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Type of publication: Baccalaureate Thesis
Type of document:

Year: 2010

Authors: Maresch, Roman

Title: Proteome analysis of Oesophagostomum dentatum.

Source: Bakkalaureatsarbeit, Vet. Med. Univ. Wien, pp. 55.


Advisor(s):

Joachim Anja
Razzazi-Fazeli Ebrahim

Reviewer(s):
Gemeiner Manfred

Vetmed Research Units:
VetCore


Abstract:
Investigations on the nematode Oesophagostomum dentatum, the nodule worm of the pig, have recently received great attention because of its worldwide occurrence and high prevalence of infection. Proteomic characterization of O. dentatum may play a key role in future control of worm populations in pig farms and may open a whole range of new antihelmintic therapy strategies. The present study describes an adapted and optimized whole proteome extraction method, which can be applied for O. dentatum as well as other nematodes. The aim of this study was the identification of different sex- as well as stage-specific protein patterns expressed in O. dentatum, using SDS-PAGE and MALDI-TOF mass spectrometry. Pigs were experimentally infected with infectious L3 larvae. Adult worms were extracted after at least 19 days from large intestines by slaughtering pigs. O. dentatum larvae were collected from the faeces of infected pigs and cleaned up by migration from agar in water and subsequently by a sedimentation step. Carrying out two different protein extraction methods for gel electrophoresis, the implications of distinct cell disruption methods based on liquid nitrogen, protease inhibitors and reducing agents were tested. Protein concentrations were obtained using the Bradford protein assay. Differences between the two extraction methods as well as the different protein expression patterns of adults and developmental stages were analyzed by performing 1D gel electrophoresis and separated proteins were silver-stained. Six distinct spots were excised from the gel and subsequently extracted by using in-gel digest. Tryptic digested proteins were identified by MALDI-TOF-TOF mass spectrometry. Re sults of the tested protein extraction methods revealed no major differences in protein concentration levels and the separation by 1D electrophoresis showed no distinct differences in protein pattern. Despite the low resolution of 1D electrophoresis, comparison of male and female O. dentatum adults indicated different protein expression profiles of the parasites. Furthermore, the comparison of adults and larvae revealed differences in protein biosynthesis of O. dentatum. Six spots partially differing between male and female worms were excised and identified by subsequent MALDI-TOF-TOF mass spectrometry. Phosphoenolpyruvate carboxykinase, Peroxiredoxin-1 and Triosephosphate isomerase from female as well as male adult worms could be positively matched after Mascot database search and exhibited a significant score.


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