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Development of an improved diagnostic test for sheep scab based on biomarkers

Sheep scab is a highly contagious ectoparasitic disease caused by the mite, Psoroptes ovis, which causes intensely pruritic lesions with severe dermatitis and is a major welfare and production issue in the UK national flock. In an attempt to improve disease control, sheep scab was recently made notifiable in Scotland indicating that early diagnostic tests will be crucial to the success of this legislation. A sensitive and specific antibody based assay has been developed which can diagnose early infestation, but which does not indicate current disease status post-treatment due to residual circulating antibody levels. However, studies into host biology and response to disease have illustrated the potential use of biomarkers (BMs) in diagnostics as indicators of disease progression and the effectiveness of treatment regimes, including a recent microarray analysis which identified over 600 host genes differentially expressed in circulating leukocytes following P. ovis infestation. As many of these genes encoded proteins known to be involved in inflammatory responses, this data was used in the search for potential BMs. Initially the genes were filtered and ranked, using bioinformatic analysis, to identify the most promising BM candidates and then evaluated using Western blot analysis against a range of sera from P. ovis infested and naïve sheep. Promising results were obtained for a complement binding protein, C4BPB, showing it was rapidly up-regulated following infestation and correlated with disease progression as determined by lesion size development. The ovine C4BPB gene was successfully sequenced for the first time and a recombinant form of this protein expressed in E. coli. Antibodies, raised in rabbits against ovine rC4BPB, were used to develop a sandwich ELISA, results from which suggested the potential of C4BPB as a BM for sheep scab as it indicated current disease status postinfestation and post-treatment. The major ruminant acute phase proteins (APPs) serum amyloid A (SAA) and haptoglobin (Hp) were then investigated using commercially available assays, as previous studies indicated they were effective markers of inflammatory disease in ruminants. Results from these analyses indicated that both APPs responded positively to infestation with P. ovis but this was not statistically significant until 4 weeks post-infestation. After treatment, the APPs declined rapidly, as described by their short half life of less than 3 days following successful treatment, compared with 56 days for the estimated half life of the host antibody against the mite antigen Pso o 2. Further statistical analysis of the APP response suggested that SAA was the more discriminatory marker, with lower pre-infestation levels and higher sensitivity at the estimated optimum cut-off values. The possibility of using a signature of BMs, as an alternative to a single BM, was discussed as a method of increasing the sensitivity and specificity of the improved test, along with the potential of combining the BM diagnostic with the existing antibody assay. It was concluded that this would provide a highly sensitive and specific test for sheep scab which would diagnose early infestation as well as indicating current disease status post-treatment, providing a highly beneficial tool to the sheep industry to aid the control of this disease.
Date January 2013
CreatorsWells, Elizabeth Anne
ContributorsHuntley, John F.; Burgess, Stewart; Hopkins, John
PublisherUniversity of Edinburgh
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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