饲料中添加侧孢短芽孢杆菌FAS05对凡纳滨对虾生长、抗病及免疫力的影响

Effects of Adding Brevibacillus laterosporu FAS05 to Feed on the Growth,Disease Resistance,and Immunity of Litopenaeus vannamei

侧孢短芽孢杆菌(Brevibacillus laterosporu)是一种重要的生物防治益生菌,但在水产养殖中鲜见报道。为研究饲料中添加侧孢短芽孢杆菌FAS05对凡纳滨对虾(Litopenaeus vannamei)生长、抗病及免疫力的影响,以体重为(1.00±0.08) g的凡纳滨对虾为研究对象,开展了28 d的养殖实验。实验分为4组,每组3个重复,每个重复50尾对虾,分别饲喂添加0 (C组,作为对照组)、10^(5)(BL1组)、10^(7)(BL2组)和10^(9)CFU/g(BL3组)侧孢短芽孢杆菌FAS05的实验饲料。结果显示,各组存活率无显著差异(P>0.05),BL1和BL2组对虾的体长、体重和特定生长率显著高于C组(P<0.05);与C组相比,BL1~BL3组养殖水体的弧菌数显著降低(P<0.05);侵染副溶血弧菌(Vibrio parahemolyticus)后,BL1~BL3组存活率显著高于C组(P<0.05),BL1~BL3组之间的差异不显著(P>0.05);与C组相比,BL1~BL3组对虾血细胞吞噬率显著增加(P<0.05),而血细胞活性氧(ROS)产量显著降低(P<0.05);BL1~BL3组对虾的溶菌酶(LZM)、过氧化氢酶(CAT)和酸性磷酸酶(ACP)均显著高于C组(P<0.05),BL1~BL3组之间的LZM和ACP活性无显著差异(P>0.05),BL2组对虾的酚氧化酶(PO)显著高于其他各组(P<0.05),BL1和BL2组中的碱性磷酸酶(ALP)和超氧化物歧化酶(SOD)显著高于C组和BL3组(P<0.05)。研究表明,侧孢短芽孢杆菌FAS05作为饲料添加剂能够促进对虾生长、激活免疫系统、提高抗病力和抑制周围环境弧菌生长,参考使用量为105CFU/g。在病害严重时,可以加大用量至107CFU/g,能够进一步提高对虾的非特异性免疫力。

Litopenaeus vannamei is one of the important crustaceans in aquaculture in China.In recent years,the shrimp culture industry has gradually developed into an intensive and high-density model,which can lead to environmental deterioration and large-scale outbreak of diseases.The widespread use of antibiotics has led to the increase of pathogen drug resistance,environmental pollution,and ecological imbalance,resulting in secondary pollution in the water.Probiotics are live microbial additives that promote good health and are environmentally sustainable and safe for use.They are often used as important substitutes for antibiotics.Brevibacillus laterosporu,as a biocontrol probiotic,is commonly used for disease control of crops,animals,and poultry,but its use is rarely reported in aquaculture.A 28-day breeding experiment was carried out with L.vannamei weighing(1.00±0.08)g,to study the effects of adding B.laterosporu FAS05 in the feed on the growth,disease resistance,and immunity of L.vannamei.The experiment was divided into four groups with three replicates in each group,and 50 shrimps in each replicate were fed with the experimental feed supplemented with 0 CFU/g(group C,as the control group),10^(5) CFU/g(group BL1),10^(7) CFU/g(group BL2),and 10^(9) CFU/g(group BL3)of B.laterosporu FAS05.B.laterosporu FAS05 was isolated from the aquaculture pond with a low number of Vibrio in summer.The bacterium was added into the basic feed with fish meal,soybean meal,and corn meal as the main protein source,fish oil and phospholipid oil were added as the fat source,wheat flour as the main sugar source,and inorganic salts and vitamins as supplements.L.vannamei was purchased from a prawn farm in Weihai City,Shandong Province.The temperature was 25-28℃and the salinity was 27-30.During the breeding experiment,eight shrimps were randomly selected in each tank every two weeks and their body length,weight,and plumpness were measured.The phagocytic activity assay was modified on the basis of the method reported by Delaporte et al(2003).Fl-1 channel flow cytometry was used to detect and analyze the offset of respiratory burst peak.The blood lymphocytes,serum,and hepatopancreas of shrimp were collected 24 hours after the experiment.The activities of immune related enzymes in hepatopancreas,such as superoxide dismutase(SOD),catalase(CAT),acid phosphatase(ACP),and alkaline phosphatase(ALP)levels,and the activities of immune related enzymes in serum,phenol oxidase(PO),and lysozyme(LZM),were determined by kits.The phagocytic activity was measured,and the assay was modified on the basis of Delaporte et al(2003).Fl-1 channel flow cytometry was used to detect and analyze the offset of respiratory burst peak.After the feeding test,a one-week infection test of Vibrio parahaemolyticus was carried out to determine disease resistance.The experimental results were expressed as Mean±SD.One-way ANOVA analysis was conducted for all data using statistical software SPSS 16.0,and significance level was defined as P<0.05.LSD homogeneity of variance test was used to compare the differences between experimental treatment groups and control groups.The results showed that there was no significant difference in survival rate among all groups(P>0.05),and the body length,body weight,and specific growth rate of shrimps in the BL1 and BL2 groups were significantly higher than those in group C(P<0.05).Compared to that in group C,the abundance of Vibrio in the aquaculture water in the BL1 to BL3 groups was significantly decreased(P<0.05).After infection with V.parahemolyticus,the survival rate of group C was about 45%,while the survival rate of the BL1 to BL3 groups was more than 80%;therefore,the survival rate of the BL1 to BL3 groups was significantly higher than that in group C(P<0.05),and no significant difference was found among the BL1 to BL3 groups(P>0.05).Compared to that of group C,the phagocytizing rate of shrimp blood cells in the BL1 to BL3 groups increased significantly(P<0.05),while the production of ROS in blood cells decreased significantly(P<0.05).The LZM,CAT and ACP activities of shrimps in the BL1 to BL3 groups were significantly higher than those in group C(P<0.05).There was no significant difference in LZM and ACP activities among the BL1 to BL3 groups(P>0.05).The PO of shrimps in the BL2 group was significantly higher than those in other groups(P<0.05).The ALP and SOD in the BL1 and BL2 groups were significantly higher than those in group C and BL3(P<0.05).The above results showed that B.laterosporu FAS05 as a feed additive could promote the growth of shrimp,activate the immune system,improve disease resistance,and inhibit the growth of Vibrio in the surrounding environment.The reference dosage was 105 CFU/g.When the infection is serious,the dosage can be increased to 107 CFU/g to further improve the non-specific immunity of shrimp.The results of this study can provide basic data for the application of B.laterosporu FAS05 in shrimp culture and production.