| Hainan Province, with its moderate natural environment and good-quality water, is one of the main tilapia (Oreochromis niloticus) farming regions in China. Though Hainan tilapia production ranks second in the country,its tilapia export volume ranks first for two consecutive years (2011and2012), with the annual output of Oreochromis niloticus reaching98,800tons. Similar to the other cultured fish pecies, with the expanding of cultured area, the increase of production and excessive use of antibiotics, diseases in Oreochromis niloticus outbreak more and more frequently in major cultural zones in China, among which bacterial diseases were the severest and brought huge economic losses to the tilapia farming industry. Aeromonas veronii, which is a new type of human-animals-fish disease pathogen, widely exists in soil, natural water bodies and aquaculture water. In recent years, the number of aquatic species infected by A.veronii is increasing. However, the domestic reports relevant to Oreochromis niloticus’infection by A.veronii are rare. No cases has not been reported in Hainan Province so far. Hence in this paper, the predominant bacteria were isolated from different infected tilapia farms artificial infection test was performed; in addition, physiological and biochemical identification are used to confirm that the disease pathogen of Oreochromis niloticus is A.veronii. Afterwards, A.veronii was isolated from ill Oreochromis niloticus of different sources (markets, farms and wild ponds). The distribution pattern of eight virulence genes in different strainswas were investigated according to ERIC-PCR experiment results. The main research methods and results are as following:From March to May in2014, in the main farming area of Oreochromis niloticus in Hainan province,12strains of predominant bacteria were isolated from different infected tilapia farms. By artificial regression infection experiments, strain HN-G-03and HN-B-02strains were isolated from the liver and brain, with strong pathogenicity, the LD50of the two strain to healthy Tilapia by intraperitoneal injection challenge were7.12×105CFU/fish and1.32×105CFU/fish, respectively. Based on the results of16S rRNA and gryB gene sequence analysis, morphological observation, physiological as well as biochemical characteristic analysis, HN-G-03was identified as Aeromonas veronii bv. Veronii and HN-B-02as Aeromonas veronii bv. Sobria. The antibiotic sensitivity analysis showed that both HN-G-03and HN-B-02were resistant to12kinds of drugs including Penicillin, Furazolidone, Ampicillin, Spectinomycin, Cefuroxim, Streptomycin, etc., highly sensitive to Levofloxacin, doxycycline, Florfenicol, Enrofloxacin and Gentamicin. The results provided a further scientific basis for the prevention and treatment of tilapia diseases. In July and August,2014, we collected diseased tilapia from six areas include Tilapia farms, markets and wild ponds. RS medium were used to isolate suspected aeromonas from the diseased tilapia and the number of isolates were152.Then we did the preliminary screening through oxidase, contact enzyme and O/F experiments and we confirmed whether they were aeromonas or not through the16S rDNA sequence analysis the numbers of aeromonas we collected were71. Finally, we found40A.veronii strains by using gyrB gene sequence. Next we detected eight common virulence genes including cytolytic enterotoxin (act), cell excitability enterotoxin (alt), healstable enterotoxin (ast), aerolysin (aerA), hemolysin (hlyA), lipase (lip), flagellar gene (fla) and pilus gene (tapA).The results showed that the positive rate of each virulence gene was35%(act),22.5%(alt),27.5%(ast),15%(hlyA),5%(lip),37.5%(fla), respectively. The positive rate of aerA and tapA were all zero. In40A.veronii strains the positive rate for genetype act+alt+ast+was7.7%, the positice rate of genetype with one or two virulence genes was50%.Based on the distribution of virulence genes, average (UPGMA) chain method was used to construct the clustering tree, The Results showed that40Averonii strains can be distributed into14virulence genetypes,8clusters. In particular, the Ⅷ genetype had the largest number of strains, containing15strains.strains from aquaculture and wild environment showed a random distribution pattern. By using ERIC-PCR technique, the electrophoresis showed4main bands, ranginging from800-6000bp; NTSYS2.1PC software were used to construct phylogenetic tree, using90%genetic similarity as standard. Results showed that isolated strains can be divided into18genotypes, with8strains being independent genotypes. Strains from different sources (farmed or wild fish) were not separated in a geological pattern. |
PDF Full Text Request