Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs

The recent development of simple, rapid genotyping techniques for Campylobacter species has enabled investigation of the determinative epidemiology of these organisms in a variety of situations. In this study we have used the technique of fla typing (PCR-restriction fragment length polymorphism analysis of the flaA and flaB genes) to identify the sources of strains contaminating the carcasses of five campylobacter-positive and two campylobacter-negative broiler flocks during abattoir processing. The results confirmed that, in the United Kingdom, individual broiler flocks are colonized by a limited number of subtypes of Campylobacter jejuni or C. coli. In some but not all cases, the same subtypes, isolated from the ceca, contaminated the end product as observed in carcass washes. However, the culture methodology, i.e, use of direct plating or enrichment, affected this subtype distribution. Moreover, the number of isolates analyzed per sample was limited. fla typing also indicated that some campylobacter subtypes survive poultry processing better than others. The extent of resistance to the environmental stresses during processing varied between strains. The more robust subtypes appeared to contaminate the abattoir environment, surviving through carcass chilling, and even carrying over onto subsequent flocks. From these studies it is confirmed that some campylobacter-negative flocks reach the abattoir but the carcasses from such flocks are rapidly contaminated by various campylobacter subtypes during processing. However, only some of these contaminating subtypes appeared to survive processing. The sources of this contamination are not clear, but in both negative flocks, campylobacters of the same subtypes as those recovered from the carcasses were isolated from the crates used to transport the birds. In one case, this crate contamination was shown to be present before the birds were loaded.     

Molecular tracking, through processing, of Campylobacter strains colonizing broiler flocks

Many of the poultry flocks produced in the United Kingdom are colonized with Campylobacter, and the intensive nature of poultry processing usually results in contaminated carcasses. In this study, a previously reported molecular oligonucleotide probe method was used to track a specific flock-colonizing strain(s) on broiler carcasses during processing in two United Kingdom commercial poultry processing plants. Five Campylobacter-positive flocks were sampled at four points along the processing line, postbleed, postpluck, prechill, and postchill, and two Campylobacter-negative flocks processed immediately after positive flocks were sampled prechill. flaA was sequenced from Campylobacter strains isolated from these flocks, and strain-specific probes were synthesized. Skin and cecal samples were plated onto selective agar to give individual colonies, which were transferred onto membranes. These were then hybridized with the strain- and genus-specific probes. For all the 5 positive flocks, there was a significant reduction in campylobacters postbleed compared to postpluck but no subsequent fall on sampling pre- and postchill, and the strain(s) predominating on the carcasses throughout processing came from the flock being processed. This indicates that strains from the abattoir environment were not a significant cause of carcass contamination in flocks with well-established campylobacter colonization. However, negative flocks that were preceded by positive flocks were contaminated by strains that did not generally originate from the predominating strains recovered from the ceca of the previous positive flocks. This suggests that the abattoir environment has a significant role in the contamination of carcasses from negative but not fully colonized flocks.     

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.     

Evidence that Certain Clones of Campylobacter jejuni Persist during Successive Broiler Flock Rotations

Through the national surveillance program for Campylobacter spp., nine broiler chicken farms that were infected with Campylobacter jejuni in at least five rotations in 1998 were identified. One additional farm, located at the island of Bornholm where divided slaughter is used extensively, was also selected. Twelve broiler houses located on 10 farms were included in the study. The C. jejuni isolates collected from the selected houses during the surveillance were typed using fla typing and macrorestriction profiling (MRP), and a subset of the isolates, representing each of the identified clones, was serotyped according to the Penner scheme. Pulsed-field gel electrophoresis typing using SmaI and KpnI revealed that the majority of houses (11 of 12) carried identical isolates in two or more broiler flocks. Such persistent clones were found in 63% of all flocks (47 of 75). The majority of persistent clones (7 of 13) had fla type 1/1, but MRPs distinguished between isolates from different houses, and fla type 1/1 clones belonged to different serotypes. Seven houses carried persistent clones that covered an interval of at least four broiler flock rotations, or at least one half year. The dominant fla type (1/1) was represented by 44% of isolates, or by at least one isolate from 31 of 62 broiler flocks. This significantly exceeded the prevalence of fla type 1/1 C. jejuni isolates that we have estimated from other studies and suggests that isolates carrying this fla type are overrepresented in flocks with recurrent Campylobacter problems. The MRPs of clones belonging to fla type 1/1 serotype O:2 isolated from persistently infected flocks shared a high percentage of bands compared to the remaining isolates, indicating that some clones that have the ability to cause persistent infections in broiler farms are highly related to each other.     

Impact of the slaughter line contamination on the presence of Salmonella on broiler carcasses

AIMS: The aim of the study was to assess the impact of Salmonella present on the slaughter line before processing on broiler carcass contamination during processing. METHODS AND RESULTS: Three Belgian broiler slaughterhouses were each visited twice. Samples were taken from the slaughter line after the cleaning and the disinfection process and before slaughter of the first flock. During the slaughter of the first flock, feathers and neck skins were collected at various points of the slaughter process. Swab samples were also taken from the crates in which the birds were transported. In two slaughterhouses, the slaughter line was contaminated with Salmonella before the onset of slaughter, especially the shackles, conveyer belt and the plucking machine in the dirty zone. During slaughter, the carcasses of the first Salmonella-free flock became contaminated with the same strains as isolated previously from the slaughter line. CONCLUSION: Contamination of the slaughter line with Salmonella leads to carcass contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of logistic slaughter is only successful when the cleaning and disinfection process completely eliminates the Salmonella contamination of the slaughter line. Only if this is achieved, will the slaughter of Salmonella-free flocks result in the absence of Salmonella on the carcasses after slaughter.     

Application of Host-Specific Bacteriophages to the Surface of Chicken Skin Leads to a Reduction in Recovery of Campylobacter jejuni

Retail poultry products are widely purported as the major infection vehicle for human campylobacteriosis. Numerous intervention strategies have sought to reduce Campylobacter contamination on broiler carcasses in the abattoir. This study reports the efficacy of bacteriophage in reducing the number of recoverable Campylobacter jejuni cells on artificially contaminated chicken skin.     

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.     

Impact of Transport Crate Reuse and of Catching and Processing on Campylobacter and Salmonella Contamination of Broiler Chickens

The influence of transport, catching, and processing on contamination of broiler chickens with Salmonella and Campylobacter was investigated. Transport crates were reused with high frequency and were often still contaminated with Salmonella and Campylobacter when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously Campylobacter- and Salmonella-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The Campylobacter types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with Campylobacter (P < 0.001).     

Enumeration of Salmonella and Campylobacter spp. in Environmental Farm Samples and Processing Plant Carcass Rinses from Commercial Broiler Chicken Flocks

A prospective cohort study was performed to evaluate the prevalences and loads of Salmonella and Campylobacter spp. in farm and processing plant samples collected from 55 commercial broiler chicken flocks. Environmental samples were collected from broiler houses within 48 h before slaughter, and carcass rinses were performed on birds from the same flocks at 4 different stages of processing. Salmonella was detected in farm samples of 50 (90.9%) flocks and in processing samples of 52 (94.5%) flocks. Campylobacter was detected in farm samples of 35 (63.6%) flocks and in processing samples of 48 (87.3%) flocks. There was a significant positive relationship between environmental farm samples and processing plant carcass rinses with respect to both Salmonella and Campylobacter prevalences and loads. Campylobacter loads were significantly higher than Salmonella loads, and the correlations between samples collected from the same flocks were higher for Campylobacter than they were for Salmonella. Boot socks were the most sensitive sample type for detection of Salmonella on the farm, whereas litter samples had the strongest association with Salmonella loads in pre- and postchill carcass rinses. Boot socks, drag swabs, and fecal samples all had similar sensitivities for detecting Campylobacter on the farm, and all were more strongly associated with Campylobacter loads in carcass rinses than were litter samples. Farm samples explained a greater proportion of the variability in carcass rinse prevalences and loads for Campylobacter than they did for Salmonella. Salmonella and Campylobacter prevalences and loads both decreased significantly as birds progressed through the processing plant.     

Enumeration of thermotolerant Campylobacter spp. from poultry carcasses at the end of the slaughter-line

AIM: To enumerate Campylobacter on poultry carcasses at the end of the slaughter-line, and investigate the extent to which Campylobacter from a positive flock were transmitted to other flocks during slaughter. METHODS AND RESULTS: The presence (in caeca) and the level (from carcasses) of Campylobacter were determined. The isolates were fingerprinted by amplified fragment length polymorphism (AFLP). A total of three of 13 broiler flocks and three of four-layer flocks harboured caecal Campylobacter. Carcasses from the caeca-positive broiler flocks were Campylobacter positive with numbers ranging from 2.6 x 10(4) to 2.6 x 10(6) CFU per carcass. Two caeca-negative broiler flocks, slaughtered directly after the positive broiler flocks, had the first carcasses contaminated with Campylobacter, with numbers below 2 x 10(4) CFU per carcass of the same AFLP haplotypes as the preceding flock. Campylobacter was detected on carcasses from only one of the caeca-positive layer flocks in numbers below 2 x 10(4) CFU per carcass. No Campylobacter was detected on carcasses from a flock succeeding the positive-layer flocks. CONCLUSION: Carcasses from Campylobacter-positive broiler flocks were heavily contaminated with Campylobacter, and transmitted low levels of Campylobacter to carcasses from negative flocks, slaughtered directly after. Campylobacter-positive layer flocks had low numbers of Campylobacter on the carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate limited cross-contamination of Campylobacter between flocks at the slaughterhouse, reducing the advantage of logistic slaughter.     

Amplified Fragment Length Polymorphism Fingerprinting of Pseudomonas Strains from a Poultry Processing Plant

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.     

Molecular characterization of the diversity of Campylobacter spp. isolates collected from a poultry slaughterhouse: analysis of cross-contamination

Investigations of a free-range broiler flock during the rearing period and at the slaughterhouse by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the flagellin (flaA) gene (flaA typing) have shown that poultry carcasses are contaminated by Campylobacter spp. strains which were previously present in the poultry faces. Moreover, the investigation of the previous and the following batches in the processing plant using flaA typing have shown that cross-contamination between batches coming from different flocks occurs and is also a risk factor for the presence of Campylobacter spp. on poultry carcasses.     

Saltelli Global Sensitivity Analysis and Simulation Modelling to Identify Intervention Strategies to Reduce the Prevalence of Escherichia coli O157 Contaminated Beef Carcasses

Introduction

Strains of Shiga-toxin producing Escherichia coli O157 (STEC O157) are important foodborne pathogens in humans, and outbreaks of illness have been associated with consumption of undercooked beef. Here, we determine the most effective intervention strategies to reduce the prevalence of STEC O157 contaminated beef carcasses using a modelling approach.

Method

A computational model simulated events and processes in the beef harvest chain. Information from empirical studies was used to parameterise the model. Variance-based global sensitivity analysis (GSA) using the Saltelli method identified variables with the greatest influence on the prevalence of STEC O157 contaminated carcasses. Following a baseline scenario (no interventions), a series of simulations systematically introduced and tested interventions based on influential variables identified by repeated Saltelli GSA, to determine the most effective intervention strategy.

Results

Transfer of STEC O157 from hide or gastro-intestinal tract to carcass (improved abattoir hygiene) had the greatest influence on the prevalence of contaminated carcases. Due to interactions between inputs (identified by Saltelli GSA), combinations of interventions based on improved abattoir hygiene achieved a greater reduction in maximum prevalence than would be expected from an additive effect of single interventions. The most effective combination was improved abattoir hygiene with vaccination, which achieved a greater than ten-fold decrease in maximum prevalence compared to the baseline scenario.

Conclusion

Study results suggest that effective interventions to reduce the prevalence of STEC O157 contaminated carcasses should initially be based on improved abattoir hygiene. However, the effect of improved abattoir hygiene on the distribution of STEC O157 concentration on carcasses is an important information gap—further empirical research is required to determine whether reduced prevalence of contaminated carcasses is likely to result in reduced incidence of STEC O157 associated illness in humans. This is the first use of variance-based GSA to assess the drivers of STEC O157 contamination of beef carcasses.     

Impact of transport crate reuse and of catching and processing on Campylobacter and Salmonella contamination of broiler chickens

The influence of transport, catching, and processing on contamination of broiler chickens with Salmonella and Campylobacter was investigated. Transport crates were reused with high frequency and were often still contaminated with Salmonella and Campylobacter when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously Campylobacter- and Salmonella-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The Campylobacter types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with Campylobacter (P < 0.001).     

May Organically Farmed Animals Pose a Risk for Campylobacter Infections in Humans?

Organic farming of meat producing poultry like broilers, means that the animals should be kept outdoors as much as possible. This pose a risk that they get infected with Campylobacter. At slaughter, carcasses may be contaminated with campylobacter. If cross contamination occurs in the kitchen or if the meat is undercooked people may ingest the bacteria and suffer from enteritis. It seems possible that close to 100 percent of organically farmed flocks may be infected with campylobacter while under Swedish conditions only 10 percent of conventionally reared flocks are infected.     

Origin of Contamination and Genetic Diversity of Escherichia coli in Beef Cattle

The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.     

Application of variable number of tandem repeat analysis to track Salmonella enterica ssp. enterica serovar Typhimurium infection of pigs reared on three British farms through the production cycle to the abattoir

Aims: This study investigated the diversity and persistence of Salmonella strains through the pork finishing cycle, from the farm into the abattoir. Methods and Results: Isolates from four batches of finishers, from farm to abattoir, were used. Salmonella Typhimurium isolates were subjected to molecular typing using pulsed‐field gel electrophoresis and variable number of tandem repeat analysis. The results demonstrated that infection was transferred from the farm to the abattoir. Within the abattoir, infection from individual pigs contaminated the exterior of the carcass and pigs exposed to Salmonella in the lairage were infected. Conclusions: Salmonella can be introduced at various points in the pig production and slaughter process. Carcass contamination may arise from infection on farm and exposure in the lairage and abattoir environment. Pigs could be contaminated by previous batches of pigs while in lairage or during the dressing process. Salmonella infection on farms is dynamic with multiple serovars present from different sources. Significance and Impact of the Study: Molecular typing methods facilitated the tracing of Salm. Typhimurium through the production cycle and differentiated some farm‐acquired from abattoir‐acquired strains. The findings emphasize the importance of integrated control strategies along the pork food chain.     

Role of batch depletion of broiler houses on the occurrence of Campylobacter spp. in chicken flocks

AIMS: The effect of batch depletion of broiler houses for campylobacter occurrence in broiler flocks was estimated in 10 flocks, each comprising a separate female and male batch. METHODS AND RESULTS: The chicks were sampled first by cloacal swabs in the broiler houses before the start of the depopulation and secondly, on arrival at the abattoir. Females were slaughtered at 5 weeks of age, males at 6 weeks. The number of campylobacter-positive batches increased from five to seven female batches, and from five to 10 male batches, between the two sampling rounds. CONCLUSION: It is concluded that batch depletion of broiler houses increased the prevalence of Campylobacter spp.-infected broilers in the flocks, that the introduction occurred when catching the first batch, and that campylobacter spreads through the entire flock within a week. SIGNIFICANCE AND IMPACT OF THE STUDY: The results from this study emphasize the need to manage depopulation of broiler houses as quickly as possible and in one batch only.     

Genotypic Analyses of Escherichia coli O157:H7 and O157 Nonmotile Isolates Recovered from Beef Cattle and Carcasses at Processing Plants in the Midwestern States of the United States

Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999–3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting. E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that most E. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing.     

Contamination of Pig Carcasses at Two Abattoirs by Escherichia coli with Special Reference to O-serotypes and Antibiotic Resistance

Evidence of the source of carcass contamination of pigs at slaughter was obtained by determining presumptive coliform counts on faeces and on carcass surfaces, and comparing the O-serotypes and antibiotic sensitivity patterns of Escherichia coli from both sites. All of the 16 pig carcasses from the slaughter line of a commercial abattoir were contaminated with presumptive coliform bacilli on most sites examined; the carcasses of six out of eight pigs slaughtered at the Meat Research Institute (MRI) abattoir were also contaminated, but only small numbers of coliforms could be detected on a few of the sites. The proportion of O-serotypes of E. coli present in faeces which were also detected on carcass surfaces, indicating faecal contamination, varied between 0 and 8.6% in MRI slaughtered pigs but reached 66.6% in one group of commercially slaughtered pigs. O-serotypes found on carcass surfaces but not in the faeces of the pigs, were used as an indication of environmental contamination and this was very evident in the commercially slaughtered pigs. A high proportion of E. coli O-serotypes in the gut were resistant to antibiotics and these were also often found on the carcass surface and, since the range of O-serotypes in the pig is similar to that reported in man, the pig must be considered to be a potential reservoir of antibiotic resistant E. coli for man.