LRRK2/NF-κB signaling modulates inflammatory responses in BCG-infected RAW264.7 macrophages

AIM: To investigate the biological function and potential mechanism of leucine-rich repeat kinase 2 (LRRK2) in RAW264.7 macrophages during Mycobacterium tuberculosis infection. METHODS: The bacillus Calmette-Guerin (BCG)-infected RAW264.7 cell model was established. Colony-forming unit (CFU) analysis was used to determine the mycobacterial viability. The releases of interleukin (IL)-1β, IL-6 and interferon-γ (IFN-γ) in the RAW264.7 cells were detected by ELISA. qPCR and Western blot were used to measure the mRNA and protein expression levels, respectively. RESULTS: LRRK2 was robustly enhanced in the RAW264.7 cells in response to BCG infection. Additionally, silencing of LRRK2 suppressed intracellular growth of mycobacteria during BCG challenge. Moreover, silencing of LRRK2 dramatically attenuated the accumulation of inflammatory cytokines IL-1β, IL-6 and IFN-γ induced by BCG infection. More importantly, LRRK2 modulated BCG-induced inflammatory responses by positively regulating the nuclear factor-κB (NF-κB) signaling pathway. CONCLUSION: LRRK2/NF-κB signaling pathway positively modulates inflammatory responses during BCG infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease.     

结核分枝杆菌PhoP-PhoR双组分系统研究进展

双组分信号转导系统广泛存在于各种原核生物中,其基本结构为1个组氨酸激酶（HK）和1个反应调节剂（RR）,该系统能够感知外界刺激并作出反应,使细菌能适应各种不利环境且能增强细菌在宿主体内的生存能力,对细菌的毒力和生长至关重要。文章概述了结核分枝杆菌DosS/DosT-DosR、MprA-MprB、PrrA-PrrB、PhoP-PhoR等12个双组分系统,重点介绍了PhoP-PhoR双组分系统的结构与功能。PhoP-PhoR是结核分枝杆菌最基本、最重要的双组分系统之一,由感受器PhoR和效应器PhoP组成,其中PhoR可以接受Mg^2＋、Cl^－或H^＋等外界刺激,进而驱动PhoP对靶基因的转录表达进行调控。PhoP作为一种效应蛋白,经过晶体结构解析发现,其可通过与DNA结合来实现对靶基因的调控,具体包括对细胞壁组成的控制、对脂类代谢和pH的调节、对结核分枝杆菌毒力网络的调控。此外,文章还介绍了PhoP突变株作为疫苗候选株所具有的优势,包括PhoP突变株在小鼠模型中毒力减弱,并且免疫恒河猴及豚鼠后均有一定的免疫保护性等,表明PhoP突变株具有较好的疫苗开发潜力。作者通过对PhoP-PhoR双组分系统的结构与功能,以及PhoP突变株作为疫苗候选株的研究进行综述,旨在为结核分枝杆菌的毒力机制和人类结核病疫苗的研究提供理论依据。     

Individual animals of a cattle herd infected with the same Mycobacterium bovis genotype shows important variations in bacteriological, histopathological and immune response parameters

Cattle are the host and main reservoir of the etiologic agent of bovine tuberculosis, Mycobacterium bovis; although other mammalian species, including humans, are susceptible. The tuberculin test and/or slaughterhouse surveillance is the diagnostic method used by control programs all around the world to control and eradicate the disease. In order to compare different tuberculosis diagnostic tests and to reach disease confirmation, a study was performed in a group of 14 steers of Friesian breed, reacting positively to tuberculin test. Three ante-mortem assays were performed according to the type of sample: the gamma interferon (IFN-gamma) test (which quantifies the release of this cytokine by sensitized lymphocytes in whole blood in response to purified protein derivative (PPD) and recombinant ESAT-6 and CFP10 proteins); PCR and bacteriologic culture from nasal swab and intradermal tuberculin test. These assays were taken at different times to assess the evolution of clinical parameters. Post-mortem examination showed macroscopic and microscopic tuberculosis lesions with acid-fast bacillus and positive cultures. By spoligotyping, we observed that all the isolates showed the same pattern. The positive results based on comparison to lesions observed ranged from 58% to 75% for the IFN-gamma assays, to 72% for cultures, and ranged from 50% to 90% for PCR in nasal swabs. In conclusion, in a herd infected by the same strain, ante-mortem direct and immune-diagnostic parameters change, suggesting that several tests are needed for a faster control of infection at herd level.     

Epidemiology and microscopic diagnosis of tuberculosis in pigs and small ruminants slaughtered at Bobo-Dioulasso abattoir,Burkina Faso

Bovine tuberculosis (bTB) is a zoonotic, infectious, chronic and contagious disease, caused by Mycobacterium bovis that mainly affects cattle. This pathology has a negative impact on animals and animal products trade. Unfortunately, in Burkina Faso where agriculture and livestock sectors represent around 80% of the socio-economic activities, the real situation of the disease is not well known especially in small ruminants and swine. Thus, our study focused on both the epidemiology and the microbiological diagnosis of tuberculosis (TB) in small ruminants and pigs slaughtered at Bobo-Dioulasso abattoir. A prospective study was conducted between August 2017 and December 2017. Epidemiological data collection was performed during routine meat inspection; moreover, samples were taken and transported to the Bacteriology laboratory of Centre Muraz for microbiological analyses. This diagnosis consisted in search of Acid Fast Bacilli (AFB) using the hot Ziehl–Neelsen staining. Out of a total of 14 648 small ruminants and 2430 pigs slaughtered during the study period, 156 and 17 had lesions suggestive of bTB with prevalence of 1.07% and 0.7%, respectively. Females and those between 2 and 4 years old were mainly infected. The most affected organs were: lungs, liver, spleen and lymph nodes. Finally, microscopy revealed 43.35% (75/173) of positive cases for AFB. These results confirm the presence of bTB in small ruminants and pigs in Burkina Faso. Efforts must still be made in the fight against this zoonosis in order to limit its economic and public health impacts.     

Detection of Mycobacterium tuberculosis complex antibodies in free-ranged wild boar and wild macaques in selected districts in Selangor and reevaluation of tuberculosis serodetection in captive Asian elephants in Pahang,Peninsular Malaysia

Tuberculosis (TB) is a chronic inflammatory and zoonotic disease caused by Mycobacterium tuberculosis complex (MTBC) members, affecting several domestic animals, wildlife species and humans. The preliminary investigation was aimed to detect antibody against MTBC among indigenous wildlife which are free-ranged wild boar, free-ranged wild macaques and captive Asian elephants in selected areas of Selangor and elephant conservation centre in Pahang, respectively. The results indicate that MTBC serodetection rate in wild boar was 16.7% (7.3–33.5 at 95% confidence interval (CI)) using an in-house ELISA bPPD IgG and 10% (3.5–25.6 at 95% CI) by DPP^®VetTB assay, while the wild macaques and Asian elephant were seronegative. The univariate analysis indicates no statistically significant difference in risk factors for sex and age of wild boar but there was a significant positive correlation (P<0.05) between bovine TB in dairy cattle and wild boar seropositivity in the Sepang district.     

牛分枝杆菌Mb1230基因的表达纯化及其活性评价

为了探究牛分枝杆菌Mb1230蛋白在牛结核病诊断中的应用价值,本试验利用PCR方法扩增出Mb1230基因,构建重组质粒pET-22b-Mb1230,经IPTG诱导表达后用亲和层析纯化重组蛋白,通过结核菌素皮内变态反应试验(TST试验)、IFN-γ释放试验(IGRA试验)和间接ELISA对重组蛋白的活性进行评价。SDS-PAGE和Western blotting结果显示,重组蛋白大小与理论值相符,且能与鼠抗His的抗体反应有特异性条带;在TST试验、IGRA试验和间接ELISA中也表现出抗原活性。结果表明,重组Mb1230蛋白具有良好的B细胞活性和T细胞活性,在牛结核病诊断中具有应用潜力。     

鹿结核病的诊断方法研究进展

鹿结核病是由结核分枝杆菌引起的鹿的一种慢性、消耗性传染病,广泛流行于世界各地,几乎每个国家的鹿场都曾发生过结核病或曾经检出过结核病菌.鹿结核病目前尚无规定性的诊断方法,本文对鹿结核病的诊断方法研究进展作综述,作为进一步研究发展鹿结核病的诊断方法的借鉴.     

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

Thirty‐five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Paraná, Brazil. Twenty‐two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff’s method and processed for acid‐fast bacilli staining, culture in Stonebrink and Lowenstein‐Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR‐positive results (54.5%) was similar to that of culture‐positive results (51.5%, P > 0.05). The percentage of PCR‐positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR‐negative were reanalysed using 2.5 μl DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.     

Defining output-based standards to achieve and maintain tuberculosis freedom in farmed deer, with reference to member states of the European Union

Within the European Union (EU), detailed legislation has been developed for cattle, but not deer, to minimise disease risks associated with trade in animals and animal products. This legislation is expressed as input-based standards, providing a detailed outline of the activity required (for example, testing of animals and application of defined control measures), on the expectation that an adequate output (for example, confidence in freedom) will be achieved. Input-based standards are at odds with the increasing shift towards output-based standards, particularly in OIE rules governing international trade. In this paper, we define output-based standards to achieve and maintain freedom from tuberculosis (TB) in farmed deer, with reference to EU member states. After considering the probability of freedom achieved for cattle under existing EU legislation, we defined a ‘free farmed deer holding’ as one with a probability of freedom from infection of at least 99%. We then developed an epidemiological model of TB surveillance systems for deer holdings, incorporating different surveillance strategies, including combinations of diagnostic tests, and a variety of different scenarios relating to the potential for introduction of infection. A range of surveillance strategies were identified to achieve and maintain a free farmed deer holding, and worked examples are presented. The surveillance system sensitivity for varying combinations of screening and confirmatory tests in live animals, animals at slaughter and on-farm deaths is also presented. Using a single test at a single point in time, none of the TB tests routinely used in farmed deer is able to achieve an acceptable probability of TB freedom. If repeat testing were undertaken, an acceptable probability of TB freedom could be achieved, with differing combinations of the surveillance system sensitivity, frequency of testing and risk of introduction. The probability of introduction of infection through the importation of infected deer was influenced by the use of a pre-movement test (assumed 90% test sensitivity and negative test results), the TB prevalence in the source herd and the number of animals imported. A surveillance system sensitivity of at least 81% was achieved with different combinations of annual live animal surveillance and surveillance of animals at slaughter or on-farm deaths. This methodology has broad applicability and could also be extended to other diseases in both deer and other species with relevance to trade in animals and animal products.     

分枝杆菌阿拉伯半乳聚糖生物合成途径研究进展

综述了阿拉伯半乳聚糖生物合成关键基因、合成步骤及其调控因素的研究进展,利用结核分枝杆菌的基因组序列,从比较基因组层面分析了阿拉伯半乳聚糖生物合成的保守性,为利用耻垢分枝杆菌模型筛选阿拉伯半乳聚糖合成过程中潜在的药物作用靶标提供了基础。