结核病与BCG疫苗接种个体TaqMan探针荧光定量PCR诊断方法的建立及初步应用

为快速鉴别诊断结核病(TB),本研究以GenBank登录的致病性结核分枝杆菌复合群、人型结核杆菌和牛分枝杆菌特有基因为对象,设计并合成引物及探针,建立TaqMan探针荧光定量PCR检测方法。实验结果表明,该方法对标准质控菌株反应呈阳性,对卡介苗(BCG)及其他微生物样品反应呈阴性;对结核分枝杆菌或牛分枝杆菌标准菌株的检测灵敏度可达单个菌细胞水平。对45份结核菌素PPD皮肤试验结果为阳性的临床样本进行TaqMan探针荧光定量PCR检测,36份为阳性;而对PPD检测为阴性的50份临床样本进行检测时,7份为阳性。本研究结果表明,所建立的方法可用于TB的鉴别诊断,可对由BCG接种或环境中分枝杆菌引起的PPD检测假阳性样本进行鉴别,对TB的快速检测和早期诊断具有重要意义。     

ELISA检测乳中牛结核抗体的研究

结核病是由结核分支杆菌引起的一种严重的慢性人畜共患传染病,曾经是人类历史上最猖獗的传染病之一。试验以牛乳为试验材料,建立一种诊断牛结核病的间接ELISA方法,目的是为诊断牛结核病找到一种更快捷、简便的方法,减少人力资源的投入,减轻医务工作者的劳动强度,便于ELISA方法在临床上应用。1材料与方法1.1材料1.1.1抗原牛结核PPD,购于黑龙江省生物制品一厂(批号为9902)。1.1.2牛乳阳性牛乳为牛结核变态反应阳性,涂片镜检或细菌分离培养呈阳性,其血清经多次ELISA检测OD值大于临界值的病牛的牛乳。阴性牛乳为牛结核变态反应阴性,细菌…     

从副结核变态反应阳性病牛群中培育健康犊牛

为了扩大健康牛群,减少牛副结核病感染、蔓延的可能性,我们对5头副结核变态反应阳性母牛采取单圈产犊、母仔分离饲养、犊牛定期检疫等措施,将病牛28头1～3代后裔犊牛全部培育为健康犊牛,经多次检疫均为副结核变态反应阴性。具体作法是:     

IP-10作为牛结核病诊断标志物的初步探究

为评价细胞因子IL-12 p40、IP-10和TNF-α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。采集田间筛选的结核病阳性牛、结核病阴性牛以及牛分枝杆菌68002人工感染牛的外周血淋巴细胞,经牛结核菌素(PPDB)、重组蛋白CFP-10-ESAT-6(CE)、MPT63、PET和PBS分别刺激6 h,提取细胞总RNA,用荧光定量PCR检测IL-12 p40、IP-10、IFN-γ和TNF-α的转录水平。结果显示结核病阳性牛的外周血淋巴细胞经PPDB和CE刺激后,其IP-10的m RNA转录水平显著高于结核病阴性牛,且与IFN-γ的m RNA转录水平具有良好的相关性;初步建立牛结核病IFN-γ和IP-10的Real-time PCR检测方法,其对临床阳性样本的检出率分别为71.45%和78.57%。因此,IP-10的m RNA转录水平与牛分枝杆菌的感染相关,有作为牛结核病诊断标志物的潜力。     

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-17mRNA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6h,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(P0.05),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-17mRNA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。     

牛结核病的流行与防控技术

一、牛结核病的防控概述1.结核病是人兽共患的慢性消耗性传染病。病原是分枝杆菌中的结核分枝杆菌复合群,其中人结核病是由结核分枝杆菌(人型菌)引起,牛结核病是由牛分枝杆菌(牛型菌)引起。2.人感染牛结核的情况。(1)发达国家。美国牛奶巴氏消毒前(1908年),10%～30%的人结核病例是由牛分枝杆菌感染所     

牛结核病PCR检测试剂盒的研制

根据GenBank中的牛结核分枝杆菌IS6110的基因片段,设计了1对引物,通过对PCR反应条件进行优化,研制了用于检测牛结核病的PCR试剂盒,该试剂盒扩增的阳性条带为317 bp;敏感性结果显示,该PCR检测试剂盒的最低核酸检测量为1.025 pg/μL;特异性试验表明,仅结核分枝杆菌扩增结果为阳性,副结核分枝杆菌、胸膜肺炎放线杆菌、大肠杆菌、巴氏杆菌、沙门氏菌、金葡萄球菌、链球菌的扩增结果均为阴性。-20 ℃至少可保存12个月,且重复性良好。应用该PCR试剂盒对24份临床样本进行了检测,其PCR检测结果与结核菌素试验检测结果相一致。结果表明,牛结核病PCR检测试剂盒能够对牛结核临床样本进行快捷、灵敏、准确的检测。     

结合皮内变态反应与γ干扰素ELISA的牛结核病检疫

巴州辖区内某奶牛场为根除牛结核病对该奶牛场的威胁,技术人员根据该奶牛场情况制定了检疫计划,选用皮内变态反应与γ干扰素ELISA检测相结合的方法,从而达到检疫的目的。初筛检疫使用牛型结核病PPD(纯化蛋白衍生物)皮内变态反应,出现阳性或可疑牛之后,再对其接触较为密切的同群牛进行γ干扰素ELISA(酶联免疫吸附试验),目的在于检出与患病动物接触的牛群是否处在牛结核感染的潜伏期。检测结果:皮内变态反应全检150头奶牛后,出现1例阳性牛和1例可疑牛,之后将阳性、可疑与同属一个圈舍的其他13头牛进行γ干扰素ELISA检测,出现1例牛型结核分枝杆菌阳性、14例阴性。表明牛结核PPD皮内变态反应结合γ干扰素ELISA可以有效提高牛结核病检疫的准确性,节省检疫时间,减少因单纯使用牛结核PPD皮内变态反应出现假阳性而造成的不必要扑杀。     

牛结核病的防治

结核分支杆菌(Mycobacterium paratuberculosis)侵入牛机体后引起病牛发生的渐进性消耗性疾病被称为牛结核病(bovine paratuberculosis)。临床发病病例中多以6月龄的牛为主,发病牛的特征性症状为病牛消瘦,顽固性腹泻,此外伴发慢性卡他性肠炎。本文分析了牛结核病的病原、流行病学、发病机理、临床症状、病理变化和诊断,并提出了治疗措施,降低此病的发生,促进养牛业发展。     

11种牛分枝杆菌抗原在牛结核病诊断中的初步评价

为筛选及评价用于牛结核病诊断的抗原,本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法,比较其对牛结核病的检出率;同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外,将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24h,检测血浆中的IFN-γ水平,评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示,不同重组蛋白对结核病阳性血清的反应活性不一,MPB70总检出率最高,为59.7%;其次是Ag85B、ESAT-6和MPB83,检出率均在45%以上;MPT51的检出率最低,仅为2.2%。豚鼠和牛皮试试验均显示,单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应(delayed type hypersensitivity,DTH),而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原,分别与CFP-10或ESAT-6混合,均可特异性地刺激结核病阳性牛产生较强的DTH反应,且与PPD-B无显著差异(P0.05)。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ,其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛(P0.05)。因此,这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验,但以CFP-10和ESAT-6为核心,TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原,均能提高检测敏感性,有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。     

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。     

The prevalence of bovine tuberculosis (Mycobacterium bovis) infections in feral populations of cats (Felis catus), ferrets (Mustela furo) and stoats (Mustela erminea) in Otago and Southland, New Zealand

Twenty-one properties in the Otago region of the South Island of New Zealand were surveyed for the presence of gross lesions due to Mycobacterium bovis infection in feral cats (Felis catus), ferrets (Mustela furo) and stoats (Mustela erminea) during 1993 and 1994. In total, 1293 cats, ferrets, stoats and weasels (Mustela nivalis) were examined for the presence of tuberculous lesions. The properties surveyed were selected according to the history and incidence of bovine tuberculosis infection in their cattle herds. Sixteen infected cattle properties were trapped in areas of Otago that were endemic for bovine tuberculosis and five properties were trapped in non-endemic areas that were considered to be free from tuberculosis infection in the cattle. No tuberculous cats, ferrets, stoats or weasels were found in non-endemic areas, and prevalence rates in the endemic areas were 0.9% for cats (n=215, 0.12相似文献   

Tuberculosis in cattle herds are sentinels for Mycobacterium bovis infection in European badgers (Meles meles): the Irish Greenfield Study

In Ireland badgers are removed in response to tuberculosis (TB) breakdowns in cattle herds (focal culling). Prevalence studies, conducted using a detailed post mortem and bacteriological examination, showed that 36-50% of badgers were infected with Mycobacterium bovis. Focal culling forms part of the medium term national strategy for the control of bovine TB in cattle and is based on the premise that badgers in areas with herd breakdowns have a higher prevalence of infection than the badger population at large. However, the hypothesis that cattle can be used as sentinels for infection in the badger population has never been formally tested. In this study we tested the hypothesis by determining the infection prevalence in badgers in areas where there had been historically, a consistently low prevalence of infection in cattle. Low cattle TB prevalence areas were defined as those herds with ≤ 2 standard reactors in the annual round of skin testing over the preceding 5 years (Greenfield sites). Using GIS, and adjusting for variation in land use, previous culling and cattle density, 198 Greenfield sites were identified and surveyed, and 138 areas with badger setts or signs of badger activity were identified. A single badger was removed from 87 sites and all were examined using detailed post mortem and bacteriological procedures. A prevalence of M. bovis infection of 14.9% was found in the Greenfield site badgers. This prevalence was significantly lower (P<0.001) than in badgers removed during focal culling (36.6%). The results validate the use of cattle as sentinels for TB in badgers and support the medium term national strategy for the control of bovine TB. The geographic variation in M. bovis infection prevalence in the Irish badger populations will be used when devising strategies for the incorporation of badger vaccination into the long term bovine TB control programme.     

Prevalence of Bovine tuberculosis in Ethiopian slaughter cattle based on post-mortem examination

A study aimed at describing the magnitude and distribution of gross lesions compatible with bovine tuberculosis (BTB) in Ethiiopian slaughter cattle in five abattoirs (four municipal and one export) located in various cattle husbandry systems in Ethiopia was carried out from July 2006 to January 2007 using detailed meat inspection procedure. Five representative abattoirs (four municipal and one export) located in distinct livestock management systems were selected. A total of 3322 cattle; 2876 (86.6%) male, 446 (13.4%) female; 3094 (93.1%) indigenous zebu, 140 (4.2%) crossbred and 88 (2.7%) pure exotic cattle were included in the study. A nine-year meat inspection record was also analyzed to elucidate the trend of BTB in the local cattle population.Of the carcasses inspected, 337 (10.2%, 95%CI= [9.2–11.2]) had lesions suggestive of tuberculosis, 69 (20.5%) generalized and 268 (79.5%) localized.TB prevalence showed a marked variation between categories of age, breed, class of animals, abattoir, geographic origin and husbandry system. It was higher in old and young animals than middle age group; in exotic than local breed; in calves than other classes of animals. The highest and lowest prevalence of TB was recorded in Adama (24.7%, 95%CI= [21.1–28.7]) and Yabello abattoirs (4.2%, 95%CI= [2.6–6.6]), respectively. Cattle whose origin was from Addis Ababa and its surrounding areas had higher prevalence of TB infection (23.9%, 95%CI= [17.6–31.5]).Cattle maintained in dairy farms had high degree of exposure (23.9%, 95%CI= [16.7–32.9]) to the infection than those maintained in other types of management system. Analysis of meat inspection records also revealed an increasing incidence of TB over the years. Our study demonstrated a high prevalence of tuberculosis in Ethiopian slaughter cattle and this could infer to similar scenario in a reference cattle population in the country. In view of Ethiopia’s increasing involvement in livestock export trade, the reported high prevalence of tuberculosis could be a major obstacle, particularly at this moment when sanitary requirements from importing countries are so much strict. Furthermore, the growing concern over increasing incidence of tuberculosis/HIV/AIDS co-infection, the high incidence of extra- pulmonary tuberculosis and a high risk of acquiring zoonotic tuberculosis among the majority of the population emphasize the need for paying the necessary attention towards the control of bovine tuberculosis.     

Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattle-specific serological assay for BTB (TB ELISA). Buffaloes (n = 997) were tested with the tuberculin skin test and TB ELISA; a subset (n = 119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes.     

牛分枝杆菌重组蛋白TB27.4的表达、纯化及活性鉴定

为探索TB27.4蛋白在牛结核病鉴别诊断中的作用,本试验以牛分枝杆菌Vallee Ⅲ株基因组DNA为模板,PCR扩增tb27.4全长基因片段,将其定向克隆到原核表达载体pET-32a(+)中,构建重组质粒pET-TB27.4,优化原核表达条件,并用AKTA Purifier对蛋白的纯化条件进行优化。SDS-PAGE结果显示重组蛋白为可溶性表达,且大小与理论值相符,用牛分枝杆菌阳性血清进行Western blotting检测有特异性条带,且可特异性地刺激牛分枝杆菌感染牛外周血淋巴细胞释放大量IFN-γ。结果表明,重组蛋白TB27.4具有良好的B细胞活性和T细胞刺激活性,为进一步研究其在牛结核病诊断中的作用奠定了基础。     

A descriptive spatial analysis of bovine tuberculosis in intensively controlled cattle farms in New Zealand

We describe the temporal and geographical distribution of confirmed cases of bovine tuberculosis (TB) in a population of cattle in the south-east of the North Island of New Zealand. Data were derived from routine TB testing conducted between 1980 and 2003 and included details for 69 farms. Four six-year periods were defined to coincide with changes in depopulation strategies against the wildlife TB reservoir, the brushtail possum Trichosurus vulpecula. For the periods 1980 to 1985 and 1986 to 1991 the median annual incidence rate of TB was 0.4 and 4.7 cases per 1000 cattle-years at risk, respectively. For the period 1992 to 2003 the median annual incidence rate of TB decreased to 1.8 cases per 1000 cattle-years at risk, coincident with the use of poisoning to control possums in the surrounding forest park (a major possum habitat area). We identified clusters of TB cases adjacent to the forest park and found no evidence of spatio-temporal interaction of TB risk among farms. Our findings support the hypothesis that possums living in the forest park are a source of bovine TB in this area and that farm-to-farm spread of disease was not an important infection mechanism.     

VPS28通过泛素化信号通路调控奶牛乳腺上皮细胞中乳蛋白的合成

旨在分析VPS28基因调控乳蛋白合成的分子机制，为奶牛泌乳性状的分子育种奠定理论基础。本研究首先利用RNA干扰（RNA interference，RNAi）技术敲降奶牛原代乳腺上皮细胞（bovine mammary epithelial cells，BMECs）中VPS28基因的表达水平，检测与乳蛋白合成、泛素化-溶酶体和泛素化-蛋白酶体通路相关的11个基因、泛素蛋白的表达水平及蛋白酶体活性；然后抑制BMECs中蛋白酶体和溶酶体的活性，检测酪蛋白相关基因、核糖体蛋白的表达水平；最后利用同位素标记相对和绝对定量（isobaric tags for relative and absolute quantification，iTRAQ）比较蛋白质组学分析敲降前后BMECs的差异表达蛋白。结果表明，敲降VPS28基因后，CSN1S1、CSN2、CSN3、RPS8、UBC、PSMC3、PSMC5基因均显著上调，PSMD12显著下调；抑制蛋白酶体后，CSN1S1、CSN2、CSN3显著上调，RPL13显著下调；抑制溶酶体活性后酪蛋白相关基因表达不显著；iTRAQ结果共筛选出129个差异表达蛋白，下调蛋白主要富集在核糖体、溶酶体、剪切体等相关通路，上调蛋白主要富集在内质网的蛋白质加工、加压素调控的水重吸收过程、RNA转运等通路中。研究表明，VPS28基因可通过泛素化信号通路影响BMECs中乳蛋白的合成。     

Risk factors of bovine tuberculosis in cattle in rural livestock production systems of Ethiopia

This study shows a representative stratified cluster sample survey of the prevalence of comparative intradermal tuberculin test in cattle from four regions in Ethiopia. Using a cut-off for positivity of 2 mm, it assesses possible risk factors for tuberculin-positive reaction in cattle. Seventy-three villages in 24 kebeles (administrative units) were randomly selected, from which 2216 cattle from 780 owners were tested. In addition, 450 of these cattle owners were interviewed for risk factor assessment. Ninety-nine percent of the tested cattle in this rural livestock production system were traditional zebus. The individual overall prevalence of cattle bovine tuberculosis (BTB)e was 3%, with the highest found in Meskan Mareko, in Central Ethiopia (7.9%) and the lowest in Woldia, in the North East edge of the Rift Valley (1.2%). Generalised Linear Mixed Models (GLMM) with random effect on kebeles was used to analyse risk factors of cattle reactors and human tuberculosis (TB) infection. Purchase of cattle and presence of other livestock in the herd were statistically significant, with OR: 1.7, p-values of 0.03 and OR: 2, p = 0.05, respectively. Family members diagnosed with TB or showing clinical signs of extra-pulmonary TB (EPTB) were reported in 86 households (19%). None of the assessed potential risk factors of disease transmission between cattle and human (food consumption, livestock husbandry and presence of BTB-positive cattle) were statistically significant.     

母猪妊娠早期血液中差异表达蛋白的鉴定

为了研究母猪妊娠早期外周血液中蛋白质的表达情况,寻找潜在的母猪妊娠早期生物标志物,本试验采集了 8头健康、生产表现正常且体况相近的配种后15 d妊娠与非妊娠的长大二元经产母猪外周血液,分离蛋白,利用iTRAQ技术及生物信息学方法筛选与胚胎附植相关的差异表达蛋白,用ELISA验证其表达情况,并进行ROC曲线分析.结果显示...