Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR.

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.

Administration of a Multi-Genus Synbiotic to Broilers: Effects on Gut Health, Microbial Composition and Performance.

In recent years, the applicability of prebiotics, probiotics and their mixtures, defined as synbiotics, in poultry production has received considerable attention. Following the increasing regulation of antibiotic use, these nutraceuticals are seen as an alternative way to sustain production efficiency and resistance to pathogens and stressors by modulating birds' gut health. The aim of this study was to evaluate the benefits provided under field conditions by administering the multi-species synbiotic PoultryStar® sol to broilers in drinking water. To this purpose, three Ross 308 broiler flocks, representing separate progenies of a breeder flock which was treated with the same synbiotic, were housed in separate farms, divided into treatment and control groups, and followed throughout the productive cycle. Synbiotic administration was shown to improve gut health even in absence of a challenge, with limited changes in terms of macroscopic intestinal lesions and more overt differences related to histopathological scores and villi length. Synbiotic-fed chickens performed consistently better in terms of body weight gain, feed conversion ratio and survivability. Lastly, the evaluation of the caecal microbiome through next-generation sequencing highlighted the effects of synbiotic supplementation on the composition of the bacterial population, the implications of which will, however, require further studies to be better comprehended.

Molecular epidemiology of fowl adenoviruses in Greece.

Outbreaks of inclusion body hepatitis (IBH) and adenoviral gizzard erosion have been anecdotally reported in Greece since approximately 2011. However, a relevant increase in clinical outbreaks compatible with IBH has been described since 2014. Unfortunately, with limited exceptions, only serological assays were performed, and involved strains were not properly characterized. In the present study, 35 outbreaks were investigated in the period between July 2017 and February 2018 in Greece. In addition to clinical and histopathological diagnosis, fowl adenovirus (FAdV) presence was investigated by PCR and sequencing. Thirty-four out of 35 samples tested FAdV positive. Twenty-nine (85.29%) and 5 (14.71%) strains were classified as FAdV-E and FAdV-D, respectively. Fowl adenovirus-E strains were genetically homogeneous and formed an independent cluster of Greek-only sequences, including the sole previously available sequence, suggesting the prolonged circulation of this species in Greece. On the contrary, FAdV-D strains were more heterogeneous and closely related to strains sampled in other European countries, testifying the occurrence of multiple introduction events. The evaluation of phylogenetic relationships, geographic clustering, age of infection, and origin of the broiler breeder flocks suggests that both vertical and horizontal transmission are important in FAdV epidemiology in Greece and highlights the limited efficacy of currently implemented control measures. Of note, a significantly higher mortality was observed in precociously infected flocks, likely because of the higher susceptibility of younger animals. This evidence stresses the need of preventing vertical and/or early infection to limit the economic impact of adenovirus-induced diseases.

Molecular epidemiology of infectious bronchitis virus and avian metapneumovirus in Greece.

Respiratory diseases like infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) have been held accountable for major losses for poultry production. Nevertheless, scarce information was present dealing with the prevalence and molecular epidemiology of these infections in Greece and the efficacy of currently applied control strategies. To fill this gap, a specific epidemiological study was designed. A total of 106 broiler and layer farms, including 10 backyard and 96 commercial flocks, were sampled between March 2016 and May 2017, and the obtained tracheal swabs were tested for IBV and aMPV using RT-PCR based techniques followed by sequencing. For each farm, data regarding production type, flock features, clinical signs, and vaccination program were also recorded. Different associations between vaccination protocol, production type, animal category, birds density, age, presence of clinical signs, and IBV and/or aMPV infection were tested. Both IBV and aMPV field strain prevalence were proven high, approximately 20 and 30%, respectively, being the GI-19 lineage (14 out of 19; 73.6%) and B subtype (30 out of 30; 100%), the most commonly detected IBV and aMPV genetic types. Infection with IBV field strains was significantly associated with clinical sign presence (odds ratio = 8.55 [95CI = 2.17-42.90]). Remarkably, only the vaccination protocol involving a double vaccination at 1 D of age was proven protective against IBV-induced symptomatology, with the odds of developing disease being 4.14 [95CI = 1.34-14.51] times lower. No association was demonstrated between aMPV infection and clinical outbreaks or between aMPV and IBV detection, suggesting the marginal role of the former pathogen in poultry farming. Globally, the present study provides the first detailed investigation of the epidemiological scenario of 2 viruses traditionally considered of pivotal relevance in poultry farming and demonstrates that remarkable benefits could be obtained with just minor adjustments in vaccination protocols.

First Identification and Molecular Characterization of Avian metapneumovirus Subtype B from Chickens in Greece.

Avian metapneumovirus (aMPV) is considered a major pathogen for turkeys but its impact on chicken production is still partially neglected, even though it is fully acknowledged as a primary pathogen in chickens as well. The lack of structured diagnostic surveys does not allow a pervasive understanding of aMPV epidemiology. Being that aMPV is almost an everyday challenge for farmers and veterinarians, a more accurate report of its presence should be detailed, posing the basis for a deep and global epidemiologic analysis. With these premises, the present work aims to report the first detection and molecular characterization of aMPV subtype B field strains from unvaccinated chickens in Greece. The Greek strains appear to be phylogenetically related among each other and with other recent Mediterranean strains while being distant from the currently applied vaccines, thus stressing once more the necessity to evaluate aMPV diffusion and evolution.