家兔斯氏艾美耳球虫杨凌株生物学特性和致病性研究
摘要
兔球虫属于艾美耳科的寄生性原虫。据报道兔球虫有17种,寄生在肠道上皮细胞。其中斯氏艾美耳球虫(Eimeria stiedai,ES)寄生在胆管上皮细胞,致病性最强,能引起严重的肝球虫病。长期以来,兔球虫病的防治以药物为主,但随着耐药虫株的不断出现,迫使人们寻求防治的新途径。近年来,杨凌地区养兔业发展比较快,但家兔球虫病时有发生,给该地区养家兔业造成很大的经济损失。本研究采用饱和食盐水漂浮法,对杨凌地区部分养兔场球虫种类和感染情况进行了调查研究,探讨了E.stiedai杨凌株的生物学特性和致病性。取得以下结果:
1杨凌地区家兔艾美耳球虫感染率高达83.5 %,且各月龄段的家兔均有不同程度的感染,其中1~2月龄的幼家兔最易感染,感染率为99.4 %,3~4月龄的感染率为97.0 %,5~7月龄的感染率为78.7 %,而12~24月龄的成年家兔的感染率为38.9 %。本次调查检出了11种家兔艾美耳球虫,其中穿孔艾美耳球虫(E.perforans)、黄艾美耳球虫(E.flsvescens)、无残艾美耳球虫(E.irrestidua)和斯氏艾美耳球虫(E.stiedai)为优势种,分别占总检出率的24.0 %,17.0 %,14.0 %和12.8 %。
2以Es YL株孢子化的卵囊口服接种试验兔,用卵囊分离沉淀法对E.stiedai卵囊进行继代增殖,分离到Es YL纯株。用分离到的Es YL株再接种试验兔,观察了Es YL株的潜伏期,镜检Es YL株的最早孢子化时间,形态特征以及孢子生殖过程等生物学特性。结果,Es的潜伏期为14~16 d,排卵囊高峰期是感染后的第21 d。卵囊呈卵圆形,大小为38.1μm~25.8μm×33.6μm~17.0μm,形状指数1.7μm。孢子囊呈卵圆形,大小为13.0μm×11.0μm,有斯氏体和孢子囊残体。29℃的培养条件下卵囊最早孢子化时间为39 h,孢子生殖过程跟艾美耳属的球虫一致。
3使用Es YL株孢子化的卵囊口服感染24只3月龄的幼兔,试验兔分为4个组(A,B、C、D),其中A组是不感染空白对照组,B、C、D为感染组,各感染组每只幼兔分别口服感染2.0×103、1.0×104和5.0×104个孢子化卵囊。以每克粪球虫卵囊数、死亡率、增重率、肝指数、血清中转氨酶水平、肝脏病变记分和肝组织病变等作为指标研究其致病性。结果,与对空白照组相比,感染的D组出现了明显的病理现象,与其它各组间均差异极显著(P﹤0.01),感染组B和C之间差异不显著(P>0.05)。说明,随着感染剂量的增加Es YL株对家兔的致病性也增强,能导致严重的肝球虫病。以上的试验对家兔肝球虫病的防治工作提供了科学依据。
Rabbit coccidian is a kind of parasitic protozoan of Eimeria. According to research, 17 species of Rabbit coccidian exist, found in the animal’s intestinal epithelial cells. Among rabbit coccidian, Eimeria stiedai is the most virulent, which parasitizes in the bile duct epithelial cells of rabbits, causing serious liver coccidiosis. Historically, chemotherapy has served as the conventional method in treating rabbit coccidiosis. Recently though, scholars have sought new ways to control the disease due to increasing problems such as resistant strains and drug residue. In recent years, while the Yangling rabbit industry has undergone rapid growth, the prevalence of rabbit coccidiosis has caused huge economic losses. This research investigates the infection rate of coccidiosis in rabbits in the Yangling area, using the saturated saline flotation method. We will discuss the biological characteristic and pathogenicity of EsYL strain.
1 The results from the experiments show the infection rate of coccidian to be 83.5%, though the infection rate in different month-age rabbits varies. The infection rate in 1~2 month old rabbits is 99.4%, higher than that of rabbits older than 2 months. The infection rate of 3~4 and 5~7 months age is 97.0% and 78.7 % respectively. The infection rate in 12~24 months age is 38.9 %. In addition, 11 species of Eimeria were detected in infected rabbits. The E.perforans, E.flsvescens, E.irrestidua, and E.stiedai were dominant species with infective rates of 24.0%, 17.0%, 14.0% and 12.8% , respectively .
2 Use of the EsYL Sporulared Oocysts orally infected to rabbits, then isolated the EsYL pure strains by the Oocysts isolate precipitation method and the generation subculture proliferation. With the isolated EsYL pure strains orally infected to rabbits again, we observe its incubation period, the first spore soocysts time, morphological characteristic, and process of morphological changes. The results tell us that Es incubation period was 14~16 d, and the Ovulation peak time was 21 d. The Oocysts is oval, with a size of 38.1μm~25.8μm×33.6μm~17.0μm, and a shape index of 1.7μm. The sporocyst is oval with stiedai body and spore angium debris, and a size of 13.0μm×11.0μm. Under the 29℃culture conditions, the first spore time was 39 h, and has the same process of morphological change as Emieria coccidia.
3 We used the EsYL strain Sporulared Oocysts by orally infected to 24 young rabbits. The test group divided into 4 group(A, B, C, D), with Group A serving as the non-infected control group and groups B, C, D as infected groups. Each infected group was orally infected with 2.0×103, 1.0×104 and 5.0×104 Sporulared Oocysts. When we study the pathogenicity index value including the OPG, mortality, weight gained, liver index, serum aminotransferase, liver lesion score, and liver lesions, the results show that, compared with the control, Group D shows obvious pathological change(P﹤0.01), while Groups C and B show no obvious change(P>0.05). The results tell us that E.stieada(iYL) strain can cause highly serious liver coccidiosis in young rabbits. The study will provide scientific basis for liver coccidiosis of rabbit.
1杨凌地区家兔艾美耳球虫感染率高达83.5 %,且各月龄段的家兔均有不同程度的感染,其中1~2月龄的幼家兔最易感染,感染率为99.4 %,3~4月龄的感染率为97.0 %,5~7月龄的感染率为78.7 %,而12~24月龄的成年家兔的感染率为38.9 %。本次调查检出了11种家兔艾美耳球虫,其中穿孔艾美耳球虫(E.perforans)、黄艾美耳球虫(E.flsvescens)、无残艾美耳球虫(E.irrestidua)和斯氏艾美耳球虫(E.stiedai)为优势种,分别占总检出率的24.0 %,17.0 %,14.0 %和12.8 %。
2以Es YL株孢子化的卵囊口服接种试验兔,用卵囊分离沉淀法对E.stiedai卵囊进行继代增殖,分离到Es YL纯株。用分离到的Es YL株再接种试验兔,观察了Es YL株的潜伏期,镜检Es YL株的最早孢子化时间,形态特征以及孢子生殖过程等生物学特性。结果,Es的潜伏期为14~16 d,排卵囊高峰期是感染后的第21 d。卵囊呈卵圆形,大小为38.1μm~25.8μm×33.6μm~17.0μm,形状指数1.7μm。孢子囊呈卵圆形,大小为13.0μm×11.0μm,有斯氏体和孢子囊残体。29℃的培养条件下卵囊最早孢子化时间为39 h,孢子生殖过程跟艾美耳属的球虫一致。
3使用Es YL株孢子化的卵囊口服感染24只3月龄的幼兔,试验兔分为4个组(A,B、C、D),其中A组是不感染空白对照组,B、C、D为感染组,各感染组每只幼兔分别口服感染2.0×103、1.0×104和5.0×104个孢子化卵囊。以每克粪球虫卵囊数、死亡率、增重率、肝指数、血清中转氨酶水平、肝脏病变记分和肝组织病变等作为指标研究其致病性。结果,与对空白照组相比,感染的D组出现了明显的病理现象,与其它各组间均差异极显著(P﹤0.01),感染组B和C之间差异不显著(P>0.05)。说明,随着感染剂量的增加Es YL株对家兔的致病性也增强,能导致严重的肝球虫病。以上的试验对家兔肝球虫病的防治工作提供了科学依据。
Rabbit coccidian is a kind of parasitic protozoan of Eimeria. According to research, 17 species of Rabbit coccidian exist, found in the animal’s intestinal epithelial cells. Among rabbit coccidian, Eimeria stiedai is the most virulent, which parasitizes in the bile duct epithelial cells of rabbits, causing serious liver coccidiosis. Historically, chemotherapy has served as the conventional method in treating rabbit coccidiosis. Recently though, scholars have sought new ways to control the disease due to increasing problems such as resistant strains and drug residue. In recent years, while the Yangling rabbit industry has undergone rapid growth, the prevalence of rabbit coccidiosis has caused huge economic losses. This research investigates the infection rate of coccidiosis in rabbits in the Yangling area, using the saturated saline flotation method. We will discuss the biological characteristic and pathogenicity of EsYL strain.
1 The results from the experiments show the infection rate of coccidian to be 83.5%, though the infection rate in different month-age rabbits varies. The infection rate in 1~2 month old rabbits is 99.4%, higher than that of rabbits older than 2 months. The infection rate of 3~4 and 5~7 months age is 97.0% and 78.7 % respectively. The infection rate in 12~24 months age is 38.9 %. In addition, 11 species of Eimeria were detected in infected rabbits. The E.perforans, E.flsvescens, E.irrestidua, and E.stiedai were dominant species with infective rates of 24.0%, 17.0%, 14.0% and 12.8% , respectively .
2 Use of the EsYL Sporulared Oocysts orally infected to rabbits, then isolated the EsYL pure strains by the Oocysts isolate precipitation method and the generation subculture proliferation. With the isolated EsYL pure strains orally infected to rabbits again, we observe its incubation period, the first spore soocysts time, morphological characteristic, and process of morphological changes. The results tell us that Es incubation period was 14~16 d, and the Ovulation peak time was 21 d. The Oocysts is oval, with a size of 38.1μm~25.8μm×33.6μm~17.0μm, and a shape index of 1.7μm. The sporocyst is oval with stiedai body and spore angium debris, and a size of 13.0μm×11.0μm. Under the 29℃culture conditions, the first spore time was 39 h, and has the same process of morphological change as Emieria coccidia.
3 We used the EsYL strain Sporulared Oocysts by orally infected to 24 young rabbits. The test group divided into 4 group(A, B, C, D), with Group A serving as the non-infected control group and groups B, C, D as infected groups. Each infected group was orally infected with 2.0×103, 1.0×104 and 5.0×104 Sporulared Oocysts. When we study the pathogenicity index value including the OPG, mortality, weight gained, liver index, serum aminotransferase, liver lesion score, and liver lesions, the results show that, compared with the control, Group D shows obvious pathological change(P﹤0.01), while Groups C and B show no obvious change(P>0.05). The results tell us that E.stieada(iYL) strain can cause highly serious liver coccidiosis in young rabbits. The study will provide scientific basis for liver coccidiosis of rabbit.
引文
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禹旺盛,殷翠琴,马永梅. 2007.呼和浩特市某养兔场球虫感染情况调查与种类鉴定.动物医学进展. 28(增): 5-8.
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尹逊河,梁京芸,李太祥. 2008. NOS底物抑制剂及NO供体在家兔斯氏艾美耳球虫感染时作用的研究.山东农业大学学报. 39(1): 54-56.
尹逊河,邱建华,王树迎. 2008. L-精氨酸和L-氨基胍在家兔斯氏艾美耳球虫感染中作用的研究.畜牧兽医学报. 39(4): 484-486.
于三科,刘蕾,林青. 2008.柔嫩艾美耳球虫YL株的生物学特性.畜牧与兽医. 40(2): 64-65.
禹旺盛,殷翠琴,马永梅. 2007.呼和浩特市某养兔场球虫感染情况调查与种类鉴定.动物医学进展. 28(增): 5-8.
袁方玉. 2001.原虫类寄生虫病分子生物学诊断技术研究进展.河北预防兽医学杂志. 12(5): 14-16.
曾智勇,刘建国,梁海英. 2003.我国肝球虫研究现状.畜牧兽医. 30(11): 28-30.
张龙现,蒋金书,刘群. 2000.毒害艾美耳球虫纯种卵囊收集鉴定及致病性测定.中国畜牧兽医杂志. 37(9): 12-15.
张龙现,宁长申,赵金凤. 1998.兔球虫病研究进展.经济动物学报. 2(3): 52-53.
张龙现,殷佩云,林昆华. 1999.我国兔球虫的研究现状.动物学杂志. 34(5): 45-52.
张龙现. 1996.兔球虫病免疫预防及血清抗体检测技术研究进展.中国养家兔杂志. 28: 31-34.
张全成,王小新. 2006.青紫兰兔暴发肝型球虫病的诊断与防治.畜牧与兽医. 38(1): 42-43.
张正黎,廖党金. 2008.球虫疫苗研究进展.中国兽医寄生虫病. 16(4): 35-38.
章怀云,刘湘新,肖洪波. 2000.吡喹酮脂质体在家兔体内的药代动力学研究.中国兽医杂志. 26(1): 55-56.
赵权,张西臣,张伟信. 2003.不同地理株鸡柔嫩艾美耳球虫致病性的研究.吉林农业大学学报. 25(5): 571-574.
赵月平,吴淑琴,穆秀明. 2008.张家口地区兔球虫感染情况初步调查.河北北方学院学报. 24(2): 48-49.
周文江,周新初. 1998.球佳治疗兔球虫病的效果.上海实验动物科学. 18(2): 107-108.
周显. 2009.优良病理组织切片制作方法.临床医学. 29(8): 108-109.
左仰贤. 1992.球虫学:第一册.天津:天津科学出版社: 23.
Bojang K A , Milligan P J M , Pinder M. 2001 . Efficacyof RTS, S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in the Gambia: a randomised trial.Lancent. 358 (9297) : 1927 - 1934.
Chapman H D, Cherry T E, Danforth H D. 2002. Sustainable coccidiosis control in poultry production: the roleof live vaccines . Int J Parasitol. 32: 617-629.
Gretchen V. 2004. A complex new vaccine show promise . Science. 306 (5696) :587 - 589.
J E P eeters, P Halen. 1980. Robenidine treatment of rabbits naturally infected withcoccidia.Laboratory Animals. 14, 53-54.
J F Diehl. Effect of Hepatic Coccidiosis Infection in Rabbits on Tissue Levels of Vitamins A and E. Nutrition. 77(60): 332-334.
Jithendran K P, Kurade N P. 1996. Rabbit coccidiosis and it’s control. World rabbit science. 4(1): 37-41.
Lillehoj H S, Lillehoj E P, Coccidiosis. 2000. A review of acquired intesitinal immunity and vaccination strategics. Avian Diseases. 44: 408-425.
Lillehoj H S. 1998. Role of T lymphocytes and cytokines incoccidiosis. International Journal for Parasitology. 28: 1071-1081.
M Elaine R O. I961. The Complement-Fixation Test in Hepatic Coccidiosis of Rabbits. Immunology. 4, 346-347.
Min W, Lillehoj H S, Burnside . 2001. Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma TGF-beta 4 and lymphotactin on DNA vaccination against Eimeriaacerv ul ina. Vaccine. 20 (12): 2672274.
Monis P T, Giglio S, Keegan A R. 2005. Emerging technologies for the detection and genetic characterization of protozoan parasites. Trends Parasitol. 21: 340-346.
Pillips E J, Keystone J S, Kain KC. 1996. Failure of combined chloro-quine and high-dose primaquine t herapy for Plasmodium vivaxmalaria acquired in Guyana. Sout h America. Clin Infect Dis . 23 :171 - 173.
Pillips E J, Keystone J S, Kain K C. 1996. Failure of combined chloro-quine and high-dose primaquine t herapy for Plasmodium vivaxmalaria acquired in Guyana. Sout h America . Clin Infect Dis. 23: 171-173.
R M Pittilo, S J Ball. 1980. The ulstrasrutural development of the macrogamete of Eiemeria stiedai. Protoplasama. 104: 34-41.
Rafael Jiménez, Roles. 1996. Emilio Acid-base disturbances in the rabbitduring experimental hepatic parasitosis. Parasitol Res. 82: 524-528.
Vermeulen A N, Schaap D C, Schetters T P M. 2001. Control of coccidiosis in chickens by vaccine . Veterinary Parasitology. 100: 13-20.
Wang J S, Tsai S F. 1992. Prevalence and pathological study on rabbit hepatic coceidiosis in Taiwan. Proc Natl Sci Counc Repub China, Part B. 15(2): 240 -243.