结核分枝杆菌耐药机制与检测技术研究进展

近年来耐药结核分枝杆菌的不断出现给结核病的治疗带来前所未有的压力。研究发现,不但牛型分枝杆菌可感染人,人型结核分枝杆菌也可感染牛,这也给养牛业的发展带来了威胁与挑战。如何建立快速、简便、敏感性强、特异性好、价格低廉的耐药性检测方法是控制该病蔓延的关键。作者阐述了常用药物异烟肼、利福平、链霉素、乙胺丁醇、吡嗪酰胺等的作用机制及传统分枝杆菌耐药性检测方法（荧光素酶系统、显微镜观察药敏检测技术（MODS）、比例法、Bactec MGIT 960系统）和以分子生物学为基础的分枝杆菌耐药性检测方法（单链探针反向杂交试验（LiPA）、Gene Xpert全自动检测系统、PCR-单链构象多态性分析（PCR-SSCP）、PCR-限制性片段长度多态性分析（PCR-RFLP）、DNA测序、基因芯片技术）的研究进展,以期为进一步的研究与应用提供参考。     

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。     

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。     

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.     

牛结核病研究进展

牛结核病是由牛分支杆菌引起的一种人畜共患慢性传染病,近年来该病的发病率有增长的趋势,不仅给我国的养殖业造成重大经济损失,而且影响到公共卫生安全。主要对牛结核病的流行病学、病原学、发病机制、诊断方法、综合防控等方面的研究进展作一介绍,以期为相关研究提供参考。     

用多重PCR方法检测奶牛场麻雀中分枝杆菌感染情况

目的：利用可以鉴别分支杆菌属中结核分枝杆菌、牛分支杆菌、非结核分枝杆菌多重PCR方法,检测奶牛场麻雀组织中分支杆菌感染情况。方法根据结核分枝杆菌种特异基因目的片段MTP40、分枝杆菌属特异基因32kD、结核分枝杆菌复合群特异基因IS6110插入序列,设计合成三对特异性引物,扩增对32kD的506bp、MTP40的396bp和IS6110的984bp片段,以此方法检测奶牛场麻雀肺组织中分支杆菌感染情况,并对阳性结果的目的片段进行克隆测序。结果在分析的21份麻雀肺组织中,检测出2例非结核分枝杆菌阳性病料,阳性率9.52%。2例阳性样本PCR扩增得到的片段与GenBank收录的32kD基因同源性分别为95.8%和97.2%。结论麻雀肺组织中存在分支杆菌感染阳性病例提示该牛场中生物体内存在分支杆菌潜伏感染,并可能导致奶牛的潜伏感染以及干扰结核检疫。     

抑制鹿源多药耐药结核菌中药的筛选

为了筛选对鹿源多药耐药结核菌有效的中药耐药抑制剂,通过MABA法检测24种中药的水和乙醇提取物对多药耐药结核菌及标准菌H37Rv的体外抗菌效果,将筛选出的抗菌效果好的黄连醇提物和黄连水提物采用紫外分光光度法进行活性成分含量测定,同时以MABA法与标准品比较体外抗结核菌活性。结果显示,黄连、独活和侧柏叶有明显的抗结核活性,其中黄连水粗提物和乙醇粗提物效果最佳,测得它们活性成分即总生物碱含量分别为22.74%和35.23%,并显示二者与小檗碱的体外抗结核作用相同。     

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.     

Assessment of diagnostic tools for eradication of bovine tuberculosis in cattle co-infected with Mycobacterium bovis and M. avium subsp. paratuberculosis

The intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-gamma) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-gamma assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-gamma test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-gamma test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-gamma tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.     

Recent advances in non-specific immune memory against bovine tuberculosis

Bovine tuberculosis is an important worldwide disease mainly related to cattle, although it also affects other mammals, including humans. In recent years, there have been considerable advances in the knowledge of the immune response mechanisms underlying the interaction of Mycobacterium bovis, the main agent of bovine tuberculosis, with its hosts. In this review we describe the most recent findings on the cattle immune response to M. bovis, particularly regarding trained innate immune responses and γδ T cells, that could support the development of vaccines and diagnostic tools to control this disease.     

Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle

Mycobacteria other than the Mycobacterium tuberculosis complex (MOTT), isolated from Northern Ireland cattle, were identified by PCR amplification of the 16S rRNA gene, and subsequent reverse cross blot hybridisation and sequence analyses. Elucidation of the MOTT species was to facilitate specificity testing of new and existing diagnostic test reagents for bovine tuberculosis. The presence of the genes for potential diagnostic antigens: MPB70, MPB64, ESAT-6 and CFP-10 in the isolated MOTT species was investigated. Molecular analyses of cultured isolates from bovine lymph node specimens of 48 cattle identified a wide variety of mycobacterial species including Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium bohemicum, Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium holsaticum, Mycobacterium palustre, Mycobacterium sp. IWGMT 90210, Mycobacterium sp. LIV-2129, a potentially novel mycobacterial species (EMBL/GenBank/DDBJ Accession Number AJ617495) and Rhodococcus equi. Apart from M. kansasii, the results of traditional (standard phenotypic and biochemical) and molecular identification methods did not correlate well, with traditional methods identifying fewer species. Most of the species identified were either recognised pathogenic or potential pathogenic species. The genes for ESAT-6, CFP-10 and, unusually, MPB64 were detected in M. kansasii only. The MPB70 gene was not detected in any of the species. This study supported restricted species distribution of these genes as well as identifying a different range of MOTT species that could be included in specificity testing of new diagnostic reagents for bovine tuberculosis.     

两种奶牛结核病检测方法的比较

本研究中采用常规皮内变态反应(TST)对广西南宁、柳州共计2 818头黑白花奶牛进行牛结核病检测,采用抗原特异性IFN-γ试验方法对PPD检测结果为阳性、可疑的牛以及部分阴性牛进行复检。将抗原特异性IFN-γ试验与TST检测结果相比,其敏感性为53.33%,特异性为70.49%,符合率为67.11%。本研究证实单独使用PPD皮内变态反应方法在牛结核病的诊断方面存在非特异性强等缺点,易造成假阳性牛的误杀;用抗原特异性IFN-γ试验对TST试验阳性和可疑牛进行复检可以提高特异性,该方法在临床上有重要应用价值。     

Diagnosis of Mycobacterium bovis infection in cattle by use of the gamma-interferon (Bovigam) assay

The strategic use of the gamma-interferon (IFN-gamma) assay (Bovigam) can provide a means for the early identification of Mycobacterium bovis infected cattle, thus ensuring their removal from an infected herd. When used in parallel with the tuberculin test, it is capable of identifying infected cattle, which might otherwise not be detected until later, if at all. The early detection and removal of these animals reduces the risk that they will become a source of infection for other cattle. When targeted in herds of high prevalence the benefits to the herd owner directly concerned can be considerable as the assay provides a means of shortening the period of restriction for such herds. This serves to generate confidence among herd owners and other stakeholders that effective schemes, based on sound scientific principles, can be developed to eradicate tuberculosis from infected cattle populations.     

Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.