【作者】 江燕；

【导师】 刘宗平； 李金贵；

【作者基本信息】 扬州大学 ， 临床兽医学， 2007， 硕士

【摘要】 球虫病历来是危害养禽业的主要疾病之一,给世界养禽业造成了巨大的经济损失。由于鸡球虫耐药虫株的频繁出现以及疫苗免疫效果的不稳定性使得该病仍较难控制,因此开发完全新型的抗球虫药有巨大的实用价值。有研究发现NO供体GSNO能够抑制E.tenella的孢子生殖。为了进一步研究明确NO对球虫孢子生殖的抑制特性并探讨NO抑制球虫孢子生殖的作用机理,本论文进行以下内容的研究。一、NO对球虫卵囊孢子生殖的抑制作用用酸化亚硝酸钠、盐酸、硫酸溶液处理新收取的未孢子化E.tenella卵囊,检查卵囊的孢子生殖情况。结果表明,20mmol/L酸化亚硝酸钠溶液对卵囊孢子生殖的抑制率可达100%;而不经酸化的亚硝酸钠和相同pH的盐酸和硫酸溶液对卵囊的孢子生殖没有明显影响。以GSNO、SNAP及Hb与新鲜球虫卵囊共孵育,结果表明,8mmol/LGSNO和SNAP能抑制球虫的孢子生殖,而NO清除剂Hb能消除GSNO和SNAP对球虫孢子生殖的抑制作用,并呈现剂量—效应关系。以上结果提示,NO能够抑制卵囊的孢子生殖。二、GSNO对孢子化卵囊及孢子囊的作用本实验研究了NO对孢子化卵囊和孢子囊的活力和致病力的影响。以卵囊活力检测实验检测GSNO处理孢子化卵囊及孢子囊的子孢子脱囊率。结果显示,对照、GSNO处理的孢子化卵囊及孢子囊的子孢子脱囊率均分别为95.51%、57.21%、47.67%。用GSNO处理的孢子化卵囊和孢子囊接种雏鸡,根据临床球虫病发生情况和盲肠病变情况来评价NO对卵囊致病力的影响。结果显示,GSNO处理的孢子化卵囊在增重、病变记分、OPG值和盲肠荷虫数等方面差异不明显(P﹥0.05);而GSNO处理的孢子囊在病变记分、OPG值和盲肠荷虫数显著低于对照(P﹤0.05)。以上结果表明,NO能降低孢子化卵囊及孢子囊的子孢子脱囊率;NO对孢子化卵囊的致病力没有明显抑制作用,但能显著降低孢子囊的致病力。三、GSNO对卵囊内与糖代谢有关酶活性的影响采用PAGE法检测GSNO对卵囊与糖代谢有关内LDH、G6PD和ACO活性的影响。酶特异染色结果显示:GSNO处理的卵囊中LDH活性无明显变化,G6PD活性有所降低;无论GSNO处理与否,卵囊的线粒体和细胞质中均有ACO活性,但线粒体内ACO活性较高。以上结果表明,GSNO对卵囊内对LDH和ACO的活性无明显抑制作用或此抑制作用是可逆的,但能降低G6PD的活性。四、GSNO对卵囊自身防御系统的影响本实验研究了NO对球虫自身防御系统的攻击作用以及对自身抗氧化能力有关的酶破坏的作用。采用ELISA和流式细胞仪检测了GSNO处理对E.tenella卵囊内HSP70表达的影响,结果表明,GSNO处理对HSP70的表达没有明显影响;卵囊孢子化6h后HSP70表达量比未孢子化卵囊明显增加。用PAGE法分析GSNO对卵囊内SOD活性的影响,酶特异染色的结果表明,与对照组一样,GSNO处理的卵囊中亦有SOD活性,此SOD初步鉴定为Cu·Zn-SOD。以上结果表明,GSNO对球虫的HSP70的表达无显著影响;GSNO对球虫内SOD的活性无明显抑制作用或具可逆抑制作用。更多还原

【Abstract】 Coccidiosis is the major parasitic disease in chickens and causes huge economic losses in poultry industry. Due to the rapid emergence of drug-resistance parasites and the instability of vaccine, the disease is difficult to control. Therefor exploiting new pattern anticoccidial drug will be great practical value. The research finded GSNO could inhibiting the sporulation of E. tenella oocysts.To further confirm the inhibitory characteristics of NO on the sporulation of E.tenella oocysts and discuss the mechanism, this paper will do the following research.1 The inhibiting effect of NO on oocystsTo study the effect of exogenous nitric oxide on E. tenella oocysts, Acidic nitrite, hydrochloric acid and sulfuric acid solution were used to treat freshly isolated oocysts in this experiment, and then the oocysts sporulation were inspected. The results showed acidic sodium nitrite (pH=1) had significant inhibiting roles on the sporulation in E.tenella oocysts, with a high inhibiting rate of 100% in the group treated by 20mmol/L acidic sodium nitrite, but the same concentration sodium nitrite solution without acid and the hydrochloric acid and sulfuric acid solution with equal the pH to acidic nitrite solution had not visible effects on the sporulation. GSNO and SNAP and Hb were used to treat freshly isolated oocysts, and then the oocysts sporulation were detected. The results showed 8mmol/L GSNO and SNAP could inhibit the sporulation of oocysts, however, NO-scavenger, Hb could significantly eliminate the inhibiting effect of GSNO and SNAP on oocysts with a dose-effect relationship. The above-mentioned results indicated No could inhibit the sporulation of E. tenella oocysts.2 The effect of GSNO on sporulated oocysts and sporocystsThe object was to evaluate the effect of NO on the livingness and pathogenicity of sporulated oocysts and sporocysts. The oocysts livingness examination of sporulated oocysts and sporocysts treated by 20mmol/L GSNO were detected by the excystation rate of sporozoites. The results showed the excystation rate of sporozoites of control group, sporulated oocysts and sporocysts treated by GSNO were 95.51%、57.21%、47.67% respectively. To evaluate the effect of NO on the pathogenicity of oocysts according to the clinical symptoms of coccidiosis and the lesion in ceca, the chickens were inoculated the sporulated oocysts or sporocysts. The results showed sporulated oocystes treated by GSNO on chicken’s weight gain, lesion scores, OPG and the parasites-burden in ceca were not higher than the sporulated oocystes group (P>0.05); compare with the sporocysts group, sporocysts treated by GSNO on lesion scores, OPG and the parasites-burden in ceca decreased statistically ( P﹤0.05 ) . The above-mentioned results indicated NO could decrease the excystation rate of sporulated oocysts and sporocysts; NO could not obviously inhibit the pathogenicity of sporulated oocystes, nevertheless, the pathogenicity of sporocysts treated by NO decreased significantly.3.The effect of GSNO on the activities of enzymes related to the metabolism of glucide of oocystsIn this study, the effects of GSNO on the activities of LDH, G6PD and ACO related to the metabolism of glucide in oocysts were detected by the PAGE. The substrate special staining results showed that the activities of LDH were similar to those of the control, but the activities of G6PD were less than those of the control, there were the activities of ACO in cytoplasm and mitochondrion of oocystes, whether treated by GSNO or not, the activities of ACO in mitochondrion were higher than those of cytoplasm. The above-mentioned results indicated GSNO had no obviously inhibiting effect on LDH and ACO, or the inhibiting effect was reversible; however GSNO could decrease the activities of G6PD.4 The effect of GSNO on the the recovery system of oocystsThe object was to evaluate the damage effect of NO on the recovery system and the enzyme related to the anti-oxidation ability of E. tenella oocysts. In order to investigate the expression of HSP70 in oocysts treated by GSNO, the ELISA and and the Flow cytometric analysis were used. The results indicated GSNO had no obviously effect on the expression of HSP70, the expression of HSP70 in oocysts sporulating for 6h was more than in the freshly collected oocysts. The PAGE method were used to analyze the effects of GSNO on the activities of SOD, The substrate special staining results showed the activities of SOD in oocystes treated by GSNO were detected respectively, and was initially distinguished as Cu·Zn-SOD. The above-mentioned results indicated GSNO had no obviously effect on the expression of HSP70; so didn’t the activities of SOD, or the inhibiting effect was reversible.更多还原