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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.

The Leadbeater's possum (Gymnobelideus leadbeateri) is a critically endangered marsupial in south-eastern Australia. Among other conservation efforts, free-ranging animals in the two remaining geographically separate populations (highland and lowland) have been extensively studied; however, little is known about their health and mortality. Although some wild populations are frequently monitored, cadavers are rarely recovered for post mortem examination. In June 2019, a recently deceased, wild, adult male lowland Leadbeater's possum was collected from a nest box and a comprehensive post mortem examination was conducted. Microfilariae of a filarioid nematode were observed in testes, liver, lung and skin samples in tissue impression smears and upon histopathological examination. No gross or histological changes were seen associated with the parasites, except for a focal area of tissue damage in the skin, suggesting that the possum is a natural host. Using a PCR-coupled sequencing method the filarioid was identified as a species of Breinlia. Species of Breinlia occur in other Australian marsupials and rodents.

Kangaroos are considered to be an important reservoir of Q fever in Australia, although there is limited knowledge on the true prevalence and distribution of coxiellosis in Australian macropod populations. Serological tests serve as useful surveillance tools, but formal test validation is needed to be able to estimate true seroprevalence rates, and few tests have been validated to screen wildlife species for Q fever. In this study, we modified and optimized a phase-specific indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in macropod sera. The assay was validated against the commercially available ID Screen Q fever indirect multispecies enzyme-linked immunosorbent assay (ELISA) kit (IDVet, Grabels, France) to estimate the diagnostic sensitivity and specificity of each assay, using Bayesian latent class analysis. A direct comparison of the two tests was performed by testing 303 serum samples from 10 macropod populations from the east coast of Australia and New Zealand. The analysis indicated that the IFA had relatively high diagnostic sensitivity (97.6% [95% credible interval [CrI], 88.0 to 99.9]) and diagnostic specificity (98.5% [95% CrI, 94.4 to 99.9]). In comparison, the ELISA had relatively poor diagnostic sensitivity (42.1% [95% CrI, 33.7 to 50.8]) and similar diagnostic specificity (99.2% [95% CrI, 96.4 to 100]) using the cutoff values recommended by the manufacturer. The estimated true seroprevalence of C. burnetii exposure in the macropod populations included in this study ranged from 0% in New Zealand and Victoria, Australia, up to 94.2% in one population from New South Wales, Australia.

BackgroundEquid gammaherpesvirus 2 (EHV2) is a gammaherpesvirus with a widespread distribution in horse populations globally. Although its pathogenic significance can be unclear in most cases of infection, EHV2 infection can cause upper respiratory tract disease in foals. Co-infection of different strains of EHV2 in an individual horse is common. Small regions of the EHV2 genome have shown considerable genetic heterogeneity. This could suggest genomic recombination between different strains of EHV2, similar to the extensive recombination networks that have been demonstrated for some alphaherpesviruses. This study examined natural recombination and genome diversity of EHV2 field isolates. ResultsWhole genome sequencing analysis of 18 EHV2 isolates, along with analysis of two publicly available EHV2 genomes, revealed variation in genomes sizes (from 173.7 to 184.8 kbp), guanine plus cytosine content (from 56.7 to 57.8 %) and the size of the terminal repeat regions (from 17,196 to 17,551 bp). The nucleotide sequence identity between the genomes ranged from 86.2 to 99.7%. The estimated average inter-strain nucleotide diversity between the 20 EHV2 genomes was 2.9%. Individual gene sequences showed varying levels of nucleotide diversity and ranged between 0 and 3.8%. The ratio of nonsynonymous substitutions, Ka, to synonymous substitutions, Ks, (Ka/Ks) suggests that over 50% of EHV2 genes are undergoing diversifying selection. Recombination analyses of the 20 EHV2 genome sequences using the recombination detection program (RDP4) and SplitsTree revealed evidence of viral recombination. ConclusionsAnalysis of the 18 new EHV2 genomes alongside the 2 previously sequenced genomes revealed a high degree of genetic diversity and extensive recombination networks. Herpesvirus genome diversification and virus evolution can be driven by recombination, and our findings are consistent with recombination being a key mechanism by which EHV2 genomes may vary and evolve.

Chlamydia psittaci was detected by PCR in the lung and equine foetal membranes of two aborted equine foetuses and one weak foal from two different studs in Victoria, Australia. The abortions occurred in September 2019 in two mares sharing a paddock northeast of Melbourne. The weak foal was born in October 2019 in a similar geographical region and died soon after birth despite receiving veterinary care. The detection of C. psittaci DNA in the lung and equine foetal membranes of the aborted or weak foals and the absence of any other factors that are commonly associated with abortion or neonatal death suggest that this pathogen may be the cause of the reproductive loss. The detection of C. psittaci in these cases is consistent with the recent detection of C. psittaci in association with equine abortion in New South Wales. These cases in Victoria show that C. psittaci, and the zoonotic risk it poses, should be considered in association with equine reproductive loss in other areas of Australia.

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.

Background Abortion in horses leads to economic and welfare losses to the equine industry. Most cases of equine abortions are sporadic, and the cause is often unknown. This study aimed to detect potential abortigenic pathogens in equine abortion cases in Australia using metagenomic deep sequencing methods. Results After sequencing and analysis, a total of 68 and 86 phyla were detected in the material originating from 49 equine abortion samples and 8 samples from normal deliveries, respectively. Most phyla were present in both groups, with the exception of Chlamydiae that were only present in abortion samples. Around 2886 genera were present in the abortion samples and samples from normal deliveries at a cut off value of 0.001% of relative abundance. Significant differences in species diversity between aborted and normal tissues was observed. Several potential abortigenic pathogens were identified at a high level of relative abundance in a number of the abortion cases, including Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus equi subspecies zooepidemicus, Pantoea agglomerans, Acinetobacter lwoffii, Acinetobacter calcoaceticus and Chlamydia psittaci. Conclusions This work revealed the presence of several potentially abortigenic pathogens in aborted specimens. No novel potential abortigenic agents were detected. The ability to screen samples for multiple pathogens that may not have been specifically targeted broadens the frontiers of diagnostic potential. The future use of metagenomic approaches for diagnostic purposes is likely to be facilitated by further improvements in deep sequencing technologies.

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.

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas (Phascolarctos cinereus) and to underlie the koala’s susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A (p`0.023) and IFNγ (p = 0.044) gene expression along with a decreased CD4:CD8 gene expression ratio (p = 0.025) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

The Leadbeater's possum (Gymnobelideus leadbeateri) is a critically endangered marsupial in south-eastern Australia. Among other conservation efforts, free-ranging animals in the two remaining geographically separate populations (highland and lowland) have been extensively studied; however, little is known about their health and mortality. Although some wild populations are frequently monitored, cadavers are rarely recovered for post mortem examination. In June 2019, a recently deceased, wild, adult male lowland Leadbeater's possum was collected from a nest box and a comprehensive post mortem examination was conducted. Microfilariae of a filarioid nematode were observed in testes, liver, lung and skin samples in tissue impression smears and upon histopathological examination. No gross or histological changes were seen associated with the parasites, except for a focal area of tissue damage in the skin, suggesting that the possum is a natural host. Using a PCR-coupled sequencing method the filarioid was identified as a species of Breinlia. Species of Breinlia occur in other Australian marsupials and rodents., Graphical abstract Image 1, Highlights • A post mortem examination of a wild endangered Leadbeater's possum was performed. • Microfilariae were observed in testes, liver, lung and skin. • The filarioid nematode was identified as Breinlia sp. Using molecular techniques. • The possum appears to be the natural host of this parasite.

Herpesviruses are attractive vaccine vector candidates due to their large double stranded DNA genome and latency characteristics. Within the scope of veterinary vaccines, herpesvirus-vectored vaccines have been well studied and commercially available vectored vaccines are used to help prevent diseases in different animal species. Felid alphaherpesvirus 1 (FHV-1) has been characterised as a vector candidate to protect against a range of feline pathogens. In this review we highlight the methods used to construct FHV-1 based vaccines and their outcomes, while also proposing alternative uses for FHV-1 as a viral vector.

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.

We present the genome sequences of macropodid alphaherpesviruses 2 and 4, two closely related pathogens of macropods. Both encoded 68 nonredundant open reading frames (ORFs) and share 90.6% genome-wide nucleotide identity. These viruses are associated with fatal outbreaks of disease in multiple marsupial species. These sequences will be important for the development of new diagnostic tools.

Chlamydia psittaci is traditionally regarded as a globally distributed avian pathogen that can cause zoonotic spill-over. Molecular research has identified an extended global host range and significant genetic diversity. However, Australia has reported a reduced host range (avian, horse, and human) with a dominance of clonal strains, denoted ST24. To better understand the widespread of this strain type in Australia, multilocus sequence typing (MLST) and ompA genotyping were applied on samples from a range of hosts (avian, equine, marsupial, and bovine) from Australia. MLST confirms that clonal ST24 strains dominate infections of Australian psittacine and equine hosts (82/88, 93.18%). However, this study also found novel hosts (Australian white ibis, King parrots, racing pigeon, bovine, and a wallaby) and demonstrated that strain diversity does exist in Australia. The discovery of a C. psittaci novel strain (ST306) in a novel host, the Western brush wallaby, is the first detection in a marsupial. Analysis of the results of this study applied a multidisciplinary approach regarding Chlamydia infections, equine infectious disease, ecology, and One Health. Recommendations include an update for the descriptive framework of C. psittaci disease and cell biology work to inform pathogenicity and complement molecular epidemiology.

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.

Wobbly possum disease virus (WPDV) is an arterivirus that was originally identified in common brushtail possums (Trichosurus vulpecula) in New Zealand, where it causes severe neurological disease. In this study, serum samples (n = 188) from Australian common brushtail, mountain brushtail (Trichosurus cunninghami) and common ringtail (Pseudocheirus peregrinus) possums were tested for antibodies to WPDV using ELISA. Antibodies to WPDV were detected in possums from all three species that were sampled in the states of Victoria and South Australia. Overall, 16% (30/188; 95% CI 11.0-22.0) of possums were seropositive for WPDV and 11.7% (22/188; 95% CI 7.5-17.2) were equivocal. The frequency of WPDV antibody detection was the highest in possums from the two brushtail species. This is the first reported serological evidence of infection with WPDV, or an antigenically similar virus, in Australian possums, and the first study to find antibodies in species other than common brushtail possums. Attempts to detect viral RNA in spleens by PCR were unsuccessful. Further research is needed to characterise the virus in Australian possums and to determine its impact on the ecology of Australian marsupials.

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.

We evaluated the health of 31 (eight males, 23 females) founder eastern quolls (

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.

A novel coccidian species was discovered in the prostate of an Antechinus flavipes (yellow-footed antechinus) in South Australia during the period of postmating male antechinus immunosuppression and mortality. This novel coccidian is unusual because it develops extraintestinally and sporulates endogenously within the prostate gland of its mammalian host. Histological examination of prostatic tissue revealed dense aggregations of spherical and thin-walled tetrasporocystic, dizoic, sporulated coccidian oocysts within tubular lumina, with unsporulated oocysts and gamogonic stages within the cytoplasm of glandular epithelial cells. This coccidian was observed occurring concurrently with dasyurid gammaherpesvirus 1 infection of the antechinus' prostate. Eimeria-specific 18S small-subunit ribosomal (r)DNA polymerase chain reaction amplification was used to obtain a partial 18S rDNA nucleotide sequence from the antechinus coccidian. Bayesian phylogenetic analysis based on 18S rDNA gene sequences revealed that the novel coccidian clusters with reptile-host coccidians, forming an ancestral basal lineage of the eimeriid clade. The species has been named Eimeria taggarti n. sp. on the basis of both sporulated oocyst morphology and molecular characterization. It is suspected that E. taggarti is sexually transmitted via excretion of sporulated oocysts or free sporocysts with prostatic secretions in semen.

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.

Coxiella burnetii may cause reproduction disorders in pregnant animals but subclinical infection in other animals. Unrecognised disease may delay implementation of control interventions, resulting in transmission of infection to other livestock and to humans. Seroreactivity to C. burnetii phase-specific antigens, is routinely used to interpret the course of human Q fever. This approach could be similarly useful in identifying new and existing infections in livestock herds to help describe risk factors or production losses associated with the infections and the implementation of disease-control interventions. This study aimed to elucidate the dynamics of C. burnetii infections using seroreactivity to phase-specific antigens and to examine the impact of infection on milk yield in goats in an endemically-infected farm that was associated with a Q fever outbreak in Australia. Seroreactivity pre- and post-partum and milk yield were studied in 164 goats (86 nulliparous and 78 parous). Post-partum, the seroprevalence of antibodies to C. burnetti increased from 4.7% to 31.4% throughout goats' first kiddings and from 47.4% to 55.1% in goats kidding for the second or greater time. Of 123 goats that were seronegative pre-partum, 26.8% seroconverted over the three-month peri-partum period, highlighting the importance of controlling infection throughout this time. The risk of seroconversion was comparable in first or later kidders, suggesting constant risk irrespective of parity. No loss in milk production associated with seroconversion to phase 2 was observed within the first nine weeks of lactation. However, seroconversion to only phase 1 was associated with extra 0.276L of milk per day (95% Confidence Interval: 0.010, 0.543; P=0.042), which warrants further investigation to ascertain whether or not the association is causal. Further studies on seroreactivity and milk production over longer periods are required, as milk production loss caused by C. burnetti may be an additional reason to control the disease in goat herds.

Recombination in alphaherpesviruses was first described more than sixty years ago. Since then, different techniques have been used to detect recombination in natural (field) and experimental settings. Over the last ten years, next-generation sequencing (NGS) technologies and bioinformatic analyses have greatly increased the accuracy of recombination detection, particularly in field settings, thus contributing greatly to the study of natural alphaherpesvirus recombination in both human and veterinary medicine. Such studies have highlighted the important role that natural recombination plays in the evolution of many alphaherpesviruses. These studies have also shown that recombination can be a safety concern for attenuated alphaherpesvirus vaccines, particularly in veterinary medicine where such vaccines are used extensively, but also potentially in human medicine where attenuated varicella zoster virus vaccines are in use. This review focuses on the contributions that NGS and sequence analysis have made over the last ten years to our understanding of recombination in mammalian and avian alphaherpesviruses, with particular focus on attenuated live vaccine use.

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.

Herpesvirus infections associated with a range of clinical findings are widespread in free-ranging and captive Australian marsupials. We report on herpesviruses identified by virus neutralization and PCR in free-ranging and captive Lumholtz's tree-kangaroos (Dendrolagus lumholtzi). Herpesvirus has not been confirmed previously by DNA testing in tree kangaroos. Virus neutralization testing for alphaherpesviruses MaHV1 and MaHV2 was positive on 4/10 captive and 0/35 free-ranging tree-kangaroo samples tested. A novel gammaherpesvirus was found on PCR in 17/20 apparently healthy individuals (11/12 free-ranging, 5/6 wild-caught, captive, and 1/2 captive-bred). One captive-bred animal that died following an acute illness was positive on PCR only for MaHV4, an alphaherpesvirus previously identified from an eastern grey kangaroo (Macropus giganteus). The detection of MaHV4, associated with morbidity and mortality in captive tree-kangaroos, raises biosecurity concerns about introducing a non-endemic alphaherpesvirus into naive wild populations through release of captive animals. We propose that: 1) further work on herpesviruses in marsupials be carried out to determine whether herpesviruses from captive individuals represent a potential threat to wild populations, particularly for endangered species in which there are captive breeding and cross-fostering programs; and 2) that captive tree kangaroos be kept in such a way that prevents cross-species transmission of herpesviruses, in particular eliminating close direct or indirect contact with other species of macropods.

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.

The iconic koala (Phascolarctos cinereus) is host to two divergent gammaherpesviruses, phascolarctid gammaherpesviruses 1 and 2 (PhaHV-1 and -2), but the clinical significance of the individual viruses is unknown and current diagnostic methods are unsuitable for differentiating between the viruses in large-scale studies. To address this, we modified a pan-herpesvirus nested PCR to incorporate high-resolution melt analysis. We applied this assay in a molecular epidemiological study of 810 koalas from disparate populations across Victoria, Australia, including isolated island populations. Animal and clinical data recorded at sampling were analyzed and compared to infection status. Between populations, the prevalence of PhaHV-1 and -2 varied significantly, ranging from 1% to 55%. Adult and older animals were 5 to 13 times more likely to be positive for PhaHV-1 than juveniles (P < 0.001), whereas PhaHV-2 detection did not change with age, suggesting differences in how these two viruses are acquired over the life of the animal. PhaHV-1 detection was uniquely associated with the detection of koala retrovirus, particularly in females (P = 0.008). Both viruses were significantly associated (P < 0.05) with the presence of genital tract abnormalities (uterine/ovarian cysts and testicular malformation), reduced fertility in females, urinary incontinence, and detection of Chlamydia pecorum, although the strength of these associations varied by sex and virus. Understanding the clinical significance of these viruses and how they interact with other pathogens will inform future management of threatened koala populations.

Detection of salmonellae within poultry environments is an important component of many food safety programs, but sampling approaches vary greatly and may not enable the detection of salmonellae when bacteria are present at a low prevalence or concentration. Intensive longitudinal sampling within caged sheds enabled us to undertake a longitudinal analysis of the spatial distribution of salmonellae in caged shed environments. Both the number of samples collected and location of sample collection within a poultry shed were important to ensure the best chance of detecting Salmonella spp. Differences in the within-shed spatial distribution of Salmonella enterica subspecies enterica serovar Typhimurium [χ(2)(27, 1,538) = 54.4; P

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.

We detected Chlamydia pecorum in two koalas (Phascolarctos cinereus) from a closed island population in Victoria, Australia, previously free of Chlamydia infection. The ompA and multilocus sequence type were most closely related to published isolates of livestock rather than koala origin, suggesting potential cross-species transmission of C. pecorum.