發(fā)布時間：2018-05-03 00:46

  本文選題：雞球蟲病 + 中藥　； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：雞球蟲病是由一種或多種艾美耳球蟲(Eimeria)寄生于雞腸上皮細(xì)胞內(nèi)所引起的嚴(yán)重危害養(yǎng)雞業(yè)發(fā)展的原蟲病。目前,雞球蟲病的治療主要依賴于化學(xué)藥物,但是化學(xué)藥物的長期使用造成耐藥蟲株產(chǎn)生和藥物殘留影響食品安全。中藥和益生菌相比化學(xué)藥物具有安全、環(huán)保、可增強(qiáng)機(jī)體免疫力且制備過程簡單等諸多優(yōu)點。因此,中藥與益生菌發(fā)酵技術(shù)在動物疫病防治方面已成為一個新的方向。為了探索中藥與益生菌發(fā)酵產(chǎn)物對雞球蟲病的治療效果,本試驗首先通過體外抑制柔嫩艾美耳球蟲(Eimeria tenella)的卵囊孢子化情況對中藥及中藥組方進(jìn)行篩選,并應(yīng)用體外抑制球蟲卵囊孢子化效果顯著的中藥組方與Lactobacillus rhamnosus ATCC53103進(jìn)行共發(fā)酵,制備復(fù)方中藥發(fā)酵制劑,最后研究其對已經(jīng)感染球蟲卵囊雛雞的治療效果,具體研究內(nèi)容及試驗結(jié)果如下:1.中藥篩選及復(fù)方中藥益生菌發(fā)酵條件確定:首先,通過體外抑制球蟲卵囊孢子化試驗,篩選出抑制球蟲卵囊孢子化效果最好的青蒿、苦參、柴胡三種單味中藥,以這三味中藥作為驅(qū)蟲主藥組成三種中藥復(fù)方,再次通過體外抑制球蟲卵囊孢子化試驗對復(fù)方中藥進(jìn)行篩選,最后得到抑制卵囊孢子化效果最好的方劑:青蒿(20%)、常山(20%)、白頭翁(15%)、仙鶴草(15%)、槐花(10%)、黃芩(10%)、甘草(10%)。使用以上方劑與Lactobacillus rhamnosus ATCC53103菌株進(jìn)行共發(fā)酵處理,以活菌數(shù)為指標(biāo)對發(fā)酵體系中的中藥添加量、發(fā)酵時間、菌株接種量、發(fā)酵溫度、發(fā)酵轉(zhuǎn)速各個條件進(jìn)行優(yōu)化。通過上述條件進(jìn)行發(fā)酵,制得復(fù)方中藥發(fā)酵制劑。使用高效液相色譜法分析發(fā)酵前后的變化。結(jié)果表明中藥添加量為100%、菌株接種量為4%、轉(zhuǎn)速為80r/min、37℃條件下培養(yǎng)12h為最佳發(fā)酵條件。高效液相色譜法分析結(jié)果發(fā)現(xiàn)發(fā)酵后與發(fā)酵前相比,其物質(zhì)成分確實發(fā)生變化,出現(xiàn)某些吸收峰不同程度的升高或降低現(xiàn)象,說明ATCC53103菌株在與復(fù)方中藥發(fā)酵的過程中也會使中藥中某些成分發(fā)生改變。最后,通過對雛雞進(jìn)行急性毒性試驗發(fā)現(xiàn),雛雞在每日灌服相當(dāng)于含有48g原藥的復(fù)方中藥發(fā)酵制劑條件下連續(xù)灌服7日,共計灌服了相當(dāng)于含有336g原藥的復(fù)方中藥發(fā)酵制劑,對雛雞的觀察以及剖檢后未見任何異�？梢宰C明該復(fù)方中藥發(fā)酵制劑具有安全性。2.復(fù)方中藥制劑與Lactobacillus rhamnosus ATCC53103協(xié)同發(fā)酵產(chǎn)物對雛雞感染E.tenella治療效果:將30只1日齡雛雞隨機(jī)分成A、B、C、D、E、F 6組,每組5只。除F組(空白對照組)以外,其余A、B、C、D、E 5組內(nèi)所有雛雞于13日齡經(jīng)口接種已孢子化的Eimeria tenella卵囊5×104個/只。自攻蟲之日起,每日檢測各組雛雞糞便中是否有球蟲卵囊排出,于攻雛雞18日齡在各攻蟲組的雛雞糞便中均檢測到球蟲卵囊,并且個別雛雞出現(xiàn)便血現(xiàn)象,可以在確定雛雞成功感染球蟲并出現(xiàn)一定臨床癥狀,對確定感染球蟲雛雞即18日齡雛雞按下述方法進(jìn)行臨床治療,A組雛雞每日每只灌服復(fù)方中藥發(fā)酵制劑200μL,B組雛雞每日每只灌服復(fù)方中藥制劑200μL,C組日每只灌服LGG ATCC53103 200μL(1×106cfu/μL),E使用地克珠利2‰飲水給藥。各組連續(xù)用藥5日,觀察分期雛雞臨床癥狀,并檢測各組雛雞的相對增重率,盲腸病變記分,盲腸病理切片,每克糞便卵囊值,并計算各組藥物抗球蟲指數(shù)ACI。同時,通過流式細(xì)胞術(shù)檢測各組雛雞外周血中CD4+和CD8+T淋巴細(xì)胞含量變化,ELISA方法檢測雛雞Ig G和SIg A含量的變化以及IFN-γ和IL-2這兩種細(xì)胞因子的表達(dá)水平變化,并對雛雞血清中一氧化氮和一氧化氮合成酶的含量進(jìn)行檢測。結(jié)果顯示,服用復(fù)方中藥發(fā)酵制劑后的感染球蟲卵囊的雛雞體重顯著上升,與對照組差異顯著(P0.05)。此外,復(fù)方中藥發(fā)酵制劑可以明顯減輕盲腸病理變化,同時可以降低受感染雛雞卵囊的排出量。復(fù)方中藥發(fā)酵制劑的ACI為130.59,達(dá)到中等抗球蟲水平。通過流式細(xì)胞術(shù)檢測結(jié)果發(fā)現(xiàn),服用復(fù)方中藥發(fā)酵制劑的雛雞外周血中CD8+T淋巴細(xì)胞含量有所升高。并且,服用復(fù)方中藥發(fā)酵制劑可以提高雛雞Ig G和SIg A含量以及IL-2的表達(dá)水平。對血清中一氧化氮和一氧化氮合成酶的檢測發(fā)現(xiàn),復(fù)方中藥發(fā)酵制劑會降低球蟲感染后的一氧化氮和一氧化氮合成酶的含量。通過以上結(jié)果表明,復(fù)方中藥制劑與Lactobacillus rhamnosus ATCC53103協(xié)同發(fā)酵產(chǎn)物對雛雞感染E.tenella具有顯著的治療效果,不但能夠提高雛雞增重,減少盲腸病變,降低卵囊排出,降低一氧化氮和一氧化氮合成酶的含量,還可以通過激特異性Ig G和SIg A抗體的分泌,提高IL-2表達(dá)水平,增強(qiáng)機(jī)體免疫功能。本研究為通過乳酸菌中藥發(fā)酵技術(shù)對雞球蟲病的防治提供理論依據(jù)。
[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.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊森;陳興平;;球蟲病對養(yǎng)雞業(yè)的危害及綜合防治[J];畜牧獸醫(yī)科技信息;2016年12期

2 王利梅;;雞球蟲病防控治療的相關(guān)研究進(jìn)展分析[J];甘肅畜牧獸醫(yī);2016年23期

3 卡馬力汗·葉斯木;;雞球蟲病的流行病學(xué)與防治措施[J];畜牧獸醫(yī)科技信息;2016年02期

4 秦波;林顯華;王麗榮;孫明杰;崔斌;陳雷;;乳酸菌發(fā)酵中藥制劑防制雞球蟲病條件的優(yōu)化及應(yīng)用[J];中國畜牧獸醫(yī);2015年11期

5 申凱;寇俊萍;余伯陽;;中藥有效成分抑制氧化應(yīng)激誘導(dǎo)的神經(jīng)細(xì)胞凋亡機(jī)制研究進(jìn)展[J];中國藥科大學(xué)學(xué)報;2015年05期

6 周芳玉;王春鳳;楊桂連;;柔嫩艾美耳球蟲保護(hù)性抗原應(yīng)用研究進(jìn)展[J];中國獸藥雜志;2015年09期

7 楊軍;趙權(quán);楊桂連;;益生菌在禽類腸道疾病防治中的應(yīng)用[J];黑龍江畜牧獸醫(yī);2015年18期

8 門薇;陳穎;李玉潔;楊慶;翁小剛;鞏仔鵬;張瑞杰;朱曉新;;腸道菌群對中藥有效成分的生物轉(zhuǎn)化研究進(jìn)展[J];中國實驗方劑學(xué)雜志;2015年02期

9 楊輝亮;劉們們;賀超;杜雪;李怡堅;楊耀景;史夢丹;宋晨;朱玲;;青蒿素及其衍生物的抗炎作用及機(jī)制研究[J];四川生理科學(xué)雜志;2014年03期

10 佳娜提·達(dá)吾列提;姜楠;寇俊萍;余伯陽;;調(diào)節(jié)細(xì)胞自噬的中藥有效成分研究進(jìn)展[J];中草藥;2014年16期

相關(guān)會議論文 前2條

1 孔靚;向誠;;中藥有效成分組的研究進(jìn)展[A];第一屆《藥學(xué)學(xué)報》藥學(xué)前沿論壇暨2015年中國藥學(xué)會中藥與天然藥物專業(yè)委員會會議論文摘要集[C];2015年

2 周芳玉;石春衛(wèi);王春鳳;趙權(quán);楊桂連;;表面展示EtMIC2蛋白重組乳酸菌抗球蟲效果研究[A];中國畜牧獸醫(yī)學(xué)會獸醫(yī)寄生蟲學(xué)分會第十三次學(xué)術(shù)研討會論文集[C];2015年

相關(guān)博士學(xué)位論文 前1條

1 劉揚(yáng);中藥有效成分分析的新方法新技術(shù)研究[D];復(fù)旦大學(xué);2012年

相關(guān)碩士學(xué)位論文 前7條

1 趙娟;青蒿素對支氣管哮喘患者外周血Th17/Treg細(xì)胞因子的影響[D];山西醫(yī)科大學(xué);2016年

2 楊軍;益生菌對柔嫩艾美爾球蟲感染雛雞免疫水平的影響及免疫保護(hù)效果評價[D];吉林農(nóng)業(yè)大學(xué);2015年

3 張云霞;pSIP409-TA4和pSIP409-IL-2復(fù)合功能型乳酸菌抗球蟲效果的檢測[D];吉林農(nóng)業(yè)大學(xué);2014年

4 王照華;傳統(tǒng)中藥厚樸主要成分厚樸酚與和厚樸酚的微生物轉(zhuǎn)化[D];中央民族大學(xué);2013年

5 張雙龍;混合乳酸菌抗雞球蟲感染作用效果的檢測[D];吉林農(nóng)業(yè)大學(xué);2012年

6 焦巧芳;中藥渣微生物轉(zhuǎn)化利用菌種篩選研究[D];浙江師范大學(xué);2010年

7 賈青輝;中藥飲水劑防治雞柔嫩艾美耳球蟲病的研究[D];河北農(nóng)業(yè)大學(xué);2006年

，

本文編號：1836232