To gather recent data regarding the infectious bronchitis (IB) and infectious bursal disease (IBD) situation in Europe, a large-scale field epidemiological survey using diagnostic samples has been implemented in 2013 for about six months in several European countries: France, Germany, Greece, Italy, the Netherlands, Poland, Portugal, the Republic of Ireland, Spain and the UK. In 234 flocks that were sampled, strains from 10 different IBV genotypes were detected: the 793B genotype was detected most frequently, followed by QX, Massachusetts (Mass) and the Xindadi-like strains. Strains belonging to the Q1, Ark, D274, D1466, Italy-02 and B1648 genotypes were detected as well, although less frequently. The separate sampling of tracheas and kidneys for IBV detection using reverse transcriptase PCR was very useful, as different genotypes or significant differences in sequences of the same genotype were detected between both organs. The data of this survey also provided valuable information about the replication of IBD vaccines and subsequent infectious bursal disease virus (IBDV) antibody responses under field conditions. The detection of five non-vvIBDV field strains of two different genotypes shows the presence of non-vvIBDV non-vaccine strains, which can easily be undetected in Europe due to the focus on sampling of clinically ill birds. Detection of vaccine virus in the bursa and antibody response to the IBD vaccination in flocks that had been vaccinated by the drinking water with a live attenuated vaccine compared to a vaccination in the hatchery using an immune-complex vaccine showed a delayed replication of the vaccines that had been applied by the drinking water, indicating mistakes in the timing and/or application of the vaccines.

Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response.

Infectious bronchitis (IB) control has a strong impact on poultry farming, because of the necessary epidemiological knowledge for planning the best strategy, the optimal strain association, the priming and boosting interventions. Broiler farming is even more problematic given the short and intense productive cycle, which requires an early onset of protection against most of the infectious threats, possibly with limited respiratory post-vaccination reactions that would have a direct impact on the bird health and productivity. For this purpose, gel vaccination has been proposed as a new approach for infectious bronchitis virus (IBV) control and vaccine intake, kinetics and compatibility of combined strains administered by gel have been analyzed in this study. After gel vaccination with single and combined 1/96 and B-48 strains on 4 groups of commercial broilers, a 21-d-long experimental trial has been conducted to monitor the vaccine safety by clinical assessment and vaccine kinetics by strain-specific real-time RT-PCR on choanal cleft swabs. The vaccine strains administered by gel were safe and negligible respiratory signs were detected, even when combined. Vaccine titers were compared among groups and within the same group among a 10-bird pooled sample and 10 swabs from individually sampled birds. 1/96 strain early reached high titers in all animals, while B-48 presence was less constant even though it was detected in almost all birds before the trial end. The individual and pooled sample comparison revealed a partial overestimation of vaccine titers in the pooled samples and the loss of the prevalence data, although the trend portrayed by the pooled swabs closely followed the individual ones.

Avian metapneumovirus (aMPV) represents one of the most prevalent diseases of turkey, especially in combination with other pathogens, and its frequency is also increasing among chickens. Despite this evidence, epidemiologic data are poor and scattered, severely preventing control of the disease even in highly developed areas such as Europe. In the present study, the detection and characterization of an aMPV subtype B strain circulating in a vaccinated but symptomatic Romanian broiler flock is reported for the first time. The phylogenetic analysis based on the partial G gene sequence demonstrates the close relationship of the Romanian virus with a group of recently emerged Italian field strains for which vaccine-induced protection was experimentally proven to be partial. These preliminary results allow us to hypothesize the spreading of vaccine-escaping aMPV subtype B strains through Europe and, consequently, dictate the carrying out of a more systematic survey to confirm this theory and enforce adequate countermeasures.

Infectious bronchitis virus is characterised by an extreme degree of variability which deeply affects the first-choice control strategies against the disease. Each country tends to adopt its own protocols and even vaccine producers themselves can also adopt different strategies in attempts to confront local epidemiological concerns. In the present study, we tested the potential environmental persistence, transmission ability and replication capability of a non-directly administrated vaccine strain at a hatchery and during transportation. To this purpose, we examined a single hatchery, where combined vaccination (Mass-like plus 793B-like strains) is commonly administered following the protectotype concept, whereas a single broiler flock receives only the Mass priming. Two groups of solely Mass-primed chicks were kept in contact with chicks vaccinated with both strains, during hatchery procedures and transportation, respectively. A regularly vaccinated control group was selected and all three were monitored by swab sampling until 11 days of age. Vaccine titres were quantified using vaccine-specific real-time RT-PCRs to check the kinetics of both strains. Mass titres were consistent among the groups, while the absence of the 1/96 vaccine strain in unvaccinated chicks confirmed the low risk of unintended vaccine transmission, which could complicate the diagnostic process and the epidemiological scenario.