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.

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.

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.

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.

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.

Evaluation of the transcriptional status of host cytokines and viral genes in the trachea of vaccinated and non-vaccinated chickens after challenge with the infectious laryngotracheitis virus.

Infectious laryngotracheitis is a highly contagious disease of chickens responsible for significant economic losses for the poultry industry worldwide. The disease is caused by Gallid herpesvirus-1 (GaHV-1) commonly known as the infectious laryngotracheitis virus. Although characterized by their potential to regain virulence, chicken embryo origin (CEO) vaccines are the most effective vaccines against laryngotracheitis as they significantly reduce the replication of challenge virus in the trachea and conjunctiva. Knowledge on the nature of protective immunity elicited by CEO vaccines is very limited. Therefore, elucidating the origin of the immune responses elicited by CEO vaccination is relevant for development of safer control strategies. In this study the transcription levels of key host immune genes (IFN-γ, IFN-ß, IL-1ß, IL-6, IL-8, IL-18) and viral genes (ICP4, ICP27, UL46, UL49), as well as viral genome loads in trachea were quantified at 6 and 12 hours post-challenge of CEO vaccinated and non-vaccinated chickens. Immediately after challenge a significant increase in IFN-γ gene expression was followed by a significant reduction in viral replication. In contrast to the rapid induction of IFN-γ, expression of the pro-inflammatory cytokines (IL-1ß, IL-6, IL-8) and type I IFN ß was either slightly reduced or remained at basal levels. These suggest that the former cytokines may not play important roles during immediate early responses induced by ILTV challenge in either vaccinated or non-vaccinated chickens. Overall, these results suggest that the rapid expression of IFN-γ may induce pathways of antiviral responses necessary for blocking early virus replication.

Infection of Broilers with Two Virulent Strains of Infectious Laryngotracheitis Virus: Criteria for Evaluation of Experimental Infections.

Infectious laryngotracheitis (ILT) is a highly contagious disease of chickens and is responsible for significant economic losses in the poultry industry worldwide; it is caused by Gallid herpesvirus-1 (GaHV-1), commonly known as infectious laryngotracheitis virus (ILTV). Experimental evaluation of ILTV strains is fundamental to identify changes in virulence that can contribute to the severity and spread of outbreaks and consequently influence the efficacy of vaccination. Several criteria had been utilized to determine the degree of virulence associated with ILTV strains. The objectives of this study were to compare the levels of virulence of the standard United States Department of Agriculture (USDA) challenge strain with a contemporary outbreak-related strain (63140) and to evaluate the efficacy of individual criteria to identify changes in virulence. Broilers were inoculated with increasing infectious doses of each strain. The criteria utilized to evaluate virulence were clinical signs of the disease, mortality, microscopic tracheal lesions, trachea genome viral loads, and antibody titers. Clinical signs scores were a useful parameter to define the peak of clinical disease but did not reveal differences in virulence between strains. Similarly, trachea microscopic lesion scores or levels of serum antibody titers were parameters that did not reveal obvious differences in virulence between strains. However, mortalities and increased viral genome loads in trachea of chickens inoculated with lower (log10 1 to 2) infectious doses clearly differentiated 63140 as a more-virulent ILTV strain. This study provides the framework to compare the virulence level of emerging ILTV isolates to the now-characterized USDA and 63140 strains.

In ovo vaccination of commercial broilers with a glycoprotein J gene-deleted strain of infectious laryngotracheitis virus.

Conventional live attenuated vaccines have been used as the main tool worldwide for the control of infectious laryngotracheitis. However, their suboptimal attenuation combined with poor mass administration practices allowed chicken embryo origin vaccine-derived isolates to circulate in the field, regain virulence, and be the cause of continuous outbreaks of the disease. Previous studies indicated that stable attenuation of infectious laryngotracheitis virus (ILTV) can be achieved by the deletion of individual viral genes that are not essential for viral replication in vitro. One of these genes is the glycoprotein J (gJ) gene. Its deletion provided significant attenuation to virulent ILTV strains from Europe and the United States. The objective of this study was to construct an attenuated gJ-deleted ILTV strain and evaluate its safety and efficacy for in ovo (IO) administration of commercial broilers. A novel gJ-deleted virus (N(delta)gJ) was constructed, and a 10(3) median tissue culture infective dose administered at 18 days of embryo age was considered safe because it did not affect hatchability or survivability of chickens during the first week posthatch. Broilers vaccinated IO and IO + eye drop at 14 days of age presented a significant reduction in clinical signs and reduction of virus loads after challenge, as compared with the nonvaccinated challenged group of chickens. Therefore, this study presents initial proof that the N(delta)gJ strain is a potential ILTV live-attenuated vaccine candidate suitable for IO vaccination of commercial broilers.

Do shade-grown coffee plantations pose a disease risk for wild birds?

Shade-grown coffee plantations are often promoted as a conservation strategy for wild birds. However, these agro-ecosystems are actively managed for food production, which may alter bird behaviors or interactions that could change bird health, compared to natural forest. To examine whether there is a difference between the health parameters of wild birds inhabiting shade-grown coffee plantations and natural forest, we evaluated birds in Costa Rica for (1) their general body condition, (2) antibodies to pathogens, (paramyxovirus and Mycoplasma spp.), and (3) the prevalence and diversity of endo-, ecto-, and hemoparasites. We measured exposure to Mycoplasma spp. and paramyxovirus because these are pathogens that could have been introduced with domestic poultry, one mechanism by which these landscapes could be detrimental to wild birds. We captured 1,561 birds representing 75 species. Although seasonal factors influenced body condition, we did not find bird general body condition to be different. A total of 556 birds of 31 species were tested for antibodies against paramyxovirus-1. Of these, five birds tested positive, four of which were from shade coffee. Out of 461 other tests for pathogens (for antibodies and nucleotide detection), none were positive. Pterolichus obtusus, the feather mite of chickens, was found on 15 birds representing two species and all were from shade-coffee plantations. Larvated eggs of Syngamus trachea, a nematode typically associated with chickens, were found in four birds captured in shade coffee and one captured in forest. For hemoparasites, a total of 1,121 blood smears from 68 bird species were examined, and only one species showed a higher prevalence of infection in shade coffee. Our results indicate that shade-coffee plantations do not pose a significant health risk to forest birds, but at least two groups of pathogens may deserve further attention: Haemoproteus spp. and the diversity and identity of endoparasites.

Genomic sequence analysis of the United States infectious laryngotracheitis vaccine strains chicken embryo origin (CEO) and tissue culture origin (TCO).

The genomic sequences of low and high passages of the United States infectious laryngotracheitis (ILT) vaccine strains CEO and TCO were determined using hybrid next generation sequencing in order to define genomic changes associated with attenuation and reversion to virulence. Phylogenetic analysis of available full genomes grouped strains into three major clades: TCO, CEO, and Australian. Comparative genomics revealed that TCO attenuation is likely the result of an ORF C truncation. Genes involved in attenuation are generally clade-specific, however four genes ORF C, UL27, UL28 and UL39 commonly contained various mutations across the CEO and TCO lineages. The Thr644 mutation in the UL27 gene encoding glycoprotein B was identified in all virulent US strains. The US10 gene was identified as a potential virulence factor for the TCO revertant 81658. The UL41 gene was responsible for the robust gain in virulence of CEO-Fowl Laryngotracheitis(®) after 20 passages in chickens.

Optimization of a duplex real-time PCR method for relative quantitation of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a highly contagious respiratory disease of chickens controlled by biosecurity and vaccination with live attenuated or recombinant vaccines. Infectious laryngotracheitis virus (ILTV) infections are characterized by a peak of viral replication in the trachea followed by a steady decrease in replication that results in the establishment of latency. Estimation of viral load is an important tool to determine the stage of ILTV infection. Here, a multiplex real-time PCR was optimized for the quantification of ILTV genomes. Quantification of viral genomes was based on the amplification of the ILTV UL44 gene, and sample variability was normalized using the chicken (Gallusgallus domesticus) alpha2-collagen gene as an endogenous control in a duplex reaction.

Protection induced by commercially available live-attenuated and recombinant viral vector vaccines against infectious laryngotracheitis virus in broiler chickens.

Viral vector vaccines using fowl poxvirus (FPV) and herpesvirus of turkey (HVT) as vectors and carrying infectious laryngotracheitis virus (ILTV) genes are commercially available to the poultry industry in the USA. Different sectors of the broiler industry have used these vaccines in ovo or subcutaneously, achieving variable results. The objective of the present study was to determine the efficacy of protection induced by viral vector vaccines as compared with live-attenuated ILTV vaccines. The HVT-LT vaccine was more effective than the FPV-LT vaccine in mitigating the disease and reducing levels of challenge virus when applied in ovo or subcutaneously, particularly when the challenge was performed at 57 days rather than 35 days of age. While the FPV-LT vaccine mitigated clinical signs more effectively when administered subcutaneously than in ovo, it did not reduce the concentration of challenge virus in the trachea by either application route. Detection of antibodies against ILTV glycoproteins expressed by the viral vectors was a useful criterion to assess the immunogenicity of the vectors. The presence of glycoprotein I antibodies detected pre-challenge and post challenge in chickens vaccinated with HVT-LT indicated that the vaccine induced a robust antibody response, which was paralleled by significant reduction of clinical signs. The chicken embryo origin vaccine provided optimal protection by significantly mitigating the disease and reducing the challenge virus in chickens vaccinated via eye drop. The viral vector vaccines, applied in ovo and subcutaneously, provided partial protection, reducing to some degree clinical signs, and challenge VIRUS replication in the trachea.

Protection induced by infectious laryngotracheitis virus vaccines alone and combined with Newcastle disease virus and/or infectious bronchitis virus vaccines.

Two types of live attenuated vaccines have been used worldwide for the control of infectious laryngotracheitis virus (ILTV): 1) chicken embryo origin (CEO) vaccines; and 2) tissue culture origin vaccines (TCO). However, the disease persists in spite of extensive use of vaccination, particularly in areas of intense broiler production. Among the factors that may influence the efficiency of ILTV live attenuated vaccines is a possible interference of Newcastle Disease virus (NDV) and infectious bronchitis virus (IBV) vaccines with the protection induced by ILTV vaccines. The protection induced by CEO and TCO vaccines was evaluated when administered at 14 days of age alone or in combination with the B1 type strain of NDV (B1) and/or the Arkansas (ARK) and Massachusetts (MASS) serotypes of IBV vaccines. Two weeks after vaccination (28 days of age), the chickens were challenged with a virulent ILTV field strain (63140 isolate, group V genotype). Protection was evaluated at 5 and 7 days postchallenge by scoring clinical signs and quantifying the challenge virus load in the trachea using real-time PCR (qPCR). In addition, the viral load of the vaccine viruses (ILTV, NDV, and IBV) was quantified 3 and 5 days postvaccination also using qPCR. The results of this study indicate that the NDV (B1) and IBV (ARK) vaccines and a multivalent vaccine constituted by NDV (B1) and IBV (ARK and MASS) did not interfere with the protection induced by the CEO ILTV vaccine. However, the NDV (BI) and the multivalent (B1/MASS/ARK) vaccines interfered with the protection induced by the TCO vaccine (P < 0.05). Either in combination or by themselves, the NDV and IBV vaccines decreased the tracheal replication of the TCO vaccine and the protection induced by this vaccine, since the ILTV-vaccinated and -challenged chickens displayed significantly more severe clinical signs and ILTV load (P < 0.05) than chickens vaccinated with the TCO vaccine alone. Although NDV and IBV challenges were not performed, the antibody responses elicited by NDV and/or the IBV vaccinations were significantly reduced (P < 0.05) when applied in combination with the CEO vaccine.

Protection against infectious laryngotracheitis by in ovo vaccination with commercially available viral vector recombinant vaccines.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines. The chicken embryo origin (CEO) vaccines, although proven to be effective in experimental settings, have limited efficacy in controlling the disease in dense broiler production sites due to unrestricted use and poor mass vaccination coverage. These factors allowed CEO vaccines to regain virulence, causing long lasting and, consequently, severe outbreaks of the disease. A new generation of viral vector fowl poxvirus (FPV) and herpesvirus of turkey (HVT) vaccines carrying ILTV genes has been developed and such vaccines are commercially available. These vaccines are characterized by their lack of transmission, lack of ILTV-associated latent infections, and no reversion to virulence. HVT-vectored ILTV recombinant vaccines were originally approved for subcutaneous HVT or transcutaneous (pox) delivery. The increased incidence of ILTV outbreaks in broiler production sites encouraged the broiler industry to deliver the FPV-LT and HVT-LT recombinant vaccines in ovo. The objective of this study was to evaluate the protection induced by ILTV viral vector recombinant vaccines after in ovo application in 18-day-old commercial broiler embryos. The protection induced by recombinant ILTV vaccines was assessed by their ability to prevent clinical signs and mortality; to reduce challenge virus replication in the trachea; to prevent an increase in body temperature; and to prevent a decrease in body weight gain after challenge. In this study, both recombinant-vectored ILTV vaccines provided partial protection, thereby mitigating the disease, but did not reduce challenge virus loads in the trachea.

Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines.

A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the DeltaC(t) values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5'-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines.

Pathogenicity and growth characteristics of selected infectious laryngotracheitis virus strains from the United States.

In a recent study, several US infectious laryngotracheitis virus (ILTV) strains and field isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) into nine different genotypes. All of the commercial poultry isolates were identified within genotypes IV, V, and VI. Based on the PCR-RFLP, Group IV isolates were characterized as genetically identical to the chicken embryo origin (CEO) vaccines, Group V as genetically closely related to the CEO vaccines, and Group VI as genetically different to the vaccine strains. The objective of this study was to determine the pathogenicity and growth characteristics of six ILTV commercial poultry isolates as compared with the CEO vaccine. Two isolates representative of PCR-RFLP Groups IV, V, and VI were selected. Differences in disease severity, viral tissue distribution in chickens, and plaque formation ability in cell culture were observed among viral genotypes IV, V, and VI, and between V-A and V-B isolates. Mild respiratory clinical signs were produced by IV-A, IV-B and the CEO vaccine, while VI-A and VI-B isolates produced severe respiratory signs and severe depression, and during the peak of clinical signs both isolates were re-isolated from the conjunctiva, sinus, trachea and thymus. Similarly to Group VI isolates, V-A and V-B produced severe respiratory signs, depression, and were re-isolated from conjunctiva, sinus, and trachea; on cell culture, both isolates produced significant larger plaques than any of the other isolates analysed. Overall, differences in pathogenicity and growth characteristics were observed among genetically closely related US ILTV isolates; however, complete genomes will be necessary to identify molecular determinants linked to the pathogenic viral phenotypes.