Field and laboratory findings following the large-scale use of intermediate type infectious bursal disease vaccines in Denmark.

Following a period of clinical outbreaks of very virulent infectious bursal disease virus (vvIBDV) in Denmark, the histological bursal lesion score (HBLS) was used on a national scale to screen broiler flocks vaccinated with intermediate IBD vaccines for lesions indicative of IBDV challenge. High lesion scores were detected in a high percentage of healthy and well performing flocks despite the lack of other indications of the presence of vvIBDV. RT-PCR and subsequent sequencing showed the frequent presence of H253Q and H253N IBDV strains that were genetically close to the sequence of the intermediate vaccines with a relative risk ratio of 13.0 (P < 0.0001) in intermediate vaccine A or B vaccinated flocks compared to unvaccinated flocks. The relevance of these H253Q and H253N strains was tested under experimental conditions using a protocol derived from the European Pharmacopoeia for safety of live IBD vaccines. The results confirmed the higher pathogenicity for the bursa of these strains compared to intermediate vaccines as well as the negative effect on antibody response to a Newcastle disease (ND) vaccination performed at the peak of the bursa damage. The efficacy of the ND vaccination was still 100% showing that the H253N and H253Q IBDV strains would be considered as safe vaccine viruses. In conclusion, the use of the HBLS to screen commercial broiler flocks vaccinated with intermediate IBD vaccines for the presence of vvIBDV does not seem to be a reliable method due to the frequent occurrence of H253N and H253Q strains in those flocks. For screening of IBD vaccinated flocks for the presence of vvIBDV or other field strains, the RT-PCR with subsequent sequencing seems to be most suitable.

Detection of different genotypes of infectious bronchitis virus and of infectious bursal disease virus in European broilers during an epidemiological study in 2013 and the consequences for the diagnostic approach.

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.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10(5) and 10(6) 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as an indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens.

The purpose of this study was to look for a reliable molecular method for confirmation of uptake of recombinant turkey herpesvirus vaccine against Newcastle disease (rHVT-F) and for use as a valuable prediction tool of Newcastle disease virus (NDV)-specific immune response in chickens deprived of maternally derived antibody (MDA). A quantitative real-time polymerase chain reaction (real-time qPCR) specific to rHVT-F was developed. The method was applied to various tissue samples taken from specific pathogen free (SPF) chickens experimentally inoculated at day-old with one dose of rHVT-F vaccine over a 6-week period. Among the tested tissues, the rHVT-F vaccine was detected predominantly in the bursa of Fabricius (BF) and the lung for the first week, followed by a progressive decline from 9 days onwards. Then, an increase of genome load was observed in the feather follicles (FF) with a peak at 2 weeks, rising to a level almost 10(3)-fold greater than in the other tissues. Importantly, the rHVT-F genome load in FF appeared to be strongly correlated to the humoral immunity specific to NDV as evaluated by haemagglutination inhibition (HI) test and NDV-specific IgG, IgM and IgA ELISAs. This is the first report of quantification of rHVT-F vaccine in FF and its correlation with the induction of ND-specific immune response in chickens with no MDA. Our data indicate that the application of this real-time qPCR assay on FF samples taken from chickens in the field may be used to confirm rHVT-F vaccine administration and uptake with the important added benefit of offering a non-disruptive sampling procedure.

The combination of attenuated Newcastle disease (ND) vaccine with rHVT-ND vaccine at 1 day old is more protective against ND virus challenge than when combined with inactivated ND vaccine.

The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks throughout the world indicate that routine vaccinations are failing to sufficiently induce the high levels of immunity necessary to control ND. There is a need for vaccination programmes that could be initiated at 1-day-old for mass application and which would induce a long-lasting immunity, with no need for a booster vaccination at a later age. In this context, the duration of immunity delivered by a vaccination programme including a recombinant herpesvirus of turkeys expressing the F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-old was compared with a classical programme that included a conventional live and an inactivated ND vaccine at the same age in commercial layer chickens. The humoral, cell-mediated and local immunity were followed weekly and birds were challenged with a viscerotropic velogenic ND virus strain at 6 and 10 weeks of age. We determined that immunity induced by the vaccination programme involving the rHVT-ND vaccine was more protective than that provided by the conventional vaccine-based regime. This might be related to a T-helper type 1 (Th1) cellular-driven immunological response, in contrast to the T-helper type 2 (Th2) humoral-oriented immune response provided by the current conventional vaccine-based vaccination programmes.

Advancement in vaccination against Newcastle disease: recombinant HVT NDV provides high clinical protection and reduces challenge virus shedding with the absence of vaccine reactions.

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to the limitation in their efficacy, current vaccination strategies against ND need improvements. This study aimed to evaluate a new-generation ND vaccine for its efficacy in providing clinical protection and reducing virus shedding after challenge. Broiler chickens were vaccinated in ovo or subcutaneously at hatch with a turkey herpesvirus-based recombinant vaccine (rHVT) expressing a key protective antigen (F glycoprotein) of Newcastle disease virus (NDV). Groups of birds were challenged at 20, 27, and 40 days of age with a genotype V viscerotropic velogenic NDV strain. Protection was 57% and 81%, 100% and 95%, and 100% and 100% after the subsequent challenges in the in ovo and subcutaneously vaccinated chickens, respectively. Humoral immune response to vaccination could be detected from 3-4 wk of age. Challenge virus shedding was lower and gradually decreased over time in the vaccinated birds compared to the unvaccinated control chickens. In spite of the phylogenetic distance between the NDV F gene inserted into the vector vaccine and the challenge virus (genotype I and V, respectively), the rHVT NDV vaccine provided good clinical protection and significantly reduced challenge virus shedding.

Efficacy of rHVT-AI vector vaccine in broilers with passive immunity against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains.

In countries where avian influenza has become endemic, early vaccination of layer pullets or broilers with classical inactivated vaccines at the hatchery is no longer an option because of interference with passive immunity indirectly induced by the necessary vaccination of the breeders. On the other hand, injection of thousands of chicks from 7 to 10 days old on farms has been determined to be unreliable and, therefore, poorly efficacious. For these reasons, interest has arisen regarding a newly developed live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 highly pathogenic avian influenza virus (HPAIV) strain (rHVT-H5), which in theory is capable of breakthrough passive immunity to both the vector (HVT) and the insert (H5) and is consequently applicable at the hatchery. The objectives of this trial were to evaluate the impact of maternally derived antibodies (MDAs) specific to H5N1 on the immunity and the efficacy (protection and virus shedding) of different vaccination programs including rHVT-H5 and inactivated H5N1 and H5N2 vaccines applied alone or in combination. Therefore, broilers carrying MDAs against both HVT and Asian H5N1 HPAIV were vaccinated on the first day of age with rHVT-H5, with or without boosting vaccination by an inactivated vaccine after 10 days. The different groups were challenged with two antigenically highly divergent Egyptian dade 2.2.1 H5N1 HPAIVs at 4 wk of age. Protection against challenge was compared with unvaccinated birds or vaccinated birds without MDAs. Between 70% and 90% clinical protection could be observed in the vaccinated groups possessing MDAs, indicating no or very low interference of MDAs with vaccination. Results regarding clinical protection, humoral, cell-mediated, and mucosal immunity, as well as re-excretion of challenge virus are presented and discussed.

Further evidence of antigenic drift and protective efficacy afforded by a recombinant HVT-H5 vaccine against challenge with two antigenically divergent Egyptian clade 2.2.1 HPAI H5N1 strains.

In this study, we have compared the protection afforded by a recombinant turkey herpesvirus vaccine expressing the H5 gene from a clade 2.2 H5N1 strain (rHVT-H5) and a Mexican-origin H5N2 inactivated vaccine, alone or in combination, against two antigenically divergent H5N1 Egyptian strains isolated in 2007 and 2008. Our results confirm the existence of a major antigenic drift among the Egyptian H5N1 strains such that, although protection against the "classical" 2007 HPAI H5N1 Egyptian strain could be obtained with both types of vaccines, only vaccination with the rHVT-H5 vaccine protected against challenge with the "variant" 2008 HPAI H5N1 Egyptian strain.

Passive protection afforded by maternally-derived antibodies in chickens and the antibodies' interference with the protection elicited by avian influenza-inactivated vaccines in progeny.

Systematic vaccination can be applied when a disease has become enzootic in a country or region. The final goal of the approach is to control or eradicate the disease within the country. This is a long-term vaccination plan that could be applied nationwide to all commercial and backyard poultry. However, after several months of vaccination in enzootic areas, maternally derived antibody (MDA) is present in young chicks, providing some protection and/or interference with vaccination. The aim of this study was to evaluate the level of protection afforded by MDA against challenge with highly pathogenic avian influenza virus (HPAIV), and its suspected interference with current inactivated vaccines in broilers under controlled laboratory conditions. In the first set of experiments, broilers were vaccinated with inactivated vaccines containing H5N2 subtype antigens in the presence or absence of homologue MDAs and challenged with a clade 2.2 H5N1 HPAIV. In the second set of experiments, day-old broilers, either with or without avian influenza MDA, received a regular-type monovalent H5N2 AI vaccine (0.5 ml) or a concentrated (0.2 ml) AL-Newcastle disease virus combined inactivated vaccine subcutaneously. They were then challenged at 11 or 35 days of age. In conclusion, our results indicate that protection induced by day-old administration of inactivated vaccine (regular or concentrated) in the presence or absence of MDA to H5N2 AIV induces poor protection against challenge with H5N1 HPAIV and should not be recommended. Based on our results, vaccination of MDA-positive chickens at a later age (10 days) seems to be a valuable recommendation, although MDAs may still interfere with vaccination to a lesser extent because they are present up to 3 wk posthatch. Therefore, in areas with high infection pressure, when possible, two vaccinations are recommended for optimal protection. Also, it might be advisable to take into account day-old AI MDA titers when one is determining the optimal age of vaccination.