Infectious Laryngotracheitis Virus Vaccine Research Articles

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.

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.

Evaluation of Recombinant Herpesvirus of Turkey Laryngotracheitis (rHVT-LT) Vaccine against Genotype VI Canadian Wild-Type Infectious Laryngotracheitis Virus (ILTV) Infection.

In Alberta, infectious laryngotracheitis virus (ILTV) infection is endemic in backyard poultry flocks; however, outbreaks are only sporadically observed in commercial flocks. In addition to ILTV vaccine revertant strains, wild-type strains are among the most common causes of infectious laryngotracheitis (ILT). Given the surge in live attenuated vaccine-related outbreaks, the goal of this study was to assess the efficacy of a recombinant herpesvirus of turkey (rHVT-LT) vaccine against a genotype VI Canadian wild-type ILTV infection. One-day-old specific pathogen-free (SPF) White Leghorn chickens were vaccinated with the rHVT-LT vaccine or mock vaccinated. At three weeks of age, half of the vaccinated and the mock-vaccinated animals were challenged. Throughout the experiment, weights were recorded, and feather tips, cloacal and oropharyngeal swabs were collected for ILTV genome quantification. Blood was collected to isolate peripheral blood mononuclear cells (PBMC) and quantify CD4+ and CD8+ T cells. At 14 dpi, the chickens were euthanized, and respiratory tissues were collected to quantify genome loads and histological examination. Results showed that the vaccine failed to decrease the clinical signs at 6 days post-infection. However, it was able to significantly reduce ILTV shedding through the oropharyngeal route. Overall, rHVT-LT produced a partial protection against genotype VI ILTV infection.

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.

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.

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.

Ocular exposure to infectious laryngotracheitis virus alters leukocyte subsets in the head-associated lymphoid tissues and trachea of 6-week-old White Leghorn chickens

ABSTRACT Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes acute respiratory disease primarily infecting the upper respiratory tract and conjunctiva. Administration of live attenuated ILTV vaccines via eye drop, drinking water, or by coarse spray elicits protective mucosal immunity in the head-associated lymphoid tissues (HALT), of which conjunctiva-associated lymphoid tissue (CALT) and the Harderian gland (HG) are important tissue components. The trachea, a non-lymphoid tissue, also receives significant influx of inflammatory cells that dictate the outcome of ILTV infection. The objective of this study was to evaluate leukocyte cellular and phenotypic changes in the CALT, HG and trachea following ocular infection with a virulent ILTV strain. At 1, 3, 5, 7 and 9 days post-infection, CALT, HG, and trachea of 6-week-old specific pathogen free (SPF) chickens ocularly-exposed to vehicle or virulent ILTV strain 63140 were dissociated, the cells enumerated and then phenotyped using flow cytometry. The CALT had the highest viral genomic load, which peaked on day 3. In ILTV-infected birds, the CALT had a decreased percentage of leukocytes. This was reflected by decreased numbers of MHCI+MHCII–, MHCI+MHCIIlow+, and CD4+ cells, while IgM+ and MHCI+MHCIIHigh+ expressing cell populations increased. In the HG, the most notable change in cells from ILTV-infected birds was a decrease in IgM expressing cells and histologically, an increase in Mott cells. In summary, an acute, ocular exposure to ILTV strain 63140 in young birds shifts subsets of lymphocyte populations in the CALT and HG with minimal impact on the trachea.

Use of degenerate oligonucleotide primed polymerase chain reaction for detection of chicken anaemia virus contamination in avian viral vaccines

For quality control of biologicals of veterinary use, the absence of extraneous agents needs to be certified. One of the requirements for quality control of avian viral vaccines is to demonstrate freedom from extraneous and adventitious pathogenic agents, like chicken anaemia virus (CAV). In this study, a degenerate oligonucleotide primed PCR (DOP-PCR) for the detection of CAV was developed. Degenerate oligonucleotide primers were selected based on sequences corresponding to conserved regions of VP1 gene. After spiking of CAV genomic DNA to an infectious laryngotracheitis virus (ILTV) vaccine, detection limit for the test was 3.056×10-9 ng/µl. To evaluate the performance of the test, 11 avian viral vaccines including infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and ILTV vaccines from 5 manufacturers were screened for CAV and no contamination was detected. The test described here may provide a rapid, sensitive and specific method for contamination detection of avian viral vaccines with CAV, and may be applied for quality control of live and killed commercial vaccines.

Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos

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

Attenuated infectious laryngotracheitis virus vaccines differ in their capacity to establish latency in the trigeminal ganglia of specific pathogen free chickens following eye drop inoculation.

Infectious laryngotracheitis (ILT) is a respiratory disease that affects chickens. It is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This virus undergoes lytic replication in the epithelial cells of the trachea and upper respiratory tract (URT) and establishes latent infection in the trigeminal ganglia (TG) and trachea. Live attenuated vaccines are widely used to control ILT. At least one of these vaccines can establish latent infections in chickens, but this has not been demonstrated for all vaccines. The aim of the current study was to determine the capacity of three commercially available vaccines (SA2, A20 and Serva) and a glycoprotein G deletion mutant vaccine candidate (ΔgG ILTV) to establish latent infection in the TG of specific pathogen free (SPF) chickens. Five groups of 7-day-old SPF chickens were eye-drop vaccinated with either one of the vaccine strains or mock-vaccinated with sterile media and followed until 20 or 21 days post-vaccination (dpv). ILTV DNA was detected at 20–21 dpv in the TG of 23/40 (57.5%) vaccinated SPF chickens (SA2 = 10/10; A20 = 6/10; Serva = 3/10; ΔgG = 4/10) by PCR, but virus could not be reactivated from TG co-cultivated with primary chicken embryo kidney cells. In the birds from which ILTV DNA was detected in the TG, ILTV DNA could not be detected in the URT or trachea of 3 birds in each of the SA2, A20 and Serva vaccinated groups, and in 4 birds in the ΔgG vaccinated group, indicating that these birds were latently infected in the absence of active lytic replication and virus shedding. Results from this study demonstrate the capacity of commercial ILTV vaccines to establish latent infections and underline their importance in the epidemiology of this disease.

Comparative genome analysis of Korean field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis (ILT) virus (ILTV) vaccines have been used to prevent and control the outbreak of ILT worldwide. Recent studies using high-throughput sequencing technology have increased the number of complete genome sequences of ILTVs, enabling comparative genome analysis. Although 37 complete genome sequences of ILTV, including vaccine strains, have been reported, the complete genome sequence of any field strain of ILTV in South Korea is yet to be published. In this study, we determined and analyzed the complete genome sequences of three virulent Korean field strains of ILTV (40798/10/Ko, 0206/14/Ko, and 30678/14/Ko). Two of the Korean field strains (40798/10/Ko and 0206/14/Ko) displayed fewer non-synonymous single nucleotide polymorphisms than those of the Serva vaccine strain, indicating that these Korean field strains of ILTV most likely originated from the vaccine strain. The third ILTV strain, 307678/14/Ko, had two regions in the genome showing recombination between the Serva vaccine-like strain and the Australian A20 vaccine-like strain. Comparative genome analysis of ILTV using the Korean field strains with variable virulence can shed light on the recent trend of the emergence of virulent ILTV strains in the field. A few amino acid changes in the genome of ILTV vaccines could enhance the virulence in the vaccine strain, and natural recombination should be considered one of the major risks for the generation of revertant strains of ILTV under field conditions.

Determination of the minimum protective dose of a glycoprotein-G-deficient infectious laryngotracheitis virus vaccine delivered via eye-drop to week-old chickens.

Infectious laryngotracheitis (ILT) is an upper respiratory tract disease of chickens that is caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. This disease causes significant economic loses in poultry industries worldwide. Despite widespread use of commercial live attenuated vaccines, many poultry industries continue to experience outbreaks of disease caused by ILTV. Efforts to improve the control of this disease have resulted in the generation of new vaccine candidates, including ILTV mutants deficient in virulence factors. A glycoprotein G deletion mutant vaccine strain of ILTV (ΔgG ILTV), recently licenced as Vaxsafe ILT (Bioproperties Pty Ltd), has been extensively characterised in vitro and in vivo, but the minimum effective dose required to protect inoculated animals has not been determined. This study performed a vaccination and challenge experiment to determine the minimum dose of ΔgG ILTV that, when delivered by eye-drop to seven-day-old specific pathogen-free chickens, would protect the birds from a robust challenge with a virulent field strain of virus (class 9 ILTV). A dose of 103.8 plaque forming units was the lowest dose capable of providing a high level of protection against challenge, as measured by clinical signs of disease, tracheal pathology and virus replication after challenge. This study has shown that the ΔgG ILTV vaccine strain is capable of inducing a high level of protection against a virulent field virus at a commercially feasible dose. These results lay the foundations upon which a commercial vaccine can be developed, thereby offering the potential to provide producers with another important tool to help control ILTV.

Development and application of high-resolution melting analysis for the classification of infectious laryngotracheitis virus strains and detection of recombinant progeny

Live attenuated vaccines against infectious laryngotracheitis virus (ILTV) are widely used in the poultry industry to control disease and help prevent economic losses. Molecular epidemiological studies of currently circulating strains of ILTV within poultry flocks in Australia have demonstrated the presence of highly virulent viruses generated by genomic recombination events between vaccine strains. In this study, high-resolution melting (HRM) analysis was used to develop a tool to classify ILTV isolates and to investigate ILTV recombination. The assay was applied to plaque-purified progeny viruses generated after co-infection of chicken embryo kidney (CEK) monolayers with the A20 and Serva ILT vaccine strains and also to viruses isolated from field samples. The results showed that the HRM analysis is a suitable tool for the classification of ILTV isolates and can be used to detect recombination between ILTV vaccine strains in vitro. This method can be used to classify a broad range of ILTV strains to facilitate the classification and genotyping of ILTV and help to further understand recombination in these viruses.

Analysis of codon usage pattern of infectious laryngotracheitis virus immunogenic glycoproteins and its biological implications

Infectious laryngotracheitis virus (ILTV) is a highly contagious acute respiratory poultry pathogen. Modified live ILTV vaccines are the only control against ILT infections. Reversions and establishment of latent infections are the major concerns imparting the need to develop safer vaccines against ILTV infection. ILTV glycoprotein B and D (gB and gD) are major protective immunogens. The factors shaping synonymous codon usage bias and nucleotide composition in ILTV glycoprotein genes have not yet been reported. In the present study, we have analyzed the synonymous codon usage indices of ILTV gB and gD genes. Variation in the codon usage was seen in both the glycoproteins majorly by mutational pressure. The pattern was determined using the correspondence analysis, effective number of codon (Nc), GC3 plot and correlation analyses among different indices. The study is a comprehensive analysis of the codon usage patterns of ILTV glycoprotein genes. This will be helpful in understanding the codon usage bias of ILTV and related DNA viruses which could further explore its biology.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome.IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease.

Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host.

To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

Early infection with Marek’s disease virus can jeopardize protection conferred by laryngotracheitis vaccines: a method to study MDV-induced immunosuppression

ABSTRACTMarek’s disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

Attenuation and protection efficacy of ORF C gene-deleted recombinant of infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by infectious laryngotracheitis virus (ILTV). The disease is controlled by the use of live-attenuated vaccines. Previously we reported the complete nucleotide sequence of the ILTV vaccine strain (TCO) and identified a nonsense mutation in the gene encoding the ORF C protein. This suggested that the ORF C protein might be associated with viral virulence. To investigate this, an ILTV recombinant with a deletion in the gene encoding ORF C was constructed using the genome of the virulent United States Department of Agriculture (USDA) challenge strain (USDAch). Compared to the parental virus, the ΔORF C recombinant replicated in chicken kidney (CK) cells with similar kinetics and generated similar titres. This demonstrated that the ORF C deletion had no deleterious effects on replication efficacy in vitro. In chickens, the recombinant induced only minor microscopic tracheal lesions when inoculated via the intra-tracheal/ocular route, while the parental strain induced moderate to severe microscopic tracheal lesions, even though virus load in the tracheas were comparable. Groups of chickens vaccinated via eye-drop with the ∆ORFC-ILTV were protected to levels comparable to those elicited by TCO vaccination. To our knowledge, this is the first report that demonstrates the suitability of ∆ORFC as a live-attenuated vaccine to prevent the losses caused by ILTV.

Protection Induced in Broiler Chickens following Drinking-Water Delivery of Live Infectious Laryngotracheitis Vaccines against Subsequent Challenge with Recombinant Field Virus.

Infectious laryngotracheitis virus (ILTV) causes acute upper respiratory tract disease in chickens. Attenuated live ILTV vaccines are often used to help control disease, but these vaccines have well documented limitations, including retention of residual virulence, incomplete protection, transmission of vaccine virus to unvaccinated birds and reversion to high levels of virulence following bird-to-bird passage. Recently, two novel ILTV field strains (class 8 and 9 ILTV viruses) emerged in Australia due to natural recombination between two genotypically distinct commercial ILTV vaccines. These recombinant field strains became dominant field strains in important poultry producing areas. In Victoria, Australia, the recombinant class 9 virus largely displaced the previously predominant class 2 ILTV strain. The ability of ILTV vaccines to protect against challenge with the novel class 9 ILTV strain has not been studied. Here, the protection induced by direct (drinking-water) and indirect (contact) exposure to four different ILTV vaccines against challenge with class 9 ILTV in commercial broilers was studied. The vaccines significantly reduced, but did not prevent, challenge virus replication in vaccinated chickens. Only one vaccine significantly reduced the severity of tracheal pathology after direct drinking-water vaccination. The results indicate that the current vaccines can be used to help control class 9 ILTV, but also indicate that these vaccines have limitations that should be considered when designing and implementing disease control programs.