牛支原体兔体攻毒模型的建立

doi:10.11843/j.issn.0366-6964.2024.03.038

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (3): 1268-1277.doi: 10.11843/j.issn.0366-6964.2024.03.038

牛支原体兔体攻毒模型的建立

武文英^1,2,3, 夏青¹, 胡萌婕¹, 赵逸轩¹, 王琛^1,2,3, 张宇豪^1,2,3, 郝成武⁴, 贺笋⁴, 郭爱珍^1,2,3, 陈建国^1,2, 陈颖钰^1,2,3*

1. 华中农业大学动物科学技术学院动物医学院, 武汉 430070;
2. 华中农业大学生猪健康养殖协同创新中心, 武汉 430070;
3. 华中农业大学农业微生物资源发掘与利用国家重点实验室, 武汉 430070;
4. 天康生物制药有限公司, 乌鲁木齐 830011

收稿日期:2023-06-12 出版日期:2024-03-23 发布日期:2024-03-27
通讯作者: 陈颖钰,主要从事动物流行病学、牛主要疾病致病机制、诊断方法及疫苗研发,E-mail:chenyingyu@mail.hzau.edu.cn
作者简介:武文英(1998-),女,河南郑州人,硕士,主要从事临床兽医学研究,E-mail:wwy.11@outlook.com
基金资助:
宁夏回族自治区重点研发计划项目（2023BCF01038）；国家现代农业（肉牛/牦牛）产业技术体系（CARS-37）

Establishment of Rabbit Challenge Model of Mycoplasma bovis

WU Wenying^1,2,3, XIA Qing¹, HU Mengjie¹, ZHAO Yixuan¹, WANG Chen^1,2,3, ZHANG Yuhao^1,2,3, HAO Chengwu⁴, HE Sun⁴, GUO Aizhen^1,2,3, CHEN Jianguo^1,2, CHEN Yingyu^1,2,3*

1. College of Animal Sciences & Technology/College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China;
2. The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China;
3. State Key Laboratory of Agricultural Microbial Resources Exploitation and Utilization, Huazhong Agricultural University, Wuhan 430070, China;
4. Tiankang Biopharmaceutical Co., Ltd., Urumqi 830011, China

Received:2023-06-12 Online:2024-03-23 Published:2024-03-27

摘要/Abstract

摘要： 牛支原体是引起牛呼吸疾病综合征（bovine respiratory disease，BRD）的重要病原之一，对养牛业造成了不可估量的经济损失。牛支原体对大多数抗生素都具有耐药性，目前没有较好的药物可以进行治疗，因此疫苗免疫是最有效的防控方法。缺乏小动物评价模型是牛支原体疫苗研发的重要制约因素之一。建立小动物攻毒模型，为后续疫苗的研究与制备提供一定的研究基础，并降低后续试验的科研成本。为建立牛支原体小动物感染模型，本研究以2月龄日本大耳白兔作为研究对象，分为4组：1）单独攻击HB0801株牛支原体，2）注射地塞米松后攻击HB0801株牛支原体，3）攻击HB0801株牛支原体后注射KLH和巯基乙酸盐培养基；4）空白对照组。通过PCR检测牛支原体排菌情况，检测血清中抗体水平，第31天后取肺制作病理切片。研究结果显示，注射地塞米松后攻击HB0801株牛支原体组的牛支原体检出率和抗体均最高，牛支原体检出率为42.11%，91.67%的动物被检测为抗体阳性。显著高于其它各组。除空白对照组外，其它攻毒组均出现了肺泡壁增厚，巨噬细胞浸润以及肺部不同程度的纤维素性渗出，符合牛支原体导致的间质性肺炎的病变特征。本研究建立了牛支原体HB0801株攻毒日本大耳白兔的感染及评价模型，地塞米松造成免疫抑制后攻毒HB0801株具有最好的造模效果。

关键词: 牛支原体, 动物模型, 兔, 地塞米松

Abstract: Mycoplasma bovis is one of the important pathogens causing bovine respiratory disease syndrome (BRD), which has caused immeasurable economic losses to the cattle industry. Mycoplasma bovis is resistant to most antibiotics, and there is no good drug to treat it at present. Therefore, vaccination is the most effective prevention and control method. The lack of a small animal evaluation model is one of the important constraints for the development of Mycoplasma bovis vaccines. The small animal challenge model was established to provide a certain research basis for the follow-up vaccine research and preparation, and reduce the scientific research cost of follow-up experiments. In order to establish a small animal model of Mycoplasma bovis infection, two-month-old Japanese big-eared white rabbits were used in this study and divided into 4 groups:1) Mycoplasma bovis strain HB0801 was challenged alone, 2) Mycoplasma bovis strain HB0801 was challenged with dexamethasone, 3) Mycoplasma bovis strain HB0801 was challenged with KLH and thioglycollate medium. 4) Blank control group. The excretion of Mycoplasma bovis was detected by PCR, and the level of antibody in serum was detected. The lungs were taken to make pathological sections after 31 days. The results showed that both the detection rate of M. bovis and the antibody against M. bovis were the highest in the group challenged with M. bovis HB0801 strain after injection of dexamethasone. The detection rate of M. bovis was 42.11%, and 91.67% of the animals tested positive for M. bovis. It was significantly higher than other groups. Except for the blank control group, the other challenge groups showed alveolar wall thickening, macrophage infiltration,and different degrees of fibrinous exudation in the lungs, which were consistent with the pathological characteristics of interstitial pneumonia caused by Mycoplasma bovis. In this study, the infection and evaluation model of Mycoplasma bovis HB0801 strain challenge in Japanese big-eared white rabbits was established, and the best modeling effect was achieved when the immunosuppression was induced by dexamethasone.

Key words: Mycoplasma bovis, animal model, rabbit, dexamethasone

中图分类号:

S855

武文英, 夏青, 胡萌婕, 赵逸轩, 王琛, 张宇豪, 郝成武, 贺笋, 郭爱珍, 陈建国, 陈颖钰. 牛支原体兔体攻毒模型的建立[J]. 畜牧兽医学报, 2024, 55(3): 1268-1277.

WU Wenying, XIA Qing, HU Mengjie, ZHAO Yixuan, WANG Chen, ZHANG Yuhao, HAO Chengwu, HE Sun, GUO Aizhen, CHEN Jianguo, CHEN Yingyu. Establishment of Rabbit Challenge Model of Mycoplasma bovis[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 1268-1277.

参考文献

[1] 刘庆国. 新疆南疆地区牛支原体的分离鉴定及致病性研究[D]. 阿拉尔:塔里木大学, 2022.
LIU Q G. Isolation, identification and pathogenicity of Mycoplasma bovis in southern Xinjiang[D]. Alaer:Tarim University, 2022. (in Chinese)
[2] 朱志刚, 蒋向君, 孙田. 犊牛支原体病的诊断与防控措施[J]. 中国乳业, 2020(11):50-51.
ZHU Z G, JIANG X J, SUN T. Diagnosis, prevention and control measures of mycoplasmosis in calves[J]. China Dairy, 2020(11):50-51. (in Chinese)
[3] 彭清洁, 胡长敏, 陈颖钰, 等. 一例奶牛牛支原体乳房炎的诊断[C]//中国奶业协会第26次繁殖学术年会暨国家肉牛牦牛/奶牛产业技术体系第3届全国牛病防治学术研讨会论文集. 兰州:全国牛病大会组委会, 2011:493-496.
PENG Q J, HU C M, CHEN Y Y, et al. Diagnosis of a case of bovine mycoplasma mastitis in a dairy cow[C]//The 26th annual meeting of China Dairy Association and the 3rd National Symposium on Bovine Disease Control of National Beef Yak/Dairy Cattle Industrial Technology System. Lanzhou:Organizing Committee of the National Bovine Disease Conference, 2011:493-496. (in Chinese)
[4] 张建华, 曹希亮, 刘超, 等. 我国奶牛牛支原体流行情况调查[J]. 中国奶牛, 2017(3):23-26.
ZHANG J H, CAO X L, LIU C, et al. Prevalence investigation of Mycoplasma bovis in China[J]. China Dairy Cattle, 2017(3):23-26. (in Chinese)
[5] HALE H H, HELMBOLDT C F, PLASTRIDGE W N, et al. Bovine mastitis caused by a Mycoplasma species[J]. Cornell Vet, 1962, 52:582-591.
[6] 辛九庆, 李媛, 郭丹, 等. 国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J]. 中国预防兽医学报, 2008, 30(9):661-664.
XIN J Q, LI Y, GUO D, et al. First isolation of Mycoplasma bovis from calf lung with pneumonia in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(9):661-664. (in Chinese)
[7] 石磊, 龚瑞, 尹争艳, 等. 肉牛传染性牛支原体肺炎流行的初步诊断[J]. 华中农业大学学报, 2008, 27(4):572.
SHI L, GONG R, YIN Z Y, et al. Preliminary diagnosis of cattle infectious Mycoplasma bovis pneumonia[J]. Journal of Huazhong Agricultural University, 2008, 27(4):572. (in Chinese)
[8] NICHOLAS R A J, AYLING R D. Mycoplasma bovis:disease, diagnosis, and control[J]. Res Vet Sci, 2003, 74(2):105-112.
[9] 胡长敏, 石磊, 龚瑞, 等. 牛支原体病研究进展[J]. 动物医学进展, 2009, 30(8):73-77.
HU C M, SHI L, GONG R, et al. Progress on bovine mycoplasmosis[J]. Progress in VeterinaryMedicine, 2009, 30(8):73-77. (in Chinese)
[10] 王天宇, 李继东, 张志诚, 等. 牛支原体病流行病学及其诊断技术研究进展[J]. 畜牧与兽医, 2021, 53(12):134-139.
WANG T Y, LI J D, ZHANG Z C, et al. Progress in research on the molecular technology of Mycoplasma bovis and its epidemic status[J]. Animal Husbandry & Veterinary Medicine, 2021, 53(12):134-139. (in Chinese)
[11] ADAMU J Y, WAWEGAMA N K, BROWNING G F, et al. Membrane proteins of Mycoplasma bovis and their role in pathogenesis[J]. Res Vet Sci, 2013, 95(2):321-325.
[12] 贺承光, 孔令聪, 孙喆. 牛支原体抗生素耐药性的研究进展[J]. 吉林畜牧兽医, 2018, 39(12):11-13.
HE C G, KONG L C, SUN Z. Research progress on antibiotic resistance of Mycoplasma bovis[J]. Jilin Animal Husbandry and Veterinary Medicine, 2018, 39(12):11-13. (in Chinese)
[13] BOKMA J, VEREECKE N, NAUWYNCK H, et al. Genome-wide association study reveals genetic markers for antimicrobial resistance in Mycoplasma bovis[J]. Microbiol Spectr, 2021, 9(2):e0026221.
[14] 东笑, 陶攀, 王贵平, 等. 牛支原体疫苗研究进展[J]. 中国动物传染病学报, 2023, doi:10. 19958/j. cnki. cn31-2031/s. 20211126. 010.
DONG X, TAO P, WANG G P, et al. Research progress of bovine Mycoplasma vaccine[J]. Chinese Journal of Animal Infectious Diseases, 2023, doi:10. 19958/j. cnki. cn31-2031/s. 20211126. 010. (in Chinese)
[15] 范媛, 陈忠琼, 谢光武, 等. 6株牛支原体分离株对鸡胚和小鼠的致病性研究[J]. 中国预防兽医学报, 2012, 34(12):963-966.
FAN Y, CHEN Z Q, XIE G W, et al. Pathogenicity of Mycoplasma bovis isolates in experimental infected chick embryos and mice[J]. Chinese Journal of Preventive Veterinary Medicine, 2012, 34(12):963-966. (in Chinese)
[16] 雷元元, 何生虎, 马烨, 等. 牛支原体感染家兔试验[J]. 动物医学进展, 2015, 36(10):53-59.
LEI Y Y, HE S H, MA Y, et al. Experiment on Mycoplasma bovis infection in rabbits[J]. Progress in Veterinary Medicine, 2015, 36(10):53-59. (in Chinese)
[17] 牛家强. 西藏牦牛源牛支原体生物学特性及感染兔肺脏转录组学研究[D]. 武汉:华中农业大学, 2021.
NIU J Q. Biological characteristics of isolated Mycoplasma bovis from yaks in Tibet and transcriptomics of lungs in infected rabbits[D]. Wuhan:Huazhong Agricultural University, 2021. (in Chinese)
[18] TURYNA B, BOŚ M, JUREK J. The influence of in vivo stimulation on functional activity of rat macrophages[J]. Folia Histochem Cytobiol, 1994, 32(2):101-105.
[19] CHU C Y, LEE S C, LIU S S, et al. Cytosine-phosphate-guanine oligodeoxynucleotides containing GACGTT motifs enhance the immune responses elicited by keyhole limpet hemocyanin antigen in dairy cattle[J]. Nucleic Acid Ther, 2011, 21(5):323-332.
[20] 郭大城, 马彦民, 许玉玲, 等. 支原体菌落形成单位与颜色改变单位定量关系研究[J]. 中国卫生检验杂志, 2020, 30(4):405-407.
GUO D C, MA Y M, XU Y L, et al. Study on quantitative relationship between colony formation unit and color changing unit for mycoplasma[J]. Chinese Journal of Health Laboratory Technology, 2020, 30(4):405-407. (in Chinese)
[21] TOWNSEND K M, BOYCE J D, CHUNG J Y, et al. Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system[J]. J Clin Microbiol, 2001, 39(3):924-929.
[22] 王贵升, 尹斐斐. 肺炎克雷伯氏菌PCR检测方法的建立[J]. 山东畜牧兽医, 2017, 38(4):6-7.
WANG G S, YIN F F. Establishment of PCR detection method for Klebsiella pneumoniae[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2017, 38(4):6-7. (in Chinese)
[23] HOZBOR D, FOUQUE F, GUISO N. Detection of Bordetella bronchiseptica by the polymerase chain reaction[J]. Res Microbiol, 1999, 150(5):333-341.
[24] THOMAS A, DIZIER I, LINDEN A, et al. Conservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis[J]. Vet J, 2004, 168(1):100-102.
[25] 徐恩红, 祁明普, 项志杰, 等. 牛呼吸疾病综合征七病原联合检测多重qPCR方法的建立[J]. 华中农业大学学报, 2023, 42(2):38-47.
XU E H, QI M P, XIANG Z J, et al. Establishment of multiple qPCR for simultaneous detection of 7 pathogens causing bovine respiratory diseases complex[J]. Journal Huazhong Agricultural University, 2023, 42(2):38-47. (in Chinese)
[26] 石刚. 牛支原体灭活疫苗佐剂的筛选与培养基的改良[D]. 石河子:石河子大学, 2013.
SHI G. Screening adjuvant for the inactivated vaccine of Mycoplasma bovis and the improvement of Mycoplasma bovis medium[D]. Shihezi:Shihezi University, 2013. (in Chinese)
[27] 胡长敏, 郭爱珍, 张文劲, 等. 抗牛支原体MbovP579蛋白的单克隆抗体及其应用:中国, 110746504A[P]. 2020-02-04.
HU C M, GUO A Z, ZHANG W J, et al. Monoclonal antibody against Mycoplasma bovis MbovP579 protein and its application:CN, 110746504A[P]. 2020-02-04. (in Chinese)
[28] SIBILA M, ARAGÓN V, FRAILE L, et al. Comparison of four lung scoring systems for the assessment of the pathological outcomes derived from Actinobacillus pleuropneumoniae experimental infections[J]. BMC Vet Res, 2014, 10:165.
[29] 庞方圆, 李洪林, 隋丽英. 一例犊牛支原体性肺炎和关节炎的病理学观察[J]. 中国畜禽种业, 2022, 18(10):132-134.
PANG F Y, LI H L, SUI L Y. Pathological observations on a case of mycoplasma pneumonia and arthritis in a calf[J]. The Chinese Livestock and Poultry Breeding, 2022, 18(10):132-134. (in Chinese)
[30] 范媛. 牛支原体生长特性及其致病性研究[D]. 重庆:西南大学, 2011.
FAN Y. The study on growth properties and pathogenesis of Mycoplasma bovis[D]. Chongqing:Southwest University, 2011. (in Chinese)
[31] 张晓宇. 奶牛犊牛主要呼吸道疾病流行病学调查及牛支体肺炎防治的研究[D]. 呼和浩特:内蒙古农业大学, 2018.
ZHANG X Y. Investigation on pathogens of main respiratory infectious diseases in dairy cows in China and rasearch on main pathogen control[D]. Hohhot:Inner Mongolia Agricultural University, 2018. (in Chinese)
[32] 周玉森. 肉牛支原体肺炎的流行病学、临床症状、诊断与防治[J]. 现代畜牧科技, 2018(3):104.
ZHOU Y S. Epidemiology, clinical symptoms, diagnosis and prevention of Mycoplasma pneumonia in beef cattle[J]. Modern Animal Husbandry Science & Technology, 2018(3):104. (in Chinese)

牛支原体兔体攻毒模型的建立

Establishment of Rabbit Challenge Model of Mycoplasma bovis

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