Veterinary and Agricultural Sciences Collected Works - Theses
Now showing items 25-36 of 51
Investigations into the critical aspects of the health and welfare of the bobby calves and dairy cows in Victorian dairy systems
Animal welfare is becoming critical to the general public, farmers and dairy industry. In Australian dairy farming systems, maintenance of good welfare of large numbers of surplus non-replacement young male dairy calves (bobby calves) that go for slaughter at an early age of 5 to 10 days old is essential. Another major area of concern for the sustainable dairy industry in Australia is the active control measures against infectious pathogens that may cause severe diseases and production loss such as enteric pathogens in neonatal calves and Mycoplasma bovis (M. bovis) infection in cattle. This thesis examines the health and welfare conditions of bobby calves after transportation for slaughter and the potential of bobby calf blood samples to detect herd level M. bovis infection in dairy cattle in Victoria, Australia. Blood samples from bobby calves were collected at a commercial abattoir in Victoria after transportation and lairage to assess their welfare by examining plasma biochemical profile. A quarter of the calves had a failure of passive transfer with a variation of passive immune status depending on which region they came from suggesting colostrum management practices are not similar in all the farms. Very few calves experienced severe hypoglycaemia and dehydration before slaughter. However, most of the calves had higher plasma creatine kinase and lactate indicative of muscular fatigue. It is not clear from this study whether increased creatine kinase was also due to the muscular bruising in calves. Distance or duration of journey and lairage time had no significant effect on energy metabolites, hydration state or muscular fatigue in bobby calves before slaughter. Most of the calves at the time of slaughter showed no evidence of substantial physiological compromise, and there was no significant association either between the transport distance and plasma analytes nor between the total duration of transport and lairage in relation with plasma analytes. These results highlight that bobby calves can be well managed from the property of origin to abattoir under existing management system in Victoria without unduly compromising their welfare. The enteric pathogen E. coli K99 was the most common pathogen (37.4%) followed by bovine rotavirus (8.1%), Salmonella spp. (5.1%) and bovine coronavirus (2.6%) in the faeces of bobby calves after their transportation. Infected calves with the higher acquisition of passive immunity had lower amounts of bovine rotavirus (BRV), bovine coronavirus (BCV) and Salmonella spp. in faeces. Hypoglycaemia was associated with increased amounts of shedding of E. coli K99 and BRV in the faeces of infected calves. Increased distance of transportation was associated with a higher excretion of BRV only. Breed and sex had no influence on pathogen prevalence in the faeces. This study highlights that the prevalence of major enteric pathogens in bobby calves is minimal except E. coli K99 compared to previously reported prevalence of enteric pathogens in Australian dairy calves with diarrhoea, and higher acquisition of passive immunity may play an important role in lowering pathogen load in faeces of infected calves. The potential for bobby calf blood samples to be used to detect maternal antibody against M. bovis for the estimation of herd-level M. bovis prevalence in dairy cattle in Victoria was also assessed. Antibodies were detected using antibody capture ELISA. Sera were evaluated for adequate transfer of passive immunity before screening for M. bovis specific maternal antibody. All the M. bovis positive samples were detected from the sera with adequate passive immunity which was consistent with M. bovis specific antibody being transferred from cows to bobby calves. A proportion of 33.3% and 32.9% positive herds against M. bovis were detected in the northern and south-eastern dairy region respectively. These results indicate that M. bovis is a common pathogen in the major dairy regions in Victoria. This study also suggests that the collection of blood from bobby calves at the abattoir is convenient and could be used as a source of samples for M. bovis prevalence and surveillance study in Victoria, Australia.
Elucidating the developmental biology of Haemonchus contortus and other nematodes using a multi-omics approach
An appraisal of current literature (Chapter 1) revealed that many parasitic worms are pathogens of animals, causing major diseases and socioeconomic losses worldwide. Efforts to control roundworms (nematodes) are often compromised by widespread resistance to currently used treatments. Thus, there is a clear need to work toward new interventions, preferably based on a deep understanding of the molecular biology of nematodes and/or the relationship that they have with their host animals. The predominant focus of the present thesis was on exploring aspects of the developmental biology of parasitic nematodes using advanced molecular (‘omic) and bioinformatic technologies, with an emphasis on the barber’s pole worm (Haemonchus contortus) - one of the most economically important parasites of ruminant livestock. The specific aims were: (1) to establish molecular data sets (resources) for H. contortus; (2) to explore RNA transcription and protein expression profiles in the developmental transition from free-living to parasitic larvae of H. contortus under well-defined conditions in vitro; (3) to construct dauer-like signalling pathways in H. contortus and some other nematodes (ascaridoids); and (4) to elucidate dauer-like signalling and the involvement of bile acid-like dafachronic acids during nematode development. All of these aims were achieved. Addressing aim 1, comprehensive molecular (transcriptomic, proteomic and lipidomic) resources were established for H. contortus using advanced nucleic acid sequencing or mass spectrometry techniques (Chapters 2-4). Addressing aim 2, these resources were utilised for in-depth explorations of molecular changes during the developmental switch from the free-living to the parasitic stage of H. contortus (Chapter 5). This work revealed extensive alterations in transcription and protein expression. There was a discordance between the mRNA transcription and protein expression changes, which appeared to relate to microRNA regulation at the post-transcriptional level and genes involved in signal transduction and signalling molecule interactions. Comparative studies with C. elegans indicated that molecules and canonical dauer signalling pathways integrate environmental cues and developmental processes in H. contortus. Addressing aim 3, gene homologues involved in the dauer signalling pathways were inferred for H. contortus using genomic, transcriptomic and proteomic data sets (Chapter 6). Transcriptomic and proteomic studies of such homologues indicated similar gene transcription and protein phosphorylation profiles between the infective stage of H. contortus and the dauer stage of Caenorhabditis elegans. Although reduced sets of genes encoding G protein-coupled receptors, insulin-like peptides and cholesterol transporters were identified in H. contortus, similar functional roles of the signalling pathways were proposed for parasitic and free-living nematodes. Addressing aim 4, the “dauer hypothesis” was tested in H. contortus using an integrated ‘omics approach (Chapter 7). The dauer-like signalling pathways were shown to be activated during the developmental transition from the free-living to the parasitic stages of H. contortus, and were linked to an amplification of a 3-keto bile acid-like steroid hormone (i.e., dafachronic acid). This hormone bound to a nuclear receptor DAF-12 in vitro, and exhibited stimulatory effects on the larval activation and development of this parasite. These stimulatory effects appeared to be associated with a modulation of the dauer-like signalling cascades and lipid (glycerolipid and glycerophospholipid) metabolism. Specific chemical inhibition of dafachronic acid biosynthesis disrupted lipid metabolism and compromised larval activation and development, suggesting key roles for the hormone signalling module in the development of H. contortus. This work “opened the door” to exploring homologous signalling pathways in biologically distinct (ascaridoid) nematodes, including Toxocara canis (causing toxocariasis) and Ascaris suum (causing acariasis) (Chapters 8 and 9). In conclusion, this thesis showed that an integrated use of these resources allows detailed explorations of signalling molecules, molecular processes and pathways likely associated with nematode development, adaptation and parasitism, and provides opportunities to identify novel intervention targets (Chapter 10). Although this work was focused mainly on H. contortus and two ascaridoid species, the multi-omics approach established herein could be readily used to explore a wide range of interesting and socioeconomically significant parasitic worms (including also trematodes and cestodes) at the molecular level, and to elucidate host-parasite interactions and disease processes.
Health survey of two subspecies of bent-winged bats: southern bent-winged bat (Miniopterus orianae bassanii) and eastern bent-winged bat (Miniopterus orianae oceanensis)
Australian bent-winged bats are small, cave-roosting, insectivorous bats. There are two subspecies: the southern bent-winged bat (Miniopterus orianae bassanii) which occurs only in south-western Victoria and south-eastern South Australia, and the eastern bent-winged bat (M. orianae oceanensis) which is more common and widespread, being distributed along the east coast of Australia. In the last 50 years, the population of southern bent-winged bats has declined to the point where the subspecies was listed as critically endangered in 2007. The cause of the population decline is not clear. This project compared the health status of both bent-winged bat subspecies to investigate the role that disease may have on the southern bent-winged bat population decline. Morphometric data was gathered for the two subspecies. Southern bent-winged bats were heavier, but forearm length was not significantly different. This suggests that southern bent-winged bats may require more energy to fly and forage than eastern bent-winged bats. Oral swabs were tested by PCR for adenoviruses, coronaviruses, filoviruses, henipaviruses, herpesviruses and lyssaviruses. Six novel herpesviruses (five betaherpesviruses and one gammaherpesvirus) were identified, with the greatest infection prevalence occurring in Victorian southern bent-winged bats. No other viruses were detected. Both bat subspecies and their environment were sampled for Pseudogymnoascus destructans, the fungus that causes white nose syndrome, which has killed millions of bats in North America. All results were negative. Bats and their environment were sampled for Histoplasma capsulatum, a potential human pathogen associated with bat caves. A prevalence of 0-19% was detected on the bats, but environmental results were negative, indicative of a low zoonotic risk. A large number of fungi were found on the skin and fur of bats, most of which were environmental or plant associated, and none of which were likely to be of significant pathogenicity for bats. A risk assessment for the introduction of P. destructans into Australia concluded that it is very likely/almost certain that P. destructans will enter Australia, and likely that bats will be exposed to the fungus over the next ten years. Eight cave-dwelling bats from southern Australia are the ones most likely to be affected. The risk was assessed as medium for the southern bent-winged bat, as any increase in mortality could impact its long term survival. The risk to the other species was deemed to range from low to very low, due to their wider distribution and/or more stable populations. Bats were examined for the presence of bat flies, mites, ticks, Riouxgolvania beveridgei (previously associated with skin nodules in bent-winged bats), Polychromophilus melanipherus and haemoplasmas. While all parasites were present in both subspecies, prevalence varied seasonally and by location group, Victorian southern bent-winged bats having a significantly greater prevalence of infection with ticks, R. beveridgei and P. melanipherus. Twenty-seven southern bent-winged bats and one eastern bent-winged bat were opportunistically necropsied and examined histologically. Trauma was the most common cause of death in the southern bent-winged bats, which mostly occurred at one site where infrastructure is positioned around a key breeding cave. In response to these findings, management actions were implemented to reduce mortality rates. The single eastern bent-winged bat examined had a severe dermatitis caused by the mite, Notoedres muris. No association was found between any of the infectious and parasitic agents surveyed and body weight, blood parameters or any signs of ill health. However, Victorian southern bent-winged bats had a greater prevalence of herpesviruses, ticks, R. beveridgei and P. melanipherus infections, possibly indicative of some type of chronic stress impacting the immune system of this population.
Cytokine expression in dogs with enteropathies
Evidence points towards immune dysregulation and altered host-antigen interactions resulting in chronic gastrointestinal inflammation. This project aims to test the hypothesis that differences in tissue and faecal cytokine profiles in dogs with chronic enteropathy (CE) exist, and also to elucidate underlying pathological mechanisms and identify potential biomarkers and therapeutic interventions. My original contribution to knowledge is that faecal cytokines are detectable via ELISA and that faecal interleukin (IL)-6 and chemokine ligand (CXCL)-8 are higher in dogs with acute diarrhoea compared to non-diarrheic dogs. However, although faecal cytokines were detected in dogs with CE, our investigations do not highlight differences in tissue and faecal cytokine expression between clinical versus non-clinical dogs with antibiotic-(ARD) and food-responsive (FRD) disease, or consistent trends between different segments of the intestine. Differences in tissue expression of cytokines in dogs with ARD vs FRD were also documented, although a distinct T-helper (Th) -1, -2, or -17 based profile for each subgroup was not found. Enzyme-linked immunosorbent assays (ELISAs) were validated according to the criteria for biomarker selection and found acceptable for faecal and tissue IL-6,-10, CXCL-8 and TNF-α ELISAs. ELISA quantification of cytokines was next performed in normal dogs and dogs with acute diarrhoea. Faecal IL-6 and CXCL-8 were significantly increased in dogs with acute diarrhoea. However, their correlation to inflammation and disease needs to be further investigated. Tissue and faecal cytokine expression were compared in dogs with chronic enteropathy pre- and post-treatment, and between dogs categorised to have FRD and ARD. No dogs with steroid-responsive disease (SRD) were assessed. Relative quantification of IL-1A,IL-1β, IL-1R1, IL-4, IL-5, IL-6, IL-10, CXCL-8, tumour necrosis factor (TNF)-α, forkhead box P3, nucleotide-binding oligomerization domain-containing protein (NOD)-1 and NOD2, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)1, NFκBIA, toll-like receptor (TLR)-2, TLR-4, TLR-5, and TLR-9 mRNA via quantitative polymerase chain reaction (qPCR), was performed using pre- and post-treatment gastric, duodenal and colonic biopsies from 6 dogs with FRD and 6 dogs with ARD. Pre- and post-treatment faecal IL-6, -10, CXCL-8 and TNF-α, colonic IL-6 and gastric CXCL-8 tissue cytokines, were also quantified via ELISA. Pre-treatment gastric CXCL-8 was higher in ARD than FRD dogs (p = 0.025). Otherwise, no differences in gastric, duodenal and colonic cytokine mRNA or faecal cytokines were found in pre- versus post-treatment samples, or when ARD and FRD dogs were compared. No correlation was found between tissue mRNA and cytokine protein expression. There was also no consistent correlation between cytokine expression and clinical or histology scores across all segments. In conclusion, faecal cytokine quantification may be performed in dogs and may be of some use in dogs with acute diarrhoea, especially if future studies show it's ability to relate to specific aetiologies. However, the role of tissue and faecal cytokine detection in the diagnosis and monitoring of CE appears limited.
Effect of elevated carbon dioxide and high temperature on major micronutrients in strawberry
In this study, four different folate derivatives (tetrahydrofolic acid – THFA, 10-formylfolic acid – 10FFA, 5-formyltetrahydrofolic acid – 5FTHFA, 5-methytetrahydrofolic acid (5MTHFA)) were identified in fresh and freeze-dried strawberry samples. The individual and interaction effects of increased [CO2] and temperature on total folates content were significant (P≤0.05), and the responses were cultivar dependant. Total folate content in strawberries varied from 52.6 ± 5.1 µg to 364.8 ± 16.0 µg/100 g FW in cultivar ‘Albion’ and from 48.6 ± 7.0 µg to 237.4 ± 23.8 µg/100 g FW in cultivar ‘San Andreas’. Although, increased temperature positively affected the total folates content under lower [CO2] levels, the effects turned negative at the highest [CO2] concentration (950 pm). Higher temperature reduced the content of total folates in strawberries by 26% and 13% in cultivar ‘Albion’ and ‘San Andreas’, respectively. Impacts of elevated [CO2], higher temperature and their interactions on total vitamin C content in strawberries were statistically significant (P≤0.05) and the responses were cultivar dependent. Vitamin C contents in cultivar ‘Albion’ and ‘SA’ fresh strawberries were in a range of 59 ± 7 mg to 133 ± 15 mg/100 g FW and 56 ± 9 mg to 132 ± 9 mg/100 g FW, respectively. Increased growth temperature to 30 °C at 650 ppm [CO2] enhanced the amounts of vitamin C significantly (P≤0.05) to a maximum by 123% and 132% in cultivars ‘Albion’ and ‘San Andreas’, respectively. However, that effect wasn’t detected when the CO2 concentration was increased further to 950 ppm, and vitamin C concentrations drastically decreased by 36% and 31% in Albion’ and ‘San Andreas’, respectively. In general, folates and vitamin C contents were significantly (P≤0.05) higher in FD strawberry than fresh fruits. The next step of the study was to study the accessibility of increased polyphenols, vitamin C and folates in the fruits of fresh and frozen strawberries using simulated in vitro gastrointestinal digestion and colonic fermentation. Elevated [CO2] (ambient to 950 ppm) and higher temperature (ambient to 30 °C) enhanced the accessibility of polyphenols, folate and vitamin C in strawberries. Bioaccessibility of Pel-3-Glu increased from 67% to 88% in fresh strawberries when exposed to elevated growth. The exact amounts of individual polyphenols in accessible fraction were significantly (P≤0.05) higher in fresh fruits of strawberries grown under elevated growth conditions. For example, the highest amounts of Pel-3-Glu (19.89±0.4 mg/100 g FW), Pel-3-Rut (2.55±0.5 mg/100 g FW), p-coumaric (0.23±0.02 mg/100 g FW), ferulic (1.33±0.05 mg / 100 g FW), quercetin (1.97±0.2 mg/100 g FW) and p- coumaroyl (0.65±0.05 mg/100 g FW) were detected in fed state simulated gastrointestinal digesta of fresh strawberry grown under elevated growth conditions. Fresh strawberries grown under ambient growth contained 93.09±6.2 µg/100g folates and 18.55±0.5 mg/100g vitamin C as bioaccessible fractions under fed state while, elevated growth enhanced soluble folates and vitamin C up to 188.63±7.5 µg/100g and 30.48±0.3 mg/100g, respectively. Fresh strawberries contained higher amounts of accessible micronutrients than frozen strawberries, while increased bile contents in intestinal fluid (fed state) facilitated the release of bioactive compounds to gastrointestinal fluid. The insoluble fraction of strawberry digests after gastrointestinal digestion was then subjected to in vitro colonic fermentation using human faecal cultures and basal media. The soluble fraction of fermented strawberry digests was extracted to analyse polyphenols, folates and vitamin C. Higher contents of folate (7.90±0.05 µg/100 g FW), vitamin C (33.6±1.0 ng/100 g FW), Pel-3-Glu (2.00±0.14 mg/100 g FW), and p-coumaric (39±5 µg/100 g FW) were observed in soluble fraction of fermented precipitate after simulated gastrointestinal digestion at fasted state in frozen strawberries. These bioactive compounds and their metabolites would play an important role in the human colon by maintaining a healthy environment via scavenging the free radicals. According to the current study, the amount of bioaccessible bioactive compounds in strawberry could vary quantitatively and qualitatively based on growth and storage conditions as well as the status of digestion (fed or fasted state). Increased carbon dioxide and temperature in the growth environment enhanced the bioaccessibility of polyphenols, folates and vitamin C in strawberries. It can be concluded that strawberry fruits grown under elevated [CO2] and temperature may not be visually attractive comparing to normal strawberries. However, considering their nutritional value, those fruits can be promoted as freeze-dried strawberry in value added foods such as dairy products. Additionally, these research outcomes would help the commercial growers to focus on the nutritional aspects of fruits and vegetables grown under such elevated and extreme environmental conditions in the future. However, as a very little information is available concerning the interactive effects of elevated [CO2] and high temperature on fruits and vegetables in the field, more researches are needed to confirm the results from glasshouse studies.
The applications of sensor-based monitoring systems for diagnosing disease in peripartum dairy cattle managed in pasture-based dairy systems in south-west Victoria
The objectives of this thesis were: (1) to quantify the association between rumination time and physical activity in pastured dairy cattle, as measured by the SCR HR-LD collar (SCR Engineers, Netanaya, Israel) and the presence of peripartum health disorders; and (2) to assess the predictive potential of measurements generated by these devices in the predictive diagnosis of postpartum disease. An observational cohort study of 148 primiparous and multiparous Holstein dairy cows fitted with the SCR HR-LD device was undertaken at a commercial dairy farm in south western Victoria. Sensor derived, 2-hourly logs records of rumination and physical activity were collected from 10 days before parturition to 14 days in milk. The results of a physical examination performed by a veterinarian on each animal at 6±3 days in milk were also recorded. These data were used to construct multivariable linear, mixed-effects models to determine the effect of common peripartum health disorders on daily rumination time (DRT) and daily physical activity (ACT). Postpartum DRT was lower in animals affected by LDA and RFM and postpartum ACT lower in animals with subclinical ketosis or left displaced abomasum. Heifers had lower levels of postpartum DRT and higher levels of ACT. To determine the predictive potential of sensor measurements, bihourly rumination and activity data from the 48 hours prior to clinical examination were used to construct classification models for LDA, subclinical ketosis and undifferentiated metritis using three algorithmic classification techniques: extreme gradient boosting, neural network analysis and the random ferns technique. Fixed-effects logistic regression models were used to describe the risk relationships between sensor measurements and health disorders for the 48 hours prior to diagnosis. Declines in rumination and activity were associated with an increased odds of being diagnosed with any health disorder. Classification models had poor sensitivity and specificity for identifying all conditions. Optimal combinations of sensitivity and specificity were for the identification of measurements associated with LDA. The utility of algorithmic classifications of health status was hampered by poor combinations of sensitivity and specificity. Cow level sensing technologies may have a role in monitoring peripartum disease in pasture-based dairies but should be viewed as a screening device to indicate which cows require further assessment before interventions are taken.
Inflammation and endothelial perturbation in canine abdominal surgery: the potential modulatory effect of lidocaine
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.
The ecology and epidemiology of Rickettsia felis in Australia
Rickettsia felis is an emerging flea-borne zoonosis that is being increasingly recognised to contribute to unspecified malaise often referred to as fevers of unknown origins. The cat flea, Ctenocephalides felis, has been most widely accepted as the biological vector for R. felis. It is an adaptable ectoparasite that readily feeds on companion animals and opportunistically on other mammalian hosts. Its ability to persist in environments and on pets despite challenges from grooming, cleaning and prophylactic medications has led to the development of innumerable commercial preventatives designed to protect against infestation. The cat flea and its endosymbiont, R. felis, have managed to disseminate along with its canine and feline hosts across continents, presenting a potential risk to human health, Australia being of no exception. Despite the widespread presence of the cat flea and its promiscuous host-feeding behaviour, the contribution of R. felis to human morbidity, resulting loss of productivity and reduced quality of life in Australia remains largely unknown. Moreover, demographic, ecological and climatic risk factors for R. felis in Australia remain uninvestigated. This study sets out the foundation to address these knowledge gaps in Australia by i) retrospectively testing sera of patients previously testing positive for murine typhus for R. felis exposure; ii) determining the prevalence and associated demographic, occupational and geographical risk factors contributing to R. felis exposure in Australian veterinarians and; iii) determining the role of rodents as peri-domestic reservoirs for R. felis, and; iv) determining the geographical and climatic risks contributing to R. felis infestation rates in cat fleas in eastern coastal Australia. Historically, a specific diagnosis of flea-borne spotted fever (FBSF) was limited, owing to immunogenic cross-reactivity with Rickettsia typhi. Thus patients who had been exposed to R. felis were not able to be specifically tested. Of 49 patient sera previously testing antibody positive for murine typhus at the Australian Rickettsial Reference Laboratory, Geelong, 14 patients yielded specific antibodies to R. felis, seven yielded specific antibodies to R. typhi, while 28 patients were classified as indeterminate (with titres reacting between two-fold across the two organisms). Of R. felis positive patients, 5 demonstrated seroconversion, indicating a recent infection that was likely contributing to clinical signs observed by their referring doctor. Despite a cluster of five individual FBSF cases being reported for the first time in Australia in 2009, it is clear that other FBSF cases have remained undiagnosed or misdiagnosed, advocating the need for medical practitioners to obtain relevant history on patient’s exposure to domestic pets and their fleas.The second study sought to address the prevalence and associated risk factors for R. felis exposure in a high-occupational risk group, veterinarians. Of 131 veterinarians tested across Australia, 16.0% were found specifically seroreactive to R. felis. Older veterinarians were significantly protected from exposure (OR=0.752,p=0.04, 95% CI=0.579−0.975), as were those that recommended flea treatment to their clients (OR=0.611,p=0.044, 95% CI=0.38−0.982). Surprisingly, participants from the cooler south-eastern states of Victoria and Tasmania were trending toward a higher odds of exposure compared with other, warmer states (OR=1.381,p=0.075, 95% CI = 0.973−1.96). This was at odds with preconceptions on the ecology of ectoparasites; warmer subtropical and tropical regions were associated with higher environmental flea burdens. Moreover, this study demonstrated the high degree of exposure of R. felis in veterinarians, suggesting incidence of FBSF may be underrepresented in Australia. For the third study, fleas collected from client-owned dogs and cats in different climatic zones along the east coast of Australia were screened for R. felis, to determine infection rates of R.felis URRWXCal2 in C. felis felis. Infection rates of 6.7%, 13.2% and 15.5% were obtained by real-time PCR (qPCR) in tropical, subtropical and temperate regions, respectively. Univariate analysis indicated that fleas from toy/small breed dogs were less likely to be harbouring R. felis (p=0.033), as were Domestic Medium Hair (DMH) and pedigree-breed cats (p=0.0002 and p=0.043 respectively). Cooler minimum temperature ranges of between 15 to 20°C and between 8 to 15°C increased the odds of R. felis positivity in fleas, as did a constrained maximum temperature range of between 27 to 30°C on multivariable analysis. In conclusion this study demonstrated that environmental conditions such as temperature play a part in influencing R. felis survival and infectivity within its flea host, which suggests that public health risk mitigation measures need to consider regional differences to adequately comprehend transmission risk of flea-borne spotted fever. Finally, the involvement of peri-domestic animals in the life cycle of R. felis was investigated. Within urban environments, movement of companion animals is limited by legislation and animal control initiatives. Rodents are able host the cat flea vector, but their participation in perpetuating the life cycle of R. felis hasn’t been conclusively demonstrated. Brain and spleen tissue collected from 256 native and introduced rats (Rattus fuscipes, Rattus norvegicus and Rattus rattus) in south-east Queensland were tested for rickettsiae by qPCR, with none detectably positive, suggesting that they do not play a significant part in maintaining R. felis. This series of studies represents an in-depth look at the ecology of R. felis in Australia, and provides an insight into characteristics that makes it such a successful but often overlooked organism. Awareness amongst the public, medical practitioners and veterinarians is currently poorly developed, and diagnosis of the condition has suffered as a result.
Use of multiplex-tandem polymerase chain reaction in an operational environment to assess cyanotoxins and taste and odour metabolites of cyanobacterial blooms in Victorian waters
Cyanobacterial blooms represent a major water quality and public health issue due to their potential to produce taste and odour (T/O) compounds, and a variety of toxic metabolites. Monitoring and management of cyanobacterial blooms in various water resources is in turn dependent on the availability of accurate and sensitive diagnostic tools, and contemporary or prior knowledge of the regional distribution of toxin and T/O types. For example, in Victoria, Australia, there is limited knowledge of the distribution/ecology of geosmin producers, such that prioritising taxa for routine monitoring is difficult. Additionally, knowledge about the prevalence of many yet to be identified or emerging toxic cyanobacteria (eg., anatoxin producers) in Australia, remains poorly understood. Due to the many limitations of commonly used detection methods (i.e., microscopy and direct detection techniques), to identify toxic and T/O producing cyanobacteria, there is a need to develop sensitive and accurate diagnostic tools. PCR-based tools have shown potential to accurately identify toxic or T/O producing cyanobacteria; however, they have important limitations for routine monitoring. Although multiplex-tandem PCR (MT-PCR) tool has shown significant potential to accurately detect four major cyanotoxins (i.e., MC, NOD, STX and CYN) in Australia, it requires additional field testing and further, this tool is not presently suitable to detect anatoxin and geosmin-producing cyanobacteria. The work presented in this study used PCR-based genetic screening tools (i.e., conventional PCR, nested-PCR or MT-PCR) for accurate diagnosis of cyanotoxins and geosmin-producing cyanobacteria on environmental cyanobacterial bloom samples collected from various parts of Victoria, Australia, over a period of eight years (i.e., from 2010 to 2018). The study also employed metagenomic approaches (eg., Illumina MiSeq and PacBio) using biomarker genes [eg., 16S ribosomal RNA (16S rRNA) and phycocyanin intergenic spacer sequence (PC-IGS or cpcBA-IGS)] to elucidate the community structure of a recent (i.e., 2016) cyanobacterial bloom in the Murray River, Victoria, Australia. Based on conventional PCR-based genetic screening (using three different primer pairs) of more than 250 cyanobacteria bloom samples collected over a period of six years (i.e., 2010-2016), we detected Dolichospermum ucrainicum as the major geosmin producer in 87% of sequenced samples. Using these data, we developed a novel, small amplicon PCR primer pair capable to broadly identify all geosmin-producing cyanobacteria identified in the current study using a single standardised protocol. Additionally, genetic screening using nested-PCR on samples (n=226) collected from 2010-2017 revealed the presence and distribution of several anatoxin-producing cyanobacteria, including Cuspidothrix issatschenkoi, Aphanizomenon sp., Dolichospermum sp., at an overall sample prevalence of 30.1%. An overwhelming majority (86.8%) of anatoxin positive nested-PCR detections were from samples collected from 2016 to 2017, as compared to only 6% of samples collected prior to 2016. Using this data obtained, novel, short amplicon, PCR primers were designed for better PCR-based detection of geosmin primers. This study is the first confirming the presence of anatoxin producers in Australia. Further, field evaluation of the efficiency of MT-PCR to assess toxic cyanobacterial blooms and analysis of total community composition on samples (n=194) collected from the Murray River bloom (March 2016 – May 2016) have shown to greatly supplement microscopic examination, and highlights the potential utility of combining targeted qPCR with metagenomic amplicon sequencing methods. Based on these findings, the MT-PCR assay was expanded to detect anatoxin and major geosmin-producing cyanobacteria. The expanded MT-PCR demonstrated superior performance in comparison to conventional or nested PCRs for all but one (anatoxin) gene target in all samples (i.e., n=91 from March 2016 – February 2018) tested. Here, we report a diagnostic specificity of 100% and diagnostic sensitivity of ≥ 81% for all gene targets, and the MT-PCR platform may provide a much-needed tool for routine monitoring of toxic and geosmin-producing cyanobacterial blooms of global relevance.
Formulating curcumin in a biodegradable polymeric composite material: a step towards wound healing applications
The natural process of wound healing typically consists of four distinct but overlapping phases which include, hemostasis (platelet aggregation and blood clot formation), inflammation (migration of blood cells), proliferation (angiogenesis or blood vessel formation), and remodelling (reorganisation of collagen and scar tissue formation). However, in diabetic patients, this elaborate well-programmed process becomes disrupted, and there is an urgent need for compounds and formulations that can improve wound healing in these cases. A variety of natural components, including curcumin, have been identified as wound-healing agents. Curcumin, is a yellow hydrophobic natural polyphenolic pigment derived from the rhizomes of the herb Carcuma longa, which has been identified as the active principal of turmeric. The inability to efficiently deliver curcumin in a soluble form presents a chief challenge for its clinical use. Here we characterised, and optimised different biodegradable and biocompatible formulations of curcumin encapsulated particles, in order to enhance the efficiency of curcumin wound healing effect. The size of the optimised curcumin particles ranged from 1286 to 1485 nm, with an encapsulation efficiency of 75%. The zeta potential exhibited values in the range of (-7.2) to (-7.96) with the PDI of 0.4. Physical characterisation using TEM imaging ensured the successful fabrication and encapsulation of curcumin in the polymeric matrix, which had been fabricated in rod shape. Release profile occurred in a biphasic manner including an initial burst, followed by a sustained release trend for curcumin particles. In vitro cytotoxicity assays along with microscopic imaging confirmed safety of the applied concentration of curcumin particles below 25 µg/ml. Moreover, the results of cellular uptake study validated the internalisation of curcumin particles. Overall this thesis, elucidated the developed biocompatible and biodegradable formulations for curcumin encapsulation do have the potential to be employed as a drug delivery vehicle for curcumin. Further validation of the potential of this preparation to enhance wound healing is still needed.
Protease-activated receptor-2 in bone morphogenesis and pathology: Is par2 essential in osteoblastic bone homeostasis?
Protease activated receptor-2 (PAR2) is a member of the small family of protease-activated G protein-coupled receptors. PAR2 is widely expressed in most tissues including bone and has been shown to regulate cell proliferation and survival, cytokine production and release as well as nociception. Recent unpublished data suggest that PAR2 plays a role in bone homeostasis by regulating the osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). Due to the potential impact of such regulation on age-related bone loss and osteoporosis, the current study undertook to investigate the hypothesis that PAR2 activation regulates the determination of mesenchymal cell fate such that osteoblastogenesis is preferred over adipogenesis both in vivo and in vitro and that such regulation impacts the potential outcome of ageing in bone. Moreover, as PAR2 is a pro-inflammatory receptor, it was also hypothesised that PAR2 is a mediator of inflammation induced osteopoenia and as such its deletion will improve the resulting bone phenotype. Comparison of the bone phenotype in ageing global PAR2 null mice with the wildtype controls showed that the global lack of PAR2 in vivo was associated with a low bone mass profile and bone tissue mineral density which was compounded by a relatively rapid structural deterioration. These observations were more pronounced in the male knockout mice. In vitro gene silencing experiments with bipotential Kusa 4b10 mesenchymal cells suggested a pro-osteogenic and anti-adipogenic role for the receptor in the differentiation of MSCs. Gene expression studies revealed a number of novel genes downstream of PAR2 that were not previously associated with these processes. These included Cnr1, Il6, Ramp3 and Enpep which were more highly expressed following PAR2 knockdown and C1qtnf3, Snorc and Gpr35, which were suppressed. Investigation of IL-6 concentrations in the medium by ELISA as well as use of anti-IL-6 neutralising antibody suggested that PAR2 promoted osteogenesis and inhibited adipogenesis partly by suppressing expression of IL-6. To further examine the role of the receptor in osteoblasts in vivo, osteoblast-specific deletion of PAR2 was achieved through the expression of Cre-recombinase that was driven by the Osterix (Osx) promoter. Surprisingly, osteoblast-specific ablation of PAR2 in growing mice not only resulted in an increased bone formation rate in 7 week old females, but also culminated in a higher bone mass profile at 13 weeks whilst males remained unaffected. The paradoxical finding may be explained by the presence of PAR2 during the critical earlier stages of MSC differentiation and its ablation only after cells have become committed Osx-expressing osteoprogenitor cells. These results suggest that PAR2 plays a role in the determination of osteoblasts prior to the stage at which gene deletion occurs in the osteoblast-specific PAR2 null mice. To investigate the role of the receptor in the pathogenesis of inflammation-induced osteopoenia, the bone phenotype of PAR2 null and wildtype dystrophin deficient mice (mdx mice) was compared. Results revealed that PAR2 deficiency protected the mdx mice from the impact of muscular dystrophy on bone structural and material properties. This study provides evidence that PAR2 slows ageing-related bone loss, possibly by promotion of osteoblast differentiation and suppression of adipogenesis in MSCs. In contrast, evidence is provided that PAR2 contributes to inflammation-induced bone loss.
The effect of Bonzi (Paclobutrazol) on height and flowering of the paper daisies Rhodanthe chlorocephala subsp.rosea and Rhodanthe manglesii
The pink paper daisies Rhodanthe chlorocephala subsp.rosea and Rhodanthe manglesii are Western Australian wild flowers bush harvested as cut flowers. They produce numbers of showy, long lasting inflorescences at the tips of 50 to 60cm tall stems in spring. The growth retardant Bonzi(R) (paclobutrazol) was applied to both species as soil drenches or whole plant sprays alone or combined seed soaks and drenches. The retardant was applied at various concentrations and times, to determine if plant height could be reduced for pot plant production. Growth was measured weekly and recorded on a graph of maximum/TinimuT desired height (20-30cm). After initial treatments on week 4, all treatments were applied using Graphical Tracking techniques, that is, when actual growth deviated above the maximum height line. Plant height was suppressed with all applications of Bonzi(R) (paclobutrazol). Increasing both the rate and number of applications of BonznK)(pac1obutrazo1) led to an increase in shoot suppression, flowering time and number. The combined seed soak (400ppm Bonzi(R)) and multiple drench application (Bonzi(R) 4mg ai/pot x 3) was most effective in suppressing shoot elongation of R.chlorocephala subsp.rosea with plants 41% shorter than untreated plants. lowering was delayed and numbers reduced, but the compact plants had sufficient numbers of flowers at the end of the trial period to appeal to consumers. Bonzi(R) caused very noticeable delays in flowering in all treated Rhodanthe manglesii plants. The 4mg drenches, (4mg ai/pot x 3) gave the most satisfactory result producing plants 38% shorter than untreated controls but some pots had not flowered by the termination of the trial. The best results, in respect to height, were again the combination seed soak plus drench, with only a single 4mg drench application required to reduce height by 48%, but germination was suppressed excessively and flowering was unacceptably delayed. Although growth was suppressed significantly by whole plant sprays none were saleable due to the unsightly chlorotic foliage effects on both species.