内蒙古地区羊源副结核分枝杆菌的分离与基因分型

doi:10.3864/j.issn.0578-1752.2023.06.015

中国农业科学 ›› 2023, Vol. 56 ›› Issue (6): 1204-1214.doi: 10.3864/j.issn.0578-1752.2023.06.015

• 畜牧·兽医 • 上一篇

内蒙古地区羊源副结核分枝杆菌的分离与基因分型

赵维宏¹(), 韩文雄², 杨波³, 孟维康¹, 柴海亮¹, 马懿敏¹, 张占胜¹, 王利峰¹, 王艳¹, 王明圆¹, 张姗¹, 丁玉林¹, 王金玲¹, 吉林台¹, 王凤龙¹, 赵丽¹(), 刘永宏¹()

¹ 内蒙古农业大学兽医学院/农业农村部动物疾病临床诊疗技术重点实验室，呼和浩特 010000
² 内蒙古赛科星繁育生物技术（集团）股份有限公司，呼和浩特 011517
³ 鄂尔多斯市动物疫病预防控制中心，内蒙古鄂尔多斯 017000

收稿日期:2021-11-11 接受日期:2022-08-30 出版日期:2023-03-16 发布日期:2023-03-23
联系方式: 赵维宏，E-mail：zhaoweihong@foxmail.com。
基金资助:
国家自然科学基金(31860698); 内蒙古农业大学高层次人才科研启动金项目(NDYB2019-3); 内蒙古农业大学高层次人才科研启动金项目(NDYB2018-5)

Isolation and Genotyping of Mycobacterium avium subsp. paratuberculosis from Sheep in Inner Mongolia

ZHAO WeiHong¹(), HAN WenXiong², YANG Bo³, MENG WeiKang¹, CHAI HaiLiang¹, MA YiMin¹, ZHANG ZhanSheng¹, WANG LiFeng¹, WANG Yan¹, WANG MingYuan¹, ZHANG Shan¹, DING YuLin¹, WANG JinLing¹, JIRINTAI Sulijid¹, WANG FengLong¹, ZHAO Li¹(), LIU YongHong¹()

¹ College of Veterinary Medicine, Inner Mongolia Agricultural University/Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture and Rural Affairs, Hohhot 010000
² Inner Mongolia Saikexing Reproductive Biotechnology (Group) Co.,Ltd., Hohhot, 011517
³ Animal Disease Control Center of Ordos, Ordos 017000, Inner Mongolia

Received:2021-11-11 Accepted:2022-08-30 Published:2023-03-16 Online:2023-03-23

摘要/Abstract

摘要：

【目的】副结核病被世界动物卫生组织（OIE）列入必须报告的《OIE疫病、感染及侵染名录》，我国将其列为二类动物疫病，引起多种反刍动物慢性、增生性肠炎，感染动物通过肠道间歇性排菌而成为养殖场的持续传染源，给养殖业带来了巨大经济损失。其病原体副结核分枝杆菌（MAP）属于胞内寄生的革兰氏阳性菌，为三类动物病原微生物，包括S型（羊型，细分为Ⅰ型、Ⅲ型和骆驼型）和C型（牛型、Ⅱ型，包括B型（野牛型））。有研究表明，各亚型MAP无宿主特异性，但具有地域性，内蒙古作为国内该病的首发地区，获得并准确鉴定内蒙古地区MAP菌株亚型及基因特征，对副结核病的预防控制意义重大。【方法】对内蒙古地区来源的28份MAP阳性的羊源病料进行MAP分离培养，菌落Ziehl-Neelsen染色，染色阳性菌扩繁，提取扩繁菌液基因组DNA，进行IS900基因、IS1311基因和DMC基因扩增、测序和序列分析，同时IS1311基因PCR产物进行Hinf I和Mse I双酶切鉴定。【结果】28份样品经过7—12周培养，共有9支培养基长出菌落，菌落半透明乳白色、表面光滑。挑取单菌落进行抗酸染色，在显微镜下观察到呈不规则（单个或分枝状）、红染的细短杆菌，符合分枝杆菌的形态学特征及抗酸染色特性。9株分离菌IS900、IS1311和DMC基因PCR扩增产物均与目的基因片段预期大小一致。确定了本研究9株分离株均为MAP菌株，分别命名为MAP-NM1至MAP-NM9。DMC基因扩增产物大小为310bp，符合Ⅱ型MAP特征；IS1311基因扩增产物经Hinf Ⅰ和Mse Ⅰ双酶切，本研究9株MAP均得到4条目的条带，与Ⅱ型MAP一致；IS1311基因测序结果与S型、C型、印度野牛型和美国野牛型MAP代表株对照分析显示，9株MAP IS1311基因片段的64、65、68、223、236、422、527、628位碱基位点符合C型和B型MAP特征；IS900基因测序结果序列分析显示，9株MAP IS900基因片段第169位及第216位碱基分别为C（胞嘧啶）和A（腺嘌呤），符合Ⅱ型和Ⅲ型MAP特征；17株来自GenBank数据库的MAP IS900基因参考序列与本研究9株分离株IS900基因系统进化树显示，本研究9株MAP均划分于Ⅱ型MAP分支；3个基因测序结果进行Blast在线分析，与本研究所得分离株同源性最高的参考序列均为Ⅱ型MAP，且同源性均高于98%。综上所述，本研究9株MAP分离株均为Ⅱ型MAP。【结论】首次分离得到内蒙古地区羊源Ⅱ型MAP菌株。

关键词: 副结核分枝杆菌, 分离培养, 亚型鉴定, 羊, 内蒙古

Abstract:

【Objective】 Paratuberculosis is listed in the must-report at the list of “World Organization for Animal Health (OIE) diseases, infections and Invasion” by OIE. It is classified as the second kind of animal disease in China. It causes chronic and proliferative enteritis in many ruminants. The infected animals become a continuous source of infection in farms through intestinal intermittent excretion, which has brought great economic losses to aquaculture. The pathogen of Mycobacterium avium subsp. paratuberculosis (MAP) belongs to intracellular parasitic Gram-positive bacteria, and is a third group of zoological pathogenic microorganisms, including type C (also designated as type II) and type S. Type C also includes type B. Type S can be further subdivided into sub-group types I and III. And sub-lineages of camelid isolates Studies have shown that each subtype of MAP has no host specificity, but is regional. Inner Mongolia is the first region of the disease in China. It is of great significance to obtain and accurately identify the subtype and genetic characteristics of MAP strains in Inner Mongolia for the prevention and control of paratuberculosis. 【Method】 28 MAP-positive sheep disease samples collected in Inner Mongolia were isolated and cultured by MAP, and the colonies were stained with Ziehl-Neelsen. The positive bacteria were propagated and the genomic DNA was extracted. IS900 gene, IS1311 gene and DMC gene were amplified, sequenced, and analyzed. The PCR products of IS1311 gene were identified by Hinf I and Mse I double digestion. 【Result】 28 samples were cultured for 7- 2 weeks, a total of 9 mediums grew colonies, and the colonies were translucent milky white smooth surface. Single colonies were selected for acid-fast staining, and irregular (single or branched), red-stained Brevibacterium was observed under the microscope, which was consistent with the morphological characteristics and acid-fast staining characteristics of Mycobacterium. The PCR products of IS900, IS1311 and DMC genes of 9 isolates were consistent with the expected size of the target gene fragment. 9 isolates were identified as MAP strains, named MAP-NM1 to MAP-NM9. DMC gene amplification product size of 310bp, which was consistent with type II MAP characteristics. IS1311 gene amplification products were digested by Hinf I and Mse I restriction endonucleases, and 4 target bands were obtained in 9 strains of MAP, which were consistent with type II MAP. The sequencing results of IS1311 gene and the analysis of MAP representative strains of type I, type II, type III, Indian Buffalo and American Buffalo showed that the nucleotide sites at positions 64, 65, 68, 223, 236, 422, 527 and 628 of the nine MAP IS1311 gene fragments were conformed to the characteristics of type C and type B MAP. Sequence analysis of IS900 gene sequencing results showed that the 169th and 216th nucleotides of the nine MAP IS900 gene fragments were C (cytosine) and A (adenine), and accorded with Type II and type III MAP. The phylogenetic tree of 17 MAP IS900 gene reference sequences from GenBank database with 9 isolates in this study showed that the 9 isolates in this study were all in the type II MAP branch. Blast online analysis was performed on the sequencing results of the three genes. The reference sequences with the highest homology with the isolates obtained in this study were all type II MAP, and the homology was higher than 98%. In conclusion, all the 9 MAP isolates were type II MAP. 【Conclusion】 To the best of our knowledge, this was the first isolate of the MAP type Ⅱ strains in sheep in Inner Mongolia.

Key words: Mycobacterium avium subsp. paratuberculosis, isolated, typing, sheep, Inner Mongolia

赵维宏, 韩文雄, 杨波, 孟维康, 柴海亮, 马懿敏, 张占胜, 王利峰, 王艳, 王明圆, 张姗, 丁玉林, 王金玲, 吉林台, 王凤龙, 赵丽, 刘永宏. 内蒙古地区羊源副结核分枝杆菌的分离与基因分型[J]. 中国农业科学, 2023, 56(6): 1204-1214.

ZHAO WeiHong, HAN WenXiong, YANG Bo, MENG WeiKang, CHAI HaiLiang, MA YiMin, ZHANG ZhanSheng, WANG LiFeng, WANG Yan, WANG MingYuan, ZHANG Shan, DING YuLin, WANG JinLing, JIRINTAI Sulijid, WANG FengLong, ZHAO Li, LIU YongHong. Isolation and Genotyping of Mycobacterium avium subsp. paratuberculosis from Sheep in Inner Mongolia[J]. Scientia Agricultura Sinica, 2023, 56(6): 1204-1214.

图/表 9

表1

表2

表3

图1

图2

表4

图3

表5

表6

参考文献 38

[1]	STABEL J R, BANNANTINE J P, HOSTETTER J M. Comparison of sheep, goats, and calves as infection models for Mycobacterium avium subsp. paratuberculosis. Veterinary Immunology and Immunopathology, 2020, 225: 110060. doi:10.1016/j.vetimm.2020.110060. doi: 10.1016/j.vetimm.2020.110060
[2]	WINDSOR P A. Managing control programs for ovine caseous lymphadenitis and paratuberculosis in Australia, and the need for persistent vaccination. Veterinary Medicine (Auckland, N Z), 2014, 5: 11-22. doi:10.2147/VMRR.S44814. doi: 10.2147/VMRR.S44814
[3]	STEVENSON K, HUGHES V M, DE JUAN L, INGLIS N F, WRIGHT F, SHARP J M. Molecular characterization of pigmented and nonpigmented isolates of Mycobacterium avium subsp. paratuberculosis. Journal of Clinical Microbiology, 2002, 40(5): 1798-1804. doi:10.1128/jcm.40.5.1798-1804.2002. doi: 10.1128/jcm.40.5.1798-1804.2002
[4]	PURDIE A C, PLAIN K M, BEGG D J, DE SILVA K, WHITTINGTON R J. Gene expression profiles during subclinical Mycobacterium avium sub species paratuberculosis infection in sheep can predict disease outcome. Scientific Reports, 2019, 9: 8245. doi:10.1038/s41598-019-44670-w. doi: 10.1038/s41598-019-44670-w
[5]	RASMUSSEN P, BARKEMA H W, MASON S, BEAULIEU E, HALL D C. Economic losses due to Johne's disease (paratuberculosis) in dairy cattle. Journal of Dairy Science, 2021, 104(3): 3123-3143. doi:10.3168/jds.2020-19381. doi: 10.3168/jds.2020-19381 pmid: 33455766
[6]	CASTELLANOS E, ARANAZ A, ROMERO B, DE JUAN L, ALVAREZ J, BEZOS J, RODRÍGUEZ S, STEVENSON K, MATEOS A, DOMÍNGUEZ L. Polymorphisms in gyrA and gyrB genes among Mycobacterium avium subsp. paratuberculosis type I, II, and III isolates. Journal of Clinical Microbiology, 2007, 45(10): 3439-3442. doi:10.1128/jcm.01411-07. doi: 10.1128/jcm.01411-07
[7]	BRYANT J M, THIBAULT V C, SMITH D G E, MCLUCKIE J, HERON I, SEVILLA I A, BIET F, HARRIS S R, MASKELL D J, BENTLEY S D, PARKHILL J, STEVENSON K. Phylogenomic exploration of the relationships between strains of Mycobacterium avium sub species paratuberculosis. BMC Genomics, 2016, 17: 79. doi:10.1186/s12864-015-2234-5. doi: 10.1186/s12864-015-2234-5
[8]	WIBBERG D, PRICE-CARTER M, RÜCKERT C, BLOM J, MÖBIUS P. Complete Genome Sequence of Ovine Mycobacterium avium subsp. paratuberculosis Strain JIII-386 (MAP-S/type III) and Its Comparison to MAP-S/type I, MAP-C, and M. avium Complex Genomes. Microorganisms, 2020, 9(1): 70. doi:10.3390/microorganisms9010070. doi: 10.3390/microorganisms9010070
[9]	HODGEMAN R, MANN R, SAVIN K, DJITRO N, ROCHFORT S, RODONI B. Molecular characterisation of Mycobacterium avium subsp. paratuberculosis in Australia. BMC Microbiology, 2021, 21(1): 101. doi:10.1186/s12866-021-02140-2. doi: 10.1186/s12866-021-02140-2
[10]	BHAT A M, MALIK H U, CHAUBEY K K, HUSSAIN T, MIR A Q, NABI S U, GUPTA S, SINGH S V. Bio-typing of Mycobacterium avium sub species paratuberculosis isolates recovered from the Himalayan sheep and goats. Tropical Animal Health and Production, 2021, 53(2): 237. doi:10.1007/s11250-021-02682-7. doi: 10.1007/s11250-021-02682-7
[11]	牟巍, 蒋菲, 何宇, 丁家波, 彭小兵, 蒋玉文. 副结核分枝杆菌map0862基因的克隆及其在大肠杆菌中的表达. 中国兽药杂志, 2010, 44(8): 10-12, 21.
	MU W, JIANG F, HE Y, DING J B, PENG X B, JIANG Y W. Cloning and expression of Mycobacterium avium subsp. paratuberculosis gene map0862 in E.coli. Chinese Journal of Veterinary Drug, 2010, 44(8): 10-12, 21. (in Chinese)
[12]	刘虹秀, 程玉笛, 党光辉, 李田田, 李鹤, 崔子寅, 宋宁宁, 陈利苹, 刘思国. 牛副结核分枝杆菌的分离及鉴定. 中国预防兽医学报, 2018, 40(12): 1177-1180.
	LIU H X, CHENG Y D, DANG G H, LI T T, LI H, CUI Z Y, SONG N N, CHEN L P, LIU S G. Isolation and identification of Mycobacterium avium subsp.paratuberculosis from cattle. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(12): 1177-1180. (in Chinese)
[13]	高建鹏, 赵鑫, 高静雯, 李慧, 张莉, 齐亚银. 马鹿源副结核分枝杆菌的分离鉴定. 动物医学进展, 2019, 40(9): 128-132. doi:10.16437/j.cnki.1007-5038.2019.09.025. doi: 10.16437/j.cnki.1007-5038.2019.09.025
	GAO J P, ZHAO X, GAO J W, LI H, ZHANG L, QI Y Y. Isolation and identification of Mycobacterium avium subsp. paratuberculosis from red deer(Cervus elaphus). Progress in Veterinary Medicine, 2019, 40(9): 128-132. doi:10.16437/j.cnki.1007-5038.2019.09.025. (in Chinese) doi: 10.16437/j.cnki.1007-5038.2019.09.025
[14]	洪都孜·波拉提, 魏玉荣, 孟肖潇, 杨学云, 古努尔·吐尔逊, 李建军, 吴建勇. 绵羊副结核分枝杆菌的分离与分子分型. 中国预防兽医学报, 2021, 43(2): 134-138.
	HONGDUZI BOLATI, WEI Y R, MENG X X, YANG X Y, GUNUER TUERXUN, LI J J, WU J Y. Isolation and molecular typing of sheep Mycobacterium avium sub species paratuberculosis. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(2): 134-138. (in Chinese)
[15]	宋先忱, 李冰, 朱延旭, 张兴会, 王世泉, 郭伶, 徐鹏, 郭维军, 刘孝刚. 辽宁绒山羊副结核分枝杆菌的分离鉴定及分子生物学检测. 中国兽医杂志, 2018, 54(6): 32-34.
	SONG X C, LI B, ZHU Y X, ZHANG X H, WANG S Q, GUO L, XU P, GUO W J, LIU X G. Isolation, identification and molecular biological detection of Mycobacterium tuberculosis from Liaoning cashmere goats. Chinese Journal of Veterinary Medicine, 2018, 54(6): 32-34. (in Chinese)
[16]	AHLSTROM C, BARKEMA H W, STEVENSON K, ZADOKS R N, BIEK R, KAO R, TREWBY H, HAUPSTEIN D, KELTON D F, FECTEAU G, LABRECQUE O, KEEFE G P, MCKENNA S L B, DE BUCK J. Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level. BMC Genomics, 2015, 16: 161. doi:10.1186/s12864-015-1387-6. doi: 10.1186/s12864-015-1387-6
[17]	MARCHESI J R, SATO T, WEIGHTMAN A J, MARTIN T A, FRY J C, HIOM S J, DYMOCK D, WADE W G. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Applied and Environmental Microbiology, 1998, 64(2): 795-799. doi:10.1128/AEM.64.2.795-799.1998. doi: 10.1128/AEM.64.2.795-799.1998 pmid: 9464425
[18]	MARSH I, WHITTINGTON R, COUSINS D. PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium aviumsubsp.paratuberculosisand Mycobacterium avium subsp. avium based on polymorphisms in IS1311. Molecular and Cellular Probes, 1999, 13(2): 115-126. doi:10.1006/mcpr.1999.0227. doi: 10.1006/mcpr.1999.0227
[19]	COLLINS D M, DE ZOETE M, CAVAIGNAC S M. Mycobacterium avium subsp. paratuberculosis strains from cattle and sheep can be distinguished by a PCR test based on a novel DNA sequence difference. Journal of Clinical Microbiology, 2002, 40(12): 4760-4762. doi:10.1128/JCM.40.12.4760-4762.2002. doi: 10.1128/JCM.40.12.4760-4762.2002
[20]	STABEL J R. An improved method for cultivation of Mycobacterium paratuberculosis from bovine fecal samples and comparison to three other methods. International Dental Journal, 1997, 9(4): 375-380. doi:10.1177/104063879700900406. doi: 10.1177/104063879700900406
[21]	WINDSOR P A. Paratuberculosis in sheep and goats. Veterinary Microbiology, 2015, 181(1/2): 161-169. doi:10.1016/j.vetmic.2015.07.019. doi: 10.1016/j.vetmic.2015.07.019
[22]	蔡珠明, 党光辉, 臧鑫鑫, 邵明珠, 唐阳阳, 崔子寅, 宋宁宁, 刘思国. 黑龙江某规模化牛场副结核分枝杆菌的分离与鉴定. 中国预防兽医学报, 2020, 42(11): 1177-1180.
	CAI Z M, DANG G H, ZANG X X, SHAO M Z, TANG Y Y, CUI Z Y, SONG N N, LIU S G. Isolation and identification of Mycobacterium avium subsp.paratuberculosis from a large-scale cattle farm in Heilongjiang. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(11): 1177-1180. (in Chinese)
[23]	常塔娜, 田莉莉, 许芳, 樊晓旭, 孙淑芳, 范伟兴. 甘肃省奶牛副结核分枝杆菌的调查与分离鉴定. 中国兽医科学, 2020, 50(7): 874-879. doi:10.16656/j.issn.1673-4696.2020.0123. doi: 10.16656/j.issn.1673-4696.2020.0123
	CHANG T N, TIAN L L, XU F, FAN X X, SUN S F, FAN W X. Investigation and isolation of Mycobacterium avium subsp. paratuberculosis in a cattle farm in Gansu Province, China. Chinese Veterinary Science, 2020, 50(7): 874-879. doi:10.16656/j.issn.1673-4696.2020.0123. (in Chinese) doi: 10.16656/j.issn.1673-4696.2020.0123
[24]	SZTEYN J, LIEDTKE K, WISZNIEWSKA-ŁASZCZYCH A, WYSOK B, WOJTACKA J. Isolation and molecular typing of Mycobacterium avium subsp. paratuberculosis from faeces of dairy cows. Polish Journal of Veterinary Sciences, 2020, 23(3): 415-422. doi:10.24425/pjvs.2020.134686. doi: 10.24425/pjvs.2020.134686
[25]	PARK H T, PARK H T, PARK W B, KIM S, HUR T Y, JUNG Y H, YOO H S. Genetic diversity of bovine Mycobacterium avium subsp. paratuberculosis discriminated by IS1311 PCR-REA, MIRU-VNTR, and MLSSR genotyping. Journal of Veterinary Science, 2018, 19(5): 627-624. DOI: https://doi.org/10.4142/jvs.2018.19.5.627. doi: https://doi.org/10.4142/jvs.2018.19.5.627
[26]	FAWZY A, ZSCHÖCK M, EWERS C, EISENBERG T. Genotyping methods and molecular epidemiology of Mycobacterium avium subsp. paratuberculosis (MAP). International Journal of Veterinary Science and Medicines, 2018, 6(2): 258-264. doi:10.1016/j.ijvsm.2018.08.001. doi: 10.1016/j.ijvsm.2018.08.001
[27]	WHITTINGTON R, MARSH I, CHOY E, COUSINS D. Polymorphisms in IS1311, an insertion sequence common to Mycobacterium avium and M. avium subsp. paratuberculosis, can be used to distinguish between and within these species. Molecular and Cellular Probes, 1998, 12(6): 349-358. doi:10.1006/mcpr.1998.0194. doi: 10.1006/mcpr.1998.0194
[28]	ZHAO L, WANG Y, WANG J L, ZHAO W H, CHENG H X, MA Y M, CHAI H L, ZHANG Z S, WANG L F, MIAO Z Q, DING Y L, SULIJID J, DANG G H, LIU S Y, WANG F L, LIU S G, LIU Y H. Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China. PLoS ONE, 2021, 16(9): e0256628. doi:10.1371/journal.pone.0256628. doi: 10.1371/journal.pone.0256628
[29]	COLLINS M T. Diagnosis of paratuberculosis. Veterinary Clinics of North America Food Animal Practice, 1996, 12(2): 357-371. DOI: 10.1016/s0749-0720(15)30411-4. doi: 10.1016/s0749-0720(15)30411-4
[30]	LIAPI M, LEONTIDES L, KOSTOULAS P, BOTSARIS G, IACOVOU Y, REES C, GEORGIOU K, SMITH G C, NASEBY D C. Bayesian estimation of the true prevalence of Mycobacterium avium subsp. paratuberculosis infection in Cypriot dairy sheep and goat flocks. Small Ruminant Research, 2011, 95(2/3): 174-178. doi:10.1016/j.smallrumres.2010.09.010. doi: 10.1016/j.smallrumres.2010.09.010
[31]	HOSSEINIPORGHAM S, CUBEDDU T, ROCCA S, SECHI L A. Identification of Mycobacterium avium subsp. paratuberculosis (MAP) in sheep milk, a zoonotic problem. Microorganisms, 2020, 8(9): E1264. doi:10.3390/microorganisms8091264. doi: 10.3390/microorganisms8091264
[32]	DZIEDZINSKA R, SLANA I. Mycobacterium avium subsp. paratuberculosis-an overview of the publications from 2011 to 2016. Current Clinical Microbiology Reports, 2017, 4(1): 19-28. doi:10.1007/s40588-017-0054-x. doi: 10.1007/s40588-017-0054-x
[33]	彭永. 副结核分枝杆菌分离及小鼠感染模型的建立[D]. 北京: 中国兽医药品监察所, 2018.
	PENG Y. Isolation of Mycobacterium avium subsp. paratuberculosis and the establishment of mouse infection model[D]. Beijing: China Institute of Veterinary Drug Control, 2018. (in Chinese)
[34]	高建鹏. 塔河马鹿源副结核分枝杆菌的分离鉴定及部分生物学特性研究[D]. 石河子: 石河子大学, 2019.
	GAO J P. Isolation, identification and some biological characteristics study of Mycobacterium avium Subsp.Paratuberculosis from Tarim red deer (Cervus elaphus yarkandensis) source[D]. Shihezi: Shihezi University, 2019. (in Chinese)
[35]	RADOSEVICH T J, REINHARDT T A, LIPPOLIS J D, BANNANTINE J P, STABEL J R. Proteome and differential expression analysis of membrane and cytosolic proteins from Mycobacterium avium subsp. paratuberculosis strains K-10 and 187. Journal of Bacteriology, 2007, 189(3): 1109-1117. doi:10.1128/JB.01420-06. doi: 10.1128/JB.01420-06
[36]	马呼和, 东风, 杨万科. 羊传染性腹泻病的调查及防治试验. 畜禽业, 2015(2): 72-74. doi:10.19567/j.cnki.1008-0414.2015.02.048. doi: 10.19567/j.cnki.1008-0414.2015.02.048
	MA H H, DONG F, YANG W K. Investigation and control of infectious diarrhea in sheep. Livestock and Poultry Industry, 2015(2): 72-74. doi:10.19567/j.cnki.1008-0414.2015.02.048. (in Chinese) doi: 10.19567/j.cnki.1008-0414.2015.02.048
[37]	ALCEDO K P, THANIGACHALAM S, NASER S A. RHB-104 triple antibiotics combination in culture is bactericidal and should be effective for treatment of Crohn's disease associated with Mycobacterium paratuberculosis. Gut Pathogens, 2016, 8: 32. doi:10.1186/s13099-016-0115-3. doi: 10.1186/s13099-016-0115-3
[38]	SHIN S J, COLLINS M T. Thiopurine drugs azathioprine and 6-mercaptopurine inhibit Mycobacterium paratuberculosis growth in vitro. Antimicrobial Agents and Chemotherapy, 2008, 52(2): 418-426. doi:10.1128/AAC.00678-07. doi: 10.1128/AAC.00678-07

编号 No.	采集地点 Place of collection	采集时间 Time of collection	物种 Species
1	呼和浩特Hohhot	2018.5	绵羊Sheep
2-4	呼和浩特Hohhot	2016.12	山羊Gost
5-7	锡林郭勒Xilingol	2015.4	绵羊Sheep
8-10	锡林郭勒Xilingol	2015.5	山羊Gost
11-12	阿拉善Alxa	2021.4	山羊Gost
A1-A16	阿拉善Alxa	2020.6	山羊Gost

引物 Primer	引物序列 Primer Sequence (5'→3')	大小Size (bp)	参考文献 References
IS900-F	GTTCGGGGCCGTCGCTTAGG	400	[17]
IS900-R	GAGGTCGATCGCCCACGTGA	400	[17]
IS1311-F	GCGTGAGGCTCTGTGGTGAA	608	[18]
IS1311-R	ATGACGACCGCTTGGGAGAC	608	[18]
DMC529	TTGACAACGTCATTGAGAATCC	146（Ⅰ型Type Ⅰ） 310（Ⅱ型Type Ⅱ）	[19]
DMC531	TCTTATCGGACTTCTTCTGGC
DMC533	CGGATTGACCTGCGTTTCAC

序号 No.	基因 Name of gene	菌株名称 Name of strain	GenBank登录号 GenBank accession number	亚型 Subtypes
1	IS900	ATCC 53950	AF305073	Ⅱ型Type Ⅱ
2		A1	MN928512	Ⅱ型Type Ⅱ
3		FA0010	MT017597	Ⅱ型Type Ⅱ
4		JTC 1285	NZ_AGAL01000886	Ⅱ型Type Ⅱ
5		316F	AF416985	Ⅱ型Type Ⅱ
6		21P	FJ775181	Ⅰ型Type Ⅰ
7		不详Unknown	AJ250015	Ⅱ型Type Ⅱ
8		不详Unknown	AJ250022	Ⅱ型Type Ⅱ
9		Ben	X16293	Ⅱ型Type Ⅱ
10		Case-2	MW546836	Ⅱ型Type Ⅱ
11		Case-7	MW546841	Ⅲ型Type Ⅲ
12		JII-1961	CP022105	Ⅱ型Type Ⅱ
13		JIII-386	CP042454	Ⅲ型Type Ⅲ
14		MAPK_CN4-13	CP033910	Ⅱ型Type Ⅱ
15		MAPK_CN9-15	CP033427	Ⅱ型Type Ⅱ
16		S397	CP053749	Ⅲ型Type Ⅲ
17		Telford	CP033688	Ⅰ型Type Ⅰ
18	IS1311	JD97/1-2	AJ223975	S型Type S
19		316V	AJ223974	C型Type C
20		98/1010	AJ308375	美国野牛型USA Bison type
21		'Bison type' S5	EF514838	印度野牛型Indian Bison type

菌株名 Name of strain	亚型 Subtypes	IS1311位点 Position on IS1311
菌株名 Name of strain	亚型 Subtypes	64	65	68	223	236	422	527	628
JD97/1-2	S型Type S	T	G	C	C	C	C	A	C
316V	C型Type C	T	G	C	Y	C	C	A	C
98/1010	B型美国野牛型 (Type B) USA Bison type	T	G	C	T	C	C	A	C
'Bison type' S5	B型印度野牛型 (Type B) Indian Bison type	-	-	C	T	C	N	N	N
MAP-NM1至MAP-NM9 MAP-NM1 to MAP-NM9	本研究分离株 The isolates in this study	T	G	C	T	C	C	A	C

序号 No.	菌株名 Name of strain	基因名称 Name of gene	同源性最高序列名称 Name of the most homologous sequence	GenBank登录号 GenBank accession number	同源性 Nucleotide homology (%)	亚型 Subtypes
1	MAP-NM1	IS900	Case-3	MW546837.1	99.45	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.47	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	98.93	Ⅱ型Type Ⅱ
2	MAP-NM2	IS900	DSM 44135	CP053068.1	99.46	Ⅱ型Type Ⅱ
		IS1311	Case-2	MW546850.1	99.31	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546844.1	98.94	Ⅱ型Type Ⅱ
3	MAP-NM3	IS900	DSM 44135	CP053068.1	99.45	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.65	Ⅱ型Type Ⅱ
		DMC	Case-4	MW546845.1	99.29	Ⅱ型Type Ⅱ
4	MAP-NM4	IS900	DSM 44135	CP053068.1	99.72	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.64	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	98.58	Ⅱ型Type Ⅱ
5	MAP-NM5	IS900	DSM 44135	CP053068.1	99.73	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.66	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546844.1	99.28	Ⅱ型Type Ⅱ
6	MAP-NM6	IS900	DSM 44135	CP053068.1	100.00	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.64	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	99.29	Ⅱ型Type Ⅱ
7	MAP-NM7	IS900	DSM 44135	CP053068.1	99.45	Ⅱ型Type Ⅱ
		IS1311	Case-4	MW546852.1	99.48	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546844.1	99.29	Ⅱ型Type Ⅱ
8	MAP-NM8	IS900	DSM 44135	CP053068.1	98.91	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.82	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	99.29	Ⅱ型Type Ⅱ
9	MAP-NM9	IS900	DSM 44135	CP053068.1	99.18	Ⅱ型Type Ⅱ
		IS1311	Case-5	MW546853.1	99.65	Ⅱ型Type Ⅱ
		DMC	Case-3	MW546851.1	98.93	Ⅱ型Type Ⅱ

内蒙古地区羊源副结核分枝杆菌的分离与基因分型

Isolation and Genotyping of Mycobacterium avium subsp. paratuberculosis from Sheep in Inner Mongolia

RichHTML

PDF

赞

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 38

相关文章 15

Metrics

本文评价

推荐阅读 0

分离株 Isolate	PCR结果 Result of PCR	IS900序列分析 Sequence analysis of IS900	IS1311序列分析 Sequence analysis of IS1311	BLAST分析 BLAST analysis	进化树 Evolutionary tree	结论 Conclusion
MAP-NM1	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM2	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM3	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM4	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM5	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM6	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM7	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM8	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ
MAP-NM9	+	Ⅱ/Ⅲ	B/C	Ⅱ	Ⅱ	Ⅱ

[1]	高云洁, 郭若男, 陈春文, 段祎凡, 张镇均, 聂蝉蝉, 李梦雨, 王慧, 冯婷婷, 崔莹莹, 党光辉, 刘思国. 羊副结核分枝杆菌间接ELISA抗体检测方法的建立与初步应用[J]. 中国农业科学, 2026, 59(3): 655-667.
[2]	贾子成, 秦冰雨, 马彩英, 杜勇, 刘统高, 薛瑞林, 王小龙, 周世卫. 饲粮营养水平对双羔陕北白绒山羊母子一体化生产性能和瘤胃微生物的影响[J]. 中国农业科学, 2026, 59(3): 668-686.
[3]	王妞, 史昕冉, 张卫东, 王昕. FGF5和FGF21对绒山羊毛乳头细胞增殖的影响[J]. 中国农业科学, 2025, 58(4): 819-830.
[4]	高奇奇, 韩哲群, 张海睿, 南珊珊, 黄炎炎, 朱恒侠, 武雪萍. 引黄灌区次生盐渍化对土壤质量与耕地产能的影响[J]. 中国农业科学, 2025, 58(20): 4272-4284.
[5]	晏川博, 宫平, 郑文新, 陈新文, 郭雷风. 复杂场景下基于改进YOLOv11的羊爬跨行为识别研究[J]. 中国农业科学, 2025, 58(18): 3783-3798.
[6]	陈凡若, 张嘉俊, 鹿萍, 崔宁, 崔莹莹, 崔子寅, 党光辉, 刘思国. 副结核分枝杆菌免疫原蛋白的筛选及免疫保护效果评价[J]. 中国农业科学, 2024, 57(6): 1204-1214.
[7]	池润清, 韩海银, 王鹏, 李凯扬, 储明星, 刘玉芳. 雌激素介导circZNF423作为ceRNA调控oar-miR-541-3p/CALM3通路促进绵羊成肌细胞增殖[J]. 中国农业科学, 2024, 57(3): 597-612.
[8]	徐乐, 张德权, 古明辉, 杨琦, 李少博, 陈鹏羽, 李欣, 陈丽. 中式烹饪羊肉气味指纹差异解析[J]. 中国农业科学, 2024, 57(24): 4964-4977.
[9]	周晓倩, 李晓贝, 张艳梅, 周昌艳, 任佳丽, 赵晓燕. 基于GC-IMS和GC×GC-ToF-MS技术分析产地对羊肚菌挥发性风味成分的影响[J]. 中国农业科学, 2024, 57(22): 4553-4567.
[10]	何军敏, 毛静艺, 魏晨, 任一帆, 张果平, 田可川, 刘桂芬. 基于miRNA测序数据解析苏博美利奴羊毛囊发育分子机制[J]. 中国农业科学, 2024, 57(19): 3917-3935.
[11]	陈恒光, 裴晓蒙, 夏雨婷, 刘静, 茆达干. 围产期日粮添加姜黄素对山羊生产性能、血液指标及炎症因子基因表达的影响[J]. 中国农业科学, 2024, 57(12): 2483-2496.
[12]	张迎鑫, 杨敏, 白雪兵, 陈畅, 吴睿智, 杨平, 陈秋生. 羊腧穴Telocytes形态特征及其与周围结构的关系[J]. 中国农业科学, 2023, 56(7): 1417-1428.
[13]	任国栋, 郝小燕, 张暄梓, 刘森, 张宏祥, 田光元, 张建新. 胍基乙酸和甜菜碱对羔羊生长性能、瘤胃发酵和血液代谢的影响[J]. 中国农业科学, 2023, 56(4): 766-778.
[14]	王鹏, 刘子嶷, 刘玉芳, 储明星. miR-535靶向GAB2基因通过激活PI3K/AKT信号通路促进山羊颗粒细胞增殖[J]. 中国农业科学, 2023, 56(23): 4757-4771.
[15]	郭泽媛, 杜张胜, 张雅琦, 陈春路, 马晓燕, 成颖, 王锴, 吕丽华. Smad7介导TGF-β信号通路对绵羊卵泡颗粒细胞增殖的影响[J]. 中国农业科学, 2023, 56(13): 2597-2608.