RESUMEN

Infectious laryngotracheitis (ILT) poses a significant threat to the poultry industry, and vaccines play an important role in protection. However, due to the increasing scale of poultry production, there is an urgent need to develop vaccines that are suitable for convenient immunization methods such as spraying. Previous studies have shown that Newcastle disease virus (NDV)-ILT vaccines administered via intranasal and intraocular routes to commercial chickens carrying maternally-derived antibodies (MDAs) are still protective against ILT. In this study, a recombinant NDV (rNDV) was generated to express infectious laryngotracheitis virus (ILTV) glycoprotein B (gB), named rLS-gB, based on a full-length cDNA clone of the LaSota strain. The protective effect of different doses of rLS-gB administered by spray vaccination to commercial chickens at 1 d of age (doa) was evaluated. The chickens were exposed to 160-µm aerosol particles for 10 min for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs and anti-ILTV MDAs in chickens, the ILTV- and NDV-specific antibody titres were significantly greater in the vaccinated groups than in the unvaccinated group. After challenge with a virulent ILTV strain, no clinical signs were observed in the 107 EID50/ml group compared to the other groups. Furthermore, vaccination with 107 EID50/ml rLS-gB significantly reduced the ILTV viral load and ameliorated gross and microscopic lesions in the trachea of chickens. Overall, these results suggested that rLS-gB is a safe and efficient candidate spray vaccine for ILT and is especially suitable for scaled chicken farms.

RESUMEN

Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant economic losses in the poultry industry globally, in addition to severe animal health and welfare concerns. To date, studying the role of ILTV genes in viral infection, replication or pathogenesis has largely been limited to genes that can be deleted from the ILTV genome and the resultant deletion mutants characterized in vitro or in vivo. However, this approach is not suitable for the study of essential genes. This study trialled two different codon deoptimization techniques that aimed to separately disrupt and downregulate the expression of two ILTV genes, ICP8 and UL12, which are essential or very important in viral replication. The target genes were partially recoded using codon usage deoptimization (CUD) and codon pair bias deoptimization (CPBD) approaches and characterized in vitro. Viruses deoptimized via CPBD showed decreased protein expression as assessed by Western blotting and/or fluorescence microscopy to measure the intensity of the fluorescent marker fused to the target protein. Viruses deoptimized by CUD showed less consistent results, with some mutants that could not be generated or isolated. The results indicate that CPBD is an attractive and viable tool for the study of essential or critically important genes in ILTV. This is the first study, to our knowledge, that utilizes CPBD and CUD techniques for the study of ILTV genes.

Asunto(s)

Infecciones por Herpesviridae , Herpesvirus Gallináceo 1 , Enfermedades de las Aves de Corral , Vacunas Virales , Animales , Pollos , Uso de Codones , Genes Esenciales , Herpesvirus Gallináceo 1/genética , Codón/genética

RESUMEN

Infectious laryngotracheitis (ILT) is a viral respiratory illness in poultry that causes massive financial losses. This research aimed to isolate and identify the ILT virus in suspected outbreaks of broiler flocks in Egypt during 2020-2021, besides investigating its genetic link with other circulating strains. Real-time-PCR was used to test 57 samples taken from unvaccinated broiler farms. Ten samples are positive for ILTV, and the virus is being isolated in SPF chicken embryos. The Sanger sequencing was used to conduct (partial) sequencing of the infected cell protein4 gene (ICP4) for eight isolates. Phylogenetic analysis conducted Maximum Likelihood, comparative sequencing analysis of ICP4 of strains under study with vaccination ILT reference strains reveled that all isolates were clustered into two major groups. The (OM291843and OM291846) clustered together with the chicken embryo origin vaccine strains (IV and V group). The remaining six strains belong to the TCO vaccine(I, II and III group). The total sequence similarity between the strains under study and the various Egyptian strains varied from (97 to 100%) while the similarity with TCO or chicken embryo origin -vaccine strains ranged from (95to 100%). There were no deletions detected in the 272-283-bp region of the ICP4 gene. Detection of arginine to methionine substitutions at position 180 (R180M) and change of Serine to Asparagine at position 227 (S227N) in the (OM291843 and OM291846) which were previously described in chicken embryo origin -vaccine strains. This reveals that field strains may have evolved from vaccine strains, notably identification of non-synonymous substitutions which might be linked to the virulence strains' attenuation. Finally, independent of geographical distribution, both chicken embryo origin-vaccine-like and TCO-Vaccine-like virus strains were circulating in Egyptian non-vaccinated broiler flocks in 2020 and 2021. Despite their genetic differences, both viruses caused significant illnesses in the field.

RESUMEN

Increasing globalization, agricultural intensification, urbanization, and climatic changes have resulted in a significant recent increase in emerging infectious zoonotic diseases. Zoonotic diseases are becoming more common, so innovative, effective, and integrative research is required to better understand their transmission, ecological implications, and dynamics at wildlife-human interfaces. High-throughput sequencing (HTS) methodologies have enormous potential for unraveling these contingencies and improving our understanding, but they are only now beginning to be realized in livestock research. This study investigates the current state of use of sequencing technologies in the detection of livestock pathogens such as bovine, dogs (Canis lupus familiaris), sheep (Ovis aries), pigs (Sus scrofa), horses (Equus caballus), chicken (Gallus gallus domesticus), and ducks (Anatidae) as well as how it can improve the monitoring and detection of zoonotic infections. We also described several high-throughput sequencing approaches for improved detection of known, unknown, and emerging infectious agents, resulting in better infectious disease diagnosis, as well as surveillance of zoonotic infectious diseases. In the coming years, the continued advancement of sequencing technologies will improve livestock research and hasten the development of various new genomic and technological studies on farm animals.

RESUMEN

Avian infectious laryngotracheitis (ILT) is a respiratory disease that causes severe economic losses in the poultry industry, mainly due to high morbidity and mortality and reduced egg production. Molecular characterization was performed on samples collected from flocks in the Brazilian States of São Paulo, Pernambuco, and Minas Gerais during 2015 and 2016 that presented clinical signs of respiratory disease. End-point PCR was used for viral detection, and DNA sequencing was used for differentiation of vaccine and field strains. Molecular analysis based on the infected cell protein (ICP4) gene separated four of the nine samples together with previous Brazilian isolates (São Paulo and Minas Gerais), one sample was grouped on the same branch as Minas Gerais strains (along with another related sample), one sample was separately branched but still related to the tissue culture origin (TCO) vaccine strain, and two samples were grouped on the same branch as the TCO vaccine strain. Molecular analysis of the thymidine kinase (TK) gene showed the existence of strains of both high and low virulence. The characterization of two fragments of the ICP4 gene and a fragment of the TK gene in this study suggested that the virus circulating in Guatapará, as well as those in Barretos and Itanhandu, that is causing respiratory problems in birds is a highly virulent field strain. The clinical signs point to a TCO vaccine strain that most likely underwent some reversal event and is a latent reactivated infection.

Asunto(s)

Infecciones por Herpesviridae , Herpesvirus Gallináceo 1 , Enfermedades de las Aves de Corral , Vacunas Virales , Animales , Pollos , Brasil/epidemiología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/genética

RESUMEN

Infectious laryngotracheitis (ILT) is an economically crucial respiratory disease of poultry that affects the industry worldwide. Vaccination is the principal tool in the control of the disease outbreak. In an earlier study, we comprehensively characterized the circulating strains in Egypt and identified both CEO-like and recombinant strains are dominant. Herein, we investigated the pathogenicity of two virulent strains representing the CEO-like (Sharkia_2018) and recombinant strain (Qalubia_2018). Additionally, we evaluated the efficacy of different commercial vaccines (HVT-LT, CEO, and TCO) against the two isolates in terms of the histopathological lesion scores and the viral (gC) gene load. A total of 270 White Leghorn-specific pathogen-free male chicks were divided into nine groups of 30 birds, each housed in separate isolators. Birds were distributed as follows; one group was non-vaccinated, non-challenged, and served as a negative control. Two groups were non-vaccinated and infected with the two isolates of interest and served as a positive control to test the pathogenicity. Six groups were vaccinated and challenged; two groups were vaccinated with vector vaccine at one day old. The other four groups were vaccinated with either the CEO- or TCO- vaccine (two groups each) at four weeks of age. Three weeks after vaccination, birds were infected with the virulent ILTV isolates. The larynx, trachea, and harderian gland samples were taken at 1, 3, and 7 days post-infection for histopathological lesion score and molecular detection. Notably, The recombinant strain was more virulent and pathogenic than CEO-like ILTV strains. Moreover, the TCO vaccine was less immunogenic than the vector and CEO vaccines.

Asunto(s)

Infecciones por Herpesviridae , Herpesvirus Gallináceo 1 , Enfermedades de las Aves de Corral , Vacunas Virales , Animales , Pollos , Egipto/epidemiología , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/genética , Masculino , Eficacia de las Vacunas , Vacunas Atenuadas , Virulencia

RESUMEN

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains.

Asunto(s)

Infecciones por Herpesviridae , Herpesvirus Gallináceo 1 , Enfermedades de las Aves de Corral , Vacunas Virales , Aminoácidos/genética , Animales , Embrión de Pollo , Pollos , Marcadores Genéticos , Infecciones por Herpesviridae/epidemiología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/genética , Ontario , Filogenia , Análisis de Secuencia de ADN , Vacunas Atenuadas/genética , Vacunas Virales/genética

RESUMEN

Infectious laryngotracheitis (ILT) is caused by Gallid herpesvirus-1 (GaHV-1) or infectious laryngotracheitis virus (ILTV) and was first described in Canadian poultry flocks. In Canada, ILTV infection is endemic in backyard flocks, and commercial poultry encounters ILT outbreaks sporadically. A common practice to control ILT is the use of live attenuated vaccines. However, outbreaks still occur in poultry flocks globally due to ILTV vaccine strains reverting to virulence and emergence of new ILTV strains due to recombination in addition to circulating wildtype strains. Recent studies reported that most of the ILT outbreaks in Canada were induced by the chicken-embryo-origin (CEO) live attenuated vaccine revertant strains with the involvement of a small percentage of wildtype ILTV. It is not known if the host responses induced by these two ILTV strains are different. The objective of the study was to compare the host responses elicited by CEO revertant and wildtype ILTV strains in chickens. We infected 3-week-old specific pathogen-free chickens with the two types of ILTV isolates and subsequently evaluated the severity of clinical and pathological manifestations, in addition to host responses. We observed that both of the isolates show high pathogenicity by inducing several clinical and pathological manifestations. A significant recruitment of immune cells at both 3 and 7 days post-infection (dpi) was observed in the tracheal mucosa and the lung tissues of the infected chickens with wildtype and CEO vaccine revertant ILTV isolates when compared to uninfected controls. Overall, this study provides a better understanding of the mechanism of host responses against ILTV infection.

RESUMEN

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.

Asunto(s)

Herpesvirus Gallináceo 1 , Vacunas , Animales , Embrión de Pollo , Pollos/inmunología , Herpesvirus Gallináceo 1/inmunología , Herpesvirus Meleágrido 1 , Membrana Mucosa , Tráquea , Vacunación/veterinaria

RESUMEN

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.

RESUMEN

The ability of infectious laryngotracheitis virus (ILTV) to replicate in organs outside of the upper respiratory tract and conjunctiva associated-lymphoid tissues is still not well understood. This study investigated the tissue distribution of an Australian field strain of ILTV (class 9) on birds experimentally inoculated via eye-drop at 7 days of age by using quantitative PCR (qPCR) and immunohistochemistry. Tissues including conjunctiva, caecal tonsil, kidney, liver, lung, spleen, thymus, trachea and blood were collected from sham-inoculated (control group; n = 2) and ILTV-inoculated (n = 8) birds at 7 days post-inoculation (dpi). Blood was collected from 13 infected birds at 14 dpi and fractionated using ficoll-paque. At 7 dpi, the highest detection rate and genomic copies (GC) were in conjunctiva (8/8; 8.08 ± 0.48 log10 GC/mg) followed by trachea (8/8; 4.64 ± 0.48) and thymus (8/8; 4.52 ± 0.48), kidney (8/8; 3.97 ± 0.48), lung (8/8; 3.65 ± 0.48), spleen (8/8; 3.55 ± 0.48), liver (8/8; 3.51 ± 0.48), caecal tonsil (7/8; 3.76 ± 0.48) and plasma (4/8; 2.40 ± 0.48 log10 GC/ml). ILTV antigen was only detected in conjunctiva (7/8), trachea (6/8) and lung (4/8) samples. At 14 dpi, ILTV detection rate and genomic copies in buffy coat cells were 12/13 and 2.86 ± 0.39 log10 GC/mg, respectively while those of plasma were 11/13 and 4.29 ± 0.39 log10 GC/ml and red blood cell were 3/13 and 0.36 ± 0.39 log10 GC/mg. In conclusion, ILTV DNA was detected in a wide range of tissues and blood fractions but ILTV antigen was only detected in respiratory organs and conjunctiva.

Asunto(s)

Pollos , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/aislamiento & purificación , Enfermedades de las Aves de Corral/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Animales , Australia , Pollos/genética , Pollos/virología , Infecciones por Herpesviridae/sangre , Infecciones por Herpesviridae/virología , Herpesvirus Gallináceo 1/genética , Herpesvirus Gallináceo 1/inmunología , Inmunohistoquímica/veterinaria , Tejido Linfoide/virología , Enfermedades de las Aves de Corral/sangre

RESUMEN

Infectious laryngotracheitis virus (ILTV) is an economically significant respiratory pathogen of poultry. Novel recombinant strains of ILTV have emerged in Australia during the last decade and currently class 9 (CL9) and class 10 (CL10) ILTV are the most prevalent circulating strains. This study conducted a comprehensive investigation of the pathogenesis of these two viral strains. Commercial broiler and specific pathogen free (SPF) chickens were inoculated with varying doses of CL9 or CL10 ILTV and subsequently evaluated for clinical and pathological signs of infection. While no difference in the levels of acute viral replication were observed across the different challenge doses, the severity of clinical signs, tracheal pathology and mortality were dose dependent. Both strains of virus persisted in the respiratory tract for up to 14 days post inoculation (dpi) and could be detected in the lung and feathers with sporadic detection in the liver, spleen or bursa. Given the prevalence of CL9 and CL10 in Australian poultry flocks, this study provides an important foundation for the development of diagnostic and therapeutic approaches for the detection and prevention of ILTV.

Asunto(s)

Pollos/virología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/patogenicidad , Enfermedades de las Aves de Corral/virología , Tropismo Viral , Animales , Australia , Plumas/virología , Genotipo , Herpesvirus Gallináceo 1/genética , Pulmón/virología , Virus Reordenados/patogenicidad , Organismos Libres de Patógenos Específicos , Replicación Viral

RESUMEN

Infectious laryngotracheitis is an important disease of chickens caused by infectious laryngotracheitis virus (ILTV). Outbreaks commonly occur in meat chicken flocks and mass vaccination with live attenuated vaccines, usually in water, is used to control the disease in these populations. Vaccination with live virus via water and nipple drinkers requires stringent adherence to protocols to ensure success, but vaccine administration monitoring is not currently assessed due to a lack of economically viable methods. Vaccinal ILTV has been shown to be detectable in dust in experimental studies and has potential as a method of assessing vaccination success. However, the pattern of vaccinal ILTV detection in dust following vaccination under commercial conditions has not been defined. We report the longitudinal profile of ILTV genome copies (GC) in poultry house dust collected on settle plates following vaccination of 8 flocks of commercial meat chickens on four farms. ILTV GC was enumerated using quantitative real-time polymerase chain reaction (qPCR). There was considerable variation between flocks in the levels of ILTV GC detected post vaccination and this variation was significantly associated with vaccine take measured in individual birds in a companion study. There was no effect of sampling location on ILTV GC in dust but the amount of dust collected was greater in locations closer to the exhaust fans in artificially ventilated houses. Results indicate that measurement of ILTV GC in single or pooled dust samples at 7-8 days post vaccination enables detection of poor vaccine takes and provides a practical means of monitoring ILT vaccination.

Asunto(s)

Pollos , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/inmunología , Vacunación Masiva/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/administración & dosificación , Animales , ADN Viral/aislamiento & purificación , Polvo , Genoma Viral , Infecciones por Herpesviridae/prevención & control , Herpesvirus Gallináceo 1/genética , Vivienda para Animales , Vacunación Masiva/métodos , Nueva Gales del Sur , Enfermedades de las Aves de Corral/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Vacunas Virales/genética

RESUMEN

Previously, we have demonstrated that the recombinant Newcastle disease virus (NDV) expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) conferred protection against both virulent NDV and ILTV challenges in chickens. In this study, we evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE eight times (EP4 to EP11). RT-PCR analysis of the vaccine seed and egg-passaged virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. Next-generation sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the EP11 virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE.

Asunto(s)

Infecciones por Herpesviridae/prevención & control , Enfermedad de Newcastle/prevención & control , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/administración & dosificación , Animales , Embrión de Pollo , Pollos , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/genética , Herpesvirus Gallináceo 1/inmunología , Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/genética , Virus de la Enfermedad de Newcastle/inmunología , Enfermedades de las Aves de Corral/inmunología , Pase Seriado , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología

RESUMEN

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in chickens. ILTV can establish latency and reactivate later in life, but there have been few investigations of ILTV latency. This study aimed to contribute to the methodologies available to detect latent ILTV. A nested PCR was developed which was more sensitive than three other molecular methods investigated in this study. This nested PCR was then used in conjunction with in vitro reactivation culture methods that were optimized and applied to trigeminal ganglia (TG) and tracheal samples from ILTV-vaccinated commercial layer birds (n = 30). ILTV DNA was detected by nested PCR in the upper respiratory tract (URT) or eye of 22 birds. Of the remaining 8 birds, ILTV could be detected by co-culture in TG of 5 birds, with reactivated virus mostly detected 6 days post-explant (dpe). ILTV was also detected in tracheal cultures by 6 dpe. In the ILTV-positive URT samples, the virus could be characterised as vaccine strains SA2 (n = 9) or A20 (n = 5). This study provides evidence for reactivation and shedding of vaccine ILTV in commercial layer birds. Moreover, this study produced a molecular and in-vitro culture method to detect latent viral infection.

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/aislamiento & purificación , Infección Latente/diagnóstico , Infección Latente/veterinaria , Animales , Pollos/virología , Herpesvirus Gallináceo 1/genética , Herpesvirus Gallináceo 1/crecimiento & desarrollo , Infección Latente/virología , Límite de Detección , Reacción en Cadena de la Polimerasa , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/virología , Tráquea/virología , Proteínas Virales/genética , Vacunas Virales/análisis

RESUMEN

Dust collected from the poultry house has been increasingly used as a population-level sample to monitor the presence of pathogens or to evaluate the administration of live vaccines. However, there are no guidelines for the storage of this sample type. This study investigated the stability of infectious laryngotracheitis virus (ILTV), a DNA virus, and infectious bronchitis virus (IBV), an RNA virus, in poultry dust kept under temperature and moisture conditions that mimic on-farm and laboratory storage. Dust samples were collected from chicks spray vaccinated with a live IBV vaccine and inoculated with a field ILTV strain via eye drop. Samples were stored under different moisture conditions (dry = 2% moisture, moist = 22%-71% moisture) and temperatures (-20, 4, 25, and 37 C) for different durations (0, 7, and 14 days, and 1, 2, 3, and 4 mo) in a factorial arrangement, followed by quantitative PCR for detection of virus genome copies (GC). The length of storage, moisture level, and storage temperature affected the viral genome load for ILTV and IBV but did not affect the number of positive samples for each virus. All treatment combinations were ILTV positive for at least 4 mo. In dry dust samples, all storage temperatures or durations had quantifiable ILTV or IBV GC. Moisture addition had a detrimental effect on viral genome load, causing an overall reduction of 0.3 log 10 for ILTV GC (7.29 and 6.97 log 10, P = 0.0001), and 1.3 log 10 for IBV GC (5.95 and 4.66 log 10, P = 0.0001), which are unlikely to have biologic significance. In conclusion, dry dust can be stored at any temperature up to 37 C for at least 4 mo without loss in qPCR detection of ILTV or IBV GC. Collection or storage of moist dust should be avoided, or air drying prior to storage is recommended if only moist dust is available.

Asunto(s)

Enfermedades de las Aves/diagnóstico , Infecciones por Coronavirus/veterinaria , Genoma Viral , Inestabilidad Genómica , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/aislamiento & purificación , Virus de la Bronquitis Infecciosa/aislamiento & purificación , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Enfermedades de las Aves/virología , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Polvo/análisis , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/virología , Herpesvirus Gallináceo 1/genética , Virus de la Bronquitis Infecciosa/genética , Manejo de Especímenes/veterinaria

RESUMEN

Infectious laryngotracheitis virus (ILTV) is a promising vaccine vector due to its heterologous gene accommodation capabilities, low pathogenicity, and potential to induce cellular and humoral arms of immunity. Owing to these characteristics, different gene-deletion versions of ILTVs have been successfully deployed as a vector platform for the development of recombinant vaccines against multiple avian viruses using conventional recombination methods, which are tedious, time-demanding, and error-prone. Here, we applied a versatile, and customisable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 accompanied with Cre-Lox system to simultaneously delete virulence factors and to insert foreign genes in the ILTV genome. Using this pipeline, we successfully deleted thymidine kinase (TK) and unique short 4 (US4) genes and inserted fusion (F) gene of the Newcastle disease virus without adversely affecting ILTV replication and expression of the F protein. Taken together, the proposed approach offers novel tools to attenuate (by deletion of virulence factor) and to generate multivalent (by insertion of heterologous genes) vaccine vectors to protect chickens against pathogens of poultry and public health importance.

RESUMEN

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.

Asunto(s)

Ojo/inmunología , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/inmunología , Tejido Linfoide/inmunología , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/inmunología , Animales , Linfocitos T CD8-positivos , Pollos , Conjuntiva/inmunología , Conjuntiva/virología , Citocinas/genética , Citocinas/inmunología , Ojo/virología , Genoma Viral , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/prevención & control , Inmunidad Celular , Interferón gamma/genética , Interferón gamma/inmunología , Tejido Linfoide/virología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Organismos Libres de Patógenos Específicos , Vacunación/veterinaria , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Carga Viral/inmunología , Vacunas Virales/administración & dosificación

RESUMEN

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.

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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.

Asunto(s)

Pollos , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/inmunología , Enfermedades de las Aves de Corral/prevención & control , Traqueítis/veterinaria , Vacunas Virales/inmunología , Animales , Infecciones por Herpesviridae/prevención & control , Infecciones por Herpesviridae/virología , Enfermedades de las Aves de Corral/virología , Traqueítis/prevención & control , Traqueítis/virología , Vacunación/veterinaria

RESUMEN

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.

Asunto(s)

Pollos/virología , Genoma Viral/genética , Técnicas de Genotipaje/veterinaria , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/genética , Polimorfismo de Nucleótido Simple/genética , Enfermedades de las Aves de Corral/virología , Alelos , Animales , Genotipo , Técnicas de Genotipaje/métodos , Infecciones por Herpesviridae/virología , Herpesvirus Gallináceo 1/clasificación , Herpesvirus Gallináceo 1/aislamiento & purificación , Tipificación de Secuencias Multilocus/veterinaria , Nanoporos , Sistemas de Lectura Abierta/genética , Filogenia