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Identifying “hidden” antigens in the liver fluke, Fasciola hepatica

McDougall, Heather C. (2012) Identifying “hidden” antigens in the liver fluke, Fasciola hepatica. PhD thesis, University of Glasgow.

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Abstract

Fasciola hepatica is responsible for substantial economic losses and animal welfare issues within the agricultural sector worldwide. The increasing incidence of fasciolosis, coupled with the emergence of flukicide resistance, makes vaccination an attractive alternative control strategy. Hidden antigens extracted from the gut of blood feeding parasites have proven to be excellent vaccine candidates against haematophagous parasites, most notably Haemonchus contortus and Rhipicephalus (Boophilus) microplus. Here, as a first step towards a prototype liverfluke vaccine an attempt to identify hidden gut antigens in F. hepatica was made. Proteomic analysis on extracts of adult F. hepatica was used to identify molecules exclusively found within the membrane-bound fraction including four proteases; cathepsin B2, legumain-2, a putative lysosomal pro-x-carboxypeptidase precursor and a saposin-like protein. Histological sections of adult F. hepatica were screened with a panel of 21 lectins to identify those with an affinity for glycoproteins on the parasite’s gut and to inform subsequent lectin affinity chromatography. Seven lectins showed affinity for the gut region, with peanut (PNA) and jacalin (JAC) lectins binding to glycoproteins on either the gastrodermal cells or gut lamellae, respectively. PNA and JAC were then used to purify glycoproteins from the crude S3 extract by affinity chromatography. The resultant fractions were separated by SDS-PAGE and the protein profiles analysed by mass spectrometry. The enriched lectin-binding fractions shared a number of proteins but one of note that was exclusively identified in the PNA-binding fraction was a cathepsin D-like aspartyl protease, which had not previously been studied in F. hepatica. The proteolytic activities of three somatic extracts of adult F. hepatica were therefore investigated. The ability of the respective fractions to digest haemoglobin, a potential food source, was measured in the presence/absence of class-specific enzyme inhibitors. These analyses confirmed the presence of cathepsin D-like aspartyl protease activity capable of digesting haemoglobin optimally at pH 2 - 2.5. Further characterisation of the cathepsin D-like aspartyl (FhCatD) protease revealed it to be highly conserved within trematodes, to be localized to the gastrodermis of immature (10 day) and adult fluke, and to be more highly expressed, at the RNA level, in the Newly Excysted Juveniles (NEJ) than adult stages. Western blot analysis of native somatic extracts, enriched lectin-binding fractions and recombinant FhCatD using antisera from naturally infected sheep, showed some recognition of the recombinant FhCatD but did not provide clear evidence that the cathepsin D is strongly antigenic during natural infection.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Fasciola hepatica; lectins; hidden antigens; vaccine; trematode; laser capture microscopy; Cathepsin D
Subjects: Q Science > QR Microbiology
S Agriculture > SF Animal culture > SF600 Veterinary Medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Veterinary Medicine
Supervisor's Name: Skuce, Dr. P.J. and Nisbet, Dr. A.J. and Devaney, Prof. E.
Date of Award: 2012
Depositing User: Mrs Heather Catherine McDougall
Unique ID: glathesis:2012-3661
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 25 Oct 2012
Last Modified: 10 Dec 2012 14:09
URI: http://theses.gla.ac.uk/id/eprint/3661

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