Advancement of Autogenous Vaccines in the Poultry Industry.

Autogenous vaccines, also known as "custom" vaccines, have become an essential instrument in the production veterinarian's toolbox for the control of emerging and evolving diseases. Autogenous vaccines require a reduced burden of U.S. Department of Agriculture licensing, making them rapidly accessible. Autogenous vaccines have made significant advancements in the ability to reduce disease within the poultry industry from a combination of several different advancements in regulation requirements, rapid and accurate diagnostic assessments, and improvements in manufacturing. The use of autogenous vaccines by poultry health professionals has also increased, and these custom-made products have been instrumental in combating diseases resulting from antigenic variants such as salmonellosis, colibacillosis, infectious coryza, infectious bursal disease, inclusion body hepatitis, viral enteritis, and viral arthritis and tenosynovitis.

Estudio recapitulativo- Avance de las vacunas autógenas en la industria avícola Las vacunas autógenas, también conocidas como vacunas "personalizadas, elaboradas de acuerdo con las necesidades del cliente" ("custom"), se han convertido en un instrumento esencial en el inventario de herramientas del veterinario de producción para el control de enfermedades emergentes y en evolución. Las vacunas autógenas requieren un procedimiento reducido para obtener la licencia por parte del Departamento de Agricultura de los Estados Unidos, lo que las hace rápidamente accesibles. Las vacunas autógenas han logrado avances significativos en la capacidad de reducir enfermedades dentro de la industria avícola gracias a una combinación de varios avances diferentes en los requisitos regulatorios, evaluaciones de diagnóstico rápidas y precisas y mejoras en la fabricación. También ha aumentado el uso de vacunas autógenas por parte de los profesionales de la salud avícola, y estos productos hechos a medida han sido fundamentales para combatir enfermedades resultantes de variantes antigénicas como la salmonelosis, la colibacilosis, la coriza infecciosa, la enfermedad infecciosa de la bolsa, hepatitis con cuerpos de inclusión, la enteritis viral y la artritis y tenosinovitis virales.

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.

Rapid Construction and Immunogenicity Testing of a Novel H5 Virus-Like Particle Prototype Vaccine Against Clade 2.3.4.4 H5N8 Highly Pathogenic Avian Influenza Virus.

From October 2016 to July 2017, 47 countries have been affected by highly pathogenic avian influenza (HPAI) viruses of the H5N8 clade 2.3.4.4 subtype, including European and African, and it has been the most severe HPAI outbreak ever in Europe. The development of effective influenza vaccines is required to combine preventive and control measures in order to avoid similar avian influenza epidemics taking place. Here we describe a novel prototype recombinant virus-like particle (VLP) vaccine based on a clade 2.3.4.4 H5 HA derived from a French duck HPAI H5N8 isolate of the 2016-2017 epidemics. Prototype vaccines with different antigen content were formulated and the immunogenicity was examined in specific-pathogen-free chickens and in ducks. Serum samples were collected at 3 and 4 weeks postvaccination, and development of the immune response was evaluated by hemagglutination inhibition test and ELISA. The VLP vaccines induced a dose-dependent and high level of antibody response in both chickens and ducks. The results of HPAI H5N8 challenge experiments in ducks are reported separately.

Construcción rápida y pruebas de inmunogenicidad de un nuevo prototipo de vacuna H5 con base en partículas similares a virus contra el clado 2.3.4.4 del virus de la influenza aviar altamente patógeno H5N8. Desde octubre del 2016 hasta julio del 2017, 47 países se han visto afectados por los virus de la influenza aviar altamente patógena del subtipo H5N8 clado 2.3.4.4, incluidos los europeos y africanos y ha sido el brote de influenza aviar de alta patogenicidad más grave en Europa. Se requiere del desarrollo de vacunas efectivas contra la influenza para combinar medidas preventivas y de control para evitar que ocurran epidemias similares de influenza aviar. En este estudio se describe un nuevo prototipo de vacuna recombinante con partículas similares a virus (VLP) basada en una hemaglutinina H5 clado 2.3.4.4 derivada de un aislamiento del virus de influenza aviar de alta patogenicidad H5N8 de patos en Francia presente en las epidemias entre los años 2016 al 2017. Se formularon prototipos de vacunas con diferente contenido de antígeno y se examinó la inmunogenicidad en pollos libres de patógenos específicos y en patos. Las muestras de suero se recolectaron a las tres y cuatro semanas después de la vacunación y el desarrollo de la respuesta inmune se evaluó mediante la prueba de inhibición de la hemaglutinación y ELISA. Las vacunas con partículas similares a virus indujeron un alto nivel de respuesta de anticuerpos dependiente de la dosis tanto en pollos como en patos. Los resultados de los experimentos de desafío con un virus de influenza aviar de alta patogenicidad H5N8 en patos se informan por separado.

Metabolic parameters linked by phenotype microarray to acid resistance profiles of poultry-associated Salmonella enterica.

Phenotype microarrays were analyzed for 51 datasets derived from Salmonella enterica. The top 4 serotypes associated with poultry products and one associated with turkey, respectively Typhimurium, Enteritidis, Heidelberg, Infantis and Senftenberg, were represented. Datasets were partitioned initially into two clusters based on ranking by values at pH 4.5 (PM10 A03). Negative control wells were used to establish 90 respiratory units as the point differentiating acid resistance from sensitive strains. Thus, 24 isolates that appeared most acid-resistant were compared initially to 27 that appeared most acid-sensitive (24 × 27 format). Paired cluster analysis was also done and it included the 7 most acid-resistant and -sensitive datasets (7 × 7 format). Statistical analyses of ranked data were then calculated in order of standard deviation, probability value by the Student's t-test and a measure of the magnitude of difference called effect size. Data were reported as significant if, by order of filtering, the following parameters were calculated: i) a standard deviation of 24 respiratory units or greater from all datasets for each chemical, ii) a probability value of less than or equal to 0.03 between clusters and iii) an effect size of at least 0.50 or greater between clusters. Results suggest that between 7.89% and 23.16% of 950 chemicals differentiated acid-resistant isolates from sensitive ones, depending on the format applied. Differences were more evident at the extremes of phenotype using the subset of data in the paired 7 × 7 format. Results thus provide a strategy for selecting compounds for additional research, which may impede the emergence of acid-resistant Salmonella enterica in food.

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.

Effects of Newcastle disease virus vaccine antibodies on the shedding and transmission of challenge viruses.

Different genotypes of avian paramyxovirus serotype-1 virus (APMV-1) circulate in many parts of the world. Traditionally, Newcastle disease virus (NDV) is recognized as having two major divisions represented by classes I and II, with class II being further divided into sixteen genotypes. Although all NDV are members of APMV-1 and are of one serotype, antigenic and genetic diversity is observed between the different genotypes. Reports of vaccine failure from many countries and reports by our lab on the reduced ability of classical vaccines to significantly decrease viral replication and shedding have created renewed interest in developing vaccines formulated with genotypes homologous to the virulent NDV (vNDV) circulating in the field. We assessed how the amount and specificity of humoral antibodies induced by inactivated vaccines affected viral replication, clinical protection and evaluated how non-homologous (heterologous) antibody levels induced by live NDV vaccines relate to transmission of vNDV. In an experimental setting, all inactivated NDV vaccines protected birds from morbidity and mortality, but higher and more specific levels of antibodies were required to significantly decrease viral replication. It was possible to significantly decrease viral replication and shedding with high levels of antibodies and those levels could be more easily reached with vaccines formulated with NDV of the same genotype as the challenge viruses. However, when the levels of heterologous antibodies were sufficiently high, it was possible to prevent transmission. As the level of humoral antibodies increase in vaccinated birds, the number of infected birds and the amount of vNDV shed decreased. Thus, in an experimental setting the effective levels of humoral antibodies could be increased by (1) increasing the homology of the vaccine to the challenge virus, or (2) allowing optimal time for the development of the immune response.

Use of FTA sampling cards for molecular detection of avian influenza virus in wild birds.

Current avian influenza (AI) virus surveillance programs involving wild birds rely on sample collection methods that require refrigeration or low temperature freezing to maintain sample integrity for virus isolation and/or reverse-transcriptase (RT) PCR. Maintaining the cold chain is critical for the success of these diagnostic assays but is not always possible under field conditions. The aim of this study was to test the utility of Finders Technology Associates (FTA) cards for reliable detection of AI virus from cloacal and oropharyngeal swabs of wild birds. The minimum detectable titer was determined, and the effect of room temperature storage was evaluated experimentally using multiple egg-propagated stock viruses (n = 6). Using real time RT-PCR, we compared results from paired cloacal swab and samples collected on FTA cards from both experimentally infected mallards (Anasplatyrhynchos) and hunter-harvested waterfowl sampled along the Texas Gulf Coast. Based on the laboratory trials, the average minimal detectable viral titer was determined to be 1 x 10(4.7) median embryo infectious dose (EID50)/ml (range: 1 x 10(4.3) to 1 x 10(5.4) EID50/ml), and viral RNA was consistently detectable on the FTA cards for a minimum of 20 days and up to 30 days for most subtypes at room temperature (23 C) storage. Real-time RT-PCR of samples collected using the FTA cards showed fair to good agreement in live birds when compared with both real-time RT-PCR and virus isolation of swabs. AI virus detection rates in samples from several wild bird species were higher when samples were collected using the FTA cards compared with cloacal swabs. These results suggest that FTA cards can be used as an alternative sample collection method when traditional surveillance methods are not possible, especially in avian populations that have historically received limited testing or situations in which field conditions limit the ability to properly store or ship swab samples.

Avian influenza surveillance in hunter-harvested waterfowl from the Gulf Coast of Texas (November 2005-January 2006).

The objectives of our study were to determine prevalence of avian influenza viruses (AIV) on wintering grounds on the Texas Gulf Coast, USA, and to compare real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) and virus isolation for detection of AIV in cloacal swabs from wild waterfowl. Cloacal swabs were collected from hunter-harvested waterfowl from November 2005 to January 2006 at four wildlife management areas. Seven AIV were isolated from four species of ducks: Green-winged Teal (Anas crecca) in November; Blue-winged Teal (Anas discors) in November; Mottled Duck (Anas fulvigula) in December, and Northern Shoveler (Anas clypeata) in January. Prevalence of AIV for each of these species during the sampling period was 1.4, 2, 6, and 0.6%, respectively. The AIV subtypes detected were H1N2, H1N4, H4N6, H6N2, and H10N7, all previously reported in North American waterfowl. Our study identified AIV subtypes not previously reported on the Texas Gulf Coast and provides baseline data for a multiyear surveillance project.

Heteroduplex mobility assay for genotyping infectious bursal disease virus.

A heteroduplex mobility assay (HMA) was developed to genotype infectious bursal disease virus (IBDV). This method analyzed 390-base pair (bp) polymerase chain reaction (PCR) products, encompassing the hypervariable region of the VP2 gene. IBDV strains from the United States and other countries were analyzed. The HMA was able to differentiate standard, antigenic variants and very virulent strains of IBDV. Minor differences between different strains from the same subtype were also detected. Close relationships between field IBDV with vaccines prepared with Delaware E strain were determined by HMA. The results obtained by HMA were confirmed by restriction fragment length polymorphism (RFLP) and phylogenetic analysis of nucleotide sequences. The HMA proved to be a useful technique to rapidly genotype different field strains of IBDV and should prove to be a useful tool in epidemiologic studies.

Development and application of reverse transcriptase nested polymerase chain reaction test for the detection of exogenous avian leukosis virus.

A polymerase chain reaction (PCR) assay that utilizes nested primers to amplify a fragment of the long terminal repeat of exogenous avian leukosis virus (ALV) was developed and evaluated for detection of ALV subgroup J directly from clinical samples. Compilation of sequence data from different endogenous and exogenous ALVs allowed the selection of a conserved set of nested primers specific for the amplification of exogenous ALV subgroups A, B, C, D, and J and excluded amplification of endogenous viruses or endogenous viral sequences within the chicken genome. The nested primers were successfully used in both PCR and reverse transcriptase (RT)-PCR assays to detect genetically diverse ALV-J field isolates. Detection limits of ALV-J isolate ADOL-Hc1 DNA by nested PCR and RNA by RT-nested PCR were superior to detection of group-specific antigen by enzyme-linked immunosorbent assay (ELISA) in cell culture. Detection of ALV-J in cloacal swabs by RT-nested PCR was compared with direct detection by antigen-capture (ac)-ELISA; RT-nested PCR detected fewer positive samples than ac-ELISA, suggesting that RT-nested PCR excluded detection of endogenous virus in clinical samples. Detection of ALV-J in plasma samples by RT-nested PCR was compared with virus isolation in C/E chicken embryo fibroblasts; the level of agreement between both assays as applied to plasma samples ranged from low to moderate. The main disagreement between both assays was observed for a group of plasma samples found positive by RT-nested PCR and negative by virus isolation, suggesting that RT-nested PCR detected ALV-J genome in plasma samples of transiently or intermittently infected birds. ALV-J transient and intermittent infection profiles are characterized by inconsistent virus isolation responses throughout the life of a naturally infected flock.

A recombination event, induced in ovo, between a low passage infectious bronchitis virus field isolate and a highly embryo adaptedvaccine strain.

The possibility of genomic recombination among different strains of infectious bronchitis virus (IBV) was examined in eve by coinfecting specific pathogen free embryonating chicken eggs with commonly used, embryo-adapted vaccine strains of IBV (Arkansas, Massachusetts, and Connecticut), and a Delaware-072-like field virus isolated from a layer farm in Minnesota. Recombination was observed between th e Massachusetts and the Delaware-072-like strains of the virus. The recombination event was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) using a combination of specific primers designed to flank a known recombination hot spot of the viral genomic sequence that codes for the S1 subunit of the spike envelope protein. The use of these primers allowed the detection of viruses that have undergone recombination around this hot spot. Cloning and sequencing of the RT-PCR product obtained was performed to confirm these results.

Chicken anemia virus and infectious bursal disease virus interfere with transcription of chicken IFN-alpha and IFN-gamma mRNA.

Chicken anemia virus (CAV) and infectious bursal disease virus (IBDV) are the two most important viruses that cause immunosuppression in commercial chickens. Because inapparent, subclinical infections by these viruses cause immunosuppression, there is need for assessment of the immune status of chickens. Interference with induction of transcription for chicken interferon-alpha (ChIFN-alpha) and ChIFN-gamma was noted after subclinical infections with either CAV or IBDV. Because the immunosuppressive viruses of chickens may interfere with transcription for ChIFN-alpha and ChIFN-gamma, we propose using this interference to assess the immune status of chickens.