An evaluation of survival and detection of Campylobacter jejuni and C. coli in broiler caecal contents using culture-based methods


J.D. Rodgers, Department of Food and Environmental Safety, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK. E-mail:


Aims:  To evaluate the culture specifications of the 2008 EU baseline survey for Campylobacter spp. in broiler flocks at slaughter, by assessing the detection of thermophilic Campylobacter in chicken caecal contents by culture on selective agar with or without enrichment culture. Additionally, to assess the impact of sample storage time on Campylobacter detection.

Methods and Results:  Serial dilutions of pooled caeca samples in phosphate-buffered saline or Campylobacter-negative caecal contents were cultured micro-aerobically at 41·5°C on mCCDA, Karmali and Preston agars before and after enrichment in Exeter broth. Direct culture on mCCDA showed a higher isolation rate than for Karmali or Preston agars, but a similar isolation rate to enrichment. Enumeration of samples showed the numbers of viable bacteria dropped slightly during storage.

Conclusions:  Direct culture on mCCDA was the most sensitive method for detection of Campylobacter, and samples with 104 CFU g−1 were still detectable after 6 days.

Significance and Impact of the Study:  Comparison of prevalence results from the 2008 EU baseline survey will need careful interpretation as the different media specified vary in their sensitivity to detect thermophilic Campylobacter. Delayed culture for up to 80 h after collection should have little impact on detection rate.


Campylobacteriosis in humans is a significant and increasing problem in the European Union and in a global context (Anon. 2009). Poultry products that are contaminated with thermophilic Campylobacter are considered one of the main sources of infection for humans, and this contamination is linked to the infection status of poultry flocks entering the abattoir (Rosenquist et al. 2006).

The detection of Campylobacter infection in broiler flocks has previously been described in many countries (Berndtson et al. 1996; Evans and Sayers 2000; Musgrove et al. 2001; Lund et al. 2003; Stern et al. 2004; Russa et al. 2005; Barrios et al. 2006; Van Overbeke et al. 2006; Arsenault et al. 2007; McDowell et al. 2008; Woldemariam et al. 2008). However, the un-harmonized sampling strategies, transport conditions and analytical methods make it difficult to compare findings between these studies (Wagenaar et al. 2008). The problems of poor harmonization and comparability were addressed in the mandatory baseline survey for the prevalence of Campylobacter spp. in broiler flocks at slaughter carried out by Member States of the European Union in 2008 (hereafter abbreviated to EU survey). This EU survey used a standardized sampling protocol and culture method laid out in the technical specifications (Anon. 2007). The methodology was based on the direct culture of a pooled caecal sample onto a selective medium incubated at 41·5°C in a micro-aerobic atmosphere. To the best of our knowledge, no validation of the methodology used for the EU survey has been described at the time of this study, and there is limited published information on the performance of direct culture for Campylobacter from chicken caecal contents using different media types. Enrichment of caecal material in Bolton broth has been shown to be less effective than direct culture for isolating thermophilic Campylobacter spp. (Musgrove et al. 2001; Williams et al. 2008); however, no comparisons with direct culture of caecal samples have been published for Exeter enrichment broth. Exeter broth has been used successfully on caecal samples (Newell et al. 2001) and was reported to maximize Campylobacter recovery from food, milk and water samples (Humphrey et al. 1995). In addition, the performance of isolation techniques for Campylobacter can be affected if the organism has reduced viability through stress encountered during sample storage or because of overgrowth of other bacterial populations (Karenlampi and Hanninen 2004). The EU methodology specified that samples should be stored at 2–8°C and analysed within a deadline (80 h from sample collection). Studies with turkey caecal material and human faecal content suggest that Campylobacter may still remain detectable if held beyond the analysis deadline (Blaser et al. 1980; Luechtefeld et al. 1981); however, this has not been shown for chicken caecal contents.

In this study, naturally contaminated chicken caecal contents were used to assess the performance of direct culture for thermophilic Campylobacter as specified in the 2008 EU survey protocol for mCCDA, Karmali and Preston agars and also to compare direct culture with enrichment culture using Exeter broth. This study also assessed the survival of Campylobacter present in caecal samples during chilled storage. The information was used to evaluate the culture specifications of the EU method for monitoring Campylobacter in broiler flocks.

Materials and methods

Caecal samples

Caecal contents from seven slaughter batches collected in the United Kingdom for the EU survey were included in the study (Table 1). For each batch, a pooled caecal sample was created by homogenizing 0·02 g of caecal content from each of ten individual caeca into 2 ml of phosphate-buffered saline (PBS), 0·1 mol l−1, pH 7·2. The Campylobacter status of these batches was initially determined by testing the caecal pool sample by using mCCDA direct culture plus enrichment, along with enumeration and confirmation methods as described later. All batches were processed within the analysis deadline of 80 h from sampling, and the day of processing was considered as day 0.

Table 1.   Initial direct culture and enumeration results from slaughter batches
BatchDirect cultureSpecies*Enumeration (CFU g−1)
  1. *Five colonies speciated per positive batch.

  2. †Sample negative after culture on mCCDA before and after enrichment.

APositiveC. jejuni4·5 × 106
BPositiveC. coli1·2 × 109
CPositiveC. jejuni2·0 × 108
DPositiveC. jejuni1·8 × 108
EPositiveC. jejuni1·4 × 108
FPositiveC. jejuni2·4 × 108

Sample preparation and storage at 4°C

Pooled caecal samples from batches A, B and C (Table 1) were diluted tenfold in caecal content that was negative for Campylobacter spp. (batch G), to create samples that contained differing levels of Campylobacter in a caecal matrix (caecal content-diluted samples). From each batch, a selection of diluted samples with high, medium and low levels of Campylobacter was split into two aliquots for storage.

Pooled caecal samples from batches D, E and F were diluted tenfold in PBS to create PBS-diluted samples that contained differing levels of Campylobacter. PBS-diluted samples containing c. 108, 106, 104 and 102 CFU g−1 from each batch were split into seven aliquots for storage.

Detection of Campylobacter spp.

The direct culture method was based on EU specification (Anon., 2007). Briefly, 10 μl of the sample was streaked directly onto selective agar plates (mCCDA, Karmali or Preston)(Table 2). All plates and broths were incubated at 41·5 ± 1°C, in a micro-aerobic atmosphere (84% N2/10% CO2/6% O2) generated in a gas charged incubator (Heraeus; Thermo, Basingstoke, UK) for 24–48 h. For enrichment culture, 10 μl of the sample was inoculated into 9 ml of Exeter broth. After 48 h of incubation, 10 μl of broth was streaked onto selective media and incubated as described. Plates were examined for grey, flat, irregular and spreading colonies typical of Campylobacter (Fitzgerald et al. 2008). One putative colony was subcultured from each plate onto sheep blood agar for confirmation as Campylobacter spp., except for the initial testing of caecal samples when five colonies were identified.

Table 2.   Composition of media used in this study
MediaBase* and blood per litreSupplements per litre
  1. *Base and supplements supplied by Oxoid, Basingstoke, UK.

mCCDA45·5 g Campylobacter blood-free selective agar base (CM0739)CCDA selective agar supplement (SR0155). Contains: 32 mg cefoperazone, 16 mg amphotericin B
Karmali43 g Campylobacter selective agar base (Karmali CM0935)Modified Karmali selective supplement (SR0205). Contains: 32 mg cefoperazone, 20 mg vancomycin, 10 mg amphotericin
Preston37 g Campylobacter agar base (CM0689) at 37 g 50 ml lysed horse bloodPreston campylobacter selective supplement (SR0117) Contains: 5000 IU polymyxin B, 10 mg rifampicin, 10 mg trimethoprim, 100 mg cyclohexamide
ExeterNutrient broth (CM0001) at 13 g 50 ml lysed horse blood250 mg sodium metabisulphite, 250 mg sodium pyruvate, 250 mg ferrous sulfate, 10 mg trimethoprim, 10 mg rifampicin, 125 000 IU polymyxin B, 2 mg fungizone, 15 mg cefoperazone
SBA40 g Blood agar base (CM0055) 70 ml sheep blood1000 mg cyclohexamide

Enumeration of Campylobacter spp.

A tenfold dilution series of the sample was made in PBS to 10−6, and 100 μl of each dilution was then plated onto duplicate mCCDA plates. To facilitate the counting of single colonies, plates were incubated for 48 h at 41·5 ± 1°C in a micro-aerobic atmosphere (87% N2/5% CO2/3% H2/5% O2) in a MACS-VA500 workstation (Don Whitley, Shipley, UK). Typical Campylobacter colonies were counted, and the least dilute pair of plates that contained an average of between 15 and 200 colonies was used to calculate the number of Campylobacter CFU g−1 of caecal content. A colony from each plate used for calculation was confirmed as Campylobacter spp.

Confirmation of Campylobacter spp.

DNA preparations of suspect colonies were prepared and tested based on the PCR method of (Best et al. 2003). Each reaction mixture contained 12·5 μl of HotStarTaq mastermix (Qiagen, Crawley,UK), 2 μl of template/target DNA, 25 mmol l−1 MgCL2, 20 μmol l−1 of mapA and ceuE primer pairs (Sigma-Aldrich, Dorset, UK) and 5 μmol l−1 of each ceuE and mapA probe (Sigma-Aldrich) and was made up to 25 μl in nuclease-free water. The reaction was performed on the Stratagene MX3005p (Stratagene, La Jolla, CA, USA), amplification consisted of an initial hold at 95°C for 15 min, then 40 cycles of 94°C for 30 s and annealing/extension at 60°C for 30 and 30 s at 72°C. Fluorescence was read at 60°C, and a cycle threshold of between 10 and 32 was viewed as a positive result for either the mapA probe (C. jejuni) or ceuE probe (C. coli).

Detection of Campylobacter spp. in caecal content-diluted samples on different media

On day 0, samples from batches A, B and C were tested by direct culture on mCCDA, Karmali and Preston agar and on each medium following Exeter broth enrichment.

Detection of Campylobacter spp. in PBS-diluted samples stored at 4°C

On day 0, samples from batches D, E and F were tested by direct culture on mCCDA and by Exeter broth enrichment followed by culture on mCCDA. Sample aliquots were tested daily from 1 to 6 days after storage.

Quantitative studies of Campylobacter spp. in samples stored at 4°C

On day 0, samples from all batches were enumerated. Aliquots of caecal content-diluted samples were enumerated after 4 days storage. Aliquots of the PBS-diluted samples were enumerated daily from 1 to 6 days after storage.

Statistical analysis

The isolation rates from direct culture on mCCDA were compared with isolation rates from the different selective media before and after enrichment by Chi-squared (χ2) analysis using the McNemar test (Feldsine et al. 2002). At a 5% level of confidence, the hypothesis that the isolation rates for mCCDA direct culture and another method could not be considered significantly different was accepted if χ2 < 3·84.


Detection of Campylobacter spp. in caecal content-diluted samples on different media

Campylobacter spp. were detectable by all media in all batches by direct culture of the undiluted sample (Table 3). Direct culture on mCCDA or Karmaili agar detected Campylobacter spp. in samples that contained c. 102–103 CFU g−1 but Preston was less sensitive with positive detection limited to samples containing c. 105 and 106 CFU g−1. Direct culture on mCCDA detected the greatest number of positive samples, Karmali showed a slightly lower detection rate, and Preston agar was markedly lower than mCCDA (Table 4).

Table 3. Campylobacter detected in caecal content-diluted samples by direct culture and following enrichment culture on different media types
Batch (initial count)DilutionDirect cultureEnrichment culture
A (4·5 × 106 CFU g−1)NeatC. jejuniC. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−1C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−2C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−3C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−4C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−5C. jejuniC. jejuni
B (1·2 × 109 CFU g−1)NeatC. coliC. coliC. coliMixedC. jejuniC. jejuni
10−1C. coliC. coliC. coliMixedMixedMixed
10−2C. coliC. coliC. coliMixedC. jejuniC. jejuni
10−3C. coliC. coliC. coliMixedMixedMixed
10−4C. coliC. coliC. coliC. jejuniMixedC. jejuni
10−5C. coliMixedC. jejuniMixedC. jejuni
10−6C. coliC. coli
10−7C. coli
C (2·0 × 108 CFU g−1)NeatC. jejuniC. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−1C. jejuniC. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−2C. jejuniC. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−3C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−4C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−5C. jejuniC. jejuniC. jejuniC. jejuniC. jejuni
10−6C. jejuniC. jejuniC. jejuni
Table 4. Campylobacter isolation by direct culture on mCCDA compared with other test methods
TestSampleNPos on mCCDA and Neg for testNeg on mCCDA and Pos for testPos on mCCDA and Pos for testχ2
  1. EN, enrichment culture; Pos, positive; Neg, negative.

  2. *Test method isolation rate significantly different from that of direct culture on mCCDA (P < 0·05).

MCCDA (EN)Caeca3021171·3
Karmali (EN)Caeca3022170·2
Preston (EN)Caeca3022170·2
MCCDA (EN)Phosphate-buffered saline9660718·2*

Enrichment followed by plating onto mCCDA, Karmali or Preston agars was able to detect similar levels of Campylobacter spp. across the batches with 29/30 test agreements (Table 3). No significant difference in detection was observed between direct culture on mCCDA and enrichment culture onto mCCDA, Karmali or Preston (Table 4). The comparison of direct culture and enrichment appeared variable between batches (Table 3). The greatest difference between direct culture and enrichment was observed in Batch B, where enrichment could not detect samples containing lower than c. 104 CFU g−1, whilst direct culture could detect as low as c. 102 CFU g−1.

Detection of Campylobacter spp. in PBS-diluted samples stored at 4°C

Prior to storage, Campylobacter spp. were detectable in all batches by both direct and enrichment culture in samples containing c. 103, 102 and 101 CFU g−1 for batches D, E and F, respectively (Table 5). Campylobacter spp. were detectable in all batches during the 7 days of the study for samples containing c. 104 CFU g−1, however, none of the samples containing c. 102 CFU g−1 tested positive from day 2 onwards. Overall, the detection rate by direct culture on mCCDA was higher than for enrichment (Table 4).

Table 5. Campylobacter detected in phosphate-buffered saline-diluted samples by direct culture and following enrichment culture on mCCDA after storage at 4°C for 0–6 days
Batch (initial count)DilutionDays of storage at 4°C
Day 1Day 2Day 3Day 4Day 5Day 6Day 7
  1. CFU g−1, colony forming units per gram of caecal content; D, direct culture; E, enrichment culture; +, positive; −, negative; n/t, not tested.

D (1·8 × 108 CFU g−1)Neat++++++++++++++
E (1·4 × 108 CFU g−1)Neat++++++++++++++
F (2·4 × 108 CFU g−1)Neat++++++++++++++

Quantitative studies of Campylobacter spp. in samples stored at 4°C

Based on the counts from day 0, the average counts of Campylobacter were slightly lower than expected after 4 days storage in both caecal-content and PBS-diluted samples (Fig. 1). The drop in count was more apparent in samples that had initially contained lower levels of Campylobacter, and the reduction was greater in PBS-diluted samples. Over the 7 days of the experiment, the average numbers of Campylobacter detected in PBS-diluted samples reduced slightly (Fig. 2).

Figure 1.

 Average observed counts vs expected counts in samples stored at 4°C for 4 days. Dashed Line, trend-line for no reduction in counts; solid circles, caecal content-diluted batches (± standard deviation); open circles, phosphate-buffered saline-diluted batches.

Figure 2.

 Average counts in phosphate-buffered saline -diluted samples stored at 4°C from zero to 6 days. Solid circles, samples with c. 108 CFU g−1 in caecal content-diluted batches (± standard deviation); open circles, samples with c. 106 CFU g−1; solid triangles, samples with c. 104 CFU g−1; open triangles, samples with c. 102 CFU g−1.

Speciation of isolates recovered from batches by the different tests

All Campylobacter isolated by either direct culture or enrichment from batches A, C, D, E and F were shown to be C. jejuni regardless of the detection method employed (Tables 1, 3 and 5). The five colonies isolated during initial testing of batch B (direct culture) were indentified as C. coli (Table 1). Most samples from batch B that tested positive by direct culture were identified as having C. coli only, except for one mixed (C. jejuni and C. coli) sample from a Karmali plate (Table 3). None of the 18 samples from Batch B that tested positive by enrichment were identified as having exclusively C. coli. Ten of the samples were identified as mixed, and in the remainder, only C. jejuni was detected.


The culture specifications for the 2008 EU survey allow a choice of selective agar to be used, mCCDA, Karmali or Preston. Modified CCDA has been used to isolate Campylobacter directly from chicken caeca (Russa et al. 2005; Williams et al. 2008; Woldemariam et al. 2008) and from caeca after enrichment in Exeter broth (Newell et al. 2001). Karmali agar has been used for direct isolation from chicken faecal samples (Denis et al. 2001), and Preston agar has been used following enrichment of chicken caecal contents (Berndtson et al. 1996). However, there is limited evidence on the performance of direct culture from caecal contents for these selective media and therefore, this study aimed to provide some indication of the sensitivity of each method.

This study has shown that direct culture of caecal contents on mCCDA on day 0 could detect Campylobacter in samples containing as low as c. 101 CFU g−1 of caecal content with c. 102 CFU g−1 being the lowest level detected in most batches. These findings are similar to detection levels reported for direct culture of chicken and human faecal samples using mCCDA (Lawson et al. 1998; Lund et al. 2003). Direct culture on Karmali agar had similar performance to mCCDA, this was not surprising as both media are similar in composition and have shown comparable performance for direct culture of chicken carcase rinses (Oyarzabal et al. 2005). Preston agar was less effective than either mCCDA or Karmali with the growth of other competing bacteria making the identification of Campylobacter colonies difficult. Preston agar has a different composition and has been reported as being less selective and less effective for direct isolation of Campylobacter from human faecal samples than mCCDA and Karmali agars (Merino et al. 1986; Gun-Munro et al. 1987).

An enrichment step may increase the sensitivity of the test for detection of Campylobacter from caecal content as it has for chicken faeces using Bolton broth and for turkey faeces samples using Preston broth and Campylobacter enrichment broth (Borck et al. 2002; Lund et al. 2004). However, other studies on broilers have suggested that enrichment of caecal samples in Bolton broth and cloacal samples in Preston broth is less sensitive than direct culture (Hald et al. 2000; Musgrove et al. 2001; Williams et al. 2008). Enrichment in Exeter broth has been used to detect Campylobacter from broiler caecal samples (Newell et al. 2001), but the method was not compared with direct culture. In this study, enrichment culture did not compare well with direct culture on mCCDA in PBS-diluted samples. This may be because of stresses incurred during PBS suspension, as stressed and injured Campylobacters can lose viability in some enrichment broth formulations (Corry et al. 1995).

The inclusion of an Exeter broth enrichment step for caecal content-diluted samples did not increase the detection rate relative to direct culture for either mCCDA or Karmali agar. However, a significant improvement was observed for Preston agar. Enrichment of a sample can allow very low levels of Campylobacter to expand rapidly to high levels (108 CFU g−1) (Morales-Rayas et al. 2008) and with Exeter enrichment competing flora should be inhibited as the broth formulation is highly selective for C. jejuni (Humphrey 1989). In this study, it is probable that most positive enriched samples contained high levels of Campylobacter in the absence of competing flora. Therefore, the less selective Preston agar displayed a similar sensitivity to mCCDA and Karmali agars for samples post-enrichment.

In general, the performance of enrichment allowed detection of some samples that contained c. 101 CFU g−1, and this is similar to studies using Preston broth (Denis et al. 2001). However, in batch B, the lowest level detected following enrichment was 104 CFU g−1. The predominant strain present in this batch may have had reduced viability in this broth as Exeter broth is highly selective for C. jejuni. Strain selection may have been on a species level as initial testing by direct culture suggested that C. coli was predominant; however, following enrichment, C. jejuni was the dominant Campylobacter isolated. The antibiotic Polymixin B is reported to be inhibitory to C. coli (Ng et al. 1988) and is present in Exeter broth. The antibiotic is present at a lower level in Preston agar and absent from mCCDA or Karmali agar. Considering the extra costs of enrichment and the potential selection against C. coli strains using Exeter enrichment, it would appear that direct culture is a more appropriate method for large scale prevalence surveys on caecal samples. However, enrichment culture could detect Campylobacter in some samples that were negative by direct culture. Therefore, using both methods in parallel could enhance sensitivity, as suggested in other studies, but at an extra cost (Hald et al. 2000; Maher et al. 2003; Habib et al. 2008).

This study has assessed the survival Campylobacter spp. in naturally contaminated caecal samples rather than samples spiked with laboratory grown cultures as there is potential for Campylobacter strains from different origins to have differing survival properties (Chan et al. 2001). The reduction in the numbers of viable Campylobacter in this study was low over the period of storage. The findings are in agreement with studies on human faeces, bovine faeces and poultry carcases known to be naturally contaminated with Campylobacter (Blaser et al. 1980; Georgsson et al. 2006; Gilpin et al. 2009). PBS-diluted samples at low contamination levels appeared to be less conducive to Campylobacter survival than caecal content-diluted samples suggesting that caecal samples are best left intact until time of testing.

An estimated 90% of Campylobacter-positive broiler flocks in the United Kingdom contain 107 CFU g−1 or higher in caecal contents (unpublished data). This study has shown direct culture on mCCDA or Karmali agar can detect between 102 and 103 CFU g−1, indicating these methods are appropriate for monitoring Campylobacter in UK broiler flocks. Based on the findings from this study, the anticipated reductions in numbers over time appear relatively low, suggesting that samples that miss the EU specified processing deadline by 1 or 2 days, could be considered for inclusion without a major impact on test sensitivity. This may ease the logistical problems associated with large national surveys but any deadline extension would be dependent on samples remaining chilled, as Campylobacter is not as resilient outside the host if stored at ambient temperatures (Blaser et al. 1980; Hudock et al. 2005).

In conclusion, the use of mCCDA or Karmali for direct culture has been demonstrated in this study to be an effective method for detecting both C. jejuni and C. coli in pooled caecal samples from broiler flocks at slaughter. Detection by direct culture on Preston agar is less sensitive, and the benefits of an Exeter broth enrichment step are equivocal. The survival of Campylobacter in chilled caecal samples suggests that the processing deadline could be extended beyond 80 h.


This work was funded by the Department for Environment, Food and Rural Affairs (project OZ0613). The authors thank the abattoirs and poultry companies for the provision of samples; they also thank colleagues from the Food and Environmental Safety Department for technical assistance.