Veterinary Science - Theses
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ItemClinical use of plasma lactate concentration and evaluation of the analytical performance of two benchtop analyzers for the measurement of L-lactate in canine plasma( 2018)Lactate has been used in small animal veterinary medicine since the 1990’s. Ongoing reviews of the literature had revealed that an up to date, comprehensive review of the use of lactate in small animal clinical practice was overdue. Consequently, this became the first objective of this thesis (Chapters 1 and 2). Through this review process, a surprising gap was identified in the veterinary literature. Despite widespread integration into clinical practice and validation of hand-held lactate analyzers against benchtop methods, there was no actual published evidence validating these benchtop methods for use in dogs (Chapter 3). Accordingly, this became the objective of the experimental component of this thesis (Chapter 4). A partial method validation study was designed to satisfy requirements outlined by the American Society of Veterinary Clinical Pathologists (ASVCP). The objective was to evaluate the analytical performance and equivalency of two commercially available benchtop lactate analyzers: a high-end point-of-care analyzer, the Radiometer ABL 800 (ABL800), and a clinical laboratory analyzer, the Cobas Integra 400 (Cobas400). As a gold-standard, definitive method for lactate measurement is poorly defined, we elected to use canine plasma manually spiked with reference standard sodium lactate as our test medium. After a preliminary assessment of linearity, intra-assay precision, and inter-assay precision using manufacturer controls, we used the spiked plasma to assess linearity, intra-assay precision, accuracy, range and equivalency of the two analyzers. Both analyzers demonstrated excellent precision. The Cobas400 was accurate over a wider range than the ABL800, however the ABL800 was more accurate within the most clinically relevant range. Method comparison was performed using the Cobas400 as the nominated reference method. Passing-Bablok linear regression and Bland-Altman analysis revealed that although the methods were in agreement, with ≈ 95% of measured differences falling within ± 1.96 standard deviations of the mean difference, they cannot be considered equivalent due to the presence of a small but clinically significant amount of constant and proportional bias. In conclusion, both analyzers demonstrated adequate analytical performance for use in clinical practice and future validation studies. However, they cannot be considered equivalent due to the presence of both constant and proportional bias and should not be used interchangeably. Consequently, analyzer specific reference intervals should be applied, and the same analyzer should be used when serially trending lactate concentrations in an individual patient.
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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.
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ItemSelect bacterial and viral pathogens of potential zoonotic or biosecurity importance in Australian brushtail (Trichosurus spp.) and ringtail (Pseudocheirus peregrinus) possums( 2018)Possums and gliders represent a diverse group of marsupial mammals native to Australia, including multiple vulnerable and endangered species. Common brushtail (Trichosurus vulpecula) and common ringtail (Pseudocheirus peregrinus) possums are two urban adapted species frequently found in gardens and parks throughout eastern Australia. Their presence in urban areas provides opportunities for transfer of zoonotic pathogens through both direct and indirect contact with humans. Diseases affecting possums are relatively poorly understood, as is the role of possums in the maintenance and transmission of zoonotic pathogens. This study aimed to investigate the significance of these possums as host species for a select range of pathogens, particularly in Victoria. Opportunistically collected serum samples from common ringtail, common brushtail and mountain brushtail possums (Trichosurus cunninghami) from Victoria and South Australia were tested for antibodies against Ross River virus (RRV) and flaviviruses. Cloacal swabs from common brushtail and ringtail possums from Victoria and New South Wales were screened for the presence of Mycobacterium ulcerans (the cause of Buruli ulcer), Campylobacter spp. and Salmonella spp. Spleen samples were screened for Francisella tularensis (the cause of tularaemia) and Coxiella burnetii (the cause of Q fever) by PCR. Antibodies to RRV were detected in 6.2% (16/259) of all possum samples, which were sampled during a concurrent epidemic of RRV in humans. Geographical areas with a moderate to high human case rate were associated with a higher seroprevalence rate in sampled possums. There was a significantly lower rate of detection in possums from urban environments compared to rural and mountainous environments. Seroconversion to an unspecified flavivirus was also present in 5.2% (5/97) of common ringtail possums and 6.5% (2/31) of mountain brushtail possums, but not in common brushtail possums (0/137). Two of the seropositive animals displayed clinical signs of encephalitis. Salmonella spp. was detected in 3.1% (7/229) of possums, while Campylobacter spp. Was found in 27.7% (41/229), with a significant predilection for common brushtail possums (38/106). The exact Campylobacter species was not identified, however, the main human pathogens C. jejuni and C. coli were excluded. No samples were positive for F. tularensis (n = 187), C. burnetii (n = 184) or M. ulcerans (n = 229). These findings indicate that, while urban-adapted possums may potentially carry zoonotic pathogens, they are unlikely to represent a high risk to public health, at least in regards to the infectious agents that were the focus of this study in the areas that were investigated. Finally, serum samples from all three possum species were screened for antibodies to Wobbly Possum Disease virus (WPDV), a recently identified virulent nidovirus in common brushtail possums in New Zealand. Although thought to be absent from Australia, antibodies to WPDV, or an antigenically similar nidovirus, were detected in all three species of possum, originating from both Victoria and South Australia. Overall, 16% (30/188) of samples were positive and 11.7% (22/188) were equivocal, with the two brushtail species most commonly affected. This is the first published report of serological evidence of WPDV, or an antigenically similar nidovirus, in Australian possums. However, attempts to detect viral RNA in spleen samples by PCR were unsuccessful.
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ItemUpdate on clinicopathological assessment of renal health in non-racing greyhounds( 2019)Background: Serum creatinine concentrations differ in greyhounds compared with non-sighthounds, but it is not known whether urine creatinine concentrations also differ and whether any difference would influence the interpretation of the urine protein to creatinine ratio (UPC). Additionally, there is some evidence for greyhounds having higher serum symmetric dimethylarginine (SDMA) than non-sighthounds, but this has yet to be confirmed in healthy non-racing greyhounds. Objectives: The objectives of this study were fourfold: (1) to compare the urine creatinine concentrations in healthy greyhounds and a control group of healthy non-sighthounds, (2) to determine the UPC reference interval in healthy greyhounds and to compare this with the UPC reference interval in a control group of healthy non-sighthounds, (3) to determine the serum SDMA concentration reference interval in healthy greyhounds and to compare this with the serum SDMA concentration reference interval in a control group of healthy non-sighthounds and with a previously established canine serum SDMA concentration reference interval, and (4) to establish whether lean body mass is correlated with serum creatinine and urine creatinine concentrations in greyhounds. Methods: The study used an observational cross-sectional design and included 98 clinically healthy non-racing greyhounds and 24 non-sighthound dogs with similar weight, age and sex distributions, as determined by t-test and chi-squared tests. SDMA, urine creatinine concentration and UPC values were measured from blood and urine samples. Linear regression was used to compare the greyhound and non-sighthound groups. Greyhound reference intervals were determined for SDMA and UPC using non-parametric methods. These were compared with the reference intervals for the non-sighthound group and with current International Renal Interest Society guidelines. In the greyhound sample, the association of urine creatinine with thigh circumference, height and weight was estimated using Pearson correlation. Statistical significance was set at P < 0.05 for all analyses. Results: Mean urine creatinine was approximately 22% higher in greyhounds than non-sighthounds after adjusting for urine concentration (P < 0.05). The upper limit of the greyhound UPC reference interval was 0.20 or 0.42, depending on whether strict or moderate exclusion criteria, respectively, were applied. The mean UPC was 29% lower in greyhounds than non-sighthounds, but this difference was not statistically significant (P = 0.1). The serum SDMA reference interval for greyhounds was 6.3–19.7 µg/dL (0.31–0.98 µmol/L). The upper end of this interval was higher than the upper limit of the published canine reference interval (6–13 µg/dL), and the mean concentration was statistically significantly higher in greyhounds (13.0 µg/dL) than non-sighthounds (10.2 µg/dL, P < 0.001). In greyhounds, there were weak correlations between the three morphometric measurements and both serum creatinine and urine creatinine after adjusting for urine concentration. Conclusions and clinical importance: These findings provide further evidence that greyhounds require several breed-specific reference intervals when evaluating renal function. Apart from having higher serum creatinine, greyhounds also have higher SDMA and higher urine creatinine when compared to non-sighthounds. Although UPC trended slightly lower in greyhounds, this finding was not significant, and therefore the threshold for non-proteinuria set by IRIS guidelines appears to be appropriate for greyhounds based on the calculated reference interval.
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ItemInvestigating the inflammatory pathways involved in placental detachment in the mare( 2018)The mechanism of placental detachment in the mare has not been investigated in detail and as such remains poorly understood. Research in other species provides strong evidence that parturition and fetal membrane detachment from the endometrium requires involvement of the immune response. Research in horses has also suggested that movement of leukocytes in the umbilical vessels, and their attraction to fetal membranes at parturition, contribute to placental detachment. The aims of this study were to 1) confirm whether a leukocyte “concentration gradient” exists between the equine neonatal umbilical artery and vein, and whether this gradient was associated with fetal membrane retention time 2) to investigate the expression of pro-inflammatory cytokines in the equine endometrium and fetal membranes at parturition using quantitative real time polymerase chain reaction (RT-qPCR) and 3) to localise pro-inflammatory cytokine proteins within the equine endometrial and fetal membrane tissues using immunohistochemistry to correlate with gene expression. Data was collected from 33 spontaneously foaling mares and their foals on a single Thoroughbred stud farm in New Zealand. Umbilical artery and vein blood, fetal membrane and endometrial biopsy tissue samples were obtained. The average fetal membrane retention time was 92 (+/-130) minutes. The median fatal membrane retention time was 47 minutes (IQR 30). There was a significantly higher number of total leukocytes, lymphocytes and neutrophils in the equine neonatal umbilical artery than the umbilical vein at parturition (p <0.001). Fetal membrane retention time tended to decrease with an increase in umbilical leukocyte concentration gradient but did not reach statistical significance (p = 0.098). The mRNA expression of IL-1, IL-6 and IL-8 was significantly greater in the endometrium of foaling mares compared to control mares (p < 0.001). The mRNA expression of IL-8 was significantly higher in the fetal membranes of foaling compared to control mares (p < 0.001) whereas mRNA expression of IL-6 in fetal membranes was significantly lower between the two groups (p< 0.001). Whilst mRNA expression for IL-1 was lower in the fetal membranes of foaling mares compared to controls, the difference was not significant (p = 0.16). This study has demonstrated the presence of an innate inflammatory immune response in the placenta of spontaneously foaling mares, suggesting the role of inflammation in the detachment of the fetal membranes from the endometrium. The results from this study have highlighted the need to further investigate the mechanism of placental detachment in the mare and also understand the pathophysiology behind abnormal fetal membrane retention.
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ItemEndothelial activation in dogs with severe sepsis( 2018)Two components of endothelial biology – endothelial glycocalyx, endothelial activation – have been explored this two-part, in-depth research project. An extensive literature review discusses the endothelial glycocalyx in health and critical illness. A prospective observational clinical research study then measures the concentration of soluble biomarkers of endothelial activation in severely septic dogs. The study found a significant difference in the concentration of biomarkers between the septic dogs and controls, a result consistent with the presence of endothelial activation in dogs with severe sepsis.
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ItemBiomechanical testing of the calcified metacarpal articular surface and its association with subchondral bone microstructure in Thoroughbred racehorses( 2018)Subchondral bone adaptation and fatigue likely alter joint surface mechanical properties. The aim of this study was to evaluate the relationship of calcified articular surface biomechanical properties in the distal palmar metacarpal condyle of Thoroughbred racehorses with subchondral bone microstructure and training history. Subchondral bone microstructure was evaluated using both micro- and clinical-computed tomography. Biomechanical properties of the calcified articular surface were tested with reference point indentation at the micron level. Articular surface toughness increased with commencement of race training, and was lower in horses that died as a result of musculoskeletal fatigue injury, had evidence of palmar osteochondral disease, had higher subchondral bone connectivity with more concave surfaces, and higher subchondral bone volume fraction. Further investigation of bone microstructural properties is warranted based on bone connectivity and surface shape findings. At clinically applicable imaging resolution, evidence of subchondral bone modelling may be an important indicator of articular surface biomechanical properties.
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ItemEffect of a novel Ovsynch program using a second treatment of prostaglandin F2Α on fixed time artificial insemination results in pasture-based dairy herds( 2018)This thesis is largely based on a clinical trial that was conducted on dairy cows in Southwest Victoria, Australia. The objective of the clinical trial was to evaluate the effect of a second prostaglandin F2α injection, within the original Ovsynch program, on the conception rate of dairy cows in a fixed time artificial insemination program. The regression of the corpus luteum was also assessed and the results between the modified and original Ovsynch programs were compared. A randomized clinical trial was conducted, which involved 1703 dairy cows selected from five split calving, pasture-fed dairy herds in Southwest Victoria, Australia. These animals included both primiparous and multiparous animals. All of the lactating cows were divided into two groups at the start of the joining period in order that artificial insemination would coincide with mating start date. This is the day on which joining starts so that the calving period coincides with the greatest seasonal pasture growth. Group 1 (n = 851), the control group, received the original Ovsynch program: Gonadotropin-releasing hormone at day 0 in the morning, prostaglandin F2α at day 7 in the morning, Gonadotropin-releasing hormone at day 9 in the afternoon and fixed time artificial insemination on day 10 in the morning, approximately 12 to 16 hours after the second Gonadotropin-releasing hormone injection. Group 2 (n = 852), the treatment group, received a modified Ovsynch program: Gonadotropin-releasing hormone at day 0 in the morning, prostaglandin F2α at day 7 in the morning, prostaglandin F2α at day 8 in the morning, Gonadotropin-releasing hormone at day 9 in the afternoon and fixed time artificial insemination on day 10 in the morning, approximately 12 to 16 hours after the second gonadotropin-releasing hormone injection. All artificial insemination was performed by experienced technicians. A subset of animals from each group, in each herd (n=95 treatment cows and n = 99 control cows), had blood samples taken at the time of the first prostaglandin injection and at the time of artificial insemination. Serum progesterone concentrations were measured at the time of the first prostaglandin injection to assess the proportion of cows likely to be cycling in each of the groups and at the time of artificial insemination to assess the proportion of cows with complete luteal regression. After adjusting for the effect of herd, conception rates were 7.0% greater in cows in the treatment group compared with cows in the control group. The proportion of cows with plasma progesterone concentrations ≥1 ng/mL in the treatment group at the time of artificial insemination was 13.5% lower than that of cows in the control group at the time of artificial insemination. Compared with the original Ovsynch program, it was estimated that a total of 14 (95% CI 8 to 43) cows need to be treated using the modified Ovsynch program to return one additional pregnancy. In conclusion, the use of the modified Ovsynch protocol is likely to increase conception rates to timed artificial insemination programs in dairy cows in Southwest Victoria.
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ItemHealth and fitness effects of Anaplasma species infection in African buffalo (Syncerus caffer)( 2017)Anaplasma marginale and A. centrale are intra-erythrocytic bacteria of domestic and wild ruminants and are mainly transmitted by ixodid ticks. Most of the work on anaplasmosis has been done on A. marginale infections in cattle, as it can cause disease with varying levels of severity, from icterus and anaemia, to abortions and death. However, wildlife, such as African buffalo (Syncerus caffer), appear to be only subclinically infected with A. marginale and A. centrale. This thesis aimed to characterise A. marginale and A. centrale in African buffalo from Kruger National Park (KNP), South Africa, and investigate the effects of the burden of Anaplasma species on the health and fitness of their host. Firstly, the major surface protein 1α (msp1α) and heat-shock protein (groEL) genes were used to characterise A. marginale and A. centrale, respectively, from African buffalo. Sequence variation and phylogenetic analyses revealed that sequences of Anaplasma spp. from African buffalo were unique and that they grouped separately when compared with previously published sequences of both species. Sequencing the same species in cattle from the same area in the future will allow for more conclusive evidence as to whether African buffalo are a reservoir for anaplasmosis, thereby providing insights into the interface of domestic and wild ruminants. Secondly, the burdens of A. marginale and A. centrale in blood samples from African buffalo were determined, using an established quantitative PCR, and then various statistical models were run to investigate associations between Anaplasma burden, co-infection dynamics and health outcomes for African buffalo. There appeared to be a time-lag between infection and host response, or co-infection response, for some of the parameters examined, showing the importance of considering such delays in studies of disease. Despite finding a positive association between the concurrent burdens of infection with the two Anaplasma species examined, once the time-lag was accounted for, there was a negative association between the species, possibly indicating resource competition or the development of cross-immunity. African buffalo did not have an anaemic response to infection with either A. marginale or A. centrale; in contrast, for animals infected with A. marginale, there was an increase in haematocrit levels in response to infection. On the other hand, there were higher serum total protein levels associated with increased burdens of A. centrale, which may be due to the development of an immune response. Host responses to infection were also affected by external factors, including season and resource availability, and host factors such as gender. Younger buffalo appeared to be infected with higher burdens of A. marginale and A. centrale. In calves, infection with A. marginale appeared to occur before infection with A. centrale, and more frequently, which could be the result of different invasion and evasion techniques of the two species in this host. This study provides an insight into the effects of a subclinical infection on a wildlife host, caused by a pathogen which may cause severe clinical disease in domesticated animals.
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ItemA bacteriological disease investigation in a captive breeding colony of Lord Howe Island Stick insects (Dryococelus australis)( 2017)The Lord Howe Island stick insect (Dryococelus australis) is critically endangered in the wild. Once driven to near-extinction, the species now exists within just a few captive breeding programs that have been established to conserve the species, to increase population numbers through breeding, and to support the eventual reintroduction of the species into the wild. In the current study, the bacteria Serratia marcescens was investigated as a possible cause of mortality in a captive breeding colony of D. australis housed at Melbourne Zoo. S. marcescens was isolated from both dead insects and from the environment. The prevalence of the bacteria in dead insects was estimated, and the gross pathological and histopathological changes in infected insects were characterised. Strain types of S. marcescens were characterised using pulsed-field gel electrophoresis, and the strain type A1 was implicated as the most likely outbreak strain associated with insect deaths. Epidemiological risk factors for mortality were also investigated, and a novel PCR test was developed to facilitate rapid detection and identification of S. marcescens. The overall findings of this research suggest that S. marcescens is a potential opportunistic pathogen of D. australis, and is associated with haemocoelomitis and death. However, high virulence was not demonstrated in infected insects under experimental conditions. Recommendations for the future management of D. australis and the future reintroduction of the species to the wild are discussed in light of the findings from this research.
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