高分辨率熔解曲线快速检测结核分枝杆菌对链霉素耐药情况的研究

探讨高分辨率熔解曲线（HRM）技术检测结核分枝杆菌对链霉素耐药性的可行性。用传统药物敏感性试验进行结核分枝杆菌链霉素耐药性检测;以Rpsl和rrs的耐药决定区为靶点设计引物进行HRM分析,判断菌株是否对链霉素耐药;以药敏结果为标准,应用SPSS17.0分析两种检测结果之间的符合率;Kappa检验分析两种结果的一致性。药敏结果显示,84株菌株中有20株对链霉素耐药,64株为敏感株;HRM检测结果显示,24株菌株对链霉素耐药,60株为敏感株;以药敏结果为参照,HRM检测结核分枝杆菌对链霉素耐药性的敏感性为75%,特异性为85.9%,经Kappa检验显示,两种方法检测结果具有中度一致性。本研究结果表明HRM可快速检测结核分枝杆菌对链霉素的耐药性,具有较好的灵敏度,在耐药结核病的早期辅助诊断方面具有一定的应用价值。     

细菌耐药性产生的分子机制与耐药基因的快速检测方法

细菌产生耐药性成为临床治疗感染性疾病失败的主要原因.本文综述了由自发基因突变和获得性细菌耐药性产生的分子机制,及目前常用的快速检测细菌耐药基因的方法,包括聚合酶链反应-单链构象多态性分析(PCR-SSCP)、质粒指纹图谱分析和PCR-限制性片段长度多肽性分析(PCR-RFLP).     

特别策划：提高对牛结核病的重视——牛结核病检测诊断技术研究进展

结核病是由结核分枝杆菌（Mycobacterium）引起的人、畜、禽共患的一种慢性传染病,由人结核分枝杆菌（MTB）、牛分枝杆菌（MB）、禽型分枝杆菌（M,avium）、非洲分枝杆菌以及田鼠分枝杆菌（M,microti）所引起,统称这些病原为结核分枝杆菌复合物（或群）。目前已知,结核病除感染人外还能使50多种哺乳动物和25种禽类感染,牛是最敏感的动物。     

海狮非结核分枝杆菌病病例

非结核分枝杆菌（NTM）是指除结核分枝杆菌和麻风分枝杆菌以外的分枝杆菌，如堪萨斯分枝杆菌（M．kansasii）、海分枝杆菌（M．marinum）等。非结核分枝杆菌感染海狮后常导致肺部感染，引起结核样病变（结核结节、干酪样坏死及空洞形成），严重者可累及全身各个脏器。进食被非结核分枝杆菌污染的冷冻海水鱼可能是主要的感染途径。笔者在临床工作中遇到2例海狮非结核分枝杆菌感染的病例，总结经验教训，报告如下。     

结核杆菌多位点环介导等温扩增检测方法的建立

为建立一种快速、高敏感、高特异、鉴别结核分枝杆菌和牛分枝杆菌的方法,本研究根据大多数致病性结核分枝杆菌共有序列esat-6,结核分枝杆菌和牛分枝杆菌gyrB共有特异性序列,以及结核分枝杆菌种特异序列mtp40设计6对特异性引物对痰液中的结核分枝杆菌进行检测,并与鉴别培养基分离培养结果以及常规PCR鉴定结果进行比较。实验结果表明,建立的LAMP检测方法具有很高的特异性。可区分致病性结核分枝杆菌和非致病结核分枝杆菌,也可鉴别结核分枝杆菌和牛分枝杆菌。LAMP检测技术的灵敏度比经典PCR技术高100倍左右,可检测到7拷贝/反应。另外,用3种方法同时检测样品发现,LAMP与细菌培养的符合率为90.91%,LAMP与常规PCR检测结果的符合率为100%。LAMP检测方法可以在流行病学调查及现场快速诊断方面广泛应用,并为临床治疗提供依据。     

牛副结核检疫中的非特异性干扰

给犊牛接种各种分枝杆菌，然后以副结核ＰＰＤ进行变态反应试验，观察各分枝杆菌感染对副结核变态反应的干扰；从各种分枝杆菌感染牛群采取血清进行副结核ＥＬＩＳＡ检测，以观察各分枝杆菌血清对牛副结核ＥＬＩＳＡ的干扰。结果：多种分枝杆菌，特别是鸟胞内分枝杆菌、瘰疬分枝杆菌的感染可干扰副结核变态反应；部分结核牛血清干扰副结核ＥＬＩＳＡ的检测     

microRNAs作为结核分枝杆菌感染标志物的研究进展

微小RNA(microRN.As)是一类长度约为22个核苷酸的非编码单链RNA分子,它参与基因转录后的表达调控,在免疫系统的调控和运行中具有重要作用。结核分枝杆菌是典型的胞内寄生菌,其感染能够引起人畜共患结核病。从microRNAs与结核免疫机制的相关性以及在结核分枝杆菌感染过程中巨噬细胞、外周血单核细胞及血清中microRNAs表达谱变化等方面,进行综述microRNAs作为结核分枝杆菌感染标志物的研究现状,以期为动物结核病检测提供理论参考。     

卡介苗与结核菌野毒株的基因差异的研究进展

人类结核已经存在数千年之久,无一个国家能幸免于难,尤其是第三国家。人们在感染结核病的同时也能感染艾滋病,这给人类的健康带来了巨大的挑战。现阶段,预防结核病的唯一方法就是接种BCG-减毒牛型结核分枝杆菌活疫苗。该疫苗是在1908年,由Leon Calmette和Camile Guerin将一株牛型结核分枝杆菌强毒株经历13年230次传代培养,使其毒力发生变异,成为对人无致病性,而保持了良好的免疫原性的菌株。本文主要是探讨结核分枝杆菌,牛型结核分枝杆菌和卡介苗之间的基因的差异。为卡介苗的改良和结核病的检测提供有益借鉴。     

牛结核病诊断技术的研究进展

牛结核病（bovine tuberculosis）主要是由牛分枝杆菌（Myeobactelium bovis）引起的一种牛的慢性消耗性传染病,该病可通过病畜传染给人类或其他动物。自从1882年Robert Koch首次分离培养并详细描述了结核分枝杆菌以来,人们开始渐渐研究和认识分枝杆菌。到1898年,Smith首次将牛分枝杆菌（M.bovis）和其它的分枝杆菌明确区分开来。牛分枝杆菌主要感染不同种属的温血动物,包括有蹄动物、有袋动物、食肉类动物、灵长类、鳍脚类动物和啮齿类动物在内的50多种哺乳动物,还包括类鹦鹉鸟、石鸽、北美洲乌鸦等20多种禽类。其中牛是最敏感的动物。人对牛结核也很敏感,且很多病例与感染动物有接触史。由于牛和人类的关系（牛奶、牛肉及制品等）较其他动物更为密切,因此约10％左右的人结核病（不同的国家和地区不同有不同的比例）足由牛分枝杆菌引起。     

CRISPR/Cas系统及其在结核分枝杆菌研究中的应用

簇状规则间隔的短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统是一种针对入侵核酸的可遗传性原核适应性免疫系统。CRISPR/Cas系统可以产生高度特异性的基因双链断裂,并通过非同源末端连接（NHEJ）或者同源重组（HR）进行修复,如今已经成为许多生物方便有效的基因组编辑工具。结核分枝杆菌因其基因操作的复杂性,其基因组研究存在诸多困难,因此,CRISPR/Cas系统的出现对结核分枝杆菌的研究具有里程碑式意义。本文围绕CRISPR/Cas系统在结核分枝杆菌中的应用,综述了国内外最新的研究进展,为结核分枝杆菌研究方法的选择提供参考。     

Research Progress on Resistance Mechanisms and Detecting Technology of Mycobacterium tuberculosis

In recent years, the constantly emerging of drug resistant Mycobacterium tuberculosis brings unprecedented pressures to treat tuberculosis. In recent studies, it has found that not only bovine-type Mycobacterium tuberculosis can infect people, but human-type Mycobacterium tuberculosis can also infect cattle, which is also brings threats and challenges to the development of the cattle industry. And how to establish quick, handy, sensitive, highly specific and inexpensive resistance testing methods is the key to control the spread of the disease.In this review, it introduced not only the mechanism of action of isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide and so on, but also the traditional detection methods of drug resistance of mycobacteria (luciferase system, microscopic observation of drug susceptibility, proportion method, Bactec MGIT 960 System)and molecular detection methods (reverse hybriditation-based line probe assay (LiPA), Gene Xpert automatic detection system, PCR-single-strand conformation polymorphism analysis(PCR-SSCP),PCR-restriction fragment length polymorphism(PCR-RFLP), DNA sequencing, gene chip, RNA/RNA mismatch assay, which may be helpful to the further and clinical treatment and research for tuberculosis.     

Characterization of selected genes upregulated in non-tuberculous European wild boar as possible correlates of resistance to Mycobacterium bovis infection

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle and wildlife worldwide. These animal hosts can serve as reservoirs of infection, thus increasing the risk of human exposure and infection. In this study we quantified by RNA macroarray fluorescent hybridization and real-time RT-PCR the mRNA levels of genes differentially expressed in oropharyngeal tonsils and mandibular lymph nodes of three and seven individual non-tuberculous and tuberculous wild boars naturally exposed to M. bovis, respectively. These results demonstrated upregulation of two genes, complement component 3 (C3) and methylmalonyl-CoA mutase (MUT), in the non-tuberculous wild boars. These upregulated genes may contribute to resistance of wild boars to bTB by modifying the innate immunity, which limits the ability of the mycobacterium to infect and persist within macrophages. The C3 and MUT genes, therefore, are likely to be good candidates to study as markers of bTB resistance using functional genomics in animal model systems. Identification of genes upregulated in wild animals resistant to bTB contributes to our understanding of the mechanisms of protective immunity and resistance to mycobacterial organisms.     

牛结核病诊断方法的研究进展

牛结核病是由牛分支杆菌、结核分支杆菌、禽分支杆菌等引起的一类慢性消耗性疾病,是牛各种传染病中致死率最高的疾病之一,该病在人畜之间可以相互传染,很难根治,是影响养牛业和全人类健康的重大疾病,因此控制本病的传播和蔓延迫在眉睫。控制牛结核病的主要措施是及时检疫和根除病牛,定期对牛群进行检疫监控。近年来,随着生命科学的发展,进一步将传统细菌检测法、免疫学检测法和分子生物学检测法3种方法相结合,特别是分子水平上科学技术的发展,使牛结核病的诊断方法逐渐得到完善,同时获得了许多牛分支杆菌的特异性抗原及其基因,对于阐明结核分支杆菌和牛分支杆菌的分子进化和遗传变异规律等方面都具有重要的流行病学意义。     

奶牛结核分枝杆菌rpoB和katG基因突变与多重耐药的相关性

本试验旨在研究奶牛结核分枝杆菌rpoB和katG基因突变与耐药性之间的关系。将临床分离的30株牛源结核分枝杆菌,利用噬菌体生物扩增法（phage amplified biologically assay,PhaB）检测其对利福平（RFP）和异烟肼（INH）的药物敏感性,采用聚合酶链式反应—DNA直接测序法（PCR-DS）扩增rpoB和katG基因并测序,分析rpoB和katG基因突变及其耐药相关性。15株表型耐药菌株中4株单耐RFP,PCR产物直接测序后发现3株在rpoB基因531位点发生突变,突变率为75.00%（3/4）;9株单耐INH菌株中7株发生突变,6株katG基因315位点基因突变,突变率为85.71%(6/7),1株发现463位点密码子基因突变,突变率为14.29%（1/7）;2株耐RFP的同时也对INH耐药,测序发现2株均在rpoB基因的531位点〖JP2〗和katG基因的315位点发生突变。所有菌株均未发现rpoB和katG基因的缺失。rpoB基因531位点和katG基因315位点的突变是RFP和INH产生耐药性的主要机制;检测牛源结核分枝杆菌RFP耐药性的同时可以初步筛选耐多药结核分枝杆菌。     

Bovine tuberculosis is more prevalent in cattle owned by farmers with active tuberculosis in central Ethiopia

A case control study was conducted between October 2004 and April 2005 to determine the prevalence of bovine tuberculosis (BTB) in cattle in central Ethiopia relative to the tuberculosis status of their owners. A total of 174 farmers (87 with active tuberculosis and 87 with no active tuberculosis), and 1041 cattle (506 owned by farmers with active tuberculosis and 535 by farmers without active tuberculosis) were included. The comparative intradermal cervical tuberculin test was used in cattle while clinical symptoms, chest X-ray and Ziehl-Neelsen staining were used for the diagnosis of tuberculosis in the farmers. In addition, mycobacterial culture, biochemical tests, and drug susceptibility tests were performed for the identification Mycobacterium spp. from both humans and cattle. The prevalence of BTB was threefold higher (odds ratio [OR]=4.2, 95% confidence interval [CI]=2.79-6.2) in cattle owned by farmers with active tuberculosis (24.3%) than in those owned by farmers who did not have active tuberculosis (8.6%). Cattle owned by farmers with active tuberculosis were four times more likely to have tuberculosis than cattle owned by farmers with no active tuberculosis. Furthermore, cattle owners who consumed raw milk were at greater risk (chi2=14.1, P<0.001, OR=3.34) of having active tuberculosis than those who consumed boiled milk. Of the 42 human isolates, 31 (74%) were Mycobacterium tuberculosis, seven (16%) were Mycobacterium bovis while four (10%) were considered a typical mycobacteria on the basis of biochemical and drug sensitivity tests. Of the 11 cattle isolates, two (18%) were Mycobacterium tuberculosis, five (46%) Mycobacterium bovis, and four (36%) were atypical mycobacteria. The prevalence of tuberculosis was higher in cattle owned by farmers with active tuberculosis than in cattle owned by farmers who did not have active tuberculosis, which could suggest possible transmission of Mycobacterium spp. between cattle and their owners.     

结核分支杆菌耐药机制的研究

结核分支杆菌（结核杆菌,Mycobacterium TuberculoSiS,M．TB）已经对多种药物产生耐药性,使得对结核病的治疗无效或者疗效降低,导致近年来结核病的发病率呈上升趋势。本文综述了结合分支杆菌对几种药物产生的耐药性的机理。     

IS900-PCR-based detection and characterization of Mycobacterium avium subsp. paratuberculosis from buffy coat of cattle and sheep

Johne's disease is one of the main causes of economic losses in ruminants and a major health hazard in the developing and developed world. Up till now, many microbiological, serological and molecular methods have been tried for the detection of Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we attempt a PCR-based detection of IS900, distinct insertion sequences of MAP from the buffy coat of cattle (n=262) and sheep (n=78), and direct genotyping by single strand conformational polymorphism (SSCP). A total of 30 (11.45%) cattle and one sheep (1.28%) were positive for MAP-IS900. This IS900-based PCR detection proved highly specific, particularly when tested on other non-MAP strains. SSCP analysis grouped the MAP-IS900 into four distinct clusters based on different band patterns. Nucleotide sequence variability between MAPs detected from sheep (GenBank accession ) and cattle (GenBank accession -) was noticed in the study. Although, in recent years IS900-PCR-based detection of MAP from WBCs is being used in human, its use in animals is still limited. Our work not only supports its use in animals but also suggests further IS900-SSCP-based MAP-genotyping, coupled with DNA sequencing, as a promising tool for rapid and effective Johne's disease surveillance.     

结核分枝杆菌疫源追踪的基因检测分析

目的探讨牛源性人结核疫源追踪的基因分析方法。方法采用复方PCR结核杆菌快速鉴定技术,针对特征性分枝杆菌标志基因(MTP40基因、α抗原基因和IS6110),对结核病人和患牛的标本,进行检测。结果患者标本结核分枝杆菌阳性;患牛标本牛分枝杆菌和结核分枝杆菌分别阳性。结论结核病奶牛不仅是人类牛型结核的可能来源,也可能成为人类人型结核的潜在来源。     

Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.     

牛结核病流行特点及防控措施

牛结核病是一种慢性消耗性人兽共患病,是OIE规定必须通报的疫病,在我国属二类动物传染病,并列为检疫扑杀对象,对养牛业发展、食品安全与人类健康构成重大威胁。目前普遍采用检疫和扑杀政策控制牛结核,但各个国家执行该政策的方式差异很大。由于牛分枝杆菌存在野生动物贮主,潜伏感染普遍、无快速高通量诊断方法等原因,控制或根除牛结核病非常困难。少数发达国家成功控制或净化了牛结核,但多耗时在二十年以上,耗资巨大。新型诊断方法如IFN-γ在当代牛结核检疫中发挥日益重要的作用,野生动物与家养牛结核的免疫预防已受关注。制约我国牛结核控制和净化的原因很多,如政策法规、国家财力、技术措施和公众认知水平等,目前尚未设定全国性的牛结核控制与净化目标。