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

  • 摘要: 本实验旨在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(2.5×10 8 cfu/mL)及低浓度(0.7×10 8 cfu/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12h、24h、3d、7d和14d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。试验结果表明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(3d)、肠(0.5、1、3、7及14 d)、肝(3d)和肾(7d)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7d及以后其死亡率显著低于对照(P < 0.05)。因此口服AI-96可以有效预防NJ-1 ≤0.7×108 cfu/mL范围内的侵袭。

     

    Abstract: 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 intestine> gill > liver > kidney. 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 2.5×108 cfu/mL NJ-1. In 0.7×10 8 cfu/mL challenge treatment, the NJ-1 number colonizing in gill (3d), 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 effect was more obviously in 0.7×10 8 cfu/mL challenge treatment after 7 d (P < 0.05). Therefore, the dietary AI-96 enzyme could improved 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.

     

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