Mucosal immune responses in the trachea after chronic infection with Mycoplasma gallisepticum in unvaccinated and vaccinated mature chickens.

Tracheitis associated with the chronic respiratory disease in chickens caused by Mycoplasma gallisepticum is marked by infiltration of leukocytes into the mucosa. Although cytokines/chemokines are known to play a key role in the recruitment, differentiation, and proliferation of leukocytes, those that are produced and secreted into the trachea during the chronic stages of infection with M. gallisepticum have not been described previously. In this study, the levels of transcription in the trachea of genes encoding a panel of 13 cytokines/chemokines were quantified after experimental infection with the M. gallisepticum wild-type strain Ap3AS in unvaccinated chickens and chickens vaccinated 40-, 48- or 57-weeks previously with the novel attenuated strain ts-304. These transcriptional levels in unvaccinated/infected and vaccinated/infected chickens were compared with those of unvaccinated/uninfected and vaccinated/uninfected chickens. Pathological changes and subsets of leukocytes infiltrating the tracheal mucosa were concurrently assessed by histopathological examination and indirect immunofluorescent staining. After infection, unvaccinated birds had a significant increase in tracheal mucosal thickness and in transcription of genes for cytokines/chemokines, including those for IFN-γ, IL-17, RANTES (CCLi4), and CXCL-14, and significant downregulation of IL-2 gene transcription. B cells, CD3+ or CD4+ cells and macrophages (KUL01+ ) accumulated in the mucosa but CD8+ cells were not detected. In vaccinated birds, the levels of transcription of the genes for IL-6, IL-2, RANTES and CXCL-14 were significantly lower after infection than in the unvaccinated/infected and/or unvaccinated/uninfected birds, while the transcription of the IFN-γ gene was significantly upregulated, and there were aggregations of B cells in the tracheal mucosa. These observations indicated that M. gallisepticum may have suppressed Th2 responses by upregulating secretion of IFN-γ and IL-17 by CD4+ cells and induced immune dysregulation characterized by depletion of CD8+ cells and downregulation of IL-2 in the tracheas of unvaccinated birds. The ts-304 vaccine appeared to induce long-term protection against this immune dysregulation. TAKE AWAY: The ts-304 vaccine-induced long-term protection against immune dysregulation caused by M. gallisepticum Detection of B cells and plasma cells in the tracheal mucosa suggested that long-term protection is mediated by mucosal B cell memory Infection of unvaccinated birds with M. gallisepticum resulted in CD8+ cell depletion and downregulation of IL-2 in the tracheal mucosa, suggestive of immune dysregulation Infection of unvaccinated birds with M. gallisepticum resulted in upregulation of IFN-γ and infiltration of CD4+ cells and antigen presenting cells (B and KUL01+ cells) into the tracheal mucosa, suggesting enhanced antigen processing and presentation during chronic infection Th2 responses to infection with M. gallisepticum may be dampened by CD4+ cells through upregulation of IFN-γ and IL-17 during chronic infection.

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

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.

Infectious Laryngotracheitis Virus Viral Chemokine-Binding Protein Glycoprotein G Alters Transcription of Key Inflammatory Mediators In Vitro and In Vivo.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and in vivo infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection.IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.

Single Nucleotide Polymorphism Genotyping Analysis Shows That Vaccination Can Limit the Number and Diversity of Recombinant Progeny of Infectious Laryngotracheitis Viruses from the United States.

Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) causes mild to severe respiratory disease in poultry worldwide. Recombination in this virus under natural (field) conditions was first described in 2012 and more recently has been studied under laboratory conditions. Previous studies have revealed that natural recombination is widespread in ILTV and have also demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In the United States, natural ILTV recombination has also been detected, but not as frequently as in Australia. To better understand recombination in ILTV strains originating from the United States, we developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two virulent U.S. field strains of ILTV (63140 and 1874c5) under experimental in vivo conditions. We also tested the capacity of the Innovax-ILT vaccine (a recombinant vaccine using herpesvirus of turkeys as a vector) and the Trachivax vaccine (a conventionally attenuated chicken embryo origin vaccine) to reduce recombination. The Trachivax vaccine prevented ILTV replication, and therefore recombination, in the trachea after challenge. The Innovax-ILT vaccine allowed the challenge viruses to replicate and to recombine, but at a significantly lower rate than in an unvaccinated group of birds. Our results demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination between these ILTV strains and also show that vaccination can limit the number and diversity of recombinant progeny viruses.IMPORTANCE Recombination allows alphaherpesviruses to evolve over time and become more virulent. Historically, characterization of viral vaccines in poultry have mainly focused on limiting clinical disease, rather than limiting virus replication, but such approaches can allow field viruses to persist and evolve in vaccinated populations. In this study, we vaccinated chickens with Gallid alphaherpesvirus 1 vaccines that are commercially available in the United States and then performed coinoculations with two field strains of virus to measure the ability of the vaccines to prevent field strains from replicating and recombining. We found that vaccination reduced viral replication, recombination, and diversity compared to those in unvaccinated chickens, although the extent to which this occurred differed between vaccines. We suggest that characterization of vaccines could include studies to examine the ability of vaccines to reduce viral recombination in order to limit the rise of new virulent field strains due to recombination, especially for those vaccines that are known not to prevent viral replication following challenge.

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.

Analysis of the complete genomic sequences of two virus subpopulations of the Australian infectious bronchitis virus vaccine VicS.

Although sequencing of the 3' end of the genome of Australian infectious bronchitis viruses (IBVs) has shown that their structural genes are distinct from those of IBVs found in other countries, their replicase genes have not been analysed. To examine this, the complete genomic sequences of the two subpopulations of the VicS vaccine, VicS-v and VicS-del, were determined. Compared with VicS-v, the more attenuated VicS-del strain had two non-synonymous changes in the non-structural protein 6 (nsp6), a transmembrane (TM) domain that may participate in autocatalytic release of the 3-chymotrypsin-like protease, a polymorphic difference at the end of the S2 gene, which coincided with the body transcription-regulating sequence (B-TRS) of mRNA 3 and a truncated open reading frame for a peptide encoded by gene 4 (4b). These genetic differences could be responsible for the differences between these variants in pathogenicity in vivo, and replication in vitro. Phylogenetic analysis of the whole genome showed that VicS-v and VicS-del did not cluster with strains from other countries, supporting the hypothesis that Australian IBV strains have been evolving independently for some time, and analyses of individual polymerase peptide and S glycoprotein genes suggested a distant common ancestor with no recent recombination. This study suggests the potential role of the TM domain in nsp6, the integrity of the S2 protein and the B-TRS 3, and the putative accessory protein 4b, as well as the 3' untranslated region, in the virulence and replication of IBV and has provided a better understanding of relationships between the Australian vaccine strain of IBV and those used elsewhere.

Cross-protective immune responses between genotypically distinct lineages of infectious laryngotracheitis viruses.

Recent phylogenetic studies have identified different genotypic lineages of infectious laryngotracheitis virus (ILTV), and these lineages can recombine in the field. The emergence of virulent recombinant field strains of ILTV by natural recombination between commercial vaccines belonging to different genotypic lineages has been reported recently. Despite the use of attenuated ILTV vaccines, these recombinant viruses were able to spread and cause disease in commercial poultry flocks, raising the question of whether the different lineages of ILTV can induce cross-protective immune responses. This study examined the capacity of the Australian-origin A20 ILTV vaccine to protect against challenge with the class 8 ILTV recombinant virus, the genome of which is predominantly derived from a heterologous genotypic lineage. Following challenge, birds vaccinated via eyedrop were protected from clinical signs of disease and pathological changes in the tracheal mucosa, although they were not completely protected from viral infection or replication. In contrast, the challenge virus induced severe clinical signs and tracheal pathology in unvaccinated birds. This is the first study to examine the ability of a vaccine from the Australian lineage to protect against challenge with a virus from a heterologous lineage. These results suggest that the two distinct genotypic lineages of ILTV can both induce cross-protection, indicating that current commercial vaccines are still likely to assist in control of ILTV in the poultry industry, in spite of the emergence of novel recombinants derived from different genotypic lineages.

Adding yeast extract to culture medium enhances replication of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells.

Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures, the first two of which are commonly used for viral isolation. Previous studies have suggested that foetal bovine serum (FBS) can inhibit coronavirus replication in cell cultures. In this study, the replication of IBV in chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was assessed using two different cell culture media containing FBS or yeast extract (YE) and two different IBV strains. The highest concentrations of viral genomes were observed when the cell culture medium (CEK) contained YE. Similar results were observed in LMH cells. Examination of the infectivity by titration demonstrated that the cell lysate from CEK cell cultures in a medium including YE contained a higher median embryo infectious dose than that from CEK cell cultures in a medium containing FBS. These results indicate that improved replication of IBV in cell cultures can be achieved by replacing FBS with YE in the cell culture medium.

Challenges and recent advancements in infectious laryngotracheitis virus vaccines.

Over the past 80 years, biosecurity measures and vaccines have been used to prevent the occurrence of outbreaks of infectious laryngotracheitis (ILT). Despite these control strategies, ILT continues to have an impact on intensive poultry industries. Attenuated vaccines, particularly those derived by passage in chicken embryos, have been associated with a number of side effects, including residual virulence, transmission to naïve birds, establishment of latent infections with subsequent reactivation and shedding of virus, and reversion to virulence after in vivo passage. Most recently, recombination between attenuated ILT vaccines in the field has been shown to be responsible for the emergence of new virulent viruses that have caused widespread disease. To address some of these issues, new-generation virally vectored recombinant vaccines have been developed and recently released in some countries. In addition, recombinant deletion mutants of ILT virus have been proposed as vaccine candidates. In this review, recent advances in the understanding of the epidemiology of traditionally attenuated ILT vaccines as well as in the development and use of new generation vaccines are examined. Next-generation vaccines, along with more appropriate immunological screening strategies, are identified as particularly promising options to enhance ILT control in the future.

Antimicrobial Consumption in the Livestock Sector in Bhutan: Volumes, Values, Rates, and Trends for the Period 2017-2021.

Data on the use of antimicrobials in humans and livestock may provide evidence to guide policy changes to mitigate the risk of antimicrobial resistance (AMR). However, there is limited information available about antimicrobial use in livestock in low- and middle-income countries, even though these nations are most vulnerable to the impact of AMR. This study aimed to assess the consumption of veterinary antimicrobials in Bhutan and identify areas for improvement to reduce the use of antimicrobials in livestock. National data on livestock numbers and annual procurement of veterinary antimicrobials over five years (2017-2021) were used to calculate rates of antimicrobial consumption and annual national expenditure on veterinary antimicrobials in Bhutan. The rate of antimicrobial consumption in Bhutan was 3.83 mg per population correction unit, which is lower than most countries in Europe, comparable with the rates of consumption in Iceland and Norway, and approximately 120-fold lower than published rates of antimicrobial consumption in South Asian countries, including Nepal and Pakistan. The low rates of antimicrobial consumption by the animal health sector in Bhutan could be attributable to stronger governance of antimicrobial use in Bhutan, higher levels of compliance with regulation, and better adherence to standard guidelines for antimicrobial treatment of livestock.

Comparison of the replication and transmissibility of an infectious laryngotracheitis virus vaccine delivered via eye-drop or drinking-water.

Live attenuated vaccines have been extensively used to control infectious laryngotracheitis (ILT). Most vaccines are registered/recommended for use via eye-drop although vaccination via drinking-water is commonly used in the field. Drinking-water vaccination has been associated with non-uniform protection. Bird-to-bird passage of chick-embryo-origin (CEO) ILT vaccines has been shown to result in reversion to virulence. The purpose of the present study was to examine the replication and transmission of a commercial CEO infectious laryngotracheitis virus (ILTV) vaccine strain following drinking-water or eye-drop inoculation. Two groups of 10 specific-pathogen-free chickens were each vaccinated with Serva ILTV vaccine strain either via eye-drop or drinking-water. Groups of four or five unvaccinated birds were placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days from vaccinated and in-contact birds to assess viral replication and transmission using quantitative polymerase chain reaction. Compared with eye-drop-vaccinated birds, drinking-water-vaccinated birds showed delayed viral replication but had detectable viral DNA for a longer period of time. Transmission to chickens exposed by contact on day 0 of the experiments was similar in both groups. Birds exposed to ILTV by contact with eye-drop vaccinated birds on days 4, 8, 12 and 16 of the experiment had detectable ILTV for up to 8 days post exposure. ILTV was not detected in chickens that were exposed by contact with drinking-water vaccinated birds on day 12 of the experiment or later. Results from this study provide valuable practical information for the use of ILT vaccine.

Comparison of the replication and transmissibility of two infectious laryngotracheitis virus chicken embryo origin vaccines delivered via drinking water.

Infectious laryngotracheitis (ILT) is an acute infectious viral disease that affects chickens, causing respiratory disease, loss of production and mortality in severe cases. Biosecurity measures and administration of attenuated viral vaccine strains are commonly used to prevent ILT. It is notable that most recent ILT outbreaks affecting the intensive poultry industry have been caused by vaccine-related virus strains. The purpose of this study was to characterize and compare viral replication and transmission patterns of two attenuated chicken embryo origin ILT vaccines delivered via the drinking water. Two groups of specific pathogen free chickens were each inoculated with SA-2 ILT or Serva ILT vaccine strains. Unvaccinated birds were then placed in contact with vaccinated birds at regular intervals. Tracheal swabs were collected every 4 days over a period of 60 days and examined for the presence and amount of virus using a quantitative polymerase chain reaction. A rapid increase in viral genome copy numbers was observed shortly after inoculation with SA-2 ILT virus. In contrast, a comparatively delayed virus replication was observed after vaccination with Serva ILT virus. Transmission to in-contact birds occurred soon after exposure to Serva ILT virus but only several days after exposure to SA-2 ILT virus. Results from this study demonstrate in vivo differences between ILT vaccine strains in virus replication and transmission patterns.

Comparative in vivo safety and efficacy of a glycoprotein G-deficient candidate vaccine strain of infectious laryngotracheitis virus delivered via eye drop.

Infectious laryngotracheitis (ILT) is an acute respiratory disease in poultry that is commonly controlled by vaccination with conventionally attenuated virus strains. Despite the use of these vaccines, ILT remains a threat to the intensive poultry industry. Our laboratory has developed a novel candidate vaccine strain of infectious laryngotracheitis virus (ILTV) lacking glycoprotein G (ΔgG-ILTV). The aim of the present study was to directly compare this candidate vaccine with three currently available commercial vaccines in vivo. Five groups of specific-pathogen-free chickens were eye-drop inoculated with one of the three commercial vaccine strains (SA2-ILTV, A20-ILTV or Serva-ILTV), or ΔgG-ILTV, or sterile medium. Vaccine safety was assessed by examining clinical signs, weight gain and persistence of virus in the trachea. Vaccine efficacy was assessed by scoring clinical signs and conducting post-mortem analyses following challenge with virulent virus. Following vaccination, birds that received ΔgG-ILTV had the highest weight gain among the vaccinated groups and had clinical scores that were significantly lower than birds vaccinated with SA2-ILTV or A20-ILTV, but not significantly different from those of birds vaccinated with Serva-ILTV. Analysis of clinical scores, weight gain, tracheal pathology and virus replication after challenge revealed a comparable level of efficacy for all vaccines. Findings from this study further demonstrate the suitability of ΔgG-ILTV as a vaccine to control ILT.

Development and application of a combined molecular and tissue culture-based approach to detect latent infectious laryngotracheitis virus (ILTV) in chickens.

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

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.

Full genomic characterisation of an emerging infectious laryngotracheitis virus class 7b from Australia linked to a vaccine strain revealed its identity.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract illness and substantial economic losses to the commercial poultry industry worldwide. Due to its geographical isolation, Australia has had a unique population of ILTV genotypes, and this has provided the researchers with an excellent opportunity to examine the evolution of herpesviruses. Recent studies on the evolution of ILTV have reported the emergence of recombinant ILTVs in Australian poultry flocks. More recently, there has been an increasing number of field outbreaks caused by ILTV isolates that are indistinguishable from serva vaccine strain using current molecular tests that rely on restriction fragment analysis of selected regions of the viral genome. In this study, whole-genome analysis of one of the field isolates revealed a new class of ILTV, identified here as class 7b, emerged as a result of recombination probably between another recombinant strain and the Serva vaccine strain (now reclassified as 7a). Interestingly, the 7b virus had the highest similarity to class 9, a virus that dominates the ILTV population in Victoria, where 7b has never been reported to date. Also, sequence analysis detected sequences unique to class 10, another recombinant virus that became predominant in some states of Australia between 2013 and 2014 but disappeared since then. These results demonstrate the influence of recombination as a continuous process towards more virulent and transmissible ILTVs.

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.

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 10(3.8) 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.

Replication-independent reduction in the number and diversity of recombinant progeny viruses in chickens vaccinated with an attenuated infectious laryngotracheitis vaccine.

Recombination is closely linked with virus replication and is an important mechanism that contributes to genome diversification and evolution in alphaherpesviruses. Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) is an alphaherpesvirus that causes respiratory disease in poultry. In the past, natural (field) recombination events between different strains of ILTV generated virulent recombinant viruses that have caused severe disease and economic loss in poultry industries. In this study, chickens were vaccinated with attenuated ILTV vaccines to examine the effect of vaccination on viral recombination and diversity following subsequent co-inoculation with two field strains of ILTV. Two of the vaccines (SA2 and A20) prevented ILTV replication in the trachea after challenge, but the level of viral replication after co-infection in birds that received the Serva ILTV vaccine strain did not differ from that of the mock-vaccinated (control) birds. Even though the levels of viral replication were similar in the two groups, the number of recombinant progeny viruses and the level of viral diversity were significantly lower in the Serva-vaccinated birds than in mock-vaccinated birds. In both the mock-vaccinated and Serva-vaccinated groups, a high proportion of recombinant viruses were detected in naïve in-contact chickens that were housed with the co-inoculated birds. Our results indicate that vaccination can limit the number and diversity of recombinant progeny viruses in a manner that is independent of the level of virus replication. It is possible that immune responses induced by vaccination can select for virus genotypes that replicate well under the pressure of the host immune response.

Vaccine efficacy against Indonesian Highly Pathogenic Avian Influenza H5N1: systematic review and meta-analysis.

Indonesia has implemented multiple strategies to control Highly Pathogenic Avian Influenza H5N1 (HPAI/H5N1), including the licensure and use of multiple vaccine formulations. The continuous drift of Indonesian HPAI/H5N1 viruses and emergence of a new clade in 2012 that became dominant in 2016, demands the assessment of commercial vaccine formulations against Indonesian field viruses. Seven databases were explored to identify relevant literature reporting performance of commercial vaccines against Indonesian HPAI/H5N1 viruses. After methodological assessment, data were collated and analyzed to report immunogenicity and vaccine efficacy (VE) to prevent respiratory and cloacal viral shedding 2-day post challenge, and death at the end of the follow-up period. Meta-analyses were performed to assess VE consistency of alternative formulations and to explore sources of heterogeneity in VE. In total, 65 studies and 46 vaccine formulations from 13 articles were grouped per OIE's VE protocols (group 1) and variations of it (groups 2,3,4). We found that concurrence of vaccine-seed and challenge-viruses in a clade designation might be a better proxy of VE than current estimates based on vaccine-homologous HI antibody titers, particularly against current fourth order clade viruses (groups 1&2). Prime-boosting was efficacious across different chicken breeds (group 3), and early vaccination may increase the risk of death (group 4). One Indonesian vaccine was tested against the new dominant clade, conferring consistent protection in chickens but not in ducks. Meta-analyses revealed high inconsistency (I2≥75%) and inefficacy of LPAI formulations against current field viruses, while potential sources of inconsistent VE were formulation of seed-homologous vaccines and the species vaccinated. We conclude that the VE of commercial vaccines in Indonesia changes as Indonesian HPAI/H5N1 evolve into new clades, which should warrant continuous matching between vaccine-seeds and emerging HPAI/H5N1. Furthermore, given the characteristics of the new Indonesian dominant HPAI/H5N1 clade, further studies to confirm VE across species are warranted.