- 學位論文
餵食乳酸菌LAB4012菌株對海鱺成長與非專一性免疫的影響
The influence of feeding lactic acid bacteria LAB4012 strain on the growth and non-specific immunity of cobia
摘要
海鱺 (Rachycentron canadum) 是臺灣重要的箱網養殖魚種，每年都為臺灣帶來高經濟效益。海鱺經常受到弧菌、發光桿菌等病原菌的危害，現行利用抗生素或疫苗來防範疫情，但抗生素的使用使得近年來不斷有抗藥性病原菌株出現，疫苗在田間實驗的效力也不穩定。Xing et al. (2013) ，以海鱺腸道分離出的乳酸菌Pediococcus pentosaceus LAB4012菌株，經餵食及感染試驗證明，可有效幫助海鱺對抗發光桿菌 (Photobacterium damselae subsp. piscicida, Pdp) ，但不會提升免疫魚的專一性抗體力價，因此推測LAB4012誘發的保護效果以非專一性免疫為主。為了探究LAB4012幫助海鱺對抗發光桿菌的方法，本研究探討LAB4012對海鱺生理與非專一性免疫的影響，包括成長率、腸道黏膜屏障、周邊白血球密度、以及發炎反應相關基因的表現等。研究結果顯示，持續兩週餵食添加LAB4012飼料的海鱺，其體重比對照組多了22%，在浸泡感染發光桿菌後，實驗組的累積死亡率比對照組低了20%。以發光桿菌感染餵食了四週LAB4012的海鱺，其腸道中分泌黏液的杯狀細胞密度比對照組高；餵食滿四週的海鱺，周邊血液中的紅血球密度和對照組沒有顯著差異，以發光桿菌感染後一天，實驗組紅血球量沒有變化，但對照組下降至與實驗組有顯著差異，第三天才又恢復；白血球密度在感染發光桿菌後持續上升，雖然和對照組白血球數目沒有顯著差異，但實驗組在測試的三個時間點的白血球密度都比對照組高。分析發炎反應相關基因IL-1β和TNF-α的表現，在持續餵食LAB4012四週後，肝臟與頭腎中的IL-1β和TNF-α基因表現都沒有顯著性影響，脾臟中的IL-1β和TNF-α的表現量則低於對照組；感染發光桿菌後，肝臟的TNF-α與頭腎中的IL-1β和TNF-α表現量和對照組相比，有顯著提高。綜合上述結果，確認以LAB4012作為益生菌餵食海鱺可以加速幼苗成長、刺激腸道黏膜細胞增加、強化感染發光桿菌後的發炎反應，這些影響將有助於提高海鱺對抗發光桿菌的感染。
並列摘要
Cobia (Rachycentron canadum) is an important fish species of cage-aquaculture industry, and has brought high economic interest in Taiwan. Cobias have been infected by many pathogens, such as vibrio and photobacteria. Antibiotics and vaccine are widely used for antagonizing bacteria pathogens, but the use of antibiotics made some drug-resistant strains of pathogens have been identified in the past few years, and the protection efficacies of vaccine were still unstable in field trials. Xing et al. (2013) demonstrated that lactic acid bacteria (LAB), Pediococcus pentosaceus LAB4012, which was derived from cobia’s intestine, could help cobia to antagonize photobacteria infection, but did not increase antibody titer of the Pdp-immunized cobia. Therefore, it is suggested that the LAB4012-induced protection against Pdp mainly rely on innate immunity. For understanding the tips of LAB4012 help cobia against Pdp, in this study, the influence of LAB4012-feeding in cobia was further examined, including the growth rate, density of intestine goblet cells, peripheral leukocytes and erythrocytes, and the expression level of inflammatory cytokines after Pdp challenge. Our results indicated that 2-week-LAB4012-feeding can significantly enhance the growth rate of smaller cobia with original body length of 12 cm, and the cumulated mortality of the fed cobia post Pdp challenge was 20% lower than that of the control group. After 4-week feeding with LAB4012, the density of goblet cells in cobia intestine villi was higher than that of control group, and the leukocyte density of the LAB-fed cobia was also higher than that of the control group. After Pdp immersion challenge, the density of the peripheral red blood cells (RBCs) in the LAB4012-fed cobia was stable. On the contrary, the RBCs level decreased in the control group at 1 day post infection (dpi) and restored at 3 dpi. Both LAB4012-fed cobia and control cobia, the leukocyte density increased after Pdp challenge, but their levels in LAB4012-fed group at 1 and 3 dpi were higher than that in the control group. The expression levels of IL-1β and TNF-α in liver and head kidney showed no difference between the LAB4012-fed group and the control group. After Pdp challenging, TNF-α expression level in the liver and both genes in head kidney were significantly up-regulated in LAB-feeding group. Therefore, we reconfirmed LAB4012 to be a probiotic for cobia due to speeding up the maturity of immune system through elevating the growth rate of smaller cobia, strengthen the intestinal mucus barrier through increasing the goblet cell density, and enhancing the expression of inflammation cytokines at the early stage of infection. In summary, LAB4012 can speed up the growth of smaller cobia and result in as a probiotic can improve non-specific immune system of cobias with increasing growth rate, inducing gut immunity, strengthening and regulating inflammation to against Pdp.