Norwegian Broiler Research Articles

A molecular epidemiological study on Escherichia coli in young chicks with colibacillosis identified two possible outbreaks across farms

Avian pathogenic Escherichia coli (APEC) is the cause of colibacillosis outbreaks in young poultry chicks, resulting in acute to peracute death. The high morbidity and mortality caused by colibacillosis results in poor animal welfare, reduced sustainability and economical loss worldwide. To advance the understanding of the molecular epidemiology, genomic relatedness and virulence traits of APEC, we performed systematic sampling from 45 confirmed colibacillosis broiler flocks with high first week mortality (FWM) during 2018–2021. From these flocks, 219 APEC isolates were whole genome sequenced (WGS) and bioinformatic analyses were performed. The bioinformatic analyses included sequence typing (ST), serotyping, detection of virulence-associated genes (VAGs) and phylogenetic analysis. Our results showed a high prevalence of ST23, ST429 and ST95 among APEC isolates from Norwegian broiler flocks, and identified ST23, ST429, ST117 and ST371 to cause disease more often alone, compared to ST95, ST69 and ST10. Phylogenetic analyses, together with associated metadata, identified two distinct outbreaks of colibacillosis across farms caused by ST429 and ST23 and gave insight into expected SNP distances within and between flocks identified with the same ST. Further, our results highlighted the need for combining two typing methods, such as serotyping and sequence typing, to better discriminate strains of APEC. Ultimately, systematic sampling of APEC from multiple birds in a flock, together with WGS as a diagnostic tool is important to identify the disease-causing APEC within a flock and to detect outbreaks of colibacillosis across farms.

The Effect of Antimicrobial Resistance Plasmids Carrying blaCMY-2 on Biofilm Formation by Escherichia coli from the Broiler Production Chain.

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.

Comparative Genome Analyses of Wild Type- and Quinolone Resistant Escherichia coli Indicate Dissemination of QREC in the Norwegian Broiler Breeding Pyramid

Quinolones are important antimicrobials for both humans and animals, and resistance toward these compounds is a serious threat to public health. In Norway, quinolone resistant E. coli (QREC) have been detected at low levels in a high proportion of broiler flocks, even without the use of quinolones in rearing of broilers. Due to the pyramidal structure of broiler breeding, QREC isolates may be disseminated from grandparent animals down through the pyramid. However, quinolone resistance can also develop in wild type E. coli through specific chromosomal mutations, and by horizontal acquisition of plasmid-mediated quinolone resistance genes. The goal of this study was to determine whether QREC is disseminated through the broiler breeding pyramid or developed locally at some stage in the broiler production chain. For this purpose, we whole genome sequenced wild type- and QREC isolates from broiler and parent flocks that had been isolated in the Norwegian monitoring program for antimicrobial resistance in feed, food and animals (NORM-VET) between 2006 and 2017, from 22 different production sites. The sequencing data was used for typing of the isolates, phylogenetic analysis and identification of relevant resistance mechanisms. Highly similar QREC isolates were identified within major sequence types from multiple production sites, suggesting dissemination of QREC isolates in the broiler production chain. The occurrence of potential resistance development among the WT E. coli was low, indicating that this may be a rare phenomenon in the Norwegian broiler production. The results indicate that the majority of the observed QREC at the bottom of the broiler production pyramid originates from parent or grandparent animals. These results highlight the importance of surveillance at all levels of the broiler production pyramid and of implementation of proper biosecurity measures to control dissemination of QREC.

Dissemination of Quinolone-Resistant Escherichia coli in the Norwegian Broiler and Pig Production Chains and Possible Persistence in the Broiler Production Environment.

In Norway, the use of quinolones in livestock populations is very low, and prophylactic use is prohibited. Despite this, quinolone-resistant Escherichia coli (QREC) isolates are present at low levels in several animal species. The source of these QREC isolates is unknown. The aim of this study was to characterize and compare QREC isolates from different animal species to identify putative factors that may promote the occurrence of QREC. A total of 280 QREC isolates, from broilers, pigs, red foxes, and wild birds, were whole-genome sequenced and analyzed. Well-known chromosomal and plasmid-mediated resistance mechanisms were identified. In addition, mutations in marR, marA, and rpoB causing novel amino acid substitutions in their respective proteins were detected. Phylogenetic analyses were used to determine the relationships between the isolates. Quinolone resistance mechanism patterns appeared to follow sequence type groups. Similar QREC isolates with similar resistance mechanism patterns were detected from the samples, and further phylogenetic analysis indicated close evolutionary relationships between specific isolates from different sources. This suggests the dissemination of highly similar QREC isolates between animal species and also the persistence of QREC strains within the broiler production chain. This highlights the importance of both control measures at the top of the production chain as well as biosecurity measures to avoid the further dissemination and persistence of QREC in these environments.IMPORTANCE Since antimicrobial usage is low in Norwegian animal husbandry, Norway is an ideal country to study antimicrobial resistance in the absence of selective pressure from antimicrobial usage. In particular, the usage of quinolones is very low, which makes it possible to investigate the spread and development of quinolone resistance in natural environments. Comparison of quinolone-resistant E. coli (QREC) isolates from livestock and wild animals in light of this low quinolone usage provides new insights into the development and dissemination of QREC in both natural and production environments. With this information, preventive measures may be taken to prevent further dissemination within Norwegian livestock and between other animals, thus maintaining the favorable situation in Norway.

BlaCTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

Escherichia coli carrying blaCTX–M–1 mediating resistance to extended-spectrum cephalosporins was recently described as a new genotype in Norwegian broiler production. The aim of this study was to characterize these isolates (n = 31) in order to determine whether the emergence of the genotype was caused by clonal expansion or horizontal dissemination of blaCTX–M–1-carrying plasmids. All included isolates were subjected to whole genome sequencing. Plasmid transferability was determined by conjugation, and plasmid replicons in the transconjugants were described using PCR-based replicon typing. Plasmid sizes were determined using S1 nuclease digestion. Plasmids in a subset of strains were reconstructed and compared to plasmids from broiler production in other European countries. The isolates belonged to nine different sequence types (STs), with the largest group being ST57 (n = 12). The vast majority of blaCTX–M–1-carrying plasmids were conjugative. All transconjugants were positive for the IncI1-Iγ replicon, and several also harbored the IncFIB replicon. Highly similar plasmids were present in different E. coli STs. Additionally, high similarity to previously published plasmids was detected. A reconstructed plasmid from an ST57 isolate harbored both IncI1-Iγ and IncFIB replicons and was considered to be co-integrated. The presence of one large plasmid was confirmed by S1 nuclease digestion. Our results show that dissemination of blaCTX–M–1 in Norwegian broiler production is due to both clonal expansion and horizontal transfer of plasmids carrying blaCTX–M–1. The blaCTX–M–1/IncI1-Iγ plasmids grouped into two main lineages, namely clonal complex (CC)-3 and CC-7. The genetic diversity at both strain and plasmid level indicates multiple introductions to Norway. We also show that the blaCTX–M–1 plasmids circulating in Norwegian broiler production are highly similar to plasmids previously described in other countries.

Biofilm forming properties of quinolone resistant Escherichia coli from the broiler production chain and their dynamics in mixed biofilms

BackgroundQuinolone resistant Escherichia coli (QREC) have been found in samples from Norwegian broiler chicken, despite quinolones not being administered to poultry in Norway. Biofilm production may be one factor contributing to the observed persistence in the broiler production chain. In the present study, 158 QREC strains from chicken caecal and retail meat samples were screened for biofilm production in microtiter plates, biofilm morphotype on Congo Red (CR) agar plates and phylotype by multiplex PCR. Furthermore, the dynamics in mixed biofilms with strains of different morphotypes were studied on glass slides and on CR agar plates.ResultsAll strains but one produced biofilm in microtiter plates and/or on CR agar plates at room temperature. There were no differences between strains from chicken caecum and chicken retail meat in the mean amount of biofilm produced in microtiter plates. Furthermore, no differences in biofilm production were observed between phylotypes. However, significant differences in biofilm production were found between biofilm morphotypes. The morphotype RDAR (red dry and rough, which has both curli and cellulose in the matrix, was displayed by 70% of the strains. Mean biofilm production by these strains were significantly higher than by strains with the morphotypes PDAR (pink dry and rough) with only cellulose or BDAR (brown dry and rough) with only curli. Interestingly, the two latter morphotypes produced biofilms with the morphotype RDAR when grown together. None of the strains achieved significantly higher numbers of colony forming units (cfu) in mixed biofilms than in single strain biofilms on glass slides.ConclusionsThe results indicate that QREC can form biofilm reservoirs on both inert and organic surfaces in production environments, as well as on meat. This may contribute to persistence and dissemination of the strains. Strains with both curli and cellulose in the biofilm matrix were significantly better biofilm formers than strains lacking one of these components. However, strains with only one of the components could compensate for this by producing mixed biofilms with strains having the other component, and thereby most likely enhance their probabilities of persistence in the production environment.

Optimisation of the broiler production supply chain

In this paper, we propose a mixed integer programming (MIP) model for the Chicken Flock Sizing, Allocation and Scheduling Problem (CFSASP), which is an important planning problem in the broiler production supply chain. To solve the CFSASP efficiently, two variants of rolling horizon heuristics (RHHs) have been developed and applied on the case of a Norwegian broiler production company. Computational results show that the RHHs successfully obtain high-quality solutions within a reasonable time. The value of optimisation is verified through comparison with the case company's plans, where the solutions from optimisation outperforms the current solutions. Sensitivity analyses are also conducted to provide managerial insights regarding certain strategic decisions, such as how many and which days to use for hatching of chickens. Due to the promising results, the case company is now implementing an optimisation-based decision support system based on the MIP model and solution methods shown in this paper.

Significant reduction of vancomycin resistant E. faecium in the Norwegian broiler population coincided with measures taken by the broiler industry to reduce antimicrobial resistant bacteria.

Vancomycin resistant enterococci (VRE) belong to the most common causes of nosocomial infections worldwide. It has been reported that use of the glycopeptide growth promoter avoparcin selected for a significant livestock-reservoir of VRE in many European countries, including Norway. However, although avoparcin was banned as a feed-additive in 1995, VRE have for unknown reasons consistently been reported in samples from Norwegian broilers. When avoparcin was banned, broiler-feed was supplemented with the polyether ionophore narasin in order to control the diseases coccidiosis and the frequent sequela necrotic enteritis. A potential link between transferrable vancomycin resistance and reduced susceptibility to narasin was recently reported. The use of narasin as a feed additive was abolished by the Norwegian broiler industry in 2016 and since then, broilers have been reared without in-feed antibacterial supplements. In this study, we demonstrate that all VRE isolates from Norwegian broilers collected in 2006–2014 displayed reduced susceptibility to narasin. Surveillance data collected two years after the narasin abolishment show a significant reduction in VRE, below the detection limit of the surveillance method, and a concurrent marked reduction in Enterococcus faecium with reduced susceptibility to narasin. The significant decline of E. faecium with reduced susceptibility to these antimicrobial compounds also coincided with an increased focus on cleaning and disinfection between broiler flocks. Furthermore, data from a controlled in vivo experiment using Ross 308 broilers indicate that the proportion of E. faecium with reduced susceptibility to narasin was heavily reduced in broilers fed a narasin-free diet compared to a diet supplemented with narasin. Our results are consistent with that the abolishment of this feed additive, possibly in combination with the increased focus on cleaning and disinfection, has had a substantial impact on the occurrence of VRE in the Norwegian broiler population.

The Risk of Development of Antimicrobial Resistance with the Use of Coccidiostats in Poultry Diets

The Risk of Development of Antimicrobial Resistance with the Use of Coccidiostats in Poultry Diets

Occurrence of and risk factors for extended-spectrum cephalosporin-resistant Enterobacteriaceae determined by sampling of all Norwegian broiler flocks during a six month period.

All broiler flocks reared and slaughtered in Norway from May-October 2016 (n = 2110) were screened for the presence of extended-spectrum cephalosporin (ESC) -resistant Enterobacteriaceae. Furthermore, we investigated possible risk factors for occurrence of such bacteria in broiler flocks. The odds of a flock being positive for ESC-resistant Enterobacteriaceae increased if the previous flock in the same house was positive, and if the flock was reared during September-October. However, we cannot exclude seasonal fluctuations in occurrence of ESC-resistant Enterobacteriaceae during the months November to April. The overall occurrence of ESC-resistant Enterobacteriaceae was 10.4%, and primarily linked to the presence of blaCMY (82.6%) in positive isolates. We describe the first findings of Escherichia coli with blaCTX-M-1, Klebsiella pneumoniae with both blaCTX-M-15 and blaSHV-12, and K. pneumoniae with blaCMY isolated from Norwegian broiler production. This study gives us a unique overview and estimate of the true occurrence of ESC-resistant Enterobacteriaceae in Norwegian broilers over a six-month period. To the best of our knowledge, this is the most comprehensive study performed on the occurrence of ESC-resistant Enterobacteriaceae in a broiler population.

On-farm broiler chicken welfare assessment using transect sampling reflects environmental inputs and production outcomes.

To evaluate the utility of transect sampling for assessing animal welfare in large chicken flocks, we quantified relationships between environmental inputs, welfare problems detected using transect sampling, and production outcomes. We hypothesised that environmental inputs including environmental complexity (i.e. number of environmental enrichment types provided), space allowance, underfloor heating (presence or absence), and photoperiod regimen (18 h continuous vs 16 h intermittent) would correspond to variations in welfare assessment findings, which would predict production outcomes. We conducted on-farm welfare assessment of Norwegian broiler flocks at approximately 28 days of age. We sampled four transects (rows between feeder and drinker lines) per flock to determine litter quality and the proportions of chickens with compromised welfare as indicated by visual signs of walking difficulties, illness, skin wounds and small bird size. Production outcome measures included mortality, reasons for carcass rejection at slaughter, footpad dermatitis, growth rate, feed conversion and an integrated production index. Greater environmental complexity was associated with a reduction in skin wounds and total welfare problems on the farm, lower mortality, fewer rejections due to wounds and underweight birds, and fewer rejections overall. Higher space allowances within levels of environmental complexity were associated with fewer walking difficulties and welfare problems overall, a reduction in rejections due to wounds, and a higher growth rate and production index. Underfloor heating was associated with a reduction in rejections due to leg deformity, and intermittent light was associated with lower illness and skin wound rates on the farm, and lower mortality. Furthermore, fewer welfare problems and better litter quality on the farm were associated with fewer carcass rejections at slaughter. Thus, data from transect sampling varied with environmental inputs and production outcomes, supporting the validity of transect sampling for practical, animal-based on-farm welfare assessment.

Associations between qualitative behaviour assessments and measures of leg health, fear and mortality in Norwegian broiler chicken flocks

Qualitative behavioural assessments (QBA) is an animal-based welfare measure that has been included in several on-farm welfare assessment protocols, including the Welfare Quality® (WQ) protocol for poultry. However, there is a scarcity of information about how it relates to other animal-based welfare indicators. The aim of this study was therefore to investigate the associations between QBA and selected animal-based welfare indicators commonly used for the assessment of broiler chicken welfare, i.e. lameness, foot pad dermatitis (FPD), fear of humans (touch test), and mortality.A total of 50 commercial broiler chicken farms were visited by one observer who conducted on-farm welfare assessments using the WQ protocol. Assessments were done close to the time of slaughter (between day 27 and 34). QBA was analysed using principal component analysis (PCA), revealing two main components, labelled arousal (PC1) and mood (PC2). The scores for the other welfare indicators were categorised into dichotomous (touch test) or ordinal scales (gait score, footpad dermatitis score and mortality) to deal with skewed distributions caused by homogenous data. To investigate the associations between QBA and the other welfare indicators, we ran logistic and ordinal logistic regression models with these welfare measures as outcomes, and the two components of QBA as the predictors.Significant negative associations were found between both components of QBA and the chickens’ fear of humans, as measured using the touch test. In other words, flocks with higher scores on both mood and arousal were less likely to have any chickens that were possible to touch by the assessor. A possible interpretation of these associations is that both QBA components may indicate greater liveliness in birds that did not accept to be touched by the observer. Flocks with a higher arousal score, as measured by the first component of QBA (PC1), were also less likely to be in a higher mortality category. For the other selected animal-based measures, there were no associations with QBA. We conclude that QBA needs thorough validation for the routine use in the assessment of broiler chicken welfare, but that the method may provide useful supplementary information in overall welfare assessments. This information may be particularly valuable in a production system, like the broiler industry, where management is highly standardised, sometimes resulting in little between-flock variation in other welfare measures.

The relationship between measures of fear of humans and lameness in broiler chicken flocks

In the Welfare Quality® assessment protocol for broilers, the touch test is included to assess the human–animal relationship in the flock. The test is designed to measure the animals’ fear of humans, assuming that broilers will withdraw from the observer if they are fearful. However, many broilers close to slaughter age have impaired walking ability, and the results from the touch test may thus be biased by lameness and poor leg health. As the touch test is currently being used in several countries to assess human–animal relationship in broilers, there is an urgent need to examine this potential relationship for a further validation of the test. In the present study, fear of humans was assessed in 50 randomly selected Norwegian broiler flocks, using the touch test as described in the Welfare Quality® protocol for ty broilers. Leg health was assessed by examining the gait of 150 random birds in each of the flocks, using a six-point gait score scale from 0 to 5. The coefficient for the relationship between touch test score and gait score was 0.034 (P<0.001), indicating that the animals express less fear as assessed by the touch test when the gait scores increase. This implies that the touch test may be confounded by impaired walking ability and therefore might be a suboptimal method of assessing fear of humans and human–animal relationship in broilers. In conclusion, the results from this study suggests that the touch test must be further validated in broilers and perhaps be replaced with a fear test that doesn’t rely on walking ability.

Associations among gait score, production data, abattoir registrations, and postmortem tibia measurements in broiler chickens

Lameness and impaired walking ability in rapidly growing meat-type broiler chickens are major welfare issues that cause economic losses. This study analyzed the prevalence of impaired walking and its associations with production data, abattoir registrations, and postmortem tibia measurements in Norwegian broiler chickens. Gait score (GS) was used to assess walking ability in 59 different commercial broiler flocks (Ross 308) close to the slaughter d, 5,900 broilers in total, in 3 different geographical regions. In each flock, 100 arbitrary broilers were gait scored and 10 random broilers were culled to harvest tibias. Abattoir registrations on flock level were collected after slaughter. A total of 24.6% of the broilers had moderate to severe gait impairment. The broilers were sampled in 2 stages, first slaughterhouse/region, and then owner/flock. The final models showed that impaired gait is associated with first-week mortality (P < 0.05), region (P < 0.001), height of tibias mid-shaft (P < 0.05), and calcium content in the tibia ash (P < 0.05), and negatively associated with DOA (P < 0.05). The prevalence of impaired gait indicates that this is a common problem in the broiler industry in Norway, although the mean slaughter age is only 31 d and the maximum allowed animal density is relatively low. Impaired walking ability could not be predicted by the welfare indicators footpad lesion score, total on-farm mortality, and decreasing DOA prevalence. Further studies are needed to explore the relationship between first-week mortality and gait score.

Transfer Potential of Plasmids Conferring Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Poultry.

ABSTRACTEscherichia coli strains resistant to extended-spectrum cephalosporins (ESC) are widely distributed in Norwegian broiler production, and the majority harbor transferable IncK or IncI1 plasmids carrying blaCMY-2. Persistent occurrence in broiler farms may occur through the survival of ESC-resistant E. coli strains in the farm environment, or by transfer and maintenance of resistance plasmids within a population of environmental bacteria with high survival abilities. The aim of this study was to determine the transferability of two successful blaCMY-2-carrying plasmids belonging to the incompatibility groups IncK and IncI1 into E. coli and Serratia species recipients. Initially, conjugative plasmid transfer from two E. coli donors to potential recipients was tested in an agar assay. Conjugation was further investigated for selected mating pairs in surface and planktonic assays at temperatures from 12°C to 37°C. Transfer of plasmids was observed on agar, in broth, and in biofilm at temperatures down to 25°C. The IncK plasmid was able to transfer into Serratia marcescens, and transconjugants were able to act as secondary plasmid donors to different E. coli and Serratia species recipients. All transconjugants displayed an AmpC phenotype corresponding to the acquisition of blaCMY-2. In summary, the results indicate that the IncK plasmid may transfer between E. coli and Serratia spp. under conditions relevant for broiler production.IMPORTANCE Certain blaCMY-2-carrying plasmids are successful and disseminated in European broiler production. Traditionally, plasmid transferability has been studied under conditions that are optimal for bacterial growth. Plasmid transfer has previously been reported between E. coli bacteria in biofilms at 37°C and in broth at temperatures ranging from 8 to 37°C. However, intergenus transfer of blaCMY-2-carrying plasmids from E. coli to environmental bacteria in the food-processing chain has not been previously studied. We demonstrate that blaCMY-2-carrying plasmids are capable of conjugative transfer between different poultry-associated bacterial genera under conditions relevant for broiler production. Transfer to Serratia spp. and to hosts with good biofilm-forming abilities and with the potential to act as secondary plasmid donors to new hosts might contribute to the persistence of these resistance plasmids. These results contribute to increased knowledge of factors affecting the persistence of ESC resistance in broiler production and can provide a basis for improvement of routines and preventive measures.

Risk factors for occurrence of cephalosporin-resistant Escherichia coli in Norwegian broiler flocks

A longitudinal study of 27 broiler farms including 182 broiler flocks was performed to determine risk factors for occurrence of cephalosporin-resistant Escherichia coli in Norwegian broiler flocks. Information regarding possible risk factors was collected by an online questionnaire and by samples obtained from broiler and parent flocks during the study period. Additional information was provided by the broiler production company. The prevalence of cephalosporin-resistant E. coli in parent flocks and broiler flocks sampled in the study was estimated.Cephalosporin-resistant E. coli was detected in 13.8% of the parent flocks and 22.5% of the broiler flocks included in the study.A multivariable generalized linear model was used to estimate risk factors. The risk for occurrence of cephalosporin-resistant E. coli was associated with the status of the previous flock in the broiler house (odds ratio=12.7), number of parent flocks supplying the broiler flock with day-old chickens (odds ratio=6.3), routines for disinfection of floor between production cycles (odds ratio=0.1), and transport personnel entering the room where the broilers are raised (odds ratio=9.3).Our findings highlights that implementation of a high level of biosecurity with a minimal number of people entering the broiler house during production cycles, as well as rigorous cleaning and disinfection routines between production cycles will contribute to a decrease in the occurrence of cephalosporin-resistant E. coli in broiler flocks provided that there is no selection pressure from antimicrobial use in the broiler production.

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production.

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the blaCMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare blaCMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying blaCMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying blaCMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of blaCMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing blaCMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.

Farm specific risk factors for Campylobacter colonisation in Danish and Norwegian broilers

Campylobacteriosis has become the leading bacterial zoonosis in humans in the European Union and other developed countries. There are many sources of human Campylobacter infections, but broilers and broiler meat have been shown to be the most important. In order to implement effective interventions that reduce the probability of Campylobacter colonisation of broiler flocks, it is essential to fully understand the risk factors involved.We present a bi-national risk factor survey comprising Campylobacter data from more than 5200 Danish and Norwegian indoor, conventional broiler flocks and the responses to a standardised questionnaire, with more than 40 explanatory variables from 277 Danish and Norwegian farms. We explored several models by using different combinations of the Danish and Norwegian data, including models with single-country datasets. All models were analysed using a generalized linear model using backwards elimination and forward selection.The results show that Norwegian broiler flocks had a lower risk of being colonised than Danish flocks. Farm specific variables that increased the risk of flocks becoming colonised with Campylobacter in both countries were: broiler houses older than five years; longer downtime (no. of days between flocks), probably a consequence of longer downtimes being associated with less focus on maintaining a high biosecurity level; broiler houses without a separate ante-room or barrier; and the use of the drinker nipples with cups or bells compared with nipples without cups.Additional country specific risk factors were also identified. For Norway, the risk of colonisation increased with increasing numbers of houses on a farm and when the water used for the broilers originated from surface water or bore holes instead of mains. For Denmark, having boot dips or low stocking density increased the risk of a flock becoming Campylobacter positive.The different model approaches allowed us to explore the effect of having a large number of data available to identify the significant variables. To a large extent, the country specific models identified risk factors that were also found in the bi-national model. However, the bi-national model identified more risk factors than the country specific models. This indicated that combining the data sets from the two countries did not disrupt the results but was beneficial due to the greater strength achieved in the statistical analyses and the possibility of examining interactions terms with the variable Country.

Emergence of AmpC-producing Escherichia coli in the broiler production chain in a country with a low antimicrobial usage profile

The aim of this study was to estimate the prevalence of cephalosporin-resistant Escherichia coli at the different levels of the Norwegian broiler production pyramid and identify the mechanisms responsible for the resistance phenotype. Samples from all levels of the broiler production pyramid and retail chicken meat (fillets) were included (n=649). The occurrence of cephalosporin-resistant E. coli at the different production levels ranged from 8 to 43%. All these isolates had an AmpC-phenotype, and the majority carried the blaCMY-2 gene. In addition, a few isolates with up-regulated chromosomal ampC were identified. The results show that Norway has a relatively high prevalence of cephalosporin-resistant E. coli in the broiler production chain in spite of a very low consumption of antimicrobial agents. Cephalosporins have not been used in the Norwegian broiler production, and it has been hypothesised that import of breeding animals and hatching eggs may be the source of these resistant bacteria. We demonstrate that these bacteria are disseminated in the production pyramid despite the lack of selection pressure from antimicrobial agents.

Effect of climate and farm environment on Campylobacter spp. colonisation in Norwegian broiler flocks

Campylobacteriosis is the most frequently reported zoonosis in the EU. A recent report states that between 50% and 80% of the human campylobacteriosis cases could be attributed to broiler as a reservoir. The current study was conducted to investigate associations between the presence of Campylobacter spp. in Norwegian broiler flocks and factors related to the climate and the farm environment. Data from 18,488 broiler flocks from 623 different farms during 2002–2007 were included in the study. A logistic regression analysis was conducted where Campylobacter spp. status of a broiler flock at the time of slaughter was defined as the dependent variable and farm was modelled as a random effect. The following factors were found to increase the probability for a broiler flock to test positive for Campylobacter spp.: daily mean temperature above 6°C during the rearing period, private water supply, presence of other livestock farms within a distance of 2km, presence of other broiler farms within a distance of 4km with flocks positive for Campylobacter spp. within 30 days prior to slaughter, heavy rainfall 11–30 days prior to slaughter, region and year. Daily mean temperature below 0°C reduced the probability. The study emphasises the importance of the farm environment and the climate for the occurrence of Campylobacter spp. in broiler flocks. The farm environment is probably a part of the Campylobacter spp. pathway into and between broiler flocks where farmyard run-off and humans or flies entering the houses might constitute vehicles transporting the organism. Fly activity is temperature-driven and flies might be a part of the explanation of the increased risk for Campylobacter spp. related to increased temperature demonstrated in the study.