS11 PT1

43. The Iceland Experiment 1.Public awareness campaign 2.Frozen chicken

44. USDA Intervention studies Norman Stern Novel biological agents to reduce or eliminate Campylobacter from chicken intestinal flora –Competitive exclusion Bacteriocins –15 trials –Administer bacteriocin 5 days before slaughter –5-fold or total reduction in Campylobacter from chicken at slaughter

45. Acknowledgements Preston PHL Eric Bolton Roisin Ure David Wareing (Dynal Biotech) WCIED, Oxford Frances Colles Kate Dingle Martin Maiden Rachel Urwin University of Staffordshire Mishele Barrigas Pete Gowland DEFRA Epidemiology Unit, University of Liverpool Nigel French Richard Kemp Howard Leatherbarrow