Campylobacter in broiler carcasses. Ingrid Hansson CRL Campylobacter Sweden

Transcription

1 Campylobacter in broiler carcasses Ingrid Hansson CRL Campylobacter Sweden

2 Sampling of broiler carcasses One whole carcass per slaughter batch shall be collected after chilling, but before processing Avoid cross-contamination during collection and transport Label the sample with a unique number

3 Transportation of carcasses The carcass shall be placed in a separate sterile plastic bag, avoid cross-contamination The samples shall be kept at +2 to 8ºC The samples shall reach the laboratory within 24 hours

4 Receipt of the samples Examine the sample and transport packaging is intact before using Check the information recorded by the sampler Samples shall be held at +2 to 8ºC Avoid cross contamination between samples and from the environment

5 Sample preparation Neck skin shall be removed together with the skin from one side of the carcass Avoid fat and make a 27g test portion and place into an empty petri dish, and further on in a stomacher bag

6 Initial suspension About 27 g test portion shall be transferred to nine volumes (about 243 ml) buffered peptone water (BPW) brought to room temperature before adding. The mixture shall be treated in a stomacher or pulsifier for approximately one minute.

7 Initial suspension shall be used as : 10 ml shall be transferred to 90 ml enrichment medium for Campylobacter spp. detection 10 ml shall be used for the enumeration of Campylobacter spp. on selective plates The rest of the initial suspension (250 ml) shall be used for the detection of Salmonella spp.

8 Detection of Campylobacter spp. 10 ml shall be transferred to 90 ml enrichment Bolton broth medium for Campylobacter spp. detection Incubate the sample in enrichment microaerobic at 37ºC for 4 to 6 hours then at 41.5ºC for 44 h ± 4h Inoculate with a sterile loop from the culture obtained in the enrichment medium to two different selective medium (mccda and Preston agar) Incubate the plates microaerobic at 41.5ºC for 44 h ± 4h

9 Quantification of Campylobacter spp. To estimate low numbers (10 cfu/g) of campylobacter, distribute 1 ml of initial suspension on a large petri dishes (14 cm Ø) of mccda, prepare duplicates by using two plates Carefully spread the inoculum uniformly over the surface of the agar plate, as quickly as possible, by using a sterile spreader until there is no longer any liquid visible on the agar surface

10 Quantification of Campylobacter spp. 1.0 ml of the 10 ml of the initial suspension shall be transferred to further dilutions to allow enumeration of up to 10 6 cfu/g Transfer 0.1 ml of the initial suspension to each of two plates of the mccd agar medium Repeat the transfer of 0.1 ml using further dilutions Carefully spread the inoculum uniformly over the surface of the agar plate, as quickly as possible, by using a sterile spreader until there is no longer any liquid visible on the agar surface

11 Campylobacter in carcasses All incubation shall be performed at 41.5 ± 1ºC for 40 to 48 hours under microaerophilic conditions (gas mixture 10% CO 2 /6% O 2 ) Campygen (Oxoid) Anoxomate

12 Counting and selection of colonies On mccda agar typical Campylobacter colonies are greish, often metallic sheen, flat and moist, with a tendency to spread Select plates containing less than 150 typical or suspect colonies. Choose at random five such colonies for subculturing for the confirmation tests. Streak selected colonies onto a blood agar plate.

13 Confirmation of Campylobacter spp Morphology and motility Suspend one colony in Brucella broth/sodium chloride and examine in microscope. Campylobacter sp is curved bacilli with a spiralling corkscrew motility

14 Confirmation of Campylobacter spp Growth at 25ºC and 41.5ºC Suspected colonies on three blood agar plates, incubate one plate at 25ºC micro-aerobic atmosphere the second 41.5ºC aerobic atmosphere and the third in 41.5ºC micro-aerobic atmosphere for further confirmation. Examine the plates for visible growth of Campylobacter colonies

15 Characteristic of Campylobacter spp Morphology: Motility: small curved bacilli characteristic corkscrew Micro-aerobic growth at 25ºC: negative Aerobic growth at 41.5ºC: Oxidase: negative positive

16 Confirmation of Campylobacter spp Detection of oxidas Take a portion of a well-isolated colony and streak selected colonies onto a filter paper moistened with oxidase reagent. Campylobacter spp are oxidase positive with a mauve, violet or deep blue colour within 10 s.

17 Identification of Campylobacter spp Detection of catalase A loop of culture into a drop of hydrogen peroxide solution on a clean microscopic slide The test is positive if bubbles appear within 30 s Confirm by using positive and negative control

18 Identification of Campylobacter spp Detection of sensitivity to nalidixic acid and to cephalotin A loop of culture into a drop a suspension in Brucella broth of a density 0.5 on the McFarland scale Dilute this suspension with the Brucella broth Flood (the suspension) the surface of a Mueller Hinton agar plate Leave in 5 min, then drain off

19 Identification of Campylobacter spp Detection of sensitivity to nalidixic acid and to cephalotin Dry the plates at 37ºC in 10 min Place a disc of nalidixic acid and a disc of cephalothin Incubate the plate microaerobic at 37ºC for 22±2 hours, with lids uppermost - Growth in the contact with the disc resistant -A zone of any size due to inhibition of growth - susceptible

20 Identification of Campylobacter spp Detection of hippurate hydrolysis A loop of culture into a 0.4 ml sodium hippurate solution Mix thoroughly and incubate in a water bath for 2 h at 37ºC Add 0.2 ml ninhydrin solution on the top. Do not shake Additional incubation of 10 min in the water bath A dark violet colour indicates a positive reaction No colour or a pale violet colour indicates a negative reaction Use negative and positive controls

21 Identification of Campylobacter spp Detection of indoxyl acetate hydrolysis Place a loop ful campylobacter colony on an indoxyl acetate disc Add a drop of sterile water If the indoxyl acetate is hydrolysed, a dark blue colour occurs within 5 to 10 min Use positive and negative controls

22 Species identification C. jejuni C. coli C. lari C. upsaliensis Catalase: or slight Nalidixic acid: S* S* R/S S Cephalothin: R R R S Hydrolysis of hippurate: Indoxyl acetate: * Both sensitive and resitant strains exist

23 Thank you for your attention Photo: Tapio Nikkilä, Leif Ljung