Bacteria Pathogeny Isolation, Identification And Primary Comparative Study Of Disease Resistance In Tilapia Species

Tilapia was an aquaculture breed recommended by the Food and Agriculture Organization (FAO). China was the largest tilapia farming county in the world, which had an annual output of more than 1,200,000 tons, consisting one half of the world total production. Tilapia culture developed into an industry chain in China. There were 11 tilapia species which had steady genetic characters, and the O.aureusO.niloticus and O.niloticus,GIFT strain were the first and second widely cultured species in China. There was few outbreak disease in tilapia culture in the past, and tilapia was considered to be a breed which had strong risistant to diseases. There were more and more outbreak diseases on large scale since 2007. According to the investigation, the incidences of diseases were related to the seasons, the size of tilapia. The main pathogeny which caused severe diseases was bacteria pathogeny, including Areomonas hydrophilia, A.sobria, Streptococcus agalactiae and Streptococcus.iniae. The bacteria pathogeny which caused large scale outbreak diseases in some areas of Guangdong province in the past two years was analysed. The bacteria pathogeny caused adult tilapia outbreak disease lasted from July to October 2009 was identified as S. agalactiae,and the pathogeny which caused large scale outbreak disease in young tilapia in Zhuhai lasted from April to May 2010 was identified as Areomonas sobria. The sensitivity to chemotherapeutants of the strains were tested, all of the bactiera pathogeny strains isolated were sensive to chloramphenicol and ofloxacin. Florfenicol, chloramphenicol and thiamphenicol were three typical pharmaceuticals, and florfenicol because of its wide antibacterial effect, weak toxity to animals and low in cost, was widely used in animal husbandry and aquaculture. The minimum inhibitory concentration(MIC) of florfenicol to S. agalactiae was 1.69mg mL^-1. Florfenicol may be a preferred chemotherapeutant for the prevention of bacterial diseases on tilapia culture.To study the influence of salinity to the growth of bacteria pathogeny, five bacteria strains were inoculated in Brain heart infusion medium (BHI) with NaCl of grade level (0.05%,0.55%,,1.05%,1.55%,2.05%,2.55%,3.05%,3.55%,4.05%,4.55%),after culturing for 15⁴8h,the biomass was detected by turbidimetry.The results show that there were significant inhibitions to the growth of S. agalactiae, S.iniae, A.sobria and Edwardsiella tarda at the range 0.05%⁰.55%.There were inhibitions to A.sobria and E.tarda at the range of 1.55%².05%. The results indicated that high salinity could inhibit the growth of bactiera pathogeny. Bacterias were sensitive to environmental changes.Tilapia cultured in high salinity could improve its taste.The development of salt water farming could be a potential way to reduce the finacial risk caused by bacterial diseases.Earlier investigations showed that the disease resistance to outbreak disease of aquarculture financial animals differed in species. To learn about the disease resistance in tilapia species, tilapia species length 5.0⁷.0cm, 9.0⁹.5cm and 11.5¹2.5cm were artificial infected with Areomonas hydrophilia by immersion method. The concentration of bacteria pathogeny were adjusted to the range of 2.7 107 cfu mL^-11.2 108cfu mL^-1. The result showed the cumulative mortality of tilapiaO.mossambica O.hornorum, O.aureus O.niloticus O.niloticus,GIFT strain (5.0⁷.0cm) were 53.33%,63.33% and 86.67% respectively. Cumulative mortality of tilapia O.mossambica O.hornorum, O.aureus O.niloticus O.niloticus,GIFT strain Oreochromis aurea and Oreochromis nilotica length 9.0⁹.5cm, were 62.2%,68.0%,78.0%,70.0% and 52% respectively. Cumulative mortality of tilapia O.mossambica O.hornorum, O.aureus O.niloticus O.niloticus ,GIFT strain and Oreochromis aurea (11.5¹2.5cm), were 32.5%, 60.0%, 78.0%, 37.5% and 26.3% respectively. After Areomonas hydrophililia infection, the serum lysozyme significantly increased in O.mossambica O.hornorum and O. aurea (11.5¹2.5cm) (p<0.05), while the APK remained unchanged. Artificial infection was carried out on tilapia O.aureus O.niloticus, O.mossambicaO.hornorum, O.niloticus,GIFT strain O. aurea and O. nilotica (8.5¹0.0cm) with S. agalactiae, and the cumulative mortality was 93.3%, 66.7%, 93.3%, 60.0%, 46.7% respectively.For establishing monoclonal antibody detecting methods of pathogeny, and further immunology study, immunoglobulin (Ig) was purified from tilapia serum by a convenient and rapid protein A affinity chromatography method. The binding buffer and washing buffer were sodium phosphate (pH 7.4) and citric acid (pH 3.0) respectively.The binding effect was superior at 4℃than 28℃. The concentation of Ig obtainded was between 1.86 mg·ml-1².95 mg·ml-1,while banding for 3.5h and the flow rate of washing buffer was 1.3 mL·min-1, The Ig purified from the tilapia species was detected through SDS-PAGE, and results showed that the molecular weight of the heavy chain and the light chain were about 80kDa and 30kDa. Protein A affinity chromatography could be a rapid and convenient method to purify serum Ig of tilapia.