Foodborne disease prevention and broiler chickens with reduced Campylobacter infection.

Studies have suggested that flies play a linking role in the epidemiology of Campylobacter spp. in broiler chickens and that fly screens can reduce the prevalence of Campylobacter spp. We examined the year-round and long-term effects of fly screens in 10 broiler chicken houses (99 flocks) in Denmark. Prevalence of Campylobacter spp.-positive flocks was significantly reduced, from 41.4% during 2003-2005 (before fly screens) to 10.3% in 2006-2009 (with fly screens). In fly screen houses, Campylobacter spp. prevalence did not peak during the summer. Nationally, prevalence of Campylobacter spp.-positive flocks in Denmark could have been reduced by an estimated 77% during summer had fly screens been part of biosecurity practices. These results imply that fly screens might help reduce prevalence of campylobacteriosis among humans, which is closely linked to Campylobacter spp. prevalence among broiler chicken flocks.

Real-time detection and identification of Chlamydophila species in veterinary specimens by using SYBR green-based PCR assays.

Infections caused by members of the Chlamydiaceae family have long been underestimated due to the requirement of special laboratory facilities for the detection of this group of intracellular pathogens. Furthermore, new studies of this group of intracellular pathogens have revealed that host specificity of different species is not as clear as recently believed. As most members of the genus Chlamydophila have shown to be transmissible from animals to humans, sensitive and fast detection methods are required. In this study, SYBR green-based real-time assays were developed that detect all members of Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp. felis was found in 10 cats, Cp. caviae was found in one guinea pig, and Cp. abortus was detected in one sheep. The screening assay appeared more sensitive than traditional microscopical examination of stained tissue smears. By combining a fast, robust, and cost-effective method for sample preparation with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays.

The influence of the cage system and colonisation of Salmonella Enteritidis on the microbial gut flora of laying hens studied by T-RFLP and 454 pyrosequencing.

BACKGROUND: In the EU conventional cages for laying hens are forbidden beginning in January 2012, however concerns about a higher transmission rate of Salmonella in alternative cages systems have been raised. The extent to which cage systems may affect the intestinal microbiota of laying hens is not known, and different microbiota may demonstrate different resistance towards colonization with Salmonella. To investigate this, ileal and caecal samples from two experimental studies where laying hens were inoculated with Salmonella Enteritidis and housed in different systems (conventional cage, furnished cage or aviary), were compared using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The distribution of genera in the microbiota in caecum was furthermore described by next generation sequencing of 16S rDNA libraries. RESULTS: Hens in the same cage type developed similar T-RFLP fingerprints of the ileal and caecal microbiota, and these could be separated from layers in the other cages types. No significant difference in the fingerprint profiles could be observed between Salmonella positive and negative samples from same cage. By deep sequencing of 16S rDNA libraries from caecum, 197 different Operational Taxonomic Units (OTU) were identified, and 195 and 196 OTU respectively, were found in hens in aviary and furnished cages, but only 178 OTU of these were recovered from conventional cages. The ratio between the dominating phyla or families and genera in the microbiota remained fairly constant throughout the study. Faecalibacterium and Butyricimonas were the most prevalent genera found in the caecal microbiota of layers irrespective of the cage type. CONCLUSIONS: Hens confined in the same cage group tend to develop similar microbiota in their ileum and caecum possibly due to isolation, while differences in the microbiota between cages may be caused by environmental or individual bird factors. Although the cages type had influence on composition of the microbiota in the layers by promoting higher diversity in furnished and aviary systems, we did not observe differences in colonization and excretion pattern of Salmonella from these groups. We suggest, that differences in group size and exposure to a more faecally contaminated environment in the alternative systems may explain the observed differences in diversity of the caecal microbiota.

A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species - C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

Estimating sensitivity and specificity of a PCR for boot socks to detect Campylobacter in broiler primary production using Bayesian latent class analysis.

The present study compares three different assays for sample collection and detection of Campylobacter spp. in broiler flocks, based on (i) the collection of faecal samples from intestinal organs (caecum), (ii) individual faecal droppings collected from the bedding and (iii) faecal material collected by socks placed on the outside of a pair of boots (boot socks) and used for walking around in the flock. The two first methods are examined for Campylobacter using a culture method (ISO-10272-2:2006), while the boot socks are tested using PCR. The PCR-assay is a genus specific multiplex PCR with primers targeting 16S rDNA in Campylobacter and primers targeting Yersinia ruckerii. Sixty-seven broiler flocks from Austria and 83 broiler flocks from Denmark were included in this prospective study and 89 of these were found to be positive in at least one method (AT: 49 samples, DK: 40 samples) whereas 61 of these were negative in all assays. In Austria samples for the three assays were collected simultaneously, which facilitates a direct comparison of the diagnostic test performance. In Denmark, however, boot socks and faecal droppings were collected three days before slaughter while caecum samples were collected at slaughter. The results were evaluated in the absence of a gold standard using a Bayesian latent class model. Austrian results showed higher sensitivity for PCR detection in sock samples (0.98; Bayesian credible interval (BCI) [0.93-1]) than for culture of faecal droppings (0.86; BCI [0.76-0.91]) or caecal samples (0.92; BCI [0.85-0.97]). The potential impact of Campylobacter introduction within the final three days before slaughter was observed in Denmark, where four flocks were tested negative three days before slaughter, but were detected positive at the slaughterhouse. Therefore the model results for the PCR sensitivity (0.88; BCI [0.83-0.97]) and cultural ISO-method in faecal samples (0.84; BCI [0.76-0.92]) are lower than for caecal samples (0.93; BCI [0.85-0.98]). In our study, PCR detection on boot sock samples is more sensitive than conventional culture. In view of the advantage of rapid results before slaughter and low costs for sampling, especially in combination with existing Salmonella surveillance systems (just another pair of boot socks needed), this method-matrix combination could be a valuable surveillance tool in the broiler primary production.

Campylobacter jejuni and Campylobacter coli in wild birds on Danish livestock farms.

BACKGROUND: Reducing the occurrence of campylobacteriosis is a food safety issue of high priority, as in recent years it has been the most commonly reported zoonosis in the EU. Livestock farms are of particular interest, since cattle, swine and poultry are common reservoirs of Campylobacter spp. The farm environment provides attractive foraging and breeding habitats for some bird species reported to carry thermophilic Campylobacter spp. We investigated the Campylobacter spp. carriage rates in 52 wild bird species present on 12 Danish farms, sampled during a winter and a summer season, in order to study the factors influencing the prevalence in wild birds according to their ecological guild. In total, 1607 individual wild bird cloacal swab samples and 386 livestock manure samples were cultured for Campylobacter spp. according to the Nordic Committee on Food Analysis method NMKL 119. RESULTS: The highest Campylobacter spp. prevalence was seen in 110 out of 178 thrushes (61.8 %), of which the majority were Common Blackbird (Turdus merula), and in 131 out of 616 sparrows (21.3 %), a guild made up of House Sparrow (Passer domesticus) and Eurasian Tree Sparrow (Passer montanus). In general, birds feeding on a diet of animal or mixed animal and vegetable origin, foraging on the ground and vegetation in close proximity to livestock stables were more likely to carry Campylobacter spp. in both summer (P < 0.001) and winter (P < 0.001) than birds foraging further away from the farm or in the air. Age, fat score, gender, and migration range were not found to be associated with Campylobacter spp. carriage. A correlation was found between the prevalence (%) of C. jejuni in wild birds and the proportions (%) of C. jejuni in both manure on cattle farms (R(2) = 0.92) and poultry farms (R(2) = 0.54), and between the prevalence (%) of C. coli in wild birds and the proportions (%) of C. coli in manure on pig farms (R(2) = 0.62). CONCLUSIONS: The ecological guild of wild birds influences the prevalence of Campylobacter spp. through the behavioural patterns of the birds. More specifically, wild birds eating food of animal or mixed animal and vegetable origin and foraging on the ground close to livestock were more likely to carry Campylobacter spp. than those foraging further away or hunting in the air. These findings suggest that wild birds may play a role in sustaining the epidemiology of Campylobacter spp. on farms.

Characterization of sulphonamide-resistant Escherichia coli using comparison of sul2 gene sequences and multilocus sequence typing.

The sul2 gene encodes sulphonamide resistance (Sul(R)) and is commonly found in Escherichia coli from different hosts. We typed E. coli isolates by multilocus sequence typing (MLST) and compared the results to sequence variation of sul2, in order to investigate the relation to host origin of pathogenic and commensal E. coli strains and to investigate whether transfer of sul2 into different genomic lineages has happened multiple times. Sixty-eight E. coli isolated in Denmark and Norway from different hosts and years were MLST typed and sul2 PCR products were sequenced and compared. PFGE was performed in a subset of isolates. All isolates were divided into 45 different sequence types (STs), with clonal complexes CC10, CC23, CC168, CC350 and CC69 being the most frequent. The sul2 gene from the majority of E. coli strains had only two point mutations, at positions 159 and 197, leading to a synonymous and a non-synonymous change, respectively. Five strains had extra single mutations. All poultry, poultry meat, and Danish human blood isolates had the same sul2 ST and some of these strains clustered under the same MLST STs, indicating that they shared habitats. Most PFGE profiles clustered according to source, but some included different sources. Sul(R) E. coli from different animals, food, human faeces and infections did not cluster according to their origin, suggesting that these habitats share E. coli and sul2 gene types. However, while pig isolates on one occasion clustered with urinary tract infection isolates, poultry isolates seemed more related to isolates from bloodstream infections in humans. Presence of mainly two types of the sul2 gene in both human and animal isolates, irrespective of date and geography, and the presence of both types in the same clonal lineages, suggest horizontal transfer of sul2.

Trends in occurrence of antimicrobial resistance in Campylobacter jejuni isolates from broiler chickens, broiler chicken meat, and human domestically acquired cases and travel associated cases in Denmark.

Campylobacter jejuni is a frequent cause of bacterial gastroenteritis. Often it causes self-limiting disease but severe or prolonged cases may require antimicrobial treatment. The agricultural use of antimicrobial agents selects for resistance among C. jejuni which is transmitted to humans via food. In Denmark, the use of fluoroquinolones in animal husbandry has been restricted since 2003. The purpose of the present study was to look at trends in occurrence of resistance among C. jejuni from broiler chickens, broiler chicken meat and human domestically acquired or travel associated cases. From 1997 through 2007, C. jejuni isolates were obtained from The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) and susceptibility tested for ciprofloxacin, erythromycin, nalidixic acid, and tetracycline. Erythromycin resistance was at a low level in all the reservoirs during the study period. Resistance to ciprofloxacin, nalidixic acid and tetracycline was significantly higher in C. jejuni from imported broiler chicken meat compared to Danish broiler chicken meat. In domestically acquired human C. jejuni isolates, resistance to ciprofloxacin and nalidixic acid was for most years significantly higher compared to the level found in isolates from Danish broiler chicken meat, whereas the resistance level was similar to the level found in isolates from imported broiler chicken meat. Imported broiler chicken meat may therefore contribute to the high level of ciprofloxacin and nalidixic acid resistance in C. jejuni isolates from domestically acquired human infections. In 2006 and 2007, the occurrence of resistance to ciprofloxacin, nalidixic acid and tetracycline was significantly higher in travel associated C. jejuni isolates compared to isolates acquired domestically. Even though the use of fluoroquinolones is restricted for animal use in Denmark, Danes are still often infected by fluoroquinolone resistant C. jejuni from imported chicken meat or by travelling.

Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark.

We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue.

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR.

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.