Highly pathogenic avian influenza management policy in domestic poultry: from reacting to preventing.

The emergence of clade 2.3.4.4b H5N1 highly pathogenic avian influenza (HPAI) viruses in 2021 has led to unprecedented epidemics in poultry, changing epidemiological patterns of year-round infections in resident wild avifauna and more frequent spill-over events to mammals. Given this situation, it is important that we recognise that traditional HPAI management strategies are no longer sufficient, and policy changes are required. Poultry vaccination has emerged as a crucial intervention in the current control of HPAI, as evidenced by France's nationwide campaign targeting domestic ducks. However, due to the logistical challenges and potential trade implications of vaccination, broader structural reforms appear also necessary. These include a shift from farm-level to territorial-level biosecurity approaches, putting into practice the concept of 'regional biosecurity'. Given the role duck farm density has played in successive HPAI epidemics in France, there is a need to think about the spatial distribution of poultry farms as a structural component of regional biosecurity and to consider the reduction of farm concentration as a measure to prevent viral spread. The integration of regional biosecurity and poultry vaccination into prevention strategies should impact the way poultry are produced and traded in the future.

Pathobiology of highly pathogenic H5 avian influenza viruses in naturally infected Galliformes and Anseriformes in France during 2015-2016.

In late 2015, an epizootic of Highly Pathogenic Avian Influenza (H5Nx) was registered in Southwestern France, including more than 70 outbreaks in commercial poultry flocks. Phylogenetic analyses suggested local emergence of H5 viruses which differed from A/goose/Guangdong/1/1996 clade 2.3.4.4b lineage and shared a unique polybasic cleavage site in their hemagglutinin protein. The present work provides an overview of the pathobiological picture associated with this epizootic in naturally infected chickens, guinea fowls and ducks. Upon necropsy examination, selected tissues were sampled for histopathology, immunohistochemistry and quantitative Real Time Polymerase Chain Reaction. In Galliformes, HPAIVs infection manifested as severe acute systemic vasculitis and parenchymal necrosis and was associated with endothelial expression of viral antigen. In ducks, lesions were mild and infrequent, with sparse antigenic detection in respiratory and digestive mucosae and leukocytes. Tissue quantifications of viral antigen and RNA were higher in chickens and guinea fowls compared to duck. Subsequently, recombinant HA (rHA) was generated from a H5 HPAIV isolated from an infected duck to investigate its glycan-binding affinity for avian mucosae. Glycan-binding analysis revealed strong affinity of rHA for 3'Sialyl-LacNAc and low affinity for Sialyl-LewisX, consistent with a duck-adapted virus similar to A/Duck/Mongolia/54/2001 (H5N2). K222R and S227R mutations on rHA sequence shifted affinity towards Sialyl-LewisX and led to an increased affinity for chicken mucosa, confirming the involvement of these two mutations in the glycan-binding specificity of the HA. Interestingly, the rHA glycan binding pattern of guinea fowl appeared intermediate between duck and chicken. The present study presents a unique pathobiological description of the H5 HPAIVs outbreaks that occurred in 2015-2016 in Southwestern France.

Guinea Fowl Coronavirus Diversity Has Phenotypic Consequences for Glycan and Tissue Binding.

Guinea fowl coronavirus (GfCoV) causes fulminating enteritis that can result in a daily death rate of 20% in guinea fowl flocks. Here, we studied GfCoV diversity and evaluated its phenotypic consequences. Over the period of 2014 to 2016, affected guinea fowl flocks were sampled in France, and avian coronavirus presence was confirmed by PCR on intestinal content and immunohistochemistry of intestinal tissue. Sequencing revealed 89% amino acid identity between the viral attachment protein S1 of GfCoV/2014 and that of the previously identified GfCoV/2011. To study the receptor interactions as a determinant for tropism and pathogenicity, recombinant S1 proteins were produced and analyzed by glycan and tissue arrays. Glycan array analysis revealed that, in addition to the previously elucidated biantennary di-N-acetyllactosamine (diLacNAc) receptor, viral attachment S1 proteins from GfCoV/2014 and GfCoV/2011 can bind to glycans capped with alpha-2,6-linked sialic acids. Interestingly, recombinant GfCoV/2014 S1 has an increased affinity for these glycans compared to that of GfCoV/2011 S1, which was in agreement with the increased avidity of GfCoV/2014 S1 for gastrointestinal tract tissues. Enzymatic removal of receptors from tissues before application of spike proteins confirmed the specificity of S1 tissue binding. Overall, we demonstrate that diversity in GfCoV S1 proteins results in differences in glycan and tissue binding properties.IMPORTANCE Avian coronaviruses cause major global problems in the poultry industry. As causative agents of huge economic losses, the detection and understanding of the molecular determinants of viral tropism are of ultimate importance. Here, we set out to study those parameters and obtained in-depth insight into the virus-host interactions of guinea fowl coronavirus (GfCoV). Our data indicate that diversity in GfCoV viral attachment proteins results in differences in degrees of affinity for glycan receptors, as well as altered avidity for intestinal tract tissues, which might have consequences for GfCoV tissue tropism and pathogenesis in guinea fowls.

Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform.

Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma spp. Escherichia coli and/or Ornithobacterium rhinotracheale in turkeys are considered as key co-infectious agents of RS. Aspergillus sp., Pasteurella multocida, Avibacterium paragallinarum or Chlamydia psittaci may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of E. coli, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.

Co-infection of turkeys with Escherichia coli (O78) and H6N1 avian influenza virus.

Respiratory diseases are responsible for major economic losses in poultry farms. While in most cases a single pathogen is not alone responsible for the clinical outcome, the impact of co-infections is not well known, especially in turkeys. The purpose of this study was to assess the possible synergism between Escherichia coli (O78) and low pathogenic avian influenza virus (LPAIV, H6N1), in the turkey model. Four-week-old commercial turkeys were inoculated with either H6N1, O78 or both agents simultaneously or three days apart. We have established an experimental infection model of turkeys using aerosolization that better mimics field infections. Birds were observed clinically and swabbed on a daily basis. Necropsies were performed at 4 and 14 days post single or dual inoculation and followed by histological and immunohistochemical analyses. Combined LPAIV/E. coli infections resulted in more severe clinical signs, were associated with higher mortality and respiratory organ lesions (mucous or fibrinous exudative material in lungs and air sacs), in comparison with the groups given single infections (P < 0.05). The time interval or the sequence between H6N1 and E. coli inoculation (none or three days) did not have a significant effect on the outcome of the dual infection and disease although slightly greater (P > 0.05) respiratory signs were observed in turkeys of the E. coli followed by H6N1 inoculated group. Microscopic lesions and immunohistochemical staining supported clinical and macroscopic findings. Efficient virus and bacteria replication was observed in all inoculated groups. E. coli and H6N1 thus exercise an additive or synergistic pathogenic effect in the reproduction of respiratory disease.

Vaccination Technology in Poultry: Principles of Vaccine Administration.

Poultry vaccines are very important tools for disease prevention and may be administered collectively by drinking water or spray or individually by injection or oculonasal drop, whereas inactivated vaccines are administered by injection only. Poultry vaccines are increasingly delivered at the hatchery to day-old chicks or in ovo, because mass vaccination is much more efficiently implemented and controlled at the hatchery than on the farm. Mass administration on the farm by drinking water or spray requires strict compliance with guidelines regarding water quality, preparation of vaccines, and application, so as to cover the whole flock. Vaccination at the hatchery uses integrated machines to deliver vaccines to day-old chicks or, increasingly, in ovo at transfer from setters to hatchers. Regardless of the route and technology, a high quality of monitoring is critically important to maintain strict compliance and best practices from the vaccine vial to the bird, to guarantee efficient administration and intake of the vaccine by the whole flock and to secure the integrity of the vaccine itself. Major recent technical innovations in poultry vaccination covering both biology and technology open a very exciting era.

Estudio recapitulativo- Tecnología de vacunación en la avicultura: principios de administración de vacunas. Las vacunas para la avicultura son herramientas muy importantes para la prevención de enfermedades y pueden administrarse colectivamente mediante agua de bebida y por aerosol, también individualmente mediante inyección o por gota oculonasal, mientras que las vacunas inactivadas se administran únicamente mediante inyección. Las vacunas avícolas se administran cada vez más en la planta de incubación a pollitos de un día o in ovo, porque la vacunación masiva se implementa y controla mucho más eficientemente en la planta de incubación que en las granjas. La administración masiva en granja mediante agua de bebida o aspersión requiere el cumplimiento estricto de las pautas relativas a la calidad del agua, preparación de vacunas y aplicación, de manera de cubrir a toda la parvada. La vacunación en la planta de incubación utiliza máquinas integradas para administrar vacunas a los pollitos de un día o, de manera más frecuente, in ovo en el momento del traslado de las incubadoras a las nacedoras. Independientemente de la ruta y la tecnología, una alta calidad del monitoreo es de vital importancia para mantener un cumplimiento estricto y las mejores prácticas desde el vial de la vacuna hasta el ave, para garantizar la administración y la captación eficiente de la vacuna por toda la parvada y para asegurar la integridad de la vacuna. Las importantes innovaciones técnicas recientes en la vacunación en avicultura que abarcan tanto la biología como la tecnología están iniciando una era fascinante.

Making the invisible visible: Effectiveness of a training program to increase awareness and biosecurity compliance in poultry barn anterooms, using glowing substances.

Biosecurity is the cornerstone of prevention strategies to limit infectious pathogen spread between poultry farms. However, poor compliance of biosecurity measures is often observed when entering or exiting barns. The lack of knowledge, understanding, risk perception or technical skill are often described for anteroom-related biosecurity errors (i.e., not respecting anteroom zoning in relation to changing boots, clothing, and hand sanitation). In this study, we designed and assessed the efficacy of an intervention aiming at improving biosecurity compliance in anterooms, using substances glowing under ultraviolet light, which mimic microbiological contamination. Training exercises offered in a single session were proposed to 145 participants with an interest in animal farming (veterinary students, agricultural students, poultry farmers and poultry farm advisors). The exercises' efficacy was assessed by measuring the change in attitude and intentions of participants about specific biosecurity measures. The participants' attitudes and intentions were quantified using a questionnaire before and after training. The change in score was assessed in a multivariate linear regression model to measure the effect of various parameters on this change. The attitudes and intentions of participants were statistically improved (p < 0.05) and were affected by the initial attitude and intention score, the gender of the participants and the status of the participant (type of student or activity in the poultry industry). The use of this exercise material, perceived as playful and engaging, could be included in the framework of biosecurity training for all stakeholders of the poultry industry.

Monitoring biosecurity in poultry production: an overview of databases reporting biosecurity compliance from seven European countries.

Compliance with required on-farm biosecurity practices reduces the risk of contamination and spread of zoonotic and economically important diseases. With repeating avian influenza epidemics in the poultry industry, the need to monitor and improve the overall level of biosecurity is increasing. In practice, biosecurity compliance is assessed by various actors (e.g., academic, private and public institutions), and the results of such assessments may be recorded and gathered in databases which are seldom shared or thoroughly analyzed. This study aimed to provide an inventory of databases related to the assessment of biosecurity in poultry farms in seven major poultry-producing European countries to highlight challenges and opportunities associated with biosecurity data collection, sharing, and use. The institutions in charge of these databases were contacted and interviewed using a structured questionnaire to gather information on the main characteristics of the databases and the context of their implementation. A total of 20 databases were identified, covering the gamut of poultry species and production types. Most databases were linked to veterinary health authorities or academia, and to a lesser extent interbranch organizations. Depending on the institutions in charge, the databases serve various purposes, from providing advice to enforcing regulations. The quality of the biosecurity data collected is believed to be quite reliable, as biosecurity is mostly assessed by trained farm advisors or official veterinarians and during a farm visit. Some of the databases are difficult to analyze and/or do not offer information concerning which biosecurity measures are most or least respected. Moreover, some key biosecurity practices are sometimes absent from certain databases. Although the databases serve a variety of purposes and cover different production types, each with specific biosecurity features, their analysis should help to improve the surveillance of biosecurity in the poultry sector and provide evidence on the benefits of biosecurity.

Backyard poultry flocks in France: A diversity of owners and biosecurity practices.

Over the past few years, the number of backyard poultry flocks has been increasing in France. A mandatory step to improve backyard poultry management is to assess health risks by characterizing the flocks and understanding the owners' motivations for keeping poultry and their husbandry practices. A survey of backyard poultry owners was conducted in France to gather information about their motivations for owning poultry, flock characteristics, and breeding and biosecurity practices. The survey was completed by 1,160 owners. The major motivations for owning poultry flocks were egg consumption (93.3 %), recycling (72.4 %) and having pet animals (53.2 %). Most owners had already heard about avian influenza (96.7 %), but were less aware about other diseases such as Newcastle Disease (41.6 %), salmonellosis (79.1 %), or campylobacteriosis (18.6 %). Owners mainly kept only egg-layers (78.4 %), and the median size flock was five egg-layers. Owners gave eggs to their relatives, occasionally or regularly, in 86.6 % of the cases. Contacts with other family poultry owners were frequent (68.9 %) and biosecurity practices were poorly implemented: 50 % of owners did not wash their hands systematically after visiting the flock and more than 60 % of owners did not wear specific shoes. Drawing from the survey data, five profiles of family poultry flocks were identified with multiple correspondence analysis and hierarchical cluster analysis. The profiles, based on flock characteristics and owners' practices and motivations, illustrate the heterogeneity of the backyard poultry sector: 1) urban poultry, 2) traditional poultry, 3) student poultry, 4) pet poultry and 5) hobby poultry. Urban poultry consisted of recently constituted (< 2 years old), small (< 3 birds) flocks of layers, and traditional poultry of older, medium-sized flocks belonging to retired and older people. These two profiles were characterized by limited contacts (direct or indirect) with other flocks and owners. Student poultry consisted of younger owners (<30 years old) with flocks over 5 years old. Pet poultry consisted of recently established, medium-size flocks of layers located in both rural or urban environments. Hobby poultry consisted of dedicated owners who breed and sell poultry and participate in exhibitions and poultry shows. Pet and hobby poultry profiles were characterized by greater knowledge of diseases and biosecurity practices, more bird movements, and reported more frequent clinical signs. The observation of different profiles can help target veterinary and public health education messages to prevent disease transmission in backyard poultry flocks in France.

Biosecurity measures in French poultry farms are associated with farm type and location.

The severe impact of recently reported Highly Pathogenic Avian Influenza (HPAI) outbreaks have emphasized the need to better monitor biosecurity practices among the different French poultry production systems. Between October 2016 and September 2018, a large national cross-sectional study was carried out in France to assess biosecurity levels in high-risk poultry farms and identify farm biosecurity profiles, using Multiple Correspondence and Hierarchical Cluster Analyses. Results indicate that there is substantial room for improvement in cleaning and disinfection, anteroom management and delimitation of farm and production units for more than 50 % of the 1,004 analyzed farms. The farm biosecurity profile showing the highest level of biosecurity included commercial intensive poultry farms (gallinaceous poultry farms, in North-Western France), and those with a recent history of HPAI outbreak (duck farms, in South-Western France). The farms biosecurity profiles showing the lowest level of biosecurity included farms with multiple species (duck and gallinaceous poultry) or multiple production types (broilers and egg-layers), located in regions with a lower poultry density and without a recent history of HPAI outbreak. Outcomes provide support to adapt biosecurity improvement and inspection plans.

Determinants of biosecurity practices in French duck farms after a H5N8 Highly Pathogenic Avian Influenza epidemic: The effect of farmer knowledge, attitudes and personality traits.

Biosecurity is crucial for infectious disease prevention, more importantly in the absence of vaccination. The need for improving the implementation of biosecurity practices was highlighted in French duck farms following the 2016-2017 H5N8 Highly Pathogenic Avian Influenza (HPAI) epidemic. Farmers have multiple reasons for not implementing biosecurity practices: external (time, money) and internal (socio-psychological). The purpose of this study was to determine how sets of socio-psychological factors (i.e. knowledge on biosecurity and avian influenza transmission, attitudes, personality traits, social background) affect the adoption of on-farm biosecurity practices. Biosecurity practices and socio-psychological determinants were assessed during 127 duck farm visits, in South West France, using both questionnaires and on-farm observations. Factorial analysis of mixed data (FAMD) and hierarchical clustering analysis (HCA) identified three groups of farmers with different socio-psychological profiles: the first group was characterized by minimal knowledge, negative attitudes towards biosecurity, little social pressure and a low level of conscientiousness. The second group was characterized by more extensive experience in poultry production, higher stress and social pressure. The third group was characterized by less experience in poultry production, better knowledge and positive attitudes towards biosecurity, increased self-confidence and orientation towards action. The first group had a significantly lower adoption of biosecurity measures than the two other groups. A better understanding of the factors involved in farmers' decision-making could improve the efficiency of interventions aiming at improving and maintaining the level of on-farm biosecurity in the duck industry.

Inferring within-flock transmission dynamics of highly pathogenic avian influenza H5N8 virus in France, 2020.

Following the emergence of highly pathogenic avian influenza (H5N8) in France in early December 2020, we used duck mortality data from the index farm to investigate within-flock transmission dynamics. A stochastic epidemic model was fitted to the daily mortality data and model parameters were estimated using an approximate Bayesian computation sequential Monte Carlo (ABC-SMC) algorithm. The model predicted that the first bird in the flock was infected 5 days (95% credible interval, CI: 3-6) prior to the day of suspicion and that the transmission rate was 4.1 new infections per day (95% CI: 2.8-5.8). On average, ducks became infectious 4.1 h (95% CI: 0.7-9.1) after infection and remained infectious for 4.3 days (95% CI: 2.8-5.7). The model also predicted that 34% (50% prediction interval: 8%-76%) of birds would already be infectious by the day of suspicion, emphasizing the substantial latent threat this virus could pose to other poultry farms and to neighbouring wild birds. This study illustrates how mechanistic models can help provide rapid relevant insights that contribute to the management of infectious disease outbreaks of farmed animals. These methods can be applied to future outbreaks and the resulting parameter estimates made available to veterinary services within a few hours.

Biosecurity risk factors for highly pathogenic avian influenza (H5N8) virus infection in duck farms, France.

Highly pathogenic avian influenza (HPAI) subtype H5N8 outbreaks occurred in poultry farms in France in 2016-2017, resulting in significant economic losses and disruption to the poultry industry. Current evidence on associations between actual on-farm biosecurity risk factors and H5N8 occurrence is limited. Therefore, a retrospective matched case-control study was undertaken to investigate the inter-relationships between on-farm biosecurity practices and H5N8 infection status to provide new insights regarding promising targets for intervention. Data were collected on 133 case and 133 control duck farms (i.e. the most affected species) located in one area of the country that was mostly affected by the disease. Data were analysed using Additive Bayesian Networks which offer a rich modelling framework by graphically illustrating the dependencies between variables. Factors indirectly and directly positively associated with farm infection were inadequate management of vehicle movements (odds ratio [OR] 9.3, 95% credible interval [CI] 4.0-22.8) and inadequate delimitation of farm and units (OR 3.0, 95% CI 1.6-5.8), respectively. Inadequate disposal of dead birds was instead negatively associated with the outcome (OR 0.1, 95% CI 0.0-0.3). The findings highlight that reinforcing farm access control systems and reducing the number of visitors are key biosecurity measures to control farm vulnerability to H5N8 infection and could help setting priorities in biosecurity practices to prevent outbreaks' re-occurrence.

Biosecurity practices on foie gras duck farms, Southwest France.

On-farm biosecurity can be assessed by analyzing patterns of practices to better tailor technical advice to producers. Given their close contact with environmental and wildlife disease reservoirs, free-range duck farms are exposed to multiple risk factors of pathogen exposure that are rare or absent in indoor production. The recurrent emergence of Highly Pathogenic Avian Influenza (HPAI) viruses in Southeast Asia and Europe has emphasized the importance of farm-level biosecurity on free-range duck farms. This study was conducted on 46 French duck farms. The farms were visited and an 80-question survey was administered to assess biosecurity practices. Patterns of practices were explored with multiple correspondence analysis and hierarchical cluster analysis. Farms were assigned to one of three clusters in which specific farm types were overrepresented: farms specialized in rearing to grow-out phases and open-circuit full cycle (i.e., all production phases on the farm) farms in cluster 1, closed-circuit full cycle farms in cluster 2, and farms specialized in gavage in cluster 3. Differences in practices might be linked with differences in production constraints. This study provides a baseline assessment of biosecurity practices on foie gras duck farms in Southwest France and will help efforts to adapt biosecurity programs to farm types.

Rapid whole-genome based typing and surveillance of avipoxviruses using nanopore sequencing.

Avian pox is an infectious disease caused by avipoxviruses (APV), resulting in cutaneous and/or tracheal lesions. Poxviruses share large genome sizes (from 130 to 360 kb), featuring repetitions, deletions or insertions as a result of a long-term recombination history. The increasing performances of next-generation sequencing (NGS) opened new opportunities for surveillance of poxviruses, based on timely and affordable workflows. We investigated the application of the 3rd generation Oxford Nanopore Minion technology to achieve real-time whole-genome sequencing directly from lesions, without any enrichment or isolation step. Fowlpox lesions were sampled on hens, total DNA was extracted and processed for sequencing on a MinION, Oxford Nanopore. We readily generated whole APV genomes from cutaneous or tracheal lesions, without any isolation or PCR-based enrichment: Fowlpox virus reads loads ranged from 0.75% to 2.62% and reads up to 61 kbp were generated and readily assembled into 3 APV complete genomes. This long read size eases the assembly step and lowers the bioinformatics capacity requirements and processing time compared to huge sets of short reads. The complete genome analysis confirmed that these Fowlpox viruses cluster within clade A1 and host full length reticuloendotheliovirus (REV) inserts. The pathobiological relevance of REV insert, although a classical feature of fowlpoxviruses (FPVs), should be further investigated. Surveillance of emerging poxviruses could greatly benefit from real-time whole genome sequencing.

Efficacy of Massachusetts and 793B Vaccines Against Infectious Bronchitis Moroccan-Italy 02 Virus in Specific-Pathogen-Free Chickens and Commercial Broilers.

The ability of commercial vaccines H120 and 4/91 to protect against Moroccan-Italy 02 infectious bronchitis virus (Mor-It02) was investigated in specific-pathogen-free (SPF) chickens and commercial broiler chickens. Commercial broiler chicks (Experiment 1) were vaccinated at the hatchery with H120 vaccine at Day 1, and challenged at Day 21 with 104 50% egg-infective dose (EID50) of Mor-It02. All chicks were observed daily for clinical signs attributable to Mor-It02 infection during the 10 days postchallenge (pc). At 5 and 10 days pc, chicks were humanely sacrificed for necropsy examination, and tissues were collected for histopathology evaluation. To better understand the findings on commercial broilers, day-old SPF chicks were divided into five groups in a second experiment: Group Mass/4-91, vaccinated with H120 and 4/91 respectively at Days 1 and 15 of age; Group Mass/Mass, vaccinated by H120 at Days 1 and 15; Group Mass, vaccinated with H120 at Day 1; Group NV, kept unvaccinated; and Group NC, kept as a negative control (unchallenged). At Day 24 of age, Groups Mass/4-91, Mass/Mass, Mass, and NV were challenged with 104 EID50 of Mor-It02. In both experiments, blood samples were collected at different periods for serologic analyses. Oropharyngeal swabs were collected for virus detection by reverse-transcription PCR. In Experiments 1 and 2, respiratory signs started as early as 24 hr pc and maximum severity was observed on Days 3 and 4 pc. The viral shedding rate was significantly lower in Group Mass/4-91 compared to other challenged groups. Serologic analysis in both experiments showed that the sera of challenged group exhibited significantly higher antibody titers than sera collected before challenge. Histopathologic investigations in SPF birds showed deciliation and hyperplasia in Group NV and less-pronounced lesions in Groups Mass/Mass and Mass. In commercial broilers vaccinated with H120 alone, hyperplasia and deciliation were observed in 90% of the tracheas. These experiments illustrated that Mor-It02 is pathogenic for chickens and a combination of live H120 and 4/91 vaccines given respectively at Day 1 and Day 15 of age confer a good protection against Mor-It02.

Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine.

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts.