The principal aims of the work presented in this thesis were to investigate the biochemical properties and protein compositions of nasal secretion (NS) from healthy cattle and to document changes in the protein expression in NS from diseased and vaccinated cattle using advanced proteomic methods. Bovine respiratory disease (BRD) is the principal source of economic loss for the cattle industry throughout the world. Even though it has been studied extensively, it still remains the number one cause of disease and death in cattle. Although the etiopathogenesis of BRD is multifactorial and complex, all of the pathogens have the common route of infection which is intranasal. Thus, NS is potentially a valuable source of biological sample in the detection of biomarkers for BRD. Therefore, a method for the collection of substantial volumes of NS from cattle was developed in this study to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L which is 15.7 fold higher compared to AP activity in serum reference ranges. This study further investigated the source of the high AP activity in bovine NS. Histochemical analysis using AP specific staining confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal mucosa. Advanced molecular methods were used to determine the characterization of nasal AP. The analysis at mRNA levels from nasal mucosa by endpoint RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant gene (ALPL) found in bovine liver, bone and kidney. Investigation using isoelectric focussing (IEF) confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that novel AP isoenzymes in NS had pIs in the same range as those of the nasal mucosa extracts (pH 4.8-6.2) but were clearly different from the extracts of other tissues. The differences in IEF migration of the AP extracts is likely to be due to post translational modifications (PTMs) such as glycosylation or phosphorylation which would be areas worthy of future research. Preliminary proteomic investigation of NS from healthy cattle using 1D gel electrophoresis, 2D gel electrophoresis and ESI-MS/MS analysis putatively identified 10 major proteins in NS consisting of 7 vascular proteins and other glandular and cellular proteins such as lactoferrin, an anti-bacterial protein commonly present in mucosal secretions, odorant binding protein known to have a role in scent recognition and glutathione-S-transferase, an enzyme capable of detoxifying noxious compounds. The final objective was therefore to compare the nasal proteins from healthy, disease and vaccinated group of animals using 2D difference gel electrophoresis (DiGE). The experimental model used for this investigation was an immunisation study against bovine malignant catarrhal fever (MCF). It was concluded that quantitative proteomics technology such as DiGE identified and measured changes in nasal protein expression in response to MCF and following vaccine protection. In conclusion, this research has contributed to the scientific knowledge regarding the biochemical properties and protein compositions that is present in bovine NS. In addition, the research also explored the use of proteomic technologies as a novel tool to analyse protein expression and identify possible biomarkers in NS. This thesis is an important step forward for a better understanding of bovine NS, and thus provides a basis for future studies involving bovine NS by way of providing reference data and alternative source of biological sample.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:637683 |
| Date | January 2015 |
| Creators | Ghazali, Mohd Faizal |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/6101/ |
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