Decoding Klebsiella pneumoniae in poultry chain: unveiling genetic landscape, antibiotic resistance, and biocide tolerance in non-clinical reservoirs.

The rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. However, limited knowledge exists about the population structure and genomic diversity of K. pneumoniae circulating in conventional poultry production. We conducted a comprehensive characterization of K. pneumoniae across the whole chicken production chain (7 farms; 14 flocks + environment + meat, 56 samples; 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features of K. pneumoniae were characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive for K. pneumoniae with a significant increase (p < 0.05) from early (n = 1/14) to pre-slaughter (n = 11/14) stages, most (n = 6/7) persisting in chicken meat batches. Colistin-resistant K. pneumoniae rates were low (4%-n = 1/24 positive samples), while most samples carried MDR strains (67%-n = 16/24) and copper-tolerant isolates (63%-n = 15/24, with sil and pco gene clusters; MICCuSO4 ≥ 16 mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity against K. pneumoniae (MIC/MBC range = 4-64 mg/L) from representative strains independently of the presence or absence of genes linked to QACs tolerance. A polyclonal K. pneumoniae population, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chicken's lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carrying sil + pco (copper) co-located with qacEΔ1 ± qacF (QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverse K. pneumoniae clones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencing K. pneumoniae persistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.

Predicting Risk of Ammonia Exposure in Broiler Housing: Correlation with Incidence of Health Issues.

The study aimed to forecast ammonia exposure risk in broiler chicken production, correlating it with health injuries using machine learning. Two chicken breeds, fast-growing (Ross®) and slow-growing (Hubbard®), were compared at different densities. Slow-growing birds had a constant density of 32 kg m-2, while fast-growing birds had low (16 kg m-2) and high (32 kg m-2) densities. Initial feeding was uniform, but nutritional demands led to varied diets later. Environmental data underwent selection, pre-processing, transformation, mining, analysis, and interpretation. Classification algorithms (decision tree, SMO, Naive Bayes, and Multilayer Perceptron) were employed for predicting ammonia risk (10-14 pmm, Moderate risk). Cross-validation was used for model parameterization. The Spearman correlation coefficient assessed the link between predicted ammonia risk and health injuries, such as pododermatitis, vision/affected, and mucosal injuries. These injuries encompassed trachea, bronchi, lungs, eyes, paws, and other issues. The Multilayer Perceptron model emerged as the best predictor, exceeding 98% accuracy in forecasting injuries caused by ammonia. The correlation coefficient demonstrated a strong association between elevated ammonia risks and chicken injuries. Birds exposed to higher ammonia concentrations exhibited a more robust correlation. In conclusion, the study effectively used machine learning to predict ammonia exposure risk and correlated it with health injuries in broiler chickens. The Multilayer Perceptron model demonstrated superior accuracy in forecasting injuries related to ammonia (10-14 pmm, Moderate risk). The findings underscored the significant association between increased ammonia exposure risks and the incidence of health injuries in broiler chicken production, shedding light on the importance of managing ammonia levels for bird welfare.

Distribution and genetic characterization of fluoroquinolone resistance gene qnr among Salmonella strains from chicken in China.

The prevalence and dissemination of the plasmid-mediated fluoroquinolone (FQ) resistance gene qnr in Salmonella are considered serious public health concerns worldwide. So far, no comprehensive large-scale studies have focused on the prevalence and genetic characteristics of the qnr gene in Salmonella isolated from chickens. Herein, this study aimed to investigate the prevalence, antimicrobial resistance (AMR) patterns, and molecular characteristics of chicken-originated qnr-positive Salmonella strains from chicken farms, slaughterhouses, and markets in 12 provinces of China in 2020-2021. The overall prevalence of the qnr gene was 21.13% (56/265), with the highest prevalence in markets (36.11%, 26/72), followed in farms (17.95%, 21/117), and slaughterhouses (10.53%, 9/76). Only the qnrS and qnrB genes were detected, and the prevalence rate of the qnrS gene (19.25%, 51/265) was higher than that of the qnrB gene (1.89%, 5/265). Whole genome sequencing identified 37 distinct AMR genes and 15 plasmid replicons, and the most frequent mutation in quinolone resistance determining regions was parC (T57S; 91.49%, 43/47). Meanwhile, four different qnrS and two qnrB genetic environments were discovered among 47 qnr-positive Salmonella strains. In total, 21.28% (10/47) of the strains were capable of conjugative transfer, and all were qnrS1-positive strains, with the majority of transferable plasmids being IncHI2 types (n = 4). Overall, the prevalence of qnr-positive Salmonella strains from chickens in China and their carriage of multiple resistance and virulence genes and transferable plasmids is a major concern, which calls for continuous surveillance of qnr-positive Salmonella and the development of measures to control its prevalence and transmission.IMPORTANCESalmonella is a common foodborne pathogen responsible for 155,000 deaths annually worldwide. Fluoroquinolones (FQs) are used as first-line drugs for the treatment of Salmonella infections in several countries and regions. However, the emergence and increasing prevalence of the FQ-resistant gene qnr in Salmonella isolated from chickens have been widely reported. Gaining insight into the genetic mechanisms of AMR genes in chicken could lead to the development of preventive measures to control and reduce the risk of drug resistance. In this study, we identified qnr-positive Salmonellae isolated from chickens in different regions of China and their AMR patterns and genome-wide characteristics, providing a theoretical basis for further control of their prevalence and transmission.

Indigenous chicken production system in different agro-ecology of Indian Himalayan Region: implication on food and economic security.

The indigenous chicken production system (ICPS) has several use values and ecosystem services. In the last few years, ICPS has been recognized for its possible contribution to household food security, income generation, wildlife protection, and bettering the women's lives. This study aimed to collect, for the first time, comprehensive information about ICPS in three different agro-ecologies (tropical, sub-tropical, and sub-temperate) of the Indian Himalayan Region (IHR) and its role in food and economic security of traditional communities. In this study region, ICPS is semi-extensive, providing homegrown feed and temporary night shelter. In sub-temperate agro-ecology, females owned non-significant (p = 0.170) more indigenous chicken flocks than males. Households in sub-temperate agro-ecologies had significantly (p ≤ 0.001) larger flock sizes and tropical livestock units (chicken-TLU). However, the livestock diversity index (LDI) was significantly higher (p ≤ 0.001) in tropical and subtropical agro-ecology. The households in the sub-temperate region highly (p ≤ 0.001) valued indigenous chicken because of its survivability and adaptability. In absolute numbers significant (p ≤ 0.001) higher numbers of adult birds died in past 1 year in sub-temperate agro-ecology. The mortality rate of adult birds in sub-temperate agro-ecology was 9%, and it was 14 and 15% in tropical and sub-tropical agro-ecologies, respectively. In sub-temperate agro-ecology, larger flock size translated into significantly higher (p ≤ 0.001) egg production and subsequently a significant (p ≤ 0.001) higher egg consumption per household per month. In sub-temperate agro-ecology, households' dietary diversity score was significantly (p ≤ 0.001) higher. Similarly, the average annual income from ICPS was significantly higher (p ≤ 0.001) in sub-temperate agro-ecology and accounted for 18% of household income. ICPS' marketing chain was relatively short in the sub-temperate region. In all agro-ecologies, indigenous chicken and egg demand was significantly higher (p ≤ 0.001) in the winter. ICPS litter is used as farmyard manure, enhancing ecological resilience. In all agro-ecologies, the three most frequently cited obstacles to extending the indigenous chicken production system are illnesses, predators, and a lack of chicks availability. ICPS contributes to food and nutritional security, economic stability, and ecological resilience in this hilly and fragile ecosystem. Even though the system is self-sustaining, management and health interventions can increase production and productivity.

Interaction between roxarsone, an organic arsenic compound, with humic substances in the soil simulating environmental conditions.

In environmental systems, the soil is a principal route of contamination by various potentially toxic species. Roxarsone (RX) is an arsenic (V) organic compound used to treat parasitic diseases and as an additive for animal fattening. When the animal excretes RX, the residues may lead to environmental contamination. Due to their physicochemical properties, the soil's humic substances (HS) are important in species distribution in the environment and are involved in various specific interaction/adsorption processes. Since RX, an arsenic (V) compound, is considered an emerging contaminant, its interaction with HS was evaluated in simulated environmental conditions. The HS-RX interaction was analyzed by monitoring intrinsic HS fluorescence intensity variations caused by complexation with RX, forming non-fluorescent supramolecular complexes that yielded a binding constant Kb (on the order of 103). The HS-RX interaction occurred through static quenching due to complex formation in the ground state, which was confirmed by spectrophotometry. The process was spontaneous (ΔG < 0), and the predominant interaction forces were van der Waals and hydrogen bonding (ΔH < 0 and ΔS < 0), with an electrostatic component evidenced by the influence of ionic strength in the interaction process. Structural changes in the HS were verified by synchronized and 3D fluorescence, with higher variation in the region referring to the protein-like fraction. In addition, metal ions (except ions Cu(II)) favored HS-RX interaction. When interacting with HS, the RX epitope was suggested by 1H NMR, which indicated that the entire molecule interacts with the superstructure. An enzyme inhibition assay verified the ability to reduce the alkaline phosphatase activity of free and complexed RX (RX-HS). Finally, this work revealed the main parameters associated with HS and RX interaction in simulated environmental conditions, thus, providing data that may help our understanding of the dynamics of organic arsenic-influenced soils.

Uncovering the effects of copper feed supplementation on the selection of copper-tolerant and antibiotic-resistant Enterococcus in poultry production for sustainable environmental practices.

The use of antibiotics in animal production is linked to the emergence and spread of antibiotic-resistant bacteria, a threat to animal, environmental and human health. Copper (Cu) is an essential element in poultry diets and an alternative to antibiotics, supplementing inorganic or organic trace mineral feeds (ITMF/OTMF). However, its contribution to select multidrug-resistant (MDR) and Cu tolerant Enterococcus, a bacteria with a human-animal-environment-food interface, remains uncertain. We evaluated whether feeding chickens with Cu-ITMF or Cu-OTMF contributes to the selection of Cu tolerant and MDR Enterococcus from rearing to slaughter. Animal faeces [2-3-days-old (n = 18); pre-slaughter (n = 16)] and their meat (n = 18), drinking-water (n = 14) and feed (n = 18) from seven intensive farms with ITMF and OTMF flocks (10.000-64.000 animals each; 2019-2020; Portugal) were sampled. Enterococcus were studied by cultural, molecular and whole-genome sequencing methods and Cu concentrations by ICP-MS. Enterococcus (n = 477; 60 % MDR) were identified in 80 % of the samples, with >50 % carrying isolates resistant to tetracycline, quinupristin-dalfopristin, erythromycin, streptomycin, ampicillin or ciprofloxacin. Enterococcus with Cu tolerance genes, especially tcrB ± cueO, were mainly found in faeces (85 %; E. faecium/E. lactis) of ITMF/OTMF flocks. Similar occurrence and load of tcrB ± cueO Enterococcus in the faeces was detected throughout the chickens' lifespan in the ITMF/OTMF flocks, decreasing in meat. Most of the polyclonal MDR Enterococcus population carrying tcrB ± cueO or only cueO (67 %) showed a wild-type phenotype (MICCuSO4 ≤ 12 mM) linked to absence of tcrYAZB or truncated variants, also detected in 85 % of Enterococcus public genomes from poultry. Finally, < 65 µg/g Cu was found in all faecal and meat samples. In conclusion, Cu present in ITMF/OTMF is not selecting Cu tolerant and MDR Enterococcus during chickens' lifespan. However, more studies are needed to assess the minimum concentration of Cu required for MDR bacterial selection and horizontal transfer of antibiotic resistance genes, which would support sustainable practices mitigating antibiotic resistance spread in animal production and the environment beyond.

Bioprotective potential of lactic acid bacteria for Salmonella biocontrol in vitro.

Lactic acid bacteria (LAB) are an important option for Salmonella control in animal production, resulting in lower antibiotic use. The objective of this research was to isolate LAB from meat products and from commercial probiotics sold as nutritional supplements for in vitro verification of their bioprotective potential. Eleven bacteria were identified as Pediococcus acidilactici, two as Lacticaseibacillus rhamnosus, one as Lacticaseibacillus paracasei paracasei, one as Limosilactobacillus fermentum, and one as a consortium of Lactobacillus delbrueckii bulgaricus and L. fermentum. All bacteria showed inhibitory activity against Salmonella, with emphasis on the inhibition of P. acidilactici PUCPR 011 against Salmonella Enteritidis 33SUSUP, S. Enteritidis 9SUSP, S. Enteritidis 56301, S. Enteritidis CRIFS 1016, Salmonella Typhimurium ATCC™ 14,028®, and Salmonella Gallinarum AL 1138, with inhibition halos of 7.3 ± 0.5 mm, 7.7 ± 1.0 mm, 9.0 ± 1.8 mm, 7.3 ± 0.5 mm, 7.7 ± 1.0 mm, and 7.3 ± 0.5, respectively. The isolates P. acidilactici PUCPR 011, P. acidilactici PUCPR 012, P. acidilactici PUCPR 014, L. fermentum PUCPR 005, L. paracasei paracasei PUCPR 013, and L. rhamnosus PUCPR 010 showed inhibition greater than 2 mm against at least 3 Salmonella and were used for encapsulation and in vitro digestion. The encapsulation efficiency ranged from 76.89 ± 1.54 to 116.48 ± 2.23%, and the population after 12 months of storage was from 5.31 ± 0.17 to 9.46 ± 0.09 log CFU/g. When simulating swine and chicken digestion, there was a large reduction in bacterial viability, stabilizing at concentrations close to 2.5 log CFU/mL after the analyses. The analyzed bacteria showed strong in vitro bioprotective potential; further analyses are required to determine in vivo effectiveness.

Environmental footprints of farmed chicken and salmon bridge the land and sea.

Food production, particularly of fed animals, is a leading cause of environmental degradation globally.1,2 Understanding where and how much environmental pressure different fed animal products exert is critical to designing effective food policies that promote sustainability.3 Here, we assess and compare the environmental footprint of farming industrial broiler chickens and farmed salmonids (salmon, marine trout, and Arctic char) to identify opportunities to reduce environmental pressures. We map cumulative environmental pressures (greenhouse gas emissions, nutrient pollution, freshwater use, and spatial disturbance), with particular focus on dynamics across the land and sea. We found that farming broiler chickens disturbs 9 times more area than farming salmon (∼924,000 vs. ∼103,500 km2) but yields 55 times greater production. The footprints of both sectors are extensive, but 95% of cumulative pressures are concentrated into <5% of total area. Surprisingly, the location of these pressures is similar (85.5% spatial overlap between chicken and salmon pressures), primarily due to shared feed ingredients. Environmental pressures from feed ingredients account for >78% and >69% of cumulative pressures of broiler chicken and farmed salmon production, respectively, and could represent a key leverage point to reduce environmental footprints. The environmental efficiency (cumulative pressures per tonne of production) also differs geographically, with areas of high efficiency revealing further potential to promote sustainability. The propagation of environmental pressures across the land and sea underscores the importance of integrating food policies across realms and sectors to advance food system sustainability.

Growth performance and meat quality from broiler chickens reared with zeolite and halloysite in feed and straw pellet.

The study's aim was to analyze the effects of different levels of halloysite (H) and zeolite (Z) in feed and pellets on the growth and meat quality in chickens; 500 Ross 308 broilers were assigned to five groups (1 g, control; 2-5 g, experimental). In Groups 2-5, different proportions of Z and H in feed (25:75 ratio; 0.5% to 2%) and pellet in kg/m2 (2, 0.650H; 3, 0.325H and 0.325Z; 4, 0.650Z; 5, 0.160H and 0.490Z) were used. Body weight was higher than 1 in all experimental groups, and carcass weight was higher, except for Group 2. The feed consumption was lower in Groups 3 and 5 than in Group 4. The breast muscle weight was higher in Group 3 than in the control group. In Groups 2, 3, and 5, the water holding capacity in the breast muscles was better than in Group 4 and in the leg muscles in Groups 3 and 4 than in Groups 1 and 5. Most of the tested characteristics indicate a beneficial effect of aluminosilicates in feed and litter on the growth and quality of meat.

Tracing Clostridium perfringens strains along the chicken production chain from farm to slaughter by multilocus sequence typing.

The current study is undertaken to characterize the prevalence, genotypes distribution, antibiotic resistance and genetic diversity of Clostridium perfringens (C. perfringens) collected from different stages of a chicken production chain. In total, 579 samples from a broiler farm and 378 samples from the slaughterhouse were collected from a large-scale rearing and slaughter one-stop enterprise in Weifang, China, between June and July 2019, of which 30.40% of the samples from farm and 54.50% of samples from slaughterhouse were determined to be positive for C. perfringens, respectively. The contamination of chicken products was relatively serious, with the total positive rate of carcasses at 59.73%; the positive rate of carcass samples was the highest in the evisceration process, which might be the critical point of C. perfringens contamination. A total of 476 isolates of C. perfringens were recovered; and 99.58% of recovered isolates were identified as type A, with the remaining isolates being type G. Antimicrobial susceptibility testing revealed that 97.15% of the isolates showed multiple antibiotic resistance and 67.89% of them were resistant to at least five classes of commonly used antibiotics. Multilocus sequence typing results of 91 representative isolates showed that the isolates can be divided into 74 sequences types (STs); 40.66% of the isolates can be included into seven clonal complexes (CCs). Although most of the isolates were classified as type A, considerable genetic diversity was observed, with the Simpson's diversity index of ST up to 0.9902. Some isolates from farm stage and slaughter stage were distributed in the same ST or CC, indicating that chicken products may be contaminated by the same ST or CC of C. perfringens originated from the farm stage. The high contamination rates of chicken products and the widespread multiple antibiotic resistance of isolates indicated potential public health risks, control measures at rearing and slaughtering stage should be considered to reduce this risk.

Longitudinal Study of the Distribution of Antimicrobial-Resistant Campylobacter Isolates from an Integrated Broiler Chicken Operation.

The aim of this study was to analyze the prevalence, antimicrobial resistance, and genetic diversity of Campylobacter isolates that were obtained from whole chicken production stages in Korea. A total of 1348 samples were collected from 10 production lines. The prevalence of Campylobacter in breeder farm, broiler farm, slaughterhouse, and retail meat products was 50.0%, 3.3%, 13.4%, and 68.4%, respectively, and Campylobacter was not detected at the hatchery stage. Resistance to quinolones/fluoroquinolones was the most prevalent at all stages. Among the multidrug-resistant isolates, 16 isolates (19.8%) from breeder farm were resistant to both azithromycin and ciprofloxacin. A total of 182 isolates were subdivided into 82 pulsed-field gel electrophoresis (PFGE) genotypes with 100% similarity. Diverse genotypes were presented with discontinuous patterns along the whole production chain. Thirty percent of Campylobacter-free flocks became positive after slaughtering. An identical genotype was simultaneously detected from both breeder farm and retail meat, even from different production lines. This study reveals that antimicrobial-resistant Campylobacter contamination can occur at all stages of the chicken supply chain. In particular, the breeder farm and slaughterhouse should be the main control points, as they are the potential stages at which antimicrobial-resistant Campylobacter could spread to retail meat products by horizontal transmission.

Characterization and Prevalence of Campylobacter spp. From Broiler Chicken Rearing Period to the Slaughtering Process in Eastern China.

Campylobacter is one of the most important foodborne pathogens worldwide, and poultry is regarded as the main reservoir of Campylobacter. The contamination of Campylobacter in broiler chickens at the farm level is closely related to the transmission of Campylobacter in the poultry production chain. This study identified 464 Campylobacter isolates from 1,534 samples from broiler rearing period and slaughtering process including 233 Campylobacter jejuni isolates and 231 Campylobacter coli isolates. We have observed a dynamic distribution of Campylobacter during broiler chicken production, that 66.3% of Campylobacter isolates were C. jejuni during broiler rearing period, while C. coli occupied 60.4% of Campylobacter isolates during the broiler slaughtering process. A tag-label method allowed us to track the dynamic of Campylobacter in each broiler chicken from 31-day age at rearing to the partition step in the slaughterhouse. At the 31-day during rearing, 150 broiler chicken were labeled, and was tracked for Campylobacter positive from rearing period to slaughtering process. Among the labeled broiler, 11 of the tracking broiler samples were able to detect Campylobacter from rearing period to slaughtering. All Campylobacter isolates from the 11 tracking samples were sequenced and analyzed. C. jejuni isolates were divided into four STs and C. coli isolates were divided into six STs. Isolates with identical core genome were observed from the same tag-labeled samples at different stages indicating a vertical transmission of Campylobacter in the early broiler meat production. Meanwhile, the core genome analysis elucidated the cross-contamination of Campylobacter during the rearing period and the slaughtering process. The virulotyping analysis revealed that all C. jejuni isolates shared the same virulotypes, while C. coli isolates were divided into three different virulotypes. The antimicrobial resistance gene analysis demonstrated that all Campylobacter isolates contained at least two antibiotic resistance genes (ARGs), and the ARG profiles were well-corresponding to each ST type. Our study observed a high prevalence of Campylobacter during the early chicken meat production, and further studies will be needed to investigate the diversity and transmission of Campylobacter in the poultry production chain.

The Costs, Benefits and Human Behaviours for Antimicrobial Use in Small Commercial Broiler Chicken Systems in Indonesia.

There are growing concerns over the threat to human health from the unregulated use of antimicrobials in livestock. Broiler production is of great economic and social importance in Indonesia. This study used a structured questionnaire approach to explore the human behaviours and economic drivers associated with antimicrobial use in small commercial broiler systems in Indonesia (n = 509). The study showed that antimicrobial use was high with farmers easily able to access antimicrobials through local animal medicine, however, it was difficult for farmers to access veterinary advice on responsible antimicrobial use. The most significant finding was that the relative cost of antimicrobials was low, and farmers observed improvements in productivity rates from routine antimicrobial administration. However, farmers seldom kept detailed records on farm productivity or economic costs; this is a hurdle to undertaking a more detailed economic analysis of antimicrobial use. There is a need for further research on the cost-effectiveness of alternative methods of preventing disease and ensuring that feasible alternatives are easily available. Farm-level economics and securing the food supply chain need to be central to any future policy interventions to reduce antimicrobial use in broiler systems in Indonesia and this observation is relevant at a regional and global level.

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.

Isolation and Identification of Arcobacter Species from Costa Rican Poultry Production and Retail Sources.

Arcobacter is a gram-negative rod recognized as a potential food- and waterborne pathogen; nevertheless, little is known about the effects of this pathogen on human and animal health. Although Arcobacter species are commonly found in nature, poultry is suspected to be the main vehicle for the transmission of this pathogen. The aims of this work were to determine the prevalence of Arcobacter spp. in broilers produced in Costa Rica for human consumption and to analyze the pathogenic capacity of the isolates through the detection of virulence genes. One hundred fifty-two samples of cecal content (87 farms), 104 samples of carcass rinse after chiller (six processing plants), and 96 carcass rinses from as many retail stores were analyzed. The suspicious isolates were identified using genus-specific PCR, and species-level identification was achieved with a multiplex PCR. Virulence genes were identified using the protocol described by L. Douidah, L. de Zutter, J. Baré, P. De Vos, P. Vandamme, O. Vandenberg, A.-M. Van den Abeele, and K. Houf (J. Clin. Microbiol. 50:735-741, 2012), which includes nine different virulence genes. The overall isolation frequency of Arcobacter was 6.5% (n = 23). Eight (34.8%) of the isolates came from cecal content, 2 (8.7%) were isolated from samples taken after chiller, and 13 (56.5%) were from retail stores. The species isolated included A. thereius (30.4%), A. butzleri (21.7%), A. skirrowii (4.3%), and A. cibarius (4.3%). The remaining samples were classified as Arcobacter sp. Gene tlyA was the most prevalent virulence gene, present in 9 of 23 samples analyzed; genes hecA and pldA were present in one only strain each. A strain of A. butzleri isolated from a retail store presented the highest number of virulence genes (five), and 11 samples did not present any of the genes analyzed. The results obtained suggest that the presence of virulent Arcobacter isolates in the poultry production chain from Costa Rica could be a risk for individuals who consume the contaminated product.

Compliance/non-compliance with biosecurity rules specified in the Danish Quality Assurance system (KIK) and Campylobacter-positive broiler flocks 2012 and 2013.

One source for Campylobacter jejuni infections in humans could be consumption of broiler meat. Transmission of Campylobacter into broiler houses/flocks occurs via many routes. A number of biosecurity rules is specified in the Quality Assurance System in Danish Chicken Production (KIK) - for which the broiler producers annually are audited for compliance with, by bureau Veritas. Multivariable logistic regression models were used to investigated the association between Compliance/non-compliance with biosecurity rules and Campylobacter-positive flocks - on KIK data from 2012 and 2013. Month and before after audit period were also included in the models. KIK rules important to comply with were: no vegetation around houses, closed systems for feed storage and distribution, and division between clean and unclean zones within broiler houses. A Campylobacter-reducing effect was observed of audit visits (in itself), indicating that there is more focus on compliance with KIK at the time of an audit visit, and that adequate daily biosecurity behavior is important.

Risk factors for Campylobacter colonization in Danish broiler flocks, 2010 to 2011.

The objectives of the two studies presented were to estimate the prevalence of Campylobacter-positive farms and flocks and to acquire updated knowledge about risk factors for the introduction of Campylobacter in Danish broiler flocks. In the first study, from September 2010 to September 2011, there were 25 Danish broiler farms visited, and a questionnaire was filled in by a veterinarian/consultant. In the second study, a similar questionnaire was distributed electronically to all Danish broiler farmers (n=164) that were on record with an email address in the Quality Assurance System in the Danish Broiler Production (KIK) database. House- and flock-specific data collected in the surveys were supplemented with information obtained from the KIK database. Data obtained from the two studies were analyzed separately by logistic regression analysis. In both models, the dependent variable was "Campylobacter flock status (positive/negative)," which was based on real-time PCR testing of fecal material from the floor of each broiler house that had been collected preslaughter using a pair of tube gauze "socks." This material was pooled into one sample. Of the 25 farms visited, 17 had delivered Campylobacter-positive flocks during the study period, and eight farms had no Campylobacter-positive flocks. Moreover, the flock prevalence of Campylobacter was 17% (n=418). Data obtained from the electronically distributed survey revealed that 63% (n=71) of the farms were Campylobacter-positive. Further, the flock prevalence of Campylobacter was 14% (n=1,286). The multivariable models from the two sets of data identified the following statistically significant risk factors: summer vs. winter; if the previous flock in the house was positive for Campylobacter vs. if the previous flock in the house was negative; and litter delivered into the house close to the time of arrival of new chickens vs. storing litter on the farm. Furthermore, the data showed that a vertically based ventilation system (where most of the air is taken into the house through chimneys) constituted a higher risk than a horizontally based system (where the air is taken in through openings in the side walls).