腹泻犊牛源大肠杆菌对小鼠的致病性

Pathogenicity of Escherichia coli strains isolated from calves with diarrhea to mice

摘要:

【背景】建立细菌性动物腹泻模型是研究细菌性腹泻机制及抗腹泻药机理的常用方法。【目的】从临床腹泻犊牛粪便中分离出5株不同血清型大肠杆菌(Escherichia coli)，经口灌服小鼠建立与临床犊牛腹泻症状近似的小鼠腹泻模型。【方法】72只昆明小鼠随机分为6组，每组12只，分为正常对照组(NC)、E. coli O1干预组、E. coli O2干预组、E. coli O8干预组、E. coli O78干预组和E. coli O86干预组，攻毒组大肠杆菌浓度均为3×10¹³ CFU/mL，每10 g体重灌服0.2 ml攻毒菌液，2次/d，连续灌服7 d，分别于灌服后3、5和7 d记录小鼠体重及腹泻率，并收集各组小鼠血清，检测白细胞介素1β (interleukin 1β，IL-1β)、IL-6、肿瘤坏死因子α (tumor necrosis factor alpha，TNF-α)及第7天血清免疫球蛋白G (immunoglobin G，IgG)、IgA和分泌型免疫球蛋白A (secretory immunoglobulin A，sIgA)的含量；于攻毒后第7天收集各组小鼠十二指肠及盲肠内容物，用于制作HE切片及16S rRNA基因分析；以E. coli O8作为攻毒菌株通过灌胃和注射的方法建立腹泻模型，使用氧氟沙星对腹泻小鼠进行治疗。【结果】攻毒5 d和7 d后，各攻毒组小鼠体重与NC组比较均显著降低(P<0.05)，腹泻率增加；HE染色结果显示，各攻毒组小鼠肠绒毛均出现不同程度的损伤，其中以E. coli O2和E. coli O8导致的损伤最为严重，E. coli O1次之；血清IL-1β、IL-6和TNF-α的含量均显著增加(P<0.05)，以E. coli O1和E. coli O8的含量增加最为显著；IgG、IgA和sIgA含量显著下降(P<0.05)，以E. coli O8下降最为显著；小鼠盲肠内容物的16S rRNA基因分析显示，与NC组比较各攻毒组小鼠菌群多样性水平降低，在门水平上攻毒小鼠厚壁菌门(Firmicutes)丰度下降，拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)和放线菌门(Actinobacteria)丰度上升；腹腔注射E. coli O8小鼠在12 h内全部死亡，并且抗生素不能逆转这种死亡，而通过灌胃造成的腹泻小鼠经抗生素治疗后可恢复正常。【结论】5株牛源大肠杆菌经口灌服小鼠后均会造成不同程度的腹泻，其中E. coli O2、E. coli O8和E. coli O1对小鼠肠绒毛破坏较为严重，E. coli O8攻毒后血清IL-1β、IL-6和TNF-α含量较高，因此，以0.2 mL/10 g，2次/d，连续灌服7 d浓度为3×10¹³ CFU/mL的E. coli O8可较好地建立小鼠腹泻模型，并可用于评价药物疗效。

关键词:大肠杆菌;腹泻;IL-1β;IL-6;TNF-α;细菌群落

Abstract:

[Background] Establishing the diarrhea model is a common method to study the mechanism of bacterial diarrhea and anti-diarrhea mechanism. [Objective] Five strains of Escherichia coli with different serotypes were isolated from the feces of calves with diarrhea. The diarrhea model of mice was established by intragastric administration. [Methods] Kunming mice were randomly divided into six groups:normal control (NC) group, E. coli O1, E. coli O2, E. coli O8, E. coli O78, and E. coli O86 groups, with 12 mice in each group. The number of E. coli in all the challenge groups was 3×10¹³ CFU/mL, and the strain liquid was administrated at 0.2 ml/10 g body weight and twice/day for 7 continuous days. The body weight and diarrhea rate of mice were recorded on days 3, 5 and 7, and the serum samples of mice in each group were collected for the determination of interleukin 1β (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), immunoglobin G (IgG), IgA, and secretory immunoglobulin A (sIgA) levels. The duodenum of mice in each group was collected on the 7th day to prepare H&E sections, and the cecum contents were collected for 16S rRNA gene analysis. A diarrhea model was established with E. coli O₈ via intragastric or intraperitoneal administration, and ofloxacin was used to treat the mice with diarrhea. [Results] On days 5 and 7, the mice in the E. coli groups showed lower body weight (P<0.05) and higher diarrhea rate than those in the NC group. HE staining results showed that the intestinal villi of mice in E. coli groups showed different degrees of damage, and E. coli O2 and O8 caused the most serious damage. The serum levels of IL-1β, IL-6, and TNF-α significantly elevated after challenge (P<0.05), especially in the E. coli O1 and O8 groups. However, the levels of IgG, IgA, and sIgA in the challenge groups significantly decreased compared with those in the NC group (P<0.05), especially in the group challenged with strain O8. The 16S rRNA gene analysis of cecum contents in mice showed that compared with the NC group, the challenge groups had decreased alpha diversity of bacteria. Specifically, the abundance of Firmicutes decreased while that of Bacteroides, Proteobacteria and Actinobacteria increased in the challenge groups. All the mice died within 12 h after intraperitoneal injection with E. coli O₈, and antibiotic did not reverse this death, while the mice with diarrhea caused by intragastric administration could restore to normal after antibiotic treatment. [Conclusion] The five strains of E. coli from dairy calves with diarrhea could cause different degrees of diarrhea in mice after intragastric administration. Among them, E. coli O2, O8, and O1 caused serious damage to the intestinal villi of mice. The serum levels of IL-1β, IL-6 and TNF-α in mice were higher after administration with E. coli O8. Therefore, intragastric administration of 3×10¹³ CFU/mL E. coli O8 at 0.2 mL/10 g and twice/day for 7 continuous days could well establish the diarrhea model of mice, which can be used to evaluate drug efficacy.

Key words:Escherichia coli;diarrhea;IL-1β;IL-6;TNF-α;bacterial colony