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Type of publication: Doctoral Thesis
Type of document:

Year: 2010

Authors: Hochwimmer, Gerald

Title: Untersuchung autochthoner und allochthoner Krebsarten heimischer Gew├Ąsser auf Pilzinfektionen unter besonderer Ber├╝cksichtigung von Aphanomyces astaci und Klassifizierung des Erregers mittels moderner molekularbiologischer Methoden (Multiplex-PCR).

Other title: Examination of authochtonous and allochthonous crayfish species for mycological infection, in special consideration of Aphanomyces astaci and classification of the germ by modern molecularbiological methodes

Source: Dissertation, Vet. Med. Univ. Wien, pp. 51.


Advisor(s):

Licek Elisabeth
Steinborn Ralf

Reviewer(s):
El-Matbouli Mansour

Vetmed Research Units:
Institute of Microbiology


Graduation date: 20.04.10


Abstract:
Background: The oomycete Aphanomyces astaci is an evident hazard for European crayfish species. As the causative agent of crayfish plague the germ causes up to 100% mortality in infected native crayfish populations. Multiple transmission paths necessitate the development of an efficient and reliable test to detect the pathogen. Results: In the present research project we succeeded in isolating the crayfish plague agent A. astaci from healthy signal crayfish (Pacifastacus leniusculus). After molecularbiological definition we used A. astaci-strain Gb04 for subsequent studies. With the aid of the 5`, 3`-RACE-PCR (rapidly amplification of cDNA ends-PCR) we were able to describe the complete sequence of two Glycosyl-Hydrolase family 18 chitinases. Furthermore we showed their expression-profile during mycel growth by RT-qPCR and their extracellular existance by Westernblot analysis. In subsequent steps we sequenced and analysed the analogue chitinase genes of different crayfish afflicting oomycetes. Based on these results we established and evaluated a multiplex qPCR-assay for the specific detection of A. astaci in moribund or perished crayfish specimens. Conclusion: The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. With the aid of this diagnostic tool we were able to detect A.astaci in clinical samples from different Austrian provinces, like Upper Austria, Carinthia and Burgenland.

Keywords:
Aphanomyces astaci / GH18-Chitinase / qPCR


Publication(s) resulting from University thesis:

Hochwimmer, G; Tober, R; Bibars-Reiter, R; Licek, E; Steinborn, R (2009): Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci. BMC Microbiol. 2009; 9:184
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