Melbourne Veterinary School - Research Publications
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ItemProtease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products(WILEY, 2022-02-15)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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ItemZoonoses and the Aboriginal and Torres Strait Islander population: A One Health scoping review.(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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ItemThe role of microbial ecology in improving the performance of anaerobic digestion of sewage sludge(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.
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ItemCRISPR interference for sequence-specific regulation of fibroblast growth factor receptor A in Schistosoma mansoni(FRONTIERS MEDIA SA, 2023-01-13)Employing the flatworm parasite Schistosoma mansoni as a model, we report the first application of CRISPR interference (CRISPRi) in parasitic helminths for loss-of-function studies targeting the SmfgfrA gene which encodes the stem cell marker, fibroblast growth factor receptor A (FGFRA). SmFGFRA is essential for maintaining schistosome stem cells and critical in the schistosome-host interplay. The SmfgfrA gene was targeted in S. mansoni adult worms, eggs and schistosomula using a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor KRAB. We showed that SmfgfrA repression resulted in considerable phenotypic differences in the modulated parasites compared with controls, including reduced levels of SmfgfrA transcription and decreased protein expression of SmFGFRA, a decline in EdU (thymidine analog 5-ethynyl-2'-deoxyuridine, which specifically stains schistosome stem cells) signal, and an increase in cell apoptosis. Notably, reduced SmfgfrA transcription was evident in miracidia hatched from SmfgfrA-repressed eggs, and resulted in a significant change in miracidial behavior, indicative of a durable repression effect caused by CRISPRi. Intravenous injection of mice with SmfgfrA-repressed eggs resulted in granulomas that were markedly reduced in size and a decline in the level of serum IgE, emphasizing the importance of SmFGFRA in regulating the host immune response induced during schistosome infection. Our findings show the feasibility of applying CRISPRi for effective, targeted transcriptional repression in schistosomes, and provide the basis for employing CRISPRi to selectively perturb gene expression in parasitic helminths on a genome-wide scale.
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ItemSchistosoma mansoni Fibroblast Growth Factor Receptor A Orchestrates Multiple Functions in Schistosome Biology and in the Host-Parasite Interplay(FRONTIERS MEDIA SA, 2022-06-22)Stem cells play significant roles in driving the complex life cycle of Schistosoma mansoni. Fibroblast growth factor (FGF) receptor A (SmFGFRA) is essential for maintaining the integrity of schistosome stem cells. Using immunolocalization, we demonstrated that SmFGFRA was distributed abundantly in germinal/stem cells of different S. mansoni life stages including eggs, miracidia, cercariae, schistosomula and adult worms. Indeed, SmFGFRA was also localized amply in embryonic cells and in the perinuclear region of immature eggs; von Lichtenberg's layer and the neural mass of mature eggs; the ciliated surface and neural mass of miracidia; the tegument cytosol of cercariae, schistosomula and adult worms; and was present in abundance in the testis and vitellaria of adult worms of S. mansoni. The distribution pattern of SmFGFRA illustrates the importance of this molecule in maintaining stem cells, development of the nervous and reproductive system of schistosomes, and in the host-parasite interplay. We showed SmFGFRA can bind human FGFs, activating the mitogen activated protein kinase (MAPK) pathway of adult worms in vitro. Inhibition of FGF signaling by the specific tyrosine kinase inhibitor BIBF 1120 significantly reduced egg hatching ability and affected the behavior of miracidia hatched from the treated eggs, emphasizing the importance of FGF signaling in driving the life cycle of S. mansoni. Our findings provide increased understanding of the complex schistosome life cycle and host-parasite interactions, indicating components of the FGF signaling pathway may represent promising targets for developing new interventions against schistosomiasis.
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ItemEstablishing haematological and biochemical reference intervals for free-ranging Scottish golden eagle nestlings (Aquila chrysaetos)(SPRINGER, 2022-06)Abstract Health assessment of individuals is an important aspect of monitoring endangered wildlife populations. Haematological and biochemical values are a common health assessment tool, and whilst reference values are well established for domestic species, they are often not available for wild animal species. This study established 31 haematological and biochemical reference intervals for golden eagle (Aquila chrysaetos) nestlings in Scotland, in order to improve the understanding of the species’ health and support conservation efforts. Reference intervals were created from 47 nestlings (ages 2–7.5 weeks old) across 37 nests, to date, the largest sample of wild individuals of this species and age cohort sampled for these purposes. Upper reference intervals for concentrations of lymphocytes, total protein, cholesterol, triglycerides, uric acid, and monocytes, calculated in this study, are higher than those found for adult raptors and the interval span is higher than that observed in adult raptors for concentrations of AST, albumin, eosinophil, LDH, and monocyte count. Statistically significant positive correlations were found with age and concentrations of haemoglobin, lymphocytes, serum pH, and creatine kinase, and significant negative correlations with age for concentrations of thrombocytes, heterophils, total protein, globulin, and lactate dehydrogenase. Packed cell volume was significantly higher for females than males, and concentration of calcium and eosinophils were higher for individuals in good body condition than those in moderate body condition. The reference intervals produced by this study will be of important use to the veterinary and conservation management communities and will aid the long-term monitoring of the Scottish golden eagle population health.
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ItemTechnology to improve reliable access to oxygen in Western Uganda: study protocol for a phased implementation trial in neonatal and paediatric wards(BMJ PUBLISHING GROUP, 2022-06-01)INTRODUCTION: Oxygen is an essential medicine for children and adults. The current systems for its delivery can be expensive and unreliable in settings where oxygen is most needed. FREO2 Foundation Australia has developed an integrated oxygen system, driven by a mains-powered oxygen concentrator, with the ability to switch automatically between low-pressure oxygen storage device and cylinder oxygen in power interruptions. The aim of this study is to assess the clinical impact and cost-effectiveness of expanding this system to 20 community and district hospitals and level IV facilities in Western Uganda. METHODS AND ANALYSIS: This will be a phased implementation with preintervention and postintervention comparison of outcomes. Standardised baseline data collection and needs assessment will be conducted, followed by implementation of the FREO2 Oxygen System in combination with pulse oximetry in 1-2 facilities per month over a 16-month period, with a total 23-month data collection period. The primary outcome will be the proportion of hypoxaemic children receiving oxygen pre and post oxygen system. Secondary outcomes will assess clinical, economic and technical aspects. Pre and post oxygen system primary and secondary outcomes will be compared using regression models and standard tests of significance. Useability will be quantitatively and qualitatively evaluated in terms of acceptability, feasibility and appropriateness, using standardised implementation outcome measure tools. ETHICS AND DISSEMINATION: Ethics approval was obtained from Mbarara University of Science and Technology (MUREC 1/7) and the University of Melbourne (2021-14489-13654-2). Outcomes will be presented to the involved facilities, and to representatives of the Ministry of Health, Uganda. Broader dissemination will include publication in peer-reviewed journals and academic conference presentations. TRIAL REGISTRATION NUMBER: ACTRN12621000241831.
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ItemSeroprevalence of Toxoplasma gondii in burrowing bettongs (Bettongia lesueur): A comparison of cat-free and cat-exposed populations(CSIRO Publishing, 2022-01-01)Toxoplasma gondii is a ubiquitous protozoan transmitted by felids and infection, morbidity, and mortality occur in numerous marsupial species. This study explores the relationship between cat exposure and Toxoplasma in burrowing bettongs (Bettongia lesueur) in the Arid Recovery Reserve (ARR), South Australia. We estimated seroprevalence, using a modified agglutination test for T. gondii-specific immunoglobulins, in cat-free and cat-exposed bettong populations. Tissue samples collected opportunistically from bettong carcasses and from cats within and around the reserve were screened for T. gondii DNA using multiplex real-time polymerase chain reaction (M-qPCR). Two cats trapped inside the ARR tested positive (50.0%; 95% CI: 15.0-85.0%). All bettongs tested from the cat-free (n = 48) and cat-exposed (n = 19) exclosures were seronegative (95% CI: 0-7.41% and 0-16.82% respectively). We found no evidence of fatal toxoplasmosis, with all bettong carcasses negative on M-qPCR (n = 11). We propose that T. gondii was not detected in bettongs coexisting with cats primarily due to low exposure of bettongs at the time of sampling, possibly due to poor oocyst viability in arid conditions or low shedding by cats. Ongoing screening throughout high and low rainfall years should be conducted to better establish the risk of Toxoplasma to bettongs in the ARR.
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ItemA wMel Wolbachia variant in Aedes aegypti from field-collected Drosophila melanogaster with increased phenotypic stability under heat stress(WILEY, 2022-04)Mosquito-borne diseases remain a major cause of morbidity and mortality. Population replacement strategies involving the wMel strain of Wolbachia are being used widely to control mosquito-borne diseases. However, these strategies may be influenced by temperature because wMel is vulnerable to heat. wMel infections in Drosophila melanogaster are genetically diverse, but few transinfections of wMel variants have been generated in Aedes aegypti. Here, we successfully transferred a wMel variant (termed wMelM) originating from a field-collected D. melanogaster into Ae. aegypti. The new wMelM variant (clade I) is genetically distinct from the original wMel transinfection (clade III), and there are no genomic differences between wMelM in its original and transinfected host. We compared wMelM with wMel in its effects on host fitness, temperature tolerance, Wolbachia density, vector competence, cytoplasmic incompatibility and maternal transmission under heat stress in a controlled background. wMelM showed a higher heat tolerance than wMel, likely due to higher overall densities within the mosquito. Both wMel variants had minimal host fitness costs, complete cytoplasmic incompatibility and maternal transmission, and dengue virus blocking under laboratory conditions. Our results highlight phenotypic differences between Wolbachia variants and wMelM shows potential as an alternative strain in areas with strong seasonal temperature fluctuations.
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ItemIntravenous Acetaminophen Does Not Provide Adequate Postoperative Analgesia in Dogs Following Ovariohysterectomy(MDPI, 2021-12-01)(1) Objective: To investigate the analgesic effects of intravenous acetaminophen after intravenous administration in dogs presenting for ovariohysterectomy. (2) Methods: 14 ASA I client-owned female entire dogs. In this randomized, blinded, clinical study, dogs were given meperidine and acepromazine intramuscularly before induction of anesthesia with intravenous propofol. Anesthesia was maintained with isoflurane in oxygen. Intravenous acetaminophen 20 mg/kg or 0.9% NaCl was administered postoperatively. Pain assessments were conducted using the Glasgow Pain Scale short form before premedication and at 10, 20, 60, 120, and 180 min post-extubation or until rescue analgesia was given. The pain scores, times, and incidences of rescue analgesia between the groups was compared. Blood was collected before and 2, 5, 10, 20, 40, and 80 min after acetaminophen administration. Acetaminophen plasma concentration was quantified by liquid chromatography-mass spectrometry. The acetaminophen plasma concentration at the time of each pain score evaluation was subsequently calculated. (3) Results: There was no significant difference in pain scores at 10 min, highest pain scores, or time of rescue analgesia between groups. In each group, 3 dogs (43%) received rescue analgesia within 20 min. (4) Conclusions: Following ovariohysterectomy in dogs, there was no detectable analgesic effect of a 20 mg/kg dosage of intravenous acetaminophen administered at the end of surgery.