Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebrafish

Abstract

To evaluate the frequency of the aerolysin (aerA), cytotoxic enterotoxin (alt) and serine protease (ahp) genes in Aeromonas hydrophila isolates from different sources, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in zebrafish. Aeromonas hydrophila isolates from clinical cases (n=40), from healthy fish (n=22) and from water environment (n=21) were analysed with respect to the prevalence of aerA, alt and ahp genes by PCR assay. These virulence factors occur among clinical isolates as well as among isolates from healthy fish and water environment. The majority (97·6%) of the strains examined carried one or more virulence genes. The isolates were divided into seven genetic profiles on the basis of PCR result: aerA(+) alt(+) ahp(+) (62·7%), aerA(+) alt(+) ahp(-) (13·3%), aerA(+) alt(-) ahp(+) (10·8%), aerA(-) alt(+) ahp(+) (4·8%), aerA(-) alt(-) ahp(+) (3·6%), aerA(+) alt(-) ahp(-) (2·4%) and aerA(-) alt(-) ahp(-) (2·4%). A higher frequency of genetic group aerA(+) alt(+) ahp(+) was determined in the isolates from diseased animals compared to those from healthy fish or water environments. Virulence properties of 26 representative strains belonging to the seven genetic profiles were further characterized. Results demonstrated that as the present of virulence genes increased, the proteolytic, haemolytic and cytotoxic activities of extracellular products also increased. And the 50% lethal doses (LD(50)s) of aerA(+) alt(+) ahp(+) isolates (<10(5)) in zebrafish were lower when compared with the strains expressing one or combinations of two virulence genes (>10(6)). Virulence properties of A. hydrophila correlated well with the presence of virulence genes tested. aerA(+) alt(+) ahp(+) was more frequent virulence genotype in A. hydrophila isolates from clinical diseases than from healthy fish and water environment, and the aerA(+) alt(+) ahp(+) isolates were more virulent to zebrafish compared to the other six genetic profiles. The detection for aerA, alt and ahp can be used for virulence typing of A. hydrophila isolates.