Infectious Laryngotracheitis Virus Research Articles

A meta-analysis for prevalence of infectious laryngotracheitis in chickens in mainland China in 1981–2022

BackgroundInfectious laryngotracheitis (ILT) is a highly infectious upper respiratory tract disease of chickens caused by infectious laryngotracheitis virus or Gallid herpesvirus 1 (GaHV-1). ILT is an important respiratory disease of chickens and annually causes significant economic losses in the chicken industry. Although numerous relevant studies have been published, the overall prevalence of ILT infection among chicken in mainland China is still unknown, and associated risk factors need to be evaluated to establish preventive measures.ResultsThe present study reviewed the literature on the prevalence of ILT in chickens in China as of December 20, 2022, retrieved from six databases—CNKI, Wanfang, VIP, PubMed, Web of Science, and ScienceDirect—were used to retrieve relevant studies published between January 1, 1981 and December 20, 2022. The literature quality of studies was assessed, and 20 studies with a total of 108,587 samples were included in the meta-analysis. Results of the meta-analysis showed that the overall prevalence of ILT was 10% (95% confidence interval: 8 –12%) through the random-effects model, which showed high heterogeneity, I2 = 99.4%. Further subgroup analyses showed that the prevalence of ILT decreased over time; furthermore, the prevalence in Northwest China was slightly lower than that in North China and South China, and the prevalence estimated using the diagnostic technique AGP was higher than that reported using other diagnostic techniques.ConclusionsILT is prevalent to some extent in mainland China. Given that the ILT attenuated live vaccine has a certain level of virulence and the prevalence differences between regions, we recommend controlling breeding density, improving immunization programs and continuously monitoring viruses and to prevent ILT prevailing in mainland China.

Seroprevalence and molecular detection of infectious laryngotracheitis virus (ILTV) in commercial chicken in Gazipur district of Bangladesh

Infectious laryngotracheitis (ILT) is one of the major diseases of chickens that cause great economic losses in commercial chicken farms. It is caused by the infectious laryngotracheitis virus (ILTV). This research work was designed to investigate the seroprevalence of ILTV in commercial chicken farms in Gazipur district. A total of 590 blood sera were collected randomly from commercial chicken farms from different locations in Gazipur district. An antigen-coated indirect ELISA was performed to determine the antibody titer against ILTV in these sera. Moreover, gross and histopathological changes in different organs were investigated. Polymerase chain reaction (PCR), a molecular detection method, was performed to detect the fragment of the glycoprotein ‘C’ (gC) gene of ILTV in the infected chicken. Overall, 195 out of 590 serum samples were positive for anti-ILTV IgG, representing 33.05% of positive cases. The highest mean titer of ILTV antibody was found in anti-ILTV-vaccinated layer chickens with a mean titer of 3554. In seasonal variation, the highest prevalence of ILTV was recorded during the winter, followed by the rainy and summer seasons. The prevalence of ILTV was the highest at the age of 10-30 weeks in layer chickens. Necropsy findings of dead chickens showed severe congestion and fibrosis in the lungs and trachea, with occlusion of the tracheal lumen by mucus, caseous exudates and blood. Microscopically, hemorrhage and huge infiltrations of inflammatory cells were found in trachea and lungs. Moreover, the nucleotide sequence of ‘gC’ gene of ILTV was amplified successfully and yielded 1.26 kbp amplicons. This study suggested that ILTV was endemic in Gazipur district and the PCR technique is a useful molecular tool for diagnosis of ILTV in the commercial chicken industry. Ann. Bangladesh Agric. (2023) 27 (1): 121-135

Based on CRISPR-Cas13a system, to establish a rapid visual detection method for avian influenza viruses.

To rapidly, specifically, and sensitively detect avian influenza virus (AIV), this research established a visual detection method of recombinase-aided amplification (RAA) based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated proteins 13a (Cas13a) system. In this study, specific primers and CRISPR RNA (crRNA) were designed according to the conservative sequence of AIV Nucleprotein (NP) gene. RAA technology was used to amplify the target sequence, and the amplification products were visually detected by lateral flow dipstick (LFD). The specificity, sensitivity, and reproducibility of RAA-CRISPR-Cas13a-LFD were evaluated. At the same time, this method and polymerase chain reaction (PCR)-agarose electrophoresis method were used to detect clinical samples, and the coincidence rate of the two detection methods was calculated. The results showed that the RAA-CRISPR-Cas13a-LFD method could achieve specific amplification of the target gene fragments, and the detection results could be visually observed through the LFD. Meanwhile, there was no cross-reaction with infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and Newcastle disease virus (NDV). The sensitivity reached 100 copies/μL, which was 1,000-fold higher than that of PCR-agarose electrophoresis method. The coincidence rate of clinical tests was 98.75 %, and the total reaction time was ~1 h. The RAA-CRISPR-Cas13a-LFD method established in this study had the advantages of rapid, simple, strong specificity, and high sensitivity, which provided a new visual method for AIV detection.

INNATE IMMUNE RESPONSES OF ILT VIRUS INFECTION IN LAYER AT PRODUCTION STAGE IN IRAQ

Since 2011, there have been epidemics of respiratory infection in Iraq's laying hen farms, including hemorrhage tracheitis, which closely resembles laryngotracheitis and exhibits symptoms such as symptoms and lesions, respiratory issues, and swelling. Infectious laryngotracheitis (ILT) in layers hen farms in Iraq had to be molecularly identified and determined, together with viral load and innate immune responses of gene transcription (INF-γ, IL1, IL6, and IL10). A total of 40 samples 20 trachea and 20 lungs were taken from various suspect contaminated layer flocks of Iraqi farms (age: 33 weeks). Real-time PCR results showed that 10 samples from both tissues tested positive for the ILT virus. The highest viral load, 5.52×103 copies/L viral nucleic acid, was found in tracheal tissue at 7 dpi, whereas the lowest viral load, 1.65×103 copies/L viral nucleic acid, was found in lung tissues. At 3 and 7 days after infection, the expression of INF-γ, IL1 and IL6 was significantly higher there in tracheal tissues (P≤0.05) than in the lung tissues, indicating a down regulation of these three molecules. In both organs at the same time, there was no up regulation of IL10. The findings of this study show that ILTv up-regulates the transcription of several cytokines in the trachea, including INF-γ, IL1, and IL6, during various stages of the cytokine production process.

Seroprevalence and Associated Risk Factors of Infectious Laryngotracheitis (ILT) from Poultry Farms in Selected Towns of East Shao Zone

Poultry is one of the largest group of livestock species in the world in which chickens largely dominate the flock composition. Although this sector has a potential in elevating poverty in one country it is seriously challenged with various factors, of which poultry disease is the main. Infectious laryngotracheitis (ILT) is a highly contagious and upper respiratory disease of chickens which is caused by a Gallid herpesvirus 1 (GaHV-1) belonging to the genus Iltovirus, and subfamily Alphaherpesvirinae within Herpesviridae family was one of a deadly poultry disease which impose to a loss of production and productivity gained from the sector. Cross-sectional study was conducted from December 2022 to April 2023 in three selected cities of Oromia region (Bishoftu, Adama and Modjo), Ethiopia. In the current study area, of the total 386 serum samples collected using simple random sampling method 14 (3.63%; 95%CI: 2.15-6.04) were found positive for anti-ILTV antibody detection using Indirect Enzyme Linked Immuno Sorbent Assay. Prevalence of the disease at flock level was found 5/17 (29.41%; 95% CI: 11.52%- 57.14%). Different risk factors (sex, age, sampling area, breed, production type, origin, management and supplementation of vitamins) were considered as covariates for this study. Of the different risk factors considered; sampling area, production type, age, origin and management were found statistically significant (p<0.05) whereas the rest factors were not significantly associated with the outcome variable using chi-square analysis. However, using logistic regression model sampling area and management were found significantly associated (p<0.05) with the occurrence of the disease. The result of this study revealed that ILT virus is circulating among commercial chickens in the selected study areas and needs due emphasis to tackle and cease its spread using vaccination, although ILT vaccine is not familiarized now days in Ethiopia. In addition, proper management and biosecurity measures will be highly recommended as the disease is a deadly disease once introduced into the farm

Molecular detection and sequence analysis of envelope glycoproteins (gC, gD) and tegument protein (UL47) of infectious laryngotracheitis virus isolated from commercial poultry flocks in Tamil Nadu, India

Molecular detection and sequence analysis of envelope glycoproteins (gC, gD) and tegument protein (UL47) of infectious laryngotracheitis virus isolated from commercial poultry flocks in Tamil Nadu, India

Attenuation of a Field Strain of Infectious Laryngotracheitis Virus in Primary Chicken Culture Cells and Adaptation to Secondary Chicken Embryo Fibroblasts

The establishment of commercial infectious laryngotracheitis virus (ILTV) live-modified vaccines has relied on serial passaging in chicken embryo (CEO) and tissue culture (TCO) for attenuation. The objective of this study was to attenuate and adapt a virulent CEO-related ILTV field strain (6340) in immortalized cells (LMH), primary chicken embryo kidney cells (CEK), chicken embryo liver cells (CEL), and chicken embryo fibroblasts (CEF). CEFs were refractory to parent ILTV, LMH cells produced low virus yields (~2.5 log10 TCID50 per mL), while CEK and CEL cells produced higher viral titers (≥log10 6.0 TCID50 per mL). After 52 passages in CELs, the cytopathic effect (CPE) was observed not only in hepatocytes but also in CEL fibroblasts. Once CPE was evident in CEL fibroblasts, 20 further passages in CEFs with viral titers reaching yields of ~4.4–5.5 log10 TCID50 per mL were performed. The attenuation of CEF-adapted viruses was evaluated after intra-tracheal and conjunctival inoculation in 28-day-old broilers by assessing clinical signs at five days post-inoculation (DPI). Virus CEL cells passages 80, 90, and 100, and CEF passages 10 and 20 were significantly attenuated compared to the parental strain. This is the first report of the attenuation of a virulent field CEO-related ILTV strain (RFLP Group V) in CEF cells—a cell type from a different embryonic germ layer (mesoderm) than ILTV target cells—the respiratory epithelium (endoderm). This finding underscores the potential use of CEF adaptation for the development of a live-attenuated ILTV vaccine.

Isolation, identification, molecular and pathogenicity characteristics of an infectious laryngotracheitis virus from Hubei province, China

Multiple outbreaks of avian infectious laryngotracheitis (ILT) in chickens, both domestically and internationally, have been directly correlate to widespread vaccine use in affected countries and regions. Phylogenetic and recombination event analyses have demonstrated that avian infectious laryngotracheitis virus (ILTV) field strains are progressively evolving toward the chicken embryo-origin (CEO) vaccine strain. Even with standardized biosecurity measures and effective prevention and control strategies implemented on large-scale farms, continuous ILT outbreaks result in significant economic losses to the poultry industry worldwide. These outbreaks undoubtedly hinder efforts to control and eradicate ILTV in the future. In this study, an ILTV isolate was successfully obtained by laboratory PCR detection and virus isolation from chickens that exhibited dyspnea and depression on a broiler farm in Hubei Province, China. The isolated strain exhibited robust propagation on chorioallantoic membranes of embryonated eggs, but failed to establish effective infection in chicken hepatocellular carcinoma (LMH) cells. Phylogenetic analysis revealed a unique T441P point mutation in the gJ protein of the isolate. Animal experiments confirmed the virulence of this strain, as it induced mortality in 6-wk-old chickens. This study expands current understanding of the epidemiology, genetic variations, and pathogenicity of ILTV isolates circulating domestically, contributing to the elucidate of ILTV molecular basis of pathogenicity and development of vaccine.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

In Ovo Vaccination with Recombinant Herpes Virus of the Turkey-Laryngotracheitis Vaccine Adjuvanted with CpG-Oligonucleotide Provides Protection against a Viral Challenge in Broiler Chickens.

Infectious laryngotracheitis (ILT) is an economically important disease in chickens. We previously showed that an in ovo adjuvantation of recombinant herpesvirus of the turkey-Laryngotracheitis (rHVT-LT) vaccine with CpG-oligonucleotides (ODN) can boost vaccine-induced responses in one-day-old broiler chickens. Here, we evaluated the protective efficacy of in ovo administered rHVT-LT + CpG-ODN vaccination against a wild-type ILT virus (ILTV) challenge at 28 days of age and assessed splenic immune gene expression as well as cellular responses. A chicken-embryo-origin (CEO)-ILT vaccine administered in water at 14 days of age was also used as a comparative control for the protection assessment. The results showed that the rHVT-LT + CpG-ODN or the CEO vaccinations provided significant protection against the ILTV challenge and that the level of protection induced by both the vaccines was statistically similar. The protected birds had a significantly upregulated expression of interferon (IFN)γ or interleukin (IL)-12 cytokine genes. Furthermore, the chickens vaccinated with the rHVT-LT + CpG-ODN or CEO vaccine had a significantly higher frequency of γδ T cells and activated CD4+ or CD8+ T cells, compared to the unvaccinated-ILTV challenge control. Collectively, our findings suggest that CpG-ODN can be used as an effective adjuvant for rHVT-LT in ovo vaccination to induce protective immunity against ILT in broiler chickens.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018–2020

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.

Questionnaire study suggests grave consequences of infectious laryngotracheitis, infectious coryza and mycoplasmosis in small chicken flocks

BackgroundA growing number of people in western countries keep small chicken flocks. In Sweden, respiratory disease is a common necropsy finding in chickens from such flocks. A respiratory real-time polymerase chain reaction (PCR) panel was applied to detect infectious laryngotracheitis virus (ILTV), Avibacterium paragallinarum (A. paragallinarum) and Mycoplasma gallisepticum (M. gallisepticum) in chickens from small flocks which underwent necropsy in 2017–2019 and had respiratory lesions. Owners (N = 100) of PCR-positive flocks were invited to reply to a web-based questionnaire about husbandry, outbreak characteristics and management.ResultsResponse rate was 61.0%. The flocks were from 18 out of Sweden’s 21 counties indicating that respiratory infections in small chicken flocks are geographically widespread in Sweden. Among participating flocks, 77.0% were coinfected by 2–3 pathogens; 91.8% tested positive for A. paragallinarum, 57.4% for M. gallisepticum and 50.8% for ILTV. Larger flock size and mixed-species flock structure were associated with PCR detection of M. gallisepticum (P = 0.00 and P = 0.02, respectively). Up to 50% mortality was reported by 63.9% of respondents. Euthanasia of some chickens was carried out in 86.9% of the flocks as a result of the outbreaks. Full clinical recovery was reported by 39.3% of owners suggesting chronic infection is a major challenge in infected flocks. Live birds had been introduced in many flocks prior to outbreaks, which suggested these as an important source of infection. Following the outbreaks, 36.1% replaced their flocks with new birds and 9.8% ceased keeping chickens.ConclusionsThis study highlights the severity of respiratory outbreaks in small non-commercial chicken flocks and points to the need for more research and veterinary assistance to prevent and manage respiratory infections in small chicken flocks.

Seroprevalence and Associated Risk Factors of Infectious Laryngotrachitis in Small Scale Chicken Production in Nekemte Town and Selectted Districts of East Wollega Zone, Oromia, Ethiopia

Infectious laryngotracheitis (ILT) is a disease of high economic consequence to the poultry sector. Infectious laryngotracheitis virus (ILTV), under the genus Iltovirus, and the family Herpesviridae, is the agent responsible for the disease. The aims of current study were, therefore, to estimate the seroprevalence of ILTV in chickens and identifying associated risk factors. A cross-sectional study was conducted from April 2022 to October 2022 in East Wollega zone specifically in Nekemte town and three surrounding districts (Diga, Guto Gida and Leka Dulecha). The districts were selected by purposive sampling, however, cluster-sampling technique was used to select the smallest administrative units (villages) and farms/households, and the study element (chickens) was selected by simple random sampling method. Logistic regression analysis was utilized in order to analyse the collated data. A total of 384 serum samples were collected from Nekemte town and the three selected districts of east Wollega zone. Indirect ELISA was used to assay for the existence of anti-ILTV antibodies in the serum. A questionnaire survey was used to identify the potential risk factors. Out of the 384 samples, 15.36% (95% CI: 12.1–19.3) tested positive for anti-ILTV antibodies, with 21.88%, 14.44%, 11.11% and 10.53% prevalence obtained from Nekemte town, Guto Gida, Diga and Leka Dulecha respectively. Univariable logistic regression analysis showed that the chicken source and Sasso breed were found to be significantly associated with ILT seropositivity. Chickens obtained from day old chicks distributers was 2.22 (COR: 2.22, 95% CI: 1.23-4.00) times more likely seropositive than those bought from market. Multivariable logistic regression analysis of potential risk factors revealed that exotic Sasso breeds of chicken were found 9.68 (AOR: 9.68, 95% CI: 1.96-47.78) more likely to be seropositive than local breed. It was concluded that the seroprevalence obtained in this study indicated the circulation of this economically important poultry disease in the study sites and, hence warrants prevention and control.

Development of interactive dashboards for monitoring endemic animal pathogens in Ontario, Canada: Ontario interactive animal pathogen dashboards.

The advancement of web-based technologies makes it possible to build user interfaces or web pages that present and summarize complex data in easy-to-read graphical formats that emphasize key information. Taking advantage of this technologic progress, we addressed the need for real-time visualizations of trends for major pathogens in the largest livestock industries in Ontario: poultry, swine, and cattle. These visualizations were built using test data from the laboratory information management system of the Animal Health Laboratory at the University of Guelph, a large veterinary diagnostic laboratory in Ontario. The data were processed using R software and used to construct interactive and dynamic visualizations using Tableau Desktop v.2021.4 (Tableau Software). We designed 12 dashboards: in chickens-influenza A virus, fowl adenovirus, infectious bronchitis virus, and infectious laryngotracheitis virus; in turkeys-influenza A virus; in swine, influenza A virus, rotavirus, and porcine reproductive and respiratory syndrome virus; in cattle-bovine viral diarrhea virus, Mycoplasma bovis, Salmonella Dublin in individual samples, and Salmonella Dublin in bulk tank milk samples. Data for each pathogen are presented in 2 dashboards. One shows the data of the last 10 y (general view) and the other the data of the last 3 y, but in more detail (comprehensive view). Information on gaining access to all dashboards is available at https://iapd.lsd.uoguelph.ca/. The visualizations provide near-real-time access to aggregated assay results for selected pathogens for veterinarians, animal health regulatory agencies, researchers, and other users who are interested in livestock pathogen surveillance.

Protection Efficacy of the Infectious Laryngotracheitis (ILT) Serva CEO Vaccine Strain in Broiler Chickens Under Different Vaccination Coverage Conditions.

Mass vaccination against infectious laryngotracheitis virus (ILTV) in drinking water can result in variable initial vaccine take. Partial initial vaccine coverage of 20% with an Australian ILT vaccine (A20) previously resulted in significant protection against virulent ILTV challenge. This follow-up study used the international Serva ILT vaccine strain in a factorial design testing four levels of vaccination coverage (0%, 10%, 20%, or 100% of chicks eye-drop vaccinated with the live vaccine at 7 days of age) and three levels of ILTV challenge (no challenge or challenge at 7 or 21 days postvaccination [DPV]). The increase in ILTV load in choanal cleft swabs detected by qPCR after challenge was significantly reduced by 20% and 100% but not by 10% vaccination coverage. Vaccination reduced weight gain in unchallenged birds. Daily weight gain of birds was not affected by ILTV challenge at 7 DPV in any group, but following challenge at 21 DPV, it was significantly reduced in unvaccinated and 10% vaccinated groups relative to 20% and 100% vaccinated groups. Vaccination of 20% of the chickens provided substantial but incomplete protection (protective index range 44%-70%) against the severity of clinical signs and mortality following challenge while 10% vaccination coverage provided limited or no protection. Clinical signs were more severe and appeared earlier following challenge at 21 DPV than at 7 DPV. Within the vaccination treatments, eye-drop-vaccinated birds were better protected than their in-contact cohorts. In conclusion, partial vaccination of 20%, but not 10% of chickens, induced substantial protection against subsequent challenge. However, the attendant risks of reduced protection against early challenge and the possible reversion to virulence of vaccine virus when transmitted to unvaccinated chickens make it essential that 100% initial vaccine take be the goal of mass vaccination programs.

Development of a One-Step Real-Time TaqMan Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay for the Detection of the Novel Variant Infectious Bursal Disease Virus (nVarIBDV) Circulating in China.

The novel variant IBDV (nVarIBDV, especially genotype A2dB1) mainly affects broilers in China. It causes an infection characterized by the atrophy of the bursa, a decrease in the level of lymphocytes, proliferation of fibrous tissue around the follicle, and severe atrophy of the follicle in the bursa. Poultry vaccinated with live IBDV vaccines do not have the challenge present with bursa atrophy, which is misdiagnosed for nVarIBDV because of the lack of other gross clinical symptoms. The present study sought to explore the potential and reliability of the real-time TaqMan analysis method for the detection and discrimination of the nVarIBDV genotype from that of the non-nVarIBDV, especially in live vaccine strains. This method will help monitor vaccinated poultry to control and manage infection with the nVarIBDV IBDVs. The nucleotide polymorphism in the 5'-UTR region and the vp5/vp2 overlapping region of the segment A sequences of IBDV were used to establish a one-step real-time TaqMan reverse transcription polymerase chain reaction (RT-PCR) method in this study. The results showed that the method accurately distinguished the nVarIBDV and non-nVarIBDV strains (especially live vaccine strains), and there were no cross-reactions with the infectious bronchitis virus (IBV), Newcastle disease virus (NDV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), fowlpox virus (FPV), Mycoplasma gallisepticum (M. gallisepticum), Mycoplasma synoviae (M. synoviae), and IBDV-negative field samples. The method showed a linear dynamic range between 102 and 107 DNA copies/reaction, with an average R2 of 0.99 and an efficiency of 93% for nVarIBDV and an average R2 of 1.00 and an efficiency of 94% for non-nVarIBDV. The method was also used for the detection of 84 clinical bursae of chickens vaccinated with the live vaccine. The results showed that this method accurately distinguished the nVarIBDV and non-nVarIBDV strains (vaccine strains), compared with a strategy based on the sequence analysis of HVRs at the vp2 gene or the reverse transcription PCR (RT-PCR) for the vp5 gene. These findings showed that this one-step real-time TaqMan RT-PCR method provides a rapid, sensitive, specific, and simple approach for detection of infections caused by nVarIBDV and is a useful clinical diagnostic tool for identifying and distinguishing nVarIBDV from non-nVarIBDV, especially live vaccine strains.

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.

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 Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale

To date, many fluorescence- and gel-based multiplex polymerase chain reaction (PCR) assays have been developed for the simultaneous detection of multiple infectious agents of respiratory disease in poultry. However, PCR assays are not available for other important emerging respiratory bacteria, such as Ornithobacterium rhinotracheale (ORT). We aimed to fill this gap by establishing a new duplex PCR method for the simultaneous detection of infectious laryngotracheitis virus (ILTV) and ORT. Multiplex primer design software was used to select the compatible multiplex primer pairs. It was determined that an annealing temperature of 65 °C and an initial concentration of 2.5 pmol/µL for each primer set were the most suitable conditions for multiplex PCR. The assay was confirmed to be specific, as it only detected the target pathogens, even in the presence of six non-target agents. The limit of detection was up to 103 copies/µL of template DNA for both ILTV and ORT. In the screening of 304 field samples, 23, 88, and 44 were positive for both ILTV and ORT, solely for ILTV, and solely ORT, respectively.

Codon pair bias deoptimization of essential genes in infectious laryngotracheitis virus reduces protein expression.

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.