Melbourne Veterinary School - Research Publications
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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, 2022-06-01)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, 2022-03-23)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.
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ItemA new Hendra virus genotype found in Australian flying foxes.(Springer Science and Business Media LLC, 2021-10-13)BACKGROUND: Hendra virus (HeV) has caused lethal disease outbreaks in humans and horses in Australia. Flying foxes are the wildlife reservoir from which the virus was first isolated in 1996. Following a heat stress mortality event in Australian flying foxes in 2013, a novel HeV variant was discovered. This study describes the subsequent surveillance of Australian flying foxes for this novel virus over a nine year period using qRT-PCR testing of tissues from flying foxes submitted primarily for Australian bat lyssavirus diagnosis. Genome sequencing and characterisation of the novel HeV variant was also undertaken. METHODS: Spleen and kidney samples harvested from flying fox carcasses were initially screened with two real-time qRT-PCR assays specific for the prototype HeV. Two additional qRT-PCR assays were developed specific for the HeV variant first detected in samples from a flying fox in 2013. Next-generation sequencing and virus isolation was attempted from selected samples to further characterise the new virus. RESULTS: Since 2013, 98 flying foxes were tested and 11 were positive for the new HeV variant. No samples were positive for the original HeV. Ten of the positive samples were from grey-headed flying foxes (GHFF, Pteropus poliocephalus), however this species was over-represented in the opportunistic sampling (83% of bats tested were GHFF). The positive GHFF samples were collected from Victoria and South Australia and one positive Little red flying fox (LRFF, Pteropus scapulatus) was collected from Western Australia. Immunohistochemistry confirmed the presence of henipavirus antigen, associated with an inflammatory lesion in cardiac blood vessels of one GHFF. Positive samples were sequenced and the complete genome was obtained from three samples. When compared to published HeV genomes, there was 84% sequence identity at the nucleotide level. Based on phylogenetic analyses, the newly detected HeV belongs to the HeV species but occupies a distinct lineage. We have therefore designated this virus HeV genotype 2 (HeV-g2). Attempts to isolate virus from PCR positive samples have not been successful. CONCLUSIONS: A novel HeV genotype (HeV-g2) has been identified in two flying fox species submitted from three states in Australia, indicating that the level of genetic diversity for HeV is broader than first recognised. Given its high genetic relatedness to HeV, HeV-g2 is a zoonotic pathogen.
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ItemThe genome sequence of the European golden eagle, Aquila chrysaetos chrysaetos Linnaeus 1758.(F1000 Research Ltd, 2021)We present a genome assembly from an individual female Aquila chrysaetos chrysaetos (the European golden eagle; Chordata; Aves; Accipitridae). The genome sequence is 1.23 gigabases in span. The majority of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the W and Z sex chromosomes.
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ItemTetrathiomolybdate Treatment Attenuates Bleomycin-Induced Angiogenesis and Lung Pathology in a Sheep Model of Pulmonary Fibrosis(FRONTIERS MEDIA SA, 2021-10-22)Idiopathic pulmonary fibrosis (IPF) is a progressive chronic lung disease characterized by excessive extracellular matrix (ECM) deposition in the parenchyma of the lung. Accompanying the fibrotic remodeling, dysregulated angiogenesis has been observed and implicated in the development and progression of pulmonary fibrosis. Copper is known to be required for key processes involved in fibrosis and angiogenesis. We therefore hypothesized that lowering bioavailable serum copper with tetrathiomolybdate could be of therapeutic value for treating pulmonary fibrosis. This study aimed to investigate the effect of tetrathiomolybdate on angiogenesis and fibrosis induced in sheep lung segments infused with bleomycin. Twenty sheep received two fortnightly infusions of either bleomycin (3U), or saline (control) into two spatially separate lung segments. A week after the final bleomycin/saline infusions, sheep were randomly assigned into two groups (n = 10 per group) and received twice-weekly intravenous administrations of either 50 mg tetrathiomolybdate, or sterile saline (vehicle control), for 6 weeks. Vascular density, expressed as the percentage of capillary area to the total area of parenchyma, was determined in lung tissue sections immuno-stained with antibodies against CD34 and collagen type IV. The degree of fibrosis was assessed by histopathology scoring of H&E stained sections and collagen content using Masson's trichrome staining. Lung compliance was measured via a wedged bronchoscope procedure prior to and 7 weeks following final bleomycin infusion. In this large animal model, we show that copper lowering by tetrathiomolybdate chelation attenuates both bleomycin-induced angiogenesis and pulmonary fibrosis. Moreover, tetrathiomolybdate treatment downregulates vascular endothelial growth factor (VEGF) expression, and improved lung function in bleomycin-induced pulmonary fibrosis. Tetrathiomolybdate also suppressed the accumulation of inflammatory cells in bronchoalveolar lavage fluid 2 weeks after bleomycin injury. The molecular mechanism(s) underpinning copper modulation of fibrotic pathways is an important area for future investigation, and it represents a potential therapeutic target for pulmonary fibrosis.