Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

Monique Mancuso,Beatriz Magariños,Alicia E Toranzo,Rubén Avendaño-Herrera,R Zaccone

doi:10.4067/s0716-97602007000100009

Abstract

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.

Highlights

Photobacterium damselae ssp. piscicida, previously known as Pasteurella piscicida, is the etiological agent of fish photobacteriosis or pasteurellosis, which is one of the most important diseases in Japan, affecting mainly yellowtail (Seriola quinqueradiata)
From a biochemical and serological point of view, Photobacterium damselae ssp. piscicida have been demonstrated to be identical, but genetic studies have supported the existence of genetic variability among the isolates which was associated with the geographical origin (Magariños et al 1997; Thyssen et al 2000)
Repetitive element PCR is a group of tecniques that generate DNA fingerprints which can be utilized for the discrimination of bacterial species and/or strains (Versalovic et al, 1991)

Summary

Introduction

Photobacterium damselae ssp. piscicida, previously known as Pasteurella piscicida, is the etiological agent of fish photobacteriosis or pasteurellosis, which is one of the most important diseases in Japan, affecting mainly yellowtail (Seriola quinqueradiata). From 1990 it has caused economic losses in the marine culture of gilthead sea bream (Sparus aurata), sea bass (Dicentrarchus labrax) and sole (Solea solea and Solea senegalensis) in the Mediterranean European countries and hybrid striped bass (Morone saxatilis x M. chrysops) in USA (Toranzo et al, 2005) This pathogen is biochemically and serologically homogeneous regardless of the geographic origin and source of isolation (Magariños et al, 1996; Bakopoulos et al, 1997), DNA fingerprinting methods such as rRNA gene restriction analysis (ribotyping) (Magariños et al, 1997), amplified fragment length polymorphism (AFLP) (Thyssen et al, 2000; Kvitt et al, 2002) and random amplified polymorphic DNA (RAPD) (Magariños et al, 2000; 2003; Hawke et al, 2003; Dalla Valle et al, 2002) have been described as powerful tools to discriminate European strains from Japanese and USA isolates. These repetitive elements, located in the intergenic regions of many bacteria genomes, are considered to be highly conserved (Stern et al, 1984; Hulton et al, 1991; Martin et al, 1992) and, due to this, they are useful for elucidating relationships within and among bacterial species

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