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“Whats new in Campylobacter infection”

“Whats new in Campylobacter infection”. Andrew Fox Health Protection /Agency NorthWest. 60. Campylobacters. Salmonellas. Rotavirus. 50. Shigellas. Cryptosporidium. 40. Norovirus. Lab reports (1000's ). 30. 20. 10. 0. 1977. 1979. 1981. 1983. 1985. 1987. 1989. 1991. 1993.

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“Whats new in Campylobacter infection”

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

  2. 60 Campylobacters Salmonellas Rotavirus 50 Shigellas Cryptosporidium 40 Norovirus Lab reports (1000's) 30 20 10 0 1977 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 Year Laboratory reporting of selected GI pathogens in England & Wales - 1977 to 2002.

  3. 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 ……

  4. There’s a lot of it about… 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.

  5. transmission FOOD Transmission pathways for Campylobacter INFECTION

  6. Campylobacter world

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

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

  9. Phenotyping • Serotyping • Penner serotyping (HS) • 65 serotypes • Lior serotyping (HL) • >100 serotypes • Colindale (modified HS) • Phagetyping • Preston/Krakhria-Lior/Grajewski/Colindale • Biotyping • Preston

  10. Problems with phenotyping • Isolates which fail to react • Need to constantly expand reagent panel • Limited availability of reagents • Lack of standardisation • Variable expression

  11. Molecular sub-typing for C.jejuni • Restriction endonuclease analysis • RFLPs • Ribotyping • PFGE • fla typing • PCR RFLP • RAPD • ERICs But problems remain, Ambiguities need combinations Standardisation Roll out

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

  13. Campylobacter isolate characterisation • Is central to disease surveillance and epidemiology, requiring methods that are • accurate • comprehensive • reproducible

  14. Chromosomal DNA Amplify  450-bp fragments of seven house-keeping genes Sequence the seven gene fragments on both strands Compare sequences of each gene fragment with the known alleles at the locus Assign 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) Multilocus sequence typing (MLST)

  15. C. jejuni sequence types Name aspA glnA gltA glyA pgm tkt uncA 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 11 12 6 ST-22 1 3 6 4 3 3 3 ST-433 2 59 4 38 17 12 35 ST-362 1 2 49 4 11 66 8 ST-179 1 6 7 2 40 32 3 ST-49 3 1 5 17 11 11 6

  16. Genetic diversity within Penner serotypes

  17. Population diversity of C. jejuni Diversity Index Serotype and phagetype 0.989 Serotype, phage and biotype 0.997 Combined genotype 0.930 MLST 0.99

  18. 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

  19. 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)

  20. Frequency of clonal complexes in different sample sources (n=160)

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

  22. Preston 2000 isolates

  23. Preston 2000 isolates ST clusters versus HS serotype

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

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

  26. Glastonbury outbreak

  27. Investigation of outbreak isolates with MLST Outbreak aspA glnA gltA glyA pgm tkt uncA ST FlaSVR Kettering 93 2 21 5 37 2 1 8 206 11 Kettering 93 2 21 3 37 2 1 5 206 11 Kettering 93 2 21 5 37 2 1 8 206 11 Kettering 93 1 7 3 4 8 9 5 42 9 Kettering 93 2 21 5 37 2 1 8 206 11 Kettering 93 2 21 3 37 2 1 5 206 11 Kettering 93 2 21 5 37 2 1 8 206 11 Kettering 93 1 7 3 4 8 9 5 42 9 Kettering 93 2 21 5 37 2 1 8 206 11 Kettering 93 2 21 3 37 2 1 5 206 11 Kettering 93 2 21 5 37 2 1 8 206 11 Kettering 93 2 21 3 37 2 1 5 206 11 France 3 1 5 17 11 11 6 49 11 France 3 1 5 17 11 11 6 49 11 France 3 1 5 17 11 11 6 49 11 France 3 1 5 17 11 11 6 49 11 France 3 1 5 17 11 11 6 49 11 France 3 1 5 17 11 11 6 49 11 France 3 1 5 17 11 11 6 49 11 Glastonbury 93 1 4 2 2 6 3 17 61 14 Glastonbury 93 1 4 2 2 6 3 17 61 14

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

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

  30. C. jejuni sequence types Name aspA glnA gltA glyA pgm tkt uncA 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 11 12 6 ST-22 1 3 6 4 3 3 3 ST-433 2 59 4 38 17 12 35 ST-362 1 2 49 4 11 66 8 ST-179 1 6 7 2 40 32 3 ST-49 3 1 5 17 11 11 6

  31. Place and Time and Type C.jejuniHS11 PT1 7 cases in 20 days 21 cases all year in NW and S Lancs HA

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

  33. 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

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

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