Effectiveness of a Simultaneous rHVT-F(ND) and rHVT-H5(AI) Vaccination of Day-Old Chickens and the Influence of NDV- and AIV-Specific MDA on Immune Response and Conferred Protection.

The recombinant herpesvirus of turkey (rHVT) vaccines targeting Newcastle disease (ND) and H5Nx avian influenza (AI) have been demonstrated efficient in chickens when used individually at day-old. Given the practical field constraints associated with administering two vaccines separately and in the absence of a currently available bivalent rHVT vector vaccine expressing both F(ND) and H5(AI) antigens, the aim of this study was to investigate whether interference occurs between the two vaccines when simultaneously administered in a single shot. The studies have been designed to determine (i) the ND and AI-specific protection and antibody response conferred by these vaccines inoculated alone or in combination at day-old, (ii) the influence of maternally-derived antibodies (MDA), and (iii) the potential interference between the two vaccine. Our results demonstrate that their combined administration is efficient to protect chickens against clinical signs of velogenic Newcastle disease virus (vNDV) and H5-highly pathogenic avian influenza virus (HPAIV) infections. Viral shedding following co-vaccination is also markedly reduced, while slightly lower NDV- and AIV-specific antibody responses are observed. NDV- and AIV-specific MDA show negative effects on the onset of the specific antibody responses. However, if AIV-specific MDA reduce the protection against H5-HPAIV induced by rHVT-H5(AI) vaccine, it was not observed for ND.

Chickens can durably clear herpesvirus vaccine infection in feathers while still carrying vaccine-induced antibodies.

Marek's disease (MD) is a major disease of chickens induced by Marek's disease virus (MDV) associated to lethal lymphomas. Current MD vaccines protect against lymphomas, but fail to prevent infection and shedding. The control of MDV shedding is crucial in order to eradicate this highly contagious virus. Like pathogenic MDV, MD vaccines infect the feather follicles of the skin before being shed into the environment. MD vaccines constitute excellent models to study virus interaction with feathers, the unique excretion source of these viruses. Herein we studied the viral persistence in feathers of a MD vaccine, the recombinant turkey herpesvirus (rHVT-ND). We report that most of the birds showed a persistent HVT infection of feathers over 41 weeks with moderate viral loads. Interestingly, 20% of the birds were identified as low HVT producers, among which six birds cleared the infection. Indeed, after week 14-26, these birds named controllers had undetectable HVT DNA in their feathers through week 41. All vaccinated birds developed antibodies to NDV, which lasted until week 41 in 95% of the birds, including the controllers. No correlation was found between HVT loads in feathers and NDV antibody titers over time. Interestingly, no HVT DNA was detected in the spleens of four controllers. This is the first description of chickens that durably cleared MD vaccine infection of feathers suggesting that control of Mardivirus shedding is achievable by the host.

Virus-Like Particle Based Vaccine Provides High Level of Protection Against Homologous H5N8 HPAIV Challenge in Mule and Pekin Duck, Including Prevention of Transmission.

The most recent pandemic clade of highly pathogenic avian influenza (HPAI) H5, clade 2.3.4.4, spread widely, with the involvement of wild birds, most importantly wild waterfowl, carrying the virus (even asymptomatically) from Asia to North America, Europe, and Africa. Domestic waterfowl being in regular contact with wild birds played a significant role in the H5Nx epizootics. Therefore, protection of domestic waterfowl from H5Nx avian influenza infection would likely cut the transmission chain of these viruses and greatly enhance efforts to control and prevent disease outbreak in other poultry and animal species, as well as infection of humans. The expectation for such a vaccine is not only to provide clinical protection, but also to control challenge virus transmission efficiently and ensure that the ability to differentiate infected from vaccinated animals is retained. A water-in-oil emulsion virus-like particle vaccine, containing homologous hemagglutinin antigen to the current European H5N8 field strains, has been developed to meet these requirements. The vaccine was tested in commercial Pekin and mule ducks by vaccinating them either once, at 3 wk of age, or twice (at 1 day and at 3 wk of age). Challenge was performed at 6 wk of age with a Hungarian HPAIV H5N8 isolate (2.3.4.4 Group B). Efficacy of vaccination was evaluated on the basis of clinical signs, amount of virus shedding, and transmission. Vaccination resulted in complete clinical protection and prevention of challenge virus transmission from the directly challenged vaccinated ducks to the vaccinated contact animals.

Una vacuna basada en partículas similares a virus proporciona un alto nivel de protección contra el desafío con un virus homólogo de influenza aviar de alta patogenicidad H5N8 en patos mula y Pekin, incluida la prevención de la transmisión. El clado pandémico más reciente de influenza aviar altamente patógena H5, clado 2.3.4.4, se diseminó ampliamente, con la participación de aves silvestres, siendo las aves acuáticas más importantes, portando el virus (incluso asintomáticamente) de Asia a América del Norte, Europa, y África. Las aves acuáticas domésticas en contacto regular con aves silvestres desempeñaron un papel importante en las epizootias H5Nx. Por lo tanto, la protección de las aves acuáticas domésticas contra la infección por influenza aviar H5Nx probablemente cortaría la cadena de transmisión de estos virus y aumentaría en gran medida los esfuerzos para controlar y prevenir brotes de enfermedades en otras aves comerciales y especies animales, así como la infección en humanos. La expectativa de una vacuna de este tipo es no solo brindar protección clínica, sino también controlar la transmisión del virus de desafío de manera eficiente y garantizar que se mantenga la capacidad de diferenciar a los animales vacunados. Se ha desarrollado una vacuna emulsionada en aceite con partículas similares al virus, que contiene el antígeno de hemaglutinina homóloga a las cepas de campo H5N8 europeas actuales, para cumplir con estos requisitos. La vacuna se probó en patos de Pekín y mulas comerciales, vacunándolos una vez, a las tres semanas de edad, o dos veces (al primer día y a las tres semanas de edad). El desafío se realizó a las seis semanas de edad con un aislado de alta patogenicidad H5N8 húngaro (2.3.4.4 Grupo B). La eficacia de la vacunación se evaluó en función de los signos clínicos, la eliminación viral y la transmisión. La vacunación dio como resultado una protección clínica completa y la prevención de la transmisión del virus de desafío de los patos vacunados.

Efficacy of a Recombinant Turkey Herpesvirus AI (H5) Vaccine in Preventing Transmission of Heterologous Highly Pathogenic H5N8 Clade 2.3.4.4b Challenge Virus in Commercial Broilers and Layer Pullets.

Outbreaks caused by the highly pathogenic avian influenza virus (HPAIV) H5N8 subtype clade 2.3.4.4 were first reported in 2014 in South Korea then spread very rapidly in Asia, to Europe, and for the first time, to North America. Efficacy of a recombinant HVT-AI (H5) vaccine (rHVT-H5) to provide clinical protection as well as to significantly reduce the shedding of an H5N8 challenge virus has already been demonstrated in SPF chickens. The aim of our studies was to test the efficacy of the same rHVT-H5 vaccine in controlling the transmission of a recent Hungarian HPAIV H5N8 challenge virus in commercial chickens. Broilers and layers were vaccinated at day old according to the manufacturer's recommendation and then challenged with a 2017 Hungarian HPAIV H5N8 (2.3.4.4b) isolate at 5 or 7 weeks of age, respectively. Evaluation of clinical protection, reduction of challenge virus shedding, and transmission to vaccinated contact birds was done on the basis of clinical signs/mortality, detection, and quantitation of challenge virus in oronasal and cloacal swabs (regularly between 1 and 14 days postchallenge). Measurement of seroconversion to AIV nucleoprotein was used as an indicator of infection and replication of challenge virus. Our results demonstrated that rHVT-H5 vaccination could prevent the development of clinical disease and suppress shedding very efficiently, resulting in the lack of challenge virus transmission to vaccinated contact chickens, regardless the type of birds. Single immunization with the tested rHVT-H5 vaccine proved to be effective to stop HPAIV H5N8 (2.3.4.4b) transmission within vaccinated poultry population under experimental conditions.

Recombinant Turkey Herpesvirus-AI Vaccine Virus Replication in Different Species of Waterfowl.

Waterfowl play a key role in the epidemiology of the H5N1 subtype of highly pathogenic avian influenza (HPAI) virus; therefore, efficient immunization of domesticated ducks and geese to maximize the impact of other control measures is of great importance. A recombinant (r)HVT-AI, expressing the HA gene of a clade 2.2 H5N1 HPAI strain had been developed and proved to be efficient against different clades of H5N1 HPAI virus in chickens after a single vaccination at 1 day old and could provide long-term immunity. We investigated whether rHVT-AI applied at 1 day old is able to replicate in different species and crossbreeds of ducks and in geese with the aim of collecting data on the possible application of rHVT-AI vaccine in different species of waterfowl for the control of H5N1 HPAI. We tested the possible differences among different waterfowl species, i.e., between geese (Anser anser, domesticated greylag goose), Muscovy ducks (Cairina moschata forma domestica), Pekin ducks (Anas platyrhynchos forma domestica), and mule ducks (Muscovy duck × Pekin duck), in their susceptibility to support the replication of rHVT-AI. Vaccine virus replication was followed by real-time PCR in spleen, bursa, and feather tip samples. Humoral immune response to vaccination was tested using the hemagglutination inhibition (HI) test and H5-specific commercial ELISA. Significant differences among the different waterfowl species regarding the rate of rHVT-AI replication was detected that were not reflected by the same difference in the immune response to vaccination. Replication of the rHVT-AI vaccine was very limited in Pekin ducks, somewhat better in mule ducks, and the vaccine virus was replicating significantly better in Muscovy ducks and geese, reaching 100% detectability at certain time points after administration at 1 day old. Results indicated that the vaccine virus could establish different levels of persistent infection in these species of waterfowl. No humoral immune response could be detected either by HI test or ELISA during the tested postvaccination period (5 wk).

Experimental and Field Results Regarding Immunity Induced by a Recombinant Turkey Herpesvirus H5 Vector Vaccine Against H5N1 and Other H5 Highly Pathogenic Avian Influenza Virus Challenges.

Vaccination against H5N1 highly pathogenic avian influenza (AI) virus (HPAIV) is one of the possible complementary means available for affected countries to control AI when the disease has become, or with a high risk of becoming, endemic. Efficacy of the vaccination against AI relies essentially, but not exclusively, on the capacity of the vaccine to induce immunity against the targeted virus (which is prone to undergo antigenic variations), as well as its capacity to overcome interference with maternal immunity transmitted by immunized breeding hens to their progeny. This property of the vaccine is a prerequisite for its administration at the hatchery, which assures higher and more reliable vaccine coverage of the populations than vaccination at the farm. A recombinant vector vaccine (Vectormune® AI), based on turkey herpesvirus expressing the hemagglutinin gene of an H5N1 HPAIV as an insert, has been used in several experiments conducted in different research laboratories, as well as in controlled field trials. The results have demonstrated a high degree of homologous and cross protection against different genetic clades of the H5N1 HPAIV. Furthermore, vaccine-induced immunity was not impaired by the presence of passive immunity, but on the contrary, cumulated with it for improved early protection. The demonstrated levels of protection against the different challenge viruses exhibited variations in terms of postchallenge mortality, as well as challenge virus shedding. The data presented here highlight the advantages of this vaccine as a useful and reliable tool to complement biosecurity and sanitary policies for better controlling the disease due to HPAIV of H5 subtypes, when the vaccination is applied as a control measure.

Vaccine Protection of Turkeys Against H5N1 Highly Pathogenic Avian Influenza Virus with a Recombinant Turkey Herpesvirus Expressing the Hemagglutinin Gene of Avian Influenza.

Outbreaks of H5 highly pathogenic avian influenza (HPAI) in commercial poultry are a constant threat to animal health and food supplies. While vaccination can enhance protection and reduce the spread of disease, there is considerable evidence that the level of immunity required for protection varies by subtype and virulence of field virus. In this study, the efficacy of a recombinant turkey herpesvirus (rHVT) vector vaccine expressing the hemagglutinin gene from a clade 2.2 AI virus (A/Swan/Hungary/4999/2006) was evaluated in turkeys for protection against challenge with A/Whooper Swan/Mongolia/L244/2005 H5N1 HPAI clade 2.2. One-day-old turkeys received a single vaccination and were challenged at 4 wk postvaccination with 2 × 10(6) 50% embryo infectious dose per bird. The results demonstrate that following H5N1 HPAI challenge 96% protection was observed in rHVT-AI vaccinated turkeys. The oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared with sham-vaccinated birds. From respiratory and gastrointestinal tracts, there was a greater than 6 log10 reduction in shedding in vaccinated birds as compared with the controls. This study provides support for the use of a commercially available rHVT-AI vaccine to protect turkeys against H5N1 HPAI.

Proposed bursa of Fabricius weight to body weight ratio standard in commercial broilers.

Several causes may induce change and atrophy in the bursa of Fabricius (BF). Databases on BF standards are available from published studies, however, updated references are needed to adjust the BF standards to present changes in highly specialized broiler genetic lines. The aim of this study was to evaluate BF-related measurements (weight and dimensions) under controlled conditions that would mimic field situations. Chickens were kept in isolation, thus avoiding exposure to disease agents by vaccination or field infections. This study was conducted using male Cobb 500 commercial broilers from the same hatch and source. Absence of disease was confirmed throughout the study. Despite the presence of individual variations, a minimum bursa-to-body weight ratio standard of 0.11 is proposed in broilers from 7 to 42 days of age.

Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus.

Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80-95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection.

Onset and long-term duration of immunity provided by a single vaccination with a turkey herpesvirus vector ND vaccine in commercial layers.

The onset and duration of immunity provided by a recombinant ND vaccine using HVT virus as vector (rHVT-ND) was followed up to 72 weeks of age in commercial layer chickens after single application or as part of two different vaccination regimes including conventional live and killed ND vaccines. Efficacy of the different vaccination programmes was checked, from 3 to 72 weeks of age, by serology as well as by challenges with a recent velogenic NDV isolate belonging to genotype VII. Assessment of protection was done based on the prevention of clinical signs and reduction of challenge virus shedding via the oro-nasal and cloacal routes. Single vaccination with the rHVT-ND vaccine at one day of age provided complete or almost complete (95-100%) clinical protection against NDV challenges from 4 weeks of age up to 72 weeks of age when the latest challenge was done. Shedding of challenge virus both by the oro-nasal and cloacal route was significantly reduced compared to the controls. Booster vaccination of rHVT-ND vaccinated birds with conventional ND vaccines significantly increased the level of anti-NDV serum antibodies and further reduced the oro-nasal excretion of challenge virus.

Protection and antibody response caused by turkey herpesvirus vector Newcastle disease vaccine.

Newcastle disease (ND) is prevalent worldwide and causes significant clinical and economic losses to the poultry industry. Current vaccine programs using live attenuated vaccines and inactivated vaccines have limitations, and new vaccines with distinct features are needed. To offer an alternative solution to control ND, a turkey herpesvirus vector Newcastle disease vaccine (HVT/ND) expressing the fusion gene of Newcastle disease virus (NDV) has been developed. First, immunogenicity of the HVT/ND was evaluated in specific-pathogen-free layer chickens after vaccination by the in ovo route to 18-day-old embryos or by the subcutaneous route to 1-day-old chicks. Antibodies against NDV were detected at 24 days of age using a commercial NDV enzyme-linked immunosorbent assay (ELISA) kit and the hemagglutination inhibition test. At least 90% of chickens were protected against challenge with velogenic neurotropic NDV Texas GB strain (genotype II; pathotype velogenic) at 4 wk of age, while none of the nonvaccinated, challenged controls were protected from challenge. Second, the age at which a vaccinated chicken elicits an immunologic response to the HVT/ND prepared for this study, and thus is protected from ND virus, was assessed in commercial broiler chickens after in ovo vaccination of 18-day-old embryos. Challenge was conducted using a low-virulence NDV strain (genotype II; pathotype lentogenic) via the respiratory tract each week between 1 and 5 wk of age, in order to mimic the situation in areas where virulent NDV strains do not normally exist and low-virulence strains cause mild respiratory symptoms leading to economic losses. Protection was evaluated by the presence or absence of isolated virus from tracheal swabs at 5 days postchallenge. Partial protection was observed at 3 wk of age, when 6 out of 10 (60%) chickens were protected. Full protection was obtained at 4 and 5 wk of age, when 9 out of 10 (90%) and 10 out of 10 (100%) chickens were protected, respectively. Finally, protection against challenge with virulent Texas GB strain at 19 wk of age was evaluated in commercial female layer chickens vaccinated at 1 day of age with HVT/ND. All of the vaccinated chickens were protected, while all of the challenge controls succumbed to the challenge. Furthermore, anti-NDV antibodies measured by ELISA were maintained through 50 wk of age.

Efficacy of a recombinant HVT-H5 vaccine against challenge with two genetically divergent Indonesian HPAI H5N1 strains.

The swift evolution rate of avian influenza (AI) H5N1 virus demands constant efforts to update inactivated vaccines to match antigenically with the emerging new field virus strains. Recently, a recombinant turkey herpesvirus (rHVT)-AI vaccine, rHVT-H5, expressing the HA gene of a highly pathogenic avian influenza (HPAI) H5N1 clade 2.2 A/Swan/Hungary/499/ 2006 strain inserted into FC-126 strain of HVT vector, has been developed to combat current threats in poultry industry. Here, we present the results of two trials where rHVT-H5 was tested alone or in combination with inactivated H5N1 vaccines (the latter vaccines contained antigens produced by using a clade 2.1.3 HPAI H5N1 virus [A/Ck/WestJava-Nagrak/2007] in the first trial or mixture of antigen produced by strain A/Ck/WestJava-Nagrak/2007 and A/Ck/Banten-Tangerang/2010 [bivalent vaccine] for second trial) in broiler chickens (Gallus gallus domesticus) carrying maternally derived antibodies to H5N1 and then challenged with Indonesian HPAI H5N1 field isolates. The effectiveness of vaccination was evaluated on the basis of clinical protection (morbidity and mortality) and measurement of virus shedding after challenge. Immune response to vaccination was followed by serology. In the first experiment, chickens were vaccinated at the day of hatch with rHVT-H5 alone (Group 1) or combined with inactivated vaccine at day old (Group 2) or at 10 days of age (Group 3). The chickens along with nonvaccinated hatch-mates were challenged at 28 days of age with the HPAI H5N1 field isolate dade 2.1.3 A/Chicken/WestJava-Subang/29/2007. Eighty, 100%, and 80% clinical protection was recorded in Group 1, 2, and 3, respectively. A similar experiment was performed a second time, but the chicks in Group 3 received the inactivated vaccine earlier, at 7 days of age. Challenge was performed at 28 days of age using a different H5N1 isolate, clade 2.1.3 A/Ck/Purwakarta-Cilingga/142/10. Clinical protection achieved in the second trial was 95%, 75%, and 90% in Group 1, 2, and 3, respectively. Shedding of challenge virus was significantly lower in the vaccinated groups compared with controls in both experiments. Vaccinated birds developed hemagglutination inhibition antibody response to H5N1 by the time of challenge. These experiments confirmed that the rHVT-H5 vaccine applied alone or in combination with inactivated H5N1 vaccines could provide high level (> 80%) clinical protection against divergent HPAI H5N1 field isolates after single immunization by 4 wk of age and a significant reduction in the excretion of challenge virus.

The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live Newcastle disease vaccine.

The development of safe, novel strong adjuvants is necessary to maximize the efficacy of and the immune response induced by new and/or available vaccines administered through the mucosal route to chickens. Chitosan is a non-toxic, biocompatible, biodegradable and natural polysaccharide derived from the exoskeleton of crustaceans and insects. It has been demonstrated to be an effective absorption enhancer to improve mucosal delivery of peptide and protein drugs in human and mice. In poultry, mucosal administration of live vaccine has been already explored with success. However, the effects of the use of the chitosan as adjuvant for mucosal vaccination in birds have not been investigated yet. To this aim, we explored its potential as adjuvant given by oculo-nasal route to one-day-old chickens with live Newcastle disease (ND) vaccine. The immune response has been evaluated during three independent vaccination experiments on specific pathogen free (SPF) chickens. It was shown that chitosan enhanced the antigen-specific cell-mediated immune response in the spleen. New protocols were developed to measure the chicken IFNgamma production after ex vivo antigen-stimulation of peripheral blood and duodenal lamina propria lymphocytes. It was then observed than the peripheral cellular immune response was earlier and stronger, while the local cellular immune response in digestive tract as shorter when chitosan was used as adjuvant. On the other hand, the chitosan had no effect on the systemic, lachrymal and digestive antibody-mediated immunity. This study indicates thus that the chitosan is a cell-promising adjuvant for the mucosal delivery of live vaccine in poultry, by enhancing the Th1 pathway of immunity. However, further investigations are required to explore its mechanism of action and to evaluate the inferred protection.

Improved vaccination against Newcastle disease by an in ovo recombinant HVT-ND combined with an adjuvanted live vaccine at day-old.

The continuous outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks demonstrate that current vaccination strategies are not fully efficacious and should be improved by new generation of vaccines. In this context, maternally immune conventional layer chickens were vaccinated in ovo with a turkey herpesvirus recombinant expressing the fusion (F) gene of NDV (rHVT-ND) and/or at day-old with an apathogenic enterotropic live ND vaccine co-administrated or not with chitosan by oculo-nasal route. The induced vaccinal immune responses and conferred protection against a challenge with a circulating NDV velogenic viscerotropic strain were evaluated. The innovative rHVT-ND/live ND-chitosan vaccination regimen provided the best protection against mortality and morbidity as well as the strongest reduction of virus shedding that could be related to the higher measured cellular immune response and digestive antibody-mediated immunity.

Efficacy of several vaccination programmes in commercial layer and broiler breeder hens against experimental challenge with Salmonella enterica serovar Enteritidis.

Two experiments were performed to evaluate the protective effect of various vaccination combinations given at 5 and 9 weeks of age against experimental challenge with Salmonella enterica serovar Enteritidis (SE) phage type 4 at 12 weeks of age. In Experiment 1, groups of commercial layers were vaccinated by one of the following programmes: Group 1, two doses of a SE bacterin (Layermune SE); Group 2, one dose of a live Salmonella enterica serovar Gallinarum vaccine (Cevac SG9R) followed by one dose of the SE bacterin; Group 3, one dose of each of two different multivalent inactivated vaccines containing SE cells (Corymune 4K and Corymune 7K; and Group 4, unvaccinated, challenged controls. In Experiment 2, groups of broiler breeders were vaccinated by the same programmes as Groups 1 and 2 above while Group 3 was an unvaccinated, challenged control group. All vaccination programmes and the challenge induced significant (P < 0.05) seroconversion as measured by enzyme-linked immunosorbent assay. Overall, in both experiments, all vaccination schemes were significantly effective in reducing organ (spleen, liver and caeca) colonization by the challenge strain as well as reducing faecal excretion for at least 3 weeks. Vaccinated layers in Groups 1 and 2 and broiler breeders in Group 2 showed the greatest reduction in organ colonization and the least faecal excretion. In Experiment 1, layers vaccinated with multivalent inactivated vaccines containing a SE component (Group 3) were only moderately protected, indicating that such a vaccination programme may be useful in farms with good husbandry and housing conditions and low environmental infectious pressure by Salmonella.

Humoral, cell-mediated and mucosal immunity induced by oculo-nasal vaccination of one-day-old SPF and conventional layer chicks with two different live Newcastle disease vaccines.

To further characterize the immune response elicited by two live Newcastle disease vaccines, humoral, cellular and mucosal immunity was evaluated after oculo-nasal vaccination of day-old chickens. The preferential replication sites for each vaccine strain were investigated by screening different tissues using quantitative real-time reverse transcription-polymerase chain reaction (QRRT-PCR). The interference of maternally derived antibody with vaccination was also considered in conventional layer chickens. In SPF chickens, similar humoral immune-response was measured in blood and tears but a differential profile of cell-mediated immunity was observed according to the vaccine strain. The lung-associated humoral immunity was higher with the tracheotropic strain while the enterotropic vaccine induced a more important specific immunity in the digestive tract. The presence of maternally derived antibody in conventional layer chickens limited, if not completely abrogated, their immune responses to vaccination. This study increases our understanding of the protective immune response against Newcastle disease virus (NDV) and provides new useful informations for the development and evaluation of new types of vaccines.

Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment-B-reassorted isolate.

The purpose of this study was to compare the molecular epidemiology of infectious bursal disease virus (IBDV) segments A and B of 50 natural or vaccine IBDV strains that were isolated or produced between 1972 and 2002 in 17 countries from four continents, with phenotypes ranging from attenuated to very virulent (vv). These strains were subjected to sequence and phylogenetic analysis based on partial sequences of genome segments A and B. Although there is co-evolution of the two genome segments (70 % of strains kept the same genetic relatives in the segment A- and B-defined consensus trees), several strains (26 %) were identified with the incongruence length difference test as exhibiting a significantly different phylogenetic relationship depending on which segment was analysed. This suggested that natural reassortment could have occurred. One of the possible naturally occurring reassortant strains, which exhibited a segment A related to the vvIBDV cluster whereas its segment B was not, was thoroughly sequenced (coding sequence of both segments) and submitted to a standardized experimental characterization of its acute pathogenicity. This strain induced significantly less mortality than typical vvIBDVs; however, the mechanisms for this reduced pathogenicity remain unknown, as no significant difference in the bursal lesions, post-infectious antibody response or virus production in the bursa was observed in challenged chickens.