Veterinary Science Collected Works - Theses
Permanent URI for this collection
Search Results
1 - 6 of 6
-
ItemLean body weight adjusted intravenous contrast medium dose for abdominal Computed Tomography in dogs( 2022-01)Contrast-enhanced computed tomography (CT) is a common diagnostic modality to investigate abdominal pathology in dogs. For the animal’s safety, the minimum contrast media (CM) dose to achieve diagnostically appropriate contrast enhancement should be administered. Although acute adverse reaction to non-ionic iodinated CM is rare, moderate (>20% from baseline) alteration of heart rate and systemic blood pressure has been reported in dogs. Use of less iodinated contrast is particularly crucial in patients with pre-existing renal dysfunction, as CM is concentrated in renal tubules, having a direct toxic effect, modulating tubular regulatory mechanisms, and producing renal vasoactive substances. Risk of contrast induced nephropathy is considered low in human patients with no history or symptoms of renal disease. In dogs, iodinated contrast dose is commonly linearly increased based on total body weight (TBW). Lean body weight (LBW) has been considered in particularly obese human patients when prescribing a dose of CM, as body fat is not metabolically active and contributes little to dispersing or diluting the CM in the blood. This thesis consists of two published articles (chapter 2 and 4), of which the final research aim was to determine if less iodinated contrast per kilogram TBW can be administered to overweight/obese dogs while maintaining adequate organ and vessel opacification to make a confident radiologic diagnosis. Chapter 2 is a retrospective study performed to determine the variability of major abdominal vessel and organ contrast enhancement and to establish any relationship with abdominal fat percentage in contrast enhanced CT studies, performed on 62 clinical patients at U-Vet Werribee Animal Hospital between February 2014 and February 2019. Intravenous Iohexol (240 or 350 mgI/ml) with a dose range of 660-880 mgI/kg TBW administered by a power injector (1.8 - 3.0 ml/s) and saline chaser was the standard injection protocol used at this institution. Findings based on a linear regression model showed a positive association of aorta (p = 0.005), liver parenchymal (p = 0.045) and portal vein (p = 0.001) enhancement to abdominal fat percentage during the portal venous phase. Following this retrospective study, variability of abdominal organ/vessel contrast enhancement contributed by CM injection method was evaluated and outlined in chapter 3. This was done in attempt to minimise contribution of varied CM injection techniques on organ/vessel contrast enhancement variances prior to the second part of the research project. Out of the three injection techniques explored, the fixed injection duration protocol was found to have the smallest organ and vessel enhancement interquartile range. The fixed injection duration protocol; Iohexol 350 mgI/ml at 700 mgI/kg TBW administration by a cephalic vein intravenous catheter, and a CT study performed with a fixed injection duration of 20 s, with the arterial and portal venous acquisition triggered 10 s and 35 s after aortic arrival respectively, was used for the subsequent part of the research project. Chapter 4 was a prospective study where we hypothesised that LBW adjusted dosing would not affect the diagnostic quality of the abdominal CT study, will minimise negative physiological effects, and reduce variability of contrast enhancement in major abdominal organs and vessels as compared to dosing according to TBW. Results from 12 dogs showed that LBW dosed abdominal CT studies were of diagnostic quality (based on subjective radiologist’s assessment) and reduced inter-individual enhancement (smaller interquartile range) variability in dogs. Contrary to our second hypothesis, there was no significant difference in the change in heart rate and blood pressure after CM administration when dosed according to TBW or LBW. Based on reports in the human literature and findings from the present study, LBW is a better body size index for determining CM dose compared to TBW and is discussed in the final chapter.
-
ItemDevelopment of a live-attenuated vaccine against canine pyometra( 2022)Pyometra is a disease affecting intact bitches and is fatal if left untreated. It is caused by the bacterial invasion of the uterus under the influence of progesterone resulting in the accumulation of pus. Uropathogenic E. coli (UPEC) are one of the most predominant pathogens isolated from the uteri of affected animals. UPEC carry uropathogenic virulence factor (UVF) genes which influence their virulence in the urogenital tract. The urogenital tract is a low iron and nutrient-deficient environment making the UPEC’s iron acquisition system and the oligopeptide uptake system critical for their virulence and survival in the urogenital tract. To date, there are no medical alternatives to ovariohysterectomy for the prophylaxis of pyometra. The main objective of the studies described in this thesis was to develop a bacterial vaccine targeting the TonB-mediated iron uptake system, the Fur regulon and the oligopeptide uptake pathway. The hypothesis was that targeting the iron uptake, iron homeostasis and oligopeptide uptake systems at once would allow the vaccine to provide protection despite the genetic diversity between UPEC strains and the redundancy of many of their metabolic systems. The first step was to assemble and characterise the complete genome of canine UPEC isolates using Illumina Miseq and Oxford Nanopore MinION sequencing technologies. Whole genome comparative genomics was carried out between canine isolates and other pathogenic and non-pathogenic Escherichia coli (E. coli) strains from various sources with a special focus on human UPEC isolates. The genomes of the canine UPEC isolates P4 and YP3 contained a single circular chromosome of 5.09 mega basepairs (Mbp) and 5.08 Mbp, respectively. The genomes were annotated using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline (PGAP) and 18 pathogenicity-associated islands carrying virulence-associated genes were predicted in both genomes. Both canine uterine isolates belonged to the phylogroup B2, multilocus sequence type ST12 and serotype O4. The evolutionary relatedness between the canine UPEC isolates and other E. coli based on the cgMLST scheme and core single nucleotide phylogeny (SNP) showed a close phylogenetic relationship between the canine pyometra strains and human UPEC strains UTI89 and NU14. The virulence gene profiles of P4 and YP3 were nearly identical to human cystitis causing UPEC subtypes. The canine faecal isolate YF8 was identified as Escherichia marmotae and contained a single chromosome of 4.55 Mbp and an extra-chromosomal plasmid pYF8. The assembled genomes of the canine UPEC isolates will improve the understanding of the disease processes involved in pyometra and help explain similarities to urinary tract infections. They also facilitated genetic manipulation of the isolates for developing vaccine candidates in this thesis. The strain P4 was selected as the parent strain for vaccine development described in this thesis. The strain P4 was subjected to mutagenesis. The genes tonB, fur and oppD were insertional inactivated by lambda red recombination to generate single (P4-ΔtonB; P4-ΔoppD; P4-Δfur), double (P4-ΔtonB::ΔoppD) and triple (P4-ΔtonB::Δfur::ΔoppD) knockout mutant strains. In vitro growth assays of the mutants were carried out in iron limiting (2,2’ dipyridyl) and iron excess (ferric chloride) conditions and in the presence of the iron-dependent antibiotic Streptonigrin, to confirm their phenotypes. Dipyridyl sensitivity assays (200 µM) showed that the tonB mutants (P4-ΔtonB, P4-ΔtonB::ΔoppD, P4-ΔtonB::Δfur::ΔoppD) did not grow as efficiently as the field strain (P<0.05). Even in iron excess conditions, these strains grew less when compared to the field strain P4 (P<0.05), indicating the mutants’ inability to utilise environmental iron. Streptonigrin sensitivity assays (5 µg/mL) showed that the tonB mutants were more resistant to the antibiotic compared to the parent strain (P<0.05). This further confirmed the mutants’ impaired iron uptake ability. Together, these in vitro growth studies demonstrated the central role of tonB in iron starvation. Following on from this, the expression of the iron regulated outer membrane proteins (OMPs) was analysed in the vaccine candidates in comparison to the parent strain P4 under iron limiting and iron excess conditions. The outer membrane fraction was isolated by sonication and high-speed centrifugation using N-lauroylsarcosine and separated by Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). To identify and evaluate the antigenicity of the OMPs, immunoblotting was carried out using monospecific polyclonal rabbit anti-IroN antibodies. The results of this study showed that OMPs were expressed in iron limiting conditions in all the vaccine strains and in the parent strain. However, when the Fur regulon was inactivated, OMPs were expressed even in iron excess conditions. This is suggestive of successful deregulation of the Fur and that fur mutant strains are capable of expressing the OMPs irrespective of the iron levels in the media. The results of Western blotting showed that anti-IroN sera bound to a 79 kDa protein, indicating that the surface-exposed iron-regulated OMPs are strong antigenic proteins. In the final study, the safety and efficacy of the vaccine candidates were evaluated in an in vivo study. To do this, a murine model was developed in C57BL/6 mice to evaluate the pathogenicity of the canine UPEC strain P4 in the urinary tract and the reproductive tract by transurethral and intravaginal inoculation, respectively. P4 was mildly to moderately pathogenic in the urogenital tract of mice and produced mild to moderate inflammatory changes in the urinary bladder and uterus of C57BL/6 mice. This model was therefore chosen to evaluate the safety and efficacy of the vaccine candidates P4-ΔtonB, P4-ΔtonB::ΔoppD and P4-ΔtonB::Δfur::ΔoppD in the reproductive tract of female mice. Intravaginal inoculation of the vaccine candidates was found to be safe with no adverse effects recorded in the vaccinated mice. While up to 60% of mice vaccinated with the single P4-ΔtonB or the double knockout vaccine candidate P4-ΔtonB::ΔoppD were protected from challenge with the parent strain P4, the triple knockout strain did not confer any protection. The humoral immune response was poor in the mice inoculated intravaginally with any of the vaccine candidates. The total serum IgG levels did not vary between vaccinated and unvaccinated mice. Subcutaneous inoculation of the single knockout strain P4-ΔtonB produced severe systemic illness in the vaccinated mice. The double P4-ΔtonB::ΔoppD and triple knockout P4-ΔtonB::Δfur::ΔoppD strains caused severe inflammatory reaction at the injection site resulting in the premature abortion of this part of the experiment. The initial humoral immune response to P4-ΔtonB::Δfur::ΔoppD inoculated subcutaneously was promising. In summary, the studies described in this thesis characterise the whole genome of canine UPEC isolates and assess their virulence in a murine model. The live attenuated vaccine candidates developed by inactivating TonB, Fur and OppD expressed multiple antigenic OMPs and showed reduced growth potential in vitro. The preliminary outcomes of the in vivo studies are promising but more work is necessary to further evaluate their potential as vaccines.
-
ItemEvaluation of the analytical performance of a clinical laboratory coagulation analyser for coagulation factor measurement in canine plasma( 2022)Abstract Objective: To evaluate the analytical performance of a clinical laboratory coagulation analyser (Stago Compact Max) for coagulation factor activity measurement in canine plasma. Design: Prospective, observational, analytical study. Methods: Whole blood was collected from 15 privately owned dogs aged between 1 and 8 years old. Dobermans and greyhounds were excluded from sample collection. Five dogs (Pool A) over 25kg had up to 175mL of blood drawn from the jugular vein, and this was processed to citrated, platelet free plasma, and combined to provide a sample pool. Ten dogs (Pool B) over 10kg had 20mL of whole blood drawn from the jugular vein, and this was processed to citrated, platelet free plasma and combined to provide a comparison pool. Complete blood count, biochemistry testing and prothrombin (PT) and activated partial thromboplastin time (aPTT) testing was performed on all dogs prior to blood draw. Samples were excluded if they were visually haemolysed, lipaemic or jaundiced. Pool A was used to assess linearity, within-run and between-run precision of fibrinogen, factor II (FII), factor V (FV), factor VII (FVII), factor VIII (FVIII) and factor X (FX). A modified one stage PT assay utilizing human factor deprived plasmas was performed for FII, FV, FVII and FX. A modified aPTT assay utilizing human factor deprived plasma was performed for FVIII. Fibrinogen was measured via the modified clotting method of Clauss. All coagulation testing was performed as per the manufacturer’s guidelines (Diagnostica Stago). Barbitone buffer was used to perform dilutions on aliquots of Pool A, and 7 dilutions were each tested 20 times on the first day of testing to supply data for within-run precision and linearity, and then tested 5 times further for 4 consecutive days to provide data for between-run precision. The following sample: buffer dilutions were tested: undiluted, 9:1, 7:3, 1:1, 3:7, 1:9, 1:19. For coagulation factor activity testing, time to clot formation was measured and interpolated with a standard curve prepared from Pool B plasma. Fibrinogen was interpolated with the calibration curve provided by the manufacturer. Limit of the blank was assessed by performing each coagulation test 5 times with barbitone buffer only. Results: Results were tabulated and mean, standard deviation and coefficient of variation calculated for each factor, for within-run and between-run precision. Concentration vs measured activity were plotted and line of best fit and linear regression analysis performed. Acceptable linearity was determined as an R squared value >0.95. R squared values were as follows: Fibrinogen 0.99, FII 0.99, FV 0.99, FVII 0.99, FVIII 0.99, FX 0.99. Acceptable coefficient of variation was determined based on manufacturer’s data and factor activity levels of importance. Coefficient of variation was above the acceptable range for FV (between-run precision) at an analyte concentration of 5%, FVII (between-run precision) at analyte concentrations of 50%, 30% and 5% and FX (between-run precision) at an analyte concentration of 5%. Relevance: Investigation of coagulation disorders in dogs is a rapidly growing field in veterinary medicine, and the effects of critical illness on coagulation factor activity are poorly understood. Evaluation of the analytical performance of a clinical laboratory coagulation analyser for coagulation factor activity measurement in dogs is the first step in increasing research and clinical assessment in this area.
-
ItemSensitivity of canine haematological cancers to BH3 mimetics( 2022)Background: Haematological cancers such non-Hodgkin lymphoma (NHL) and acute and chronic leukaemias are common in both humans and dogs. Whilst these cancers can be treated with cytotoxic chemotherapy (and immunotherapy in people), development of treatment resistance is common. A frequently identified mechanism associated with resistance to chemotherapy-induced cell death is overexpression of the antiapoptotic B cell lymphoma 2 (BCL2) protein. Highly specific small molecule inhibitors of antiapoptotic BCL2 proteins, known as B cell lymphoma Homology 3 (BH3) mimetics, result in rapid induction of apoptosis in vitro and in vivo in human haematological cancer cells. This has led to the approval of the BCL2-specific inhibitor, venetoclax (VEN), for the treatment of chronic lymphocytic leukaemia (CLL) and acute myeloid leukaemia (AML) in Australia, North America and Europe. Expression of BCL2 has been reported in canine nodal lymphoma, however sensitivity of primary canine cells to BH3 mimetics has not been evaluated. Objectives: This study aimed to assess the in vitro sensitivity of non-neoplastic lymphocytes and primary haematological cancer cells from dogs to VEN or the dual BCL2/BCLxL inhibitor, navitoclax (NAV). The second aim was to evaluate the association between BCL2 protein expression and sensitivity to VEN. Methods: Nine dogs without cancer and 30 dogs with haematological cancers were recruited. Lymphocytes were isolated from peripheral blood, lymph node and/or bone marrow and incubated with VEN or NAV for 24 hours. Viable cells were enumerated using flow cytometry and the half maximal effective concentration (EC50) was calculated; BCL2 protein from whole cell lysates was assessed via immunoblotting. Results: Non-neoplastic lymph node-derived B and T canine lymphocytes were more sensitive to VEN than circulating lymphocytes (P = 0.02). Eighteen dogs with haematological cancers were included in the final analysis, including six cases of non-indolent multicentric B cell lymphoma, four cases of acute leukaemia, three cases of non-indolent multicentric T cell lymphoma, two cases each of indolent T-zone lymphoma and T-cell CLL, and one case of multiple myeloma. Neoplastic T lymphocytes (7/7) showed marked sensitivity to BH3 mimetics, with an EC50 <100nM, whilst 6/7 samples of non-indolent B cell cancers were resistant to VEN, with an EC50 >1000nM. All samples of acute leukaemia showed sensitivity to NAV, however sensitivity to VEN varied. Canine BCL2 protein was detected in all samples sensitive to VEN and was variably detected in resistant samples. All samples that lacked BCL2 were resistant to VEN. Conclusion and Clinical Importance: Neoplastic canine T lymphocytes are sensitive to VEN at concentrations achievable in vivo, thus VEN may be a novel therapeutic agent for treatment of canine T cell cancers. Detection of BCL2 protein is insufficient to predict in vitro sensitivity to VEN.
-
ItemDevelopment of methods to evaluate therapy for spinal cord trauma in dogs( 2020)Spinal cord injury (SCI) is a common cause of neurological deficits in dogs and a major cause of morbidity and decreased quality of life in people. In dogs, the most common causes of SCI are intervertebral disc diseases, particularly 'type I' intervertebral disc herniation knowns as intervertebral disc extrusion (IVDE), which occurs predominantly in chondrodystrophic breeds. The IVDE commonly causes contusion and compression to the spinal cord, which in most severe cases leads to complete loss of voluntary motor functions, bladder control and pain sensation distal to the level of the lesion. For decades, researchers have used animal models in their studies of SCI. However, limited progress has been achieved in translating any beneficial findings from experimental animal models to human SCI. Moreover, experimental studies in rodent models may not closely represent clinical SCI in human and canine patients. Naturally occurring compressive and contusive lesion of SCI associated with IVDE in dogs may be more analogous to human disease than induced experimental animal models. Canine naturally occurring SCI could simulate the injury in human patients including; mechanism of damage, pathophysiological events, healing processes, functional recovery, measures of evaluating outcomes, and effectiveness of the candidate therapies. Therefore, recruiting dogs of naturally occurring IVDE associated with SCI has become a common practice in clinical research trials. To date, no therapeutic method has been shown to treat SCI successfully in dogs with severe lesions. Therefore, the long-term outcome in these cases is chronic paraplegia with loss of sensation distal to the level of the lesion. Since this is true in both human and canine patients, novel therapies must be investigated, including promising neural stem cells (NSCs) therapy. The principal objective of this thesis was to study canine SCI and develop a reliable and reproducible paradigm for evaluating the effect of candidate therapies, using human neural precursor cells (hNPCs) in dogs as an example. This was approached by; 1) evaluating different MRI measures to monitor the spinal cord lesion in putatively naturally occurring canine spinal cord trauma, pre- and post- surgical or non-surgical treatments of IVDE, 2) ex vivo examination of hNPCs’ impact on inflammatory factors in stimulated whole blood (WB) samples from healthy dogs, 3) evaluating methods to measure responses to transplant of hNPCs into the spinal cord of the paraplegic dog with grade 5 SCI, as a pilot clinical study, 4) exploring the fate of hNPCs at 18 months post-xenotransplantation into spinal cord tissue (SCT) of a dog with grade 5 SCI, and 5) investigating the possibility of isolate and culture of canine NSCs from SCT of the dog. The findings of this thesis showed that the pathological and morphometric alterations of the objective and quantifiable parameters of the MRI measures of the spinal cord and vertebral canal post-SCI and following treatments are important prognostic indicators. The interaction effect of time and surgery is an important factor in decreasing spinal cord compression and vertebral canal narrowing. The results of the ex vivo study suggested that hNPCs could modulate the inflammatory responses in stimulated WB by reducing the production of tumour necrosis factor-alpha. The in vivo clinical pilot study concluded that neither adverse effects nor immunological reactions were developed following transplantation of hNPCs into the spinal cord of the dog. Although recovery of the cutaneous trunci muscle reflex was identified, no improvements of the deep pain sensation or locomotion and urinary functions were shown at the end of the 6 months observation period post-therapy. The immunohistochemical findings revealed that the grafted hNPCs had survived and migrated, after xenotransplantation into SCT of the host dog. The gross and histopathological examination showed neither significant pathological changes nor tumour formation at 18 months following xenotransplantation in the dog with grade 5 SCI. This research work reported that it is possible to isolate, and culture canine derived NSCs from SCT. Therefore, this shows promise for future research using of dog-to-dog allotransplantation of canine derived NSCs, which may minimise the associated complications of xenotransplantation. In summary, the observations of the studies in this thesis offer insight into the methods developed to evaluate potential benefits of using hNPCs for transplantation therapy in dogs with grade 5 SCI. The current research work helps understanding of the pathological and morphometrics alterations of spinal cord lesion pre and post treatment of IVDE. The findings are of value in understanding the mechanism of potential therapeutic effects of hNPCs, including the anti-inflammatory effect and the fate of the cells following transplantation into the spinal cord of the host. Finally, this research represents an initial step towards investigating the efficacy and potential benefits of hNPCs in a preclinical study in a large number of dogs with naturally occurring SCI.
-
ItemInflammation and endothelial perturbation in canine abdominal surgery: the potential modulatory effect of lidocaine( 2019)Complication rates following emergency laparotomy surgery are high, with organ dysfunction being a commonly encountered post-operative complication. Given the endothelium acts as the interface between the systemic circulation and the organs, its function is vital to maintaining organ health. The endothelium is in a constant state of flux, impacted largely by the local environment of which it is a part. In the presence of wide-spread systemic inflammation, inflammatory mediators precipitate change to the structure of the endothelial glycocalyx. These changes result in shedding of the endothelial glycocalyx and alteration of the endothelial phenotype. The endothelium may, as a result, lose the capacity to regulate vasomotor tone, and shift toward a pro-inflammatory and pro-coagulant state. This predisposes to reduced tissue oxygen delivery, and organ dysfunction may ensue. This thesis aimed to answer two key questions: does surgical trauma induced in canine patients undergoing emergent abdominal surgery invoke a systemic inflammatory response and subsequent endothelial activation? And if so, does lidocaine, a proposed immunomodulatory drug, mitigate this effect when given in the post-operative period? Chapter two provides a detailed review of endothelial structure and function, and current literature pertaining to systemic inflammation and endothelial activation in the context of abdominal surgery. Chapter two also examines the literature regarding the proposed mechanisms through which lidocaine acts as an immunomodulatory drug, and reviews publications that investigate the use of lidocaine as an anti-inflammatory drug in human patients after abdominal surgery. Chapter three is a randomized, blinded clinical trial quantifying the effect of emergency abdominal surgery on the concentration of markers of systemic inflammation and endothelial perturbation in canine patients in the post-operative period. The trial also assessed the potential use of lidocaine as a post-operative immunomodulatory therapy in dogs having undergone laparotomy. Fifty canine patients undergoing abdominal surgery were enrolled in the study. Patients were randomized into two separate groups: a study group receiving lidocaine intravenously, and a control group receiving 0.9% NaCl intravenously for a twelve-hour period following abdominal surgery. Blood samples were gathered prior to surgery, followed by six and twelve hours post-operatively. Concentrations of markers of systemic inflammation (IL-6) and markers of endothelial perturbation (VEGF and HA) were quantified via means of ELISA at each time point. Results revealed a significant increase in the concentration of markers of systemic inflammation and endothelial perturbation in post-operative blood samples. No immunomodulatory or endothelial preserving effect of lidocaine was appreciated.