张黎;郑龙龙;白冰;谭凡;王辰;吕晓玲;白瑞;郑明学;
ZHANG Li;ZHENG Long-long;BAI Bing;TAN Fan;WANG Chen;LYU Xiao-ling;BAI Rui;ZHENG Ming-xue;College of Veterinary Medicine, Shanxi Agricultural University;

• 1:山西农业大学动物医学学院

摘要(Abstract)：

为了鉴定造成太谷区某鸡场57日龄蛋鸡死亡的鸡球虫种类，明确其生物学特性，本试验采用饱和食盐水漂浮法和单卵囊分离技术从病死鸡粪便中分离纯化得到1株球虫；将分离球虫口服接种SPF鸡，观察并测定卵囊最短孢子化时间、裂殖体寄生部位和大小、卵囊寄生部位和大小、潜隐期、死亡率、增重情况、肠道病变记分等指标，了解该虫株的生物学特点并初步鉴定其类型，随后采用序列特征性扩增区域(SCAR)标记技术进一步确定虫株类型。结果显示，分离虫株的裂殖体寄生于空肠，平均大小为(63.17±7.66)μm×(47.12±6.91)μm,卵囊仅盲肠可见，平均大小为(19.92±0.20)μm×(19.27±0.11)μm,虫株潜隐期为152 h,最短孢子化时间为18 h,接种5.0×10⁴个卵囊/羽和1.1×104个卵囊/羽和1.1×10⁵个卵囊/羽的分离虫株可分别造成40%和50%的实验鸡死亡，相对增重率下降至31.61%和20.87%,病变记分分别达到3.9和4.0;SCAR标记扩增显示，分离虫株与毒害艾美耳球虫(E.necatrix)的同源性达97.1%,命名为E.necatrix-CN-SX-2018。结果表明，本试验分离得到1株致病性较强的E.necatrix,不仅丰富了山西省E.necatrix流行毒株的生物信息，也为鸡球虫疫苗评价提供了良好的种子资源。
This study aimed to identify the species of chicken coccidia that caused the death of 57-day-old layers in a chicken farm in Taigu District, and to determine its biological characteristics. A strain of coccidia was isolated and purified from the feces of dead chicken by saturated saline water floating method and single oocyst separation technique. After oral inoculation of the coccidia to SPF chickens, the shortest sporulation time, the parasitic position and size of the schizont, the parasitic position and size of the oocyst, the latent period, mortality, weight gain and the score of intestinal lesions were measured to assess the biological characteristics of the coccidia strain, and to preliminarily identify the type of the coccidia. Sequence characterized amplified region(SCAR) marker technology was used to determine its strain type. The results showed the schizonts parasitized the jejunum, with an average size of(63.17±7.66) μm×(47.12±6.91) μm, the oocysts were only visible in the cecum, with an average size of(19.92±0.20) μm×(19.27±0.11) μm, the latent period of the coccidia was 152 h, and the shortest sporulation time was 18 h. Inoculation of experimental chickens with 5.0×105个卵囊/羽的分离虫株可分别造成40%和50%的实验鸡死亡，相对增重率下降至31.61%和20.87%,病变记分分别达到3.9和4.0;SCAR标记扩增显示，分离虫株与毒害艾美耳球虫(E.necatrix)的同源性达97.1%,命名为E.necatrix-CN-SX-2018。结果表明，本试验分离得到1株致病性较强的E.necatrix,不仅丰富了山西省E.necatrix流行毒株的生物信息，也为鸡球虫疫苗评价提供了良好的种子资源。
This study aimed to identify the species of chicken coccidia that caused the death of 57-day-old layers in a chicken farm in Taigu District, and to determine its biological characteristics. A strain of coccidia was isolated and purified from the feces of dead chicken by saturated saline water floating method and single oocyst separation technique. After oral inoculation of the coccidia to SPF chickens, the shortest sporulation time, the parasitic position and size of the schizont, the parasitic position and size of the oocyst, the latent period, mortality, weight gain and the score of intestinal lesions were measured to assess the biological characteristics of the coccidia strain, and to preliminarily identify the type of the coccidia. Sequence characterized amplified region(SCAR) marker technology was used to determine its strain type. The results showed the schizonts parasitized the jejunum, with an average size of(63.17±7.66) μm×(47.12±6.91) μm, the oocysts were only visible in the cecum, with an average size of(19.92±0.20) μm×(19.27±0.11) μm, the latent period of the coccidia was 152 h, and the shortest sporulation time was 18 h. Inoculation of experimental chickens with 5.0×10⁴ oocysts/feather and 1.1×104 oocysts/feather and 1.1×10⁵ oocysts/feather of the coccidia strain caused 40% and 50% mortality, the relative weight gain rate dropped to 31.61% and 20.87% compared to control chickens, the lesion score reached 3.9 and 4.0,respectively. SCAR marker amplification showed that the homology between the isolated coccidia and Eimeria necatrix(E. necatrix) was 97.1%,and the isolated coccidia strain was thus named E. necatrix-CN-SX-2018. The isolation of a highly pathogenic strain of E. necatrix not only enriches the collection of the epidemic strains of chicken coccidiosis in Shanxi Province, but also provides a valuable strain for the evaluation of chicken coccidiosis vaccines.

关键词(KeyWords)： 鸡球虫病;生物学特性;SCAR标记;毒害艾美耳球虫
chicken coccidiosis;biological characteristic;SCAR marker;Eimeria necatrix

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(31972647);; 山西省博士毕业生、博士后研究人员来晋工作奖励资金科研项目(SXBYKY2021041);; 山西农业大学动物医学学院科研创新项目(DY-Q002);山西农业大学高等学校科技创新项目(2021L136)

作者(Authors): 张黎;郑龙龙;白冰;谭凡;王辰;吕晓玲;白瑞;郑明学;
ZHANG Li;ZHENG Long-long;BAI Bing;TAN Fan;WANG Chen;LYU Xiao-ling;BAI Rui;ZHENG Ming-xue;College of Veterinary Medicine, Shanxi Agricultural University;

参考文献(References)：

• [1] 龚振兴，魏红梅，刘百林，等.饱和食盐水漂浮鸡球虫卵囊的用量和处理时间研究[J].湖南农业科学，2019,6:77- 79.
• [2] 方素芳，关琛，崔平，等.不同地理株大型艾美耳球虫单卵囊分离及卵囊产量的比较[J].河北北方学院学报(自然科学版),2019,35(11):32- 35+39.
• [3] Rao P V,Raman M,Dhinakarraj G,et al.Multiplex PCR assay using SCAR primers to detect Eimeria spp.in chicken[J].J Parasit Dis,2013,37(1):110- 113.
• [4] Hinsu A T,Thakkar J R,Koringa P G,et al.Illumina next generation sequencing for the analysis of Eimeria populations in commercial broilers and indigenous chickens[J].Frontiers in Veterinary Science,2018,5:176.
• [5] Johnson J,Reid W M.Anticoccidial drugs:lesion scoring techniques in battery and floor-pen experiments with chickens[J].Experimental Parasitology,1970,28(1):30- 36.
• [6] Taha S,Nguyen-Ho-Bao T,Daugschies A,et al.In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites:quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR)[J].Parasitology Research,2021,120(7):2689- 2693.
• [7] Gadelhaq S M,Arafa W M,Aboelhadid S M.Molecular characterization of Eimeria species naturally infecting Egyptian Baldi chickens[J].Iranian Journal of Parasitology,2015,10(1):87- 95.
• [8] Kumar S,Garg R,Moftah A,et al.An optimised protocol for molecular identification of Eimeria from chickens[J].Veterinary Parasitology,2014,199(1-2):24- 31.
• [9] Gawel A,Bobusia K,Bobrek K.Identification of Eimeria spp.occurring in hens and broiler chickens in Poland[J].Medycyna Weterynaryjna,2015,71(6):382- 385.
• [10] 黄兵，吴薛忠，史天卫，等.毒害艾美球虫纯种分离的初步鉴定和致病性研究[J].中国兽医寄生虫病，1993,1(4):24- 28.
• [11] Geng T T,Zhao J L,Zhou Y J,et al.Prevalence of Eimeria parasites in the Hubei and Henan Provinces of China[J].Parasitol Res,2021,120(2):655- 663.
• [12] Fan X C,Liu T L,Wang Y,et al.Genome-wide analysis of differentially expressed profiles of mRNAs,lncRNAs and circRNAs in chickens during Eimeria necatrix infection[J].Parasites & Vectors,2020,13(1):167.
• [13] 董青.毒害艾美耳球虫河南分离株早熟系选育及生物学特性研究[J].中国兽医杂志，2021,57(4):5- 9.
• [14] 颜静蕊.鸡E.necatrix早熟株疫苗的免疫效力研究[D].太谷：山西农业大学，2013.
• [15] 刘国辉.不同球虫疫苗商品肉鸡免疫效果研究[D].青岛：青岛农业大学，2019.