| Vibrio anguillarum, a halophilic Gram-negative bacterium, is a worldwide causative agent of vibriosis, which may infect more than 80 different marine and estuarine fish species. In our country's fish aquaculture, V. anguillarum is an important pathogen to sea bass (Lateolabrax japanicus), flounder (Paralichthys olivaceus), and turbot (Scophthalmus maximus), so it is important to elucidate the various virulence factors involved. Several factors like metalloprotease, siderophore, outer membrane protein have been been demonstrated to be an important virulence component of this bacterium. However, on the whole, not much is known about the molecular basis of the putative virulence factors of this bacterium, especially in the low virulent or avirulent strains. Furthermore, the virulence differences between high virulent strain and low virulent strain can not be fully explained by ang of these known factors, so we believe there must be other putative virulence factors cause differences in their virulence.Comparison of the genome sequences of non-pathogenic and pathogenic strains can provide useful information on genes that are specific for highly virulent isolates. Some of these differences may determine strain-specific characteristics such as virulence factors, which can be used as a valuable tool to establish the nature and severity of disease. Currently, the amount of sequence information available in the database required to perform such analysis is too limited. As an alternative approach suppression subtractive hybridization (SSH) technique is a powerful for analyzing the differences between two complex genomes, and for identifying DNA sequences that are present in one strain (the tester), but absent in another strain (the driver). This technique has been used in different bacterial species to identify genomic differences between virulent and avirulent strainsV. anguillarum strain VIB72 was defined as having high virulence whereas strain CW1 was defined as having low virulence on the basis of their different LD50 values to zebrafish (Brachydanio rerio). Suppression subtractive hybridization was used to identify genetic differences between these two strains. After screening, 59 subtracted library clones were isolated which were specific for strain VIB72, and the DNA sequences of these clones were determined. Seventeen fragments showed high homology to the genes of known functions in other bacteria. This includes soluble lytic murein transglycosylase, mobilization protein (MobA, MobC), transposase (IS66), resistance-related protein (metallo-beta-lactamase and acetyltransferase family), toxin protein (DT-201 and alveicin A immunity protein), ATP-dependent endonuclease of OLD family like protein, SocE and GTP-binding protein HflX (high frequency of lysogenization). These fragments may represent parts of putative pathogenicity islands (PAIs) in V. anguillarum. The remaining fragments showed no significant homology to any known genes. The results indicated that SSH was successful in identifying genetic differences and putative virulence genes among different strains of V. anguillarum. The potential biological functions of these genes will form a foundation for further elucidating how V. anguillarum causes disease in fish. In addition, these sets of specific DNA fragments offer a large choice of candidates for the development of new molecular diagnostic and epidemiological tools. |
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