绵羊肺炎支原体人工感染SPF鸡胚的研究

doi:10.16431/j.cnki.1671-7236.2020.09.034

摘要/Abstract

摘要： 为探索SPF鸡胚作为绵羊肺炎支原体实验室感染模型的可行性，本试验用不同浓度绵羊肺炎支原体（10⁸、10⁹、10¹⁰ ccu/mL）经由卵黄囊和尿囊腔两个部位接种7日龄SPF鸡胚，通过统计鸡胚死亡情况和不同鸡胚组织样品中绵羊肺炎支原体检测阳性率（PCR检测和支原体分离鉴定），确定绵羊肺炎支原体鸡胚感染方式、感染剂量和最佳分离部位，再用3株不同来源的绵羊肺炎支原体分离株感染鸡胚，观察其对鸡胚的致病力。结果表明，绵羊肺炎支原体感染鸡胚最佳接种途径为卵黄囊接种，感染剂量为10⁹ ccu/mL、0.2 mL/只，最佳分离部位为卵黄液。3株支原体均能感染和致死鸡胚，并均能从卵黄液中分离到绵羊肺炎支原体，但对鸡胚的致病性存在一定差异：FL3株致鸡胚死亡率为45%，略高于MoGH3-3株（40%），二者均高于A3株（25%），但差异均不显著（P>0.05）；FL3株鸡胚检测阳率为100%，高于MoGH3-3株（85%）及A3株（90%），但差异也不显著（P>0.05）。本试验初步确定了绵羊肺炎支原体可感染和致死SPF鸡胚，不同分离株对SPF鸡胚致病力有差异，表明SPF鸡胚可作为下一步建立绵羊肺炎支原体实验室感染模型的候选，为绵羊肺炎支原体致病性研究和疫苗研制奠定基础。

关键词: 绵羊肺炎支原体; 感染; SPF鸡胚

Abstract: In order to evaluate the SPF chicken embryos as a candidate of the experimental model of Mycoplasma ovipneumoniae (Mo) infection,the 7-day-old SPF chicken embryos were infected with different concentrations of Mo(10⁸,10⁹ and 10¹⁰ ccu/mL) by injection of vitelline fluid and allantoic cavity.The mortality and Mo detection rate (PCR detection and isolation) of infected chicken embryos were employed to evaluate the optimal inoculation route,dose and sampling site.Three different isolates of Mo were submitted to injection of chicken embryos for pathogenicity comparison.The results showed that the optimal inoculation route and dose were vitelline fluid injection with 0.2 mL (10⁹ ccu/mL) Mo per embryo and the sampling site for detection was also vitelline fluid.Three Mo isolates revealed good infectivity and lethality to the chicken embryo.The chicken embryos mortality caused by FL3 strain (45%) and MoGH3-3 (40%) were both higher than that by A3 strain (25%),but with no significance (P>0.05).The detection rate of Mo from the chicken embryos in FL3 group (100%) was higher than that of MoGH3-3 strain(85%) and that of A3 strain (90%),but with no significance(P>0.05).This experiment preliminarily proved that the SPF chicken embryos could be infected and partially killed by Mo,and the virulence of different strains of Mo to the chicken embryos was different.The results provided the data for further establishment of the chicken embryo infection model of Mo,which would benefit the research on Mo pathogenicity and vaccine development.

Key words: Mycoplasma ovipneumoniae; infection; SPF chicken embryos

中图分类号:

S858.26

袁婷, 郝华芳, 马丽娜, 陈胜利, 颜新敏, 吴娅琴, 蔡亚婷, 孙延鸣, 储岳峰. 绵羊肺炎支原体人工感染SPF鸡胚的研究[J]. 中国畜牧兽医, 2020, 47(9): 2988-2996.

YUAN Ting, HAO Huafang, MA Lina, CHEN Shengli, YAN Xinmin, WU Yaqin, CAI Yating, SUN Yanming, CHU Yuefeng. Experimental Infection of Mycoplasma ovipneumoniae in SPF Chicken Embryos[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2988-2996.

参考文献

[1] 储岳峰,逯忠新,赵萍,等.绵羊霉形体病研究现状[J].畜牧与兽医,2008,40(11):103-105. CHU Y F,LU Z X,ZHAO P,et al.Advances in sheep mycoplasmosis[J].Animal Husbandry and Veterinary Medicine,2008,40(11):103-105.(in Chinese)
[2] EINARSDOTTIR T,GUNNARSSON E,HJARTARDO-TTIR S.Icelandic ovine Mycoplasma ovipneumoniae are variable bacteria that induce limited immune responses in vitro and in vivo[J].Journal of Medical Microbio-logy,2018,67(10):1480-1490.
[3] ALLEY M R,IONAS G,CLARKE J K.Chronic non-progressive pneumonia of sheep in New Zealand——A review of the role of Mycoplasma ovipneumoniae[J].New Zealand Veterinary Journal,1999,47(5):155-160.
[4] 倪文浩,赵亚南,刘宜勇,等.新疆地区不同绵羊品种支原体肺炎血清学调查[J].草食家畜,2019,2:53-58. NI W H,ZHAO Y N,LIU Y Y,et al.Serological investigation of Mycoplasma pneumonia in different sheep breeds in Xinjiang[J].Grass-Feeding Livestock,2019,2:53-58.(in Chinese)
[5] 陆承平.兽医微生物[M].北京:中国农业出版社,2001. LU C P.Veterinary Microbiology[M].Beijing:China Agriculture Press,2001.(in Chinese)
[6] 王岩.羊支原体肺炎综合性防治措施[J].畜牧兽医科技信息,2015,6:49-50. WANG Y.Comprehensive control measures for Mycoplasma pneumonia in sheep[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2015,6:49-50.(in Chinese)
[7] JIANG F,HE J,NAVARRO-VARRO N,et al.Elongation factor Tu and heat shock protein 70 are membrane associated proteins from Mycoplasma ovipneumoniae capable of inducing strong immune response in mice[J].PLoS One,2016,11(8):e0161170.
[8] XUE D,MA Y,LI M,et al.Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway[J].Veterinary Immunology and Immunopathology,2015,163(1-2):57-66.
[9] 王恒,殷慧群,章孝荣,等.鸡胚模型在生物研究中的应用进展[J].生命科学,2009,21(2):312-314. WANG H,YIN H Q,ZHANG X R,et al.Research advances of chick embryo model in biological experiments[J].Chinese Bulletin of Life Scinences,2009,21(2):312-314.(in Chinese)
[10] MCAULIFE L,HATCHELL F M,AYLING R D,et al.Detection of Mycoplasma ovipneumoniae in pasteurella vaccinated sheep flocks with respiratory disease in England[J].Veterinary Record,2003,153:687-688.
[11] 杜鲜.绵羊肺炎支原体免疫层析血清学和病原分子诊断技术的研究[D].石河子:石河子大学,2017. DU X.The study of serological immunochromato-graphy and pathogenic molecular diagnostic techniques in Mycoplasma ovipneumoniae[D].Shihezi:Shihezi Univer-sity,2017.(in Chinese)
[12] YANG F L,TANG C,WANG Y,et al.Genorae sequence of Mycoplasma ovipneumoniae strain SC01[J].Journal of Bacteriology,2011,193(18):5018.
[13] 胡景韶,蒋学良,胡诚隆,等.一种由支原体感染的绵羊增生性间质性肺炎的研究[J].中国兽医杂志,1982,5:2-6. HU J S,JIANG X L,HU C L,et al.Research on a hyperplastic interstitial pneumonia of sheep with Mycoplasma[J].Chinese Journal of Veterinary Medicine,1982,5:2-6.(in Chinese)
[14] 谢秀兰,额尔和花,黎帅,等.宁夏不同绵羊品种中绵羊肺炎支原体的调查[J].中国兽医杂志,2017,53(6):11-13. XIE X L,E ER H H,LI S,et al.The survey of Mycoplasma ovipneumoniae in diferent sheep breeds in Ningxia area[J].Chinese Journal of Veterinary Medicine,2017,53(6):11-13.(in Chinese)
[15] 杜奕洲,郗宇,赵金玉,等.新疆南疆部分地区绵羊支原体感染的分子流行病学调查[J].中国兽医科学,2019,49(11):1427-1433. DU Y Z,XI Y,ZHAO J Y,et al.Molecular epidemiological investigation of sheep Mycoplasma infection in parts of Southern Xinjiang[J].Chinese Veterinary Science,2019,49(11):14273-1433.(in Chinese)
[16] 朱琳,常卫华,张勇,等.甘肃地区绵羊支原体病原菌的分离培养[J].草业科学,2013,30(9):1428-1432. ZHU L,CHANG W H,ZHANG Y,et al.Isolation and culture of sheep Mycoplasma pneumoniae in Gansu province[J].Pratacul Tural Science,2013,30(9):1428-1432.(in Chinese)
[17] 王华,杨发龙,王永,等.山羊支原体性肺炎流行病学调查[J].中国畜牧兽医,2011,38(1):210-213. WANG H,YANG F L,WANG Y,et al.Epidemiological investigation of caprine Mycoplasma pneumoniae in Sichuan province[J].China Animal Husbandry & Veterinary Medicine,2011,38(1):210-213.(in Chinese)
[18] 江锦秀,林裕胜,游伟,等.福建省羊支原体性肺炎分子流行病学研究[J].福建农业学报,2017,32(1):12-16. JIANG J X,LIN Y S,YOU W,et al.A molecular epidemiological study on Mycoplasma pneumonia of sheep and goats in Fujian[J].Fujian Journal of Agricultural Science,2017,32(1):12-16.(in Chinese)
[19] BESSER T E,CASSIRER E F,HIGHLAND M A,et al.Bighora sheep pneumonia sorting out the cause of apply microbial disease[J].Preventive Veterinary Medicine,2013,108(2-3):85-93.
[20] 许建,储岳峰,赵萍,等.中国部分地区绵羊肺炎支原体的基因多态性分析[J].畜牧兽医学报,2011,42(9):1295-1301. XU J,CHU Y F,ZHAO P,et al.Investigation of gene polymorphism on Mycoplasma ovipneumoniae isolates from some areas of China[J].Chinese Journal of Animal and Veterinary Sciences,2011,42(9):1295-1301.(in Chinese)
[21] THIRKELL D,SPOONER R K,JONES G E,et al.Polypeptide and antigenic variability among strains of Mycoplasma ovipneumoniae demonstrated by SDS-PAGE and immunoblotting[J].Veterinary Microbiology,1990,21(3):241-254.
[22] IONAS G C,CLARKE J K,MARSHALL R B.The isolation of multiple stains of Mycoplasma ovipneu-moniae from individual pneumonic sheep lungs[J].Veterinary Microbiology, 1991,29(3-4):349-360.
[23] PARHAM K,CHURCHEWARD C P,MCAULIFFE L,et al.A high level of strain variation within the Mycoplasma ovipneumoniae population of the UK has implication of disease diagnosis and management[J].Veterinary Microbiology,2006,118(1):83-90.
[24] 王小义,张红梅,古丽汗·阿布都热木,等.人工感染绵羊肺炎支原体后绵羊甘露聚集结合凝集素变化及组织病理学[J].中国兽医杂志,2015,51(2):9-12. WANG X Y,ZHANG H M,ABUDUREMU G,et al.Change of MBL in serum and histopathologic analysis after artificially infecting Mycoplasma ovipneu-moniae[J].Chinese Journal of Veterinary Medicine,2015,51(2):9-12.(in Chinese)
[25] 王建昌.绵羊肺炎支原体山东株的分离鉴定和标准株外膜蛋白的特性研究[D].泰安:山东农业大学,2007. WANG J C.Isolation and identification of Mycoplasma ovipneumoniae from Shandong and studies on out membrane protein of standard strain Y-98[D].Tai'an:Shandong Agricultural University,2007.(in Chinese)