Infectious Laryngotracheitis Virus Strains Research Articles

Spray vaccination with a Newcastle disease virus (NDV)-vectored infectious laryngotracheitis (ILT) vaccine protects commercial chickens from ILT in the presence of maternally-derived antibodies

ABSTRACT 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.

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.

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.

Comparative full genome sequence analysis of wild-type and chicken embryo origin vaccine-like infectious laryngotracheitis virus field isolates from Canada.

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.

Pathogenicity and vaccine efficacy of two virulent infectious laryngotracheitis virus strains in Egypt.

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. Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-022-03458-3.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

Host Responses Following Infection with Canadian-Origin Wildtype and Vaccine Revertant Infectious Laryngotracheitis Virus.

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.

Assessing the infiltration of immune cells in the upper trachea mucosa after infectious laryngotracheitis virus (ILTV) vaccination and challenge

ABSTRACT 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+, CD8β+, 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+, CD8β+, 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 CD8β+ 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+, CD8β+, 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.

A Cell Line Adapted Infectious Laryngotracheitis Virus Strain (BΔORFC) for in ovo and Hatchery Spray Vaccination Alone or in Combination with a Recombinant HVT-LT Vaccine.

To produce more-stable, live attenuated vaccines for infectious laryngotracheitis virus (ILTV), deletion of genes related to virulence has been extensively pursued. Although its function remains unknown, the open reading frame C (ORF C) is among the genes potentially associated with viral virulence that is nonessential for replication in vitro. Earlier results indicated that the ILT virus with deletion of the ORF C gene (BΔORFC) was suitable and safe for eye drop administration but was not sufficiently attenuated for in ovo administration. The objective of this study was to evaluate the safety and protection efficacy of a cell line-adapted, gene-deleted strain (BΔORFC) of ILTV when administered in ovo and/or spray (SP) by itself, or in combination with the recombinant HVT-LT (rHVT-LT) vaccine. Results indicated that vaccination with the BΔORFC strain, either by itself or in combination with an rHVT-LT vaccine, did not affect hatchability, and only marginal signs of respiratory distress were recorded for groups of chickens that received the BΔORFC strain via SP. The replication and seroconversion induced by the BΔORFC strain after in ovo and SP administration was very limited, whereas the replication of the rHVT-LT vaccine was delayed when combined with the BΔORFC strain in ovo. Compared to rHVT-LT or BΔORFC when administered alone, dual vaccination with rHVT-LT + BΔORFC was more effective in mitigating clinical signs of the disease and reducing challenge virus load in the trachea. To our knowledge, this study provides the first proof of concept that ILTV strains can be sufficiently attenuated for early vaccination in ovo or at hatch; also, this study documented the benefits of using a dual (recombinant and live attenuated) hatchery vaccination strategy for ILTV.

Transmission of infectious laryngotracheitis virus vaccine and field strains: the role of degree of contact and transmission by whole blood, plasma and poultry dust

Understanding the mechanisms of transmission of infectious laryngotracheitis virus (ILTV) is critical to proper control as both vaccine and wild-type strains circulate within chicken flocks with potential adverse consequences. The relative efficiency of transmission by direct contact between chickens and airborne transmission has not been investigated. Furthermore, relatively high levels of ILTV DNA have been detected in poultry dust and blood but the infectivity of these is unknown. In this study, comparison of in-contact and airborne transmission of two vaccine and one field strain of ILTV revealed that all transmitted to 100% of in-contact birds by 6 days post-exposure (dpe). Airborne transmission without contact resulted in 100% transmission by 14 and 17 dpe for the wild-type and Serva vaccine virus but only 27% transmission by 21 dpe for the A20 vaccine virus. The infectivity of dust or extracts of dust and blood or plasma from infected chickens at various stages of infection was assessed by inoculation into susceptible chickens. There was no transmission by any of these materials. In conclusion, direct contact facilitated efficient ILTV transmission but the virus was unable to be transmitted by dust from infected chickens suggestive of a limited role in the epidemiology of ILTV.

Molecular and genetic characterization of avian laryngotracheitis virus isolates obtained in Ukraine

Aim. To conduct a virological, PCR, PCR-RFLP and sequencing study of infectious laryngotracheitis virus (ILTV) isolates obtained from sick and dead chickens at industrial and backyard poultry farms in the eastern region of Ukraine collected over the years 2010–2019 and to establish their pathotype and relationship with internationally occurring strains. Methods. Material for virological studies was collected in the framework of research program of the NSC IEСVM during 2010-2019 in the poultry farms in the North-Eastern region of Ukraine, where the birds with the respiratory clinical signs were found. In total, 28 poultry farms were observed. ILTV isolates were obtained with conventional methods, using 10–12-day-old chicken embryos. A 0,2 ml of 10–20 % suspension of pathological material in PBS was used for inoculation. For in-depth studies, we used 4 isolates of ILTV obtained from sick and dead chickens from industrial and backyard poultry farms in Kharkiv, Luhansk, Donetsk, and Sumy regions from 2010–2019. The identification of ILTV isolates was performed via conventional PCR. The pathotype of ILTV strains was determined using PCR-RFLP (polymerase chain reaction – restriction fragment length polymorphism) analysis. The PCR-RFLP was performed at Royal GD, the Netherlands. The (partial) sequencing of the US8 gene was performed using Sanger sequencing method. The phylogenetic analysis, using sequences of 2 Ukrainian strains (MZ323228, MZ333273) and 17 international gene sequences present in GenBank, was performed using the Maximum Likelihood method. For comparative analysis, sequences of vaccine ILT virus strains were used. Results. Over the years 2010-2019, 7 isolates of ILTV were obtained from sick and dead poultry with typical clinical signs and internal lesions at industrial and backyard farms of the Kharkiv, Donetsk, Luhansk and Sumy regions, and the Autonomous Republic of Crimea. Other avian respiratory viral and bacterial pathogens were not detected. Five isolates were obtained from poultry of industrial holdings where vaccination against ILT is carried out. Using PCR-RFLP analysis of 4 isolates, we found that three of them (Sumy 6-11/19, A 04-12, B 2-10) to belong to vaccine-type ILTV strains and only one, B 59-11strain, belongs to wild-type ILTV. Vaccine-type ILTV strains circulated and possibly still circulate in Ukraine in industrial and backyard poultry farms among both vaccinated and non- vaccinated poultry. An ILTV wild-type strain was obtained from non-vaccinated chickens from a backyard farm, which may indicate an important role of backyard farms in maintaining the circulation of the virus. After partial sequencing and phylogenetic analysis of the ILTV US8 gene the two Ukrainian strains studied were placed into two different clusters: The vaccine-type B 2-10 strain, obtained from sick vaccinated chickens from an industrial farm, was close to vaccine-type strains circulating in, China, Italy and the USA. The wild-type B 59-11strain, obtained from sick non-vaccinated backyard chickens, was located in another cluster and closest to a the wild-type B 59-11 ILTV strain from Brazil. Conclusions. In this article we describe for the first time the characterization of vaccine-type and wild-type isolates of ILTV in industrial and backyard poultry farms, proving their relevance for the poultry production in Ukraine. The results obtained show the need and prospects for further monitoring of ILTV circulation in small backyard poultry farms and in industrial poultry farms, especially following the frequent use for vaccination of live attenuated wild-type ILTV strains in Ukraine. Further molecular, phylogenetic and epidemiological characterization of the strains obtained should be performed in the near future to further precise their attributes, epidemiology and origin.

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.

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).

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.

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.

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.

Airborne Transmission of Vaccinal and Wild Type Infectious Laryngotracheitis Virus and Noninfectivity of Extracts of Excreta from Infected Chickens.

Infectious laryngotracheitis virus (ILTV) is thought to exit the host in respiratory aerosols and enter by inhalation of these. High levels of ILTV DNA have been detected in excreta, raising the possibility of alternative routes of shedding from the host. However, it is not known whether or not the ILTV DNA in excreta represents infective virus. This study investigated transmission of wild type and vaccinal ILTV from infected to susceptible commercial meat chickens. Airborne- and excreta-mediated transmission of two field isolates of ILTV (Classes 9 and 10) and three vaccine strains (SA2, A20, and Serva) were tested. To test airborne transmission, air from isolators containing infected birds was ducted through a paired isolator containing uninfected chickens. To test excreta transmission, aliquots were prepared from excreta containing a high level of ILTV DNA within the first week after infection. Chicks were infected bilaterally by eye drop. Clinical signs were monitored daily and choanal cleft swab samples for ILTV detection by quantitative PCR were collected at 4, 8, 15, 22, and 28 days postinfection (DPI) in the airborne transmission study and at 7 and 14 DPI from the excreta transmission studies. There was no transmission of ILTV from excreta, suggesting that ILTV is inactivated during passage through the gut. All strains of ILTV were transmitted by the airborne route but only to a limited extent for the vaccine viruses. The field viruses induced clinical signs, pathology, and greatly elevated ILTV genome copies in swabs. In summary, these findings confirm the suspected airborne transmission of ILTV, demonstrate differential transmission potential between wild type and vaccine strains by this route, and indicate that excreta is unlikely to be important in the transmission of ILTV and the epidemiology of ILT.

Genomic Stability for PCR Detection of Infectious Laryngotracheitis Virus and Infectious Bronchitis Virus in Poultry Dust Samples Stored Under Different Conditions.

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.

Latency characteristics in specific pathogen-free chickens 21 and 35 days after intra-tracheal inoculation with vaccine or field strains of infectious laryngotracheitis virus

ABSTRACT Latency is an important feature of infectious laryngotracheitis virus (ILTV) yet is poorly understood. This study aimed to compare latency characteristics of vaccine (SA2) and field (CL9) strains of ILTV, establish an in vitro reactivation system and examine ILTV infection in peripheral blood mononuclear cells (PBMC) in specific pathogen-free chickens. Birds were inoculated with SA2 or CL9 ILTV and then bled and culled at 21 or 35 days post-inoculation (dpi). Swabs (conjunctiva, palatine cleft, trachea) and trigeminal ganglia (TG) were examined for ILTV DNA using PCR. Half of the TG, trachea and PBMC were co-cultivated with cell monolayers to assess in vitro reactivation of ILTV infection. ILTV DNA was detected in the trachea of approximately 50% of ILTV‐inoculated birds at both timepoints. At 21 dpi, ILTV was detected in the TG only in 29% and 17% of CL9- and SA2-infected birds, respectively. At 35 dpi, ILTV was detected in the TG only in 30% and 10% of CL9- and SA2-infected birds, respectively. Tracheal organ co-cultures from 30% and 70% of CL9- and SA2-infected birds, respectively, were negative for ILTV DNA at cull but yielded quantifiable DNA within 6 days post-explant (dpe). TG co-cultivation from 30% and 40% of CL9-and SA2-infected birds, respectively, had detectable ILTV DNA within 6 dpe. Latency characteristics did not substantially vary based on the strain of virus inoculated or between sampling timepoints. These results advance our understanding of ILTV latency and reactivation. RESEARCH HIGHLIGHTS Following inoculation, latent ILTV infection was detected in a large proportion of chickens, irrespective of whether a field or vaccine strain was inoculated. In vitro reactivation of latent ILTV was readily detected in tracheal and trigeminal ganglia co-cultures using PCR. ILTV latency observed in SPF chickens at 21 days post-infection was not substantially different to 35 days post-infection.