The poultry red mite Dermanyssus gallinae (De Geer) is a blood feeding ectoparasite that infests many bird species. Economically it is the most important parasite affecting egg-laying hens. The aim of this study was to identify vaccine candidate proteins from D. gallinae using a number of approaches. An immunisation trial was conducted using four protein fractions derived from D. gallinae. The fractions were injected into hens and immunoglobulin Y was purified from the yolk of eggs laid by the hens. An in vitro feeding assay in which mites were fed these antibodies showed a significant increase (p = 0.013) in mortality of mites fed with antibodies against a PBS-soluble fraction of D. gallinae. The PBS-soluble proteins were then fractionated by anion exchange chromatography into three fractions, which were then used to immunise hens. One fraction produced antibodies that caused the greatest percentage mortality of mites, based on the results from a single feeding assay. To identify further potential vaccine antigens, three expressed sequence tag (EST) databases were produced. A complementary DNA (cDNA) library was prepared from a mixed gender and life stage population and 46 contigs were identified with significant homology to proteins from other organisms. A second database was generated using a suppression subtractive hybridisation approach and 133 “fed mite enriched” contigs identified with significant homology to proteins in either the NCBI non-redundant (nr) protein, KEGG databases or had a domain predicted by InterProScan. The most abundant proteins in this database were vitellogenin and GP80, a precursor molecule of vitellogenin. Roche 454 sequencing technology was used to generate an EST database of 13,363 contigs, of which 26 % had significant homology to a protein in the NCBI nr protein database. The majority of contigs (45 %) were classified as Cellular Processes and Signalling proteins. Illumina Solexa sequencing technology was also used to analyse the expression levels of genes in fed and starved mites. A total of 66 contigs were obtained with a significant and greater than three-fold change in expression level between the two groups. The contig with the largest fold change was homologous to vitellogenin (fold change 110). Paramyosin and tropomyosin have been used successfully to immunise hosts against other parasitic species. Tropomyosin had previously been characterised in D. gallinae. In this study paramyosin was characterised and recombinant versions of both proteins were used in an immunisation trial. Anti-tropomyosin (p < 0.001) and anti-paramyosin (p = 0.004) antibodies fed to mites in the in vitro feeding assay caused a significant increase in the mortality of the mites when compared to controls. An in vivo challenge was performed injecting three groups of hens with the PBS-soluble native protein fraction, a recombinant protein cocktail, consisting of paramyosin, tropomyosin, cathepsin L, cathepsin D and histamine release factor, and an adjuvant only control, which were subjected to a challenge of D. gallinae. Each group consisted of three replicates based on weight. Western blot analysis of hen serum showed a significant increase (p < 0.05) in the titre of antibody from the hens immunised with the recombinant cocktail compared with controls. One of the replicates immunised with the PBS-soluble protein fraction showed a strong response but this was lacking in the other two replicates and no significant difference in IgY titre was found. No significant differences were found between the number of mites collected from the test or control groups following a large mite challenge. In conclusion the PBS-soluble protein fraction, paramyosin and tropomyosin have been shown to have potential as vaccine candidates based on in vitro studies. The bioinformatic analysis of D. gallinae has provided a large EST database and a list of 66 proteins that had a significant difference in expression levels in fed and starved mites, which can be further mined for potential vaccine candidates.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:563511 |
| Date | January 2011 |
| Creators | Wright, Harry Watmore |
| Contributors | Nisbet, Alasdair. : Huntley, John |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/5703 |
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