Functions of the structural domains of cartilage superficial zone proteoglycan/proteoglycan 4 (SZP/PRG4).

Proteoglycan 4 (PRG4) is a mucinous proteoglycan with an apparent molecular weight of -345 kDa. It has been detected in a variety of tissues including cartilage, tendon, bone heart and liver, and is also known as cartilage superficial zone protein (SZP), lubricin, megakaryocyte stimulating factor (MSF) precursor protein and camptodactyly-arthropathy-coxa vara pericarditis (CACP) protein. PRG4 has been shown to reduce friction in joints, and immunological studies have located PRG4 on the surface of articular cartilage, in synovial fluid, on the surface of meniscus and on the surface of mature compressed tendon. PRG4 is involved in the congenital joint pathology known as CACP. Analyses of the PRG4 sequence reveal a propensity for a diverse number of functions including lubrication, matrix-binding, self-aggregation, cytoprotection and cell proliferation. This study set out to investigate the potential functions of the N- (exons 2-5) and C-terminal (exons 7-12) structural domains of human and bovine PRG4, facilitated by recombinant protein expression. Preliminary results from solid-phase binding assays showed an interaction between the N- terminal domain of PRG4 and plasminogen activator inhibitor-1 (PAI-1), type II collagen and a 70kDa fibronectin fragment. Immunoprecipitaion experiments demonstrated a potential interaction between the PRG4 C-terminal domain and fibronectin fragments. Both the N- and C- terminal contain functional heparin binding sites. The C-terminal domain was shown to interact with superficial zone chondrocytes, an interaction that was perturbed by heparin. The study also used purified full-length PRG4 from three sources: human recombinant, bovine articular cartilage and bovine tendon. These PRG4 species bound to heparin and displayed similar glycosylation profiles. This study also shows that PRG4 is susceptible to degradation by a number of matrix proteinases including elastase, plasmin, matrix metalloproteinase-7 and cathepsin B. Collectively, the data presented in this thesis provides novel information concerning the biochemistry, susceptibility to digestion and functional capabilities of PRG4

Identification of factors associated with Fasciola hepatica infection risk areas on pastures via an environmental DNA survey of Galba truncatula distribution using droplet digital and quantitative real-time PCR assays

Abstract Environmental DNA (eDNA) is a powerful tool for identifying the spatial and temporal presence and density of species in a range of aquatic habitats. The analysis of eDNA has a wide range of application, one of which may be to inform of Fasciola hepatica infection risk on pastures based on the detection of its eDNA as well as that of its intermediate snail host, Galba truncatula eDNA. Here, droplet digital PCR (ddPCR) and quantitative real‐time PCR (qPCR) assays were developed to detect the eDNA of F. hepatica, and its intermediate snail host, G. truncatula in water samples collected from pastures grazed by cattle and/or sheep. Environmental factors associated with species presence, as detected via an eDNA survey, were identified using zero‐inflated linear mixed models. Sixty‐four habitats were sampled across six farms in Ceredigion, Wales, UK, with ddPCR and qPCR identifying 42 and 33 habitats to be positive for G. truncatula eDNA, respectively. G. truncatula eDNA was significantly less likely to be detected in habitats fully shaded by trees, those that contained black or dark brown soils and habitats that contained deep water pools (p < 0.05). Significantly higher G. truncatula eDNA concentrations were observed in habitats that tend to dry up during Summer (i.e., temporary habitats) (p < 0.05). ddPCR also identified five habitats to be positive for F. hepatica eDNA; however, questions remain regarding the utility of F. hepatica eDNA detection due to a lack of specificity toward infective F. hepatica larval stages. The results of this study inform of factors which influences G. truncatula distribution and ecology on pastures and also provided practical information for farmers to aid F. hepatica control in their flocks and herds

Associations between Gastrointestinal Nematode Infection Burden and Lying Behaviour as Measured by Accelerometers in Periparturient Ewes

The application of precision livestock farming (PLF) technologies will underpin new strategies to support the control of livestock disease. However, PLF technology is underexploited within the sheep industry compared to other livestock sectors, and research is essential to identify opportunities for PLF applications. These opportunities include the control of endemic sheep disease such as parasitic gastroenteritis, caused by gastrointestinal nematode infections, which is estimated to cost the European sheep industry EUR 120 million annually. In this study, tri-axial accelerometers recorded the behaviour of 54 periparturient Welsh Mule ewes to discover if gastrointestinal nematode (GIN) infection burden, as measured by faecal egg count (FEC), was associated with behavioural variation. Linear mixed models identified that increasing FECs in periparturient ewes were significantly associated with a greater number of lying bouts per day and lower bout durations (p = 0.013 and p = 0.010, respectively). The results demonstrate that FECs of housed periparturient ewes are associated with detectable variations in ewe behaviour, and as such, with further investigation there is potential to develop future targeted selective treatment protocols against GIN in sheep based on behaviour as measured by PLF technologies