Veterinary Biosciences - Theses
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ItemEvaluation of Ki-67, goblet cell and MUC2 mucin RNA expression in dogs with lymphoplasmacytic and granulomatous colitis( 2023-08)Background: Intestinal mucus barrier disruption occurs with chronic inflammatory enteropathies. The lack of studies evaluating changes in mucus health in dogs with chronic colitis arises from inherent challenges with assessment of the intestinal mucus layer. It is therefore unknown if reduced expression of goblet cells and/or mucin 2 (MUC2), which are key players in mucus production and secretion, correlate with inflammation severity in dogs with granulomatous colitis (GC) or lymphocytic-plasmacytic colitis (LPC). It is also unknown if Ki-67 expression, which has been evaluated in dogs with small intestinal inflammation, similarly correlates to severity of colonic inflammation in dogs with GC and LPC. Objectives: Study objectives included assessing if Ki-67 expression is upregulated in dogs with GC compared to LPC and dogs without colitis; comparing GBC and MUC2 expression among GC, LPC and non-inflamed colon; and exploring the use of RNAscope in-situ hybridization (ISH) to evaluate MUC2 expression in canine colon. Methods: Formalin-fixed endoscopic colonic biopsies were obtained from 48 dogs between January 2015 and March 2022. A blinded pathologist reviewed all biopsies. Dogs were classified into the GC (n=19), LPC (n=19) or no colonic signs (NC) (n=10) group based on the final histopathological diagnosis. Ki-67 immunohistochemistry, Alcian-Blue/PAS staining to highlight GBCs, and RNAscope ISH using customized canine MUC2-targeted probes were performed. At least five microscopic fields per slide were selected to measure Ki-67 labelling index (KI67%), GBC staining percentage (GBC%) and MUC2 expression (MUC2%) using computer image analysis. Pearson correlation coefficients were used to determine associations between World Small Animal Veterinary Association histologic (WH) score and measured variables. Linear regression models were used to compare the relationships between WH scores with KI67%, GBC%, and MUC2%; and between GBC% and MUC2% across groups. Results: Median WH scores were highest in dogs with GC. Median KI67% was highest in the NC group (6.69%; range, 1.70-23.60%). Median GBC% did not correlate with colonic inflammation in all groups. Median MUC2% normalised to WH score (MUC2%*) was highest in the NC group (10.02%; range, 3.05-39.09%), which was two and three-fold higher compared to the GC and LPC groups respectively. Mucin 2 expression had a strongly positive correlation with GBC% in the NC group (5.71% increase in MUC2%; 95%-CI, 0.503–0.987, p =.12), while no significant trends were observed in the GC and LPC groups. With increased severity of colonic inflammation, MUC2 expression sharply declined in the LPC group (-8.90%; 95%-CI, -17.98-0.19, p =.05) but remained static in the GC group. Conclusion and relevance: Dogs with GC had the most severe histologic colonic inflammation. Dogs without colitis had the highest Ki-67 expression. Goblet cell expression did not correlate with histologic severity of colonic inflammation overall. Dogs with GC and LPC likely have differences in pathways regulating MUC2 biosynthesis and secretion. Changes in MUC2 expression appear influenced by pathways regulating GBC activity rather than quantity in GC and LPC. Development of therapeutic strategies aimed at modifying GBC function and MUC2 expression may help improve mucus barrier integrity and mucosal healing in dogs with chronic colitis.
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ItemThe health of the captive Leadbeater's possum, (Gymnobelideus leadbeateri): implications for the conservation of the wild population.( 2023-05)The Leadbeater’s possum, Gymnobelideus leadbeateri, is a critically endangered marsupial with a range-restricted habitat and high risk of extinction. Two genetically distinct populations of the possums are described, the highland population found in the Victorian Central Highlands, Australia, and the lowland population, found in a single remnant of swamp forest. The highland Leadbeater’s possums entered captivity within Zoos Victoria in 1970. In Australia, highland animals were kept in captivity from 1970-2012 with smaller numbers kept internationally. Institutional memory within Zoos Victoria suggested that the highland population bred without difficulty and often in excess of enclosure space available and that the species had very few health issues with simple husbandry requirements. This captive population inbred with limited founders and senesced in the late 1980s and early 1990s. The last highland animal of this population died in 2012. In 2012, the wild remnant lowlands population was noted to have rapidly decreased from ~100 animals to approximately 60 animals over four years (2012-2016). From 2012-2021, 21 animals were brought into captivity to form a captive breeding program and insurance population. Despite ongoing pairing of these animals, and breeding continuing in the slowly disappearing wild population, no lowland animal has produced young in captivity. This study aimed to investigate Leadbeater’s possum health and disease, focusing on captive populations, with the aim of informing species recovery efforts and conservation measures. Fecundity and longevity in captive Leadbeater’s possums were studied using detailed analysis of historical records. Fecundity can be used as a broad indicator of health or adaptation to captivity. Institutional memory of keeping this species in captivity suggested that the breeding population of highland possums was ‘healthier’ than lowland animals, which may have corresponded with increased longevity. Analysis via Kaplan Meier survival graphs indicated that there was no significant difference in longevity regardless of sex, institution, captive conditions, or population groups, with some longer-lived outliers in the highland population. If longevity is a metric for health or adaption to captivity, the study findings suggest no appreciable differences. Fecund animals lived significantly longer – perhaps suggesting a correlation between adaptation to captivity and being able to breed in these conditions. Fecundity of the highland’s population was examined and revealed that the population had limited wild-born founder animals and fewer still that bred. Animals that did breed successfully were either wild born or of the first filial generation, and a small subset of these animals were highly fecund with six males and six females each producing greater than ten offspring. This resulted in a closely related population that petered out due to heavy inbreeding and lack of interest in a species thought to be abundant. A review of all mortality data was undertaken with a particular focus on diseases of significance to the population and to reproduction. Females had a range of pathology. Pyometra or cystic ovaries or uteri were commonly observed – all potentially associated with nulliparous. Many lowland possum males had histopathology consistent with infertility with no obvious aetiology, as a diagnosis of exclusion, mineral deficiency is likely. Chronic nephropathy was the most frequently clinically significant post-mortem finding, with unknown aetiology and an increase in diagnosis with age, suggesting a species predilection. Cardiovascular disease was the second most common pathology, and appeared to be associated with advanced age, and with diagnoses including atherosclerosis and arteriosclerosis. It is likely these findings are associated with obesity and increased caloric intake. It was important to consider these diseases in a holistic framework however as organ systems do not differentiate their borders. Leadbeater’s possums appeared very sensitive to disseminated infection, with the reproductive system being a common seed point. Possum health and diet are inextricably linked. The gut microbiome of three captive lowland female possums was characterised over a temporal diet change using next generation sequencing to analyse diversity. The possums had a narrow ‘core’ biome of bacteria that remained consistent across time and diet changes. When the diet altered over the opportunistic study period, the microbiome shifted in diversity and abundance with a similar trend across all three animals. There appeared to be some consistencies with the way each possum’s biome changed when diet changed and there were fluctuations within the most abundant species that appeared to be related to the fluctuation of macro-nutrients, particularly energy density. Opportunistic comparison with the gut microbiome of wild caught animals as they adjusted to a captive diet provided context for understanding the microbiome of this species, but further work is needed to elucidate the links between diet, microbiome, health, and disease. Examination of ectoparasite assemblages of wild highland and lowland populations revealed that flea assemblage varied by habitat type and /or elevation and thus population of possums. Two host-specific flea species, Stephanocircus domrowi and Wurunjerria warnekei were detected only on possums in highland habitats while generalist marsupial fleas Acanthopsylla r. rothschildii and Choristopsylla tristis were found in lowland habitat. Wurunjerria warnekei was suspected to be extinct prior to this study. A novel haemoparasite nematode was described, Breinlia sp. from an aged lowland Leadbeater’s possum found deceased in a nest box. Histology indicated that this haemoparasite was likely largely benign, with mild skin pathology found from migration of the nematode to the dermis. Translocation has already occurred within the two populations of Leadbeater’s possum, with little known about the microbiome, health and resilience to stressors, viruses, and parasites and the impact of change on the host’s health; this research has filled knowledge gaps and informed conservation actions.
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ItemD-Dimer in dogs, cats and horses: Investigations into immunoreactivity and structure( 2023)Background: D-dimer is a degradation product of cross-linked fibrin generated during thrombolysis. D-dimer is measured in human medicine for the early detection and exclusion of thrombotic syndromes. Commercially available D-dimer assays use monoclonal antibodies against human D-dimer, and there is limited sensitivity and specificity data for these antibodies in companion animals, thus the accuracy of human assays in veterinary species remains uncertain. Little is known about the sequence and structure of D-dimer in companion animals. Objectives: To evaluate the immunoreactivity of D-dimer in dogs, horses and cats with commercially available antibodies, and investigate potential differences in sequence and structure between species. Methods: A cross-linked fibrin lysate was prepared from canine, feline and equine plasma samples, and immunoblotting performed with a variety of commercially available D-dimer antibodies. Amino acid sequences of fibrinogen fragments involved in D-dimer were compared between species (cat, horse, dog and human), using available sequences for fibrinogen chains, and these were used for multiple sequence alignment, estimation of physiochemical properties, analysis of conserved structural domains, and prediction of three-dimensional structure by homology modelling. Results: The antibodies evaluated demonstrated variable reactivity with D-dimer in each species. The monoclonal antibody DD44 bound canine D-dimer with good specificity and sensitivity, but this antibody did not react with feline or equine D-dimer. The polyclonal antibody D2D bound putative D-dimer in dogs, cats and horses with good specificity, and increased sensitivity compared to human D-dimer. Analysis of sequence and structure revealed high inter-species homology, and three-dimensional models were prepared using a human template. Conclusions: Commercially available antibodies to human D-dimer have variable immunoreactivity with D-dimer in companion animals, suggesting that the use of human assays is inappropriate until analytical and/or clinical validation of the antibody used in that assay has been performed for each species. There are no overt structural variations to explain the variation in immunoreactivity between species, and this could reflect minor variations in structure at the site of binding for each antibody, which is largely unknown.