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Joint disease has a major impact on the athletic performance in horses, thus representing not only a welfare issue but also a source of economic loss due to veterinary expenses, limited performance, and early retirements. Its diagnosis is routinely established on the basis of clinical lameness examination, radiography, and conventional synovial fluid analysis. Accurate assessment of joint disease is essential to determine adequate treatment and prognosis. However, definite diagnosis often remains difficult. Biomarkers in body fluids could offer clinicians a valuable tool to identify advanced stages of joint diseases more accurately. Various marker molecules have already been used successfully in research settings, but to date none has been introduced and validated in everyday horse practice.
The aim of this study was to evaluate the possible use of selective parameters, namely MPO activity and MPO concentration, MMP-2 and -9 activities, C2C, CTXII, CPII, and CS846 concentrations, in equine synovial fluid and serum as diagnostic markers of joint diseases. Equine samples were taken from horses affected with septic arthritis, osteoarthritis (OA), chronic arthritis, osteochondrosis dissecans (OCD), and from healthy controls. For the determination of MPO activity a specifically modified MPO assay was established in this study. The results show this assay to be a reliable and feasible tool for a specific detection of MPO activity in equine synovial fluid and serum. Furthermore, MPO concentration in synovial fluid was determined using an equine MPO immunoassay. MPO activity and MPO concentration were significantly increased in synovial fluids from septic arthritis compared to all other diagnostic groups.
Using gelatin zymography, this study demonstrates that MMP-2 and -9 activities increase considerably under pathological conditions. For a precise quantification of enzyme activities, MMP-2 and -9 immunocapture activity assays were introduced.
MMP-2 and -9 activities were significantly elevated in synovial fluids from septic arthritis compared to all other diagnostic groups, and MMP-2 activity was significantly increased in synovial fluids from OA and chronic arthritis compared to controls and OCD samples.
For the evaluation of markers of cartilage metabolism, C2C, CTXII, CPII, and CS846 immunoassays were applied on equine samples. CTXII concentration was significantly increased in synovial fluids from septic arthritis compared to all other diagnostic groups and in synovial fluids from OA and chronic arthritis compared to controls. Serum CTXII levels were significantly elevated in septic arthritis specimens compared to controls. No significant differences for C2C concentrations could be detected between any of the diagnostic groups. CPII and CS846 concentrations were significantly decreased in synovial fluids from OA and chronic arthritis compared to all other diagnostic groups. Serum CS846 concentration was significantly increased in septic arthritis specimens compared to all other diagnostic groups, and serum CPII concentration was significantly elevated in septic arthritis specimens relative to the chronic arthritis group.
Equine samples were further classified into predefined diagnostic groups based on certain parameter(s) using discriminant analysis. Serum parameters investigated in this study showed correct classification rates below 50% indicating that they are of less clinical value to indicate joint disease. Synovial CTXII concentration showed the greatest correct classification rate of 68.4% for a single parameter. A combination of biomarkers seems to be most promising for an accurate assessment of joint condition. The highest overall correct classification rate of 86.7% was found for a three-combination system of synovial CTXII, CS846, and CPII concentrations. It appears that synovial markers of cartilage metabolism, particularly CTXII, CS846, and CPII concentrations may have the greatest potential to discriminate between different joint diseases.
These observations provide new insights into the diagnostic potential of diverse biomarkers in equine joint diseases. The results presented here may help to refine the diagnosis of distinct joint diseases and provide a profound basis for further studies on biomarkers for equine joint disease.