Protection Efficacy of Recombinant HVT-ND-LT and the Live Attenuated Tissue Culture Origin Vaccines Against Infectious Laryngotracheitis Virus When Administered Individually or in Combination.

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses to the poultry industry. Control of the disease is achieved by vaccination and implementation of biosecurity measures. The use of bivalent and trivalent recombinant herpesvirus of turkey (rHVT) vaccines expressing infectious laryngotracheitis virus (ILTV) genes has increased worldwide. In the United States, vaccination programs of long-lived birds (broiler breeders and commercial layers) against ILT include immunizations with either HVT recombinant vector vaccines, in ovo or at hatch, or live attenuated vaccines administered via drinking water (chicken embryo origin [CEO]) or eye drop (tissue culture origin [TCO]). The efficacy of bivalent rHVT-LT at hatch followed by drinking water or eye-drop CEO vaccination has been shown to provide more robust protection than rHVT-LT alone. The objective of this study was to evaluate the protection efficacy of a commercial trivalent rHVT-ND-LT when administered at 1 day of age followed by TCO vaccination via eye drop at 10 wk of age. Groups vaccinated with only rHVT-ND-LT or TCO, the combination of rHVT-ND-LT + TCO, and one nonvaccinated group of chickens were challenged with a virulent ILTV strain at 15 wk of age. After challenge, mortalities were prevented only in the group of chickens vaccinated with the rHVT-ND-LT + TCO. Clinical signs of the disease and challenge virus replication in the trachea were significantly reduced for both the rHVT-ND-LT + TCO- and TCO-vaccinated groups of chickens. To assess challenge virus transmission, contact-naive chickens were introduced to all vaccinated groups immediately after challenge. At 8 days postintroduction, infection of contact-naive chickens was evidenced in those introduced to the rHVT-ND-LT and TCO group but prevented in the rHVT-ND-LT + TCO group. Overall, these results indicated that compared to rHVT-ND-LT or TCO when administered alone, the rHVT-ND-LT + TCO vaccination strategy improved protection against disease and reduced shedding of the challenge virus.

Eficacia protectora de las vacunas recombinantes HVT-ND-LT y las vacunas con virus vivo atenuado con origen en cultivo de tejidos contra el virus de la laringotraqueítis infecciosa cuando son administradas individualmente o en combinación. La laringotraqueítis infecciosa (ILT) es una enfermedad respiratoria que causa importantes pérdidas económicas a la industria avícola. El control de la enfermedad se logra mediante la vacunación y la implementación de medidas de bioseguridad. El uso de vacunas con el herpesvirus de pavo recombinante (rHVT) bivalentes y trivalentes que expresan genes del virus de la laringotraqueítis infecciosa (ILTV) ha aumentado en todo el mundo. En los Estados Unidos, los programas de vacunación de aves de larga vida (reproductoras pesadas y aves de postura comerciales) contra la laringotraqueítis incluyen inmunizaciones con vacunas con vector HVT recombinante, ya sea in ovo o al día de edad en la planta incubadora, o la aplicación de vacunas vivas atenuadas administradas a través del agua de bebida (origen en embrión de pollo [CEO]) o por gota ocular (origen en cultivo de tejidos [TCO]). Se ha demostrado que la eficacia de la vacuna rHVT-LT bivalente aplicada al día de edad en incubadora, seguida de la inmunización con la vacuna CEO en el agua de bebida o por gota ocular, proporciona una protección más sólida que la aplicación únicamente de la vacuna rHVT-LT. El objetivo de este estudio fue evaluar la eficacia protectora de una vacuna recombinante rHVT-ND-LT trivalente comercial cuando se administró al día de vida seguido de la vacunación con la vacuna TCO mediante gota ocular a las 10 semanas de edad. Los grupos vacunados únicamente con la vacuna rHVT-ND-LT, con TCO, la combinación con rHVT-ND-LT + TCO y un grupo de pollos no vacunados fueron desafiados con una cepa virulenta del virus de la laringotraqueítis a las 15 semanas de edad. Después del desafío, se previno la mortalidad únicamente en el grupo de pollos vacunados con la combinación rHVT-ND-LT + TCO. Los signos clínicos de la enfermedad y la replicación del virus de desafío en la tráquea se redujeron significativamente en los grupos de pollos vacunados con la combinación rHVT-ND-LT + TCO y con la vacuna TCO. Para evaluar la transmisión del virus de desafío, pollos sin contacto previo al virus se introdujeron en todos los grupos vacunados inmediatamente después del desafío. A los 8 días posteriores a la introducción, se evidenció la infección de los pollos sin contacto previo que se introdujeron en los grupos que recibieron únicamente la vacuna rHVT-ND-LT o la vacuna TCO, pero se previno en el grupo con la combinación rHVT-ND-LT + TCO. En general, estos resultados indicaron que, en comparación con las vacunas rHVT-ND-LT o TCO cuando se administran solas, la estrategia de vacunación rHVT-ND-LT + TCO mejoró la protección contra la enfermedad y redujo la diseminación del virus de desafío.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Review of Poultry Recombinant Vector Vaccines.

The control of poultry diseases has relied heavily on the use of many live and inactivated vaccines. However, over the last 30 yr, recombinant DNA technology has been used to generate many novel poultry vaccines. Fowlpox virus and turkey herpesvirus are the two main vectors currently used to construct recombinant vaccines for poultry. With the use of these two vectors, more than 15 recombinant viral vector vaccines against Newcastle disease, infectious laryngotracheitis, infectious bursal disease, avian influenza, and Mycoplasma gallisepticum have been developed and are commercially available. This review focuses on current knowledge about the safety and efficacy of recombinant viral vectored vaccines and the mechanisms by which they facilitate the control of multiple diseases. Additionally, the development of new recombinant vaccines with novel vectors will be briefly discussed.

Estudio Recapitulativo- Revisión acerca de las vacunas con vectores recombinantes para la avicultura. El control de las enfermedades en la avicultura se ha basado en gran medida en el uso de varias vacunas vivas e inactivadas. Sin embargo, durante los últimos 30 años, la tecnología de ADN recombinante se ha utilizado para generar nuevas vacunas avícolas. El virus de la viruela aviar y el virus del herpes del pavo son los dos vectores principales que se utilizan actualmente para construir vacunas recombinantes para la avicultura. Con el uso de estos dos vectores, se han desarrollado y están disponibles comercialmente más de 15 vacunas con vectores virales recombinantes contra la enfermedad de Newcastle, la laringotraqueítis infecciosa, enfermedad infecciosa de la bolsa, influenza aviar y Mycoplasma gallisepticum. Esta revisión se enfoca en el conocimiento actual sobre la seguridad y eficacia de las vacunas con vectores virales recombinantes y los mecanismos por los cuales facilitan el control de múltiples enfermedades. Además, se discutirá brevemente el desarrollo de nuevas vacunas recombinantes con nuevos vectores.

A Cell Line Adapted Infectious Laryngotracheitis Virus Strain (BΔORFC) for in ovo and Hatchery Spray Vaccination Alone or in Combination with a Recombinant HVT-LT Vaccine.

To produce more-stable, live attenuated vaccines for infectious laryngotracheitis virus (ILTV), deletion of genes related to virulence has been extensively pursued. Although its function remains unknown, the open reading frame C (ORF C) is among the genes potentially associated with viral virulence that is nonessential for replication in vitro. Earlier results indicated that the ILT virus with deletion of the ORF C gene (BΔORFC) was suitable and safe for eye drop administration but was not sufficiently attenuated for in ovo administration. The objective of this study was to evaluate the safety and protection efficacy of a cell line-adapted, gene-deleted strain (BΔORFC) of ILTV when administered in ovo and/or spray (SP) by itself, or in combination with the recombinant HVT-LT (rHVT-LT) vaccine. Results indicated that vaccination with the BΔORFC strain, either by itself or in combination with an rHVT-LT vaccine, did not affect hatchability, and only marginal signs of respiratory distress were recorded for groups of chickens that received the BΔORFC strain via SP. The replication and seroconversion induced by the BΔORFC strain after in ovo and SP administration was very limited, whereas the replication of the rHVT-LT vaccine was delayed when combined with the BΔORFC strain in ovo. Compared to rHVT-LT or BΔORFC when administered alone, dual vaccination with rHVT-LT + BΔORFC was more effective in mitigating clinical signs of the disease and reducing challenge virus load in the trachea. To our knowledge, this study provides the first proof of concept that ILTV strains can be sufficiently attenuated for early vaccination in ovo or at hatch; also, this study documented the benefits of using a dual (recombinant and live attenuated) hatchery vaccination strategy for ILTV.

Una cepa del virus de la laringotraqueítis infecciosa adaptada a una línea celular (BΔORFC) para vacunación in ovo y en aerosol en incubadora aplicada por sí sola o en combinación con una vacuna recombinante para laringotraqueítis con el vector HVT. Para producir vacunas vivas atenuadas más estables contra el virus de la laringotraqueítis infecciosa (ILTV), se ha buscado ampliamente la eliminación de genes relacionados con la virulencia. Aunque su función sigue siendo desconocida, el marco de lectura continuo C (ORF C) se encuentra entre los genes potencialmente asociados con la virulencia viral que no es esencial para la replicación in vitro. Resultados anteriores indican que el virus de la laringotraqueítis infecciosa con deleción del gene ORF C (BΔORFC) era adecuado y seguro para la administración ocular, pero no estaba lo suficientemente atenuado para su administración in ovo. El objetivo de este estudio fue evaluar la seguridad y la eficacia de la protección de una cepa del virus de la laringotraqueítis infecciosa con deleción genética y adaptada a una línea celular (BΔORFC) cuando se administra in ovo y/o en aerosol por sí sola, o en combinación con una vacuna recombinante con el vector HVT (vacuna rHVT-LT). Los resultados indicaron que la vacunación con la cepa BΔORFC, ya sea sola o en combinación con la vacuna rHVT-LT, no afectó la incubabilidad, y solo se registraron signos marginales de dificultad respiratoria para los grupos de pollos que recibieron la cepa BΔORFC por aspersión. La replicación y seroconversión inducida por la cepa BΔORFC después de la administración in ovo y por aspersión fue muy limitada, mientras que la replicación de la vacuna rHVT-LT se retrasó cuando se combinó con la cepa BΔORFC in ovo. En comparación con las vacunas rHVT-LT o BΔORFC administradas por sí solas, la vacunación dual con rHVT-LT + BΔORFC fue más eficaz para mitigar los signos clínicos de la enfermedad y reducir la carga del virus de desafío en la tráquea. Hasta donde se conoce, este estudio proporciona la primera prueba del concepto de que las cepas del virus de la laringotraqueítis infecciosa pueden atenuarse lo suficiente para la vacunación temprana in ovo o en la incubadora. Además, este estudio documentó los beneficios de utilizar una estrategia de vacunación de incubadora dual (vacuna recombinante y viva atenuada) para la laringotraqueítis infecciosa.

Assessing the infiltration of immune cells in the upper trachea mucosa after infectious laryngotracheitis virus (ILTV) vaccination and challenge.

The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8ß+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8ß+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8ß+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

Activation of Cytotoxic Lymphocytes and Presence of Regulatory T Cells in the Trachea of Non-Vaccinated and Vaccinated Chickens as a Recall to an Infectious Laryngotracheitis Virus (ILTV) Challenge.

While the protective efficacy of the infectious laryngotracheitis virus (ILTV) vaccines is well established, little is known about which components of the immune response are associated with effective resistance and vaccine protection. Early studies have pointed to the importance of the T cell-mediated immune responses. This study aimed to evaluate the activation of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and to quantify the presence of regulatory T cells (Tregs) in the larynx-trachea of chickens vaccinated with chicken embryo origin (CEO), tissue culture origin (TCO) and recombinant Herpesvirus of Turkey-laryngotracheitis (rHVT-LT) vaccines after challenge. Our results indicated that CEO vaccine protection was characterized by early CTLs and activated CTLs enhanced responses. TCO and rHVT-LT protection were associated with a moderate increase in resting and activated CTLs followed by an enhanced NK cell response. Tregs increase was only detected in the non-vaccinated challenged group, probably to support healing of the severe trachea epithelial damage. Taken together, our results revealed main differences in the cellular immune responses elicited by CEO, TCO, and rHVT-LT vaccination in the upper respiratory tract after challenge, and that activated CTLs rather than NK cells play a main role in vaccine protection.

The Requirement of Glycoprotein C for Interindividual Spread Is Functionally Conserved within the Alphaherpesvirus Genus (Mardivirus), but Not the Host (Gallid).

Marek's disease (MD) in chickens is caused by Gallid alphaherpesvirus 2, better known as MD herpesvirus (MDV). Current vaccines do not block interindividual spread from chicken-to-chicken, therefore, understanding MDV interindividual spread provides important information for the development of potential therapies to protect against MD, while also providing a natural host to study herpesvirus dissemination. It has long been thought that glycoprotein C (gC) of alphaherpesviruses evolved with their host based on their ability to bind and inhibit complement in a species-selective manner. Here, we tested the functional importance of gC during interindividual spread and host specificity using the natural model system of MDV in chickens through classical compensation experiments. By exchanging MDV gC with another chicken alphaherpesvirus (Gallid alphaherpesvirus 1 or infectious laryngotracheitis virus; ILTV) gC, we determined that ILTV gC could not compensate for MDV gC during interindividual spread. In contrast, exchanging turkey herpesvirus (Meleagrid alphaherpesvirus 1 or HVT) gC could compensate for chicken MDV gC. Both ILTV and MDV are Gallid alphaherpesviruses; however, ILTV is a member of the Iltovirus genus, while MDV is classified as a Mardivirus along with HVT. These results suggest that gC is functionally conserved based on the virus genera (Mardivirus vs. Iltovirus) and not the host (Gallid vs. Meleagrid).

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major.

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

Evaluation of vaccination against infectious laryngotracheitis (ILT) with recombinant herpesvirus of turkey (rHVT-LT) and chicken embryo origin (CEO) vaccines applied alone or in combination.

The chicken embryo origin (CEO) infectious laryngotracheitis (ILT) live attenuated vaccines, although capable of protecting against disease and reducing challenge virus replication, can regain virulence. Recombinant ILT vaccines do not regain virulence but are partially successful at blocking challenge virus replication. The objective of this study was to evaluate the effect of rHVT-LT vaccination on CEO replication and how this vaccination strategy enhances protection and limits challenge virus transmission to naïve contact chickens. The rHVT-LT vaccine was administered at 1 day of age subcutaneously and the CEO vaccine was administered at 6 weeks of age via eye-drop or drinking water. CEO vaccine replication post vaccination, challenge virus replication and transmission post challenge were evaluated. After vaccination, only the group that received the CEO via eye-drop developed transient conjunctivitis. A significant decrease in CEO replication was detected for the rHVT-LT + CEO groups as compared to groups that received CEO alone. After challenge, reduction in clinical signs and challenge virus replication were observed in all vaccinated groups. However, among the vaccinated groups, the rHVT-LT group presented higher clinical signs and challenge virus replication. Transmission of the challenge virus to naïve contact chickens was only observed in the rHVT-LT vaccinated group of chickens. Overall, this study found that priming with rHVT-LT reduced CEO virus replication and the addition of a CEO vaccination provided a more robust protection than rHVT alone. Therefore, rHVT-LT + CEO vaccination strategy constitutes an alternative approach to gain better control of the disease.

Immune Responses in the Eye-Associated Lymphoid Tissues of Chickens after Ocular Inoculation with Vaccine and Virulent Strains of the Respiratory Infectious Laryngotracheitis Virus (ILTV).

Infectious laryngotracheitis (ILT) is an acute respiratory disease of poultry caused by infectious laryngotracheitis virus (ILTV). Control of the disease with live attenuated vaccines administered via eye drop build upon immune responses generated by the eye-associated lymphoid tissues. The aim of this study was to assess cytokine and lymphocyte changes in the conjunctiva-associated lymphoid tissues (CALT) and Harderian gland (HG) stimulated by the ocular inoculation of the ILTV chicken embryo origin (CEO) vaccine strain and virulent strain 63140. This study offers strong evidence to support the roles that the CALT and HG play in the development of protective ILTV immune responses. It supports the premise that ILTV-mediated immunomodulation favors the B cell response over those of T cells. Further, it provides evidence that expansions of CD8α+ cells, with the concomitant expression of the Granzyme A gene, are key to reducing viral genomes in the CALT and halting ILTV cytolytic replication in the conjunctiva. Ultimately, this study revealed that the early upregulation of interleukin (IL)-12p40 and Interferon (IFN)-γ cytokine genes, which shape the antigen-specific cell-mediated immune responses, retarded the decline of virus replication, and enhanced the development of lesions in the conjunctiva epithelium.

Evaluation of Protective Efficacy When Combining Turkey Herpesvirus-Vector Vaccines.

Turkey herpesvirus (HVT) is widely used as a vaccine against Marek's disease in chickens and recently as a vector for foreign genes from infectious bursal disease virus, Newcastle disease (ND) virus, infectious laryngotracheitis (ILT) virus, and avian influenza virus. Advantages of HVT-vector vaccines are that the vaccines do not contain live respiratory viruses or live infectious bursal disease virus able to replicate and cause disease or embryo mortality, they can be administered at hatch or in ovo, and they are relatively insensitive to interference from maternally derived antibodies. As producers have tried to combine HVT-vector vaccines to protect against additional diseases, reports have indicated that applying two vectored vaccines using the same HVT vector is reported to reduce the efficacy of one or both vaccines. To confirm this interference, we evaluated commercial vaccines from multiple companies, including products with inserts designed to protect against ND, infectious ILT, and infectious bursal disease (IBD). Using a standard dosage, we found that the ILT product was most severely affected by the addition of other vaccines, as demonstrated by a significant increase in clinical signs, significant decrease in weight gain, and increase in quantity of challenge virus observed from tracheal swabs collected from Days 3-5 postchallenge. The ND and IBD products were also affected by the addition of other vaccines, although in most cases differences compared to vaccination with the vector alone were not statistically significant. This study demonstrates the importance of following manufacturer guidelines and the need for validating alternative strategies to benefit from the high level of protection offered by vector vaccines.

Evaluación de la eficacia de la protección cuando se combinan vacunas recombinantes con base en el virus herpes del pavo como vector. El virus herpes de los pavos (HVT) se usa ampliamente como una vacuna contra la enfermedad de Marek en pollos y recientemente como un vector para genes externos como del virus de la enfermedad infecciosa de la bolsa, del virus de la enfermedad de Newcastle (ND), del virus de la laringotraqueítis infecciosa (ILT) y del virus de la influenza aviar. Las ventajas de las vacunas con vector de HVT consisten en que las vacunas no contienen virus vivos respiratorios o virus de la enfermedad infecciosa de la bolsa, no son capaces de replicarse y causar enfermedad o mortalidad embrionaria, pueden administrarse en el momento de la eclosión o in ovo y son relativamente insensibles a la interferencia de anticuerpos de origen materno. A medida que los productores han intentado combinar las vacunas con el vector HVT para inducir protección contra enfermedades adicionales, los informes han indicado que la aplicación de dos vacunas vectorizadas utilizando el mismo vector HVT reduce la eficacia de una o de ambas vacunas. Para confirmar esta interferencia, se evaluaron las vacunas comerciales de múltiples compañías, incluidos los productos con inserciones diseñadas para proteger contra la enfermedad de Newcastle, la laringotraqueítis infecciosa aviar y contra la enfermedad infecciosa de la bolsa. Utilizando una dosis estándar, se encontró que el producto para la laringotraqueítis infecciosa se vio más afectado por la adición de otras vacunas, como lo demuestra un aumento significativo en los signos clínicos, una disminución significativa en el aumento de peso y un aumento en la cantidad de virus de desafío observados en los hisopos traqueales recolectados de tres a cinco días después del desafío. Los productos para la enfermedad de Newcastle y para la enfermedad de Gumboro también se vieron afectados por la adición de otras vacunas, aunque en la mayoría de los casos las diferencias en comparación con la vacunación únicamente con el vector no fueron estadísticamente significativas. Este estudio demuestra la importancia de seguir las pautas del fabricante y la necesidad de validar estrategias alternativas para beneficiarse del alto nivel de protección ofrecido por las vacunas con vectores.

Commercial Vaccines and Vaccination Strategies Against Infectious Laryngotracheitis: What We Have Learned and Knowledge Gaps That Remain.

Infectious laryngotracheitis (ILT) is an upper respiratory disease of chickens, pheasants, and peafowl caused by the alphaherpesvirus Gallid alpha herpesvirus 1 (GaHV-1), commonly known as infectious laryngotracheitis virus. ILT is an acute respiratory disease characterized by clinical signs of conjunctivitis, nasal discharge, dyspnea, and lethargy. In severe forms of the disease, hemorrhagic tracheitis together with gasping, coughing, and expectoration of bloody mucus are common. The morbidity and mortality rates of the disease vary depending on the virulence of the strain circulating, the level of virus circulating in the field, and the presence of other respiratory infections. Since the identification of the disease in the 1920s, ILT continues to affect the poultry industry negatively across the globe. The disease is primarily controlled by a combination of biosecurity and vaccination. The first commercial vaccines, introduced in the late 1950s and early 1960s, were the chicken embryo origin live attenuated vaccines. The tissue culture origin vaccine was introduced in late 1970s. Recombinant viral vector ILT vaccines were first introduced in the United States in the 2000s, and now they are being used worldwide, alone or in combination with live attenuated vaccines. This review article provides a synopsis of what we have learned about vaccines and vaccination strategies used around the world and addresses knowledge gaps about the virus and host interactions that remain unknown.

Estudio recapitulativo. Vacunas comerciales y estrategias de vacunación contra la laringotraqueitis infecciosa: lo que se ha aprendido y los vacíos de conocimiento que persisten La laringotraqueítis infecciosa (ILT, por sus siglas en inglés) es una enfermedad del tracto respiratorio superior de pollos, faisanes y pavos reales, causada por el alfaherpesvirus herpesvirus del pollo 1 (GaHV-1), conocido comúnmente como virus de la laringotraqueitis infecciosa. La laringotraqueitis infecciosa es una enfermedad respiratoria aguda caracterizada por signos clínicos de conjuntivitis, secreción nasal, disnea y letargo. En las formas severas de la enfermedad, son comunes la traqueítis hemorrágica junto con jadeo, tos y expectoración de moco con sangre. Las tasas de morbilidad y mortalidad de la enfermedad varían según la virulencia de la cepa que está circulando, el nivel de virus que circula en el campo y la presencia de otras infecciones respiratorias. Desde la identificación de la enfermedad en la década de los 1920's, la laringotraqueitis infecciosa continúa afectando negativamente a la industria avícola en todo el mundo. La enfermedad se controla principalmente mediante una combinación de bioseguridad y vacunación. Las primeras vacunas comerciales introducidas a fines de los años cincuenta y principios de los sesenta, fueron las vacunas atenuadas vivas con origen en embrión de pollo. La vacuna con origen en cultivo de células se introdujo a fines de los años 70 en los Estados Unidos. Las vacunas contra la laringotraqueitis infecciosa desarrolladas con vectores virales recombinantes se introdujeron por primera vez en los Estados Unidos en la década de 2000's y ahora se están utilizando en todo el mundo, solas o en combinación con vacunas atenuadas vivas. Este artículo recapitulativo proporciona una sinopsis de lo que se ha aprendido sobre las vacunas contra la laringotraqueitis infecciosa, las estrategias de vacunación utilizadas en todo el mundo y aborda los vacíos en el conocimiento sobre el virus y las interacciones con el huésped que siguen siendo desconocidas.

Protection Efficacy of a Recombinant Herpesvirus of Turkey Vaccine Against Infectious Laryngotracheitis Virus Administered In Ovo to Broilers at Three Standardized Doses.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. The disease is caused by Gallid alpha herpesvirus-1 (GaHV-1), commonly known as the infectious laryngotracheitis virus (ILTV). Vaccination remains necessary for the control of the disease. Due to the inherent virulence of live attenuated vaccines, in particular that of the chicken embryo origin (CEO) vaccines, the use of ILT viral vector recombinant vaccines has significantly expanded worldwide as a safer vaccination strategy. However, the protective efficacy of recombinant ILT vaccines can be compromised by the use of fractional doses and improper handling and administration of the vaccine. The objective of this study was twofold: 1) to evaluate the protection efficacy induced by a commercial recombinant HVT-LT (rHVT-LT) vaccine when administered in ovo to broilers at three standardized doses (6000 plaque-forming units [PFU], 3000 PFU, and 1000 PFU), and 2) to assess the potential of rHVT-LT-vaccinated chickens to spread virus to contact chickens after challenge. Independently of the vaccine dose, vaccinated chickens showed reduction in clinical signs, maintained body weight gain after challenge, and lessened the challenge virus replication in the trachea at a rate of 52%-65%. However, in spite of this reduction, transmission of challenge virus from rHVT-LT-vaccinated (6000/Ch, 3000/Ch) to contact-naive chickens was evident. This study is the first to support that rHVT-LT vaccination did not prevent spread of challenge virus to contact birds.

Eficacia de la protección de una vacuna con un herpesvirus de los pavos (HVT) recombinante contra el virus de la laringotraqueitis infecciosa (ILTV) administrada in ovo en pollos de engorde en tres dosis estandarizadas. La laringotraqueítis infecciosa (ILT, por sus siglas en inglés) es una enfermedad respiratoria altamente contagiosa de los pollos que produce importantes pérdidas económicas para la industria avícola. La enfermedad es causada por el alfa herpesvirus-1 del pollo (GaHV-1), conocido comúnmente como el virus de la laringotraqueitis infecciosa (ILTV). La vacunación sigue siendo necesaria para el control de la enfermedad. Debido a la virulencia inherente de las vacunas atenuadas vivas, en particular la de las vacunas con origen embrion de pollo (CEO), el uso de vacunas contra la laringotraqueítis con vectores virales recombinantes se ha extendido significativamente en todo el mundo como una estrategia de vacunación más segura. Sin embargo, la eficacia protectora de las vacunas recombinantes contra la laringotraqueítis puede verse comprometida por el uso de dosis fraccionarias y por el manejo y administración inadecuados de la vacuna. El objetivo de este estudio fue doble: 1) evaluar la eficacia de la protección inducida por una vacuna comercial recombinante HVT-LT (rHVT-LT) cuando se administró in ovo en pollos de engorde en tres dosis estandarizadas (6000 unidades formadoras de placa [PFU], 3000 PFU y 1000 PFU), y 2) para evaluar el potencial de los pollos vacunados con rHVT-LT para propagar el virus a los pollos en contacto después del desafío. Independientemente de la dosis de la vacuna, los pollos vacunados mostraron una reducción en los signos clínicos, mantuvieron el aumento de peso corporal después del desafío y disminuyeron la replicación del virus de desafío en la tráquea a una tasa de 52% -65%. Sin embargo, a pesar de esta reducción, la transmisión del virus de desafío de los pollos vacunados con rHVT-LT con 6000 unidades formadoras de placa y desafiados o con 3000 unidades formadoras de placa y también desafiados a los pollos susceptibles en contacto fue evidente. Este estudio es el primero en demostrar que la vacunación con rHVT-LT no impidió la propagación del virus de desafío a las aves en contacto.

MinION sequencing to genotype US strains of infectious laryngotracheitis virus.

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing. RESEARCH HIGHLIGHTS A multi-allelic assay identified nine ILTV genotypes circulating in the US Single-allele genotyping is congruent with whole genome phylogenetic partitioning US ILTV strains can be grouped into five genotypes using the single-allele assay The single-allele assay can be done using MinION sequencing of barcoded amplicons.

Effect of Pullet Vaccination on Development and Longevity of Immunity.

Avian respiratory disease causes significant economic losses in commercial poultry. Because of the need to protect long-lived poultry against respiratory tract pathogens from an early age, vaccination programs for pullets typically involve serial administration of a variety of vaccines, including infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and infectious laryngotracheitis virus (ILTV). Often the interval between vaccinations is only a matter of weeks, yet it is unknown whether the development of immunity and protection against challenge when vaccines are given in short succession occurs in these birds, something known as viral interference. Our objective was to determine whether serially administered, live attenuated vaccines against IBV, NDV, and ILTV influence the development and longevity of immunity and protection against challenge in long-lived birds. Based on a typical pullet vaccination program, specific-pathogen-free white leghorns were administered multiple live attenuated vaccines against IBV, NDV, and ILTV until 16 weeks of age (WOA), after which certain groups were challenged with IBV, NDV, or ILTV at 20, 24, 28, 32, and 36 WOA. Five days post-challenge, viral load, clinical signs, ciliostasis, tracheal histopathology, and antibody titers in serum and tears were evaluated. We demonstrate that pullets serially administered live attenuated vaccines against IBV, NDV, and ILTV were protected against homologous challenge with IBV, NDV, or ILTV for at least 36 weeks, and conclude that the interval between vaccinations used in this study (at least 2 weeks) did not interfere with protection. This information is important because it shows that a typical pullet vaccination program consisting of serially administered live attenuated vaccines against multiple respiratory pathogens can result in the development of protective immunity against each disease agent.

Long-term protection against a virulent field isolate of infectious laryngotracheitis virus induced by inactivated, recombinant, and modified live virus vaccines in commercial layers.

Infectious laryngotracheitis (ILT) is an acute respiratory disease of chickens controlled through vaccination with live-modified attenuated vaccines, the chicken embryo origin (CEO) vaccines and the tissue-culture origin (TCO) vaccines. Recently, novel recombinant vaccines have been developed using herpesvirus of turkey (HVT) and fowl pox virus (FPV) as vectors to express ILTV immunogens for protection against ILT. The objective of this study was to assess the protection efficacy against ILT induced by recombinants, live-modified attenuated, and inactivated virus vaccines when administered alone or in combination. Commercial layer pullets were vaccinated with one or more vaccines and challenged at 35 (35 WCH) or 74 weeks of age (74 WCH). Protection was assessed by scoring clinical signs; and by determining the challenge viral load in the trachea at five days post-challenge. The FPV-LT vaccinated birds were not protected when challenged at 35 weeks; the HVT-LT and TCO vaccines in combination provided protection similar to that observed in chickens vaccinated with either HVT-LT or TCO vaccines when challenged at 35 weeks, whereas protection induced by vaccination with HVT-LT followed by TCO was superior in the 74 WCH group compared with the 35 WCH group. Birds given the inactivated ILT vaccine had fewer clinical signs and/or lower viral replication at 74 WCH when combined with TCO or HVT-LT, but not when given alone. Finally, the CEO-vaccinated birds had top protection as indicated by reduction of clinical signs and viral replication when challenged at 35 weeks (74 weeks not done). These results suggest that certain vaccine combinations may be successful to produce long-term protection up to 74 weeks of age against ILT.

Single Nucleotide Polymorphism Genotyping Analysis Shows That Vaccination Can Limit the Number and Diversity of Recombinant Progeny of Infectious Laryngotracheitis Viruses from the United States.

Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) causes mild to severe respiratory disease in poultry worldwide. Recombination in this virus under natural (field) conditions was first described in 2012 and more recently has been studied under laboratory conditions. Previous studies have revealed that natural recombination is widespread in ILTV and have also demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In the United States, natural ILTV recombination has also been detected, but not as frequently as in Australia. To better understand recombination in ILTV strains originating from the United States, we developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two virulent U.S. field strains of ILTV (63140 and 1874c5) under experimental in vivo conditions. We also tested the capacity of the Innovax-ILT vaccine (a recombinant vaccine using herpesvirus of turkeys as a vector) and the Trachivax vaccine (a conventionally attenuated chicken embryo origin vaccine) to reduce recombination. The Trachivax vaccine prevented ILTV replication, and therefore recombination, in the trachea after challenge. The Innovax-ILT vaccine allowed the challenge viruses to replicate and to recombine, but at a significantly lower rate than in an unvaccinated group of birds. Our results demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination between these ILTV strains and also show that vaccination can limit the number and diversity of recombinant progeny viruses.IMPORTANCE Recombination allows alphaherpesviruses to evolve over time and become more virulent. Historically, characterization of viral vaccines in poultry have mainly focused on limiting clinical disease, rather than limiting virus replication, but such approaches can allow field viruses to persist and evolve in vaccinated populations. In this study, we vaccinated chickens with Gallid alphaherpesvirus 1 vaccines that are commercially available in the United States and then performed coinoculations with two field strains of virus to measure the ability of the vaccines to prevent field strains from replicating and recombining. We found that vaccination reduced viral replication, recombination, and diversity compared to those in unvaccinated chickens, although the extent to which this occurred differed between vaccines. We suggest that characterization of vaccines could include studies to examine the ability of vaccines to reduce viral recombination in order to limit the rise of new virulent field strains due to recombination, especially for those vaccines that are known not to prevent viral replication following challenge.

Cytokine gene transcription in the trachea, Harderian gland, and trigeminal ganglia of chickens inoculated with virulent infectious laryngotracheitis virus (ILTV) strain.

The objective of this study was to determine how cytokine transcription profiles correlate with patterns of infectious laryngotracheitis virus (ILTV) replication in the trachea, Harderian gland, and trigeminal ganglia during the early and late stages of infection after intratracheal inoculation. Viral genomes and transcripts were detected in the trachea and Harderian gland but not in trigeminal ganglia. The onset of viral replication in the trachea was detected at day one post-infection and peaked by day three post-infection. The peak of pro-inflammatory (CXCLi2, IL-1ß, IFN-γ) and anti-inflammatory (IL-13, IL-10) cytokine gene transcription, 5 days post-infection, coincided with the increased recruitment of inflammatory cells, extensive tissue damage, and limiting of virus replication in the trachea. In contrast, transcription of the IFN-ß gene in the trachea remained unaffected suggesting that ILTV infection blocks type I interferon responses. In the Harderian gland, the most evident transcription change was the early and transient upregulation of the IFN-γ gene at 1 day post-infection, which suggests that the Harderian gland is prepared to rapidly respond to ILTV infection. Overall, results from this study suggest that regulation of Th1 effector cells and macrophage activity by Th1/2 cytokines was pertinent to maintain a balanced immune response capable of providing an adequate Th1-mediated protective immunity, while sustaining some immune homeostasis in preparation for the regeneration of the tracheal mucosa.

Attenuation and Protection Efficacy of a Recombinant Infectious Laryngotracheitis Virus (ILTV) Depleted of Open Reading Frame C (ΔORFC) when Delivered in ovo.

In an effort to produce more stable vaccines for infectious laryngotracheitis virus (ILTV), recombinant strains with deletion of genes associated with virulence have been evaluated for attenuation and protection efficacy. Among viral genes associated with virulence, a cluster of five open reading frames (ORFs; A through E) have been identified. An attenuated ILTV recombinant strain with deletion of the ORF C gene induced protection comparable to that elicited by the tissue culture origin (TCO) vaccine when administered via eyedrop. The objective of this study was to evaluate the attenuation and protection efficacy of the ΔORF C strain when delivered in ovo to maternal antibody negative (MAb-) and maternal antibody positive (MAb+) embryos. In ovo delivery of the ΔORF C strain did not affected hatchability or body weight gain, while virus transmission to contact chickens was minor. Nevertheless, nine of ninety (10%) of MAb- chickens vaccinated with the ΔORF C strain showed marked dyspnea, and upon postmortem examination bloody mucoid plugs and high viral genome load were detected in their tracheas. Moreover, the ΔORF C strain induced satisfactory protection in MAb- chickens, but marginal protection in MAb+ chickens after challenge. The reduced protection observed for MAb+ groups of chickens was likely caused by the interference of maternally derived antibodies. This report presents the use of a genetically attenuated ILTV strain delivered in ovo as a potential new approach in the control of ILTV.

Seeding of the mucosal leukocytes in the HALT and trachea of White Leghorn chickens.

Characterising the immune cells of the head-associated lymphoid tissues (HALT) and trachea during maturation in young birds is critical to understanding the immunological responses to avian respiratory diseases and vaccines. Selected mucosal leukocytes of the conjunctiva-associated lymphoid tissue (CALT), Harderian gland (HG), nasal-associated lymphoid tissue (NALT) and trachea from 4-, 6-, 8-, and 10-week-old chickens were enumerated and phenotyped. HG, NALT, and trachea cellularity increased as the birds aged with cell viability varying by tissue. The results showed that the T cell subset numbers, but not B cell numbers, increased in the mucosal tissues of chickens during aging.