Probiotic Yoghurt Enriched with Mango Peel Powder: Biotransformation of Phenolics and Modulation of Metabolomic Outputs after In Vitro Digestion and Colonic Fermentation.

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.

A RETROSPECTIVE ANALYSIS OF MORBIDITY AND MORTALITY IN THE CAPTIVE LEADBEATER'S POSSUM (GYMNOBELIDEUS LEADBEATERI) POPULATION FROM 1970 TO 2021.

The Leadbeater's possum (Gymnobelideus leadbeateri) is a critically endangered nocturnal marsupial with a restricted range in the Central Highlands of Victoria, Australia. There are two genetically distinct populations divided by location: highland and lowland. Lowland possums exist in one remnant swamp forest and entered captivity in 2012 when ∼60 individuals remained. Today, with less than 20 lowland individuals remaining, any information that informs the yet-unsuccessful breeding program is critical. This study encompasses a retrospective analysis of the causes of mortality and significant histological lesions in captive highland and lowland individuals across seven institutions internationally from 1970 to 2021. During this time, 245 possums lived in captivity. Postmortem records exist for 99 animals, including 349 histopathology diagnoses from 80 reports and 264 gross necropsy diagnoses from 78 reports. Diagnoses were assigned into two categories based on the importance to the individual (causing death or morbidity to a single animal [n = 194]), or importance to the wider population (causing death or morbidity to more than one animal or was related to reproduction [n = 155]). Individual animals had multiple diagnoses, which were tallied as individual data points. Renal disease was diagnosed 57 times; the most common finding was chronic nephropathy (43/57). Cardiovascular disease was diagnosed 33 times; atherosclerosis associated with obesity was common (n = 10/33). Both categories suggest causal association with captive husbandry but elicit no comment on the lack of success of the breeding program. Reproductive disease was diagnosed 36 times in 24 animals (14 females and 10 males). In females, 11 cases of uterine inflammation and associated clinical signs were associated with ascending infection or neoplasia. Of the seven lowland male possums with mortality data, five were infertile (azoospermia or testicular atrophy). More investigation into the reproductive health of this population is indicated to understand the lack of success in the current breeding program.

Whole genome sequence analysis of equid gammaherpesvirus -2 field isolates reveals high levels of genomic diversity and recombination.

BACKGROUND: Equid 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. RESULTS: Whole 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 38.1%. 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. CONCLUSIONS: Analysis 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.

Rapid typing of infectious laryngotracheitis virus directly from tracheal tissues based on next-generation sequencing.

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.

Metagenomic investigation of potential abortigenic pathogens in foetal tissues from Australian horses.

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.

Differential Response of the Chicken Trachea to Chronic Infection with Virulent Mycoplasma gallisepticum Strain Ap3AS and Vaxsafe MG (Strain ts-304): a Transcriptional Profile.

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.

Infectious Laryngotracheitis Virus Viral Chemokine-Binding Protein Glycoprotein G Alters Transcription of Key Inflammatory Mediators In Vitro and In Vivo.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and in vivo infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection.IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.

A Novel Glaesserella sp. Isolated from Pigs with Severe Respiratory Infections Has a Mosaic Genome with Virulence Factors Putatively Acquired by Horizontal Transfer.

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.

Analysis of the complete genomic sequences of two virus subpopulations of the Australian infectious bronchitis virus vaccine VicS.

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

Development and Validation of TaqMan Real-Time Polymerase Chain Reaction Assays for the Quantitative and Differential Detection of Wild-Type Infectious Laryngotracheitis Viruses from a Glycoprotein G-Deficient Candidate Vaccine Strain.

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.

Cross-protective immune responses between genotypically distinct lineages of infectious laryngotracheitis viruses.

Recent phylogenetic studies have identified different genotypic lineages of infectious laryngotracheitis virus (ILTV), and these lineages can recombine in the field. The emergence of virulent recombinant field strains of ILTV by natural recombination between commercial vaccines belonging to different genotypic lineages has been reported recently. Despite the use of attenuated ILTV vaccines, these recombinant viruses were able to spread and cause disease in commercial poultry flocks, raising the question of whether the different lineages of ILTV can induce cross-protective immune responses. This study examined the capacity of the Australian-origin A20 ILTV vaccine to protect against challenge with the class 8 ILTV recombinant virus, the genome of which is predominantly derived from a heterologous genotypic lineage. Following challenge, birds vaccinated via eyedrop were protected from clinical signs of disease and pathological changes in the tracheal mucosa, although they were not completely protected from viral infection or replication. In contrast, the challenge virus induced severe clinical signs and tracheal pathology in unvaccinated birds. This is the first study to examine the ability of a vaccine from the Australian lineage to protect against challenge with a virus from a heterologous lineage. These results suggest that the two distinct genotypic lineages of ILTV can both induce cross-protection, indicating that current commercial vaccines are still likely to assist in control of ILTV in the poultry industry, in spite of the emergence of novel recombinants derived from different genotypic lineages.

Unveiling genome plasticity and a novel phage in Mycoplasma felis: Genomic investigations of four feline isolates.

Mycoplasma felis has been isolated from diseased cats and horses, but to date only a single fully assembled genome of this species, of an isolate from a horse, has been characterized. This study aimed to characterize and compare the completely assembled genomes of four clinical isolates of M. felis from three domestic cats, assembled with the aid of short- and long-read sequencing methods. The completed genomes encoded a median of 759 ORFs (range 743-777) and had a median average nucleotide identity of 98.2 % with the genome of the available equid origin reference strain. Comparative genomic analysis revealed the occurrence of multiple horizontal gene transfer events and significant genome reassortment. This had resulted in the acquisition or loss of numerous genes within the Australian felid isolate genomes, encoding putative proteins involved in DNA transfer, metabolism, DNA replication, host cell interaction and restriction modification systems. Additionally, a novel mycoplasma phage was detected in one Australian felid M. felis isolate by genomic analysis and visualized using cryo-transmission electron microscopy. This study has highlighted the complex genomic dynamics in different host environments. Furthermore, the sequences obtained in this work will enable the development of new diagnostic tools, and identification of future infection control and treatment options for the respiratory disease complex in cats.

Genomic characterization of Mycoplasma edwardii isolated from a dog bite induced cat wound reveals multiple horizontal gene transfer events and loss of the CRISPR/Cas system.

A domestic short hair cat (Felis catus) suffering from a purulent wound infection resulting from a dog bite was sampled for bacterial culture and isolation as the wound had been unresponsive to prolonged antimicrobial treatment. A mycoplasma was isolated from the wound. Whole genome sequencing of the isolate was performed using short-read Illumina and long-read Oxford Nanopore chemistry, and the organism was identified as Mycoplasma edwardii. Comparison of the genome sequence of the isolate to a reference M. edwardii genome sequence (canid isolate) identified the loss of several key bacterial factors involved in genome editing, as well the insertion of several novel ORFs most closely related to those found in other canine mycoplasmas, specifically Mycoplasma canis, M. cynos, M. molare and M. maculosa. This is only the second known report of disease caused by M. edwardii in a non-canid species, and the first report of it infecting and causing clinical disease in a cat.

Expanding the known distribution of phascolartid gammaherpesvirus 1 in koalas to populations across Queensland and New South Wales.

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

Genomic diversity and natural recombination of equid gammaherpesvirus 5 isolates.

BACKGROUND: Equid gammaherpesvirus 5 (EHV5) is closely related to equid gammaherpesvirus 2 (EHV2). Detection of EHV5 is frequent in horse populations worldwide, but it is often without a clear and significant clinical impact. Infection in horses can often present as subclinical disease; however, it has been associated with respiratory disease, including equine multinodular pulmonary fibrosis (EMPF). Genetic heterogeneity within small regions of the EHV5 glycoprotein B (gB) sequences have been reported and multiple genotypes of this virus have been identified within individual horses, but full genome sequence data for these viruses is limited. The primary focus of this study was to assess the genomic diversity and natural recombination among EHV5 isolates. RESULTS: The genome size of EHV5 prototype strain and the five EHV5 isolates cultured for this study, including four isolates from the same horse, ranged from 181,929 to 183,428 base pairs (bp), with the sizes of terminal repeat regions varying from 0 to 10 bp. The nucleotide sequence identity between the six EHV5 genomes ranged from 95.5 to 99.1%, and the estimated average nucleotide diversity between isolates was 1%. Individual genes displayed varying levels of nucleotide diversity that ranged from 0 to 19%. The analysis of nonsynonymous substitution (Ka > 0.025) revealed high diversity in eight genes. Genome analysis using RDP4 and SplitsTree programs detected evidence of past recombination events between EHV5 isolates. CONCLUSION: Genomic diversity and recombination hotspots were identified among EHV5 strains. Recombination can drive genetic diversity, particularly in viruses that have a low rate of nucleotide substitutions. Therefore, the results from this study suggest that recombination is an important contributing factor to EHV5 genomic diversity. The findings from this study provide additional insights into the genetic heterogeneity of the EHV5 genome.

Development of diagnostic and point of care assays for a gammaherpesvirus infecting koalas.

The recent listing of koala populations as endangered across much of their range has highlighted the need for better management interventions. Disease is a key threat to koala populations but currently there is no information across the threatened populations on the distribution or impact of a gammaherpesvirus, phascolarctid gammaherpesvirus 1 (PhaHV-1). PhaHV-1 is known to infect koalas in southern populations which are, at present, not threatened. Current testing for PhaHV-1 involves lengthy laboratory techniques that do not permit quantification of viral load. In order to better understand distribution, prevalence and impacts of PhaHV-1 infections across koala populations, diagnostic and rapid point of care tests are required. We have developed two novel assays, a qPCR assay and an isothermal assay, that will enable researchers, clinicians and wildlife managers to reliably and rapidly test for PhaHV-1 in koalas. The ability to rapidly diagnose and quantify viral load will aid quarantine practices, inform translocation management and guide research into the clinical significance and impacts of PhaHV-1 infection in koalas.

Molecular characterisation of the Australian and New Zealand livestock Chlamydia pecorum strains confirms novel but clonal ST23 in association with ovine foetal loss.

Chlamydia pecorum is a veterinary pathogen associated with abortions and perinatal mortality in sheep. Recent studies investigating foetal and perinatal lamb mortality in sheep from Australia and New Zealand identified C. pecorum clonal sequence type (ST)23 strains in aborted and stillborn lambs. Presently, there is limited genotypic information on C. pecorum strains associated with reproductive disease, although whole genome sequencing (WGS) of one abortigenic ST23 C. pecorum strain identified unique features, including a deletion in the CDS1 locus of the chlamydial plasmid. We applied WGS on two ST23 strains detected in aborted and stillborn lambs from Australia and used phylogenetic and comparative analyses to compare these to the other available C. pecorum genomes. To re-evaluate the genetic diversity of contemporary strains, we applied C. pecorum genotyping, and chlamydial plasmid sequencing to a range of C. pecorum positive samples and isolates from ewes, aborted foetuses and stillborn lambs, cattle and a goat from diverse geographical regions across Australia and New Zealand.The two new C. pecorum genomes are nearly identical to the genome of the Australian abortigenic strain including the unique deletion in the chlamydial plasmid. Genotyping revealed that these novel C. pecorum ST23 strains are widespread and associated with sheep abortions on Australian and New Zealand farms. In addition, a goat C. pecorum strain (denoted ST 304) from New Zealand was also characterised. This study expands the C. pecorum genome catalogue and describes a comprehensive molecular characterisation of the novel livestock ST23 strains associated with foetal and lamb mortality.

An assessment of ectoparasites across highland and lowland populations of Leadbeater's possum (Gymnobelideus leadbeateri): Implications for genetic rescue translocations.

Leadbeater's possum (Gymnobelideus leadbeateri) is a nocturnal arboreal marsupial with a restricted range centered on the Victorian Central Highlands, south-eastern Australia. Most populations inhabit wet montane ash forest and subalpine woodland, with one notable exception - a small, outlying and genetically-distinct lowland population inhabiting swamp forest at Yellingbo, Victoria. The species has been listed as critically endangered since 2015. Translocations are the mainstay of critical genetic rescue and this study explores the ectoparasites that are 'along for the ride' during translocation activities. Ectoparasites (133 fleas, 15 ticks and 76 mites) were collected opportunistically from 24 Leadbeater's possum colonies during population monitoring and genetic sampling across the lowland and highland populations. The composition of the flea assemblage varied by habitat type. Significantly greater numbers of the general marsupial fleas Acanthopsylla r. rothschildii. and Choristopsylla tristis (as a proportion of total flea numbers) were detected in lowland habitats, compared to highland habitats (Fishers exact test, P < 0.0001). Two host-specific flea species, Stephanocircus domrowi and Wurunjerria warnekei were detected only on possums in highland habitats. As a proportion of total fleas this was significantly different to possums in lowland habitats (Fisher's exact test, P = 0.0042 and P < 0.0001, respectively). Wurunjerria warnekei was suspected to be extinct prior to this study. Ticks (Ixodes tasmanii, n = 15) and mites (Haemdoelaps cleptus, n = 47 and H. anticlea, n = 29) have been identified in Leadbeater's possums historically. The possible causes of the different flea assemblages may be environmental/climatic, or due to the historic geographic division between highland and lowland animals. The planned translocations of highland individuals to lowland habitats will expose lowland individuals to novel species of previously exclusively highland fleas with unknown indirect consequences, thus careful monitoring will be required to manage any potential risks.

Detection of Breinlia sp. (Nematoda) in the Leadbeater's possum (Gymnobelideus leadbeateri).

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.