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.

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.

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.

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.