【作者】 李可；

【导师】 冯新；

【作者基本信息】 吉林大学 ， 兽医硕士（专业学位）， 2023， 硕士

【摘要】 胃肠道(GIT)中定居的微生物群和肠粘膜共同促进宿主免疫系统的发育。共生的微生物通过竞争受体和肠道营养物质、产生抗菌物质、创造微环境和刺激先天免疫系统来影响宿主动物的抗病性能。在竞争性定植过程中,微生物群中优势成分的出现被认为是宿主免疫系统进化的潜在驱动力。乳杆菌作为其中的优势成分之一,它们和宿主的共同进化为促进宿主健康提供了有利的基础。因乳杆菌属的多样化及复杂性,想要进一步描述它们与宿主之间的相互作用,探讨利用不同源性的乳杆菌对宿主肠道疾病进行治疗和预防的可能性,就需要证明乳杆菌属中哪些菌种是宿主GIT系统中主要和关键的参与者。开发这类益生菌并及时回补这些乳酸菌对预防宿主肠道疾病有着重要作用。本研究基于微生物组学分析仔猪肠道中乳酸菌在不同条件下的丰度演替规律,并分型定位到乳杆菌属。分别测定了饲料添加剂、品种、健康状态和地域因素对仔猪肠道中不同乳杆菌种类、平均相对丰度和菌株持久性的影响,结果显示仔猪肠道中主要乳杆菌种类基本一致,按平均相对丰度由高至低依次排列的顺序基本一致,瑞士乳杆菌>阴道乳杆菌>德氏乳杆菌>粘膜乳杆菌>罗伊氏乳杆菌>约氏乳杆菌>仓鼠乳杆菌>唾液乳杆菌>脑桥乳杆菌。瑞士乳杆菌和阴道乳杆菌丰度居高(P<0.01)。腹泻组仔猪瑞士乳杆菌丰度随时间先降低后回升,自身可以进行补给调控,治疗后可与对照组恢复一致。阴道乳杆菌腹泻状态下丰度下降,在治疗后回升。与空白组相比,腹泻组和腹泻治疗组罗伊氏乳杆菌的丰度比例显著上升,产生的大量抑菌物质有利于清除肠道致病菌,维护肠道健康。初步确定瑞士乳杆菌和阴道乳杆菌为仔猪肠道持久性菌株,且瑞士乳杆菌的自身调节性能优于阴道乳杆菌。基于上述结果对目标菌株进行分离鉴定,共得到34株猪源乳杆菌临床分离菌株。评价分离菌株对胆盐和低p H值的抗性、抑菌力和体外对上皮细胞的粘附能力,其中,罗伊氏乳杆菌LJ-E6对模拟胃肠道的耐受存活率为70.00%且对猪肠上皮细胞(IPEC-J2)的黏附率为51.16%;用乳杆菌上清进行的体外抑菌效果显示,阴道乳杆菌的抑菌活性好,且阴道乳杆菌G1903的p H6.0上清对产气荚膜梭菌有抑菌效果。结合小鼠肠道荧光成像实验检测菌株肠道内定植力,罗伊氏乳杆菌LJ-E6和道乳杆菌G1903可在体内定植12 h以上。筛选出这两种菌株作为饲料添加剂开展仔猪饲喂效果研究。在仔猪饲喂试验中,断奶仔猪分为对照组、罗伊氏乳杆菌组和阴道乳杆菌组,在对照组中,仔猪饲喂不含任何抗生素或益生菌的基础日粮,在罗伊氏乳杆菌组中,以基础日粮添加罗伊氏乳杆菌LJ-E6,在阴道乳杆菌组中,以基础日粮添加阴道乳杆菌G1903。所有试验断奶仔猪饲喂28天后对每组仔猪的生长性能、腹泻率、免疫因子和肠道菌群进行测定和分析。发现乳酸菌均可提高仔猪的生长性能,优化仔猪肠道菌群结构,降低了志贺菌属(Shigella)、梭菌属(Clostridium)、拟杆菌属(Bacteroide)、链球菌属(Streptococcus)丰度;提高了乳酸菌属(Lactobacillus)、普氏菌属(Prevotella)等有益菌属的丰度。综上,本研究证实猪源乳杆菌罗伊氏乳杆菌LJ-E6和阴道乳杆菌G1903可在仔猪肠道内持久定植,且日粮中添加猪源性乳酸菌可提高仔猪的生长性能,优化健康仔猪肠道菌群。因此这两种乳酸菌可作为饲料添加剂,在仔猪断奶初期使用来调控仔猪肠道菌群以及预防仔猪肠道疾病。更多还原

【Abstract】 The microbiota resident in the GIT and the intestinal mucosa jointly promote the development of the host immune system.Symbiotic microorganisms affect the disease resistance of host animals by competing with receptors and intestinal nutrients,producing antibacterial substances,creating microenvironments,and stimulating the innate immune system.The emergence of dominant components of the microbiota during competitive colonisation is thought to be a potential driver of the evolution of the host immune system.Lactobacillus is one of the dominant components,and their co-evolution with the host provides a good basis for promoting host health.Due to the diversity and complexity of the genus Lactobacillus,it is necessary to demonstrate which Lactobacillus strains are the most important and key players in the host GIT system in order to further describe their interactions with the host and explore the possibility of using different sources of Lactobacillus for the treatment and prevention of host intestinal diseases.This study was based on a microbiomic analysis of the abundance of Lactobacillus in the piglet gut under different conditions and typing to the genus Lactobacillus.The effects of feed additives,varieties,health status and regional factors on different Lactobacillus species,average relative abundance and strain persistence in piglet intestines were measured respectively.The results show that the main Lactobacillus species in the intestine of piglets are basically the same,and the order of average relative abundance from high to low is basically the same.Lactobacillus helveticus >Lactobacillus vaginalis > Lactobacillus delbrueckii > Lactobacillus mucosae > Lactobacillus reuteri >Lactobacillus johnsonii > Lactobacillus hamsteri > Lactobacillus salivarius > Lactobacillus pontis.The abundance of Lactobacillus helveticus and Lactobacillus vaginalis was high(P < 0.01).The abundance of Lactobacillus helveticus in diarrhea piglets decreased first and then increased.It can regulate the supply itself,and it can be restored to the control group after treatment.The abundance of Lactobacillus vaginalis decreased in the diarrhea state and increased after treatment.The abundance of Lactobacillus reuteri increased significantly in the diarrhea group and the diarrhea treatment group compared to the control group,and the production of antibacterial substances was beneficial to remove intestinal pathogens and maintain intestinal health.It was preliminarily determined that Lactobacillus helveticus and Lactobacillus vaginalis were persistent strains in the intestine of piglets,and that Lactobacillus helveticus had a better self-adjusting performance than Lactobacillus vaginalis.Based on the above results,isolate and identify the target strain,a total of 34 clinical isolates of Lactobacillus from pigs were isolated and identified.The resistance of the isolated strains to bile salt and low p H,the antibacterial power and the adhesion ability to epithelial cells in vitro were evaluated.Lactobacillus reuteri LJ-E6 showed 70.00% tolerance to the simulated gastrointestinal tract and 51.16% adherence to porcine intestinal epithelial cells(IPEC-J2).In vitro antibacterial effect of the supernatant of Lactobacillus showed that Lactobacillus vaginalis had the strongest antibacterial activity,and the supernatant of Lactobacillus vaginalis G1903(p H6.0)had an antibacterial effect on Clostridium perfringens.Combined with the intestinal fluorescence imaging experiment in mice,Lactobacillus reuteri LJ-E6 and Lactobacillus vaginalis G1903 could colonize in vivo for more than 12 h.These two strains were selected as feed additives to study the feeding effect of piglets.piglets were fed a basic diet without any antibiotics or probiotics.In the piglet feeding experiment,weaned piglets were divided into control group,Lactobacillus reuteri group and Lactobacillus vaginalis group.In the control group,piglets were fed a basal diet without any antibiotics or probiotics.The Lactobacillus reuteri and Lactobacillus vaginalis groups added Lactobacillus reuteri LJ-E6 and Lactobacillus vaginalis G1903 to the basal diet respectively.The growth performance,diarrhea rate,immune factors and intestinal flora of each group of piglets were measured and analyzed after 28 days of weaned piglets.It was found that the feeding of Lactobacillus could improve the growth performance of piglets,optimize the structure of intestinal flora,and reduce the abundance of Shigella,Clostridium,Bacteroide and Streptococcus;it improves the abundance of beneficial bacteria such as Lactobacillus and Prevotella in the gut.In summary,this study confirmed that Lactobacillus reuteri LJ-E6 and Lactobacillus vaginalis G1903 can be persistently colonise the intestinal tract of piglets.Adding pig-derived Lactobacillus to the diet can improve the growth performance of piglets and optimize the intestinal flora of healthy piglets.These two Lactobacillus can be used as feed additives to regulate the intestinal flora of piglets and prevent intestinal diseases in piglets at the early stage of weaning.更多还原