Ring test No Detection and enumeration of Campylobacter spp. in broiler meat

Transcription

1 EU-RL- Campylobacter Ring test No Detection and enumeration of Campylobacter spp. in broiler meat Ingrid Hansson, EU-RL Campylobacter

2 Ring test No 7 April 2010 The ring test involved: Detection of Campylobacter spp. by enrichment and culture on selective agar plates Subculture of suspected Campylobacter colonies on non selective agar plates Confirmation and speciation by phenotypic and/or molecular methods Enumeration by direct culture on selective agar plates

3 Ringtest No Culture media; mccda and other selective media Incubation time and temperatures; according to ISO 10272, Part 1: Detection method and Part 2: Colonycount technique.0 Confirmation tests; morphology, motility, growth at 25ºC (microaerobic) and oxidase Species identification; Catalase, Hippurate hydrolysis, Indoxyl acetate hydrolysis, Nalidixic acid (optional), Cephalotin (optional), PCR (optional)

4 No of days for transport of the ring tests to the NRLs Days Transport of the ring test: No of NRLs Starting the analysis: No of NRLs The samples were distributed from the CRL on the 12 th of April. The 10 vials and the broiler meat were placed in a plastic container (DG container 500, Noax Sys AB), which were placed in a cool insulation box (Noax Sys AB) and delivered by CLX Cargo Logistics Express AB.

5 Ring test No 7, April 2010 The ring test included detection and enumeration of Campylobacter in 10 samples consisting of meat (breast fillets) from broilers. The meat should be used together with 10 vials, each containing one capsule with or without Campylobacter spp. Three of the vials contained a strain of E. coli resistance to some microbial substances

6 Description of the 10 vials included in ring test 2010, with the mean number of Campylobacter/samples, (vial together with broiler meat), when testing the samples, before the test. Sample No Species Inoculation Mean No of Campylobacter 1 Campylobacter coli medium dose 3.0 log cfu/g 2 Campylobacter jejuni low dose 1.5 log cfu/g 3 Escherichia coli Campylobacter lari low dose 1.2 log cfu/g 5 Uninoculated - 6 C. jejuni + E. coli medium dose 2.7 log cfu/g 7 Campylobacter coli low dose 1.8 log cfu/g 8 Campylobacter jejuni medium dose 2.7 log cfu/g 9 C. coli + E. coli medium dose 3.4 log cfu/g 10 Campylobacter lari medium dose 2.6 log cfu/g

7 Ringtest; Detection and enumeration Second selective media used by NRLs; 12 Karmali 7 Skirrow 4 Preston agar 4 Butzler 4 Campyfood ID 1 AHB 1 Campy Cefex 1 CAT

8 Ringtest; Detection and enumeration Microaerobic atmosphere ; 23 Gas-generating kits 11 Microaerobic incubator 5 Anoxomat system 1 Zip-lock bags filled with gas 1 Injections in jars.

9 Ringtest; Detection and enumeration Microaerobic atmosphere ; 23 Gas-generating kits 11 Microaerobic incubator 5 Anoxomat system 1 Zip-lock bags filled with gas 1 Injections in jars.

10 Ring test No No of participted NRL Enrichment in Bolton broth 24 hours* Enrichment in Bolton broth 48 hours Enrichment in Preston broth 24 hours mccda 2 nd selective medium mccda 2 nd selective medium mccda 2 nd selective medium* No of NRL s = Optional

11 Correct detection in the ring test 2010 No of correct samples BB 24h mccda NRL s BB 24h 2 nd NRL s BB 48h mccda NRL s BB 48h 2 nd NRL s PB 24h mccda NRL s PB 24h 2 nd NRL s Not done

12 Reported results from detection Sample No BB 24h mccda +/ng*/other BB 24h 2 nd +/ng*/other BB 48h mccda +/ng*/other BB 48h 2 nd +/ng*/other 1. C. coli 19 / - / 1 19 / - / - 33 / - / 1 33 / - / - 2. C. jejuni 18 / 2 / - 18 / 1 / - 34 / - / - 32 / 1 / - 3. E.coli 2 / - / 18 1 / 4 / 14 3 / 2 / 29 3 / 4 / C. lari 17 / 3/ - 17 / 2/ - 32 / 2 / - 31 / 2 / - 5. Neg - / 20/ - - / 19 / - - / 34 / - - / 33 / - 6. C. j + E.c 11 / 1 / 8 13 / 1 / 5 24 / 2 / 8 25 / 3 / 5 7. C. coli 20 / - / - 19 / - / - 34 / - / - 32 / 1 / - 8. C. jejuni 19 / - / 1 17 / 1 / 1 33 / - / 1 32 / - / 1 9. C.c + E.c 16 / 1 / 3 15 / 2 / 2 31 / 1 / 2 27 / 2 / C. lari 19 / 1 / - 19 / - / - 33 / 1 / - 31 / - / 2

13 Reported results from detection Sample No PB 24h mccda +/no growth/other PB 24h 2 nd +/no growth/other 1. C. coli 29/ 2 / - 21 / 3 / - 2. C. jejuni 28 / 2 / 1 22 / 1 / 1 3. E.coli 3 / 13 / 15 2 / 9 / C. lari 24 / 7 / - 17 / 7 / - 5. Neg - / 31 / - - / 24 / - 6. C. j + E.c 28 / 1 / 2 22 / - / 2 7. C. coli 27 / 4 / - 20 / 4 / - 8. C. jejuni 30 / 1 / - 23 / 1 / - 9. C.c + E.c 29 / 1 / 1 22 / 1 / C. lari 26 / 5 / - 20 / 4 / -

14 Reported results from detection Sample BB 48h mccda BB 48h 2 nd PB 24h mccda No +/no growth/other +/no growth/other +/no growth/other 1. C. coli 33 / - / 1 33 / - / - 29 / 2 / - 2. C. jejuni 34 / - / - 32 / 1 / - 28 / 2 / 1 3. E.coli 3 / 2 / 29 3 / 4 / 26 3 / 13 / C. lari 32 / 2 / - 31 / 2 / - 24 / 7 / - 5. Neg - / 34 / - - / 33 / - - / 31 / - 6. C. j + E.c 24 / 2 / 8 25 / 3 / 5 28 / 1 / 2 7. C. coli 34 / - / - 32 / 1 / - 27 / 4 / - 8. C. jejuni 33 / - / 1 32 / - / 1 30 / 1 / - 9. C.c + E.c 31 / 1 / 2 27 / 2 / 4 29 / 1 / C. lari 33 / 1 / - 31 / - / 2 26 / 5 / -

15 05F1 2A8D D 37CF B5B 07AB 344C 2C14 26 E3 E8C4 31FA EA3F 085B F135 2E 41 3E2C 39EA 3C9D 063A F0C1 36D B DCF D13 F5C9 EB0C Results; Detected/Not detected Campylobacter Results from 34 NRLs BB 48h mccda BB 48h 2nd PB 24h mccda 0

16 1. C. coli med 2. C. jejuni low 3. E. coli med 4. C. lari med 5. Neg 6. C. jejuni + E. coli 7. C. coli low 8. C. jejuni med 9. C. coli + E. coli 10. C. lari med Species identification, phenotyping after enrichment in Bolton Broth 48h and mccda Reported species C. jejuni C. coli C. lari Other No Growth NRLs reported species identification from mccda by phenotyping

17 1. C. coli med 2. C. jejuni low 3. E. coli med 4. C. lari med 5. Neg 6. C. jejuni + E. coli 7. C. coli low 8. C. jejuni med 9. C. coli + E. coli 10. C. lari med Species identification, phenotyping after enrichment in Bolton Broth 48h and 2 nd selective Reported species C. jejuni C. coli C. lari Other No Growth NRLs reported species identification from 2 nd selective agar by phenotyping

18 1. C. coli med 2. C. jejuni low 3. E. coli med 4. C. lari med 5. Neg 6. C. jejuni + E. coli 7. C. coli low 8. C. jejuni med 9. C. coli + E. coli 10. C. lari med Species identification, molecular methods (PCR) Reported species C. jejuni C. coli C. lari Camp spp Negative NRLs reported species identification from mccda by phenotyping

19 05F1 2A8D D 37CF B5B 07AB 344C 2C14 26 E3 E8C4 31FA EA3F 085B F135 2E 41 3E2C 39EA 3C9D 063A F0C1 36D B DCF D13 F5C9 EB0C Correct species identification of Campylobacter* Phenot mccda Phenot 2nd PCR Spec * Spec id after enrichment in BB at 48 h and then selective agar

20 1. C. coli M 2. C. jejuni L 3. E. coli 4. C. lari L 5. Neg 6. C. j. + E. c. 7. C. coli L 8. C. jejuni M 9. C. c. + E. c. 10. C. lari M No of Campylobacter spp. enumerated by 31 NRLs in the 10 samples in Ring Test No 7, April ,5 5 4,5 4 3,5 3 2,5 2 1,5 1,5 False pos: 4 False neg:

21 Ring test No 7, April 2010 Acknowledgement To all Member States, Norway, Switzerland and Iceland and FYROM for participating in the ring test To the staff of the CRL-Campylobacter To the Commission for funding the work of the CRL