复方中草药与鼠李糖乳杆菌协同发酵产物对柔嫩艾美耳球虫治疗效果研究

发布时间：2018-05-03 00:46

本文选题：鸡球虫病 + 中药　；参考：《吉林农业大学》2017年硕士论文

【摘要】：鸡球虫病是由一种或多种艾美耳球虫(Eimeria)寄生于鸡肠上皮细胞内所引起的严重危害养鸡业发展的原虫病。目前,鸡球虫病的治疗主要依赖于化学药物,但是化学药物的长期使用造成耐药虫株产生和药物残留影响食品安全。中药和益生菌相比化学药物具有安全、环保、可增强机体免疫力且制备过程简单等诸多优点。因此,中药与益生菌发酵技术在动物疫病防治方面已成为一个新的方向。为了探索中药与益生菌发酵产物对鸡球虫病的治疗效果,本试验首先通过体外抑制柔嫩艾美耳球虫(Eimeria tenella)的卵囊孢子化情况对中药及中药组方进行筛选,并应用体外抑制球虫卵囊孢子化效果显著的中药组方与Lactobacillus rhamnosus ATCC53103进行共发酵,制备复方中药发酵制剂,最后研究其对已经感染球虫卵囊雏鸡的治疗效果,具体研究内容及试验结果如下:1.中药筛选及复方中药益生菌发酵条件确定:首先,通过体外抑制球虫卵囊孢子化试验,筛选出抑制球虫卵囊孢子化效果最好的青蒿、苦参、柴胡三种单味中药,以这三味中药作为驱虫主药组成三种中药复方,再次通过体外抑制球虫卵囊孢子化试验对复方中药进行筛选,最后得到抑制卵囊孢子化效果最好的方剂:青蒿(20%)、常山(20%)、白头翁(15%)、仙鹤草(15%)、槐花(10%)、黄芩(10%)、甘草(10%)。使用以上方剂与Lactobacillus rhamnosus ATCC53103菌株进行共发酵处理,以活菌数为指标对发酵体系中的中药添加量、发酵时间、菌株接种量、发酵温度、发酵转速各个条件进行优化。通过上述条件进行发酵,制得复方中药发酵制剂。使用高效液相色谱法分析发酵前后的变化。结果表明中药添加量为100%、菌株接种量为4%、转速为80r/min、37℃条件下培养12h为最佳发酵条件。高效液相色谱法分析结果发现发酵后与发酵前相比,其物质成分确实发生变化,出现某些吸收峰不同程度的升高或降低现象,说明ATCC53103菌株在与复方中药发酵的过程中也会使中药中某些成分发生改变。最后,通过对雏鸡进行急性毒性试验发现,雏鸡在每日灌服相当于含有48g原药的复方中药发酵制剂条件下连续灌服7日,共计灌服了相当于含有336g原药的复方中药发酵制剂,对雏鸡的观察以及剖检后未见任何异常可以证明该复方中药发酵制剂具有安全性。2.复方中药制剂与Lactobacillus rhamnosus ATCC53103协同发酵产物对雏鸡感染E.tenella治疗效果:将30只1日龄雏鸡随机分成A、B、C、D、E、F 6组,每组5只。除F组(空白对照组)以外,其余A、B、C、D、E 5组内所有雏鸡于13日龄经口接种已孢子化的Eimeria tenella卵囊5×104个/只。自攻虫之日起,每日检测各组雏鸡粪便中是否有球虫卵囊排出,于攻雏鸡18日龄在各攻虫组的雏鸡粪便中均检测到球虫卵囊,并且个别雏鸡出现便血现象,可以在确定雏鸡成功感染球虫并出现一定临床症状,对确定感染球虫雏鸡即18日龄雏鸡按下述方法进行临床治疗,A组雏鸡每日每只灌服复方中药发酵制剂200μL,B组雏鸡每日每只灌服复方中药制剂200μL,C组日每只灌服LGG ATCC53103 200μL(1×106cfu/μL),E使用地克珠利2‰饮水给药。各组连续用药5日,观察分期雏鸡临床症状,并检测各组雏鸡的相对增重率,盲肠病变记分,盲肠病理切片,每克粪便卵囊值,并计算各组药物抗球虫指数ACI。同时,通过流式细胞术检测各组雏鸡外周血中CD4+和CD8+T淋巴细胞含量变化,ELISA方法检测雏鸡Ig G和SIg A含量的变化以及IFN-γ和IL-2这两种细胞因子的表达水平变化,并对雏鸡血清中一氧化氮和一氧化氮合成酶的含量进行检测。结果显示,服用复方中药发酵制剂后的感染球虫卵囊的雏鸡体重显著上升,与对照组差异显著(P0.05)。此外,复方中药发酵制剂可以明显减轻盲肠病理变化,同时可以降低受感染雏鸡卵囊的排出量。复方中药发酵制剂的ACI为130.59,达到中等抗球虫水平。通过流式细胞术检测结果发现,服用复方中药发酵制剂的雏鸡外周血中CD8+T淋巴细胞含量有所升高。并且,服用复方中药发酵制剂可以提高雏鸡Ig G和SIg A含量以及IL-2的表达水平。对血清中一氧化氮和一氧化氮合成酶的检测发现,复方中药发酵制剂会降低球虫感染后的一氧化氮和一氧化氮合成酶的含量。通过以上结果表明,复方中药制剂与Lactobacillus rhamnosus ATCC53103协同发酵产物对雏鸡感染E.tenella具有显著的治疗效果,不但能够提高雏鸡增重,减少盲肠病变,降低卵囊排出,降低一氧化氮和一氧化氮合成酶的含量,还可以通过激特异性Ig G和SIg A抗体的分泌,提高IL-2表达水平,增强机体免疫功能。本研究为通过乳酸菌中药发酵技术对鸡球虫病的防治提供理论依据。
[Abstract]:Coccidiosis is a kind of protozoon which is caused by one or more Eimeria (Eimeria) parasitic on the epithelial cells of chicken intestines. At present, the treatment of coccidiosis is mainly dependent on chemical drugs, but the long-term use of chemical drugs causes the production of drug-resistant strains and drug residues to affect food safety. As a result, the fermentation technology of Chinese medicine and probiotics has become a new direction in the prevention and treatment of animal blight. In order to explore the therapeutic effect of Chinese medicine and probiotic fermentation products on the treatment of coccidiosis, the experiment first passed the outside in vitro. To inhibit the sporulation of oocyst of Eimeria tenella (Eimeria tenella), the traditional Chinese medicine and traditional Chinese medicine were screened, and the traditional Chinese medicine group was co fermented with Lactobacillus rhamnosus ATCC53103 in vitro to inhibit the sporulation of coccidiococcium oocyst, and the preparation of compound Chinese medicine was prepared. Finally, the infection of coccidia oocyst had been studied. The treatment effect of chicks, specific research contents and experimental results are as follows: 1. screening of Chinese medicine and determination of the fermentation conditions of probiotics in compound Chinese medicine: first, through the inhibition of coccidian oocyst sporulation test in vitro, three kinds of Artemisia Artemisia, Sophora flavescens and Chai Hu, which can inhibit the sporulation of coccidia oocysts, are selected as the main medicine of the three herbs. Three kinds of Chinese medicine compound were formed, and the compound traditional Chinese medicine was screened by the test of coccidiococcium oocyst sporulation in vitro. Finally, the best prescription to inhibit the sporulation of oocyst was obtained: Artemisia annua (20%), Changshan (20%), white headed wpulon (15%), cranes (15%), Sophora japonica (10%), Radix Scutellariae (10%) and licorice (10%). The use of the above prescription and Lactobacillus rhamnosus ATCC5 The 3103 strains were treated by CO fermentation. With the number of living bacteria as the index, the dosage of traditional Chinese medicine in the fermentation system, the fermentation time, the inoculation amount of the strain, the fermentation temperature and the fermentation speed were optimized. The fermentation of the compound Chinese medicine was fermented through the above conditions. The change of the fermentation before and after the high performance liquid chromatography was analyzed. The results showed that the changes were analyzed before and after the fermentation. The dosage of Chinese medicine was 100%, the inoculation amount of the strain was 4%, the speed was 80r/min, the 12h was the best fermentation condition under the condition of 37 C. The analysis results of high performance liquid chromatography found that the material composition changed after fermentation, and some absorption peaks were raised or reduced in different degrees, indicating that the ATCC53103 strain was in the compound with the compound. In the process of traditional Chinese medicine fermentation, some components of traditional Chinese medicine will be changed. Finally, through the acute toxicity test on chickens, it is found that the chicks are filled with the fermenting preparation of the compound traditional Chinese medicine containing 48g medicine daily for 7 days. Observation and no abnormality after examination, the efficacy of the compound Chinese medicine fermenting preparation was proved to be safe.2. compound Chinese medicine preparation and Lactobacillus rhamnosus ATCC53103 co fermentation product to treat E.tenella infection of chicks. 30 1 day old chicks were randomly divided into A, B, C, D, E, F 6 groups, 5 in each group except the F group (blank control group). The remaining A, B, C, D, E 5 groups of chickens were inoculated with sporulated Eimeria tenella oocyst at 13 days of age, 5 * 104 / only. On the day of the attack, the oocysts were detected in the feces of each chick each day. The coccidiococcium oocysts were detected in the chicks of the chicks at 18 days of age at the age of attack. The chickens can be successfully infected with coccidia and have certain clinical symptoms, and the chicks infected with coccidiosis, that is, 18 day old chicks, are treated according to the following methods. The A group chicks are given 200 mu L each day, and each of the B chicks is filled with 200 mu L each day, and each of the C group is filled with LGG ATCC53103 200 mu each day. L (1 * 106cfu/ mu L), E used dikzali 2 per thousand drinking water. The clinical symptoms of chicks were observed in each group for 5 days. The relative weight gain rate of chicks, cecum pathological sections, cecum pathological sections, each gram of fecal oocyst value were measured, and the anti coccidian index ACI. of each group was calculated, and the chicks were detected by flow cytometry. The changes in the content of CD4+ and CD8+T lymphocytes in blood, the changes in the content of Ig G and SIg A in chicks and the changes of the expression level of IFN- gamma and IL-2, and the content of nitric oxide and nitric oxide synthetase in serum of chicks were detected by ELISA method, and the results showed that the eggs were infected with the eggs after taking compound Chinese medicine. The body weight of the chicks increased significantly, and the difference was significant (P0.05). In addition, the compound Chinese medicine fermentation preparation can obviously reduce the pathological changes of the cecum and reduce the excretion of the oocyst in the infected chicks. The ACI of the compound Chinese medicine fermenting preparation is 130.59, reaching the medium level of the anti coccidiosis. The content of CD8+T lymphocyte in the peripheral blood of chicks of the Chinese traditional Chinese medicine fermented preparation has been increased. Moreover, the content of Ig G and SIg A and the expression level of IL-2 can be improved by the use of the compound Chinese medicine. The detection of nitric oxide and nitric oxide synthase in the serum shows that the compound Chinese medicine fermentation preparation will reduce the infection of coccidiosis after the first. Nitrogen oxide and nitric oxide synthase content. Through the above results, the results showed that the synergistic fermentation products of compound Chinese medicine preparation and Lactobacillus rhamnosus ATCC53103 have significant therapeutic effect on chickens infected with E.tenella. It can not only improve the weight increase of chicks, reduce the cecum disease, reduce the excretion of oocyst, reduce nitric oxide and nitric oxide synthase. The content of the enzyme can also increase the expression of IL-2 and enhance the immune function of the body through the secretion of the specific Ig G and SIg A antibodies. This study provides a theoretical basis for the prevention and control of coccidiosis in chicken by the fermentation technology of lactic acid bacteria.

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

【参考文献】