Search

Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.

Chlamydia psittaci is an avian pathogen with zoonotic potential. In Australia, C. psittaci has been well reported as a cause of reproductive loss in mares which subsequently have been the source of infection and illness in some in-contact humans. To date, molecular typing studies describe the predominant and clonal C. psittaci sequence type (ST)24 strains in horse, psittacine, and human infections. We sought to assess the clonality between ST24 strains and the emergence of equine ST24 with a comprehensive genomics approach. We used culture-independent probe-based and metagenomic whole-genome sequencing to investigate 13 C . psittaci genomes from horses, psittacines, and a pigeon from Australia. Published genomes of 36 C . psittaci strains were also used to contextualise our Australian dataset and investigate lineage diversity. We utilised a single-nucleotide polymorphism (SNP) based clustering and multi-locus sequence typing (MLST) approach. C. psittaci has four major phylogenetic groups (PG1-4) based on core-genome SNP-based phylogeny. PG1 contained clonal global and Australian equine, psittacine, and human ST24 genomes, with a median pairwise SNP distance of 68 SNPs. PG2, PG3, and PG4 had greater genomic diversity, including diverse STs collected from birds, livestock, human, and horse hosts from Europe and North America and a racing pigeon from Australia. We show that the clustering of C. psittaci by MLST was congruent with SNP-based phylogeny. The monophyletic ST24 clade has four major sub-lineages. The genomes of 17 Australian human, equine, and psittacine strains collected between 2008 and 2021 formed the predominant ST24 sub-lineage 1 (emerged circa 1979). Despite a temporal distribution of 13 years, the genomes within sub-lineage 1 had a median pairwise SNP distance of 32 SNPs, suggesting a recent population expansion or potential cross-host transmission. However, two C. psittaci genomes collected in 2015 from Victorian parrots clustered into distinct ST24 sub-lineage 4 (emerged circa 1965) with ovine strain C19/98 from Germany. This work describes a comprehensive phylogenomic characterisation of ST24 and identifies a timeline of potential bird-to-equine spillover events.

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.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of ‘southern’ (south Australian) and ‘northern’ (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of ‘southern’ (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, ‘RecKoRV’ variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

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.

This study investigated the health-promoting effects and prebiotic functions of mango peel powder (MPP) both as a plain individual ingredient and when incorporated in yoghurt during simulated digestion and fermentation. The treatments included plain MPP, plain yoghurt (YA), yoghurt fortified with MPP (YB), and yoghurt fortified with MPP and lactic acid bacteria (YC), along with a blank (BL). The identification of polyphenols in the extracts of insoluble digesta and phenolic metabolites after the in vitro colonic fermentation were performed employing LC-ESI-QTOF-MS2. These extracts were also subjected to pH, microbial count, production of SCFA, and 16S rRNA analyses. The characterisation of phenolic profiles identified 62 phenolic compounds. Among these compounds, phenolic acids were the major compounds that underwent biotransformation via catabolic pathways such as ring fission, decarboxylation, and dehydroxylation. Changes in pH indicated that YC and MPP reduced the media pH from 6.27 and 6.33 to 4.50 and 4.53, respectively. This decline in pH was associated with significant increases in the LAB counts of these samples. The Bifidobacteria counts were 8.11 ± 0.89 and 8.02 ± 1.01 log CFU/g in YC and MPP, respectively, after 72 h of colonic fermentation. Results also showed that the presence of MPP imparted significant variations in the contents and profiles of individual short chain fatty acids (SCFA) with more predominant production of most SCFA in the MPP and YC treatments. The 16s rRNA sequencing data indicated a highly distinctive microbial population associated with YC in terms of relative abundance. These findings suggested MPP as a promising ingredient for utilisation in functional food formulations aiming to enhance gut health.

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.

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.

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.

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.

While bats are often viewed as carriers of infectious disease agents, little research has been conducted on the effects these pathogens may have on the bat populations themselves. The southern bent-winged bat (Miniopterus orianae bassanii) is a critically endangered subspecies endemic to south-eastern Australia. Population numbers of this bat have declined over the past 50 years, but the reasons for this are unclear. As part of a larger study to determine if disease could be a contributing factor to this decline, southern bent-winged bats from several locations in Victoria and South Australia were captured and examined for the presence of the blood parasite, Polychromophilus melanipherus, and haemoplasmas (Mycoplasma sp.). Results were compared with those obtained from populations of the more common, partially sympatric, eastern bent-winged bat (Miniopterus orianae oceanensis) from three different locations in Victoria. Both organisms were found in both subspecies (prevalence of P. melanipherus 60% by PCR for southern bent-winged bats compared with 46% for eastern bent-winged bats; prevalence of haemoplasmas 10% for southern bent-winged bats compared with 8% for eastern bent-winged bats), with no association between the probability of infection, body weight, abnormal blood parameters or any other indicators of ill health. However, Victorian southern bent-winged bats had heavier burdens of P. melanipherus than both the South Australian southern bent-winged bats and eastern bent-winged bats. Further investigations are required to determine if these differences are impacting population health., Graphical abstract Image 1, Highlights • Polychromophilus melanipherus not associated with ill health in southern or eastern bent-winged bats. • Haemoplasma infection not associated with ill health in southern or eastern bent-winged bats. • Greater prevalence and intensity of Polychromophilus melanipherus infection in Victorian southern bent-winged bats.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum. Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

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.

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.

Highlights • Vaccines contributed to emergence of recombinant IBV strains. • Exotic avian coronaviruses have played a decisive role in recombination events. • Recombination breakpoints have been in genes for nsp 2, 3 and 16 and the S protein. • Avian coronavirus of unknown origin have been recombining with IBV for 20 years., Australian strains of infectious bronchitis virus (IBV) have been evolving independently for many years, with control achieved by vaccination with local attenuated strains. Previous studies have documented the emergence of recombinants over the last 20 years, with the most recent one, Ck/Aus/N1/08, detected in 2008. These recombinants did not appear to be controlled by the vaccines currently in use. In this study we sequenced the complete genomes of three emergent Australian strains of IBV (IBV/Ck/Aus/N1/88, IBV/Ck/Aus/N1/03 and IBV/Ck/Aus/N1/08) and a previously incompletely characterised vaccine strain, IBV/Ck/Aus/Armidale, and compared them to the genome of the vaccine strain VicS. We detected multiple recombination events throughout the genome between wild type viruses and the vaccine strains in all three emergent isolates. Moreover, we found that strain N1/88 was not entirely exogenous, as was previously hypothesised. Rather, it originated from a recombination event involving the VicS vaccine strain. The S glycoprotein genes of N1/88 and N1/03 were known to be genetically distinct from previously characterised circulating strains and from each other, and the original donors of these genes remains unknown. The S1 glycoprotein gene of N1/88, a subgroup 2 strain, shares a high nucleotide identity with the sequence of the S1 gene of the recent isolate N1/08. As the subgroup 2 strains have not been isolated for at least 20 years, it appears likely that an unknown avian coronavirus that was the donor of the S1 glycoprotein sequence of N1/88 in the 1980s is still recombining with IBV strains in the field.

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.

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.

An unknown member of the family Pasteurellaceae was repeatedly isolated from 20- to 24-week-old pigs with severe pulmonary lesions reared on the same farm in Victoria, Australia. The etiological diagnosis of the disease was inconclusive. The complete genome sequence analysis of one strain, 15-184, revealed some phylogenic proximity to Glaesserella ( Haemophilus ) parasuis , the cause of Glasser's disease. However, the sequences of the 16S rRNA and housekeeping genes, as well as the average nucleotide identity scores, differed from those of all other known species in the family Pasteurellaceae . The protein content of 15-184 was composite, with 60% of coding sequences matching known G. parasuis products, while more than 20% had a closer relative in the genera Actinobacillus , Mannheimia , Pasteurella , and Bibersteinia . Several putative virulence genes absent from G. parasuis but present in other Pasteurellaceae were also found, including the apxIII RTX toxin gene from Actinobacillus pleuropneumoniae , ABC transporters from Actinobacillus minor , and iron transporters from various species. Three prophages and one integrative conjugative element were present in the isolate. Horizontal gene transfers might explain the mosaic genomic structure and atypical metabolic and virulence characteristics of 15-184. This organism has not been assigned a taxonomic position in the family, but this study underlines the need for a large-scale epidemiological and clinical characterization of this novel pathogen in swine populations, as a genomic analysis suggests it could have a severe impact on pig health. IMPORTANCE Several species of Pasteurellaceae cause a range of significant diseases in pigs. A novel member of this family was recently isolated from Australian pigs suffering from severe respiratory infections. Comparative whole-genome analyses suggest that this bacterium represents a new species, which possesses a number of virulence genes horizontally acquired from a diverse range of other Pasteurellaceae . While the possible contribution of other coinfecting noncultivable agents to the disease has not been ruled out in this study, the repertoire of virulence genes found in this organism may nevertheless explain some aspects of the associated pathology observed on the farm. The prevalence of this novel pathogen within pig populations is currently unknown. This finding is of particular importance for the pig industry, as this organism can have a serious impact on the health of these animals.

Chlamydia infection is known to impact the health of koalas (Phascolarctos cinereus) in New South Wales (NSW) and Queensland, but the clinical significance of Chlamydia infections in Victorian koalas is not well described. We examined the prevalence of Chlamydia infection and assessed associated health parameters in two Victorian koala populations known to be Chlamydia positive. The same testing regimen was applied to a third Victorian population in which Chlamydia had not been detected. We examined 288 koalas and collected samples from the urogenital sinus and conjunctival sacs. Detection and differentiation of Chlamydia species utilized real-time PCR and high-resolution melting curve analysis. Chlamydia pecorum was detected in two populations (prevalences: 25% and 41%, respectively) but only from urogenital sinus swabs. Chlamydia was not detected in the third population. Chlamydia pneumoniae was not detected. Chlamydia pecorum infection was positively associated with wet bottom (indicating chronic urinary tract disease) in one Chlamydia-positive population and with abnormal urogenital ultrasound findings in the other Chlamydia-positive population. The prevalence of wet bottom was similar in all populations (including the Chlamydia-free population), suggesting there is another significant cause (or causes) of wet bottom in Victorian koalas. Ocular disease was not observed. This is the largest study of Chlamydia infection in Victorian koalas, and the results suggest the potential for epidemiologic differences related to Chlamydia infections between Victorian koalas and koalas in Queensland and NSW and also between geographically distinct Victorian populations. Further studies to investigate the genotypes of C. pecorum present in Victorian koalas and to identify additional causes of wet bottom in koalas are indicated.

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

Koalas (Phascolarctos cinereus) are iconic Australian marsupials currently threatened by several processes, including infectious diseases and ecological disruption. Infection with Chlamydia pecorum, is considered a key driver of population decline. The clinical sign of 'wet bottom', a staining of the rump associated with urinary incontinence, is often caused by chlamydial urinary tract infections. However, wet bottom has been recorded in koalas free of C. pecorum, suggesting other causative agents in those individuals. We used 16S rRNA diversity profiling to investigate the microbiome of the urogenital tract of ten female koalas in order to identify potential causative agents of wet bottom, other than C. pecorum. Five urogenital samples were processed from koalas presenting with wet bottom and five were clinically normal. All koalas were negative for C. pecorum infection. We detected thirteen phyla across the ten samples, with Firmicutes occurring at the highest relative abundance (77.6%). The order Lactobacillales, within the Firmicutes, comprised 70.3% of the reads from all samples. After normalising reads using DESeq2 and testing for significant differences (P < 0.05), there were 25 operational taxonomic units (OTUs) more commonly found in one group over the other. The families Aerococcaceae and Tissierellaceae both had four significantly differentially abundant OTUs. These four Tissierellaceae OTUs were all significantly more abundant in koalas with wet bottom. This study provides the foundation for future investigations of causes of koala wet bottom, other than C. pecorum infection. This is of clinical relevance as wet bottom is often assumed to be caused by C. pecorum and treated accordingly. Our research highlights that other organisms may be causing wet bottom, and these potential aetiological agents need to be further investigated to fully address the problems this species faces.

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.

While bats are often viewed as carriers of infectious disease agents, little research has been conducted on the effects these potential pathogens may have on the bat populations themselves. The southern bent-winged bat (Miniopterus orianae bassanii) is a critically endangered subspecies endemic to south-eastern Australia. Population numbers of this bat have been declining for the past 50 years, but the reasons for this are unclear. As part of a larger study to determine if disease could be a contributing factor to this decline, 351 southern bent-winged bats from four locations were captured, and oral swabs were collected and tested for the presence of potentially pathogenic viruses. Results were compared with those obtained from 116 eastern bent-winged bats (Miniopterus orianae oceanensis) from three different locations. The eastern bent-winged bat is a related but more common and widespread subspecies whose geographical range overlaps partly with southern bent-winged bats. Herpesviruses were detected in bent-winged bats from all seven locations. At least six novel herpesviruses (five betaherpesviruses and one gammaherpesvirus) were identified. The prevalence of herpesvirus infection was higher in eastern bent-winged bats (44%, 51/116), compared to southern bent-winged bats (27%, 95/351), although this varied across the locations and sampling periods. Adenoviruses and a range of different RNA viruses (lyssaviruses, filoviruses, coronaviruses and henipaviruses) were also tested for but not detected. The detected herpesviruses did not appear to be associated with obvious ill health, and may thus not be playing a role in the population decline of the southern bent-winged bat. The detection of multiple novel herpesviruses at a high prevalence of infection is consistent with our understanding of bats as hosts to a rich diversity of viruses.

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.

Koalas (Phascolarctos cinereus) are iconic Australian marsupials currently threatened by several processes. Infectious reproductive tract disease, caused byChlamydia pecorum, and koala retrovirus infection are considered key drivers of population decline. The clinical sign of ‘wet bottom’, a staining of the rump associated with urinary incontinence, is often caused by chlamydial urogenital tract infections. However, wet bottom has been recorded in koalas free ofC. pecorum, suggesting other causative agents in those individuals. Current understanding of the bacterial community of the koala urogenital tract is limited. We used 16S rRNA diversity profiling to investigate the microbiome of the urogenital tract of ten female koalas. This was to produce baseline data on the female koala urogenital tract microbiome, and to undertake preliminary investigations of potential causative agents of wet bottom, other thanC. pecorum. Five urogenital samples were processed from koalas presenting with wet bottom and five were clinically normal. We detected thirteen phyla across the ten samples, withFirmicutesoccurring at the highest relative abundance (77.6%). The orderLactobacillales, within theFirmicutes, comprised 70.3% of the reads from all samples. After normalising reads using DESeq2 and testing for significant differences (P< 0.05), there were 25 operational taxonomic units (OTUs) more commonly found in one group over the other. The familiesAerococcaceaeandTissierellaceaeboth had four significantly differentially abundant OTUs. These fourTissierellaceaeOTUs were all significantly more abundant in koalas with wet bottom.ImportanceThis study provides an essential foundation for future investigations of both the normal microflora of the koala urogenital tract, and better understanding of the causes of koala urogenital tract disease. Koalas in the states of Queensland and New South Wales are currently undergoing decline, and have been classified as vulnerable populations. Urogenital tract disease is a leading cause of hospital admissions in these states, yet previously little was known of the normal flora of this site. Wet bottom is a clinical sign of urogenital tract disease, which is often assumed to be caused byC. pecorumand treated accordingly. Our research highlights that other organisms may be causing wet bottom, and these potential aetiological agents need to be further investigated to fully address the problems this species faces.

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.

Localised infection caused by Mycobacterium ulcerans is described in two Kelpies, a Whippet and a Koolie domiciled on the Bellarine Peninsula, Victoria, Australia. The diagnosis was confirmed using real-time polymerase chain reaction (PCR) targeting the M. ulcerans-specific insertion sequence (IS2404) in DNA extracted from swabs of ulcerated lesions in all cases. Where available, molecular typing confirmed that three of the dogs were infected with a strain of M. ulcerans that was indistinguishable from a disease-causing strain in people and other animals in Victoria. One dog was still undergoing treatment at the time of writing, but the remaining three dogs were successfully treated with a combination of surgical debridement and medical therapy in one case, and medical therapy alone in the other two. Investigation of the home environs of three of the dogs using real-time PCR revealed low amounts of M. ulcerans DNA in various environmental samples. Mycobacterium ulcerans infection should be included in the differential diagnoses of any ulcerated skin lesions in dogs that live in or visit endemic areas of Victoria and Queensland.

Chlamydia pecorum infection is a threat to the health of free-ranging koalas (Phascolarctos cinereus) in Australia. Utilizing an extensive sample archive we determined the prevalence of C. pecorum in koalas within six regions of Victoria, Australia. The ompA genotypes of the detected C. pecorum were characterized to better understand the epidemiology of this pathogen in Victorian koalas. Despite many studies in northern Australia (i.e. Queensland and New South Wales), prior Chlamydia studies in Victorian koalas are limited. We detected C. pecorum in 125/820 (15 %) urogenital swabs, but in only one ocular swab. Nucleotide sequencing of the molecular marker C. pecorum ompA revealed that the majority (90/114) of C. pecorum samples typed were genotype B. This genotype has not been reported in northern koalas. In general, Chlamydia infection in Victorian koalas is associated with milder clinical signs compared with infection in koalas in northern populations. Although disease pathogenesis is likely to be multifactorial, the high prevalence of genotype B in Victoria may suggest it is less pathogenic. All but three koalas had C. pecorum genotypes unique to southern koala populations (i.e. Victoria and South Australia). These included a novel C. pecorum ompA genotype and two genotypes associated with livestock. Regression analysis determined that significant factors for the presence of C. pecorum infection were sex and geographical location. The presence of 'wet bottom' in males and the presence of reproductive tract pathology in females were significantly associated with C. pecorum infection, suggesting variation in clinical disease manifestations between sexes.

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.

Background Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans, with endemicity predominantly in sub-Saharan Africa and south-eastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium and remain elusive. Real-time PCR investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. This report seeks to expand on previously published findings by the authors' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria, Australia. Methodology/Principal Findings Twenty-seven clinical cases of M. ulcerans infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998–2011. Common ringtail possums (Pseudocheirus peregrinus), a common brushtail possum (Trichosurus vulpecula) and a mountain brushtail possum (Trichosurus cunninghami) were included in the clinically affected cohort. Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions, generally confined to the face, limbs and/or tail. The disease was minor and self-limiting in the case of both Trichosurus spp. possums. In contrast, many of the common ringtail possums had cutaneous disease involving disparate anatomical sites, and in four cases there was evidence of systemic disease at post mortem examination. Where tested using real-time PCR targeted at IS2404, animals typically had significant levels of M. ulcerans DNA throughout the gut and/or faeces. A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces (subclinical cases), and in one of these the organism was cultured from liver tissue. Comparisons were made between clinically and subclinically affected possums, and 61 PCR-negative, non-affected individuals, with regards to disease category and the categorical variables of species (common ringtail possums v others) and sex. Animals with clinical lesions were significantly more likely to be male common ringtail possums. Conclusions/Significance There is significant disease burden in common ringtail possums (especially males) in some areas of Victoria endemic for M. ulcerans disease. The natural history of the disease generally remains unknown, however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection, whereas some severely affected animals, especially common ringtail possums, may become systemically, and potentially fatally affected. Subclinical gut carriage of M. ulcerans DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient. Further work is required to determine whether M. ulcerans infection poses a potential threat to possum populations, and whether these animals are acting as environmental reservoirs in certain geographical areas., Author Summary Mycobacterium ulcerans causes skin disease predominantly in sub-Saharan Africa and southeastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium are unknown. Investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums. This report expands on these studies by detailing the clinical, pathological and microbiological findings in affected wild possum species in endemic areas. Twenty-seven clinically and 12 subclinically affected individuals were identified. Most clinical cases were adults with skin ulcers of the face, limbs and/or tail. The disease was mild and self-limiting in both Trichosurus spp. possums. In contrast, many of the common ringtail possums had multiple skin ulcers and in some there was evidence of internal disease. There were also significant levels of M. ulcerans DNA throughout the gut. Comparisons were made with regards to disease category, species and sex; with clinical cases more likely to be male common ringtail possums. Asymptomatic gut carriage of M. ulcerans DNA is quite common and may be transient in some individuals. Further work is needed to determine whether M. ulcerans infection poses a potential threat to possum populations, and whether these animals are acting as reservoirs in some areas.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated vaccines. Glycoprotein G (gG) is a virulence factor in ILTV and a gG deficient strain of ILTV (ΔgG-ILTV) has shown potential for use as a vaccine. In the poultry industry vaccination via drinking water is common, but technology is now available to allow quicker and more accurate in ovo vaccination of embryos at 18 days of incubation. In this study ΔgG-ILTV was delivered to chicken embryos at three different doses (10(2), 10(3) and 10(4) plaque forming units per egg) using manual in ovo vaccination. At 20 days after hatching, birds were challenged intra-tracheally with wild type ILTV and protection was measured. In ovo vaccination was shown to be safe, as there were no developmental differences between birds from hatching up to 20 days of age, as measured by weight gain. The highest dose of vaccine was the most efficacious, resulting in a weight gain not significantly different from unvaccinated/unchallenged birds seven days after challenge. In contrast, birds vaccinated with the lowest dose showed weight gains not significantly different from unvaccinated/challenged birds. Gross pathology and histopathology of the trachea reflected these observations, with birds vaccinated with the highest dose having less severe lesions. However, qPCR results suggested the vaccine did not prevent the challenge virus replicating in the trachea. This study is the first to assess in ovo delivery of a live attenuated ILTV vaccine and shows that in ovo vaccination with ΔgG-ILTV can be both safe and efficacious.

Localised infection caused by Mycobacterium ulcerans is described in two Kelpies, a Whippet and a Koolie domiciled on the Bellarine Peninsula, Victoria, Australia. The diagnosis was confirmed using real-time polymerase chain reaction (PCR) targeting the M. ulcerans-specific insertion sequence (IS2404) in DNA extracted from swabs of ulcerated lesions in all cases. Where available, molecular typing confirmed that three of the dogs were infected with a strain of M. ulcerans that was indistinguishable from a disease-causing strain in people and other animals in Victoria. One dog was still undergoing treatment at the time of writing, but the remaining three dogs were successfully treated with a combination of surgical debridement and medical therapy in one case, and medical therapy alone in the other two. Investigation of the home environs of three of the dogs using real-time PCR revealed low amounts of M. ulcerans DNA in various environmental samples. Mycobacterium ulcerans infection should be included in the differential diagnoses of any ulcerated skin lesions in dogs that live in or visit endemic areas of Victoria and Queensland.