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Keywords:

  • biosecurity;
  • Campylobacter spp.;
  • environment;
  • poultry;
  • recovery

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aim:  The aim of this study was to investigate the prevalence of Campylobacter species in a subset of intensive poultry flocks by examining samples collected in geographically disparate areas on the island of Ireland.

Methods and Results:  Faecal, water and environmental samples were collected from the interior of poultry houses on nine farms. Three cultural methods were used for Campylobacter isolation: direct plating, enrichment culture and a recovery method for emerging Campylobacter spp. Presumptive Campylobacter isolates were confirmed using biochemical tests and further identified to species level by multiplex PCR. All flocks sampled in this study were found to be contaminated with Campylobacter at the time of sampling. Structural and air samples taken from the interior of broiler houses were also found to be Campylobacter positive. All water samples were found to be Campylobacter negative. The Campycheck method was used for the isolation of emerging Campylobacter spp.

Conclusions: Campylobacter spp. were recovered (as contaminants) from the poultry house interior, air and environmental samples in all intensive poultry flocks surveyed.

Significance and Impact of the Study:  This study highlights the need for improved biosecurity on selected poultry farms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Over the last number of years, Campylobacter spp. have emerged as the most common cause of acute bacterial enteritis in humans. In Ireland, campylobacteriosis is the most common cause of bacterial gastroenteritis, and 1815 cases of campylobacteriosis were notified in 2006 (crude incidence rate: 42·8/100 000) (HPSC 2006). This is the highest reported rate in nine years and represents an upward trend in incidence since 2001. Thus, it is clear that this pathogen represents a significant burden to the public health system.

Poultry is regarded as one of the most important reservoirs for Campylobacter and constitutes a very significant vehicle for the transmission of Campylobacter to humans (Humphrey et al. 2007). Various studies have demonstrated high levels of Campylobacter on broiler chickens from poultry farms (Stern et al. 1995) and on retail chickens (Zhao et al. 2001). Consequently, undercooked and raw poultry meats are commonly associated with human campylobacteriosis by cross-contamination (Wingstrand et al. 2006; Lindqvist and Lindblad 2008). The occurrence of Campylobacter remains at high levels in broiler meat and broiler flocks in most European countries (EFSA Journal, 2007). Thus, control strategies aimed at reducing the incidence of campylobacteriosis have focused on reducing the occurrence of Campylobacter in broiler flocks. Biosecurity measures at farm level are key to this type of strategy, and included among these measures are monitoring of personal hygiene, effective cleaning of farm buildings and their immediate environment and water treatment. The prevalence of Campylobacter spp. detected on a poultry farm may give an indication of the effectiveness of biosecurity measures in place at a particular location, in this instance, in intensive poultry flocks at diverse locations throughout Ireland. Biosecurity measures in the broiler house have been addressed in a number of previous studies, and several risk factors for the contamination of broiler flocks have been highlighted (Engvall et al. 1986; Pearson et al. 1993; Berndtson et al. 1995; Evans and Sayers 2000). Despite preventive biosecurity measures, contamination of intensive poultry flocks with Campylobacter is an ongoing problem, and it is generally recognized that the source of contamination and the conduit for initial infection of broiler flocks with Campylobacter are not as yet fully defined. Thus, epidemiological studies addressing the prevalence and potential transmission routes of Campylobacter spp. at farm level continue to be important.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sampling procedure

Three different geographical areas of Ireland (denoted 1, 2 and 3) were chosen for this study, and three farms per location were selected giving nine farms sampled (denoted A to I). Sampling was carried out when the flock age was between four and six weeks. A total of 108 samples (faecal, water and internal environment) were collected from each of the nine farms during the sampling period from July to September, 2006. Sixty fresh faecal samples were aggregated with five pooled samples on site at each farm, four litres of water from supply tanks and four samples of broiler house air were also collected at each farm. All samples were transported to the laboratory under chilled conditions and processed immediately.

Isolation of Campylobacter spp. from faeces

Methodology based on the horizontal method for detection and enumeration of Campylobacter spp. (ISO 10272-1:2006; International Organisation for Standardisation 2006, Switzerland) was used to isolate Campylobacter spp. from faeces by direct plating. Briefly, 25 g of faecal material was weighed and diluted with 225 ml of maximum recovery diluent (MDR, CM733; Oxoid Ltd, Basingstoke, UK), transferred to sterile filter bags and pulsified (Pulsifier, Microgen Bioproducts) for 15 s. Serial dilution of the pulsified material was performed, and 100 μl of each dilution was plated on Campylobacter blood-free selective agar base (CCDA, CM0739; Oxoid) supplemented with CCDA selective supplement (SR0115; Oxoid). Plates were incubated at 37°C for 48 h under microaerophilic conditions in Anaeropack jars (Mitsubishi Gas Chemical Co.). Following incubation, five colonies were randomly selected from plates and subcultured to obtain pure colonies. Purified isolates were retained for biochemical analysis.

A methodology designed to isolate emerging Campylobacteraceae was also used as described by Lynch et al. (2007) but with minor modifications, subsequently referred to as the ‘Campycheck method’. Briefly, 25 g of faecal material was weighed, diluted with 225 ml of Campylobacter enrichment broth (CEB; Lab-M Ltd, Lancs, UK) containing 5% (v/v) lysed horse blood and pulsified for 15 s in sterile filter bags. Filter bags were incubated at 37°C for 24 h in a defined atmosphere (80·5% N2, 2·5% O2, 7% H2, 10% CO2). Ten millilitres of incubated broth was centrifuged at 800 g for 10 s, and the supernatant was serially diluted with MRD. Two-hundred microlitres of each dilution was pipetted into 0·6-μm membrane filters (Whatman, UK), placed on the surface of anaerobe basal agar (ABA; Oxoid) plates containing 5% lysed horse blood and supplemented with 10 μg ml−1 trimethoprim (Sigma). Filters were removed from the agar surface after 15 min; the area directly beneath the filter was distributed by spreading and the plates were incubated at 37°C for up to 6 days under defined atmospheric conditions as described previously. Five colonies were randomly selected from plates and subcultured on tryptone soya agar (TSA; Oxoid) to obtain pure colonies.

Isolation of Campylobacter spp. from water

An enrichment culture method for the detection and semiquantitative enumeration of thermotolerant Campylobacter spp. (ISO 17995:2005; International Organisation for Standardisation 2005, Switzerland) was used. Briefly, collection of water samples (4 l) from supply tanks was carried out by manual siphoning. Samples (n = 18) were filtered through 0·45-μm filters (Millipore, UK), and the filter discs were placed in 30-ml sterile containers to which 20 ml of CEB + supplement (CVTC Supplement; Lab-M) was added. Containers were incubated at 37°C for 16 h and further incubated at 42°C for 32 h. Following incubation, 100 μl and 1 ml of samples from each container were spread on CCDA plates supplemented with CVTC and incubated at 37°C for 48 h under microaerophilic conditions. Five colonies were randomly selected from plates and subcultured on CCDA to obtain pure colonies.

Isolation of Campylobacter spp. from interior poultry house air and environment

Air samples (n = 18) were subjected to direct plating and enrichment culture recovery methods as described above. A Sampl`air MK2 (AES Chemunex, Bruz, France) double agar plate sampler was used to collect aerosol samples directly onto either CCDA agar plates supplemented with CVTC or TSA plates. Structural samples (n = 27) were collected from inside the poultry houses, walls (n = 9), structural columns (n = 9) and feeders (n = 9) using sterile swabs premoistened with 10 ml MRD. An area of 0·1 m2 was swabbed, and swabs were recovered from sterile filter bags containing 100 ml of CEB containing 5% (v/v) lysed horse blood and CVTC supplement. Samples were then processed as for the air enrichment culture method.

Biochemical tests

Presumptive Campylobacter isolates were confirmed using standard biochemical procedures including the KOH, catalase (3% H2O2) and oxidase (tetramethyl-p-phenylenediamine; Sigma) reactions. l-Alanine aminopeptidase was assayed using commercially available strips (Aminopeptidase test; Fluka). A latex agglutination test was also used (DrySpot Campylobacter test kit; Oxoid). Following biochemical analysis, isolates were stored at −80°C until subtype analysis could be performed.

Multiplex PCR

DNA extraction was carried out using a commercial kit (DNeasy blood and tissue kit; Qiagen). Isolates were identified to the species level using two genotyping methods as described by Wang et al. (2002) and Klena et al. (2004). Presumptive Helicobacter isolates were confirmed as Helicobacter pullorum by 16S rRNA PCR using primers described by Relman (1993) and 16S rRNA sequencing. DNA amplification was carried out in a PTC-200 Peltier thermal cycler (MJ Research). Amplicon sizes were determined in comparison with a molecular weight marker (110 bp ladder; Qiagen) following migration on a 1·5% agarose gel.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation and identification of Campylobacter spp.

By the end of the sampling period, Campylobacter spp. were detected on all poultry farms included in the study. Campylobacter was most frequently isolated from faecal samples, with 82·2% (37/45) of samples testing positive for the presence of the pathogen. Two (2/27) structural swabs and two (2/18) air samples taken from the inside of the poultry houses were also positive for the presence of Campylobacter (Table 1). Campylobacter was not recovered from the water supply to the poultry houses on any of the nine farms. The prevalence of Campylobacter did vary with geographic location, as the percentage of positive samples for locations 1, 2 and 3 were 38·8%, 25% and 50%, respectively (Fig. 1). Isolation of Campylobacter from air samples also showed a geographic bias with the positive samples detected on farms G and H in location 3 and not at any of the other farms (Table 1).

Table 1.   Number of Campylobacteraceae isolated during the sampling period
LocationFarm* (day)No. of positive samples/ no. of samples tested
Faeces†Water Air‡Structural swabs (column, feeder, wall)
  1. *Farms sampled are designated from A to I.

  2. †Faecal samples were processed using direct plating and the Campycheck method.

  3. ‡Air samples were processed using direct plating and enrichment culture.

  4. §Boldface type indicates samples identified as Campylobacteraceae.

1A (25) 5/5§0/20/20/3
B (25) 3/50/20/21/3 (column)
C (24) 5/50/20/20/3
2D (26) 3/50/20/20/3
E (35) 3/50/20/20/3
F (31) 3/50/20/20/3
3G (31) 5/50/21/20/3
H (31) 5/50/21/20/3
I (34) 5/50/20/21/3 (wall)
Total 37/450/182/182/27
image

Figure 1.  Percentage of positive samples for Campylobacter spp. recovered from different sources at different geographic locations during the sampling period. Grey bars, faecal samples; white bars, air samples; black bars, structural samples.

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Identification of isolates by multiplex PCR

When identification to species level was carried out by multiplex PCR analysis, the most prevalent species identified was C. jejuni (57%) followed by C. coli (36·6%). In addition, other members of the Campylobacteraceae family along with H. pullorum were recovered over the course of the survey. Of 172 isolates speciated, 95·3% (164/172) were confirmed as Campylobacteraceae, 3·5% (6/172) as Helicobacter and 1·2% (2/172) were non-Campylobacteraceae. All Helicobacter isolates found were identified as H. pullorum by 16S rRNA PCR and sequencing. Speciation revealed C. jejuni to be the predominant but not the only species present in faecal samples from this study (Table 2). By direct plating, C. jejuni and C. coli were recovered from faecal samples from farms in location 1 and location 3; however, using the same method, only C. coli and not C. jejuni was recovered from farms in location 2. In contrast, when the Campycheck method was used to recover Campylobacter from faecal samples, only C. jejuni and C. coli were recovered from farms in location 3, while in locations 1 and 2, no C. coli were recovered. However, in these two locations, faecal samples processed using the Campycheck method detected H. pullorum in both locations and Campylobacteraceae in location 2 alone (Table 2). When air samples were processed by direct plating, only C. jejuni was recovered from farms (G and H) in location 3; when air samples from location 3 were processed using enrichment culture, C. jejuni was once again the only species detected. Enrichment culture alone was used to process interior environment (structural) samples; using this recovery method, C. coli was recovered from a poultry house internal structural column at farm B in location 1. C. jejuni was recovered from a wall swab at farm I in location 3.

Table 2.   Number of species identified in relation to source and recovery method
Location*SourceSpecies identified
Recovery method
DirectEnrichmentEmerging
  1. *A total of nine farms have been sampled, three at each location 1, 2 and 3.

  2. †ND, not determined.

1FaecesC. jejuni (13)ND†C. jejuni (17)
C. coli (17)Helicobacter (1)
SwabNDC. coli (1)ND
2FaecesC. coli (16)NDC. jejuni
Campylobacteraceae (3)
3FaecesC. jejuni (60)NDC. jejuni (2)
C. coli (6)C. coli (23)
AirC. jejuni (1)C. jejuni (3) 
SwabC. jejuni (1)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Reduction of Campylobacter carriage in live flocks is a key strategy in the prevention of human Campylobacter-related illness. Addressing this issue requires intervention at the early stages of commercial poultry production. When one examines the farm to fork route, it is apparent that a reduction in flock contamination rates would result in reduced rates of contamination at critical points along the food chain.

Over the last number of years, studies conducted on poultry farms have highlighted several risk factors that may contribute to flock contamination (Kapperud et al. 1993; Guerin et al. 2007). In this study, the potential of in-house water, air and environmental structures have been assessed as possible sources of Campylobacter contamination of poultry flocks. Results reveal that Campylobacter spp. were recovered from all nine poultry farms surveyed, suggesting the widespread presence of this pathogen in poultry farms at diverse locations. Samples from distinct poultry house interior locations were found to be contaminated with Campylobacter spp., underscoring the need for rigorous decontamination procedures before introduction of new flocks into poultry houses. C. jejuni was recovered from poultry house interior air samples, although positive samples were only recovered from two of the nine farms. The potential of air to act as a conduit for microbial transmission within a poultry flock cannot be disregarded, perhaps influencing the rate at which colonization occurs once the pathogen is introduced. The significance of airborne transmission in the spread of Campylobacter has not been frequently addressed in the literature to date, although in a study by Bull et al. (2006), the bacterium has been detected in the air up to thirty metres downstream of a broiler house. Campylobacter can survive in aqueous environments for limited periods outside the host (Korhonen and Martikainen 1991). To optimize the potential of recovering Campylobacter spp. from water, enrichment culture was used; however, no Campylobacter spp. were recovered from any water samples from any of the farms surveyed. Results from this study suggest that water is not acting as conduit for transmission of the pathogen in the flocks sampled and, therefore, that adequate biosecurity measures at a least in relation to the water supply appear to be in place at all locations. As vertical transmission appears increasingly unlikely to be a significant source of broiler flock contamination (Callicott et al. 2006; Workman et al. 2008), focus on reducing horizontal transmission risks should be valuable in the protection of poultry flocks. Intervention strategies including biosecurity measures at farm level have been reported as being successful in decreasing the percentage of Campylobacter-positive broiler flocks in certain EU countries such as Denmark (Borck et al. 2007). A high level of biosecurity should theoretically protect against Campylobacter, and consistent application of such measures should ensure the prevention of initial colonization.

The most prevalent Campylobacter species isolated during this study was C. jejuni with C. coli less frequently isolated. Currently, routine recovery methods used for the surveillance of Campylobacter look solely for these two species. Therefore, in this study, a recently described method for the recovery and identification of emerging Campylobacteraceae (Lynch et al. 2007) was employed.

An improvement in the understanding of Campylobacter epidemiology in intensive poultry flocks in Ireland should contribute towards innovative measures being implemented at the farm level, which will result in downstream improvements in public health. Thus, epidemiological studies addressing the prevalence of Campylobacter spp. at farm level continue to be important to support a reduction in prevalence of this pathogen along the food chain.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors acknowledge the financial support for this project provided by Safefood (04-RESR-04). We also acknowledge the co-operation of the poultry farmers involved in this study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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