高丝氨酸内酯酶AI-96对嗜水气单胞菌NJ-1浸浴攻毒斑马鱼保护效应的研究

Effect of quorum-quenching enzyme from Bacillus sp AI-96 on Aeromonas hydrophila NJ-1 bath challenge in zebrafish

为评价斑马鱼口服高丝氨酸内脂酶AI-96对嗜水气单胞菌NJ-1浸浴攻毒的保护效应,实验设置基础料与实验料两组饲料,实验料是在基础料中按3 U/g饲料添加N酰基高丝氨酸内酯酶AI-96(以下简称AI-96),通过高浓度(2.5×108cfu/mL)及低浓度(0.7×108cfu/mL)两组剂量的嗜水气单胞菌NJ-1(以下简称NJ-1)分别浸浴攻毒斑马鱼,在12 h、24 h、3 d、7 d和14 d取鳃丝、肠道壁、肝和肾样,采用实时荧光定量PCR法检测取样器官中NJ-1量,并统计攻毒周期内的死亡率,来评价高丝氨酸内酯酶AI-96的保护力。结果显示:在攻毒周期内所取组织内均检测到NJ-1,按菌数肠>鳃>肝>肾,其中高NJ-1剂量未加酶组各组织NJ-1数量均分别明显高于低剂量处理组。在高剂量攻毒条件下,加酶组各组织NJ-1数量均显著低于未加酶处理组(P<0.05),鳃除外;在低剂量攻毒条件下,未加酶组的NJ-1数量在鳃(3 d)、肠(0.5、1、3、7及14 d)、肝(3 d)和肾(7 d)显著高于加酶组(P<0.05),其余差异不显著(P>0.05)。此外无论在高、低剂量攻毒条件下,加酶组的死亡率均低于未加酶组,其中低剂量攻毒7 d及以后其死亡率显著低于对照(P<0.05)。结果表明,口服AI-96可以有效预防NJ-1≤0.7×108cfu/mL范围内的侵袭。

The aim of the present experiment was to study the effects of quorum-quenching enzyme AI-96 against Aeromonas hydrophila NJ-1 attack with the bath challenge in zebrafish.The experiments set two dietary groups for zebrafish,1）basal diet as control,2）enzyme-containing diet with 3 U/g food of AI-96.Zebrafish tissues（gill,liver,kidney and intestine）were sampled at day 0.5,1,3,7 and 14 after co-bath challenge with live Aeromonas hydrophila NJ-1 at two doses（high 2.5×10^8 cfu/mL,low 0.7×10^8 cfu/mL）.The populations of NJ-1 colonizing in the tissues were quantified by real time PCR,and accumulated mortality of zebrafish were recorded everyday.Results showed that after challenge,the NJ-1 were detectable in the tissues of the attacked zebrafish in the total population order of intestinegillliverkidney.More NJ-1 colonized in the tissues in the high dosage challenge treatment than the low group.Interestingly,the diets containing AI-96 enzmye significantly decreased the colonizing populations of NJ-1 in the zebrafish（P〈0.05）,compared with the counterparts fed with basal diets when the fish was chanllenged with 2.5×10^8 cfu/mL NJ-1.In 0.7×10^8 cfu/mL challenge treatment,the NJ-1 number colonizing in gill（3 d）,intestine（0.5,1,3,7,14 d）,liver（3 d）and kidney（7 d）were also lower in the fish treated with AI-96 containing diets than the ones with basal diets（P〈0.05）.Moreover,these effects were more obvious in 0.7×10^8 cfu/mL challenge treatment after 7 d（P〈0.05）.Therefore,the dietary AI-96 enzyme could improve the survival rate against NJ-1 attack in both challenge levels.For this reason,quorum-quenching enzyme could effectively protect zebrafish against A.hydrophila at 0.7×10^8 cfu/mL level in zebrafish.