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dc.contributor.authorDelroux, Karineen
dc.date.accessioned2018-05-14T10:12:19Z
dc.date.available2018-05-14T10:12:19Z
dc.date.issued2005
dc.identifier.urihttp://hdl.handle.net/1842/29727
dc.description.abstracten
dc.description.abstractTicks are hematophagous arthropod ectoparasites that feed on their vertebrate hosts for prolonged periods from days to several weeks. To successfully complete their blood meal they developed strategies to secure attachment on their host's skin, to ease blood flow and to evade their host's inflammatory and acquired immune responses. Ticks are also vectors of a number of major human and animal diseases and salivary gland secretions have been implicated in transmission and establishment of such infections.en
dc.description.abstractAmblyomma variegatum is a hard tick, found in tropical areas. Infestation with A variegatum is associated with exacerbation (not transmission) of dermatophilosis, a skin disease of cattle caused by the actinomycete Dermatophilus congolensis. This observation suggests that A variegatum ticks secrete molecules that can modulate the host immune response in such a way as to both prevent potentially harmful responses against themselves and change the pattern of infection and disease caused by unrelated pathogens.en
dc.description.abstractThe purpose of this study is to identify and characterise saliva and salivary gland molecules that could contribute to the development of novel anti-tick vaccines or have potential therapeutical value for medical application such as non-steroid anti-inflammatory drugs. To identify such molecules, three methodological approaches have been used.en
dc.description.abstractA proteomics approach was carried out to identify proteins contained in saliva and salivary glands of partially fed A variegatum ticks. A combination of protein separation on one- and two-dimensional SDS-PAGE and peptide mass fingerprint analysis of selected proteins through MALDI-ToF/MS (Matrix-Assisted Laser Desorption/Ionisation- Time of Flight/ Mass Spectrometry) technology was undertaken. Resulting monoisotopic masses of 16 selected spots (9) and bands (7) were compared with proteins of Swissprot database, and with the 6 frame-translated of deposited EST of Amblyomma variegatum, Amblyomma americanum, Rhipicephalus appendiculatus, Ixodes scapularis and Ixodes pacificus (TIGR) databases. A 48 kDa band matched a cathepsin L like proteinase previously identified in B microplus and H longicornis ticks, and a 45 kDa protein matched an EST which was homologous to silk fibroin of Bombyx mori. A 86 kDa band and a 66 kDa spot matched myosin of mammalian origin, the other proteins matched EST of the tick databases but the functions and identities of those matches remain to be determined. However, comparative analysis of protein pattern of saliva and salivary glands of unfed and partially fed female A variegatum confirmed that feeding induced the expression of a large number of proteins.en
dc.description.abstractA genomics approach was also undertaken to identify salivary gland genes, which are specifically expressed during feeding of female A variegatum-.en
dc.description.abstractA cDNA library of salivary glands of partially fed female A variegatum was constructed in the bacteriophage A,TriplEx2. The titre of the library was 8x10 pfu/ml with 90% recombinants. Immunoscreening was carried out with a hyperimmune antiserum obtained from a rabbit immunised with salivary gland extracts of unfed female A variegatum and with antiserum prepared from a calf repeatedly exposed to feeding A variegatum. While both antisera recognised saliva and salivary gland protein extracts on Western blot analysis, they failed to detect expressed recombinant proteins after many attempts of screening the cDNA library.en
dc.description.abstractSequence comparison with GenBank® non-redundant (NCBI) and with Amblyomma variegatum Gene Index (TIGR) databases of 33 randomly selected cDNA clones was carried out. The comparative analysis revealed that one clone shared sequence identity with boophilin, a thrombin inhibitor of B microplus; and another cDNA displayed 31% homology with TCI83 of AvGI, which shared itself 28% sequence identity with an immunomodulator of Dermacentor andersoni. Six cDNA clones shared sequence homology with molecules which functions are related to wound healing, anticoagulation and cement cone formation. While several cDNA clones did not match any sequences of either database others matched sequences of genes, which functions, have yet to be ascribed.en
dc.description.abstractA Signal Sequence Trap (SST) technique, using alkaline phosphatase as reporter gene and COS-7 cells as the host, was used to isolate genes encoding type I membrane and secreted proteins from a cDNA library of salivary glands of partially fed female A variegatum. A total of 8832 clones were screened and 13 positive clones were isolated amongst them, 6 displayed a signal peptide at the N terminal end of their deduced amino acid sequence. Sequence comparison with sequences of the GenBank® nr (NCBI) and AvGI (TIGR) databases revealed that one clone shared 99% identity with TC3, a glycine rich protein from the AvGI database that is believed to be a cement cone protein. The full-length sequence of this clone, FLAv41E was isolated from the >,TriplEx2 cDNA library of salivary glands of partially fed female A variegatum. FLAv41E encoded a 1221 bp cDNA with a 1053 bp open reading frame. The deduced protein contained 351 aa with 20 aa predicted signal sequence its theoretical molecular weight was 30.9 kDa with a pi of 8.1. Its secondary structure revealed domain similarities with a 29 kDa cement protein previously characterised in salivary gland of Haemaphysalis longicornis ticks. Reverse transcription PCR (polymerase chain reaction) amplification of FLAv4IE gene revealed that it was expressed in the salivary glands of female A variegatum from day 3 to 10 of feeding. A his-tagged FLAv41E protein minus its signal peptide and the C terminal end of the FLAv41E protein were successfully expressed in Sf-21 insect cells using a baculovirus system. Both constructs were detected in the cell pellet extracts by Western blot analysis.en
dc.description.abstractThe results obtained are discussed with regard to comparative analysis of the three different approaches used to identify and characterise salivary glands and saliva molecules for anti-tick vaccine development.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2018 Block 18en
dc.relation.isreferencedbyAlready catalogueden
dc.titleCloning of genes expressed in the salivary glands during feeding of the tick Amblyomma variegatumen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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