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Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant economic losses in the poultry industry globally, in addition to severe animal health and welfare concerns. To date, studying the role of ILTV genes in viral infection, replication or pathogenesis has largely been limited to genes that can be deleted from the ILTV genome and the resultant deletion mutants characterized in vitro or in vivo. However, this approach is not suitable for the study of essential genes. This study trialled two different codon deoptimization techniques that aimed to separately disrupt and downregulate the expression of two ILTV genes, ICP8 and UL12, which are essential or very important in viral replication. The target genes were partially recoded using codon usage deoptimization (CUD) and codon pair bias deoptimization (CPBD) approaches and characterized in vitro. Viruses deoptimized via CPBD showed decreased protein expression as assessed by Western blotting and/or fluorescence microscopy to measure the intensity of the fluorescent marker fused to the target protein. Viruses deoptimized by CUD showed less consistent results, with some mutants that could not be generated or isolated. The results indicate that CPBD is an attractive and viable tool for the study of essential or critically important genes in ILTV. This is the first study, to our knowledge, that utilizes CPBD and CUD techniques for the study of ILTV genes.

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

Wild birds are known reservoirs of bacterial and viral pathogens, some of which have zoonotic potential. This poses a risk to both avian and human health, since spillover into domestic bird populations may occur. In Victoria, wild-caught cockatoos trapped under licence routinely enter commercial trade. The circovirus Beak and Feather Disease Virus (BFDV), herpesviruses, adenoviruses and Chlamydia psittaci have been identified as significant pathogens of parrots globally, with impacts on both aviculture and the conservation efforts of endangered species. In this study, we describe the results of surveillance for psittacid herpesviruses (PsHVs), psittacine adenovirus (PsAdV), BFDV and C. psittaci in wild cacatuids in Victoria, Australia. Samples were collected from 55 birds of four species, and tested using genus or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of known and novel pathogens. There were no clinically observed signs of illness in most of the live birds in this study (96.3%; n = 53). Beak and Feather Disease Virus was detected with a prevalence of 69.6% (95% CI 55.2-80.9). Low prevalences of PsHV (1.81%; 95% CI 0.3-9.6), PsAdV (1.81%; 95% CI 0.3-9.6), and C. psittaci (1.81%; 95% CI 0.3-9.6) was detected. Importantly, a novel avian alphaherpesvirus and a novel avian adenovirus were detected in a little corella (Cacatua sanguinea) co-infected with BFDV and C. psittaci. The presence of multiple potential pathogens detected in a single bird presents an example of the ease with which such infectious agents may enter the pet trade and how novel viruses circulating in wild populations have the potential for transmission into captive birds. Genomic identification of previously undescribed avian viruses is important to further our understanding of their epidemiology, facilitating management of biosecurity aspects of the domestic and international bird trade, and conservation efforts of vulnerable species.

Introduction. Chlamydia psittaci is primarily a pathogen of birds but can also cause disease in other species. Equine reproductive loss caused by C. psittaci has recently been identified in Australia where cases of human disease were also reported in individuals exposed to foetal membranes from an ill neonatal foal in New South Wales. Hypothesis/Gap Statement. The prevalence of C. psittaci in association with equine reproductive over time and in different regions of Australia is not known. Aim. This study was conducted to detect C. psittaci in equine abortion cases in Australia using archived samples spanning 25 years. Methodology. We tested for C. psittaci in 600 equine abortion cases reported in Australia between 1994 to 2019 using a Chlamydiaceae real-time quantitative PCR assay targeting the 16S rRNA gene followed by high-resolution melt curve analysis. Genotyping and phylogenetic analysis was performed on positive samples. Results. The overall prevalence of C. psittaci in material from equine abortion cases was 6.5 %. C. psittaci -positive cases were detected in most years that were represented in this study and occurred in Victoria (prevalence of 7.6 %), New South Wales (prevalence of 3.9 %) and South Australia (prevalence of 15.4 %). Genotyping and phylogenetic analysis showed that the C. psittaci detected in the equine abortion cases clustered with the parrot-associated 6BC clade (genotype A/ST24), indicating that infection of horses may be due to spillover from native Australian parrots. Conclusion. This work suggests that C. psittaci has been a significant agent of equine abortion in Australia for several decades and underscores the importance of taking appropriate protective measures to avoid infection when handling equine aborted material.

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.

Coxiella burnetii causes Q fever in individuals exposed to infected ruminants. Vaccination in 3-4-month-old goats, has been reported to result in significantly greater reduction in C. burnetii shedding compared to goats vaccinated one month before breeding, the most commonly used strategy of controlling Q fever on infected intensively-managed herds. It is possible that an even greater reduction in the number of animals shedding C. burnetii could be achieved if vaccination were administered shortly after protection from colostrum antibodies wanes and animals become susceptible to infection with C. burnetii. This study aimed to evaluate the immunogenicity and safety of a formaldehyde-inactivated phase 1 C. burnetii vaccine in 8-week-old goats. Two injections, four weeks apart, elicited specific IgM and IgG responses in all vaccinated goats (n = 6), while no antibodies were detected in two control groups (n = 12). Swelling at the site of inoculation was observed in all the vaccinated and in 10/11 of the placebo-treated goats but receded after 3 weeks. Weight change and rectal temperatures were also comparable between vaccinated and control goats. The data indicated that this vaccine could be suitable for immunising 8-week-old goats, although further trials to determine level of protection against challenge are required.

The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging, therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.

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 HIGHLIGHTSFollowing 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. 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.

Equine abortion is a cause of severe economic loss to the equine industry. Equine herpesvirus 1 is considered a primary cause of infectious abortion in horses, however other infectious agents can also cause abortion. Abortions due to zoonotic pathogens have implications for both human and animal health. We determined the prevalence of Coxiella burnetii, Leptospira spp. and Toxoplasma gondii in 600 aborted equine foetal tissues that were submitted to our diagnostic laboratories at the University of Melbourne from 1994 to 2019. Using qPCR we found that the prevalence of C. burnetii was 4%. The highest annual incidence of C. burnetii was observed between 1997–2003 and 2016–2018. The prevalence of C. burnetii in Victoria and New South Wales was 3% and 6% respectively. All the samples tested negative for Leptospira spp. and Toxoplasma gondii DNA. Equine herpesvirus 1 DNA was detected at a prevalence of 3%. This study has provided evidence for the presence of C. burnetii in equine aborted foetal tissues in Australia, but the role of C. burnetii as potential cause of abortion in Australia requires further investigation. C. burnetii is a zoonotic disease agent that causes the disease ‘Q fever’ in humans. We recommend that appropriate protective measures should be considered when handling material associated with equine abortions to reduce the risk of becoming infected with C. burnetii.

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. Electronic supplementary material The online version of this article (10.1007/s00705-018-4086-1) contains supplementary material, which is available to authorized users.

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.

Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose-response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

Infectious laryngotracheitis virus (ILTV) encodes several unique genes, including a pair of unique nuclear proteins UL0 and UL[-1] that are expressed during replication in cell culture. Although the UL0 gene has been shown to be dispensable for replication, the role of UL[-1] has not been elucidated. In this study a deletion mutant of ILTV lacking the UL[-1] gene was constructed using homologous recombination. The coding sequences of the gene were replaced with the gene for enhanced green fluorescent protein and the cytomegalovirus major immediate early promoter element. The progeny virus carrying the reporter gene was readily identified using fluorescent microscopy, but was unable to propagate in the permissive cells in the absence of wild type ILTV. Even after plaque purification and fluorescent associated cell sorting the recombinant virus deficient in UL[-1] gene could not be successfully isolated. Our findings suggest that the UL[-1] gene has an important role in ILTV replication.

Purpose. Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas. Methodology. Blood or spleen samples from 648 koalas where tested for KoRV provirus, and subsequently genotyped, using PCRs to detect the pol and env genes respectively. Clinical data was also recorded where possible and analysed in comparison to infection status. Results. The prevalence of KoRV was 24.7 % (160/648). KoRV-A was detected in 141/160 cases, but KoRV-B, a genotype associated with neoplasia in captive koalas, was not detected. The genotype in 19 cases could not be determined. Genomic differences between KoRV in Victoria and type strains may have impacted genotyping. Factors associated with KoRV infection, based on multivariable analysis, were low body condition score, region sampled, and ‘wet bottom’ (a staining of the fur around the rump associated with chronic urinary incontinence). Koalas with wet bottom were nearly twice as likely to have KoRV provirus detected than those without wet bottom (odds ratio=1.90, 95 % confidence interval 1.21, 2.98). Conclusion. Our findings have important implications for the conservation of this iconic species, particularly regarding translocation potential of Victorian koalas.

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.

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.

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

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.

Infectious laryngotracheitis (ILT) is a significant viral disease of chickens in many countries around the globe. In this report the status of ILT in Australia has been used as a model to evaluate the evolution of the ILT viruses (ILTVs). Due to its geographical isolation, Australia harbored a distinct lineage of ILT viruses (ILTV) up to 2007. However examination of the ILT viruses (ILTV) involved in outbreaks between 2007 and 2009 has revealed that many of the outbreaks were caused by two new viral genotypes, class 8 and class 9. These two recombinant viruses were found to emerge as a result of recombination between previously existing live vaccine strains (SA2 and A20), and another live vaccine strain (Serva) introduced into the country in 2007. The new recombinant ILTVs were also shown to possess significantly higher virulence and replication capacity compared with a previously predominant ILTV, class 2. In the current study, examination of a large number of ILTVs isolated from outbreaks between 2009 and 2015 revealed the emergence of yet another recombinant virus (class 10) that appears to have become a predominant genotype in New South Wales. In Victoria however, the recombinant class 9 gradually became the predominant virus, replacing class 2. Therefore, there was an unusual pattern in geographical spread of the newly emerged viruses in different states of the country. These results suggest that ILTV is fast evolving towards a greater transmissibility and therefore greater capacity to spread into ILTV-free areas.

Recombination in alphaherpesviruses allows evolution to occur in viruses that have an otherwise stable DNA genome with a low rate of nucleotide substitution. High-throughput sequencing of complete viral genomes has recently allowed natural (field) recombination to be studied in a number of different alphaherpesviruses, however, such studies have not been applied to equine herpesvirus 1 (EHV-1) or equine herpesvirus 4 (EHV-4). These two equine alphaherpesviruses are genetically similar, but differ in their pathogenesis and epidemiology. Both cause economically significant disease in horse populations worldwide. This study used high-throughput sequencing to determine the full genome sequences of EHV-1 and EHV-4 isolates (11 and 14 isolates, respectively) from Australian or New Zealand horses. These sequences were then analysed and examined for evidence of recombination. Evidence of widespread recombination was detected in the genomes of the EHV-4 isolates. Only one potential recombination event was detected in the genomes of the EHV-1 isolates, even when the genomes from an additional 11 international EHV-1 isolates were analysed. The results from this study reveal another fundamental difference between the biology of EHV-1 and EHV-4. The results may also be used to help inform the future safe use of attenuated equine herpesvirus vaccines.

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.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes upper respiratory tract disease in chickens and significant losses to the poultry industry worldwide. Both antibody and cell-mediated responses are generated against ILTV infection; however, the correlation of humoral immune response with protection against ILTV infection is debatable. To examine if whether antibody responses to individual ILTV glycoproteins are correlated with disease and protection, four ILTV glycoproteins (gD, gE, gG and gJ) were expressed as recombinant proteins and used in conjunction with commercially available recombinant gC and gI in indirect ELISAs to measure post-vaccination and/or post-challenge chicken serum antibodies. Serum optical density (OD) values detected by the whole virus, gC, gI and gJ were significantly higher in birds vaccinated with the Serva vaccine strain compared to the SA2 vaccine strain. However, the mean ODs detected by gD, gE and gG were not significantly different between the vaccine strains. Examination of post-ILTV vaccination sera found that gE was the most antigenic glycoprotein and that gC ODs were strongly correlated with those of gI and gJ, while ODs to gG had a relatively poor correlation with those of other glycoproteins. Moderate to poor correlations were found between microscopic tracheal lesion scores and ODs to individual glycoproteins. Examination of post-vaccination pre-challenge antibodies to individual glycoproteins did not find a strong correlation with protective immunity as measured by the severity of clinical signs, gross lesions, and tracheal viral load. Results from this study demonstrated that systemic antibody titers to individual ILTV glycoproteins C, D, E, G, I and J had a relatively poor correlation to protective immunity.

Viruses in avian hosts can pose threats to avian health and some have zoonotic potential. Hospitals that provide veterinary care for avian patients may serve as a site of exposure of other birds and human staff in the facility to these viruses. They can also provide a useful location to collect samples from avian patients in order to examine the viruses present in wild birds. This study aimed to investigate viruses of biosecurity and/or zoonotic significance in Australian birds by screening samples collected from 409 birds presented to the Australian Wildlife Health Centre at Zoos Victoria's Healesville Sanctuary for veterinary care between December 2014 and December 2015. Samples were tested for avian influenza viruses, herpesviruses, paramyxoviruses and coronaviruses, using genus- or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of both known and novel viruses. A very low prevalence of viruses was detected. Columbid alphaherpesvirus 1 was detected from a powerful owl (Ninox strenua) with inclusion body hepatitis, and an avian paramyxovirus most similar to Avian avulavirus 5 was detected from a musk lorikeet (Glossopsitta concinna). Two distinct novel avian alphaherpesviruses were detected in samples from a sulphur-crested cockatoo (Cacatua galerita) and a tawny frogmouth (Podargus strigoides). Avian influenza viruses and avian coronaviruses were not detected. The clinical significance of the newly detected viruses remains undetermined. Further studies are needed to assess the host specificity, epidemiology, pathogenicity and host-pathogen relationships of these novel viruses. Further genome characterization is also indicated, and would be required before these viruses can be formally classified taxonomically. The detection of these viruses contributes to our knowledge on avian virodiversity. The low level of avian virus detection, and the absence of any viruses with zoonotic potential, suggests low risk to biosecurity and human health.

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.

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.

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens with a worldwide distribution. Differentiating between wild-type and vaccine strains of ILT virus (ILTV) would be useful for enhancing disease control, and in the early stages of a disease outbreak molecular diagnostic tools for the detection and differentiation of the circulating virus could be applied. This study developed TaqMan real-time PCR (qPCR) assays to detect and differentiate the glycoprotein G (gG)-deficient (ΔgG) ILTV candidate vaccine strain of ILTV from ILTV strains that contain the gG gene. The gG+ve and gG-ve ILTV TaqMan assays were used in individual and multiplex format to detect, differentiate, and quantitate ILTV DNA in laboratory and clinical samples. The assays were highly sensitive and highly specific, with a detection limit of 10 viral template copies for each assay. Low interassay coefficients of variation were recorded (0.021-0.042 and 0.013-0.039) for gG+ve and gG-ve TaqMan assays, respectively. The multiplex assay was successfully used to examine the replication kinetics of wild-type and ΔgG strains of ILTV in cultured leghorn male hepatoma cells and embryonated hen eggs under coinfection conditions. The results showed that the TaqMan qPCR assay, along with the ΔgG ILTV vaccine, has the potential to be used in a "Differentiating Infected from Vaccinated Animals" strategy for the control and eradication of ILT.

Vaccination against Coxiella burnetii, the cause of Q fever, is reportedly the only feasible strategy of eradicating infection in ruminant herds. Preventive vaccination of seronegative goats is more effective in reducing shedding of C. burnetii than vaccinating seropositive goats. The age at which goats born on heavily-contaminated farms first seroconvert to C. burnetii has not yet been documented. In a 16-month birth cohort study, the age at which goats seroconverted against C. burnetii was investigated; 95 goats were bled every 2 weeks and tested for antibodies against C. burnetii. Risk factors for seroconversion were explored and goats shedding C. burnetii were identified by testing vaginal swabs taken at the goats’ first kidding using a com1 polymerase chain reaction assay. The first surge in the number of goats with IgM to C. burnetii was observed at week 9. Thus, a first vaccination not later than 8 weeks of age to control C. burnetii in highly contaminated environments is indicated. The odds of seroconversion were 2.0 times higher [95% confidence interval (CI) 1.2, 3.5] in kids born by does with serological evidence of recent infection (IgM seropositive) compared to kids born by IgM seronegative does, suggesting either in utero transmission or peri-parturient infection. The rate of seroconversion was 4.5 times higher (95% CI 2.1, 9.8) during than outside the kidding season, highlighting the risk posed by C. burnetii shed during kidding, even to goats outside the kidding herd. Shedding of C. burnetii at kidding was detected in 15 out of 41 goats infected before breeding. Electronic supplementary material The online version of this article (doi:10.1186/s13567-017-0452-3) contains supplementary material, which is available to authorized users.

Wild populations of the critically endangered woylie (Bettongia penicillata) recently declined by 90% in southwest Western Australia. Increased predation is the leading hypothesis for decline, but disease may be playing a role increasing susceptibility to predation. To explore this possibility, we surveyed woylie populations in the wild, in captivity and in a predator-free sanctuary for exposure to, and infection with, four known pathogens of macropods: herpesviruses, Wallal and Warrego orbiviruses, and Toxoplasma gondii. Our study found two of 68 individuals positive for neutralizing antibodies against known macropodid alphaherpesviruses. Three of 45 individuals were PCR positive for a herpesvirus that was shown to be a novel gammaherpesvirus or a new strain/variant of Potoroid Herpesvirus 1. Further sequence information is required to definitively determine its correct classification. There was no evidence of antibodies to orbivirus Wallal and Warrego serogroups, and all serological samples tested for T. gondii were negative. This is the first report of PCR and serological detection of herpesviruses in the woylie. Positive individuals did not demonstrate clinical signs of herpesviral diseases; therefore, the clinical significance of herpesviruses to wild woylie populations remains unclear. Further monitoring for herpesvirus infections will be important to inform disease risk analysis for this virus and determine temporal trends in herpesvirus activity that may relate to population health and conservation outcomes.

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.

We detected herpesvirus infection in a male yellow-footed antechinus (Antechinus flavipes) and male agile antechinus (Antechinus agilis) during the period of postmating male antechinus immunosuppression and mortality. Histopathologic examination of tissues revealed lesions consistent with herpesvirus infection in the prostate of both animals. Herpesvirus virions were observed by transmission electron microscopy in the prostate tissue collected from the male yellow-footed antechinus. Herpesvirus DNA was detected in prostate, liver, lung, kidney, spleen, and ocular/nasal tissues using a pan-herpesvirus PCR targeting the viral DNA polymerase. Nucleotide sequencing identified a novel herpesvirus from the Gammaherpesvirinae subfamily that we have tentatively designated dasyurid herpesvirus 1 (DaHV-1).

A single nucleotide polymorphism (SNP) has been previously associated with EHV-1 neurological disease in several countries around the world. This disease is very uncommon in Australia and little information is available about the presence of this SNP in Australian EHV-1 isolates. The ORF30 sequence of 66 Australian EHV-1 isolates was determined and the genotype was compared to the disease manifestation of the case from which the virus was isolated. Of the 66 isolates, 61 were from cases of abortion and 5 were cases associated with equine herpesvirus myeloencephalopathy (EHM). There was no association between pathotype and genotype in these isolates. In total, 64 of the 66 isolates encoded N752, including 4 isolates from EHM cases. The ORF30 sequence was also determined for 14 EHV-4 isolates, including 2 isolates from confirmed EHV-4 abortion cases. All 14 EHV-4 isolates had aspartic acid at the position equivalent to EHV-1 AA752. Aspartic acid was also confirmed in this position for the single isolate of AHV-3 sequenced in this study. The nucleotide sequence of ORF68 was also determined and showed considerable genetic heterogeneity in the EHV-1 isolates, however, this ORF was highly conserved among the 14 EHV-4 isolates sequenced, with only one SNP identified among 7 isolates. These results confirm that the EHV1 ORF30 N752 is unique and that the D752 sequence is most likely to be the true parent strain of this virus. We suggest that the abortigenic form of EHV-1 should be considered to be the more recently emerged mutant.

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.

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.

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.

Infectious laryngotracheitis virus (ILTV) causes severe respiratory disease in poultry throughout the world. Recently the role of glycoprotein G (gG) in ILTV pathogenesis has been investigated and it has been shown to have chemokine-binding activity. An ILTV vaccine candidate deficient in gG has been developed and the deletion has been shown to alter the host's immune response to the virus. To understand the effect of the gG gene on transcription of other viral genes, the global expression profile of 72 ILTV genes in gG-deleted and wild-type ILTVs were investigated both in vivo and in vitro using quantitative reverse transcription-polymerase chain reaction. Several genes were differentially expressed in the different viruses in LMH cell cultures or in the tracheas of infected birds, and the expression of a number of genes, including ICP27, gC, gJ, Ul7 and UL40, differed significantly both in vivo and in vitro, suggesting that they had direct or indirect roles in virulence. This study has provided insights into the interactions between gG and other ILTV genes that may have a role in virulence.

Background Felid herpesvirus 1 (FHV-1) causes upper respiratory tract diseases in cats worldwide, including nasal and ocular discharge, conjunctivitis and oral ulceration. The nature and severity of disease can vary between clinical cases. Genetic determinants of virulence are likely to contribute to differences in the in vivo phenotype of FHV-1 isolates, but to date there have been limited studies investigating FHV-1 genetic diversity. This study used next generation sequencing to compare the genomes of contemporary Australian clinical isolates of FHV-1, vaccine isolates and historical clinical isolates, including isolates that predated the introduction of live attenuated vaccines into Australia. Analysis of the genome sequences aimed to assess the level of genetic diversity, identify potential genetic markers that could influence the in vivo phenotype of the isolates and examine the sequences for evidence of recombination. Results The full genome sequences of 26 isolates of FHV-1 were determined, including two vaccine isolates and 24 clinical isolates that were collected over a period of approximately 40 years. Analysis of the genome sequences revealed a remarkably low level of diversity (0.0–0.01 %) between the isolates. No potential genetic determinants of virulence were identified, but unique single nucleotide polymorphisms (SNPs) in the UL28 and UL44 genes were detected in the vaccine isolates that were not present in the clinical isolates. No evidence of FHV-1 recombination was detected using multiple methods of recombination detection, even though many of the isolates originated from cats housed in a shelter environment where high infective pressures were likely to exist. Evidence of displacement of dominant FHV-1 isolates with other (genetically distinct) FHV-1 isolates over time was observed amongst the isolates obtained from the shelter-housed animals. Conclusions The results show that FHV-1 genomes are highly conserved. The lack of recombination detected in the FHV-1 genomes suggests that the risk of attenuated vaccines recombining to generate virulent field viruses is lower than has been suggested for some other herpesviruses. The SNPs detected only in the vaccine isolates offer the potential to develop PCR-based methods of differentiating vaccine and clinical isolates of FHV-1 in order to facilitate future epidemiological studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3050-2) contains supplementary material, which is available to authorized users.

Spillover of viruses from farmed poultry into wild birds is a relatively new area of study at the livestock-wildlife interface. These transmission events can threaten the health of wild birds. There is growing evidence of transmission of vaccine viruses from poultry to wild birds, including attenuated vaccine strains of Newcastle disease virus and infectious bronchitis virus, and also spread of virulent viruses that may have evolved under the pressure of vaccine use, such as Marek's disease virus. Viral contaminants of poultry vaccines, including reticuloendotheliosis virus, may also be transmitted to wild birds and result in disease. New, vectored vaccines are less likely to directly spread to wild birds but this risk may rise as a result of recombination.

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.

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.

A second novel gammaherpesvirus was detected in a free-ranging koala (Phascolarctos cinereus) shown previously to be infected with phascolarctid herpesvirus 1. Analysis of the DNA polymerase gene showed that the virus was genetically distinct from all known gammaherpesviruses. This is the first reported dual gammaherpesvirus infection in an Australian marsupial.

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. The virus is horizontally transmitted and causes large outbreaks of disease. Recent studies have shown that a glycoprotein G deficient candidate vaccine strain of ILTV (ΔgG ILTV) is safe and protects birds from disease following challenge with virulent virus. This study examined the transmission dynamics of this candidate vaccine and of ILTV in field and experimental settings. The reproduction ratio (R₀, average number of secondary infectious cases from a typical infectious case) was calculated from the growth rate of disease epidemics in broiler flocks. Assuming a latent period of 2 days and an infectious period of 4 days R₀ was estimated to be 2.43 (95% CI 2.25-2.69). In experimental settings the transmission characteristics of ΔgG ILTV were similar to those of wildtype virus, and importantly ΔgG ILTV remained safe following one in vivo passage and subsequent infection via contact-exposure. There was minimal transmission of wildtype virus in vaccinated birds. The findings from this study further demonstrate the suitability of ΔgG ILTV for use as a live attenuated vaccine. Knowledge of the basic reproduction ratio of ILTV will be valuable for future studies that aim to improve disease control using vaccination programs.

Infectious bronchitis viruses (IBVs) are group III coronaviruses that infect poultry worldwide. Genetic variations, including whole-gene deletions, are key to IBV evolution. Australian subgroup 2 IBVs contain sequence insertions and multiple gene deletions that have resulted in a substantial genomic divergence from international IBVs. The genomic variations present in Australian IBVs were investigated and compared to those of another group III coronavirus, turkey coronavirus (TCoV). Open reading frames (ORFs) found throughout the genome of Australian IBVs were analogous in sequence and position to TCoV ORFs, except for ORF 4b, which appeared to be translocated to a different position in the subgroup 2 strains. Subgroup 2 strains were previously reported to lack genes 3a, 3b and 5a, with some also lacking 5b. Of these, however, genes 3b and 5b were found to be present but contained various mutations that may affect transcription. In this study, it was found that subgroup 2 IBVs have undergone a more substantial genomic rearrangements than previously thought.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations due to residual pathogenicity and reversion to virulence. To avoid these problems and to better control disease, attention has recently turned towards developing a novel vaccine strain that lacks virulence gene(s). Glycoprotein G (gG) is a virulence factor in ILTV. A gG-deficient strain of ILTV has been shown to be less pathogenic than currently available vaccine strains following intratracheal inoculation of specific pathogen free chickens. Intratracheal inoculation of gG-deficient ILTV has also been shown to induce protection against disease following challenge with virulent virus. Intratracheal inoculation, however, is not suitable for large-scale vaccination of commercial poultry flocks. In this study, inoculation of gG-deficient ILTV via eye-drop, drinking water and aerosol were investigated. Aerosol inoculation resulted in undesirably low levels of safety and protective efficacy. Inoculation via eye-drop and drinking water was safe, and the levels of protective efficacy were comparable with intratracheal inoculation. Thus, gG-deficient ILTV appears to have potential for use in large-scale poultry vaccination programmes when administered via eye-drop or in drinking water.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is currently controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations because of residual pathogenicity and reversion to virulence, suggesting that a novel vaccine strain that lacks virulence gene(s) may enhance disease control. Glycoprotein G (gG) has recently been identified as a virulence factor in ILTV. In this study the immunogenicity and relative pathogenicity of gG deficient ILTV was investigated in SPF chickens. Birds vaccinated with gG deficient ILTV were protected against clinical signs of disease following challenge with virulent ILTV and gG deficient ILTV was also shown to be less pathogenic than currently available commercial vaccine strains. Thus gG deficient ILTV appears to have potential as a vaccine candidate.

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.

The emergence of new variant strains of the poultry pathogen infectious bronchitis virus (IBV) is continually reported worldwide, owing to the labile nature of the large single-stranded RNA IBV genome. High resolution melt curve analysis previously detected a variant strain, N1/08, and the present study confirmed that this strain had emerged as a result of recombination between Australian subgroup 2 and 3 strains in the spike gene region, in a similar manner reported for turkey coronaviruses. The S1 gene for N1/08 had highest nucleotide similarity with subgroup 2 strains, which is interesting considering subgroup 2 strains have not been detected since the early 1990s. SimPlot analysis of the 7.2-kb 3' end of the N1/08 genome with the same region for other Australian reference strains identified the sites of recombination as immediately upstream and downstream of the S1 gene. A pathogenicity study in 2-week-old chickens found that N1/08 had similar pathogenicity for chicken respiratory tissues to that reported for subgroup 2 strains rather than subgroup 3 strains. The results of this study demonstrate that recombination is a mechanism utilized for the emergence of new strains of IBV, with the ability to alter strain pathogenicity in a single generation.

Infectious laryngotracheitis (ILT) is an upper respiratory tract disease in chickens caused by infectious laryngotracheitis virus (ILTV), an alphaherpesvirus. Despite the extensive use of attenuated, and more recently recombinant, vaccines for the control of this disease, ILT continues to affect the intensive poultry industries worldwide. Innate and cell-mediated, rather than humoral immune responses, have been identified as responsible for protection against disease. This review examines the current understandings in innate and adaptive immune responses towards ILTV, as well as the role of ILTV glycoprotein G in modulating the host immune response towards infection. Protective immunity induced by ILT vaccines is also examined. The increasing availability of tools and reagents for the characterisation of avian innate and cell-mediated immune responses are expected to further our understanding of immunity against ILTV and drive the development of new generation vaccines towards enhanced control of this disease.

We isolated a macropodid herpesvirus from a free-ranging eastern grey kangaroo (Macropus giganteous) displaying clinical signs of respiratory disease and possibly neurologic disease. Sequence analysis of the herpesvirus glycoprotein G (gG) and glycoprotein B (gB) genes revealed that the virus was an alphaherpesvirus most closely related to macropodid herpesvirus 2 (MaHV-2) with 82.7% gG and 94.6% gB amino acid sequence identity. Serologic analyses showed similar cross-neutralization patterns to those of MaHV-2. The two viruses had different growth characteristics in cell culture. Most notably, this virus formed significantly larger plaques and extensive syncytia when compared with MaHV-2. No syncytia were observed for MaHV-2. Restriction endonuclease analysis of whole viral genomes demonstrated distinct restriction endonuclease cleavage patterns for all three macropodid herpesviruses. These studies suggest that a distinct macropodid alphaherpesvirus may be capable of infecting and causing disease in eastern grey kangaroos.

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction with the gG-ve ILTV vaccine in a 'differentiating infected from vaccinated animals' approach to the control of ILT.