山羊淋巴结炎病原菌的分离鉴定及多重PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2017.02.016

摘要/Abstract

摘要：

旨在对重庆地区山羊体表型淋巴结炎病原菌进行分离鉴定，并建立相应检测方法。无菌采集山羊患病部位脓液，进行病原菌分离鉴定，依据菌株16S RNA基因序列构建系统进化发育树，同时设计特异性引物，建立PCR检测方法。结果表明，从病料中主要分离到3种病原菌，分别为伪结核棒状杆菌、化脓隐秘杆菌和金黄色葡萄球菌；依据伪结核棒状杆菌磷脂酶D、化脓隐秘杆菌16S RNA和金黄色葡萄球菌nuc保守核苷酸序列设计引物，建立的多重PCR检测方法，可以从3种菌株混合基因组中扩增出大小分别为507、1 378和278 bp的对应菌株特异性序列，该方法最低检出浓度为10³ cfu•mL^-1；对采集的80份临床样品进行多重PCR检测，发现伪结核棒状杆菌与金黄色葡萄球菌混合感染检出率为12.5%，伪结核棒状杆菌与化脓隐秘杆菌混合感染检出率为3.75%，三种菌混合感染检出率为7.5%。成功分离到山羊淋巴结炎的3种主要病原菌，并建立多重PCR检测方法，为该病的防控及治病机制研究奠定了基础。

关键词: 山羊, 淋巴结炎, 多重PCR, 伪结核棒状杆菌, 化脓隐秘杆菌, 金黄色葡萄球菌

Abstract:

The purpose of this paper was to isolate and identify the pathogenic bacteria of lymphadenitis in goat of Chongqing area and establish the test method. Sterile separation operation was carried out to isolate pathogenic bacteria from fester of the goat. The phylogenetic tree was founded according to the gene sequence of 16S RNA. And the specific primers were designed to establish PCR test method. The result showed that Corynebacterium pseudotuberculosis, Arcanobacterium pyogenes and Staphylococcus aureus were isolated from the samples, and three pairs of specific oligonucleotide primers were respectively designed according to the gene sequence of Phospholipases D, 16S RNA and nuc for multiple PCR detection. The multiple PCR detection showed that a fragment of 507, 1 378 and 278 bp amplified from the mixture genome .The sensitivity test showed that the multiplex PCR could detect minimum detectable concentration of 10³ cfu•mL^-1.The detection of 80 clinical samples indicated that, the detection rates of mixed infections of Corynebacterium pseudotuberculosis and Staphylococcus aureus, Corynebacterium pseudotuberculosis and Arcanobacterium pyogenes, three kinds of bacterias were 12.5%, 3.75% and 7.5%, respectively. Three pathogenic bacterias of lymphadenitis in goat were isolated successfully and the multiplex PCR detection method was detected in this study, it laid a foundation for the research of the prevention and control and the pathogenesis for lymphadenitis in goat.

Key words: goat, lymphadenitis, multiplex PCR, Corynebacterium pseudotuberculosis, Arcanobacterium pyogenes, Staphylococcus aureus

中图分类号:

许国洋，付利芝，杨金龙，杨柳，徐登峰，张邑帆，沈克飞，张素辉. 山羊淋巴结炎病原菌的分离鉴定及多重PCR检测方法的建立[J]. 畜牧兽医学报, 2017, 48(2): 324-330.

XU Guo-yang, FU Li-zhi, YANG Jin-long, YANG Liu,XU Deng-feng, ZHANG Yi-fan, SHEN Ke-fei, ZHANG Su-hui. Isolation and Identification of Pathogenic Bacteria of Lymphadenitis in Goat and Establishment of Multiplex PCR Detection Method[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(2): 324-330.

参考文献

［1］邢福珊,张彦明,王韡，等.羊干酪样淋巴结炎PCR检测方法的建立［J］.中国兽医学报,2008,28(2):149-151.
XING F S, ZHANG Y M , WANG W, et al. Establishment of CLA PCR detection method［J］. Chinese Journal of Veterinary Science, 2008,28(2):149-151. (in Chinese)
［2］蔡俊波,王爱玲,李海敏，等.陕南白山羊伪结核棒状杆菌分离鉴定及灭活铝胶疫苗研制［J］.动物医学进展,2016,37(7):49-52.
CAI J B, WANG A L, LI H M, et al. Isolation and identification of Corynebacterium pseudotuberculosis from white goat in south Shaanxi and development of an inactivated vaccine with aluminum hydroxide adjuvant［J］. Progress in Veterinary Medicine, 2016,37(7):49-52. (in Chinese)
［3］COLOM-CADENA A, VELARED R, SALINAS J, et al. Management of a caseous lymphadenitis outbreak in a new Iberian ibex (Capra pyrenaica) stock reservoir［J］. Acta Vet Scand, 2014,56:83.
［4］OREIBY A F, HEGAZY Y M, OSMAN S A, et al. Caseous lymphadenitis in small ruminants in Egypt. Clinical, epidemiological and prophylactic aspects ［J］. Tierarztl Prax Ausg G Grosstiere Nutztiere,2014, 42(5): 271-277.
［5］OLIVERIA M, BARROCO C, MOTTOLA C, et al. First report of Corynebacterium pseudotuberculosis from caseous lymphadenitis lesions in Black Alentejano pig (Sus scrofa domesticus)［J］. BMC Vet Res, 2014,10: 218.
［6］BASTOS B L, LOUREIRO D, RAYNAL J T, et al. Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep ［J］. BMC Vet Res, 2013, 9:254.
［7］邢福珊,张彦明,郭燕，等.山羊干酪性淋巴结炎病原的分离鉴定［J］.中国兽医科技,2003,33(7):65-67．
XING Ｆ S, ZHANG Ｙ M, GUO Ｙ, et al. Isolation and identification of pathogenic bacteria of lymphadenitis in goat［J］.Chinese Journal of Veterinary Science and Technology, 2003,33(7):65-67．(in Chinese)
［8］刘吉山,张韩杰,王艳，等.波尔山羊干酪样淋巴结炎的病原分离与初步鉴定［J］.动物医学进展,2005,26(2):101-103.
LIU J S,ZHANG H J,WANG Y, et al. Isolation and idendification of pathogeny of boer goat caseous lymphadenitis［J］.Progress in Veterinary Medicine, 2005, 26(2):101-103. (in Chinese)
［9］张素辉，付利芝，许国洋，等.山羊源化脓隐秘杆菌的分离与鉴定及自然感染病例的病理学观察［J］.畜牧兽医学报,2015,46(12):2251-2257.
ZHANG S H, FU L Z, XU G Y, et al. Isolation and identification of Arcanobacterium pyogenes from goats and pathological of naturally infected case［J］.Acta Veterinaria et Zootechnica Sinica,2015,46(12):2251-2257. (in Chinese)
［10］MALONE F E, FEE S A, KAMP E M, et al. A serological investigation of caseous lymphadenitis in four flocks of sheep ［J］. Ir Vet J, 2006, 59(1):19-21.
［11］WASHBURN K E, BISSETT W T, FAJT V R, et al. Comparison of three treatment regimens for sheep and goats with caseous lymphadenitis ［J］. J Am Vet Med Assoc, 2009,234(9):1162-1166.
［12］KUMAR J, SINGH F, TRIPATHI B N, et al. Epidemiological, bacteriological and molecular studies on caseous lymphadenitis in Sirohi goats of Rajasthan, India［J］. Trop Anim Health Prod, 2012, 44(7):1319-1322.
［13］潘淑惠,王璇,万晴姣，等.山羊伪结核棒状杆菌感染途径及病理变化观察［J］.上海畜牧兽医通讯,2007(5):24-25.
PAN S Ｈ,WANG X,WAN Q J, et al. The route of infection and pathological change observation of Corynebacterium pseudotuberculosis in goat［J］. Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2007(5):24-25. (in Chinese)
［14］ZAVOSHTI F R, KHOOJINE A B, HELAN J A, et al. Frequency of caseous lymphadenitis (CLA) in sheep slaughtered in an abattoir in Tabriz: comparison of bacterial culture and pathological study ［J］ . Comp Clin Path, 2012, 21(5): 667-671.
［15］JORDAN D, SETANCE C B, SPOONCER W F, et al. Inspection of lymph nodes for caseous lymphadenitis and its effect on the density of microbes on sheep carcasses ［J］. Meat Sci, 2012, 92(4):837-840.
［16］BASTOS B L, MEYER R, GUIMARES J E. Haptoglobin and fibrinogen concentrations and leukocyte counts in the clinical investigation of caseouslymphadenitis in sheep ［J］. Vet Clin Pathol,2011, 40(4): 496-503.
［17］张素辉，黄勇富，付利芝，等.山羊源奥斯陆莫拉菌的分子鉴定与分析［J］. 中国人兽共患病学报, 2014, 30(1):12-16.
ZHANG S H, HUANG Y F, FU L Z, et al. Isolation and identification of Moraxella osloensis in goat［J］. Chinese Journal of Zoonoses, 2014, 30(1):12-16. (in Chinese)
［18］ESTEVAO B S, GALLARDO A, ABALOS A, et al. Diagnosis of caseous lymphadenitis in sheep from Patagonia ［J］. Rev Argent Microbiol, 2007, 39(1):44-46.
［19］ALMEIDA S, TIWARI S, MARIAON D,et al. The genome anatomy of Corynebacterium pseudotuberculosis VD57 a highly virulent strain causing Caseouslymphadenitis［J］. Stand Genomic Sci, 2016, 11:29.
［20］NAGEL-ALNE G E, VALLE P S, KRONTVEIT R, et al. Caprine arthritis encephalitis and caseous lymphadenitis in goats: use of bulk tank milk ELISAs for herd-level surveillance［J］. Vet Rec, 2015,176(7):173.
［21］KURIAJ K N, MBUTHIA P G, KAMG’ETHE E K, et al. Caseous lymphadenitis in goats: the pathogenesis, incubation period and serological response after experimental infection［J］. Vet Res Comm. 2001, 25(25):89-97.
［22］SOARES S C,TROST E,RAMOS R T J, et al. Genome sequence of Corynebacterium pseudotuberculosis biovar equi strain 258 and prediction of antigenic targets to improve biotechnological vaccine production. (Special Issue: Research on industrial biotechnology within CLIB-Graduate Cluster-Part III.)［J］.J Biotechnol, 2013, 167(2):135-141.
［23］张素辉,付利芝,黄勇富，等.山羊化脓隐秘杆菌的分离鉴定与分析［J］.中国兽医杂志,2015,51(6):37-39.
ZHANG S H, FU L Z, HUANG Y F, et al. Separation identification and analysis of Trueperella (Arcanobacterium) pyogenes in goats［J］. Chinese Journal of Veterinary Medicine, 2015, 51 ( 6 ) :37-39. (in Chinese)
［24］冯峰.山羊化脓隐秘杆菌的分离鉴定及其对小鼠的致病性试验［D］.成都：四川农业大学, 2010.
FENF F. Isolation and Identification of Arcanobacterium pyogenes from goat and it's pathogenicity experiment on mice ［D］.Chengdu：Sichuan Agricultural University，2010. (in Chinese)
［25］SON H, PARK S, BEUCHAT L R, et al. Inhibition of Staphylococcus aureus by antimicrobial biofilms formed by competitive exclusion microorganisms on stainless steel［J］. Int J Food Microbiol, 2016, 238:165-171.
［26］崔新洁.金黄色葡萄球菌诱导牛乳腺上皮细胞凋亡及其信号通路的研究［D］.呼和浩特：内蒙古大学,2013.
CUI X J. The study on apoptosis and its signaling in bovine mammary epithelial cell induced by Staphylococcus aureus ［D］. Hohhot: Inner Mongolia University, 2013. (in Chinese)

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