Alistair Raymond Legione

Background: Elephant endotheliotropic herpesviruses (EEHVs) can cause fatal haemorrhagic disease (EEHV-HD) in young Asian elephants ( Elephas maximus ), posing a significant threat to the conservation of the species. Understanding the dynamics of EEHV infection within a herd may help to understand risk factors associated with EEHV-HD, however, only few longitudinal EEHV surveillance studies exist. Methods: This study documented EEHV viral dynamics in nine Asian elephants over a two-year period spanning an anticipated major stressor: relocation from Melbourne Zoo to Werribee Open Range Zoo. Antibodies specific to each EEHV (sub)species were measured by enzyme-linked immunosorbent assays (ELISA). Viral DNA was detected in blood and trunk wash samples by quantitative PCR (qPCR). Results: Contrary to our hypothesis, EEHV detection by qPCR was more common prior to relocation than after relocation. Pre-relocation viral detection was largely attributable to a series of systemic EEHV5 infections in five elephants, unrelated to any changes in herd management or dynamics. Prospective serological testing demonstrated an increase in EEHV5A-specific antibodies in three young elephants with low pre-existing antibodies in response to systemic EEHV5 infection. Trunk wash detection of virus preceded viraemia in four out of five elephants with systemic EEHV5 infection, including two of the animals with low EEHV5A antibodies, indicating virus exposure or localised replication. Conclusions: Transport and relocation of the entire herd were not associated with widespread viral reactivation. In this herd, combined serology testing and trunk wash surveillance identified animals at risk of systemic EEHV infection and provided early evidence of viral exposure prior to viraemia. Integration of serology and trunk wash qPCR with routine haematology and blood qPCR screening may enhance early detection of EEHV infection in susceptible elephants.

Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants ( Elephas maximus ) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 x 10 ⁶ viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at −80 °C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398) and the previously reported EEHV1B sequence (KC462164), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants ( Elephas maximus ) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 × 10 ⁶ viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at -80 °C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398 ) and the previously reported EEHV1B sequence ( KC462164 ), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

Coronaviruses evolve rapidly, with recombination and mutation fostering the emergence of variant strains. The avian coronavirus infectious bronchitis virus (IBV) is an important poultry pathogen and a valuable natural model for studying coronaviruses. Australian strains have evolved independently of those infecting chickens elsewhere in the world, so understanding the biology and evolution of these strains can further our understanding of factors driving the emergence of novel coronaviruses. We infected groups of specific pathogen-free Leghorn chickens with six Australian IBVs (from five distinct genotypes) isolated between 1962 and 2013. All six affected the respiratory tract, but only one was nephropathogenic (N1/62). All six induced significant lesions and actively replicated in the upper respiratory tract, but they had lower levels of replication and induced less severe lesions in the middle and lower trachea. There were significant differences between the six strains in the severity of the lesions they induced and in their tissue tropism and effect on tracheal ciliary motility. Strains N1/62 (strain T) and N1/03 caused the most severe tracheal ciliostasis and replicated to the highest levels in tissues. Strain N1/03 caused the most severe lesions at 9 days post-infection. Only strain N1/03 caused lesions in the lower trachea. Overall, strains N1/03 and N1/62 were the most virulent. This study is the first to characterize the histological changes induced by the recently isolated Australian IBVs and compare them directly with older strains. Recombination between field and vaccine strains of IBV has yielded emergent IBVs in Australia that appear to have enhanced virulence for the respiratory tract.

Recombinant koala retrovirus (recKoRV) is a recently discovered variant of koala retrovirus (KoRV), which likely emerged due to recombination with another retrovirus (such as Phascolarctos endogenous retrovirus). KoRV spread and endogenization in Australia were thought to be ongoing in a north to south direction given the low prevalence of the virus in southern koala populations, based on molecular detection of the pol gene. However, recKoRV has highlighted that fragments of KoRV with the pol region missing are present within southern koalas. In this study, a new 5′-region-based KoRV PCR assay was developed, capable of detecting both intact KoRV and all known variants of recKoRV. Using this assay, 319 archived DNA samples from 287 Victorian koalas were retested to investigate KoRV endogenization. We found 98.3% (282/287) of these samples were positive for the KoRV-5′ fragment, the majority of which were KoRV-pol negative (222/287) on prior testing. Our findings demonstrate extensive KoRV integration into the Victorian koala populations, suggestive of a historic presence of KoRV in Victorian koalas. This finding makes biological sense relative to the translocation history of Victorian koalas, compared to the prior paradigm of low virus prevalence, and provides new epidemiological and practical management implications.

Recombinant koala retrovirus (recKoRV) is a recently discovered variant of koala retrovirus (KoRV), which likely emerged due to the insertion of another retrovirus (likely Phascolarctos endogenous retrovirus) into the backbone of KoRV. KoRV endogenisation was thought to be ongoing in Victoria based on the low prevalence of the virus based on molecular detection of the pol gene, however recKoRV was not incorporated into the previous KoRV diagnostic test results. In this study, a new 5’-region-based PCR assay was developed, capable of detecting both intact KoRV and recKoRV. Using this assay, 319 archived DNA samples from 287 Victorian koalas were retested to investigate KoRV endogenisation. We found a 98.3% (282/287) of these samples were positive for the KoRV-5’ fragment, the majority of which were KoRV- pol negative (222/287) on prior testing. Our findings demonstrate extensive KoRV integration into the Victorian koala populations, suggestive of a historic presence of KoRV in Victorian koalas. This finding makes biological sense relative to the translocation history of Victorian koalas, compared to the prior paradigm of ongoing endogenisation, and provides new epidemiological and practical management implications.

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

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.

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.

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 characterised. This study aimed to characterise 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 open reading frames (min, 743, max 777) and had a median average nucleotide identity (ANI) of 98.2% with the genome of the available equid origin reference strain. Comparative genomic analysis revealed the occurrence of multiple horizontal gene transfer (HGT) 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 visualised 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. Data summary: All genome data for this study have been deposited in GenBank under BioProject PRJNA906261. Genome assemblies, as well as Illumina and Oxford Nanopore sequence reads for each isolate, can be found under their respective BioSamples: SAMN32182834 (isolate 047), SAMN32182835 (isolate 219), SAMN32182836 (isolate 329 and associated phage), and SAMN32182837 (isolate 632). The authors confirm all supporting data and protocols have been provided within the article. Impact statement: Mycoplasma felis is commonly associated with clinical cases of conjunctivitis and feline respiratory disease complex in cats, the leading cause of euthanasia in animal shelters. In the absence of vaccines, infection control is currently limited to the prolonged treatment with antimicrobials. Prior to this study there was only one complete genome assembly of an isolate of M. felis , which had been obtained from a horse. This study has provided the first high quality hybrid assembled genomes of M. felis isolates from cats. This work adds four new genomes from clinical cases, as well as the identification and validation of the presence of a novel phage that utilises the mycoplasma translation code. The genomic data presented here can assist future projects investigating improved diagnostics and development of new treatment options for this significant feline pathogen.