The use of filamentous hemagglutinin adhesin to detect immune responses to Campylobacter hepaticus infections in layer hens.

Campylobacter hepaticus is the aetiological agent of Spotty Liver Disease (SLD). SLD can cause significant production loss and mortalities among layer hens at and around peak of lay. We previously developed an enzyme linked immunosorbent assay (ELISA), SLD-ELISA1, to detect C. hepaticus specific antibodies from bird sera using C. hepaticus total proteins and sera pre-absorbed with Campylobacter jejuni proteins. The high specificity achieved with SLD-ELISA1 indicated the presence of C. hepaticus specific antibodies in sera of infected birds. However, some of the reagents used in SLD-ELISA1 are time consuming to prepare and difficult to quality control. This understanding led to the search for C. hepaticus specific immunogenic proteins that could be used in recombinant forms as antibody capture antigens in immunoassay design. In this study, an immunoproteomic approach that combined bioinformatics analysis, western blotting, and LC MS/MS protein profiling was used, and a fragment of filamentous hemagglutinin adhesin (FHA), FHA1,628-1,899 with C. hepaticus specific antigenicity was identified. Recombinant FHA1,628-1,899 was used as antigen coating on ELISA plates to capture FHA1,628-1,899 specific antibodies in sera of infected birds. SLD-ELISA2, based on the purified recombinant FHA fragment, is more user-friendly and standardizable than SLD-ELISA1 for screening antibody responses to C. hepaticus exposure in hens. This study is the first report of the use of FHA from a Campylobacter species in immunoassays, and it also opens future research directions to investigate the role of FHA in C. hepaticus pathogenesis and its effectiveness as a vaccine candidate.

Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications.

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise L-cysteine, L-lysine and L-arginine. It was found that L-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but L-lysine or L-arginine addition did not enhance growth. Brucella broth supplemented with L-cysteine (0.4 mM), L-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Isoquinoline alkaloids induce partial protection of laying hens from the impact of Campylobacter hepaticus (spotty liver disease) challenge.

Spotty liver disease (SLD) is a serious condition affecting extensively housed laying hens. The causative bacterium was described in 2015 and characterized in 2016 and named Campylobacter hepaticus. Antibiotics are the only tool currently available to combat SLD. However, antimicrobial resistance has already been detected, so finding therapeutic alternatives is imperative. Isoquinoline alkaloids (IQA), such as sanguinarine and chelerythrine, have been shown to have immunomodulatory effects. It has been hypothesized that IQA could ameliorate some of the deleterious effects of SLD. This study aimed to address that hypothesis in an experimental disease induction model. Birds were fed with diets containing 2 different doses of an IQA containing product, 100 mg of product/kg of feed (0.5 ppm of sanguinarine) and 200 mg of product/kg of feed (1.0 ppm of sanguinarine). Two additional groups remained untreated (a challenged positive control and an unchallenged negative control). After 4 wk of treatment, birds from all groups except the negative control group were exposed to C. hepaticus strain HV10. The IQA treated groups showed a reduction in the number of miliary lesions on the liver surface and reduced lesion scores compared with untreated hens. A significant reduction of egg mass was detected 6 d after exposure to C. hepaticus in the untreated group (P = 0.02). However, there was not a significant drop in egg-mass in the IQA groups, especially those fed with a high dose of IQA (P = 0.93). IQA supplementation did not produce significant changes in intestinal villus height and crypt depth but did result in a significant reduction in the proinflammatory cytokine, interleukin-8, in the blood (P < 0.01). Microbiota analysis showed that IQA treatment did not alter the alpha diversity of the cecal microbiota but did produce changes in the phylogenetic structure, with the higher dose of IQA increasing the Firmicutes/Bacteroidetes ratio. Other minor changes in production indicators included an increase in feed consumption (P < 0.01) and an increase in body weight of the treated hens (P < 0.0001). The present study has demonstrated that IQA confers some protection of chickens from the impact of SLD.

Infectious bronchitis virus in Australia: a model of coronavirus evolution - a review.

Infectious bronchitis virus (IBV) was first isolated in Australia in 1962. Ongoing surveillance and characterization of Australian IBVs have shown that they have evolved separately from strains found throughout the rest of the world, resulting in the evolution of a range of unique strains and changes in the dominant wild-type strains, affecting tissue tropism, pathogenicity, antigenicity, and gene arrangement. Between 1961 and 1976 highly nephropathogenic genotype GI-5 and GI-6 strains, causing mortalities of 40% to 100%, predominated, while strains causing mainly respiratory disease, with lower mortality rates, have predominated since then. Since 1988, viruses belonging to two distinct and novel genotypes, GIII and GV, have been detected. The genome organization of the GIII strains has not been seen in any other gammacoronavirus. Mutations that emerged soon after the introduction of vaccination, incursion of strains with a novel lineage from unknown sources, recombination between IBVs from different genetic lineages, and gene translocations and deletions have contributed to an increasingly complex IBV population. These processes and the consequences of this variation for the biology of these viruses provide an insight into the evolution of endemic coronaviruses during their control by vaccination and may provide a better understanding of the potential for evolution of other coronaviruses, including SARS-CoV-2. Furthermore, the continuing capacity of attenuated IBV vaccines developed over 40 years ago to provide protection against viruses in the same genetic lineage provides some assurance that coronavirus vaccines developed to control other coronaviruses may continue to be effective for an extended period.

Evaluation of Residues in Hen Eggs After Exposure of Laying Hens to Water Containing Per- and Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFAS) have been used in aqueous film-forming foams used in firefighting, resulting in soil and groundwater contamination and leading to human exposure via animal products grown in contaminated areas. The present study reports the relationship between PFAS intake by hens and the PFAS concentrations in the edible parts of eggs. Laying hens were exposed via drinking water to different concentrations of 4 PFAS compounds (perfluorooctane sulfonate [PFOS], perfluorohexane sulfonate [PFHxS], perfluorooctanoic acid [PFOA], and perfluorohexanoic acid) over 61 d. Egg PFAS residues were assessed for a further 30 d after exposure ceased. The target concentrations of PFAS were 0, 0.3, 3, 30, and 300 µg/L for the treatment groups T1-T5, respectively; and PFAS residues were determined from the eggs collected every second day. There was a linear correlation between the PFAS concentrations in the drinking water of hens and those detected in the egg, which could be useful in estimating PFAS concentrations in the egg by measuring water concentrations. Exposure of hens to drinking water with PFAS concentrations below the Australian Government Department of Health limits (PFOS and PFHxS, 0.07 µg/L; PFOA, 0.56 µg/L), and with no other sources of PFAS exposure, is unlikely to result in egg PFAS concentrations that would exceed the 10% limit set by Food Standards Australia New Zealand for human consumption. Environ Toxicol Chem 2021;40:735-743. © 2020 SETAC.

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.

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.

Pathogenesis and tissue tropism of natural field recombinants of infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV) is an economically significant respiratory pathogen of poultry. Novel recombinant strains of ILTV have emerged in Australia during the last decade and currently class 9 (CL9) and class 10 (CL10) ILTV are the most prevalent circulating strains. This study conducted a comprehensive investigation of the pathogenesis of these two viral strains. Commercial broiler and specific pathogen free (SPF) chickens were inoculated with varying doses of CL9 or CL10 ILTV and subsequently evaluated for clinical and pathological signs of infection. While no difference in the levels of acute viral replication were observed across the different challenge doses, the severity of clinical signs, tracheal pathology and mortality were dose dependent. Both strains of virus persisted in the respiratory tract for up to 14 days post inoculation (dpi) and could be detected in the lung and feathers with sporadic detection in the liver, spleen or bursa. Given the prevalence of CL9 and CL10 in Australian poultry flocks, this study provides an important foundation for the development of diagnostic and therapeutic approaches for the detection and prevention of ILTV.

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.

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.

Full genome analysis of Australian infectious bronchitis viruses suggests frequent recombination events between vaccine strains and multiple phylogenetically distant avian coronaviruses of unknown origin.

Australian strains of infectious bronchitis virus (IBV) have been evolving independently for many years, with control achieved by vaccination with local attenuated strains. Previous studies have documented the emergence of recombinants over the last 20 years, with the most recent one, Ck/Aus/N1/08, detected in 2008. These recombinants did not appear to be controlled by the vaccines currently in use. In this study we sequenced the complete genomes of three emergent Australian strains of IBV (IBV/Ck/Aus/N1/88, IBV/Ck/Aus/N1/03 and IBV/Ck/Aus/N1/08) and a previously incompletely characterised vaccine strain, IBV/Ck/Aus/Armidale, and compared them to the genome of the vaccine strain VicS. We detected multiple recombination events throughout the genome between wild type viruses and the vaccine strains in all three emergent isolates. Moreover, we found that strain N1/88 was not entirely exogenous, as was previously hypothesised. Rather, it originated from a recombination event involving the VicS vaccine strain. The S glycoprotein genes of N1/88 and N1/03 were known to be genetically distinct from previously characterised circulating strains and from each other, and the original donors of these genes remains unknown. The S1 glycoprotein gene of N1/88, a subgroup 2 strain, shares a high nucleotide identity with the sequence of the S1 gene of the recent isolate N1/08. As the subgroup 2 strains have not been isolated for at least 20 years, it appears likely that an unknown avian coronavirus that was the donor of the S1 glycoprotein sequence of N1/88 in the 1980s is still recombining with IBV strains in the field.

Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR-restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 10(2) or 10(3) plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 10(4) PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain.

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.