使用多株益生菌透過調節烏魚免疫反應及腸道微生物群提升生長性能和抗奴卡氏菌(Nocardia seriolae)能力

Abstract

烏魚 (Mugil cephalus) 是台灣重要的養殖魚類。近年烏魚許多嚴重疾病如: 奴卡氏菌症造成嚴重損失。目前在烏魚養殖上仍沒有有效的奴卡氏菌症的防治方式，加上由於益生菌已被證實能幫助魚類增加抗病能力，因此本研究旨以添加多株益生菌的方式，來對抗奴卡氏菌的感染。研究中以不同組合的Lactobacillus rhamnosus、Lactobacillus reuteri和Bacillus subtilis natto三株益生菌以1*109CFU/g添加於飼料，投餵35天後始使用奴卡氏菌進行攻毒。結果顯示，餵食L. reuteri和B. subtilis natto的混菌組別中在增重率、特殊成長率以及飼料轉換率都有所提升；免疫基因表現結果顯示，添加三株益生菌的飼料提升了促發炎因子IL-8和IL-1β，此外抗奴卡相關基因 TNFRSF25、PANX1表現也有顯著上升。攻毒後，也以混合L. rhamnosus，L. reuteri和B. subtilis natto的組別活存率最高，達到86%，顯著高於控制組。在菌相組成方面，Alpha diversity沒有顯著的差異，但在Beta diversity中，有添加益生菌的組別其菌相組成與控制組相比具有明顯的分群，從LefSE分析中，在控制組中，大多與病原菌有關，例如:Cytophagales、Erysipelothrix、Pseudomonas fragi、Streptococcus、Staphylococcus及Nocardioides，在有添加益生菌組別中大多發現有益菌的存在，Lre+Bsn組發現Bacillus；Lrh+Bsn組發現Sphingobacteriales、Dysgonomonadaceae；Lre+Lrh+Bsn組發現Lysinibacillus豐度的提高，綜合上述結果，在經過35天的多株益生菌投餵後，這三株菌益生菌可以藉由改變腸道中的微生物相來降低腸道中病原菌的數量，從而提升烏魚的免疫能力以及生長性能。因此未來可以針對這三株益生菌的使用作為烏魚養殖抗奴卡氏菌症的防治策略之方法，提升經濟效益。

Grey mullet (Mugil cephalus) is an important aquaculture species in Taiwan. In recent years, serious diseases such as infections caused by Nocardia seriolae have resulted in significant losses. Currently, there is no effective method to prevent and control N. seriolae infection in grey mullet aquaculture. Given that probiotics have been proven to enhance disease resistance in fish, this study aims to protect against N. seriolae infection by treating fish with multiple probiotic strains. In the study, different combinations of three probiotic strains Lactobacillus rhamnosus, Lactobacillus reuteri, and Bacillus subtilis natto were added to the feed at a concentration of 1*109 CFU/g. After 35 days of feeding, the growth rate, immunity, and gut microbiota were analyzed followed by a challenge test using N. seriolae. The results showed that the group fed with the combination of L. reuteri and B. subtilis natto exhibited improvements in weight gain rate, specific growth rate, and feed conversion ratio. The results of immune gene expression revealed that feeding with diet containing the three probiotic strains enhanced the expression of pro-inflammatory factors IL-8 and IL-1β. Additionally, the expression of anti-N. seriolae related genes TNFRSF25 and PANX1 also significantly increased. After the challenge, the group supplemented with a combination of the three probiotics also showed the highest survival rate at 86%, which was significantly higher than that of the control group. In microbial composition, there was no significant difference observed in alpha diversity. However, in beta diversity, the group supplemented with probiotics showed distinct clustering compared to the control group, indicating notable differences in microbial composition. From LefSE analysis, most of enriched taxa in the control group were associated with pathogens such as Cytophagales, Erysipelothrix, Pseudomonas fragi, Streptococcus, Staphylococcus, and Nocardioides. In contrast, in groups supplemented with probiotics, beneficial bacteria were predominantly found: the Lre+Bsn group showed Bacillus; the Lrh+Bsn group showed Sphingobacteriales and Dysgonomonadaceae; and the Lre+Lrh+Bsn group showed increased abundance of Lysinibacillus. Combining the above results, after 35 days of feeding with a multi-strain probiotic, these three probiotic strains can reduce the abundance of pathogens in the intestine by altering the gut microbiota. This process enhances the immune response and growth performance of grey mullet. Therefore, in the future, utilizing these three probiotic strains could serve as a strategy to combat N. seriolae infections in grey mullet aquaculture, potentially enhancing economic benefits.