石斑魚消化道細菌分泌抗神經壞死症病毒 物質之研究

Abstract

病毒性神經壞死症（viral nervous necrosis disease，VNN）是近年來海水養殖魚的重要疾病，可引起三十幾種海水養殖魚苗死亡。本實驗分離石斑魚腸內菌株，測試作為生物性防疫工具的可能性，並在活體餵食實驗中評估保護效果。由石斑魚腸道分離之菌叢，以GFB-14E生化系統進行初步菌種鑑定，再依照菌液上清液對石斑神經壞死症病毒（Grouper nervous necrosis virus, GNNV）的中和能力，挑選出三株具有抗GNNV活性的菌株，三株細菌之培養上清液對GNNV的中和力價指數（Log NI）都大於5。以不同孔徑分子篩及不同高溫來處理菌液，發現這三株細菌所分泌的抗病毒物質皆能耐受100 °C高溫處理10分鐘，其中一株分子量介於 5-10 kDa，另兩株則小於 3 kDa。細菌上清液經100 °C高溫及分子篩處理後的蛋白質濃度降至0，但仍保有抗病毒活性，顯示抗病毒物質為耐熱性的小分子，這些物理特性可作為日後分離純化該物質的依據。將細菌與飼料混合後，餵食石斑魚苗四星期，再以腹腔攻毒GNNV，結果負對照組的累計死亡率為24%，且50%以上的負對照組魚苗有明顯病癥，而餵食細菌組的累計死亡率為0，且無任何外觀與行為上的病癥。分離、鑑定餵食細菌組之魚苗消化道菌相，發現負對照組與餵食細菌組的腸內菌相組成明顯不同。

Nervous necrosis virus （NNV） is a critically important pathogen for reared marine fish, and has caused mass mortality in more than 30 fish species in recent 10 years. In this study, we cloned anti-NNV bacteria from the intestinal microflora of grouper, and investigated the possibility of using intestinal bacteria as a biological control for viral nervous necrosis（VNN） disease. The normal flora from grouper intestine was first isolated and preliminary identified by GFB-14E system. The bacteria secreting substance against grouper NNV（GNNV） were selected according to the result of neutralization test. Three bacterial strains with high neutralization titers（Log NI >5） against GNNV were selected for further characterization. It was found that the anti-GNNV substances in the culture supernatants of these selected clones were stable after 100 °C for 10 minutes treatment. The size range of anti-GNNV substances was estimanated by microfilter with different pore sizes. The size of anti-GNNV substance of one bacteria strain is between 3-5 kDa, and of another two strains is smaller than 3 kDa. After 100 °C 10 min and microfilter treatment, the protein concentration of bacterial supernatant declined to zero, but their anti-NNV activities were still high. Therefore, it is revealed that the anti-GNNV substances are small molecule and heat-resistant. In the feeding test, the cultured-bacteria were mixed with commercial food and fed grouper larvae for 4 weeks and then challenged with GNNV by intra-peritoneal injection. The accumulated mortality of negative control fish was 24%, and over 50% of fishes showed clinical symptom of VNN. However, the accumulative mortality of the fed groups was zero, and the fishes were very healthy. The intestinal flora of groupers was examined, and the compositions of intestinal flora of the fed groupers were different from that of the negative control fish.