Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment

Cloning and Detection of the β-Hemolysin Gene by Polymerase Chain Reaction from Aeromonas hydrophila Isolated from Fish and Soft-Shelled Turtle in China

Chun Xia, Zhi-hong Ma, Zhi-guang Wu, M. Habibur Rahman, Kenji Kawai


Aeromonas hydrophila (Ah) is considered one of the most important pathogenic bacteria for fish and soft-shelled turtles in China because it has caused serious damage in the aquaculture industry (Qian et al. 1995; Ma et al. 1998). Its virulence is closely related to the toxins produced, especially β-hemolysin. β-hemolysin genes exist in all the pathogenic strains that have been isolated from human beings and some animals (Howard et al. 1987; Pollard et al. 1990). Few data are available concerning β-hemolysin genes of virulent strains in relation to pathogenicity, although the hemolysin exists in all of the pathogenic strains from diseased fishes (Hirono and Aoki 1991; Hirono et al. 1992; Qian et al. 1995; Ma et al. 1998).

To characterize β-hemolysin protein and to develop a toxin vaccine for Ah infections, the β-hemolysin gene from a virulent AhTPS30 strain was cloned and analyzed, then detected in pathogenic β-hemolytic Ah isolated from cultured fish and soft-shelled turtle (Qian et al. 1995; Ma et al. 1998).

The bacterial strain TPS30 was isolated from kidney of moribund silver carp Hypothalmichthys molitrix collected in ZheJian Province, China. The strain was highly virulent (the dose with 50% probability of causing death was 5 × 103 colony-forming units/fish), contained β-hemolysin, and had some characteristics similar to those of Ah (Qian et al. 1995). Others Ah strains also were isolated.

Genomic DNA was isolated according to Frederick et al. (1994). One pair of primers was designed based on the number of AH28SAHEM (Hirono et al. 1992). The forward primer was 5′-GCTATGAAAAAACTAAAAATAACTG-3′ at region 423–448. The reverse primer was 5′-CAGTATAA GTGGGGAAATGGAAAG-3′ at region 1,983– 2,007. The gene was amplified using a Taq PCR Kit. Thirty-two cycles were performed under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 3 min. After thermal cycling, the samples were postheated at 72°C for 10 min. The recombinant plasmid DNA was sequenced. This nucleotide sequence was designated as AHTPS30HEM.

To detect pathogenic β-hemolytic Ah isolates, polymerase chain reaction (PCR) and nested PCR were completed using oligonucleotides corresponding to nucleotide (nt) 119–143 (forward primer AhP1: 5′-CAAGAGGTCTGTGGCGACA-3′), nt 307–327 (reverse primers AhP2:5′-TTTCACCGGTAGCAGGATTG-3′), and nt 1,084–1,092 (reverse primers AhP3:5′-AAGGTGTGGTTCC AGTTC-3′) according to AHTPS30HEM. After the first PCR with strains using AhP1/AhP3, 1 µL of PCR product was subjected to a second PCR with AhP1/AhP2. These PCR products were 948 base pair (bp) and 208 bp, respectively. A PCR was preformatted by using AhP1/AhP2.