枯草芽孢杆菌、发酵乳杆菌和乳酸片球菌复合制剂对兔子宫内膜炎治疗效果的研究
发布时间：2018-08-13 09:18
【摘要】：[目的]子宫内膜炎是奶牛常见的一种产后期疾病,且多数为隐性,该病给养殖业的发展带来严重的威胁。多年以来,研究者们一直在寻求高效安全的治疗方法。益生菌复合后仍是无毒安全的,即不会产生药物残留,同时还可以增强动物机体的免疫力。本试验通过从奶牛阴道分泌物中提取益生菌,将其与乳酸片球菌等比例混合制备复合益生菌,研究该复合制剂对子宫内膜炎的治疗效果。[方法]试验一:取健康奶牛阴道的分泌物,分别采用厌氧和需氧的培养方式,经过分离纯化、染色镜检、生理生化、16SrDNA序列分析及同源性比较,确定分离得到的菌株种属。试验二:(1)测定菌株不同时间的生长情况及不同培养基pH、不同接种量和不同液体培养基盛液量对菌株生长的影响,确定各菌的最适生长条件;(2)测定三株菌体外抑菌效果及相容性;(3)选取40只昆明系SPF小鼠,平均分成4组。分别是空白组(K)、枯草芽孢杆菌组(S1)、发酵乳杆菌组(S2)和乳酸片球菌组(S3),7 d后剖检察看脏器病变并计算脏器系数,以此评价三株菌的安全性。试验三:(1)选取20只昆明系SPF小鼠,随机分为2组,试验组(复合益生菌)和空白组(生理盐水),7d后剖检察看脏器病变并计算脏器系数,同时检测血液中主要的炎性细胞数变化,以此判断复合益生菌是否安全。(2)取15只母兔,平均分成3组,分为空白组、感染组和治疗组,通过子宫灌注致病菌和复合益生菌的方法分别进行造模和治疗,均为隔天一次,连续三次。观察临床表现、检测尿液及主要炎性细胞数变化、观察剖检后子宫形态及子宫组织病理学切片、检测兔血清中抗体和相关炎性因子水平,根据这些指标来评价该复合制剂治疗子宫内膜炎的效果。[结果]1、奶牛阴道分泌物经分离纯化、染色镜检、生理生化、16SrDNA序列分析和同源性比较,得到两株益生菌。一株是枯草芽孢杆菌,另一株是发酵乳杆菌。2、均在37℃条件下探究菌株生物学特性,发现枯草芽孢杆菌以6%的接种量接种于20 mL pH为6.8的LB液体培养基中,培养18 h后活力最强;乳酸片球菌和发酵乳杆菌分别以2%和5%的接种量接种于30 mL pH为6.4的MRS液体培养基中,厌氧培养24 h后生长状态最好。牛津杯法检测体外抑菌效果,发现三株益生菌都有抑菌性。通过菌株间的相容性检测,发现三株益生菌可在同一环境中生长,无拮抗作用。通过毒性试验检测,发现三株益生菌不会引起小鼠主要脏器的病变;与空白组相比,脏器系数差异不显著(p0.05),说明三株益生菌单独使用时是安全的。3、通过毒性试验检测,发现使用的三株益生菌经复合后不会引起小鼠主要脏器的病变;与空白组相比,脏器系数差异不显著(p0.05);血常规检测发现,与空白组相比,血液中主要的炎性细胞数变化差异不显著(p0.05)。说明三株益生菌复合后是安全的、无致病性。4、经复合益生菌治疗后,动物的精神状态良好;硝酸银法检测尿液,结果为阴性或弱阳性;与感染组相比,治疗组血液中主要的炎性细胞数量显著减少(p0.01,p0.05);观察子宫形态,发现肿胀及充血程度减弱;观察子宫组织病理学切片发现,治疗组子宫内膜上皮细胞排列较为整齐,中性粒细胞浸润数量明显减少,子宫腺分布均匀,空泡化程度显著减轻。5、用ELISA方法检测血清中主要的抗体和相关炎性因子水平,发现与感染组相比,治疗组血清中的IgA、IgG和IgM的水平显著升高(p0.01,p0.05);而治疗组血清中的促炎因子IL-1β和相关炎性因子CRP的水平显著下降(0.01p0.05)。[结论]枯草芽孢杆菌、发酵乳杆菌和乳酸片球菌经复合后是安全的,并可以提高机体免疫力,缓解炎症,对子宫内膜炎有治疗作用。
[Abstract]:[Objective] Endometritis is a common postpartum disease in dairy cows, and most of them are recessive. This disease poses a serious threat to the development of animal husbandry. For many years, researchers have been searching for efficient and safe treatment methods. The effect of compound probiotics on endometritis was studied by extracting probiotics from the vaginal secretions of dairy cows and mixing them with P. lactis in the same proportion. Purification, staining microscopy, physiology and biochemistry, 16S rDNA sequence analysis and homology comparison were used to determine the strain genus. Experiment 2: (1) Determine the growth of the strain at different time and the effects of pH, inoculation amount and liquid volume on the growth of the strain, and determine the optimum growth conditions; (2) Test 3 (3) Forty Kunming SPF mice were divided into four groups, namely blank group (K), Bacillus subtilis group (S1), Lactobacillus fermentation group (S2) and Lactococcus lactis group (S3). After 7 days, the organ lesions were examined and the organ coefficients were calculated to evaluate the safety of the three strains. SPF mice were randomly divided into two groups, the experimental group (compound probiotics) and the blank group (normal saline). After 7 days, the pathological changes of the organs were observed and the coefficients of the organs were calculated, and the changes of the main inflammatory cells in the blood were detected to determine whether the compound probiotics were safe or not. (2) 15 female rabbits were divided into three groups, which were divided into blank group, infection group and treatment group. Pathogenic bacteria and compound probiotics were perfused into the uterus to make models and treat them once every other day for three consecutive times.Clinical manifestations were observed,urine and the number of major inflammatory cells were detected,uterine morphology and pathological sections were observed after dissection,and the levels of antibodies and related inflammatory factors in the serum of rabbits were detected. [Results] 1. Two strains of probiotics were obtained from the vaginal secretion of dairy cows by isolation and purification, staining microscopy, physiological and biochemical analysis, 16S rDNA sequence analysis and homology comparison. One strain was Bacillus subtilis, the other was Lactobacillus fermentum. 2. The biological characteristics of the strains were studied at 37 C. It was found that Bacillus subtilis was inoculated with 6% inoculum in LB liquid medium with 20 mL pH of 6.8, and its activity was the strongest after 18 hours of culture. The growth state of Lactobacillus lactis and Lactobacillus fermentum was the best when inoculated with 2% and 5% inoculum respectively in MRS liquid medium with 30 mL pH of 6.4 after 24 hours of anaerobic culture. Three probiotics could grow in the same environment without any antagonism. Toxicity test showed that the three probiotics did not cause pathological changes in the main organs of mice. Compared with the blank group, there was no significant difference in organ coefficient (p0.05), indicating that the three probiotics could grow in the same environment without antagonism. It is safe to use alone. 3. Through toxicity test, it was found that the three probiotics did not cause the main organ lesions in mice; compared with the blank group, there was no significant difference in organ coefficient (p0.05); blood routine test found that there was no significant difference in the number of major inflammatory cells in the blood compared with the blank group (p0.05). Ming three strains of probiotics compound is safe, non-pathogenic. Observing the pathological section of the uterus, we found that the number of endometrial epithelial cells in the treatment group was more orderly, the infiltration of neutrophils was significantly reduced, the distribution of uterine glands was uniform, and the degree of vacuolization was significantly reduced. The serum levels of IgA, IgG and IgM were significantly increased in the treatment group (p0.01, p0.05), while the serum levels of IL-1beta and CRP were significantly decreased in the treatment group (0.01 p0.05). [Conclusion] Bacillus subtilis, Lactobacillus fermentans and Lactococcus lactis are safe after being combined, and can improve the immunity, alleviate inflammation and antibiotics. Endometritis has therapeutic effects.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23
，
本文编号：2180547
[Abstract]:[Objective] Endometritis is a common postpartum disease in dairy cows, and most of them are recessive. This disease poses a serious threat to the development of animal husbandry. For many years, researchers have been searching for efficient and safe treatment methods. The effect of compound probiotics on endometritis was studied by extracting probiotics from the vaginal secretions of dairy cows and mixing them with P. lactis in the same proportion. Purification, staining microscopy, physiology and biochemistry, 16S rDNA sequence analysis and homology comparison were used to determine the strain genus. Experiment 2: (1) Determine the growth of the strain at different time and the effects of pH, inoculation amount and liquid volume on the growth of the strain, and determine the optimum growth conditions; (2) Test 3 (3) Forty Kunming SPF mice were divided into four groups, namely blank group (K), Bacillus subtilis group (S1), Lactobacillus fermentation group (S2) and Lactococcus lactis group (S3). After 7 days, the organ lesions were examined and the organ coefficients were calculated to evaluate the safety of the three strains. SPF mice were randomly divided into two groups, the experimental group (compound probiotics) and the blank group (normal saline). After 7 days, the pathological changes of the organs were observed and the coefficients of the organs were calculated, and the changes of the main inflammatory cells in the blood were detected to determine whether the compound probiotics were safe or not. (2) 15 female rabbits were divided into three groups, which were divided into blank group, infection group and treatment group. Pathogenic bacteria and compound probiotics were perfused into the uterus to make models and treat them once every other day for three consecutive times.Clinical manifestations were observed,urine and the number of major inflammatory cells were detected,uterine morphology and pathological sections were observed after dissection,and the levels of antibodies and related inflammatory factors in the serum of rabbits were detected. [Results] 1. Two strains of probiotics were obtained from the vaginal secretion of dairy cows by isolation and purification, staining microscopy, physiological and biochemical analysis, 16S rDNA sequence analysis and homology comparison. One strain was Bacillus subtilis, the other was Lactobacillus fermentum. 2. The biological characteristics of the strains were studied at 37 C. It was found that Bacillus subtilis was inoculated with 6% inoculum in LB liquid medium with 20 mL pH of 6.8, and its activity was the strongest after 18 hours of culture. The growth state of Lactobacillus lactis and Lactobacillus fermentum was the best when inoculated with 2% and 5% inoculum respectively in MRS liquid medium with 30 mL pH of 6.4 after 24 hours of anaerobic culture. Three probiotics could grow in the same environment without any antagonism. Toxicity test showed that the three probiotics did not cause pathological changes in the main organs of mice. Compared with the blank group, there was no significant difference in organ coefficient (p0.05), indicating that the three probiotics could grow in the same environment without antagonism. It is safe to use alone. 3. Through toxicity test, it was found that the three probiotics did not cause the main organ lesions in mice; compared with the blank group, there was no significant difference in organ coefficient (p0.05); blood routine test found that there was no significant difference in the number of major inflammatory cells in the blood compared with the blank group (p0.05). Ming three strains of probiotics compound is safe, non-pathogenic. Observing the pathological section of the uterus, we found that the number of endometrial epithelial cells in the treatment group was more orderly, the infiltration of neutrophils was significantly reduced, the distribution of uterine glands was uniform, and the degree of vacuolization was significantly reduced. The serum levels of IgA, IgG and IgM were significantly increased in the treatment group (p0.01, p0.05), while the serum levels of IL-1beta and CRP were significantly decreased in the treatment group (0.01 p0.05). [Conclusion] Bacillus subtilis, Lactobacillus fermentans and Lactococcus lactis are safe after being combined, and can improve the immunity, alleviate inflammation and antibiotics. Endometritis has therapeutic effects.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23
，
本文编号：2180547
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