枯草芽孢桿菌、發(fā)酵乳桿菌和乳酸片球菌復(fù)合制劑對(duì)兔子宮內(nèi)膜炎治療效果的研究
發(fā)布時(shí)間：2018-08-13 09:18
【摘要】：[目的]子宮內(nèi)膜炎是奶牛常見(jiàn)的一種產(chǎn)后期疾病,且多數(shù)為隱性,該病給養(yǎng)殖業(yè)的發(fā)展帶來(lái)嚴(yán)重的威脅。多年以來(lái),研究者們一直在尋求高效安全的治療方法。益生菌復(fù)合后仍是無(wú)毒安全的,即不會(huì)產(chǎn)生藥物殘留,同時(shí)還可以增強(qiáng)動(dòng)物機(jī)體的免疫力。本試驗(yàn)通過(guò)從奶牛陰道分泌物中提取益生菌,將其與乳酸片球菌等比例混合制備復(fù)合益生菌,研究該復(fù)合制劑對(duì)子宮內(nèi)膜炎的治療效果。[方法]試驗(yàn)一:取健康奶牛陰道的分泌物,分別采用厭氧和需氧的培養(yǎng)方式,經(jīng)過(guò)分離純化、染色鏡檢、生理生化、16SrDNA序列分析及同源性比較,確定分離得到的菌株種屬。試驗(yàn)二:(1)測(cè)定菌株不同時(shí)間的生長(zhǎng)情況及不同培養(yǎng)基pH、不同接種量和不同液體培養(yǎng)基盛液量對(duì)菌株生長(zhǎng)的影響,確定各菌的最適生長(zhǎng)條件;(2)測(cè)定三株菌體外抑菌效果及相容性;(3)選取40只昆明系SPF小鼠,平均分成4組。分別是空白組(K)、枯草芽孢桿菌組(S1)、發(fā)酵乳桿菌組(S2)和乳酸片球菌組(S3),7 d后剖檢察看臟器病變并計(jì)算臟器系數(shù),以此評(píng)價(jià)三株菌的安全性。試驗(yàn)三:(1)選取20只昆明系SPF小鼠,隨機(jī)分為2組,試驗(yàn)組(復(fù)合益生菌)和空白組(生理鹽水),7d后剖檢察看臟器病變并計(jì)算臟器系數(shù),同時(shí)檢測(cè)血液中主要的炎性細(xì)胞數(shù)變化,以此判斷復(fù)合益生菌是否安全。(2)取15只母兔,平均分成3組,分為空白組、感染組和治療組,通過(guò)子宮灌注致病菌和復(fù)合益生菌的方法分別進(jìn)行造模和治療,均為隔天一次,連續(xù)三次。觀察臨床表現(xiàn)、檢測(cè)尿液及主要炎性細(xì)胞數(shù)變化、觀察剖檢后子宮形態(tài)及子宮組織病理學(xué)切片、檢測(cè)兔血清中抗體和相關(guān)炎性因子水平,根據(jù)這些指標(biāo)來(lái)評(píng)價(jià)該復(fù)合制劑治療子宮內(nèi)膜炎的效果。[結(jié)果]1、奶牛陰道分泌物經(jīng)分離純化、染色鏡檢、生理生化、16SrDNA序列分析和同源性比較,得到兩株益生菌。一株是枯草芽孢桿菌,另一株是發(fā)酵乳桿菌。2、均在37℃條件下探究菌株生物學(xué)特性,發(fā)現(xiàn)枯草芽孢桿菌以6%的接種量接種于20 mL pH為6.8的LB液體培養(yǎng)基中,培養(yǎng)18 h后活力最強(qiáng);乳酸片球菌和發(fā)酵乳桿菌分別以2%和5%的接種量接種于30 mL pH為6.4的MRS液體培養(yǎng)基中,厭氧培養(yǎng)24 h后生長(zhǎng)狀態(tài)最好。牛津杯法檢測(cè)體外抑菌效果,發(fā)現(xiàn)三株益生菌都有抑菌性。通過(guò)菌株間的相容性檢測(cè),發(fā)現(xiàn)三株益生菌可在同一環(huán)境中生長(zhǎng),無(wú)拮抗作用。通過(guò)毒性試驗(yàn)檢測(cè),發(fā)現(xiàn)三株益生菌不會(huì)引起小鼠主要臟器的病變;與空白組相比,臟器系數(shù)差異不顯著(p0.05),說(shuō)明三株益生菌單獨(dú)使用時(shí)是安全的。3、通過(guò)毒性試驗(yàn)檢測(cè),發(fā)現(xiàn)使用的三株益生菌經(jīng)復(fù)合后不會(huì)引起小鼠主要臟器的病變;與空白組相比,臟器系數(shù)差異不顯著(p0.05);血常規(guī)檢測(cè)發(fā)現(xiàn),與空白組相比,血液中主要的炎性細(xì)胞數(shù)變化差異不顯著(p0.05)。說(shuō)明三株益生菌復(fù)合后是安全的、無(wú)致病性。4、經(jīng)復(fù)合益生菌治療后,動(dòng)物的精神狀態(tài)良好;硝酸銀法檢測(cè)尿液,結(jié)果為陰性或弱陽(yáng)性;與感染組相比,治療組血液中主要的炎性細(xì)胞數(shù)量顯著減少(p0.01,p0.05);觀察子宮形態(tài),發(fā)現(xiàn)腫脹及充血程度減弱;觀察子宮組織病理學(xué)切片發(fā)現(xiàn),治療組子宮內(nèi)膜上皮細(xì)胞排列較為整齊,中性粒細(xì)胞浸潤(rùn)數(shù)量明顯減少,子宮腺分布均勻,空泡化程度顯著減輕。5、用ELISA方法檢測(cè)血清中主要的抗體和相關(guān)炎性因子水平,發(fā)現(xiàn)與感染組相比,治療組血清中的IgA、IgG和IgM的水平顯著升高(p0.01,p0.05);而治療組血清中的促炎因子IL-1β和相關(guān)炎性因子CRP的水平顯著下降(0.01p0.05)。[結(jié)論]枯草芽孢桿菌、發(fā)酵乳桿菌和乳酸片球菌經(jīng)復(fù)合后是安全的,并可以提高機(jī)體免疫力,緩解炎癥,對(duì)子宮內(nèi)膜炎有治療作用。
[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.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23
，
本文編號(hào)：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.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23
，
本文編號(hào)：2180547
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