沙门氏菌菌蜕佐剂增强结核分枝杆菌4种蛋白的免疫作用研究

探究肠炎沙门氏菌菌蜕(ghost)佐剂对结核分枝杆菌(MTB)4种抗原的免疫增强作用,本研究以MTB中的Rv3802、Rv0394c(SEC)、Rv0199及E6c10(ESAT6与CFP10融合蛋白)蛋白为免疫原,应用小鼠模型评价沙门氏菌菌蜕对MTB4种抗原的免疫佐剂效应。结果显示,蛋白加菌蜕佐剂组可以刺激小鼠产生持久高水平的抗体,并且菌蜕佐剂的免疫增强效果高于弗氏不完全佐剂;IgG1/IgG2a的比率增高,提示蛋白加菌蜕佐剂组小鼠趋向于Th2型免疫反应,同时可以诱导淋巴细胞分化为CD4+T细胞亚群并产生IFN-γ及IL-4细胞因子;病理组织学显示以菌蜕佐剂配伍蛋白制备的亚单位疫苗具有良好的安全性。本研究为结核病的预防和新型疫苗的研发奠定了一定的基础。     

Study on Optimization of the Conditions of Detecting Mycobacterium tuberculosis in Milk by Technique of Loop-mediated Isothermal Amplification

To optimize the loop-mediated isothermal amplification (LAMP) method to detect Mycobacterium tuberculosis in milk,the highly conservative 16S rRNA gene of Mycobacterium tuberculosis was selected as a target to design specific primers.Compared the modified method of CATB/NaCl,bacterial genome DNA extraction kit and thermal cracking method to extract the DNA of Mycobacterium tuberculosis,the best approach was chosen.Mycobacterium tuberculosis suspension liquid was diluted with sterilization milk to confirm the susceptibility of this assay.And then Brucella,Escherichia coli,Staphylococcus aureus,Listeria monocytogenes,Bacillus pasteurii and Salmonella were used for specificity detection.The results showed that the modified method of CATB/NaCl was better than the others.The sensitivity of LAMP was 3×10⁰ CFU/mL,and the specificity was 100%.The sensitivity of detecting Mycobacterium tuberculosis in milk by LAMP was 3×10¹ CFU/mL.     

Molecular Epidemiology of Mycobacterium bovis in Humans and Cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is a serious re‐emerging disease in both animals and humans. The evolution of the Multi‐ and Extensively drug‐resistant M. bovis strains (MDR‐TB and XDR‐TB) represents a global threat to public health. Worldwide, the disease is responsible for great economic losses in the veterinary field, serious threat to the ecosystem, and about 3.1% of human TB cases, up to 16% in Tanzania. Only thorough investigation to understand the pathogen's epidemiology can help in controlling the disease and minimizing its threat. For this purpose, various tools have been developed for use in advanced molecular epidemiological studies of bTB, either alone or in combination with standard conventional epidemiological approaches. These techniques enable the analysis of the intra‐ and inter‐species transmission dynamics of bTB. The delivered data can reveal detailed insights into the source of infection, correlations among human and bovine isolates, strain diversity and evolution, spread, geographical localization, host preference, tracing of certain virulence factors such as antibiotic resistance genes, and finally the risk factors for the maintenance and spread of M. bovis. They also allow for the determination of epidemic and endemic strains. This, in turn, has a significant diagnostic impact and helps in vaccine development for bTB eradication programs. The present review discusses many topics including the aetiology, epidemiology and importance of M. bovis, the prevalence of bTB in humans and animals in various countries, the molecular epidemiology of M. bovis, and finally applied molecular epidemiological techniques.     

细胞因子IL-17作为牛结核病诊断标志物的研究及实时荧光定量PCR检测方法的建立

试验旨在评价细胞因子IL-6和IL-17 mRNA转录水平与牛分枝杆菌感染之间的关系,及其在牛结核病诊断中的应用潜力。通过皮内变态反应试验和IFN-γ释放试验临床筛选结核病阳性牛和结核病阴性牛,采集试验动物抗凝全血,分离、收集外周血淋巴细胞,分别用牛结核菌素（PPD-B）、禽结核菌素（PPD-A）、重组蛋白CFP-10-ESAT-6（CE）、pET-32a载体标签蛋白（PET）或PBS 37℃培养6 h,用实时荧光定量PCR检测细胞因子IL-6、IL-17和IFN-γ的mRNA相对转录水平。结果显示,PET和空白对照PBS类似,不能刺激细胞因子mRNA转录水平的提高,表明CE中包含的PET对试验的影响可忽略不计;牛外周血淋巴细胞经PPD-B、PPD-A或CE刺激后,结核病阳性牛样品中IL-17和IFN-γ的mRNA转录水平均显著高于结核病阴性牛（P<0.05）,其中PPD-B刺激效果强于CE和PPD-A,而CE刺激的特异性更好;选取CE作为最佳刺激源,结果显示,IL-17和IFN-γ的mRNA转录水平之间相关性良好（spearman r=0.79）,并初步建立了基于IL-17和IFN-γ转录水平的实时荧光定量PCR检测方法;以此方法对14头结核病阳性牛进行临床检验,IL-17实时荧光定量PCR法的阳性样本检出率为85.7%,高于IFN-γ（71.4%）。本研究结果初步表明,牛分枝杆菌特异性抗原（PPD-B、CE）诱导的IL-17 mRNA转录水平与牛结核病相关,以CE为刺激源建立的IL-17实时荧光定量PCR检测方法具有用于牛结核病诊断的潜力。     

Potential novel markers to discriminate between active and latent tuberculosis infection in Chinese individuals

Latent tuberculosis infection (LTBI) constitutes the main reservoir for reactivation tuberculosis. The finding of potential biomarkers for differentiating between TB and LTBI is very necessary. In this study, the immunological characteristics and potential diagnostic utility of Rv2029c, Rv2628 and Rv1813c proteins were assessed. These three proteins stimulated PBMCs from ELISPOT-positive LTBI subjects produced higher levels of IFN-γ in comparison with TB patients and ELISPOT-negative healthy subjects (p < 0.05). BCG vaccination and non-TB respiratory disease had little influence on the immunological responses of Rv2029c and Rv2628 proteins (p > 0.05). The LTBI diagnostic performance of Rv2029c was higher than Rv2628 and Rv1813c by ROC evaluation. But Rv2628 had much higher specificity than Rv2029c in active TB patients and uninfected healthy subjects. The IgG level against Rv1813c was higher in the TB group than in LTBI and uninfected healthy subjects (p < 0.05). These results suggest that T cell response to Rv2628 and antibody against Rv1813c might be applicable as biomarkers to distinguish TB from LTBI and uninfected individuals.     

应用胶体金试纸条诊断方法检测牛结核病的试验

采用牛结核MPB70/83抗体检测胶体金试验方法对贵阳周边及某县奶牛场共309头奶牛进行牛结核病检测,同时采用结核菌素(PPD)皮试进行对比试验,结果显示,该病的感染率为4.48%~6.47%,胶体金试纸条试验与PPD反应具有一定的符合率[75%(15/20)],采用生物统计分析,进行χ2检验,计算得χ2c=0.703,χc2<χ20.05,显示两种试验差异极不显著。同时试验还证明了牛结核MPB70/83抗体检测胶体金试纸条试验方法具有快速、简便、特异、敏感的特点,并且可作为PPD反应的辅助诊断方法。因此,该方法具有良好的应用前景。     

Responses to tuberculin among Zebu cattle in the transhumance regions of Karamoja and Nakasongola district of Uganda

Responses to tuberculin in Zebu cattle of the transhumant pastoral farming system in Karamoja region and Nakasongola district in the north-eastern and mid-central regions in Uganda, respectively, were investigated using a comparative intradermal tuberculin skin test. Of the 1864 cattle tested from 30 large units (superherds) in Karamoja and 7 herds in Nakasongola, a total of 28 animals from 19 herds (51.4%) tested positive. Inter-district tuberculin reactor prevalence variations seemed to be influenced by climate, with impact on both the management patterns and transmissibility of agent. High herd tuberculin reactor prevalence (51.4%) was attributed to widespread contacts and mixing of animals between herds. Low individual animal tuberculin test positivity (mean = 1.4%) was attributed to low transmissibility of the agent under the Karamoja climate, which is semi-arid, and to increased resistance due to non-specific response to environmental mycobacteria and natural selection, since there was no active control against bovine tuberculosis. Owing to similarities in management practices in Karamoja and widespread risk factors, it was difficult to identify which were more important, but variations in sources of drinking water pointed to provision of lake and borehole water during dry season as reducing the risk. Positive bovine tuberculin reactor prevalence and skin reactor status were related to age.     

陕西省关中地区奶牛结核病调查

2006-2008年对陕西关中部分地区(西安、宝鸡、咸阳和渭南)奶牛结核病依据我国国家标准《GB/T118645-2002》"结核菌素试验"进行调查,调查数据显示2006年-2008年检测奶牛分别为6090头、5328头、4286头,阳性率分别为0.77%、1.05%、1.42%,陕西关中地区奶牛结核病阳性率有明显上升趋势。西安、宝鸡、咸阳和渭南地区的检测头数分别为4129头、4680头、3797头、3058头,阳性率分别为0.90%、0.80%、1.18%、1.43%,其中渭南地区的阳性率最高。从养殖规模上来看,10头以下养殖户奶牛结核阳性率最高为1.48%。     

扑杀病害奶牛时静松灵和敌敌畏联合应用效果观察

布鲁氏菌病（以下简称布病）和牛结核病为人畜共患病,我国将＂两病＂规定为二类动物疫病。尤其是布病对人畜普遍易感,且经接触、空气、黏膜多途径传播、感染,疫情发生后,按照＂检、杀、免、消＂的防治原则,进行科学的果断处置,使疫情得到控制,对提高人口质量,促进畜牧业健康发展具有极为重要的意义。在扑杀过程中,静松灵（Xylazine Hydrochloride Injection）和敌敌畏（DD-VP）的联合应用,成本低,省时省力,符合动物福利原则,虽不是完全的安乐死,但使动物痛苦得到了降低,值得推广。     

IMAGING DIAGNOSIS—MAGNETIC RESONANCE IMAGING FINDINGS OF AN INTRACRANIAL EPIDURAL TUBERCULOMA IN A DOG

Magnetic resonance (MR) imaging is highly sensitive for detecting tuberculomas in human patients but the specificity of the MR imaging features is low. Misdiagnosis with intracranial neoplasia is common, especially with dural‐based lesions or lesions located in the epidural space. We describe the MR imaging characteristics of an intracranial epidural tuberculoma caused by Mycobacterium tuberculosis infection in a dog. The intracranial mass and skull flat bone lysis and erosion are similar to those described in human caseating tuberculomas and can mimic intracranial neoplastic disease.