評估餵食乳酸菌對石斑魚抗鰻弧菌之效果

Abstract

石斑魚為台灣主要養殖經濟魚種之一，但集約式養殖，使石斑魚養殖遭受細菌性疾病的威脅，例如弧菌症。在許多的研究發現益生菌具有增強魚類免疫力及抗病原菌的能力。本實驗室先前研究發現，於海鱺腸道中分離出一株乳酸菌 Pediococcus pentosaceus strain 4012 (LAB4012)，經由餵食後可幫助海鱺抵抗發光桿菌症。在本研究的體外實驗中發現LAB4012之上清液可以抑制鰻弧菌生長，更進一步發現LAB4012分泌的乳酸在抑制鰻弧菌生長上扮演了重要的角色。在餵食實驗中發現LAB4012可以增加石斑魚的成長率及血液中紅血球的數量。另外以鰻弧菌感染一天後，餵食組的石斑魚白血球數量及頭腎吞噬細胞的吞噬活性皆有顯著性的上升。而在基因表現方面，餵食LAB4012或LAB4012混合培養乳酸菌之上清液，其對發炎因子，抗發炎因子及抗菌肽基因皆無影響。但經由餵食LAB4012後再感染鰻弧菌，發現LAB4012可降低頭腎的促發炎因子TNF-1、TNF-2和IL-1β，並增加抗發炎因子TGF-β1的表現。此外在鰻弧菌攻毒實驗中發現，LAB4012餵食組之累積死亡率(15%)低於控制組(65%)，根據以上結果可確定LAB4012可作為石斑魚的益生菌，其可調節石斑魚免疫力及幫助石斑魚抵抗鰻弧菌的感染。

Groupers (Epinephelus spp.) are economically important fish species in aquaculture in China and Taiwan. Due to intensive culture, groupers have suffered from some bacterial diseases, such as vibriosis. Many studies reveal that probiotics have the potential to enhance fish immunity and to antagonize pathogens. In our previous study, a lactic acid bacterium Pediococcus pentosaceus strain 4012 (LAB4012) isolated from cobia intestine, can protect the cobia from photobacteriosis after 2-week feeding. In this study, we examined the potential of LAB4012 to be a probiotic for the orange-spotted grouper through feeding and to protect the groupers away from vibriosis. In vitro, LAB4012 culture supernatant with low pH value antagonized the growth of Vibrio anguillarum, and lactic acid in the metabolite of LAB4012 was found to be major factor for the growth inhibition of Vibrio anguillarum. In vivo, supplementation of LAB4012 in the commercial diet enhanced the growth rate and erythrocyte numbers of the groupers. One day after the infection of Vibrio anguillarum, the leukocyte numbers and the phagocytic activity of the head-kidney phagocytes in the LAB4012-fed groupers significantly increased compared to those in groupers without LAB4012-feeding. After 4-week feeding with LAB4012 or the mixture of LAB4012 and its culture supernatant, the gene expression levels of the pro-/anti-inflammatory cytokines and antimicrobial peptides did not significantly change; however, in the head kidney of the LAB4012-fed groupers post Vibrio anguillarum infection, the gene expressions of TNF-1, TNF-2 and IL-1β were down-regulated and of TGF-β1 was up-regulated. Moreover, the accumulated mortality of the LAB4012-fed groupers was 50% lower than that of the control fish post Vibrio anguillarum infection (6x105 CFU per fish). According to these results, we suggested that LAB4012 can be a dietary probiotic for groupers to modulate the fish immunity and protect the groupers against Vibrio anguillarum infection.