分枝杆菌分型三重PCR检测方法的建立及应用

本研究旨在建立一种快速鉴定分枝杆菌的三重PCR方法,并比较分析其在临床检测中的可靠性。根据已发表的结核分枝杆菌、牛分枝杆菌和非洲分枝杆菌rv 3036c基因,结核分枝杆菌rv 1970f基因(RD7)和牛分枝杆菌pncA基因的序列,改造并设计合成了3对特异性扩增引物,建立了一种能对分枝杆菌样品进行初步鉴定的三重PCR方法。结果显示该方法可针对rv 3036c、rv 1970f和pncA基因分别扩增出大小为500、125和249 bp的目的片段,能特异性检测出结核分枝杆菌(500和125 bp两条带)和牛分枝杆菌(500和249 bp两条带),并可将结核分枝杆菌、牛分枝杆菌与其他分枝杆菌加以区分。本方法的检测灵敏度为50 pg/μL模板基因组DNA。对86株抗酸染色阳性菌进行三重PCR鉴定,鉴定结果与细菌16S rDNA和ITS序列测定结果一致,检测准确度为100%,优于生长特征和生化试验鉴定。     

Establishment and Application of Mycobacterium Typing Triple PCR Detection Method

This study was aimed to establish a triple PCR method to rapidly identify Mycobacterium species, and evaluate its testing reliability.Three pairs of primer that were respectively specific to rv 3036c, rv 1970f and pncA genes of Mycobacterium were designed to establish a triple PCR for preliminary identification of Mycobacterium tuberculosis(M.tuberculosis), Mycobacterium bovis(M.bovis) and other Mycobacterium spp.PCR products were the expected sizes of 500(rv3036c), 125(rv1970f) and 249 bp(pncA), and contained two DNA bands(500 and 125 bp) with M.tuberculosis DNA template, two DNA bands(500 and 249 bp) with M.bovis DNA template.No band or non-specific band appeared with Mycobacterium spp.except M.tuberculosis and M.bovis DNA templates.The sensitivity of the triple PCR was calculated to 50 pg/μL template of genomic DNA.86 acid-fast bacteria were detected by the triple PCR, 16S rDNA and ITS gene sequencing, growth test and biochemical test, and the results were consistent between triple PCR and 16S rDNA and ITS gene sequencing.The detecting accuracy of triple PCR was 100%, and higher than growth test and biochemical test.     

Serological reactivity to Mycobacterium bovis protein antigens in cattle.

The serological response to 12 purified Mycobacterium bovis antigens were examined in an ELISA assay. These antigens included the majority of M. bovis protein antigens described to date and in most cases they were very similar to the M. tuberculosis antigens of the same molecular mass.

The purified antigens were tested against sera from M. bovis infected cattle, M. bovis culture-negative cattle from infected herds and animals infected with related microorganisms, mainly other mycobacterial species. All the antigens gave strong reactions with at least some sera from the M. bovis infected group and showed cross-reactivity with some of the sera from the other two groups. The antigen with the highest specificity reacted strongly with only 60% of the M. bovis infected sera. Antigens that reacted with most or all of the M. bovis infected sera also gave the highest cross-reactivity with sera from the other two groups. These results indicate that a serological test based on any one or a combination of these antigens, without removal of the cross-reacting epitopes, would be unsatisfactory.     

Mapping of IS6110 insertion sites in Mycobacterium bovis isolates in relation to adaptation from the animal to human host

The physiological role and impact of IS6110 insertions on the biology of Mycobacterium tuberculosis complex is not well understood. Insertion of IS6110 in coding regions can cause loss of gene activity, while homologous recombination between two copies of IS6110 can result in the deletion of genes or in rearrangement of genomic regions involved. In addition to these genomic changes, IS6110 can also activate flanking genes through acting as a mobile promoter.

In order to determine the possible role of IS6110 transposition in the adaptation to humans, we selected Mycobacterium bovis isolates from endogenous reactivation cases in elderly people in The Netherlands. The human isolates contained higher number of IS6110 copies in comparison to the bovine M. bovis strains. These additional integration sites of IS6110 were sequenced and analyzed. From 12 of such IS6110 insertion sites, 6 loci were located in the intergenic regions, and 6 other occurred within coding regions. IS6110 was inserted in a position where it might serve as a promoter in two cases. We conclude that IS6110 transpositions in M. bovis may be a driving force in the adaptation from the animal to the human host.     

Interaction of antigen presenting cells with mycobacteria

The interaction of mycobacteria with antigen presenting cells is a key feature in the pathogenesis of tuberculosis and the outcome of this interaction is pivotal in determining whether immunity or disease ensues. Human and mouse macrophages and dendritic cells (DC) have been shown to become infected with mycobacteria and to produce a response to infection that reflects their suggested role in immunity. Thus, macrophages elicit anti-microbial mechanisms for elimination of mycobacteria and DC up-regulate expression of molecules that aid their stimulation of T lymphocytes. We have examined the effects of infection with the avirulent strain Mycobacterium bovis BCG and with virulent M. bovis on bovine antigen presenting cells. Differences in the intracellular survival of bacteria within DC and macrophages were observed with higher numbers of bacteria maintained within DC following infection compared to macrophages. BCG was killed more effectively than M. bovis. Alterations in the expression of cell surface molecules involved in antigen presentation and the stimulation of T cells, including MHC II and CD40, were observed following infection of bovine antigen presenting cells. In addition infected DC secreted IL-12, TNF and IL-10 whereas macrophages produced TNF, IL-10 and little IL-12. Generally responses were more marked when virulent M. bovis was used compared to BCG. These studies indicate that infection of bovine antigen presenting cells by mycobacterial species results in the induction of both innate and adaptive immune responses that are critical for the outcome of infection.     

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: a first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n = 207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09–22.99, P < 0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002–2003, O.R. 3.69; 95% CI 1.27–11.9, P < 0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.     

Bovine TB and the development of new vaccines

Bovine tuberculosis (bTB) is caused by Mycobacterium bovis. The incidence of bTB is increasing in cattle herds of developed countries that have a wild life reservoir of M. bovis, such as the UK, New Zealand and the USA. The increase in the incidence of bTB is thought to be due, at least in part, to a wildlife reservoir of M. bovis. M. bovis is also capable of infecting humans and on a worldwide basis, M. bovis is thought to account for up to 10% of cases of human TB [Cosivi O, Grange JM, Daborn CJ et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998;4(1):59–70]. Thus, the increased incidence of bTB, besides being a major economic problem, poses an increased risk to human health. In the UK, the incidence of bTB continues to rise despite the use of the tuberculin test and slaughter control policy, highlighting the need for improved control strategies. Vaccination of cattle, in combination with more specific and sensitive diagnostic tests, is suggested as the most effective strategy for bovine TB control. The only vaccine currently available for human and bovine TB is the live attenuated Bacille Calmette Guerin (BCG). BCG is thought to confer protection through the induction of Th1 responses against mycobacteria. However, protection against TB conferred by BCG is variable and to this date the reasons for the successes and failures of BCG are not clear. Therefore, there is a need to develop vaccines that confer greater and more consistent protection against bTB than that afforded by BCG. Given that BCG is currently the only licensed vaccine against human TB, it is likely that any new vaccine or vaccination strategy will be based around BCG. In this review we discuss immune responses elicited by mycobacteria in cattle and the novel approaches emerging for the control of bovine TB based on our increasing knowledge of protective immune responses.     

Evaluation of MPB70, bovine PPD and lipoarabinomannan as antigens in ELISA for the serodiagnosis of bovine tuberculosis

The performance of the secretory protein MPB70 of Mycobacterium bovis, bovine PPD, and lipoarabinomannan (LAM) were evaluated as antigens in ELISA for detection of tuberculosis (TB) infected cattle. Sera were from 120 M. bovis infected cattle and 223 cattle from a TB free herd. ELISA results were analyzed using receiver operating characteristic (ROC) curves in relation to culture results. The areas under the three ROC curves were 71 ± 49% SE (MPB70), 71 ± 27% SE (bovine PPD), and 56 ± 4% SE (LAM).     

Simulation model of within-herd transmission of bovine tuberculosis in Argentine dairy herds

Transmission of bovine tuberculosis was quantified in three dairy herds located in south Santa Fe Province, Argentina. Using estimates of Mycobacterium bovis transmission (β) and a Reed–Frost simulation model, the prevalence of tuberculosis infection in the study herds over time was investigated. The Reed–Frost model was modified by incorporating randomness in both β and the incubation period () of M. bovis. The mean estimated herd β was 2.2 infective contacts per year and did not differ significantly between the study herds. Modeling as Poisson distributed (mean 24 months) best fit the observed prevalences. Infection was predicted by the model either to spread quickly (<10 years) within a herd and reach a high prevalence (>50%), or to persist at a low prevalence (<5–10%). The model was robust, predictions were realistic and the mean β estimated was consistent with previous studies of bovine tuberculosis.     

Genotyping of Mycobacterium bovis by geographic location within Mexico

The spacer oligonucleotide typing (spoligotyping) method was used to differentiate 62 Mycobacterium bovis isolates obtained from tissues with macroscopic lesions typical of tuberculosis in dairy cattle from different regions of Mexico. Our purpose was to see if a strain from one region was genetically different from those of other regions (with the long-term aim of doing molecular trace back of isolates obtained in the laboratory). Results from the genetic analysis indicate that M. bovis isolates cannot be grouped by geographic location due to a wide range of genetic types involved in dairy cattle infections. Isolates even from the same herd showed different spoligotypes but some isolates from different region had similar genetic patterns. Genetic typing without epidemiologic information does not seem to be a plausible method to trace back animals to source of origin to detect and eliminate sources of infection.     

CRISPR/Cas System and Its Application in the Study of Mycobacterium tuberculosis

Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-related protein (Cas) system is a heritable prokaryotic adaptive immune system against invading nucleic acids. It is an editing tool for many organisms that can generate highly specific gene double-strand breaks and repair them by non-homologous end joining (NHEJ) or homologous recombination (HR). Due to the complexity of its genetic manipulation, Mycobacterium tuberculosis genome research has many difficulties. Therefore, the emergence of the CRISPR/Cas system has a milestone significance for the research of Mycobacterium tuberculosis. In this review, we focus on the application of the CRISPR/Cas system in Mycobacterium tuberculosis and summarizes the present research progress in order to provide a reference for the selection of Mycobacterium tuberculosis research methods.     

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。     

Development and Application of FQ-PCR Assay for Detecting Mycobacterium bovis

To develop a detection method for rapid diagnosis of dairy cow tuberculosis, evaluation of the raw milk contamination status and tracing the route of transmission, specific primers of Mycobacterium bovis were designed to develop the FQ-PCR assay and the reaction conditions were optimized.Standard curve of the FQ-PCR test was developed and its properties were evaluated.The results showed that the developed FQ-PCR test could be used to detect the Mycobacterium bovis.The best primer concentration and annealing temperature were 400 nmol/L and 52 ℃, respectively.Properties evaluation showed that the method had good specificity, sensitivity, repeatability and clinical application.The test results indicated this method could be used to qualitatively and quantitatively detect Mycobacterium bovis.It would be an important technology for diagnosis and decontamination of dairy cow tuberculosis and safety assessment of raw milk.     

牛结核分枝杆菌实时荧光定量PCR检测方法的建立及应用

为建立可应用于快速检测奶牛结核病、评估鲜乳污染状况、追溯传播途径的试验方法,本研究根据牛结核分枝杆菌基因组设计合成特异性引物,建立实时荧光定量PCR方法,并对反应条件进行优化,构建标准曲线,评价该方法的性能。结果显示,本研究所建立的实时荧光定量PCR方法能有效检测牛结核分枝杆菌目的基因,其最佳引物浓度为400 nmol/L,最佳退火温度为52 ℃。所构建的标准曲线相关性好,可用于样本的定量检测。该方法的性能评价显示,其最小检出模板浓度为80.24 ng/L,且该方法具有较好的特异性、可重复性,可对鲜乳样本进行检测。试验结果表明,本研究所建方法可用于牛结核分枝杆菌的定性和定量检测,这为奶牛结核病的诊断与净化及鲜乳食品安全评估提供重要技术。     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

犊牛支原体肺炎继发牛A型多杀性巴氏杆菌感染的诊治

某奶牛场犊牛相继发生肺炎和关节炎,为确诊该牛场犊牛群发病的原因并提出防控方案,本试验剖检新生犊牛并采集病料,分别开展牛支原体及其他病原菌的分离培养、PCR鉴定及药敏分析;进行牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒PCR检测;制作犊牛肺脏组织病理切片并进行观察和评估。从犊牛肺脏组织分离到牛支原体和牛A型多杀性巴氏杆菌;牛病毒性腹泻病毒、牛口蹄疫病毒和牛传染性鼻气管炎病毒检测均为阴性;肺脏组织病理切片可见肺泡结构破坏、出血及大量炎性细胞浸润;药敏试验结果显示,牛支原体和牛A型多杀性巴氏杆菌分别对泰乐菌素和头孢唑啉敏感,但对青霉素、庆大霉素、林可霉素和氨苄西林均呈现耐药。该犊牛群确诊为牛支原体肺炎继发牛A型多杀性巴氏杆菌感染,采用泰乐菌素联合头孢唑啉肌肉注射,配合对症治疗和规范管理,有效控制了该场犊牛疾病。     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.     

新疆石河子地区牛支原体的分离鉴定及药物敏感性分析

【目的】确定引起新疆石河子地区集约化牛场常发性肺炎的主要病原同时进行病原的体外药物敏感性分析。【方法】采集有典型咳嗽、流涕症状的牛鼻拭子10份和病死牛肺脏组织1份,用牛支原体特异性引物进行PCR检测,将检测为阳性的样本进行病原培养纯化,对纯化后的分离株菌落进行形态学观察、Dienes染色、生化试验及16S rRNA测序和进化分析,通过测定颜色变化单位(CCU)测定分离株生长曲线,并对分离株进行药物敏感性试验。【结果】PCR结果显示,10份鼻拭子中检测出7份牛支原体阳性样本,1份病死牛肺脏组织也检测为阳性;在涂有肺脏组织研磨液培养液的PPLO固体培养基上长出针尖状的菌落,纯化后分离株菌落形态为典型的煎蛋状;Dienes染色可见明显的深蓝色中心脐;生化试验结果显示,分离株不水解明胶、精氨酸、七叶苷,不发酵乳糖、葡萄糖和甘露醇,不分解尿素,可还原氯化三苯基四氮唑;16S rRNA测序结果显示,分离株与牛支原体国际标准株PG45相似性为99.7%,与国内牛支原体地方流行株XBY01、Ningxia-1、NM2012、Tibet-10的相似性最高,均为99.9%;生长曲线测定结果显示,分离株在培...     

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS–PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.     

Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.