Occurrence and enumeration of Campylobacter spp. during the processing of Chilean broilers

Background  

Thermotolerant Campylobacter is among the more prevalent bacterial pathogens that cause foodborne diseases. This study aimed at evaluating the occurrence of thermotolerant Campylobacter contamination in chicken carcasses and processing plant stations (chilling water, scalding water, defeathering machinery, evisceration machine, and transport crates) in two of the Chilean main slaughterhouses. In addition, the isolation rates of thermotolerant Campylobacter during evisceration and following chiller processing were compared.     

Quantifying transmission of Campylobacter spp. among broilers

Campylobacter species are frequently identified as a cause of human gastroenteritis, often from eating or mishandling contaminated poultry products. Quantitative knowledge of transmission of Campylobacter in broiler flocks is necessary, as this may help to determine the moment of introduction of Campylobacter in broiler flocks more precisely. The aim of this study was to determine the transmission rate parameter in broiler flocks. Four experiments were performed, each with four Campylobacter-inoculated chicks housed with 396 contact chicks per group. Colonization was monitored by regularly testing fecal samples for Campylobacter. A mathematical model was used to quantify the transmission rate, which was determined to be 1.04 new cases per colonized chick per day. This would imply that, for example, in a flock of 20,000 broilers, the prevalence of Campylobacter would increase from 5% to 95% within 6 days after Campylobacter introduction. The model and the estimated transmission rate parameter can be used to develop a suitable sampling scheme to determine transmission in commercial broiler flocks, to estimate whether control measures can reduce the transmission rate, or to estimate when Campylobacter was introduced into a colonized broiler flock on the basis of the time course of transmission in the flock.     

Evaluation of novel agars for the enumeration of Campylobacter spp. in poultry retail samples

The purpose of this study was to examine the performance of novel agars for the identification and enumeration of Campylobacter species. The analytical sensitivity and specificity of Campylobacter Selective agar (CASA), Brilliance CampyCount agar (BCCA) and CampyFoodIDagar (CFA) for 84 Campylobacter spp. isolates and 50 non-Campylobacter spp. isolates from 37 distinct genera were of 100% sensitivity, with a 98% specificity for BCCA and CFA, and a 100% specificity for CASA. The application of these selective agars for Campylobacter spp. enumeration in comparison to the conventional agars, modified charcoal cefoperazonedeoxycholate agar (mCCDA) and Campy-Cefex (CCA) was examined using Campylobacter jejuni and Campylobacter coli inoculated samples. From C. jejuni inoculated samples, recovery on BCCA was significantly greater than other media (p < 0.05). Recovery on CASA was not significantly different from mCCDA and CCA (p > 0.05). With C. coli inoculated samples, recovery was significantly greater on BCCA and CASA than with other media (p < 0.05). The recovery of both C. jejuni and C. coli from inoculated samples with CFA was significantly less than with other media (P < 0.05). CASA was able to effectively inhibit and differentiate Campylobacter spp. from background microflora while false positive organisms occurred with BCCA and CFA. An examination of 483 randomly selected suspect Campylobacter colonies from naturally contaminated samples demonstrated a colony confirmation rate for CCA, CFA, BCCA, mCCDA, and CASA, of 84%, 87%, 88%, 90%, and 100%, respectively. The media evaluated present an alternative to conventional selective agars for the identification and enumeration of thermotolerant Campylobacter spp. from samples of poultry origin through the farm to fork continuum.     

Evaluation of Chromocult enterococci agar for the isolation and selective enumeration of Enterococcus spp. in broilers

AIMS: To investigate the productivity and specificity of a new chromogenic enterococci selective medium (Chromocult enterococci agar) recently developed by Merck. METHODS AND RESULTS: The study was carried out comparing Chromocult enterococci agar with MRS agar (Merck), a basal lactic acid bacteria medium in current use. A total of 216 faecal samples from poultry were collected and enterococci populations were counted. Likewise, 100 randomly selected strains were identified for each medium. The differences found between the two media were analysed and discussed. CONCLUSIONS: A good sensitivity of 98% was obtained for Chromocult agar and all false-positive isolates obtained were identified as Leuconostoc spp. However significant differences (P<0.01) were obtained between the enterococci species isolation rates identified from these two media, suggesting the poor growth of some species in Chromocult enterococci agar. Viable counts of Enterococcus spp. obtained with MRS agar were significantly higher than those obtained with Chromocult enterococci agar. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of chromogenic media for microbiological analysis is increasing. Independent studies are important to evaluate newly developed chromogenic media.     

Evaluation of agar plates for direct enumeration of Campylobacter spp. from poultry carcass rinses

Campy-Cefex, a modification of Campy-Cefex, modified charcoal cefoperazone deoxycholate (mCCDA), Karmali, CAMPY, and Campy-Line agars were evaluated for their efficiency to isolate and enumerate Campylobacter spp. from poultry carcass rinses. Campy-Cefex and its modification produced the best results but were statistically similar to CAMPY, mCCDA, and Karmali.     

Use of multilocus sequence typing to investigate the association between the presence of Campylobacter spp. in broiler drinking water and Campylobacter colonization in broilers

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.     

Transmission of Campylobacter spp. to chickens during transport to slaughter

AIMS: To determine the prevalence of Campylobacter-contaminated transport crates and to determine whether contaminated crates represent a risk for contamination of chickens during transport to slaughter. METHODS AND RESULTS: Samples were collected from cleaned transport crates before they were dispatched to the farms. Chicken groups were sampled within 24 h before transport to slaughter and at the slaughterhouse. Campylobacter spp. were isolated from 69 of 122 (57%) sampled batches of transport crates. Twenty-six slaughter groups, negative at farm level, were transported in batches of crates from which Campylobacter spp. had been isolated. In 11 (42%) of these 26 slaughter groups, Campylobacter spp. were found in samples taken at slaughter. The corresponding figure for at-farm-negative slaughter groups transported in negative crates was four (15%) testing positive at slaughterhouse of 27 slaughter groups [relative risk (RR) = 2.9, 95% CI 1.1-7.3]. In four of 11 slaughter groups, genetic subtyping by pulsed-field gel electrophoresis was able to support the hypothesis of contamination from crates to chickens during transport to slaughter. CONCLUSIONS: Despite washing and disinfection, crates were frequently contaminated with Campylobacter and it could have contaminated chickens during transport to slaughter. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter-positive crates are a risk factor for chickens testing campylobacter-positive at slaughter.     

Sources of Campylobacter spp. Colonizing Housed Broiler Flocks during Rearing

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.     

Sources of Campylobacter spp. colonizing housed broiler flocks during rearing

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.     

Evaluation of methods for the enumeration of Campylobacter jejuni and Campylobacter coli bacteriophages

The Spiral Plater System used for enumeration of bacteria was evaluated for the titration of Carnpylobacter phages. Twelve phages of C. jejuni were titrated using the conventional surface droplet technique, soft agar overlay-pour plate technique, and the Spiral Plater System. Phage counts obtained by the three methods were similar but the Spiral Plater System showed greater precision than the other methods.     

Selective medium for the isolation and enumeration of Klebsiella spp.

A highly selective medium for the enumeration and isolation of Klebsiella pneumoniae and Klebsiella oxytoca was developed in which the typical colonies were convex and 1 to 2 mm in diameter. Their pigment was either a mucoid pink-red color or a more watery pale red with a dark red center. Relatively little colonial growth occurred for any other bacterial genera, and where such colonies did grow, they could be easily differentiated since the form was atypical. The medium already appears to have potential value as a means of assessing the efficiency of treating sewage and monitoring the microbiological quality of vegetables.     

The incidence of Campylobacter spp. on processed turkey from processing plants in the midwestern United States

AIM: The primary aim of this study was to determine the incidence of Campylobacter spp. on turkey, presented for processing at participating production plants located in the midwest region of the United States. METHODS AND RESULTS: The two participating plants were visited on a monthly basis for a period of 1 year. Sampling of carcasses was carried out using a surface swab technique. Swabs were obtained from carcasses at two points on the production line - prechill and postchill. In addition, samples of chill water were also obtained for examination. Isolation and detection of Campylobacter was carried out using enrichment in Preston broth with recovery of the organism on blood free Campylobacter selective agar (CCDA). Isolates recovered were screened and identified using the API Campy identification system. The study found that 34.9% of all samples tested were positive for Campylobacter spp. The overall, contamination rates observed for both plants were relatively similar (39.2% for plant A and 30.6% for plant B). Differences were observed in the incidence of Campylobacter spp. on prechill vs postchill carcasses (i.e. 40.8% prechill vs 37.6% postchill for plant A and 41.8% prechill vs 19.8% postchill for plant B). Campylobacter species most often isolated included Camp. jejuni and Camp. coli. Other species recovered were Camp. fetus fetus, Camp. upsaliensis and Camp. lari. CONCLUSIONS: The incidence of Campylobacter spp. on processed poultry was relatively common. Factors such as the processing plant examined, season and the farms presenting birds for processing influenced the incidence of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences were observed in the prevalence of Campylobacter spp. isolated from the two plants examined. The study suggests a seasonal prevalence of Campylobacter in the cooler months with processing conditions also influencing the overall occurrence of the organism. The incidence, isolation and detection of Campylobacter spp. from processed poultry are discussed.     

Selective medium for isolation and enumeration of Bifidobacterium spp.

A new method was developed for the isolation and enumeration of Bifidobacterium spp. from natural aquatic environments. The method was based on the utilization of a new medium, Bifidobacterium iodoacetate medium 25, and resuscitation techniques were used to isolate injured bifidobacteria. The new medium was tested with a nonselective reference medium on sewage and sewage-polluted surface waters. Relatively little colonial growth of any other bacterial genera occurred; when such colonies did grow, Bifidobacterium could be easily differentiated by its colonial morphology or, after Gram staining, by its typical bifidobacterial morphology.     

Restoring the selectivity of Bolton broth during enrichment for Campylobacter spp. from raw chicken

Aims: When isolating Campylobacter spp. from retail raw chicken using BS EN ISO 10272‐1:2006, contaminants frequently cause overgrowth on mCCDA plates. Therefore, these organisms proliferate in the enrichment medium, Bolton broth, indicating a lack of selectivity in this medium. This study sought to characterize the contaminant flora and to devise a modified Bolton broth to inhibit their growth. Methods and results: Contaminants (n = 30) from separate samples were identified and antibiotic resistances determined. Most (93%) were extended spectrum β‐lactamase (ESBL) producing Escherichia coli, able to hydrolyse the cefoperazone present in Bolton broth and mCCDA. To inhibit these organisms, original formulation Bolton broth was supplemented with potassium clavulanate, at three concentrations, and recoveries of campylobacters from raw chicken were determined. Using standard Bolton broth, only 49% of samples (n = 104) yielded campylobacters, but supplementation with 2 mg l^?1 potassium clavulanate increased this significantly (P < 0·05), with 91% of samples positive. Conclusions: Potassium clavulanate can restore the selectivity of Bolton broth when isolating Campylobacter spp. from raw chicken. Significance and Impact of the Study: Raw chicken is often contaminated with the pathogen Campylobacter, but the ISO methodology for its detection is becoming compromised by the increasing presence of antibiotic‐resistant bacteria. A simple modification ensures effective detection of this pathogen.     

Frequency and Spatial Distribution of Environmental Campylobacter spp.

Humans are exposed to Campylobacter spp. in a range of sources via both food and environmental pathways. For this study, we explored the frequency and distribution of thermophilic Campylobacter spp. in a 10- by 10-km square rural area of Cheshire, United Kingdom. The area contains approximately 70, mainly dairy, farms and is used extensively for outdoor recreational activities. Campylobacter spp. were isolated from a range of environmental samples by use of a systematic sampling grid. Livestock (mainly cattle) and wildlife feces and environmental water and soil samples were cultured, and isolates were presumptively identified by standard techniques. These isolates were further characterized by PCR. Campylobacter jejuni was the most prevalent species in all animal samples, ranging from 11% in samples from nonavian wildlife to 36% in cattle feces, and was isolated from 15% of water samples. Campylobacter coli was commonly found in water (17%) and sheep (21%) samples, but rarely in other samples. Campylobacter lari was recovered from all sample types, with the exception of sheep feces, and was found in moderate numbers in birds (7%) and water (5%). Campylobacter hyointestinalis was only recovered from cattle (7%) and birds (1%). The spatial distribution and determinants of C. jejuni in cattle feces were examined by the use of model-based spatial statistics. The distribution was consistent with very localized within-farm or within-field transmission and showed little evidence of any larger-scale spatial dependence. We concluded that there is a potentially high risk of human exposure to Campylobacter spp., particularly C. jejuni, in the environment of our study area. The prevalence and likely risk posed by C. jejuni-positive cattle feces in the environment diminished as the fecal material aged. After we took into account the age of the fecal material, the absence or presence of rain, and the presence of bird feces, there was evidence of significant variation in the prevalence of C. jejuni-positive cattle feces between grazing fields but no evidence of spatial clustering beyond this resolution. The spatial pattern of C. jejuni is therefore consistent with that for an organism that is ubiquitous in areas contaminated with cattle feces, with a short-scale variation in infection intensity that cannot be explained solely by variations in the age of the fecal material. The observed pattern is not consistent with large-scale transmission attributable to watercourses, wildlife territories, or other geographical features that transcend field and farm boundaries.