The Preston broth was prepared according to the recommendations of the Nordic Committee on Food Analysis (1
). Briefly, 25 g of nutrient broth no. 2 (catalog no. CM67; Oxoid, Basingstoke, United Kingdom) was dissolved in 1,000 ml of distilled water, autoclaved for 15 min at 121°C, and, after cooling to 50°C, was combined aseptically with a mixture of 50 ml lysed horse blood (Danish Institute for Food and Veterinary Research [DFVF], Copenhagen, Denmark), 4 ml of modified Preston Campylobacter
selective supplement (catalog no. SR204E; Oxoid), and 4 ml of Campylobacter
growth supplement (catalog no. SR084E; Oxoid).
The Bolton broth was prepared according to the manufacturer's recommendations. Briefly, 13.8 g of Bolton broth (catalog no. CM0983; Oxoid) was dissolved in 500 ml of distilled water, autoclaved for 15 min at 121°C, and, after cooling to 50°C was combined aseptically with a mixture of 25 ml lysed horse blood (DFVF) and one vial of Bolton broth selective supplement (SR0183E) reconstituted as directed.
The modified charcoal cefoperazone deoxycholate agar (mCCDA) was prepared according to the manufacturer's recommendations. Briefly, 45.5 g of Campylobacter blood-free selective agar (catalog no. CM0739; Oxoid) was suspended in 1,000 ml of distilled water and brought to boil to dissolve completely. The agar was autoclaved at 121°C for 15 min and, after cooling to 50°C, was combined aseptically with two vials of CCDA selective supplement (catalog no. SR0155; Oxoid) reconstituted twice with 2 ml sterile distilled water.
The Preston agar was prepared according to the manufacturer's recommendations. Briefly, 18.5 g of Campylobacter agar base (catalog no. CM0689; Oxoid) was suspended in 475 ml of distilled water and brought to boil to dissolve completely. The agar was autoclaved at 121°C for 15 min, and after cooling to 50°C was combined with a mixture of 25 ml lysed horse blood (DFVF) and one vial of Preston Campylobacter selective supplement (catalog no. SR0117; Oxoid) reconstituted with 2 ml of 50/50 acetone/sterile distilled water.
The brain heart infusion medium (BHI) was prepared according to the manufacturer's recommendations and consisted of 37 g/liter BHI (Difco, Sparks, MD) with 5% (vol/vol) calf blood (DFVF) and 0.5% agar added.
Campylobacter real-time PCR.
Real-time TaqMan PCR was performed in a RotorGene 3000 (Corbett Research, Australia) in 0.2-ml PCR tubes as described previously (12
), except for the addition of 2.0 μl/reaction of 87% pure glycerol (Merck A/S, Denmark) and 1.0 μl/reaction of 12.5 mM dNTP mix with dUTP (Applied Biosystems, Foster City, CA), enabling uracil-N
-glycosylase treatment to prevent carryover contamination. Each PCR analysis included a positive DNA control, a negative DNA control, a nontemplate control (NTC), and an IAC. The cutoff level and definition of positive/negative responses were exactly as described previously (12
). Samples with a threshold cycle (CT
) response below 40 were considered to be positive.
As shown in Table , the following samples were collected in Denmark: 99 pooled fecal samples on shoe covers from rearing houses, 101 cloacal swab samples from an abattoir, 102 samples of neck skin from an abattoir, and 100 samples of neck skin from the retail sector. The shoe cover, cloacal swab, and neck skin samples from the abattoir originated from flocks at 34 different farms. Approximately half of the samples were taken in the spring, when the prevalence of Campylobacter spp. in chicken flocks is expected to be low in Denmark (approximately 20%), and the other half were taken in the early autumn, when the prevalence is expected to be high (approximately 60%). Approximately half of the neck skin samples from the retailers were taken from chicken flocks that were reported to be Campylobacter positive by the supplier, and the other half from flocks that had tested negative.
Samples from the chicken production chain with positive isolates of thermotolerant Campylobacter
Fecal samples were collected on disposable shoe covers in the rearing houses and shipped at ambient temperature to DFVF. On arrival, the shoe cover samples were weighed and added to 1:10 (wt/vol) physiological saline and homogenized for 60 s in a stomacher.
The cloacal swab samples were taken at the abattoir from 12 or 13 individual broilers on arrival at the abattoir. The swabs were stored in tightly capped 15-ml plastic tubes with BHI and shipped to DFVF. On arrival, 20 samples were pooled into 1 sample and homogenized manually in 30 ml physiological saline for 60 s. Because of the high background flora in the fecal samples, growth during transportation (one day) was regarded as insignificant to the outcome of the test.
Approximately 40 g of chicken neck skin was cut and pooled into a plastic bag at the slaughter line. The samples were transferred to a stomacher bag, sealed, and shipped on ice packs to DFVF by mail. On arrival, the samples were diluted 1:1 in physiological saline and homogenized for 60 s in a stomacher.
For retail samples, fresh (nonfrozen) chickens were purchased; 40 g neck skin was cut at the laboratory and diluted 1:1 in physiological saline and homogenized for 60 s in a stomacher.
Reference culture methods.
The detection of Campylobacter
spp. was conducted in accordance with the recommendations from the Nordic Committee on Food Analyses (1
) and the International Organization for Standardization (3
). All samples were enriched in Bolton and Preston broths (1:10) at 42 ± 0.5°C in a microaerobic atmosphere (6% O2
, 7% CO2
, 7% H2
, and 80% N2
) for 24 h before 100 μl was plated onto Preston agar and mCCDA. The agar plates were incubated at 42 ± 0.5°C in a microaerobic atmosphere for 48 h. From the selective agar, five typical thermotolerant Campylobacter
colonies were selected for verification by an internationally validated gel-based PCR assay for the identification of Campylobacter jejuni
, C. coli
, and C. lari
). In the absence of typical colonies, five nontypical colonies were selected for PCR verification.
DNA extraction by magnetic beads.
From the fecal sample suspensions (cloacal swabs and shoe covers), 1-ml aliquots were drawn for DNA extraction before enrichment. From the neck skin samples, 1-ml aliquots were drawn for DNA extraction after overnight enrichment in Bolton broth. The samples were centrifuged at 16,000 × g
for 7 min at 4°C, and DNA extraction was performed on a KingFisher processor (Thermo Lab Systems, Helsinki, Finland) using an automated, magnetically based separation and DNA isolation kit for blood, cells, and tissue (Thermo Lab Systems) as specified by the manufacturer. Briefly, the sample pellet was resuspended in lysis buffer and transferred to a 96-well plate (Thermo Lab Systems) containing magnetic particles, washing buffers, and elution buffer. The DNA extraction program consisted of two salt-buffer washing steps and two alcohol-buffer washing steps, followed by a final elution step (for a detailed protocol, see the Rapid Diagnostic Group website [http://www.pcr.dk/Innovations_pcr/innovations_pcr_startside.htm
]). A total of 5 μl of the extracted DNA was used as the template in the real-time PCR.
Statistical data analysis.
The comparative validation study included three test characteristics: relative accuracy, sensitivity, and specificity (5
) (see Table ). The relative accuracy is defined as the degree of correspondence between the response obtained by the alternative method and the reference method on identical samples, as follows: (PA + NA + FP) × 100/(PA + NA + TP + FN + FP), where PA refers to positive agreement, NA to negative agreement, FP to false positives, TP to true positives, and FN to false negatives. The relative sensitivity is defined as the ability of the alternative method to detect the target microorganism compared to the reference method, as follows: (PA + TP) × 100/(PA + FN). The relative specificity is defined as the ability of the alternative method not to detect the target microorganism when it is not detected by the reference method, as follows: (NA × 100)/(NA + FP).
Comparison of the results obtained by the real-time PCR and the reference culture methods
To compare the performances of Bolton and Preston broths, the number of positive responses obtained from each was subtracted, giving one difference for each combination of sample type and agar type. The Wilcoxon signed-rank test with a continuity correction was applied to these differences to test whether the two enrichment broths differed significantly from each other (14
). The calculations where performed using Splus software, professional edition version 6.1.
A collaborative trial involving nine national laboratories was performed to evaluate the robustness and reproducibility of the real-time PCR method testing identical samples.
The collaborative trial was designed and conducted according to the recommendations from NordVal (5
). The nine participating laboratories received pellets from 18 coded 1-ml samples, including 6 chicken neck skin samples, 6 shoe cover samples, and 6 cloacal swab samples (see Table ). The samples were spiked in duplicate with C. jejuni
CCUG 11284 at three levels, making it possible to assess the usefulness of the method at various infection levels. The shipment included a positive DNA control (1 μg/ml C. jejuni
CCUG 11284) and a negative DNA control (1 μg/ml Arcobacter butzleri
CCUG 30485), a ready-to-use PCR mixture with added IAC, and reagents for the magnetically based DNA extraction. To minimize any interlaboratory variability (not attributable to the method performance), we supplied all the reagents necessary. Each participant received a detailed protocol describing the DNA extraction, real-time PCR setup, real-time PCR run, and data analysis and a reporting form to record the obtained PCR results to return to DFVF. The participants were also asked to return a file containing the real-time PCR runs.
Collaborative trial: real-time PCR results for the detection of thermotolerant Campylobacter spp. in spiked samples from chickensa
A second collaborative trial, comprising eight participating laboratories, was subsequently performed only on shoe cover samples. The second trial was performed exactly as the first one, except for using a modified DNA extraction protocol, with an increased amount of paramagnetic particles.
The samples for the collaborative trial were prepared as described above (“Sample preparation”). Regarding the neck skin samples, one broth was left unspiked, one was spiked with 1 to 10 CFU/100 ml, and one with 10 to 100 CFU/100 ml, and incubated at 42 ± 0.5°C for 24 h in a microaerobic atmosphere. After the enrichment, 1-ml aliquots were drawn and centrifuged at 16,000 × g for 7 min at 4°C. The supernatant was discarded, and the pellet kept at −80°C until shipped on ice to the trial participants.
From both shoe cover and cloacal swab samples, 1-ml aliquots were drawn, spiked with 0, 100 to 500, or 1,000 to 2,000 CFU/ml, and centrifuged at 16,000 × g for 7 min at 4°C. The supernatant was discarded and the pellet kept at −80°C until shipped on ice to the trial participants.
status of all samples was confirmed at DFVF by the reference culture method according to International Organization for Standardization publication no. 10272-1 (3
) and Nordic Committee on Food Analyses publication no. 119 (1
) prior to and after spiking. The stability of the samples was examined using real-time PCR (12
) immediately after spiking, prior to commencement of the collaborative trial, and during the period of analysis, to verify the continued detection of Campylobacter
. The possible detrimental effect of shipping time at ambient temperature on the real-time PCR results was investigated, and no detrimental effect was found.
At the participating laboratories, DNA was extracted from the samples by using the Magnesil KF genomic system (Promega, Madison, WI) on an automated DNA extraction platform of the laboratories choosing but following the KingFisher protocol described above. In the second trial, the amount of paramagnetic particles was increased from 20 μl/sample to 75 μl/sample. A total of 5 μl purified DNA was used as the template in the real-time PCR.
Real-time PCR at the participating laboratories was performed on a Mx3000 or Mx4000 real-time PCR system (Stratagene, La Jolla, CA), ABI-PRISM 7000 or 7900 (Applied Biosystems, Foster City, CA), RotorGene 3000 (Corbett Research, Mortlake, Australia), or an iCycler thermal cycler (Bio-Rad, Hercules, CA). The participating laboratories were asked to use the blank (NTC), the process blank (a Campylobacter-negative sample processed throughout the entire protocol), and the negative control to assign the cutoff line and report back the CT values.
The test reports and the real-time PCR analyses from the participating laboratories were carefully evaluated on return to the expert laboratory, and the results were approved for inclusion in the statistical analysis, unless they fell into at least one of the following two categories: (i) obvious performance deviation from the protocol and (ii) presence of target amplicons in the negative control results, indicating cross contamination at the participating laboratory.
The results obtained in the collaborative trial were analyzed according to the recommendations from NordVal (5
). The relative specificity was calculated for the unspiked samples by the following equation: (1 − [FP/N
−]) × 100%, where N
− refers to the total number of unspiked samples. The relative sensitivity was calculated for each level of spiking by the following equation: (TP/N
+) × 100%, where N
+ refers to the number of spiked samples. The relative accuracy was calculated for all levels of spiking by the following equation: ([PA + NA + FP]/N
) × 100%, where N
refers to the number of samples tested.
On-site validation on flock basis at abattoir.
At a major abattoir, the real-time PCR method was validated against a routinely used gel-based PCR method approved previously by the Danish authorities to monitor production. The two methods were performed in parallel on 400 pooled cloacal swab samples collected from 20 chicken flocks. Each cloacal swab sample was a pool of 25 swabs. Thus, 20 pooled samples represented 500 chickens per flock. The samples giving a positive signal had CT values between 17 and 39. All samples giving negative signals had CT values of >40 (see Table ).
On-site results of the real-time PCR method on cloacal swabs, validated against an approved gel-based PCRa