Danish Chickens Research Articles

Vancomycin resistance in Enterococcus faecium isolated from Danish chicken meat is located on a pVEF4-like plasmid persisting in poultry for 18 years

The occurrence of vancomycin-resistant Enterococcus faecium (VREfm) in food is relevant to public health as foodborne VREfm may colonize the gut of consumers and transfer vancomycin resistance genes to the indigenous gut microbiota. Therefore, we determined occurrence and elucidated genetic traits of VREfm in Danish retail chicken meat. Three out of 40 samples (7.5%) from two slaughterhouses yielded VREfm (vancomycin MIC > 32 mg/L). This is the first report of VREfm in Danish retail poultry meat since 2010 (DANMAP). All three VREfm belonged to the sequence type ST32, cluster type CT1068. Using whole genome sequencing, we detected transposon Tn1546 harbouring the vanA operon encoding vancomycin resistance. The vanA operon was located on a 43.4 kb plasmid highly similar (99.9% identity across 97.5% of the sequence) to pVEF4, which was observed in VREfm in Norwegian poultry in 1998 and in Danish poultry in 2010. The remarkable persistence of a pVEF4-like plasmid in enterococcal populations may be explained by the presence of two independent plasmid stability systems, the ω/ε/ζ toxin-antitoxin system and the prgOPN gene cluster. Filter mating experiments showed that the pVEF4-like plasmid could transfer between E. faecium strains in vitro and that transfer occurred concomitantly with a larger, co-residing plasmid. The data presented here indicate that poultry meat constitutes a reservoir of VREfm and further investigations are needed to assess the risk of foodborne transmission to humans.

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.

Source attribution of human campylobacteriosis in Denmark

SUMMARY This study assesses the contribution of different sources of human campylobacteriosis in Denmark using two different source-attribution approaches. In total, 794 non-human isolates and 406 isolates from human cases (domestic, travel related, and cases with unknown travel history) were collected. Isolates were characterized by multilocus sequence typing, flaA typing and susceptibility to antibiotics. Both models used indicate that the major burden of human campylobacteriosis in Denmark originates from the domestic broiler chicken reservoir. The second most important reservoir was found to be cattle. The Asymmetric Island model attributed 52% [95% credibility interval (CrI) 37-67] to Danish chicken, 17% (95% CrI 3-33) to imported chicken, and 17% (95% CrI 7-28) to cattle. Similarly, the Campylobacter source-attribution model apportioned 38% (95% CrI 28-47) to Danish chicken, 14% (95% CrI 10-18) to imported chicken, and 16% (95% CrI 7-25) to cattle. The addition of flaA type as an extra discriminatory typing parameter did not change the attribution of cases markedly.

Escherichia coli Producing CTX-M-1, -2, and -9 Group β-Lactamases in Organic Chicken Egg Production

A zoonotic contribution to the spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli at the community level has been proposed repeatedly (3), but information on the origin and frequency of ESBLs within different animal reservoirs is still limited. To improve the current understanding of the evolution and epidemiology of ESBLs in poultry farming, we investigated the occurrence of ESBL-producing E. coli in flocks of chicken egg layers reared in Danish organic systems, where low flock density, restricted antimicrobial use, and access to outdoor areas are regulated at the European level (4). From June to August 2009, we visited four randomly selected healthy layer flocks (flocks A, B, C, and D), reared at independent farms with no history of antimicrobial and biocide use. E. coli bacteria with reduced susceptibility to cefotaxime were recovered from flocks B and C using sock samples enriched in MacConkey broth supplemented with cefotaxime (2 μg/ml) (1). PCR analysis (6, 7) followed by partial sequencing revealed the presence of blaTEM-1 (n = 7), blaCTX-M-1 group (compatible with blaCTX-M-1/61) (n = 9), blaCTX-M-2 group (compatible with blaCTX-M-2/20/44) (n = 3), and blaCTX-M-9 group (compatible with blaCTX-M-14/17) (n = 4) genes (http://www.ncbi.nlm.nih.gov/ and http://www.lahey.org/Studies/) in randomly selected colonies (Table ​(Table1).1). We revisited farms B and C after 10 and 11 weeks, respectively, aiming to quantify the prevalence of CTX-M-positive chickens and the concentration of CTX-M-producing E. coli in feces. The study population remained unchanged, since no chickens had entered the farms in the interval between the two sampling points. At both farms, 2 (3%) out of 60 cloacal swabs collected from individual animals and processed as described previously (1) were positive for CTX-M-producing E. coli. Of 10 fresh fecal droppings collected from the floor on each farm, 7 (farm B) and 3 (farm C) samples yielded CTX-M-producing E. coli at concentrations ranging between 102 and 103 CFU per gram of feces, which accounted for ≤0.03% of total E. coli. By XbaI pulsed-field gel electrophoresis (PFGE) analysis, 30 CTX-M producers obtained from different sample types (sock samples, swabs, and fecal droppings) displayed 16 epidemiologically unrelated band patterns according to Tenover et al. (8). Two isolates were untypeable. Twenty strains representing distinct PFGE profiles and sample types carried blaCTX-M on transferable IncI1 (n = 7), IncN (n = 3), and untypeable (n = 10) plasmids, often containing additional determinants conferring resistance to tetracycline, sulfonamides, and trimethoprim, as demonstrated by conjugation and PCR-based replicon typing (PBRT) (2, 5) (Table ​(Table1).1). Restriction fragment length polymorphism (RFLP) analysis using ClaI and BglII showed that farm-specific IncI1 and IncN plasmids were widespread among different E. coli lineages in association with specific blaCTX-M genes (Table ​(Table1).1). All PBRT-untypeable plasmids, which demonstrated poor quality images by RFLP analysis, were approximately 149 kb in size. TABLE 1. Genetic and phenotypic traits of CTX-M-producing Escherichia coli from organic layers To our knowledge, this is the first description of CTX-M-producing E. coli in the Danish chicken production. The factors leading to the origin and persistence of these resistance genes of high clinical relevance in organic poultry not exposed to antimicrobial agents remain unknown. Further research based on plasmid sequencing and gene characterization is needed to elucidate the nature of the nonantimicrobial resistance genes located on blaCTX-M gene-carrying plasmids and their possible role in favoring the spread and maintenance of such plasmids in the absence of antimicrobial selective pressure.

Antimicrobial resistance among Campylobacter jejuni isolated from raw poultry meat at retail level in Denmark

Campylobacter jejuni isolated from raw poultry meat collected at retail shops in Denmark in the period 1996–2003 were tested for susceptibility to seven antimicrobial agents. The food samples consisted of raw chicken meat and other raw poultry meat of domestic or imported origin. The highest levels of resistance among C. jejuni were observed for tetracycline, nalidixic acid and ciprofloxacin, whereas macrolide resistance was rarely detected. C. jejuni originating from other poultry meat (mainly duck and turkey meat) exhibited the highest occurrences of antimicrobial resistance monitored; approximately one third of the isolates were tetracycline resistant ( N = 100). Among chicken meat isolates, the occurrence of tetracycline resistance was significantly higher ( P < 0.005) in C. jejuni isolated from imported chicken meat ( N = 88) than in C. jejuni from Danish chicken meat ( N = 367). The same tendency was observed for chloramphenicol, nalidixic acid and ciprofloxacin ( P < 0.05). The trends in resistance in the period 1996–2003 among C. jejuni isolates from chicken meat indicate a decrease in the occurrence of resistance towards fluoroquinolones. This may be due to reduced application of fluoroquinolones for food animals. Monitoring of the occurrence of antimicrobial resistance in C. jejuni isolated from raw uncooked poultry has been performed on a yearly basis since 1996, thus providing useful insight into consumer exposure to antimicrobial-resistant C. jejuni.

Antibiotic-resistant strains of Enterococcus isolated from Swedish and Danish retailed chicken and pork

Two hundred and seventy-seven Enterococcus isolates representing the predominating enterococcal flora of retailed chicken and pork were identified by phenotypical features, and by random amplified polymorphic DNA. The resistance to nine different antibiotics was determined. Enterococcus faecium was the most frequently occurring species in both Swedish and Danish chicken. Enterococcus faecalis and unidentified groups of Enterococcus dominated in pork. Seventy-three per cent of the Enterococcus isolates from Swedish chicken were resistant to one or more of the tested antibiotics. The corresponding values for Swedish pork, Danish chicken and Danish pork were 9%, 55% and 14%, respectively. Tetracycline resistance was most frequent in isolates from Danish pork and Swedish chicken, while erythromycin resistance was most frequent in Danish chicken. Strains with acquired vancomycin resistance, mainly Ent. faecium, were found only on Danish chicken, except for one isolate from Danish pork. All vancomycin-resistant isolates contained the vanA gene. Vancomycin resistance could be transferred to a vancomycin-sensitive Ent. faecium strain from four of nine tested donor strains.

Exposure to air contaminants in chicken catching.

A cross-sectional study of chicken catchers was carried out by personal sampling of air contaminants. The catchers used either the drawer method (DM) or truck method (TM) for loading chickens into cages. DM catchers were exposed to higher concentrations of hazardous substances than TM catchers, except for ammonia. In terms of geometric means total dust concentrations were 11.3 mg/m3 (DM catchers) and 8.1 mg/m3 (TM catchers). Full-shift dust exposure exceeded the Danish occupational exposure limit (OEL) of 3 mg/m3 for organic dust by at least a factor of 2. From paired personal sampling data the concentration of respirable dust ranged from 18% to 28% of the total dust concentration. The average exposure level to bacterial endotoxin in total dust at 82 ng/m3 for DM catchers and 42 ng/m3 for TM catchers exceeded a limit of 10 ng/m3 recommended for poultry processing industry workers in the United States. Endotoxin content of airborne dust averaged 6.5 +/- 4.9 ng/mg. Exposure to microorganisms was determined by microscopy (total count) and by culturing (viable count). The geometric means for total count (microorganisms/m3) were 7.0 x 10(8) (DM catchers) and 4.9 x 10(8) (TM catchers) and for viable count 3.2 x 10(7) cfu/m3 (DM catchers) and 1.4 x 10(7) cfu/m3 (TM catchers). The ratio of microorganisms counted by microscopy to levels of viable microorganisms ranged from 5 to 200, implying a strong underestimation of exposure levels from viable counts alone. It was concluded that Danish chicken catchers were exposed beyond recommended limits for the air contaminants evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)

A micro‐scale serum neutralisation test for the detection and titration of antibodies to chicken anaemia agent ‐ prevalence of antibodies in Danish chickens

A micro-scale screening neutralisation test (SNT) and a method for end point titration of sera for antibodies to chicken anaemia agent (CAA) were developed. All 25 known positive sera gave positive results by the SNT, while 24 of 25 sera, supposed to be negative according to an alternative neutralisation test method, gave negative results by the SNT. The remaining one sample was repeatedly positive by the SNT and by endpoint titration. A total of 1161 chicken serum samples from 97 flocks, representing the different categories of the commercial chicken population in all regions of Denmark, were tested for the presence of neutralising antibodies against CAA. Of these 861 (74%) samples representing 87 (90%) flocks gave positive results. Generally the proportions of positive samples per flock and positive flocks increased with increasing age of birds. Neutralising titres ranged from 3.1 to 5.8 log(10) per ml. Again values tended to increase with increasing age, though maternal levels in 1-day-old chicks were usually high.