Effects of Mcl-1 silencing on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis

AIM: To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference. METHODS: The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang, H37Rv, H37Ra and BCG. Mcl-1-shRNA was applied to the mouse model of infection, and the control groups were set up. On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected. The expression of Mcl-1 at mRNA and protein levels was determined by real-time PCR and Western blot. The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry. RESULTS: The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05). The expression of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group (P<0.05). Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice. CONCLUSION: The Mcl-1 expression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

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.     

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid–fluorescence in situ hybridization

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid–fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.     

Characterization of Mycobacterium salmoniphilum as causal agent of mycobacteriosis in Atlantic salmon,Salmo salar L., from a freshwater recirculation system

Thirty Atlantic salmon, Salmo salar L., with low corporal condition relative to other fish present in the culture system, were sampled from a freshwater recirculation pisciculture located in Chile. The most characteristic signs and lesions were cachexia and presence of multiple greyish‐white granulomas within internal organs. The external and internal lesions, along with the microscopic, histologic and biochemical findings, were consistent with mycobacteriosis. The identification of Mycobacterium salmoniphilum as the causal agent of the lesions was possible through the use of molecular analyses. This study represents the first report of Mycobacterium salmoniphilum in a freshwater recirculation system and the first case of fish mycobacteriosis described in Chile.     

Estimates of within-herd incidence rates of Mycobacterium bovis in Canadian cattle and cervids between 1985 and 1994

We analysed the individual-animal data from six of the nine outbreaks of tuberculosis in Canadian cattle and cervids from 1985 to 1994. A “positive/reactor” animal was one which had either a positive culture or a positive or suspicious reaction on a mid-cervical, comparative cervical, or gross or histopathological test for tuberculosis. Individual-animal data were collected only for herds which had one or more positive/reactor animals. Data were collected from the outbreak records in the Regional or District offices of Agriculture and Agri-food Canada’s Animal and Plant Health Directorate. The within-herd spread of Mycobacterium bovis was studied by determining the most-likely date at which the herd was first exposed to M. bovis and the number of reactions which had developed by the time the herd was investigated. The animal-time units at risk in the herd were probably overestimated, resulting in conservative estimates of the within-herd incidence rates. Negative-binomial regression was used to investigate factors which might have influenced the within-herd spread of tuberculosis. Increasing age appeared to be a risk factor for being a positive/reactor animal. When compared to animals 0–12 months old, animals 13–24 months old had an incidence rate ratio (IRR) of 7.6, while animals >24 months old had an IRR of 10.4 (p=0.009). Actual and predicted incidence rates for tuberculosis in mature (>24 months old) animals were calculated. Actual and predicted incidence rates were similar for cervids, within an outbreak. There was more variability between actual and predicted rates in the dairy and beef animals. In the one outbreak (Ontario) where there were positive/reactor cervid, dairy and beef herds, the actual incidence rate for cervids (IR=9.3 cases per 100 animal-years) was almost twice that of dairy cattle (IR=5.0) and three times that of beef cattle (IR=3.1).     

结核分枝杆菌(H37Rv和BCG)在感染AECⅡ细胞系A549中对TLRs信号通路和促炎因子表达的影响

肺泡Ⅱ型上皮细胞(alveolar epithelial typeⅡcells,AECⅡ)是由呼吸道吸入的结核病原菌最先侵染的靶细胞.本研究通过建立结核分枝杆菌(Mycobacterium tuberculosis,Mtb)强毒株(H37Rv)、弱毒株(Bacillus Calmette-Guérin,BCG)感染AECⅡ细胞模型,分析不同感染时间Toll样受体(toll-liking receptors,TLRs)信号通路活化和炎症细胞因子分泌的差异,探讨AECⅡ细胞在Mtb感染时的免疫应答和持续感染的分子机制.利用H37Rv和BCG分别感染AECⅡ细胞系A549,通过荧光定量PCR和Western-blot等技术在核酸和蛋白水平分析感染6、12和24h时TLRs信号通路信号分子和促炎细胞因子的表达变化.结果表明,mRNA表达水平检测发现,在H37Rv感染A549细胞中,TLR2、TLR4、MyD88和NFκB在6h时显著高于BCG感染组;在感染24h时,两组TLR2、TLR4表达均上调,且差异不显著,而H37Rv感染组NFκB呈上调表达;在蛋白水平分析发现,在H37Rv感染引起A549细胞中MyD88和磷酸化NFκB表达呈显著下调趋势,且高于BCG感染组;而促炎细胞因子IL-1a、IL-6、IL-12a、IL-8、TNF-α和CSF2 mRNA表达水平随着感染时间升高,且H37Rv感染组IL-1a、IL-6 1L-8、TNF-α、和CSF2的表达显著高于BCG感染组,IL-12a差异不显著.本研究发现,Mtb强毒株感染AECⅡ细胞对TLRs信号通路表现为抑制趋势,且引起细胞的炎症反应明显强于弱毒株,这为阐明AECⅡ细胞在不同毒力结核分枝杆菌感染中的免疫调控机制研究和临床肺结核的发病机制研究提供了参考依据.     

荧光定量聚合酶链式反应检测乳及乳制品中结核分枝杆菌方法的建立与评价

建立一种快速检测乳及乳制品中结核分枝杆菌的荧光定量聚合酶链式反应法.根据结核分枝杆菌保守序列设计特异引物,建立结核分枝杆菌的实时荧光定量聚合酶链式反应技术(polymerase chain reaction,PCR)检测法.对该方法的灵敏度、特异度和重复性进行考察.结果表明:建立的实时荧光定量PCR方法标准曲线线性关系良好,得到标准曲线方程为y=-3.29x+38.60 (R2=0.998 0);敏感性高,可达1×102 copies/μL;抗干扰能力强,仅结核分枝杆菌出现特异性扩增曲线;重复性良好,分别用不同质量浓度的标准质粒进行组内和组间平行实验,批内和批间的变异系数均小于1％.因此,实时荧光定量PCR法能准确快速检测乳及乳制品中结核分枝杆菌.     

环介导等温扩增法检测牛奶中结核分枝杆菌条件的优化

为优化环介导等温扩增（loop-mediated isothermal amplification,LAMP）法检测牛奶中结核分枝杆菌的条件,以结核分枝杆菌高度保守的16S rRNA基因为靶基因设计3对特异性引物（F3-B3、FIP-BIP、FLP-BLP）进行试验。比较改良后的CATB/NaCl法、细菌基因组DNA提取试剂盒及热裂解法提取其DNA的效率,确定最佳的提取方法。用灭菌处理过的牛奶对结核分枝杆菌悬液进行倍比稀释以确定该检测方法的敏感度。以牛奶中常见的布鲁氏菌、大肠杆菌、金黄色葡萄球菌、李斯特菌、巴氏杆菌、沙门氏菌为对照,对该方法进行特异性测试。结果表明,改良后的CTAB/NaCl法对牛奶中结核分枝杆菌DNA的提取效果要优于其他两种方法。LAMP法检测结核分枝杆菌的灵敏度为3×10⁰ CFU/mL,对人工阳性乳中结核分枝杆菌检测的灵敏度为3×10¹ CFU/mL。