NO对E.tenella卵囊的抑制特性及其机理研究
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。
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 oocysts
To 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 sporocysts
The 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 oocysts
In 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 oocysts
The 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.
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。
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 oocysts
To 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 sporocysts
The 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 oocysts
In 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 oocysts
The 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.
引文
[1] 刘贤勇 , 索勋 . 鸡球虫病及其控制策略 [J]. 中国农业科技导报 , 2006, 8 (5): 31-37.
[2] 孔繁瑶, 宁长申, 殷佩石, 等. 15 株柔嫩艾美耳球虫 Eimeria tenella 对 5 种抗球虫药的耐药性调查 [J]. 北京农业大学学报,1994, 20 (3): 302-307.
[3] 汪明, 孔繁瑶, 殷佩石, 等. 10 株柔嫩艾美耳球虫对四种药物的耐药性检测[J]. 中国农业大学学报,1996,1 (5):110-114.
[4] 曾明华,胡志平. 柔嫩艾美耳球虫野外株对 3 种聚醚类离子载体抗生素的敏感性试验 [J]. 中国兽医学报, 1996, 16 (4): 390-393.
[5] Moncada S R, Palmer M J, Higgs E A. Nitric oxide: Physiology, pathophysiology and pharmacology. [J] Pharmacol Rev. 1991, 43:109~142.
[6] 李金贵,朱蓓蕾,蒋金书. 外源性一氧化氮对雏鸡柔嫩艾美耳球虫卵囊作用的研究 [J]. 中国农业科, 2003, 36 (3): 336-341.
[7] Furchgott RF and Zawadzki JV, The obligatory role of endothelial cells in the relaxation of arterial smooth muscel by acetylcholine [J]. Nature, 1980, 283: 373-376.
[8] Cherry PD, Furchgott RF, Zwadzki JV, et al. The role of endothelial cells in the relaxation of isolated arteries by bradykinin [J]. Proc Natl Acad Sci USA, 1982, 79: 2106-2110.
[9] 王韵, 韩济生. 一氧化氮在医学中的现在和将来[J]. 生理科学进展,1999, 30 (1): 94-95.
[10] Palmer RMJ, Ferrige AG and Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor [J]. Nature, 1987, 327: 524-526.
[11] Moncada S, Higgs A. The L-arginine-nitric oxide pathway [J]. N Eng J Med, 1993, 329: 2002-2012.
[12] Mayer B and Hemmena B. Biosynthesis and action of nitric oxide in mammalian cells [J]. Trends Binchem.Sci, 1997, 22: 477-481.
[13] Carry JS, Lsaba S, Christoph T. Isothioureas: potent inhibitors of nitric oxide synthase with variable isoform selectivity [J]. Br J Pharmacol, 1995,114: 510-516.
[14] Rdomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive nitric oxide synthase in vascular endothelial cells [J]. Proc Natl Acad Sci USA, 1990, 87: 10043-10047.
[15] Li G, Regunathan S, Barrow CJ, et al. Agmatine: an endogenous clonidine-displacing substance in the brain [J]. Science, 1994, 263: 966-969.
[16] Bogdan C, Nathan C, Barrow C J, et al. Modulation of macrophage function by transforming factor β, interleukin-4 and interleukin-10 [J]. Ann Ny Acad Sci, 1993, 685: 713-719.
[17] 刘昭前, 周洪灏, 一氧化氮合酶抑制剂的研究进展[J]. 中国药理学通报, 1999, 15 (1): 11-14.
[18] Green SJ, Crawford RM, Hockmeyer JT, et al. Leishmania major amastigotes initiate the L-arginine dependent killing mechanism in IFN- γ stimulated macrophage by induction of TNF-α[J]. J Immunol, 1990, 145: 4290-4297.
[19] Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: approprite choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions [J]. Journal of cardiovascular pharmacology, 1991, 17(suppl 3): s25-s33.
[20] Ignarro L J, Cirino G, Casini A, et al. Nitric oxide as a signaling molecule in the vascular system: an overview [J]. Journal of cardiovascular pharmacology, 1999, 34: 879-886.
[21] Maragos C M. Complexes of NO with nuclephiles as agents for the controlled biological release of nitric oxide, vasorelaxant effects [J]. J Med Chem, 1991, 34: 3242-3247.
[22] Morely D, Keefer L K. Nitric oxide/nucleophile complexs: a unique class of nitric oxide-based vasodilators [J]. J Cardiovasc Pharmacol, 1993, 22 (suppl 7): S3-S9.
[23] 周建涛, 吴有才, 何香. S-亚硝基硫醇的生理意义及分析方法 [J].国外医学临床生物化学与检验学分册, 1999, 20 (2): 49-51.
[24] Van GE, Agteresch H J, et al. The effect of nitric oxide donors on hamodynamics and blood flow distribution in the porcine carotid circulation [J]. Br J Pharmacol, 1995, 114: 1303-1309.
[25] Bialecki R A, Stinson FC. Kca channel antagonists reduce No donor-mediated relaxation of vascular and tracheal smooth muscle [J]. Am J Physiol, 1995, 268: L152-L159.
[26] Taylor-Robinson A M, Liew FY, Severn A, et al. Regulation of the immune response by nitric oxide differentially produced by T helper typeⅠand T helper typeⅡcells [J]. Eue J Immunol, 1994, 24: 980-984.
[27] Mooradian D L, Hutsell TC, Keefer LK. Nitric oxide donor molecules: effect of NO release on vascular smooth muscle cell proliferation in vitro [J]. J Cardiovasc Pharmacol, 1995, 25: 674-678.
[28] 钟慈生, 孙安阳. 一氧化氮的生物医学 [M].上海: 上海医科大学出版社, 1997.
[29] Nussler A, Drapier J C, Renia L, et al. L-arginine-dependent destuction of intrahepatic malaria parasites in response to tumor necrosis factor and /orinterleukin-6 stimulation [J]. Eue J Immunol, 1991, 21: 227-230.
[30] Melkowa Z, Esteban M. Inhibition of caccina virus DNA replication by inducible expression of niric oxide synthase [J]. J Immunol, 1995, 155 (12): 5711-571.
[31] Radi R, Beckman J S, Bush KM, et al. Peroxynitrite oxidantion of sulfhydryls [J]. J Bi1o Chem, 1991, 266 (7): 4244-4250.
[32] Allen PC. Plasma nitric oxide increases during cecal coccidiosis in chickens [J]. FASEBS J, 1994, 8: A1415.
[33] Allen PC. Production of free radical species during Eimeria maxima infections in chickens [J]. 1997, 76: 814-821.
[34] Allen PC. Nitric oxide production during Eimeria tenella infection in chickens [J]. Poultry Science, 1997, 76: 810-813.
[35] Allen PC and Lillenhojh S. Genetic influence on nitric oxide production during E.tenella infections in chickens [J]. Avian Dis, 1998, 42 (2): 397-403.
[36] Allen PC. Effect of daily oral dose of L-arginine on coccidiosis infections in chickens [J]. Poultry Science, 1997, 78 (11): 1506-1509.
[37] Hussian I and Qureshi MA. Nitric oxide synthase activity and mRNA expression in chicken macrophages [J]. Poultry Science, 1997, 76: 1524-1530.
[38] 申兆菊, 朱蓓蕾, 蒋金书. 雏鸡柔嫩艾关耳球虫感染与体内 NO 的生成 [J]. 畜牧兽医学报,2001,32 (3): 270-276.
[39] 申兆菊, 朱蓓蕾, 蒋金书. NO 在雏鸡球虫感染过程中的作用 [J]. 畜牧兽医学报, 2002, 33 (4): 395-399.
[40] 李金贵,朱蓓蕾,蒋金书. 内源性 NO 在雏鸡球虫感染中的作用 [J]. 中国兽医学, 2002, 22 (6): 601-603.
[41] 傅小平,李银,沈永林等. 一氧化氮( NO)在鸡感染柔嫩艾美球虫中的作用 [J]. 南京农业大学学报 2001, 24 (3): 77-80.
[42] 包永占, 史万玉, 秦建华,等. 不同剂量柔嫩艾美耳球虫感染对雏鸡脾重与血清NO 含量的影响 [J]. 中国兽医杂志. 2005, 41(10): 20-21.
[43] Moncada S R, Palmer M J and Higgs E A. Nitric oxide: physiology, pathophysiology and pharmacology [J]. Pharmacol Rev, 1991, 43: 109-142.
[44] Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: approprite choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions [J]. Journal of cardiovascular pharmacology, 1991, 17(suppl 3): s25-s33.
[45] Stefan F, Birgit S, Heiko K. Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells [J]. Biochem J, 1999, 338: 367-374.
[46] Balcioglu A, Watkins CJ, Maher TJ. Use of a hemoglobin-trapping approach in thedetermination of nitric oxide in vitro and in vivo systems [J]. Neurochem Res, 1998, 23 (5):815～820.
[47] Leitch G J, Qing H E. Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum [J]. Infection and Immunity, 1994, 62 (11): 5173~5176.
[48] Glaucia N R V, Cunha J S. Nitric oxide is involved in control of Trypansoma cruzi-induced parasitemia and directly kills the parasite in vivo [J]. Infection and Immunity, 1994, 62 (11): 5177~5182.
[49] Rocket K A, Awburn M M, Cowden W B, et al. Killing of Plasmodium falciparum in vivo by nitric oxide derivatives [J], Infection and Immunity, 1991, 59 (9): 3280~3283.
[50] 索勋,李国清. 鸡球虫病学[M]. 北京:中国农业大学出版社, 1998.
[51] 蒋建林, 蒋金书. 柔嫩艾美耳球虫各阶段虫体纯化方法的改进. 中国农业大学学报. 1996, 1 (5): 99-102.
[52] Jonson J, Reid WM. Anticoccidial drugs: lesion scoring techniques in battery and floor-open experiments with chickens [J]. Exp Parasitol, 1970, 28: 30-36.
[53] 黄兵,赵其平,吴薛忠,等. 冷冻保存卵囊及繁殖一代的活性测定 [J]. 上海畜牧兽医通讯. 1995, 3: 14-14.
[54] Sofia G. Konstantinova, Elevter M. Russanow. Aconitase Acivity in Rat Liver [J]. Comp. Biochem. Physiol, 1996, 113B (1): 125-130.
[55] 刘毓秀, 程桂芳译. 蛋白质的凝胶电泳 [M]. 北京: 科学出版社, 1986.
[56] Shirley M W. Enzyme variation in Eimeria species of the chicken [J]. Parasitology, 1975, 71: 369-376.
[57] Avi Sadka, Esther Dahan, Lidya Cohen, et al. Aconitase activity and expression during the development of lemon fruit [J]. Physiologia Plantarum, 2000, 108: 255-262.
[58] A T Eprintsev, E V Semenova, V N Popov. Induction of Aconitase Hydratase in Hepatocytes of Starving Rats [J]. Biochemistry (Moscow), 2002, 67 (7): 795-801.
[59] Mei-ling Cheng, Hung-yao Ho, Chi-ming Liang,et al. Cellular glucose-6-phosphate dehydrogenase (G6PD) status modulatesthe effects of nitric oxide (NO) on human foreskin fibroblasts [J]. FEBS Letters 2000, 475: 257-262.
[60] Ezdihar A. Hassoun, Sidney J. Stohs. Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures [J]. Toxicology, 1996, 112: 219-226.
[61] Eanes RZ, Kun E. Separation and characterization of aconitase hydrate isoenymes from pig tissues [J]. Biochim Biophys Acta, 1971, 227: 204-210.
[62] Bellis CD, Tsugeki R, Alpi A, et al. Purification and characterization of aconitaseisoforms from etiolated pumpkin cotyledons [J]. Physiol Plant, 1993, 88: 485-492.
[63] Drapier J C, Hirling H, Wietzerbin J, et al. Biosynthesis of nitric oxide activated iron regulatory factor in macrophages [J]. EMBO J, 1993, 12: 3643-3649.
[64] Stadler J, Billiar TR, Curran RD, et al. Effector of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes [J]. Am J Physiol, 1991, 260: C910-916.
[65] Lyons. R. E. and A. M. Johnson. Gene sequence and transcription differences in 70kDa heat shock protein correlate with murine virulence of Toxoplasma gondi [J]. International Journal for Parasitology, 1998, 28: 1041-1045.
[66] Emillo del Cacho, Mergarita Gallego, Desire Pereboom, et al. Eimeria tenella: hsp70 expression during sporogony [J]. J Parasitol, 2001, 87 (5): 946-950.
[67] E del Cacho, M.Gallego, F. Lopez-bernad, et al. Differences in hsp70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella [J]. J Parasitol, 2005, 91(5): 1127-1131.
[68] Michalski W P, Prowse S J. Superoxide dismutases in Emeria tenella [J]. Mol Biochem Parasitol, 1991, 47(2): 189-195.
[69] 陈胜, 邬堂春, 海涛,等. 用酶联免疫吸附法测定血浆 hsp70 抗体滴度 [J]. 中国劳动卫生职业病杂志, 1994,17 (2): 100-101.
[70] Beauchamp C O, I Fridovich. Superoxide dismutase: improved assays and an assay applicable to acrylamide Gels [J]. Analytical Biochemistry, 1971, 44: 276-287.
[71] 王爱国, 罗广华, 邵丛林. 大豆种子超氧化物歧化酶的研究 [J]. 植物生理学报, 1983, 9: 77-84.
[72] Adhuna, P Salotra, R Bhatnagar. Nitric oxide induced expression of stress proteins in virulent and avurient promastigotes of Leishmania donovani [J]. Immunology Letters, 2000, 71: 171-176.
[73] 安立敏, 胡健, 夏经纲, 等. 一氧化氮释放剂对心肌热体克蛋白 70 诱导的实验研究[J]. 中国循环杂志, 2005, 20 (4): 305-308.
[74] 彼得 L 朗 主编. 球虫生物学 [M]. 广西: 广西科学技术出版社, 1990.
[75] Bina Joe, Belur R. lokesh. Studies on the inaction of superoxide dismutase acitivity by nitric oxide from rat peritoneal macrophages [J]. Molecular and Cellular Biochemistry, 1997, 168: 87-93.
[76] Luca Salvati, Marco Mattu, Marco Colasanti, et al. NO donors inhibit Leishmania infantum cysteine proteinase activity [J]. Biochimica et Biophysica Acta, 2001, 1545: 357-366.
[2] 孔繁瑶, 宁长申, 殷佩石, 等. 15 株柔嫩艾美耳球虫 Eimeria tenella 对 5 种抗球虫药的耐药性调查 [J]. 北京农业大学学报,1994, 20 (3): 302-307.
[3] 汪明, 孔繁瑶, 殷佩石, 等. 10 株柔嫩艾美耳球虫对四种药物的耐药性检测[J]. 中国农业大学学报,1996,1 (5):110-114.
[4] 曾明华,胡志平. 柔嫩艾美耳球虫野外株对 3 种聚醚类离子载体抗生素的敏感性试验 [J]. 中国兽医学报, 1996, 16 (4): 390-393.
[5] Moncada S R, Palmer M J, Higgs E A. Nitric oxide: Physiology, pathophysiology and pharmacology. [J] Pharmacol Rev. 1991, 43:109~142.
[6] 李金贵,朱蓓蕾,蒋金书. 外源性一氧化氮对雏鸡柔嫩艾美耳球虫卵囊作用的研究 [J]. 中国农业科, 2003, 36 (3): 336-341.
[7] Furchgott RF and Zawadzki JV, The obligatory role of endothelial cells in the relaxation of arterial smooth muscel by acetylcholine [J]. Nature, 1980, 283: 373-376.
[8] Cherry PD, Furchgott RF, Zwadzki JV, et al. The role of endothelial cells in the relaxation of isolated arteries by bradykinin [J]. Proc Natl Acad Sci USA, 1982, 79: 2106-2110.
[9] 王韵, 韩济生. 一氧化氮在医学中的现在和将来[J]. 生理科学进展,1999, 30 (1): 94-95.
[10] Palmer RMJ, Ferrige AG and Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor [J]. Nature, 1987, 327: 524-526.
[11] Moncada S, Higgs A. The L-arginine-nitric oxide pathway [J]. N Eng J Med, 1993, 329: 2002-2012.
[12] Mayer B and Hemmena B. Biosynthesis and action of nitric oxide in mammalian cells [J]. Trends Binchem.Sci, 1997, 22: 477-481.
[13] Carry JS, Lsaba S, Christoph T. Isothioureas: potent inhibitors of nitric oxide synthase with variable isoform selectivity [J]. Br J Pharmacol, 1995,114: 510-516.
[14] Rdomski MW, Palmer RMJ, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive nitric oxide synthase in vascular endothelial cells [J]. Proc Natl Acad Sci USA, 1990, 87: 10043-10047.
[15] Li G, Regunathan S, Barrow CJ, et al. Agmatine: an endogenous clonidine-displacing substance in the brain [J]. Science, 1994, 263: 966-969.
[16] Bogdan C, Nathan C, Barrow C J, et al. Modulation of macrophage function by transforming factor β, interleukin-4 and interleukin-10 [J]. Ann Ny Acad Sci, 1993, 685: 713-719.
[17] 刘昭前, 周洪灏, 一氧化氮合酶抑制剂的研究进展[J]. 中国药理学通报, 1999, 15 (1): 11-14.
[18] Green SJ, Crawford RM, Hockmeyer JT, et al. Leishmania major amastigotes initiate the L-arginine dependent killing mechanism in IFN- γ stimulated macrophage by induction of TNF-α[J]. J Immunol, 1990, 145: 4290-4297.
[19] Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: approprite choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions [J]. Journal of cardiovascular pharmacology, 1991, 17(suppl 3): s25-s33.
[20] Ignarro L J, Cirino G, Casini A, et al. Nitric oxide as a signaling molecule in the vascular system: an overview [J]. Journal of cardiovascular pharmacology, 1999, 34: 879-886.
[21] Maragos C M. Complexes of NO with nuclephiles as agents for the controlled biological release of nitric oxide, vasorelaxant effects [J]. J Med Chem, 1991, 34: 3242-3247.
[22] Morely D, Keefer L K. Nitric oxide/nucleophile complexs: a unique class of nitric oxide-based vasodilators [J]. J Cardiovasc Pharmacol, 1993, 22 (suppl 7): S3-S9.
[23] 周建涛, 吴有才, 何香. S-亚硝基硫醇的生理意义及分析方法 [J].国外医学临床生物化学与检验学分册, 1999, 20 (2): 49-51.
[24] Van GE, Agteresch H J, et al. The effect of nitric oxide donors on hamodynamics and blood flow distribution in the porcine carotid circulation [J]. Br J Pharmacol, 1995, 114: 1303-1309.
[25] Bialecki R A, Stinson FC. Kca channel antagonists reduce No donor-mediated relaxation of vascular and tracheal smooth muscle [J]. Am J Physiol, 1995, 268: L152-L159.
[26] Taylor-Robinson A M, Liew FY, Severn A, et al. Regulation of the immune response by nitric oxide differentially produced by T helper typeⅠand T helper typeⅡcells [J]. Eue J Immunol, 1994, 24: 980-984.
[27] Mooradian D L, Hutsell TC, Keefer LK. Nitric oxide donor molecules: effect of NO release on vascular smooth muscle cell proliferation in vitro [J]. J Cardiovasc Pharmacol, 1995, 25: 674-678.
[28] 钟慈生, 孙安阳. 一氧化氮的生物医学 [M].上海: 上海医科大学出版社, 1997.
[29] Nussler A, Drapier J C, Renia L, et al. L-arginine-dependent destuction of intrahepatic malaria parasites in response to tumor necrosis factor and /orinterleukin-6 stimulation [J]. Eue J Immunol, 1991, 21: 227-230.
[30] Melkowa Z, Esteban M. Inhibition of caccina virus DNA replication by inducible expression of niric oxide synthase [J]. J Immunol, 1995, 155 (12): 5711-571.
[31] Radi R, Beckman J S, Bush KM, et al. Peroxynitrite oxidantion of sulfhydryls [J]. J Bi1o Chem, 1991, 266 (7): 4244-4250.
[32] Allen PC. Plasma nitric oxide increases during cecal coccidiosis in chickens [J]. FASEBS J, 1994, 8: A1415.
[33] Allen PC. Production of free radical species during Eimeria maxima infections in chickens [J]. 1997, 76: 814-821.
[34] Allen PC. Nitric oxide production during Eimeria tenella infection in chickens [J]. Poultry Science, 1997, 76: 810-813.
[35] Allen PC and Lillenhojh S. Genetic influence on nitric oxide production during E.tenella infections in chickens [J]. Avian Dis, 1998, 42 (2): 397-403.
[36] Allen PC. Effect of daily oral dose of L-arginine on coccidiosis infections in chickens [J]. Poultry Science, 1997, 78 (11): 1506-1509.
[37] Hussian I and Qureshi MA. Nitric oxide synthase activity and mRNA expression in chicken macrophages [J]. Poultry Science, 1997, 76: 1524-1530.
[38] 申兆菊, 朱蓓蕾, 蒋金书. 雏鸡柔嫩艾关耳球虫感染与体内 NO 的生成 [J]. 畜牧兽医学报,2001,32 (3): 270-276.
[39] 申兆菊, 朱蓓蕾, 蒋金书. NO 在雏鸡球虫感染过程中的作用 [J]. 畜牧兽医学报, 2002, 33 (4): 395-399.
[40] 李金贵,朱蓓蕾,蒋金书. 内源性 NO 在雏鸡球虫感染中的作用 [J]. 中国兽医学, 2002, 22 (6): 601-603.
[41] 傅小平,李银,沈永林等. 一氧化氮( NO)在鸡感染柔嫩艾美球虫中的作用 [J]. 南京农业大学学报 2001, 24 (3): 77-80.
[42] 包永占, 史万玉, 秦建华,等. 不同剂量柔嫩艾美耳球虫感染对雏鸡脾重与血清NO 含量的影响 [J]. 中国兽医杂志. 2005, 41(10): 20-21.
[43] Moncada S R, Palmer M J and Higgs E A. Nitric oxide: physiology, pathophysiology and pharmacology [J]. Pharmacol Rev, 1991, 43: 109-142.
[44] Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: approprite choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions [J]. Journal of cardiovascular pharmacology, 1991, 17(suppl 3): s25-s33.
[45] Stefan F, Birgit S, Heiko K. Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells [J]. Biochem J, 1999, 338: 367-374.
[46] Balcioglu A, Watkins CJ, Maher TJ. Use of a hemoglobin-trapping approach in thedetermination of nitric oxide in vitro and in vivo systems [J]. Neurochem Res, 1998, 23 (5):815～820.
[47] Leitch G J, Qing H E. Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum [J]. Infection and Immunity, 1994, 62 (11): 5173~5176.
[48] Glaucia N R V, Cunha J S. Nitric oxide is involved in control of Trypansoma cruzi-induced parasitemia and directly kills the parasite in vivo [J]. Infection and Immunity, 1994, 62 (11): 5177~5182.
[49] Rocket K A, Awburn M M, Cowden W B, et al. Killing of Plasmodium falciparum in vivo by nitric oxide derivatives [J], Infection and Immunity, 1991, 59 (9): 3280~3283.
[50] 索勋,李国清. 鸡球虫病学[M]. 北京:中国农业大学出版社, 1998.
[51] 蒋建林, 蒋金书. 柔嫩艾美耳球虫各阶段虫体纯化方法的改进. 中国农业大学学报. 1996, 1 (5): 99-102.
[52] Jonson J, Reid WM. Anticoccidial drugs: lesion scoring techniques in battery and floor-open experiments with chickens [J]. Exp Parasitol, 1970, 28: 30-36.
[53] 黄兵,赵其平,吴薛忠,等. 冷冻保存卵囊及繁殖一代的活性测定 [J]. 上海畜牧兽医通讯. 1995, 3: 14-14.
[54] Sofia G. Konstantinova, Elevter M. Russanow. Aconitase Acivity in Rat Liver [J]. Comp. Biochem. Physiol, 1996, 113B (1): 125-130.
[55] 刘毓秀, 程桂芳译. 蛋白质的凝胶电泳 [M]. 北京: 科学出版社, 1986.
[56] Shirley M W. Enzyme variation in Eimeria species of the chicken [J]. Parasitology, 1975, 71: 369-376.
[57] Avi Sadka, Esther Dahan, Lidya Cohen, et al. Aconitase activity and expression during the development of lemon fruit [J]. Physiologia Plantarum, 2000, 108: 255-262.
[58] A T Eprintsev, E V Semenova, V N Popov. Induction of Aconitase Hydratase in Hepatocytes of Starving Rats [J]. Biochemistry (Moscow), 2002, 67 (7): 795-801.
[59] Mei-ling Cheng, Hung-yao Ho, Chi-ming Liang,et al. Cellular glucose-6-phosphate dehydrogenase (G6PD) status modulatesthe effects of nitric oxide (NO) on human foreskin fibroblasts [J]. FEBS Letters 2000, 475: 257-262.
[60] Ezdihar A. Hassoun, Sidney J. Stohs. Cadmium-induced production of superoxide anion and nitric oxide, DNA single strand breaks and lactate dehydrogenase leakage in J774A.1 cell cultures [J]. Toxicology, 1996, 112: 219-226.
[61] Eanes RZ, Kun E. Separation and characterization of aconitase hydrate isoenymes from pig tissues [J]. Biochim Biophys Acta, 1971, 227: 204-210.
[62] Bellis CD, Tsugeki R, Alpi A, et al. Purification and characterization of aconitaseisoforms from etiolated pumpkin cotyledons [J]. Physiol Plant, 1993, 88: 485-492.
[63] Drapier J C, Hirling H, Wietzerbin J, et al. Biosynthesis of nitric oxide activated iron regulatory factor in macrophages [J]. EMBO J, 1993, 12: 3643-3649.
[64] Stadler J, Billiar TR, Curran RD, et al. Effector of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes [J]. Am J Physiol, 1991, 260: C910-916.
[65] Lyons. R. E. and A. M. Johnson. Gene sequence and transcription differences in 70kDa heat shock protein correlate with murine virulence of Toxoplasma gondi [J]. International Journal for Parasitology, 1998, 28: 1041-1045.
[66] Emillo del Cacho, Mergarita Gallego, Desire Pereboom, et al. Eimeria tenella: hsp70 expression during sporogony [J]. J Parasitol, 2001, 87 (5): 946-950.
[67] E del Cacho, M.Gallego, F. Lopez-bernad, et al. Differences in hsp70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella [J]. J Parasitol, 2005, 91(5): 1127-1131.
[68] Michalski W P, Prowse S J. Superoxide dismutases in Emeria tenella [J]. Mol Biochem Parasitol, 1991, 47(2): 189-195.
[69] 陈胜, 邬堂春, 海涛,等. 用酶联免疫吸附法测定血浆 hsp70 抗体滴度 [J]. 中国劳动卫生职业病杂志, 1994,17 (2): 100-101.
[70] Beauchamp C O, I Fridovich. Superoxide dismutase: improved assays and an assay applicable to acrylamide Gels [J]. Analytical Biochemistry, 1971, 44: 276-287.
[71] 王爱国, 罗广华, 邵丛林. 大豆种子超氧化物歧化酶的研究 [J]. 植物生理学报, 1983, 9: 77-84.
[72] Adhuna, P Salotra, R Bhatnagar. Nitric oxide induced expression of stress proteins in virulent and avurient promastigotes of Leishmania donovani [J]. Immunology Letters, 2000, 71: 171-176.
[73] 安立敏, 胡健, 夏经纲, 等. 一氧化氮释放剂对心肌热体克蛋白 70 诱导的实验研究[J]. 中国循环杂志, 2005, 20 (4): 305-308.
[74] 彼得 L 朗 主编. 球虫生物学 [M]. 广西: 广西科学技术出版社, 1990.
[75] Bina Joe, Belur R. lokesh. Studies on the inaction of superoxide dismutase acitivity by nitric oxide from rat peritoneal macrophages [J]. Molecular and Cellular Biochemistry, 1997, 168: 87-93.
[76] Luca Salvati, Marco Mattu, Marco Colasanti, et al. NO donors inhibit Leishmania infantum cysteine proteinase activity [J]. Biochimica et Biophysica Acta, 2001, 1545: 357-366.