Studies on Stage Specific Genes in Brugia pahangi

Hunter, Sarah J (1997) Studies on Stage Specific Genes in Brugia pahangi. Master of Veterinary Science thesis, University of Glasgow.

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Abstract

Lymphatic filarial worms are mosquito-borne parasites, with a complex life cycle culminating in the mammalian host, where the adult nematodes can live stably in the lymphatics for several years. The L3, or infective stage larvae, is the transmission stage, passing from the arthropod vector to the definitive host and so is the first stage of the parasite to be encountered by the host immune system. It has been shown that the L3 is responsible for eliciting a stage-specific protective immune response and so any antigens which are up-regulated or distinct to this life cycle stage are worthy of further study. This project set out to identify L3 genes of Brugia pahangi using two different approaches. The first involved the immunoscreening of an adult cDNA expression library, using a rabbit anti-serum raised against live L3. The serum was first pre-adsorbed against adult antigens in an attempt to make the screen more specific for L3 antigens. However, the degree of cross-reactivity between the different life cycle stages and the immunodominance of a small subset of antigens rendered this method unsuitable for identifying L3-specific genes. The second approach was to differentially screen an L3 cDNA library. The library was made by SL-PCR in an attempt to overcome the problems associated with the limited amount of parasite material available. The library was made from L3 which were removed from the jird at three days post-infection, and was differentially screened using PCR derived probes from mosquito-derived L3, 3d pi L3 and adult worms. Plaques which were positive only on the mosquito-derived L3 filters were chosen for further investigation. This method produced four positive clones which were characterized by sequence analysis and Northern and Southern blotting. The four genes were all found to code for structural proteins, and although highly expressed at the L3 stage were also present in the L4. However, all four cDNAs were only found at a very low level, if at all, in the adult parasite. In order to further examine the expression pattern of these genes, semi-quantitative RT-PCR was carried out, using mosquito and mammalian stages of the parasite. In general, the results confirmed the Northern expression pattern in the post-infective stages, with signals obtained at the L2 stage for three of the genes, and at the pretransmission L3 stage for all four of the genes. These studies demonstrate the feasibility of adopting a differential screening approach for the identification of stage-specific transcripts from filarial worms.

Item Type: Thesis (Master of Veterinary Science)
Qualification Level: Masters
Additional Information: Adviser: Eileen Devaney
Keywords: Veterinary science, Parasitology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-74742
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 16:42
Last Modified: 27 Sep 2019 16:42
URI: http://theses.gla.ac.uk/id/eprint/74742

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