Melbourne Veterinary School - Research Publications

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    Genomic diversity and natural recombination of equid gammaherpesvirus 5 isolates
    Onasanya, AE ; El-Hage, C ; Diaz-Mendez, A ; Vaz, PK ; Legione, AR ; Devlin, JM ; Hartley, CA (ELSEVIER, 2023-11)
    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.
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    Synthetic 5-amino-6-D-ribitylaminouracil paired with inflammatory stimuli facilitates MAIT cell expansion in vivo
    Nelson, AG ; Wang, H ; Dewar, PM ; Eddy, EM ; Li, S ; Lim, XY ; Patton, T ; Zhou, Y ; Pediongco, TJ ; Meehan, LJ ; Meehan, BS ; Mak, JYW ; Fairlie, DP ; Stent, AW ; Kjer-Nielsen, L ; Mccluskey, J ; Eckle, SBG ; Corbett, AJ ; Souter, MNT ; Chen, Z (FRONTIERS MEDIA SA, 2023-08-31)
    INTRODUCTION: Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which mediate host immunity to microbial infection by recognizing metabolite antigens derived from microbial riboflavin synthesis presented by the MHC-I-related protein 1 (MR1). Namely, the potent MAIT cell antigens, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), form via the condensation of the riboflavin precursor 5-amino-6-D-ribitylaminouracil (5-A-RU) with the reactive carbonyl species (RCS) methylglyoxal (MG) and glyoxal (G), respectively. Although MAIT cells are abundant in humans, they are rare in mice, and increasing their abundance using expansion protocols with antigen and adjuvant has been shown to facilitate their study in mouse models of infection and disease. METHODS: Here, we outline three methods to increase the abundance of MAIT cells in C57BL/6 mice using a combination of inflammatory stimuli, 5-A-RU and MG. RESULTS: Our data demonstrate that the administration of synthetic 5-A-RU in combination with one of three different inflammatory stimuli is sufficient to increase the frequency and absolute numbers of MAIT cells in C57BL/6 mice. The resultant boosted MAIT cells are functional and can provide protection against a lethal infection of Legionella longbeachae. CONCLUSION: These results provide alternative methods for expanding MAIT cells with high doses of commercially available 5-A-RU (± MG) in the presence of various danger signals.
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    Association between insulin dysregulation and adrenocorticotropic hormone in aged horses and ponies with no clinical signs of pituitary pars intermedia dysfunction
    Li, FI ; Spence, RJ ; de Laat, MA ; Harris, PA ; Sonntag, J ; Menzies-Gow, NJ ; Durham, AE ; Bailey, SR ; Sillence, MN (WILEY, 2023-11)
    BACKGROUND: High concentrations of adrenocorticotropic hormone (ACTH) are used as an indicator of pituitary pars intermedia dysfunction (PPID), but other factors that may influence ACTH need to be understood, if diagnostic reference ranges for ACTH are to be used with confidence. Insulin dysregulation (ID) could be one such factor, as insulin affects pituitary hormones in other species. OBJECTIVES: To test the hypothesis that a relationship exists between high insulin and high ACTH in aged (>15-year-old) animals with no clinical signs of PPID. STUDY DESIGN: A cohort study. METHODS: Thirteen horses and eleven ponies (17-25 years-old; mares and geldings) were clinically examined for signs of PPID in the spring (November 2020) and autumn (April 2021). On the same day, blood samples were taken before and 2 h after an oral glucose test (OGT). Concentrations of insulin, glucose, ACTH and cortisol were measured. RESULTS: There was no association between ACTH and cortisol. However, there was a positive linear correlation between ACTH and post-OGT (insulin in the autumn (r = 0.427, p = 0.04). Two horses and six ponies had ACTH above the cut-off value for PPID diagnosis, and of these eight animals, six also had insulin concentrations above the cut-off value for ID. MAIN LIMITATIONS: The cohort was small and thyrotropin-releasing hormone (TRH) stimulation tests were not performed. CONCLUSIONS: In autumn, high ACTH was associated with ID, when no clinical signs of PPID were present. Because ACTH is used in PPID diagnosis, further work is required to understand this interaction.
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    Development of a body condition index to estimate adiposity in ponies and horses from morphometric measurements
    Potter, SJJ ; Erdody, MLL ; Bamford, NJJ ; Knowles, EJJ ; Menzies-Gow, N ; Morrison, PKK ; Argo, CM ; McIntosh, BJJ ; Kaufman, K ; Harris, PAA ; Bailey, SRR (WILEY, 2024-03)
    BACKGROUND: There is a high prevalence of obesity in ponies and pleasure horses. This may be associated with equine metabolic syndrome and an increased risk of laminitis. Body condition scoring (BCS) systems are widely used but are subjective and not very sensitive. OBJECTIVES: To derive a body condition index (BCI), based on objective morphometric measurements, that correlates with % body fat. STUDY DESIGN: Retrospective cohort study. METHODS: Morphometric measurements were obtained from 21 ponies and horses in obese and moderate body condition. Percentage body fat was determined using the deuterium dilution method and the BCI was derived to give the optimal correlation with body fat, applying appropriate weightings. The index was then validated by assessing inter-observer variation and correlation with % body fat in a separate population of Welsh ponies; and finally, the correlation between BCI and BCS was evaluated in larger populations from studies undertaken in Australia, the United Kingdom and the United States. RESULTS: The BCI correlated well with adiposity in the ponies and horses, giving a Pearson r value of 0.74 (P < 0.001); however, it was found to slightly overestimate the % body fat in leaner animals and underestimate in more obese animals. In field studies, the correlation between BCI and BCS varied particularly in Shetlands and miniature ponies, presumably due to differences in body shape. MAIN LIMITATIONS: Further work may be required to adapt the BCI to a method that is more applicable for Shetlands and miniature ponies. CONCLUSIONS: This BCI was able to provide an index of adiposity which compared favourably with condition scoring in terms of accuracy of estimating adiposity; and was more consistent and repeatable when used by inexperienced assessors. Therefore, this may be a useful tool for assessing adiposity; and may be more sensitive than condition scoring for tracking weight gain or weight loss in individual animals.
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    Renal arterial infusion of tempol prevents medullary hypoperfusion, hypoxia, and acute kidney injury in ovine Gram-negative sepsis
    Betrie, AH ; Ma, S ; Ow, CPC ; Peiris, RM ; Evans, RG ; Ayton, S ; Lane, DJR ; Southon, A ; Bailey, SR ; Bellomo, R ; May, CN ; Lankadeva, YR (WILEY, 2023-09)
    AIM: Renal medullary hypoperfusion and hypoxia precede acute kidney injury (AKI) in ovine sepsis. Oxidative/nitrosative stress, inflammation, and impaired nitric oxide generation may contribute to such pathophysiology. We tested whether the antioxidant and anti-inflammatory drug, tempol, may modify these responses. METHODS: Following unilateral nephrectomy, we inserted renal arterial catheters and laser-Doppler/oxygen-sensing probes in the renal cortex and medulla. Noanesthetized sheep were administered intravenous (IV) Escherichia coli and, at sepsis onset, IV tempol (IVT; 30 mg kg-1  h-1 ), renal arterial tempol (RAT; 3 mg kg-1  h-1 ), or vehicle. RESULTS: Septic sheep receiving vehicle developed renal medullary hypoperfusion (76 ± 16% decrease in perfusion), hypoxia (70 ± 13% decrease in oxygenation), and AKI (87 ± 8% decrease in creatinine clearance) with similar changes during IVT. However, RAT preserved medullary perfusion (1072 ± 307 to 1005 ± 271 units), oxygenation (46 ± 8 to 43 ± 6 mmHg), and creatinine clearance (61 ± 10 to 66 ± 20 mL min-1 ). Plasma, renal medullary, and cortical tissue malonaldehyde and medullary 3-nitrotyrosine decreased significantly with sepsis but were unaffected by IVT or RAT. Consistent with decreased oxidative/nitrosative stress markers, cortical and medullary nuclear factor-erythroid-related factor-2 increased significantly and were unaffected by IVT or RAT. However, RAT prevented sepsis-induced overexpression of cortical tissue tumor necrosis factor alpha (TNF-α; 51 ± 16% decrease; p = 0.003) and medullary Thr-495 phosphorylation of endothelial nitric oxide synthase (eNOS; 63 ± 18% decrease; p = 0.015). CONCLUSIONS: In ovine Gram-negative sepsis, renal arterial infusion of tempol prevented renal medullary hypoperfusion and hypoxia and AKI and decreased TNF-α expression and uncoupling of eNOS. However, it did not affect markers of oxidative/nitrosative stress, which were significantly decreased by Gram-negative sepsis.
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    Nodular hyperplasia of lymphoglandular complexes in dogs: A potential diagnostic pitfall for rectal masses
    Stent, AW ; Kiupel, M ; Dandrieux, JRS ; Liffman, R ; Bera, MM (SAGE PUBLICATIONS INC, 2024-03)
    Lymphoglandular complexes are components of the gut-associated lymphoid tissue that are characterized by submucosal lymphoid aggregates invested by projections of mucosal epithelium. Reports of pathology involving these structures are rare in both human and veterinary literature. Here, the authors report 2 cases of rectal masses excised from dogs following a period of tenesmus and hematochezia. In both animals, the masses were composed of lymphoid tissue closely encompassing tubuloacinar structures. Immunohistochemistry and polymerase chain reaction antigen receptor rearrangement testing demonstrated that the lymphoid population was polyclonal, comprising T and B cells arranged in loosely follicular aggregates centered on the epithelial foci. In light of these findings, a diagnosis of lymphoglandular complex nodular hyperplasia was reported. To the authors' knowledge, this is the first report of this condition in dogs.
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    Elevated lead exposure in Australian hunting dogs during a deer hunting season
    Hampton, JO ; Cobb, ML ; Toop, SD ; Flesch, JS ; Hyndman, TH (ELSEVIER SCI LTD, 2023-04-15)
    There is growing recognition of the threat posed by toxic lead-based ammunition. One group of domestic animals known to be susceptible to harmful lead exposure via this route is hunting dogs. Scent-trailing dogs ('hounds') are used to hunt introduced sambar deer (Cervus unicolor) during a prescribed eight-month (April-November) annual hunting season, during which they are fed fresh venison, in Victoria, south-eastern Australia. We used this annual season as a natural experiment to undertake longitudinal sampling of dogs for lead exposure. Blood was collected from 27 dogs owned by four different deer hunters and comprising three different breeds just prior to the start of the hound hunting season (March 2022) and in the middle of the season (August 2022), and blood lead levels (BLLs) (μg/dL) were determined via inductively coupled plasma mass spectrometry (ICP-MS). Using Tobit regression, the expected BLLs across all dogs were significantly lower before the season (0.50 μg/dL, standard error [SE] = 0.32 μg/dL) than during the season (1.39 μg/dL, SE = 0.35 μg/dL) (p = 0.01). However, when the breed of dog was included in the analyses, this effect was only significant in beagles (P < 0.001), not bloodhounds (p = 0.73) or harriers (p = 0.43). For 32% of the dogs before the season, and 56% during the season, BLLs exceeded the established threshold concentration for developmental neurotoxicity in humans (1.2 μg/dL). Time since most recent venison feeding, sex of dog and owner were not associated with BLLs. The finding that BLLs more than doubled during the hunting season indicates that lead exposure is a risk in this context. These results expand the sphere of impact from environmental lead in Australia from wild animals and humans, to include some groups of domestic animals, a textbook example of a One Health issue.
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    Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products
    Pagel, CN ; Kularathna, PK ; Sanaei, R ; Young, ND ; Hooper, JD ; Mackie, EJ (WILEY, 2022-05)
    BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.
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    Zoonoses and the Aboriginal and Torres Strait Islander population: A One Health scoping review.
    Riley, T ; Anderson, NE ; Lovett, R ; Meredith, A ; Cumming, B ; Srinivas, PN (Public Library of Science (PLoS), 2022)
    With limited access to animal health services, and high disease burdens among domesticated animals, Aboriginal and Torres Strait Islander communities in Australia face higher risk of disease including zoonoses. However, we lack understanding of the contribution of often preventable zoonoses to the health of these communities, which would enable us to enhance public health strategies and improve health outcomes. We conducted a scoping review to identify the current state of evidence on zoonoses in the Aboriginal and Torres Strait Islander population. We examined the size, scope and characteristics of the evidence base and analysed the zoonoses detected in the studies within a One Health framework. We identified 18 studies that detected 22 zoonotic pathogens in animals, people, and the environment, with most studies detecting pathogens in a single One Health sector and no studies investigating pathogens in all three sectors. Findings indicate that despite the strong conceptual foundations of One Health throughout the evidence base, evidence is lacking in application of this concept. There is a need to undertake further research that prioritises Aboriginal and Torres Strait Islander leadership, considers the contribution of human, animal and environmental health factors, and investigates the prevalence and impact of zoonoses in communities through a One Health approach.
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    The role of microbial ecology in improving the performance of anaerobic digestion of sewage sludge
    Krohn, C ; Khudur, L ; Dias, DA ; van den Akker, B ; Rees, CAA ; Crosbie, NDD ; Surapaneni, A ; O'Carroll, DMM ; Stuetz, RMM ; Batstone, DJJ ; Ball, ASS (FRONTIERS MEDIA SA, 2022-12-14)
    The use of next-generation diagnostic tools to optimise the anaerobic digestion of municipal sewage sludge has the potential to increase renewable natural gas recovery, improve the reuse of biosolid fertilisers and help operators expand circular economies globally. This review aims to provide perspectives on the role of microbial ecology in improving digester performance in wastewater treatment plants, highlighting that a systems biology approach is fundamental for monitoring mesophilic anaerobic sewage sludge in continuously stirred reactor tanks. We further highlight the potential applications arising from investigations into sludge ecology. The principal limitation for improvements in methane recoveries or in process stability of anaerobic digestion, especially after pre-treatment or during co-digestion, are ecological knowledge gaps related to the front-end metabolism (hydrolysis and fermentation). Operational problems such as stable biological foaming are a key problem, for which ecological markers are a suitable approach. However, no biomarkers exist yet to assist in monitoring and management of clade-specific foaming potentials along with other risks, such as pollutants and pathogens. Fundamental ecological principles apply to anaerobic digestion, which presents opportunities to predict and manipulate reactor functions. The path ahead for mapping ecological markers on process endpoints and risk factors of anaerobic digestion will involve numerical ecology, an expanding field that employs metrics derived from alpha, beta, phylogenetic, taxonomic, and functional diversity, as well as from phenotypes or life strategies derived from genetic potentials. In contrast to addressing operational issues (as noted above), which are effectively addressed by whole population or individual biomarkers, broad improvement and optimisation of function will require enhancement of hydrolysis and acidogenic processes. This will require a discovery-based approach, which will involve integrative research involving the proteome and metabolome. This will utilise, but overcome current limitations of DNA-centric approaches, and likely have broad application outside the specific field of anaerobic digestion.