Comparisons of Sampling Procedures and Time of Sampling for the Detection of Salmonella in Danish Infected Chicken Flocks Raised in Floor Systems

Bacteriological follow-up samples were taken from 41 chicken (Gallus gallus) flocks in floor systems, where Salmonella enterica (Salmonella) had been detected either directly in bacteriological samples or indirectly by serological samples. Three types of follow-up samples were compared to each other within each flock: 1) 5 pairs of socks, analysed as 5 samples, 2) 2 pairs of socks, analysed as one sample, and 3) 60 faecal samples, analysed as one pooled sample. Agreement between sampling methods was evaluated by the following statistical tests: 'Kappa', 'The adjusted rand', McNemar's test for marginal symmetry, Proportion of agreement P₀, P₊, P_-, and Odds Ratio. The highest agreement was found between the 2 types of sock sampling, while the lowest agreement was found by comparing 60 faecal samples with 5 pairs of socks. Two pairs of socks analysed as one pool appeared to be just as effective in detecting S. enterica as the 60 faecal samples. In broiler flocks, 5 pairs of socks were used both in the routine samples taken at about 3 weeks of age for the establishment of infection of the flock, and as one of the follow-up samples taken shortly before slaughter age, which means that the only notable differences between the 2 sampling rounds were the age of the broilers and of their litter. S. enterica was detected more frequently in samples from broilers about 3 weeks old, than in similar samples taken from broilers a few days prior to slaughter at ca. 33–40 days of age.     

The estimation of pooled-sample sensitivity for detection of Salmonella in turkey flocks

Aims:  To investigate the effectiveness of pooled sampling methods for detection of Salmonella in turkey flocks.
Methods and Results:  Individual turkey droppings were taken from 43 flocks, with half the dropping tested for S almonella as an individual sample and the other half included in a pool of five. A pair of boot swabs and a dust sample were also taken from each flock. The results were analysed using Bayesian methods in the absence of a gold standard. This showed a dilution effect of mixing true-positive with negative samples, but even with this the pooled faecal samples were found to be a highly efficient method of testing compared with individual faecal samples. The more samples included in the pool, the more sensitive the pooled sampling method was predicted to be. The sensitivity of dust sampling was much more sensitive than faecal sampling at low prevalence.
Conclusions:  Pooled faecal sampling is an efficient method of Salmonella detection in turkey flocks. The additional testing of a dust sample greatly increased the effectiveness of sampling, especially at low prevalence.
Significance and Impact of the Study:  This is the first study to relate the sensitivity of the sampling methods to the within-flock prevalence.     

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.     

Comparison of sampling methods to detect Salmonella infection of turkey flocks

Aims:  To compare the efficiency of various sampling methods for detection of Salmonella in turkey flocks.
Methods and Results:  In a field study that compared various sampling methods one pair of boot swabs taken from the whole turkey house provided suitably sensitive results for fattening and rearing flocks and was no less sensitive than two pairs, each from half the house, tested as a pooled sample. The sensitivity was further enhanced by adding a dust sample. The dust sample appeared to be particularly useful in flocks with a low prevalence, especially in breeding flocks, and was more sensitive than a method which used five pairs of boot swabs per flock. Combined incubation of a boot swab and a dust sample showed no interference between the two sample types and a maximum sensitivity of detection. Litter samples and commercial sponge drag swabs provided a lower level of detection.
Conclusions:  A single pair of boot swabs taken from the whole house is recommended for routine sampling of commercial rearing or fattening flocks. An additional dust sample could be added to increase detection in flocks with a low prevalence or in breeding flocks, but adding an additional pair of boot swabs would not increase detection compared with a single pair.
Significance and Impact of the Study:  This study demonstrates that significant efficiencies can be made in sampling programmes for detection of Salmonella in turkey flocks without detracting from the sensitivity. Similar studies are recommended for other poultry sectors, particularly in chicken breeding flocks.     

Comparison of selective enrichment media for the detection of Salmonella in poultry faeces

AIMS: The aim of this study was to compare the results of semisolid media and Rappaport-Vassiliadis (RV) medium for the detection of Salmonella in faecal samples from broiler and layer flocks. METHODS AND RESULTS: Three different selective enrichment media were used: (a) RV medium; (b) diagnostic semisolid Salmonella medium (DIASALM) and (c) modified semisolid RV (MSRV) medium. The performance of DIASALM and MSRV was significantly better compared with RV. CONCLUSION: The results of this study indicate that approximately 95% of the samples containing Salmonella would be detected by a combination of a semisolid medium (MSRV or DIASALM) and RV. SIGNIFICANCE AND IMPACT OF THE STUDY: The International Standard method ISO 6579, including RV and selenite cystine broth as selective enrichment media, is most frequently used for the isolation of Salmonella from poultry faeces. This study reveals that there are more suitable combinations of selective enrichment media.     

A longitudinal study to assess the persistence of vancomycin-resistant Enterococcus faecium (VREF) on an intensive broiler farm in the United Kingdom

Seven years after the ban of avoparcin, VREF could still be isolated within sectors of the UK broiler industry. The aim of this study was to assess whether there is a carryover of VREF between consecutive flocks of birds, to conduct a preliminary investigation of possible routes of entry of VREF into broiler houses and to follow the dynamics of VREF shed by growing birds. A series of nine visits were made to two of six houses on a conventional broiler farm. A total of 343 vanA VREF were recovered from environmental (95/843) and faecal (248/416) samples. Significant differences were observed in the carryover of VREF between pre- and postcohort postcleaning and disinfection visits (RR 0.57, P=0.006). Ninety-nine percent of the VREF isolates were resistant to more than five antimicrobials, with 42 isolates (n=49) positive for erm(B) and 32 (n=40) for vat(E). Pulsed field gel electrophoresis (PFGE) typing identified 50 PFGE types within 15 different PFGE clusters of 90% similarity, demonstrating a high level of genetic diversity within VREF populations from epidemiologically related broiler flocks and broiler houses. Further characterization of Tn1546 from different clones showed a low diversity of Tn-types, suggesting horizontal transfer of resistance determinants between different genetic clones. Thus, this study does not only show the persistence of VREF but also of multi-drug resistant lineages of VREF.     

Rapid detection of Salmonella enterica in primary production samples by eliminating DNA amplification inhibitors using an improved sample pre-treatment method

Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples.     

Evaluation of PCR for detection of Campylobacter in a national broiler surveillance programme in Denmark

AIMS: To develop and evaluate a rapid and sensitive PCR method for detection of Campylobacter spp. directly from chicken faeces. METHODS AND RESULTS: DNA was isolated from faecal swabs using magnetic beads followed by PCR using a prealiquoted PCR mixture, which had been stored in the freezer. The result could be obtained in <6 h. The method was evaluated on 1282 samples from the Danish surveillance programme for Campylobacter in broilers by comparing with conventional culture. The diagnostic specificity was calculated to be 0.99. The detection limits of the PCR method and of the conventional culture were compared using spiked control material. For both methods the detection limit was 36 CFU ml-1. CONCLUSIONS: It was concluded that the PCR proved useful for detection of Campylobacter in pooled cloacal swabs from broilers. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking cloacal samples in the broiler flocks the technique can be used as an important tool for planning and directing the broiler slaughtering process. This will be a great help in minimizing the risk of contaminating Campylobacter-free flocks at the abattoir.     

A novel multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces

AIMS: To develop a multiplex PCR assay for the detection of Salmonella enterica serovar Enteritidis in human faeces. METHODS AND RESULTS: A total of 54 Salmonella strains representing 19 serovars and non-Salmonella strains representing 11 different genera were used. Five primer pairs were employed in the assay. Three of them targeted to the genes hilA, spvA and invA that encode virulence-associated factors. A fourth primer pair amplified a fragment of a unique sequence within S. enterica serovar Enteritidis genomes. An internal amplification control (a fragment of a conservative sequence within the 16S rRNA genes) was targeted by a fifth primer pair. The assay produced two or three amplicons from the invA, hilA and 16S rRNA genes for 19 Salmonella serovars. All Salmonella and non-Salmonella strains yielded a band of an internal amplification control. For S. enterica serovar Typhimurium, four products (the fourth from the spvA gene), and for S. enterica serovar Enteritidis five amplicons (the fifth from the sdf gene) were observed. S. enterica serovar Enteritidis was cultured from three of 71 rectal swabs from diarrhoeal patients. Five specific amplicons were generated with the multiplex PCR assay only from culture-positive faecal samples. CONCLUSION: The multiplex PCR assay specifically detects S. enterica serovar Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel multiplex PCR assay, which contains an internal amplification control and enables concurrent survey for Salmonella virulence genes.     

Development of a combined selective enrichment method and polymerase chain reaction (PCR) assay for sensitive detection of Salmonella in food samples

PCR assays were formatted using primer pairs homologous to phoE and invA genes. The amplification conditions were optimized with pure cultures and reactions were carried out to define selectivity, specificity and sensitivity of both primer pairs. The performance of the invA primer pair was better than that of the phoE pair, making the specific detection of Salmonella serovars and strains isolated from different food samples possible. Using the invA primer pair, the combined selective enrichment method with the polymerase chain reaction assay was established and used to detect Salmonella from artificially multi-contaminated food samples. The complete procedure detected as few as three cells of Salmonella (3 c.f.u.) from milk and meat samples.     

Use of multiple‐locus variable‐number tandem‐repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections

Aims: To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple‐locus variable‐number tandem‐repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients. Methods and Results: A total of 102 isolates of Salm. Typhimurium phage type DT41 were MLVA typed. MLVA typing showed 4, 12, 25, 9 and 8 different alleles at the five MLVA loci 9, 5, 6, 10 and 3, respectively. A dendrogram based on MLVA types was constructed, and one large group, nine minor groups and 29 more unrelated MLVA types were obtained. The major group included 20 of the 30 human isolates. Isolates obtained from broiler breeders demonstrated major diversity, indicating the existence of several independent introductions of DT41 at farm level. When comparison was made to isolates included from Germany and England, DT41 seems to be ubiquitous in the wild fauna which might represent a risk factor for poultry. Conclusions: Transmission from Danish broilers to humans was not demonstrated, neither was the transmission from rearing farms to broiler breeder farms. Sources of infection at broiler breeder farm level remained unidentified. Significance and Impact of the Study: Major diversity was demonstrated for DT41 MLVA types. A persisting problem with infection of broiler breeder flocks with DT41 was not reflected in broiler flocks originating from these flocks.     

Identification by subtractive hybridization of sequences specific for Salmonella enterica serovar enteritidis.

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1‐year period in Lithuania

Aims: The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. Methods and Results: During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter‐positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73·8%) were positive for Camp. jejuni and 17 flocks (40·48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1‐year period. Conclusions: Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm‐specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. Significance and Impact of the Study: For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.     

Lipopolysaccharide-specific IgG in egg yolk from two chicken flocks infected with Salmonella enteritidis

An indirect enzyme-linked immunosorbent assay (ELISA) was used to determine levels of IgG reacting with lipopolysaccharide from Salmonella enteritidis in samples of egg yolk from broiler breeder and egg-laying flocks, some of which were known to be infected with S. enteritidis. There was positive correlation between infection with salmonella and a high reading in the ELISA.     

Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods

AIM: To develop and validate high throughput methods for the direct enumeration of viable and culturable Salmonella and Escherichia coli O157:H7 in ground beef, carcass, hide and faecal (GCHF) samples from cattle. METHODS AND RESULTS: The hydrophobic grid membrane filtration (HGMF) method and the spiral plate count method (SPCM) were evaluated as rapid tools for the estimation of pathogen load using GCHF samples spiked with known levels of Salmonella serotype Typhimurium. Validation studies showed that for a single determination of each sample type the low end of the detection limits were approx. 2.0 x 10(0) CFU g(-1) for ground beef, 5.0 x 10(-1) CFU (100 cm(2))(-1) for Salmonella and 8.0 x 10(-1) CFU (100 cm(2))(-1) for E. coli O157:H7 on carcasses, 4.0 x 10(1) CFU (100 cm(2))(-1) for hide and 2.0 x 10(2) CFU g(-1) for faecal samples. In addition, ground beef (n = 609), carcass (n = 1520) and hide (n = 3038) samples were collected from beef-processing plants and faecal samples (n = 3190) were collected from feed-lot cattle, and these samples were tested for the presence of Salmonella and E. coli O157:H7 by enrichment and enumeration methods. CONCLUSIONS: The direct enumeration methods described here are amenable to high throughput sample processing and were found to be cost-effective alternatives to other enumeration methods for the estimation of Salmonella and E. coli O157:H7, in samples collected during cattle production and beef processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of the methods described here would allow for more routine testing and quantification data collection, providing useful information about the effectiveness of beef processing intervention strategies.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

Effect of Salmonella vaccination of breeder chickens on contamination of broiler chicken carcasses in integrated poultry operations

While measures to control carcass contamination with Salmonella at the processing plant have been implemented with some success, on-farm interventions that reduce Salmonella prevalence in meat birds entering the processing plant have not translated well on a commercial scale. We determined the impact of Salmonella vaccination on commercial poultry operations by monitoring four vaccinated and four nonvaccinated breeder (parental) chicken flocks and comparing Salmonella prevalences in these flocks and their broiler, meat bird progeny. For one poultry company, their young breeders were vaccinated by using a live-attenuated Salmonella enterica serovar Typhimurium vaccine (Megan VAC-1) followed by a killed Salmonella bacterin consisting of S. enterica serovar Berta and S. enterica serovar Kentucky. The other participating poultry company did not vaccinate their breeders or broilers. The analysis revealed that vaccinated hens had a lower prevalence of Salmonella in the ceca (38.3% versus 64.2%; P < 0.001) and the reproductive tracts (14.22% versus 51.7%; P < 0.001). We also observed a lower Salmonella prevalence in broiler chicks (18.1% versus 33.5%; P < 0.001), acquired from vaccinated breeders, when placed at the broiler farms contracted with the poultry company. Broiler chicken farms populated with chicks from vaccinated breeders also tended to have fewer environmental samples containing Salmonella (14.4% versus 30.1%; P < 0.001). There was a lower Salmonella prevalence in broilers entering the processing plants (23.4% versus 33.5%; P < 0.001) for the poultry company that utilized this Salmonella vaccination program for its breeders. Investigation of other company-associated factors did not indicate that the difference between companies could be attributed to measures other than the vaccination program.     

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.     

Efficacy of flagellin gene typing for epidemiological studies of Campylobacter jejuni in poultry estimated by comparison with macrorestriction profiling

Thirty isolates of Campylobacter jejuni isolated from 29 different Danish broiler flocks were chosen for the evaluation of PCR-Fla typing as a genotyping tool. Except for two isolates that originated from the same broiler flock, the isolates were clearly distinguishable on basis of their macrorestriction profiles using the restriction endonucleases SmaI and KpnI. PCR-Fla typing of the 30 isolates yielded 16 distinct genotypes, whereas one isolate was untypeable by this method. The dominant PCR-Fla type (1/1) was shared by eight isolates, and five additional Fla groups containing two or three isolates were obtained. The PCR-Fla type of one isolate changed spontaneously after five subcultures, illustrating the relative plasticity of the gene locus. Comparison of MRPs within and between Fla-types support the view that some PCR-Fla types may be conserved within clonal lines. It is concluded that PCR-Fla typing is useful as a genotyping tool in large-scale epidemiological studies but that additional analyses with other methods are required to properly define interstrain relationships.     

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.