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.

Escherichia coli isolates from femoral bone marrow of broilers exhibit diverse pheno- and genotypic characteristics that do not correlate with macroscopic lesions of bacterial chondronecrosis with osteomyelitis.

The pheno- and genotypic relatedness among Escherichia coli isolates from broilers with and without macroscopic lesions of the femoral head were investigated. In total, 219 isolates obtained from the bone marrow were characterized by serotyping, antimicrobial resistance (AMR) profiles, phylogenetic grouping, detection of virulence-associated genes (VAGs) and pulsed-field gel electrophoresis (PFGE). Serotyping revealed that 48.4% of the isolates were assigned to one of the three serotypes (O78:K80: 21.0%, O2:K1: 18.7%, O1:K1: 8.7%). Substantial phenotypic variation was also noticed in AMR testing as most of the birds harboured E. coli isolates with different AMR profiles, which is of high clinical relevance. The majority of isolates could be classified into phylogenetic groups D (54.3%) and B2 (25.6%), followed by A (11.4%) and B1 (8.7%). Virulotyping showed that the highest number of isolates contained genes iucD (86.8%) and iss (84.9%), whereas papC (16.0%) and astA (12.3%) were present in least number of isolates. PFGE resulted in 58 different profiles from 200 typeable isolates. No correlation was found between specific serotypes, AMR profiles, phylogenetic groups, PFGE types or VAG profiles of E. coli and the occurrence of bacterial chondronecrosis with osteomyelitis, contradicting the hypothesis of a specific bacterial pheno- or genotype being involved in the disease.

Evidence of genotypes 1 and 3 of avian hepatitis E virus in wild birds.

Although the presence of four genotypes of avian hepatitis E virus (HEV) in chickens has been demonstrated, its natural host range is still barely known. In this study, swab samples from 626 wild birds originating from 62 bird species were investigated for HEV detection by molecular methods. The aim was to explore the cross-species infection of avian HEV and to compare the genetic diversity between strains infecting chicken and wild birds. In total, 8 positive samples from 4 different bird species (song thrush, little owl, feral pigeon and common buzzard) were identified and further confirmed by partial sequencing of ORF3. Based on a 237bp fragment of the capsid gene retrieved from 5 samples, phylogenetic analysis revealed the presence of avian HEV genotypes 1 and 3 in wild birds. The wild bird isolates shared 82.7-84.8% and 85.7-100% nucleotide sequence identity, respectively, to chicken isolates from the corresponding genotype. For two of the genotype 1 samples (14-2901 and 14-2906), from feral pigeons, genotype assignment could be also confirmed by phylogenetic analysis based on partial nucleotide sequence of the helicase gene. For the first time, the appearance of genotype 1 in Europe was detected, which together with close genetic relationship between HEVs present in chickens and wild birds indicates cross-species transmission.

DETECTION OF ZOONOTIC PATHOGENS IN WILD BIRDS IN THE CROSS-BORDER REGION AUSTRIA - CZECH REPUBLIC.

To assess the importance of wild birds as a reservoir of zoonotic pathogens in Austria and the Czech Republic, we sampled 1,325 wild birds representing 13 orders, 32 families, and 81 species. The majority belonged to orders Columbiformes (43%), Passeriformes (25%), and to birds of prey: Accipitriformes, Strigiformes, and Falconiformes (15%). We collected cloacal swabs from 1,191 birds for bacterial culture and 1,214 triple swabs (conjunctiva, choana, cloaca) for DNA and RNA isolation. The cloacal swabs were processed by classical bacteriologic methods for isolation of Escherichia coli , Salmonella spp., methicillin-resistant Staphylococcus aureus (MRSA), and thermophilic Campylobacter spp. Nucleic acids isolated from triple swabs were investigated by PCR for West Nile virus, avian influenza viruses, and Chlamydia spp. We also tested tissue samples from 110 fresh carcasses for Mycobacterium spp. by PCR and we cultured fresh droppings from 114 birds for Cryptococcus spp. The most-frequently detected zoonotic bacteria were thermophilic Campylobacter spp. (12.5%) and Chlamydia spp. (10.3%). From 79.2% of the sampled birds we isolated E. coli , while 8.7% and 0.2% of E. coli isolates possessed the virulence genes for intimin (eaeA) and Shiga toxins (stx1 and stx2), respectively. Salmonella spp. were rarely found in the sampled birds (2.2%), similar to findings of MRSA (0.3%). None of the samples were positive for Cryptococcus neoformans , Mycobacterium spp., avian influenza viruses, or West Nile virus.

Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus.

In a prospective longitudinal study, a broiler breeder flock and its progeny were monitored for the presence of avian hepatitis E virus (HEV) RNA and antibodies. The flock was part of a multiple-age farm where the presence of avian HEV with clinical signs (increased mortality and decreased egg production) was demonstrated in several previous production cycles. Samples were taken twice at the rearing site and several times at the production site from broiler breeders including cockerels and day-old chicks. The samples were investigated by conventional and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and histological methods. At all time points, samples from the hens were positive for avian HEV RNA. The birds did not show any clinical signs, even though histopathological lesions of non-specific aetiology in the liver and spleen could be demonstrated. A significant increase in the number of positive birds and viral load was seen in week 45, in accordance with an increase in antibody titres. In comparison, cockerels investigated in week 62 tested negative by RT-PCR and ELISA. Avian HEV RNA was also detected in day-old chicks hatched from eggs laid in week 25, indicating vertical transmission. All partial helicase and capsid sequences retrieved within this study clustered together and were identical to previous sequences obtained from the same multiple-age farm. In conclusion, avian HEV persisted on the farm over years and circulated between the rearing and the production sites without causing any clinical signs although high viral loads in the adult hens were observed.

TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA.

Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.