Horsing around – Using equine facilitated learning to support the development of social-emotional competence of students at risk of school failure

The social-emotional issues some students experience can place them at risk of school failure. Traditional methods of support can be ineffective or not sustainable and new alternative approaches need to be attempted to support social-emotional competency, school engagement and success for students at risk. This paper discusses preliminary outcomes of an equine facilitated learning (EFL) programme specifically designed to focus on using horses to improve the resilience and social-emotional competency in students perceived as ‘at risk’ of school failure. This qualitative exploratory study used interviews and observations over a six month period to listen to the voices of the students themselves about their experiences of EFL. Initial findings from the pilot study suggest that EFL programmes can be a novel and motivating way to promote resilience training and social-emotional development of students at risk of failure and, in turn, improve their level of engagement and connection with school environments.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Multi-events stadium Buchanan Park redevelopment

Multi-purpose Community Entertainment and Recreation Venue, catering for the Mount Isa Rodeo; including campdraft, equine sports, shows, exhibition, trade events, concerts and other community activities. The design involved redevelopment of a portion of the Buchanan Park Race Course located in Mount Isa. The project included community infrastructure planning, major landscaping and the construction of built facilities.

In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece®, (2) acellular dermal replacement, Alloderm®, (3) knitted poly(lactic-co-glycolic acid) (10:90)–poly(-caprolactone) (PLGA–PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [3H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece®, Alloderm® and PLGA–PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece® contracted to 10% of the original size while Alloderm® supported cell proliferation predominantly on the surface of the material. PLGA–PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.

Chlamydia pneumoniae is genetically diverse in animals and appears to have crossed the host barrier to humans on (at least) two occasions

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non- Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

A Bayesian approach for estimating detection times in horses : exploring the pharmacokinetics of a urinary acepromazine metabolite

We describe the population pharmacokinetics of an acepromazine (ACP) metabolite (2-(1-hydroxyethyl)promazine) (HEPS) in horses for the estimation of likely detection times in plasma and urine. Acepromazine (30 mg) was administered to 12 horses, and blood and urine samples were taken at frequent intervals for chemical analysis. A Bayesian hierarchical model was fitted to describe concentration-time data and cumulative urine amounts for HEPS. The metabolite HEPS was modelled separately from the parent ACP as the half-life of the parent was considerably less than that of the metabolite. The clearance ($Cl/F_{PM}$) and volume of distribution ($V/F_{PM}$), scaled by the fraction of parent converted to metabolite, were estimated as 769 L/h and 6874 L, respectively. For a typical horse in the study, after receiving 30 mg of ACP, the upper limit of the detection time was 35 hours in plasma and 100 hours in urine, assuming an arbitrary limit of detection of 1 $\mu$g/L, and a small ($\approx 0.01$) probability of detection. The model derived allowed the probability of detection to be estimated at the population level. This analysis was conducted on data collected from only 12 horses, but we assume that this is representative of the wider population.

Persistent digital hyperthermia over a 48 h period does not induce laminitis in horses

Persistent digital hyperthermia, presumably due to vasodilation, occurs during the developmental and acute stages of insulin-induced laminitis. The objectives of this study were to determine if persistent digital hyperthermia is the principal pathogenic mechanism responsible for the development of laminitis. The potent vasodilator, ATP-MgCl 2 was infused continuously into the distal phalanx of the left forefoot of six Standardbred racehorses for 48h via intra-osseous infusion to promote persistent digital hyperthermia. The right forefoot was infused with saline solution and acted as an internal control. Clinical signs of lameness at the walk were not detected at 0h, 24h or 48h post-infusion. Mean±SE hoof wall temperatures of the left forefoot (29.4±0.25°C) were higher (P<0.05) than those on the right (27.5±0.38°C). Serum insulin (15.0±2.89μIU/mL) and blood glucose (5.4±0.22mM) concentrations remained unchanged during the experiment. Histopathological evidence of laminitis was not detected in any horse. The results demonstrated that digital vasodilation up to 30 °C for a period of 48. h does not trigger laminitis in the absence of hyperinsulinaemia. Thus, although digital hyperthermia may play a role in the pathogenesis of laminitis, it is not the sole mechanism involved.

Continuous intravenous infusion of glucose induces endogenous hyperinsulinaemia and lamellar histopathology in Standardbred horses

Endocrinopathic laminitis is frequently associated with hyperinsulinaemia but the role of glucose in the pathogenesis of the disease has not been fully investigated. This study aimed to determine the endogenous insulin response to a quantity of glucose equivalent to that administered during a laminitis-inducing, euglycaemic, hyperinsulinaemic clamp, over 48. h in insulin-sensitive Standardbred racehorses. In addition, the study investigated whether glucose infusion, in the absence of exogenous insulin administration, would result in the development of clinical and histopathological evidence of laminitis. Glucose (50% dextrose) was infused intravenously at a rate of 0.68 mL/kg/h for 48. h in treated horses (n = 4) and control horses (n = 3) received a balanced electrolyte solution (0.68 mL/kg/h). Lamellar histology was examined at the conclusion of the experiment. Horses in the treatment group were insulin sensitive (M value 0.039 ± 0.0012. mmol/kg/min and M-to-I ratio (100×) 0.014 ± 0.002) as determined by an approximated hyperglycaemic clamp. Treated horses developed glycosuria, hyperglycaemia (10.7 ± 0.78. mmol/L) and hyperinsulinaemia (208 ± 26.1. μIU/mL), whereas control horses did not. None of the horses became lame as a consequence of the experiment but all of the treated horses developed histopathological evidence of laminitis in at least one foot. Combined with earlier studies, the results showed that laminitis may be induced by either insulin alone or a combination of insulin and glucose, but that it is unlikely to be due to a glucose overload mechanism. Based on the histopathological data, the potential threshold for insulin toxicity (i.e. laminitis) in horses may be at or below a serum concentration of ∼200. μIU/mL.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.