Escherichia coli Isolated from Organic Laying Hens Reveal a High Level of Antimicrobial Resistance despite No Antimicrobial Treatments.

The present study investigated the resistance characteristics of E. coli isolates originating from 18 organic laying hen flocks. E. coli was isolated from different organs at three different time points, resulting in 209 E. coli isolates. The antibiotic susceptibility was determined by applying a microdilution assay. General, a high resistance rate was found. The antibiotic susceptibility was independent from the presence of pathological lesions, the isolation site, or the affiliation to a pathogenic serogroup. The majority of the isolates proved to be multi-drug-resistant (95.70%), of which 36.84% could be categorized as extensively drug-resistant. All isolates were resistant to oxacillin and tylosin. Resistance rates to amoxicillin (67.94%), cefoxitin (55.98%), ceftazidime (82.30%), colistin (73.68%), nalidixic acid (91.87%), streptomycin (42.58%), tetracycline (53.59%), and sulfamethoxazole (95.22%) were high. None of the isolates revealed pan-drug-resistance. A great heterogeneity of resistance profiles was found between isolates within a flock or from different organs of the same bird, even when isolates originated from the same organ. An increase in antimicrobial resistance was found to be correlated with the age of the birds. The fact, that no antibiotic treatment was applied except in two flocks, indicates that resistant bacteria circulating in the environment pose a threat to organic systems.

In addition to birds' age and outdoor access, the detection method is of high importance to determine the prevalence of gastrointestinal helminths in laying hens kept in alternative husbandry systems.

The prevalence of gastrointestinal helminths was investigated in sixty-six commercial non-caged layer flocks. Twenty-nine flocks were housed indoors in aviaries or floor systems, nineteen flocks were kept in conventional free-range systems with outdoor access, and eighteen flocks in organic free-range systems. Flocks were investigated at end of rearing (mean age 17 weeks), peak of egg production (mean age 38 weeks) and before slaughter (mean age 74 weeks). Four different methods were applied to determine worm infestation. During necropsies, worm infestations were recorded and mucosal scrapings were evaluated for the presence of worm eggs. Faecal samples from each flock were investigated by simple flotation method and McMaster counting technique. No gastrointestinal helminths were found in pullets. During production, 87.9 % of the layer flocks were infected with at least one nematode species at the peak of production. The prevalence further increased significantly up to 98.5 % at the end of production (p=0.05). This increase could be ascribed mainly to infections with Ascaridia (A.) galli and/or Heterakis (H.) gallinarum which were most prevalent in all husbandry systems. Furthermore, their prevalence increased significantly with the age of birds (p=0.023; p < 0.001). With regard to the husbandry system, the prevalence of Capillaria spp. was significantly higher in flocks from outdoor systems compared to flocks that were kept indoors. Cestodes were only detected at the end of production with a prevalence of 15.2 % and significantly more flocks with access to outdoor run were found positive. Interestingly, H. gallinarum was found with a high prevalence indoor and in outdoor systems. Anthelminthic treatment did not impact the prevalence of nematode infections. Comparing four different methods for the detection of helminths it was revealed that their efficiencies varied depending on the worm species. Overall, the simple flotation method was superior to detect A. galli and Capillaria spp. This method proved also very efficient for the detection of H. gallinarum but the additional evaluation of the worm infestation during necropsy increased the level of prevalence. Cestodes were mainly found during necropsies when the worm infestation was evaluated. The detection of parasite eggs in mucosal scrapings from the intestines was the least effective method for all helminths. These findings lead to the recommendation to combine faecal investigations with an evaluation of the worm infestation during necropsy of at least five birds.

Antimicrobial Resistance Profiling of Gallibacterium anatis from Layers Reveals High Number of Multiresistant Strains and Substantial Variability Even Between Isolates from the Same Organ.

In this investigation, data on antimicrobial resistance (AMR) profiles of 213 Gallibacterium anatis isolates were determined from 93 laying hens originating from 39 flocks. Each flock was sampled three times during its life time for the presence of G. anatis. The broth microdilution method was applied comprising 21 antimicrobial substances. Multidrug resistance was found in 96.2% of the G. anatis isolates. Most of the isolates were resistant to tetracycline (89.2%), tylosin (94.8%), enrofloxacin (58.2%), nalidixic acid (77.4%), and sulfamethoxazole (77.0%). Resistance against antimicrobial substances increased significantly with the age of birds. A total of 99 different AMR profiles were detected. On flock level, different AMR profiles were found in 71.8% of the flocks independent of the sampling time point. On bird level, identical AMR profiles were mostly found in isolates originating from the same organ of a single bird, but 22 such paired isolates differed in their AMR profile. Variations of AMR profiles were found within isolates from a single bird, but from different organs. Isolates from systemic organs were significantly more resistant to different antimicrobial substances compared to isolates from the reproductive tract. No influence could be found in regard to an increase of resistance and applied antibiotic treatment.

Influence of alternative husbandry systems on postmortem findings and prevalence of important bacteria and parasites in layers monitored from end of rearing until slaughter.

In the present study 66 layer flocks housed in different alternative husbandry systems were monitored from placement of birds on the farm until slaughter to evaluate a possible influence on the occurrence of selected non-infectious as well as infectious diseases. Postmortems were performed and the occurrence of extraintestinal Escherichia coli and Gallibacterium anatis was investigated. No specific postmortem findings were seen in pullets. Non-infectious diseases were mainly found in layers. Most prominent postmortem findings in layers were reproductive tract lesions and the presence of intestinal helminths. From each flock E coli and from approximately 65 per centof the flocks G anatis were isolated. No significant differences were seen in regard to the housing system, but the prevalence of G anatis increased with the age of birds. The majority of reproductive tract lesions could be associated with E coli alone or G anatis co-infections. The prevalence of Ascaridia galli and Heterakis gallinarum was not influenced by the housing system, but significantly increased with age. Cestodes were present in six flocks. Histomonosis was detected twice. Dermanyssus gallinae was found in 5 pullet and 20 layer flocks. Additional investigations were performed on demand. Again, reproductive tract lesions were the most prominent postmortem findings. In one flock each histomonosis or erysipelas was diagnosed, respectively. Severe affection by D gallinae was found once. Necrotic enteritis was seen in two layer flocks.

High prevalence of Brachyspira spp. in layers kept in alternative husbandry systems associated with frequent species variations from end of rearing to slaughter.

A longitudinal survey was conducted to investigate the presence of Brachyspira species in layer flocks. A total of 66 layer flocks kept in alternative husbandry systems were sampled at three time points: end of rearing, at peak of lay and at end of lay. Content from caecal samples of freshly killed birds was cultured at each sampling time point and processed for further investigations. Gross pathological lesions in caeca were recorded during post mortem investigation. Spirochaetes were isolated from 50 flocks: three flocks were positive at all three sampling points, 25 flocks at two and 22 flocks at one sampling point, respectively. The presence of Brachyspira spp. could not be related to specific gross pathological caecal lesions or antibiotic treatments. The number of positive flocks increased with the age of birds. Furthermore, organic flocks were more often positive than flocks from barn systems. In total 80 spirochaetal cultures were obtained. B. intermedia (43.8%) was the most common species, followed by B. pulli (13.8%) and B. pilosicoli (12.5%). Brachyspira murdochii and B. innocens were found in 5.0% and 2.5%, respectively, whereas 11.3% of Brachyspira isolates could not be identified to species level. In 11.3% of the samples mixed infections were detected. Finally, the longitudinal survey revealed for the first time a possible shift in the Brachyspira species in a substantial number (32%) of layer flocks during their lifetime.

High genetic diversity among extraintestinal Escherichia coli isolates in pullets and layers revealed by a longitudinal study.

BACKGROUND: Various information about the genetic diversity of Escherichia coli isolates from chickens are available but a detailed epidemiological investigation based upon isolates obtained from interrelated pullet and layer flocks is still missing. Therefore, in the course of a longitudinal epidemiological study on pullets and layers, 144 E. coli isolates from chickens with or without pathological lesions of the reproductive tract were serotyped and genotyped with pulsed-field gel electrophoresis (PFGE). These isolates were collected during rearing, peak and at the end of production. The actual study is the first of its kind so as to elucidate genetic relatedness among extraintestinal E. coli isolated from chickens with varying pathological conditions in interrelated layer farms/flocks at different stages of rearing. RESULTS: Serotyping revealed that 63.19 % of the isolates could not be assigned to any of the three serotypes tested whereas 30.55 % of the isolates belonged to serotype O1:K1, 4.86 % to O2:K1 and 1.38 % to O78:K80. After macrorestriction digest with XbaI, 91.66 % of the isolates were typeable resulting in 96 distinct PFGE profiles. Among them, five PFGE types included isolates collected from diseased chickens as well as from birds without pathological lesions. This finding shows that pathogenicity of E. coli in layers seems to be largely influenced by concurrent susceptibility factors. Furthermore, in six out of eight cases where two isolates were collected from each of eight birds, different PFGE types were found in the same or different organs of the same bird. The existence of predominant or persistent E. coli genotypes was only observed in two cases. CONCLUSIONS: It is concluded that extraintestinal E. coli genotypes and serotypes in pullets and layers are heterogenous and also do not maintain a single clonality within the same bird. The facts that E. coli strains did not show any definite clonal population structure based on geographical region, age of the host and pathological lesions should have relevance in further epidemiological studies and control strategies.