Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants (Elephas maximus) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 x 106 viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at -80 °C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398) and the previously reported EEHV1B sequence (KC462164), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

Chlamydiosis poses a significant threat to koala health and population viability. Accurate diagnosis of chlamydial infection in koalas at veterinary hospitals informs patient prognosis, biosecurity and determines treatment. However, there is no standardised approach to the diagnosis of chlamydiosis in wildlife hospitals. The performance of available diagnostic tests has not previously been rigorously assessed, and there is a lack of sufficient well-characterised reference samples. This study aimed to estimate the diagnostic accuracy of four molecular detection assays for Chlamydia pecorum infection in koalas using Bayesian latent class analysis (BLCA). Swabs were collected from koalas presented to a wildlife hospital (n = 45) and historical population surveys (n = 48). The four assays that were compared were a qPCR with melt curve that targets 16S rRNA (16SG), a probe-based qPCR assay that targets ompB, and two loop-mediated isothermal amplification (LAMP) assays, targeting MreC and a hypothetical protein (CpecG_0573). The 16SG qPCR and CpecG_0573 LAMP showed the highest overall diagnostic accuracy, with Youden's indices of 83.8 % (95 % posterior credible interval [95 % PCI]: 66.8 %, 93.4 %), and 81.5 % (95 % PCI: 62.5 %, 93.7 %) respectively. The underlying true prevalence of infection in hospitalised koalas (i.e., the latent class) was estimated to be 35.2 % (95 % PCI 21.4, 51.2 %). Combining the 16SG qPCR with the CpecG_0573 LAMP assay at this estimated infection prevalence yielded an excellent positive predictive value (PPV) > 96 %. Therefore, a testing protocol applying these tests in series is recommended for diagnosing chlamydiosis in this population of hospitalised koalas.

BACKGROUND:Varicellovirus felidalpha-1 (FHV-1, previously Felid alphaherpesvirus-1) is a significant cause of upper respiratory tract disease in feline populations. Cats infected with FHV-1 show clinical signs that vary in severity. This can be due to differences in host responses and virus strain virulence. Investigating the gene transcription profiles during infections using FHV-1 strains could inform our understanding of host and viral factors contributing to disease outcomes. This study characterised the transcriptomes of Crandell-Rees feline kidney (CRFK) cells infected with field or vaccine FHV-1 strains to better understand the host response during infection. METHODS:Crandell-Rees feline kidney cells were infected with either the FHV-1 F2 vaccine strain or the 384/75 field strain associated with severe disease. The transcriptomes were characterised using RNA-sequencing. To determine the host cellular transcription profile, the total transcripts were mapped to the cat genome and compared to uninfected cells. To characterise the viral transcription profile, the total reads were mapped to each FHV-1 strain. The differentially expressed host genes between infection strains were compared and further analysed using the PANTHER database to examine host pathway regulation. RESULTS:The findings in this study show the differential host gene expressions induced by FHV-1 compared to uninfected CRFK cells. Genes encoding histone proteins were upregulated, while genes involved in cell adhesion and migration processes were downregulated during infections with FHV-1. Comparative analysis between field and vaccine strains showed similarities and differences in host gene expressions. Notably, upregulated genes unique to the field strain were associated with regulatory proteins involved in the cell cycle, while downregulated host genes in field and vaccine strains showed distinct host gene and pathway expressions involved in immune activation. CONCLUSIONS:This study demonstrates the host and viral gene expressions during FHV-1 infection shows the distinct host responses to field and vaccine strains using an in vitro model. These findings provide a foundation for future transcriptomic investigations in other cell types, including ex-vivo explants systems, to enhance our understanding of host and viral factors contributing to disease outcomes.

Feline upper respiratory tract disease (URTD) is a significant health concern in crowded environments, such as catteries and shelters. Feline calicivirus (FCV), which is endemic in domestic cats, is a major contributor to URTD and can cause a range of diseases that vary in severity. Unlike many other caliciviruses, including human caliciviruses and noroviruses, FCV replicates efficiently in cell culture and is a well-established model for studying calicivirus-host cell interactions. In this study, RNA-sequencing was used to characterise host and viral transcription profiles in Crandell-Rees feline kidney (CRFK) cells infected with either the attenuated F9 vaccine strain or a virulent field strain associated with virulent systemic FCV (VS-FCV) disease. At six hours post-infection, both strains induced the upregulation of inflammatory and stress response pathways, while genes involved in metabolism, cell signalling, and extracellular matrix maintenance were downregulated. Between field and vaccine strains, pathways related to membrane signalling and cytoskeletal organisation were uniquely altered in the field strain, while the F9 vaccine strain distinctly altered chromatin organisation, cytokine signalling and mitochondrial metabolism. These findings demonstrate the virulence of FCV strains differentially influences host gene expression in CRFK cells, which may inform host-pathogen interactions that contribute to FCV pathogenesis and variations in disease outcomes.

ABSTRACT Previous studies have demonstrated the safety and efficacy of a live-attenuated glycoprotein G (gG) deletion mutant vaccine strain of ILTV (∆gG-ILTV). In the current study, transcriptional profiles of chicken tracheal organ cultures (TOCs), 24 h post inoculation with ∆gG-ILTV or the gG-expressing parent wild-type strain, CSW-1 ILTV were explored and compared with the mock-infected TOCs using RNA-seq analysis. Transcriptomes of the vaccine and wild-type ILTV were also compared with each other. Although no viral genes (except for gG) were differentially regulated between the two ILTV-infected TOCs, pair-wise comparison of the transcriptomes of the ∆gG-ILTV or the CSW-1 ILTV-infected TOCs (each compared with mock-infected TOCs) identified the similarities and differences in host gene transcription between them. Several immune checkpoint inhibitors with likely roles in ILTV-mediated immune augmentation, and gene ontologies indicating cytokine response, and cytokine signaling were upregulated in both TOCs. Additionally, several other biological processes, molecular functions, and cellular components were enriched uniquely in the ∆gG-ILTV-infected TOCs, including those that indicated modifications to tracheal extracellular matrix (ECM) structural components, which may have a role in immune modulation in vivo . This study has revealed that the modifications of transcription of host genes during the early stages of ILTV infection are not limited to changes in cytokine or chemokine gene transcription, but several other immune-related genes and ECM components. Moreover, their differential regulation in the ex vivo system appears to be influenced by gG expression, potentially affecting the outcome of ILTV infection in vivo . IMPORTANCE Infectious laryngotracheitis virus (ILTV) remains a serious threat to poultry industries worldwide, causing significant economic losses. The glycoprotein G (gG) of ILTV is a virulence factor and a chemokine-binding protein with immunoregulatory functions. The influence of gG on the transcription of select host chemokine and cytokine genes has been demonstrated previously. This study extends our understanding of the early and localized host–ILTV interactions using genome-wide transcriptome analysis of ILTV-infected chicken tracheal organ cultures, and the role of gG during the process. Differential regulation of genes encoding immune checkpoint inhibitors observed in this study may have a role in ILTV-induced inhibition of type I interferon response, or negative regulation of T cell responses, bringing clarity to these ILTV immune-evasion mechanisms. Furthermore, differential regulation of genes encoding certain structural components and receptors with roles in cell migration, in the absence of gG, is consistent with the immunomodulatory role of ILTV gG.

Antimicrobial resistance (AMR) is a major global threat to human health, animal welfare and sustainable development. Whilst resistance can arise naturally, inappropriate antimicrobial exposure accelerates its emergence. Recognizing this risk, international One Health initiatives emphasize reducing antimicrobial use, especially of drugs considered critical for human medicine. Much of the evidence guiding these efforts comes from food-producing and companion animals, but antimicrobial use in laboratory animals, particularly rodents, remains poorly described. To address this gap, we surveyed all veterinarians certified by the American College of Laboratory Animal Medicine in the United States (921) and Canada (10). A total of 157 veterinarians completed the survey, resulting in a 16.9% overall response rate. The response rate was 16.6% for participants in the United States and 40.0% for those in Canada. Despite a strong response, the small number of eligible veterinarians restricts data extrapolation in the Canadian context. Survey results showed that antimicrobial use is common and routine in rodent vivaria. Overall, 91.7% of respondents reported regular antimicrobial use. Applications included both clinical treatment and research purposes such as induction of microbiome dysbiosis, prophylaxis in immunocompromised rodents and gene induction. Reported drug classes included fluoroquinolones and third-generation cephalosporins, with some use of glycopeptides and carbapenems, all considered critically important to human health. Disposal practices often involved release into sewerage or landfill without inactivation, raising environmental concerns. These findings suggest that antimicrobial use in laboratory rodents may represent an underrecognized contributor to AMR and highlight the importance of targeted stewardship.

A glycoprotein-G-deleted live-attenuated vaccine strain of the infectious laryngotracheitis virus (ILTV), ΔgG-ILTV, is safe and efficacious against ILTV challenge. In the current study, the transcriptome of peripheral blood mononuclear cells (PBMCs) of the ΔgG-ILTV-vaccinated group of specific-pathogen-free chickens were compared to those of the nonvaccinated group at 7 days post-vaccination. Tracheal transcriptomes after challenge with virulent ILTV were compared between groups of the non-vaccinated-challenged and the vaccinated-challenged as well as the non-vaccinated-challenged and the uninfected chickens at 4 to 5 days post-challenge. The clinical outcomes after challenge between these groups were also evaluated. Significant differences were observed in the tracheal transcriptome of the non-vaccinated-challenged birds compared to the other two groups. Enriched gene ontologies and pathways that indicated heightened immune responses and impairments to ciliary and neuronal functions, cell junction components, and potential damages to cartilaginous and extracellular components in the trachea of the non-vaccinated-challenged birds were consistent with their severe tracheal pathology compared to the other two groups. On the contrary, the absence of any difference in the tracheal transcriptome between the vaccinated-challenged and the uninfected birds were reflected by the preservation of tracheal mucosal integrity in both groups and mild infiltration of leukocytes in the vaccinated-challenged birds. The results from this study demonstrated that vaccination with ΔgG-ILTV prevented the changes in tracheal transcriptome induced during ILTV challenge, resulting in clinical protection. Additionally, these results also provide insights into the molecular mechanisms underlying the tracheal pathology induced by ILTV infection.

Q Fever is a zoonotic disease caused by Coxiella burnetii, which can infect a wide range of host species, including humans, domestic animals and wild animals. Domestic livestock are the primary reservoir for human infections and humans are usually considered incidental hosts, with human-to-human infection being exceedingly rare. Because livestock are reservoirs, at-risk groups for Q fever have been considered to be abattoir workers, veterinary personnel, farm workers, livestock handlers and wool shearers. However, there has been an increasing prevalence of human cases that have a history of direct or indirect exposure to wildlife, pointing towards likely wildlife reservoirs. Coxiellosis can be diagnosed using indirect and direct methods. Specific antibodies against C. burnetii are usually detectable within 1–3 weeks of infection in humans and experimental animal models. Anti-phase 2 immunoglobin M (IgM) and immunoglobin G (IgG) antibodies appear first in the early stages of acute infections, followed by a delayed and less pronounced phase 1 IgM and IgG antibody response. Interpretation of polymerase chain reaction (PCR) and serology test results are useful for estimating the date of onset of symptoms or clinical signs and the date of exposure. This allows a time window of exposure to be determined and may assist with identification of likely sources of infection.

Infectious laryngotracheitis virus (ILTV) remains a significant viral disease in the poultry industry worldwide and vaccination has proven to be an invaluable tool for disease control. Vaccine type, dose and route of administration are important parameters that determine the success of vaccination programmes and control strategies. The current study aimed to investigate the optimal dose for drinking-water vaccination with ΔgG-ILTV, an attenuated glycoprotein G-deficient ILTV vaccine that is efficacious when administered by eye-drop. Three groups of 1-week old specific-pathogen-free chickens were vaccinated with increasing doses of ΔgG-ILTV (103.8, 104.3 and 105.0 plaque-forming units per bird) via the drinking-water. Additional groups of birds included an eye-drop vaccination control (n = 20), and two unvaccinated control groups (n = 20 and 10, respectively). Three weeks after vaccination, all groups, except one unvaccinated control group (n = 10), were challenged with virulent ILTV. Vaccine efficacy was assessed after challenge by recording mortality rate and scoring of clinical signs and gross tracheal pathology. Challenge resulted in severe clinical disease and a high mortality rate in unvaccinated birds. Eye-drop vaccination resulted in complete clinical protection against this specific challenge. The efficacy of drinking-water vaccination showed a direct association with the administered vaccine dose. Results from this study highlight the need for improved understanding of virus-host interactions and immunological responses that occur following drinking-water vaccination, in order to improve the efficacy of vaccination strategies that use this route.

Chlamydia psittaci is an obligate, intracellular, bacterial pathogen generally associated with clinical and subclinical infection of birds. It is a zoonotic pathogen in humans causing psittacosis a serious respiratory disease and reported to cause infection in animals including cattle, sheep and horses. Although there have been sporadic reports of disease due to C. psittaci in horses since the last century, reports from Australia over the last decade have highlighted the potential of disease in horses and zoonotic transfer. Epizootics of abortions and stillbirths in mares and serious neonatal disease, termed equine psittacosis (EP), have highlighted the potential of C. psittaci to both cause disease in horses and recognise them as major mammalian vectors for zoonotic transmission. Molecular characterisation techniques for these Australian isolates have demonstrated that the majority of equine associated C. psittaci strains have identified the globally disseminated pathogenic 6BC/ST24 type. Diagnosis is primarily through molecular techniques to identify C. psittaci genomically as seroconversion has not been reliably observed in horses. In tissues from abortion samples histopathological changes typically include lymphohistiocytic placentitis though this is neither a sensitive or specific finding in cases of EP. Neonatal foal illness is characterised by severe interstitial pneumonia and disease is generally fatal. Recognition of EP has heightened both awareness of C. psittaci as an equine abortigenic pathogen and the zoonotic threat that infected horses pose. Personal protective equipment should be donned by exposed personnel and strict biosecurity and control measures should be enforced following equine abortion or foetal loss cases and neonatal illness pending diagnostic evaluation.

INTRODUCTION:Feline calicivirus (FCV) commonly causes upper respiratory tract, oral and ocular infections in species of the family Felidae, with high prevalence amongst domestic cat (Felis catus) populations worldwide. Detection of FCV-specific antibodies in serum provides evidence of previous infection with FCV and an indication of whether a cat may be protected against clinical FCV disease. This study describes the most extensive sampling for anti-FCV antibodies in feral and stray cat populations in Australia, and examines variation in prevalence associated with cat age, sex and location. METHODS:Blood samples were opportunistically collected from 669 feral, stray or Indigenous community cats from the Northern Territory, South Australia, Victoria, south-east Tasmania and south-west New South Wales. The sera were harvested and tested for antibodies capable of neutralising the FCV vaccine strain F9 by serum-virus neutralisation assay. RESULTS:Of the 669 cats tested, 69.7% had detectable FCV-F9-neutralising antibodies (titres ≥5). Maturity was significantly associated with higher seroprevalence and higher antibody titres, with adult cats being more than twice as likely to have detectable FCV-neutralising antibodies than subadults. Male cats had a higher seroprevalence and slightly higher antibody titres than females. Cats living in closer proximity to humans had significantly higher seroprevalences and higher FCV-neutralising antibody titres than feral cats from more remote regions of Australia. CONCLUSION:Australian feral and stray cats have a high risk of natural exposure to and infection with FCV, with the prevalence and levels of pre-existing immunity to FCV being highest amongst adult cats living in highly modified urban, peri-urban and agricultural environments.

This cross-sectional study surveyed veterinarians and facility managers to characterise the use of antimicrobials in laboratory rodent facilities within Australia and New Zealand. Most facilities (71%) reported routine administration of antimicrobials. The indications for antibiotic use reflected those described in publications and differed significantly to reasons for use in non-laboratory animals. Antimicrobials used include those of critical importance to human health, and access to these drugs is unregulated, as prescription-only classes are ordered through research catalogues, without human or veterinary physician prescriptions. The ways in which antimicrobials are used in Australian and New Zealand rodent facilities are likely contributing to antimicrobial resistance within rodent populations, particularly as they are largely administered in drinking water, risking subtherapeutic dosing. Much antimicrobial use reported is unnecessary and could be replaced with changes to husbandry and handling. The generation of resistance in both pathogenic and commensal microbes may also represent a work health and safety issue for humans working with these animals. Reported disposal of antimicrobials included discharge into wastewater, without inactivation, and some respondents reported disposal of substrate, or soiled bedding, nesting material, and disposable enrichment items, from treated animals and medicated feed into landfill, without prior inactivation. Environmental contamination with resistant microbes and antimicrobials is a significant driver of antimicrobial resistance. As such, significant opportunities exist to implement judicious and responsible use of antimicrobials within research rodent facilities in Australia and New Zealand, with a particular focus on instituting aseptic surgery, optimising dosing regimens, and inactivation of medicated water and substrate before disposal.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic bacteria of global public health significance. The organism has a complex, diverse, and relatively poorly understood animal reservoir but there is increasing evidence that macropods play some part in the epidemiology of Q fever in Australia. The aim of this cross-sectional survey was to estimate the animal- and tissue-level prevalence of coxiellosis amongst eastern grey (Macropus giganteus) and red (Osphranter rufus) kangaroos co-grazing with domestic cattle in a Q fever endemic area in Queensland. Serum, faeces and tissue samples from a range of organs were collected from 50 kangaroos. A total of 537 tissue samples were tested by real-time PCR, of which 99 specimens from 42 kangaroos (84% of animals, 95% confidence interval [CI], 71% to 93%) were positive for the C. burnetii IS1111 gene when tested in duplicate. Twenty of these specimens from 16 kangaroos (32%, 95% CI 20% to 47%) were also positive for the com1 or htpAB genes. Serum antibodies were present in 24 (57%, 95% CI 41% to 72%) of the PCR positive animals. There was no statistically significant difference in PCR positivity between organs and no single sample type consistently identified C. burnetii positive kangaroos. The results from this study identify a high apparent prevalence of C. burnetii amongst macropods in the study area, albeit seemingly with an inconsistent distribution within tissues and in relatively small quantities, often verging on the limits of detection. We recommend Q fever surveillance in macropods should involve a combination of serosurveys and molecular testing to increase chances of detection in a population, noting that a range of tissues would likely need to be sampled to confirm the diagnosis in a suspect positive animal.

Abstract Leadbeater's possums (Gymnobelideus leadbeateri) are a critically endangered marsupial found in a restricted area of cold, wet forest in South‐Eastern Australia. The majority of Leadbeater's possums inhabit highland forest, with one outlying lowland population. In 2012, a breeding program was established for the lowland Leadbeater's possums when this genetically distinct population faced imminent extinction. Successful reproduction by highland Leadbeater's possums in the international zoo‐based population between 1970 and 2010 led to the widespread belief that the species bred readily in captivity. Lowland possums have not bred in the 2012–2021 contemporary captive conservation breeding program. This study reviewed the historic captive‐breeding data and found that of the 84% (162/194) that reached reproductive maturity; 37% of males (n = 30) and 39.5% of females (n = 32) bred, and this success was highly skewed towards a subset of highly fecund individuals (14% of females and 15% of males produced 75% and 80% of all offspring). Although lack of reproductive output in the captive lowland animals could be explained if age at mortality was lower than that of highlands possums, comparison of the longevity of highland and lowland animals had no significant difference. Conservation objectives that specify how captive breeding may support in situ recovery of wild populations are integral to the success of captive programs. A lack of reflective analysis of past husbandry records allowed misconceptions of success and approaches implemented in the management of the breeding program, reducing the benefits for the conservation of this high profile threatened species. This case study provides a lesson for the management of conservation breeding programs and illustrates the importance of well‐defined conservation objectives, integration of in situ and ex situ strategies, and the importance of objective, systematic and timely analysis of available evidence to inform management objectives and improve conservation outcomes in real time.

There is a pressing need for effective feral cat management globally due to overabundant feline populations, disease transmission and their destructive impact on biodiversity. Virus-vectored immunocontraception (VVIC) is an attractive method for cat population management. Virus-vectored immunocontraceptives could be self-disseminating through horizontal transmission of the VVIC in feral cat populations, or they may be modified to act as non-transmissible vaccine-type immunocontraceptives for delivery to individual cats. These later constructs may be particularly attractive for use in owned (pet) cats and stray cats but could also be used for feral cats that are caught, vaccinated, and released.Here, we report the construction of three felid alphaherpesvirus 1 (FHV-1) derived immunocontraceptive candidates containing genes that encode for feline zona pellucida subunit 3 (ZP3) and gonadotropin-releasing hormone (GnRH). Two of the vaccine candidates were engineered to include disruptions to the thymidine kinase viral virulence gene to reduce the ability of the vaccines to be horizontally transmitted. Analysis of in vitro growth characteristics and protein expression are reported, and their potential for use as a population management tool for cats is discussed.

Coinfections are known to play an important role in disease progression and severity. Coinfections are common in cats, but no coinfection studies have investigated the in vitro dynamics between feline viral and bacterial pathogens. In this study, we performed co-culture and invasion assays to investigate the ability of common feline bacterial respiratory pathogens, Chlamydia felis and Mycoplasma felis, to replicate in and invade into Crandell-Rees feline kidney cells. We subsequently investigated how coinfection of these feline cells with each bacterium (C. felis or M. felis) and the common feline viral pathogen, felid alphaherpesvirus 1 (FHV-1), affects replication of each agent in this cell culture system. We also investigated the metabolic impact of each co-pathogen using metabolomic analysis of infected and coinfected cells. C. felis was able to invade and replicate in CRFKs, while M. felis had little capacity to invade. During coinfection, FHV-1 replication was minimally affected by the presence of either bacterial pathogen, but bacterial replication kinetics were more affected, particularly in M. felis. Both C. felis and M. felis replicated to higher levels in the presence of a secondary pathogen. Coinfections resulted in reprogramming of the glycolysis pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle. The distinct metabolic profiles of coinfected cells compared to those of cells infected with just one of these three pathogens, as well as the impact of coinfections on viral or bacterial load, suggest strong interactions between these three pathogens and possible synergistic mechanisms enhancing virulence that need further investigation.IMPORTANCEIn the natural world, respiratory pathogens coexist within their hosts, but their dynamics and interactions remain largely unexplored. Herpesviruses, mycoplasmas, and chlamydias are common and significant causes of acute and chronic respiratory and system disease in animals and people, and these diseases are increasingly found to be polymicrobial. This study investigates how coinfection of feline cells between three respiratory pathogens of cats impact each other as well as the host innate metabolic response to infection. Each of these pathogens have been implicated in the induction of feline upper respiratory tract disease in cats, which is the leading cause of euthanasia in shelters. Understanding how coinfection impacts co-pathogenesis and host responses is critical for improving disease management.

Koala populations across the east coast of Australia are under threat of extinction with little known about the presence or distribution of a potential pathogen, phascolartid gammaherpesvirus 1 (PhaHV-1) across these threatened populations. Co-infections with PhaHV-1 and Chlamydia pecorum may be common and there is currently a limited understanding of the impact of these co-infections on koala health. To address these knowledge gaps, archived clinical and field-collected koala samples were examined by quantitative polymerase chain reaction to determine the distribution of PhaHV-1 in previously untested populations across New South Wales and Queensland. We detected PhaHV-1 in all regions surveyed with differences in detection rate between clinical samples from rescued koalas (26%) and field-collected samples from free-living koalas (8%). This may reflect increased viral shedding in koalas that have been admitted into care. We have corroborated previous work indicating greater detection of PhaHV-1 with increasing age in koalas and an association between PhaHV-1 and C. pecorum detection. Our work highlights the need for continued surveillance of PhaHV-1 in koala populations to inform management interventions, and targeted research to understand the pathogenesis of PhaHV-1 and determine the impact of infection and co-infection with C. pecorum.

Infectious laryngotracheitis (ILT) remains a significant concern for the poultry industry worldwide due to its impact on animal welfare and its substantial economic consequences. The disease is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This study investigated in vitro host-virus interactions of a glycoprotein G (gG) deletion mutant vaccine strain of ILTV (ΔgG ILTV), and its parent wild-type strain (CSW-1 ILTV). Inoculations were performed separately for the two strains of ILTV using both a primary (chicken embryonic kidney, CEK) and a continuous culture (leghorn male hepatoma, LMH) of chicken cells. Transcriptome analysis was performed at 12 hours post infection. Each cell-type displayed distinct effects on host and viral gene transcription, with a greater number of viral and host genes differentially transcribed in CEK cells and LMH cells, respectively. Both cell-types infected with either strain demonstrated enrichment of pathways related to signalling, and gene ontologies (GO) associated with chemotaxis. Infection with either strain upregulated both SOCS proteins and certain proto-oncogenes, which may contribute to prolonged viral persistence by promoting immunosuppression and preventing apoptosis, respectively. Patterns of gene transcription related to cytokines, chemokines, endosomal TLRs, and interferon responses, as well as pathways associated with histone acetylation, transport, and extracellular matrix organization were similar within each cell type, regardless of the viral strain. In CEK cells, GO terms and pathways were downregulated uniquely after CSW-1 ILTV infection, indicating a viral-strain specific effect in this cell-type. Overall, this study highlights that the observed differences in host and ILTV gene transcription in vitro were more strongly influenced by the cell-types used rather than the presence or absence of gG. This underscores the importance of cell-line selection in studying host-virus interactions and interpreting experimental results.

Macropods are often implicated as the main native Australian reservoir hosts of Coxiella burnetii (Q fever); however, the maintenance and transmission capacity of these species are poorly understood. The objective of this cross-sectional study was to describe the epidemiology of C. burnetii in a high-density population of eastern grey kangaroos (Macropus giganteus) in a peri-urban coastal nature reserve in New South Wales, Australia. Blood, faeces and swabs were collected from forty kangaroos as part of a population health assessment. Frozen and formalin-fixed tissues were also collected from 12 kangaroos euthanised on welfare grounds. Specimens were tested for C. burnetii using PCR, serology, histopathology and immunohistochemistry. A total of 33/40 kangaroos were seropositive by immunofluorescence assay (estimated true seroprevalence 84%, 95% confidence interval [CI] 69% to 93%), with evidence of rising titres in two animals that had been tested four years earlier. The PCR prevalence was 65% (95% CI 48% to 79%), with positive detection in most sample types. There was no evidence of pathology consistent with C. burnetii, and immunohistochemistry of PCR-positive tissues was negative. These findings indicate that kangaroos are competent maintenance hosts of C. burnetii, likely forming a significant part of its animal reservoir at the study site.

Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant eco-nomic 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.