Infectious Laryngotracheitis Virus Research Articles

Detection and Molecular Characterization of Avian Infectious Laryngotracheitis Virus Isolated from a Breeder Flock

Detection and Molecular Characterization of Avian Infectious Laryngotracheitis Virus Isolated from a Breeder Flock

Detection of Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Slovenia.

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains—three detected in non-vaccinated flocks and one in a flock vaccinated against ILT—were identical or very similar to the chicken embryo–origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections.

Metagenomic Analysis of the Respiratory Microbiome of a Broiler Flock from Hatching to Processing

Elucidating the complex microbial interactions in biological environments requires the identification and characterization of not only the bacterial component but also the eukaryotic viruses, bacteriophage, and fungi. In a proof of concept experiment, next generation sequencing approaches, accompanied by the development of novel computational and bioinformatics tools, were utilized to examine the evolution of the microbial ecology of the avian trachea during the growth of a healthy commercial broiler flock. The flock was sampled weekly, beginning at placement and concluding at 49 days, the day before processing. Metagenomic sequencing of DNA and RNA was utilized to examine the bacteria, virus, bacteriophage, and fungal components during flock growth. The utility of using a metagenomic approach to study the avian respiratory virome was confirmed by detecting the dysbiosis in the avian respiratory virome of broiler chickens diagnosed with infection with infectious laryngotracheitis virus. This study provides the first comprehensive analysis of the ecology of the avian respiratory microbiome and demonstrates the feasibility for the use of this approach in future investigations of avian respiratory diseases.

Pathogenic and Transmission Potential of Wildtype and Chicken Embryo Origin (CEO) Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

Control, Elimination, and Monitoring of Infectious Laryngotracheitis in a Multiage Commercial Layer Pullet Farm in Canada.

A multiage commercial layer pullet operation with a history of chicken embryo (CEO) modified live infectious laryngotracheitis (ILT) virus vaccination suffered severe ILT breaks in 2017. The initial sequencing revealed that the circulating virus was of vaccine origin. Changes to the timing and dosage of CEO ILT vaccine failed to control the outbreak. The clinical resolution of the outbreak occurred with the transition to a turkey herpesvirus vector vaccine given in hatchery followed by a tissue culture origin (TCO) vaccine given before the onset of symptoms on the farm. The circulating ILT viruses were monitored periodically using next generation sequencing. This site became free of ILT virus within one year after implementing the new vaccination program.

Control, Suppression, and Monitoring of Infectious Laryngotracheitis in a Multiage Commercial Layer Pullet Farm in Canada.

A multiage commercial layer pullet operation with a history of chicken embryo-origin (CEO) modified live infectious laryngotracheitis (ILT) virus vaccination suffered severe ILT outbreaks in 2017. The initial sequencing revealed that the circulating virus was of vaccine origin. Changes to the timing and dosage of CEO ILT vaccine failed to control the outbreak. The clinical resolution of the outbreak occurred with the transition to a turkey herpesvirus vector vaccine given in-hatchery, followed by a tissue culture-origin vaccine given on the farm. The circulating ILT viruses were monitored periodically by next-generation sequencing. This site became free of ILT virus within 1 yr after implementing the new vaccination program.

Pathobiological and molecular studies on infectious Laryngotracheitis virus (ILTV) infection in commercial chicken flocks in India

Infectious laryngotracheitis (ILT) is one of the highly infectious and contagious diseases of chickens caused by Gallid Herpesvirus 1, a member of the genus Iltovirus. It is an economically important disease in major poultry producing countries, including India. Despite India having large population of poultry birds, very limited information is available on the disease magnitude and molecular epidemiology. We screened 560 tissue samples collected from necropsied cases of tracheitis and conjunctivitis to diagnose ILT by histopathology, immunohistochemistry and polymerase chain reaction. Out of 560 samples, ILT was diagnosed in 90 (16.07%) cases by histopathology; however, polymerase chain reaction detected ILT virus (ILTV) in 128 (22.85%). Immu-nohistochemistry of representative PCR positive tissues (n=30) from trachea, lungs and conjunctiva detected viral antigen in the lining or denuded epithelial cells in 76.66% (23/30) cases. Analysis of age wise occurrence of ILT cases in our study revealed 47.65% in adult birds over 18 weeks of age, 35.15% in grower birds aged between 7 and18 weeks of age, 17.18% in chicks aged less than 6 weeks. Nucleotide sequence of gene encoding viral thymidine kinase revealed involvement of vaccine and virulent strains of ILTV in the investigated cases. Based on our findings and recent publications, we conclude that there is resurgence or re-emergence of ILT in the chicken flocks in different parts of India, indicating a possible biosecurity breach. Further genetic characterization of virus by whole genome sequencing technologies would decipher the genotype circulating in India, and help to design suitable native vaccine candidate(s).

Циркуляція вірусу інфекційного ларинготрахеїту в птахівничих господарствах України в 2009–2020 роках

Циркуляція вірусу інфекційного ларинготрахеїту в птахівничих господарствах України в 2009–2020 роках

Recombinase-aided amplification–lateral flow dipstick assay—a specific and sensitive method for visual detection of avian infectious laryngotracheitis virus

The purpose of this study was to explore a specific, simple, and sensitive method for diagnosis of avian infectious laryngotracheitis virus. Recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) were combined for labeling the optimized RAA probe with 6-carboxyfluorescein (FAM) and the 5′-end of the downstream primer with biotin, respectively. By optimizing the reaction time, temperature, and primer concentration of RAA, a RAA–LFD assay, which could be used for detection of infectious laryngotracheitis, was established. After the specificity and sensitivity test, the target gene fragments could be amplified by RAA–LFD assay in 20 min under isothermal conditions (37°C), and the amplification products could be visually observed and determined by LFD within 3 min. There was no cross-reaction with nucleic acids of other avian pathogens, the lowest detectable limit of RAA–LFD was 102 copies/μL, and the sensitivity of this method was 100 times higher than that of conventional PCR with the lowest detectable limit of 104 copies/μL. The results showed that RAA–LFD assay was highly sensitive, easy to use, and more suitable for clinical detection.

Molecular detection and genetic characterization of infectious laryngotracheitis virus in poultry in Myanmar

BackgroundAvian infectious laryngotracheitis (ILT) is a highly contagious viral disease that causes severe economic losses to the poultry industry worldwide. In Southeast Asian countries, including Myanmar, poultry farming is a major industry. Although it is known that infectious respiratory pathogens, including infectious laryngotracheitis virus (ILTV), are a major threat to poultry farms, there are no data currently available on the epidemiology of ILTV in Myanmar. Therefore, in this study, we conducted a molecular detection of ILTV in 20 poultry farms in Myanmar.ResultsOf the 57 tested oropharyngeal swabs, 10 were positive for ILTV by polymerase chain reaction of a 647 bp region of the thymidine kinase (TK) gene, giving a prevalence of ILTV of 17.5% (10/57). Further sequencing analysis of infected cell protein 4 (ICP4) gene and glycoprotein B, G, and J (gB, gG, and gJ) genes indicated that these isolates were field strains. Phylogenetic analysis revealed that the Myanmar strains clustered together in a single branch and were closely related to other reference strains isolated from Asian countries.ConclusionsThis study demonstrated the presence of ILTV in poultry farms in Myanmar. The genetic characterization analysis performed provides the fundamental data for epidemiological studies that monitor circulating strains of ILTV in Myanmar.

Analysis of Whole-Genome Sequences of Infectious laryngotracheitis Virus Isolates from Poultry Flocks in Canada: Evidence of Recombination.

Infectious laryngotracheitis virus (ILTV) is a herpes virus that causes an acute respiratory disease of poultry known as infectious laryngotracheitis (ILT). Chicken embryo origin (CEO) and tissue culture origin (TCO) live attenuated vaccines are routinely used for the control of ILT. However, vaccine virus is known to revert to virulence, and it has been recently shown that ILT field viral strains can undergo recombination with vaccinal ILTV and such recombinant ILT viruses possess greater transmission and pathogenicity potential. Based on complete or partial genes of the ILTV genome, few studies genotyped ILTV strains circulating in Canada, and so far, information is scarce on whole-genome sequencing or the presence of recombination in Canadian ILTV isolates. The objective of this study was to genetically characterize the 14 ILTV isolates that originated from three provinces in Canada (Alberta, British Columbia and Quebec). To this end, a phylogenetic analysis of 50 ILTV complete genome sequences, including 14 sequences of Canadian origin, was carried out. Additional phylogenetic analysis of the unique long, unique short and inverted repeat regions of the ILTV genome was also performed. We observed that 71%, 21% and 7% of the ILTV isolates were categorized as CEO revertant, wild-type and TCO vaccine-related, respectively. The sequences were also analyzed for potential recombination events, which included evidence in the British Columbia ILTV isolate. This event involved two ILTV vaccine (CEO) strains as parental strains. Recombination analysis also identified that one ILTV isolate from Alberta as a potential parental strain for a United States origin ILTV isolate. The positions of the possible recombination breakpoints were identified. These results indicate that the ILTV wild-type strains can recombine with vaccinal strains complicating vaccine-mediated control of ILT. Further studies on the pathogenicity of these ILTV strains, including the recombinant ILTV isolate are currently ongoing.

Assessment of A20 infectious laryngotracheitis vaccine take in meat chickens using swab and dust samples following mass vaccination in drinking water

Infectious laryngotracheitis, caused by the alphaherpesvirus infectious laryngotracheitis virus (ILTV), is an important disease of chickens. Partial control of this disease in meat chickens is commonly achieved by mass vaccination with live virus in drinking water. There is a need for a practical test to evaluate vaccination outcomes. For the Serva ILTV vaccine, quantitative real-time PCR (qPCR) enumeration of ILTV genome copies (GC) in flock level dust samples collected at 7–8 days post vaccination (dpv) can be used to differentiate flocks with poor and better vaccine take. This study aimed to validate this approach for A20, another widely used ILT vaccine in Australia. In four meat chicken flocks vaccinated with A20 in water using two different water stabilization times (20 or 40 min), swabs from the trachea and choanal cleft and dust samples were collected at 0, 7, 14 and 21 dpv. ILTV GC detection in swabs and dust was highest at 7 dpv and at this time ILTV GC load in dust was strongly and positively associated with vaccine take in individual birds assessed by swab samples. Choanal cleft swabs provided significantly fewer ILTV positive results than paired tracheal swab samples but the level of ILTV GC detected was similar. Water stabilization time had only minor effects on vaccination response in favour of the shorter time. Location of dust collection had no effect on viral load measured in dust samples. Dust samples collected at 0 and 7 dpv can be used to assess the vaccination status of flocks.

Recombinant Fowlpox Virus Expressing gB Gene from Predominantly Epidemic Infectious Larygnotracheitis Virus Strain Demonstrates Better Immune Protection in SPF Chickens.

Background: Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens. Antigenic mutation of infectious laryngotracheitis virus (ILTV) may result in a vaccination failure in the poultry industry and thus a protective vaccine against predominant ILTV strains is highly desirable. Methods: The full-length glycoprotein B (gB) gene of ILTV with the two mutated synonymous sites of fowlpox virus (FPV) transcription termination signal sequence was cloned into the insertion vector p12LS, which was co-transfected with wild-type (wt) FPV into chicken embryo fibroblast (CEF) to develop a recombinant fowlpox virus-gB (rFPV-gB) candidate vaccine strain. Furthermore, its biological and immunological characteristics were evaluated. Results: The results indicated that gB gene was expressed correctly in the rFPV by indirect immunofluorescent assay and Western blot, and the rFPV-gB provided a 100% protection in immunized chickens against the challenge of predominant ILTV strains that were screened by pathogenicity assay when compared with the commercialized rFPV vaccine, which only provided 83.3%. Conclusion: rFPV-gB can be used as a potential vaccine against predominant ILTV strains.

Isolation, identification and chemical inactivation of infectious laryngotracheitis virus for use as a vaccine candidate

Infectious laryngotracheitis (ILT) is a poultry respiratory disease associated with considerable mortality in chicken and decreasing egg production. Vaccination along with biosecurity measures are considered as the main strategy for ILT control. This study was aimed to evaluate the potency of an inactive ILT vaccine candidate derived from a local ILTV isolate. The isolated virus was characterized and treated with various chemicals and their concentrations. The virus infectivity was entirely abolished by treatment of 3 mM binary ethylene imine following 16 h incubation. The immune response of inactivated ILTV suspension with adjuvans was evaluated in both SPF chickens (experiment-I) and Hyline pullets (experiment-II). Efficacy of the inactivated and live ILT vaccines combination was compared. The results of experiment-I showed that the inactivated antigen induced specific antibody titers against ILTV. In experiment-II, despite the increase in serum antibody level administration of the inactivated antigen alone did not offer sufficient protection. The full protection was found in chickens that received the combination regimen. We conclude that simultaneous administration of the inactivated and live ILT vaccines was efficient for induction of immunity against ILTV. Keywords: infectious laryngotracheitis virus; vaccine; inactivation; immune response.

Superinfection and recombination of infectious laryngotracheitis virus vaccines in the natural host

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1–4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.

Molecular detection and characterization of infectious laryngotracheitis virus in backyard chickens in Turkey

Gallid herpesvirus I (GaHV1) is etiological agent of infectious laryngotracheitis (ILT) and ILT is one of the important disease that included in respiratory infections of chickens. Few studies have been conducted in Turkey and there is no data about the existence of ILT in backyards. The purpose of the study was to document the detection and characterization of GaHV1 in backyard chickens using PCR, RFLP and sequencing. Of the163 tracheal swap samples which were taken from 43 backyard flocks 5 (3.07%) were found to be positive for ILT infection. Positivity was 4.65 % (2/43) at flock level. The nucleic acid sequences of the ICP4 gene compared with ILTV sequences in GenBank and the level of identity differed from 96.82 to 100%. When the sequences of samples compared with TCO strains 99.77-100% homology was observed. The virtual RFLP analysis with the HgaI restriction enzyme characterized strains as having a pattern similar to the vaccine strain TCO. This is the first study that presents presence and characterization of GaHV1 in backyards. Large scale studies are needed to estimate prevalence of ILT in Turkey. Chickens should be monitored and growers should avoid to contacting vaccinated birds with non-vaccinated chickens, to control ILTV outbreaks.

Genotyping of Infectious Laryngotracheitis Virus (ILTV) Isolates from Western Canadian Provinces of Alberta and British Columbia Based on Partial Open Reading Frame (ORF) a and b.

Simple SummaryInfectious laryngotracheitis virus is an economically important acute upper respiratory tract disease in chickens. To control the disease, there are two types of vaccines commercially available, the recombinant viral vector and the live attenuated vaccines. The live attenuated vaccines are effective in disease control, but because of their residual virulence, they can replicate, cause disease, and revert to their original virulent form. Strains of the virus can be categorized as vaccine-related or wild type related. Information is scarce on the type of strains that are circulating in Canada. This study aims to discriminate between wild type and vaccine strains associated with infectious laryngotracheitis cases in the provinces of Alberta and British Columbia between the years 2009–2018. To accomplish this objective, the sequencing of two specific partial genes was performed. As a result, 27 samples from Alberta, and 5 samples from British Columbia were successfully sequenced. From the total samples, ~85% were related to vaccine strains and the rest categorized as wild type. These results reinforce the concern on current practices surrounding vaccination and the need to implement better biosecurity measures.Infectious laryngotracheitis virus (ILTV) causes an acute upper respiratory disease in chickens called infectious laryngotracheitis (ILT). Live attenuated vaccines are effective in disease control; however, they have residual virulence, which makes them able to replicate, cause disease and revert to the original virulent form. Information is scarce on the molecular nature of ILTV that is linked to ILT in Canada. This study aims to determine whether isolates originating from ILT cases in Western Canada are a wild type or vaccine origin. Samples submitted for the diagnosis of ILT between 2009–2018 were obtained from Alberta (AB, n = 46) and British Columbia (BC, n = 9). For genotyping, a Sanger sequencing of open reading frame (ORF) a and b was used. A total of 27 from AB, and 5 from BC samples yielded a fragment of 1751 base pairs (bp). Three of the BC samples classified as group IV (CEO vaccine strains) and 2 as group V (CEO revertant). Of the AB samples, 22 samples clustered with group V, 3 with group VI (wild type), and 2 with group VII, VIII, and IX (wild type). Overall, 17 non-synonymous single nucleotide polymorphisms (SNPs) were detected. Further studies are underway to ascertain the virulence and transmission potential of these isolates.

Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus

A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.

Marked differences in virulence of three Australian field isolates of infectious laryngotracheitis virus in meat and layer chickens

ABSTRACT The objectives of this study were to compare the virulence of contemporary infectious laryngotracheitis virus (ILTV) field isolates of classes 9, 10, and 14 in meat and layer chickens, and to evaluate cloacal and oropharyngeal swabs and dust as sample types for ILTV detection. A total of 211 chickens were divided into groups and inoculated with ILTV class 9, 10, or 14, or sham-inoculated via eye drop at 15 or 22 days of age. Chickens were euthanized at 5 and 9 days post-infection. Virulence was assessed by scoring of clinical signs (conjunctivitis, dyspnoea, and demeanour), ILTV genomic copies (GC) in oropharyngeal and cloacal swabs, mortality and microscopic lesions in conjunctiva and trachea. Class 14 caused subclinical infection, while inoculation with class 9 or class 10 resulted in severe clinical signs and microscopic lesions. Compared to class 14 (2.25 ± 0.36 log10 GC), higher viral load was observed in oropharyngeal swabs of classes 9 (7.86 ± 0.48) and 10 (7.53 ± 0.36), with a higher proportion of positive oropharyngeal and cloacal swabs in the latter groups (P < 0.0001). Viral detection in cloacal swabs was delayed at early stages of infection compared to oropharyngeal swabs. Dust samples from class 9- and class 10-inoculated groups showed a trend towards higher GC than that of class 14. Overall, clinical scores, mortality, viral load, and microscopic lesions were similar for classes 9 and 10, but class 9 caused more severe disease in layer chickens than meat chickens. In summary, ILTV classes 9 and 10 exhibited severe virulence, while class 14 exhibited very mild virulence. RESEARCH HIGHLIGHTS Wide variation in the virulence of three field Australian field ILTV strains. Class 9 and class 10 strains were highly virulent, while class 14 was mildly virulent. The highly virulent strains were associated with significantly higher viral genome copies in various sample types than the mildly virulent strain.

Glycoprotein-C-gene-deleted recombinant infectious laryngotracheitis virus expressing a genotype VII Newcastle disease virus fusion protein protects against virulent infectious laryngotracheitis virus and Newcastle disease virus

To develop an alternative vectored vaccine against both Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV), the glycoprotein C (gC) gene was first deleted from an avirulent ILTV. Based on this gC-deleted ILTV mutant, a recombinant ILTV expressing the fusion protein (F) of a genotype VII NDV (designated ILTV-ΔgC-F) was then constructed. Expression of the NDV F protein in ILTV-ΔgC-F-infected LMH cells was examined with an immunofluorescence assay and western blotting. The F gene was stably maintained in the genome of ILTV-ΔgC-F and the F protein was stably expressed. Compared with the parental virus, ILTV-ΔgC-F demonstrated an increased penetration capacity in vitro, and an increased replication rate in vitro and in vivo. Both the parental virus and ILTV-ΔgC-F were avirulent in chickens. Vaccination of specific-pathogen-free chickens with ILTV-ΔgC-F induced ILTV-specific antibodies, detected with an enzyme-linked immunosorbent assay (ELISA), and provided complete clinical protection against virulent ILTV, although viral shedding and replication were detected in the respiratory tract in the early stage of infection in a very small number of birds. Vaccination with ILTV-ΔgC-F also provided significant protection against challenge with a virulent genotype VII NDV, although the level of NDV-specific antibodies detected with an ELISA was low. Notably, the numbers of birds that were positive for the virulent genotype VII NDV and the replication of the challenge virus NDV in selected target tissues were significantly lower in the ILTV-ΔgC-F-vaccinated chickens than in the control birds. Our results indicate that ILTV-ΔgC-F has potential utility as a bivalent candidate vaccine against both infectious laryngotracheitis and Newcastle disease.