鹿结核病分子诊断技术的研究进展

鹿结核病是一种由结核分枝杆菌引发的慢性、消耗性传染病,多年来世界各国均受到结核病的困扰。结核病通常以结核杆菌形式感染人类及以牛结核杆菌形式感染奶牛,与其他同属结核菌相比,牛分枝杆菌可感染更多种类动物致病,如牛、羊、鹿等动物。由于鹿结核病目前尚无规定性的诊断方法,同时鹿结核病主要是牛型结核分枝杆菌感染所致,一般参照牛结核病的部分诊断方法进行诊断。分子诊断技术能快速高效地诊断结核分枝杆菌,对进一步治疗、扑灭结核病起至关重要的作用。因此,文章对近年来鹿常见的结核病分子诊断技术的研究进展进行综述。     

鹿结核病及其防治

鹿结核病是由结核杆菌引起的人兽共患慢性传染病,其病理特点是在机体组织中形成结核结节性肉芽肿和干酪样坏死灶。鹿结核病在集体和个体养鹿场流行较广,是危害养鹿业较为严重的一种传染病。 1.流行病学:患有结核病的病鹿、动物和人是该病的传染源。对结核病鹿应隔离饲养或淘汰,鹿场严禁其它结核动物接触。结核的传染     

鹿结核分枝杆菌MPB70蛋白的表达与免疫原性鉴定

本研究以分离的鹿结核分枝杆菌DNA为模板扩增免疫原性蛋白MPB70基因,获得约590 bp片段,并将其克隆,构建原核表达载体pET-30a-MPB70,将重组质粒转入大肠杆菌BL21（DE3）,经IPTG诱导后纯化和SDS-PAGE分析,在20 ku处可见特异性蛋白条带。利用鹿结核阳性血清进行Western blotting鉴定,原核表达的融合蛋白可与鹿结核阳性血清抗体结合,并出现特异的免疫反应。该蛋白可作为特异性抗原进行鹿结核病的检测,从而为鹿结核病诊断方法的研究奠定基础。     

鹿结核病的诊断、病原分离及鉴定

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 ,不仅影响养鹿业的发展 ,也威胁人类健康。结核分枝杆菌有牛型结核分枝杆菌、人型结核分枝杆菌和禽型结核分枝杆菌三个型。据报道[1] 引起鹿结核病的主要病原体是牛型分枝杆菌 (40 %～ 5 9% )和鸟—胞内分枝杆菌 (30 %～ 4 0 % ) ,在鹿中存在结核菌素假变态反应和副变态反应 ,因此 ,不能单纯依靠变态反应方法进行初次定性 ,必须进行综合检查。为确诊本病我们采用病原体检查、变态反应学诊断及动物试验等方法 ,进行综合检查 ,将结果报告如下。1　流行情况20 0…     

应用琼脂扩散试验诊断绵羊副结核的研究报告

副结核病是由副结核分枝杆菌引起的慢性传染病。主要侵害牛、绵羊、山羊、鹿等反刍动物。为了更好的防治本病,研究一种简便易行、特异性高的诊断方法是十分必要的。我们于1986年应用琼脂扩散试验做了绵羊副结核病的诊断研究,现报告如下。材料与方法菌种:牛副结核分枝杆菌P18菌株,由中国兽药监察所惠赠。琼扩抗原制备     

鹿结核病的流行及防控进展

鹿结核病是由牛分枝杆菌引起的鹿的慢性、消耗性传染病,广泛流行于世界各地,严重危害着养鹿业的发展,同时也威胁着人类健康和其他动物的安全。为保证养鹿业的发展和人类的健康,对该病进行有效防控显得十分重要。美国、新西兰等国家通过对可疑感染的鹿群进行持续性监测和利用捕猎季节减少感染鹿群的数量等策略,成功实现了对鹿结核的控制。现对国内外鹿结核病的流行现状、诊断技术及疫苗研究进行综述,以期为鹿结核病的防控提供借鉴。     

鹿结核自然病例结核分枝杆菌复合物的鉴定与分型

对收集的5例鹿结核自然病例进行结核分枝杆菌复合物分子生物学鉴定和基因分型,探索结核分枝杆菌传播过程中人与其他动物间的关系。从而掌握结核病的传播规律,并为动物结核病的防控提供临床数据。试验中以常规病理组织学方法制作切片观察病灶组织学变化,应用Oligo(7.0)设计特异性引物,以IS6110插入序列设计引物进行结核分枝杆菌复合物的鉴定。采用单管多重PCR方法,对所分离的结核分枝杆菌复合物PCR鉴定阳性菌株,进行牛分枝杆菌和结核分枝杆菌菌种鉴定。结果所采集的鹿结核自然病例中人型结核分枝杆菌2例,牛型结核分枝杆菌3例。表明结核病在人与其他动物间相互传染,并且,人传染鹿的可能性更大。     

评价荧光探针PCR方法在鹿副结核病检测中的应用

副结核病又称约内氏病,病原菌为副结核分枝杆菌,世界各地的野生和驯养鹿群中都有该病发生.病鹿表现出慢性持续性腹泻、体重迅速减轻、皮下水肿,严重时出现死亡,对养鹿业造成极大经济损失,同时副结核分枝杆菌还是人克罗恩氏病罪魁祸首.研究表明,水是人克罗恩氏病与动物副结核病的主要传染源,国内外已有大量文献报道水中副结核分枝杆菌的检测,但副结核病在动物和人之间的传播关系尚未明确[1-2].     

野生动物结核病感染的研究进展

野外和圈养野生动物的结核病疫情呈现增高的趋势,不但造成重大经济损失,而且影响到公共卫生安全。野生动物的结核病的病原是结核分枝杆菌复合菌群,但主要是由结核分枝杆菌(M.tuberculosis)和牛分枝杆菌(M.bovis)所引发,其感染宿主谱很广,已知可感染灵长类、野牛、野猪、鹿、大象、鹦鹉、负鼠、獾、貘、羚羊等动物。本文从野生动物结核病的流行病学、症状和病变、诊断方法、防治措施等方面进行了综述,表明许多野生动物具有易感性并表现不同的结核病临床病症。由于野生动物结核病的发生与家畜和人的结核病防控密切相关,必须予以必要的关注和防范。     

国外近年来牛付结核病研究情况介绍

国外近年来对牛付结核病的研究进展较快。现仅就七十年代以来国外对本病的研究情况做一简要介绍: 流行病学七十年前报导过牛、羊、猪、鹿、骆驼等家畜发生付结核病。1950年马斯凯(Maokic)和卡锡(Carber)把豚鼠,鸡不发生牛付结核实验感染“作为付结核     

塔里木马鹿结核病的诊治

2007年新疆某规模马鹿场部分马鹿发病,仔鹿多表现为消瘦、食欲下降,营养消耗性死亡;成年鹿多表现为咳嗽、喘气、体温升高、突然喘气死亡。通过病理解剖,细菌培养,接种动物等方法确诊该鹿场马鹿死亡原因是感染了牛型结核分枝杆菌,说明牛型结核分枝杆菌可以感染马鹿。     

Expression of MPB70 Protein and Identification the Immunogenicity of Deer Mycobacterium tuberculosis

In this study,the immunogenicity protein MPB70 gene was amplified from Mycobacterium tuberculosis genome DNA which separated from deer, and about 590 bp fragment was obtained. Then the fragment was cloned and constructed prokaryotic expression vector of pET-30a-MPB70, and the recombinant plasmid was put into E. coli BL21(DE3).Purified after IPTG induction, and analyzed by SDS-PAGE, a specificity protein band was observed at 20 ku. Using the deer serum positive of tuberculosis in Western blotting, the fusion protein could be combined with deer serum positive of tuberculosis antibody and arise specific immune response. The protein could be used as a specific antigen to test the deer tuberculosis. The study laid a foundation for further studying the deer tuberculosis appraisal method.     

吉林省鹿结核病的流行病学调查

为掌握结核病在吉林省鹿群中的流行传播状况,从吉林省养鹿业富有代表性地区的18个鹿场随机采集血清样本1856份作为研究对象,利用结核杆菌Ag85-East6-mpt70抗原多联表达蛋白作为检测抗原,进行酶联免疫检测,调查吉林省鹿结核病流行情况,结果发现,吉林省地区鹿群中存在结核病病例,且各地区间阳性率差异显著或极显著（P<0.05或P<0.01）,防疫部门应加强防控,防止疫病扩散。     

Diagnosis of tuberculosis due to Mycobacterium bovis in New Zealand red deer (Cervus elaphus) using a composite blood test and antibody assays

A blood test for tuberculosis in deer was developed as an ancillary test to clarify the status of skin test-positive deer, with non-specific sensitisation following exposure to saprophytic mycobacteria. The blood test incorporates the measurement of the relative humoral and cellular immunological responses to Mycobacterium bovis and M. avium antigens to provide a composite test with high levels of sensitivity (>95%) and specificity (>98%). The specificity of the test has allowed it to be used in parallel with the skin test to salvage thousands of tuberculosis-free deer with non-specific skin test-positive reactions, while its high sensitivity has consistently identified M. bovis-specific reactivity in tuberculous skin test-positive animals. The rules for establishing the diagnostic parameters for the cellular and antibody assays were developed by retrospective analysis of the laboratory results using blood samples from many thousand tuberculous or disease-free deer. The sensitivity of the blood test was tested in this study using 150 animals with tuberculosis diagnosed by the isolation of M. bovis. It had sensitivity values of 95.7-95.9% in herds with a low (<2.0%) or a high (>30.0%) incidence of tuberculosis. The test had a specificity of 98.0% when tested on 218 disease-free animals, 118 of which were skin test-positive. An antibody test was developed to diagnose M. bovis in skin test-negative anergic deer from tuberculosis infected herds. When this test was used with deer blood taken 10 days after reading the skin test, it had a sensitivity of 85.3% for 102 M. bovis-positive deer. When used in combination with skin test, the antibody test complemented the skin test to raise the sensitivity of the combined tests to 95.0%, when antibody-positive or skin test-positive tests were used to diagnose tuberculosis. The specificity of the antibody test was 100% when used to evaluate 218 disease-free deer from non-infected herds.     

Diagnosis of tuberculosis due to Mycobacterium bovis in New Zealand red deer (Cervus elaphus) using a composite blood test and antibody assays

A blood test for tuberculosis in deer was developed as an ancillary test to clarify the status of skin test-positive deer, with non-specific sensitisation following exposure to saprophytic mycobacteria. The blood test incorporates the measurement of the relative humoral and cellular immunological responses to Mycobacterium bovis and M. avium antigens to provia composite test with high levels of sensitivity (>95%) and specificity (>98%). The specificity of the test has allowed it to be used in parallel with the skin test to salvage thousands of tuberculosis-free deer with non-specific skin test-positive reactions, while its high sensitivity has consistently identified M. bovis-specific reactivity in tuberculous skin test-positive animals. The rules for establishing the diagnostic parameters for the cellular and antibody assays were developed by retrospective analysis of the laboratory results using blood samples from many thousand tuberculous or disease-free deer. The sensitivity of the blood test was tested in this study using 150 animals with tuberculosis diagnosed by the isolation of M. bovis. It had sensitivity values of 95.7–95.9% in herds with a low (<2.0% ) or a high (>30.0%) incidence of tuberculosis. The test had a specificity of 98.0% when tested on 218 disease-free animals, 118 of which were skin test-positive.

An antibody test was developed to diagnose M. bovis in skin test-negative “anergic” deer from tuberculosis infected herds. When this test was used with deer blood taken 10 days after reading the skin test, it had a sensitivity of 85.3% for 102 M. bovis-positive deer. When used in combination with skin test, the antibody test complemented the skin test to raise the sensitivity of the combined tests to 95.0%) when antibody-positive or skin test-positive tests were used to diagnose tuberculosis. The specificity of the antibody test was 100% when used to evaluate 218 disease-free deer from non-infected herds.     

鹿结核病诊断与免疫方法的研究

从５１头临床病鹿中分离出３９株分枝杆菌（Ｍｙｃｏｂａｃｔｅｒｉｕｍ），其中牛分枝杆菌（Ｍ．ｂｏｖｉｓ）与鸟－胞内分枝杆菌（Ｍ，ａｖｉｕｍ－ｉｎｔｒａｃｅｌｌｕｌａｒｅ）的分离率最高，分别为２５．０％和２０．５％。建立了以牛型ＰＰＤ为抗原的间接ＥＬＩＳＡ和变态反应ＯＴ点眼法两种诊断方法。两种诊断方法比较，结果有交叉，总符合率为８７．３８％。鹿对ＢＣＧ皮下接种有良好的免疫应答，最佳免疫程序为：仔鹿出生２４ｈ内第１次皮下免疫接种冻干ＢＣＧ０．７５ｍｇ；第２次免疫接种为１２月龄，方法同前；第３次免疫接种为２４月龄，方法同前。应用此免疫程序在５个结核污染场的３８０８头阴性鹿免疫试验证明，对仔鹿保护率为９９．７％，成鹿保护率为９９．９％。     

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White‐tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programmes. One approach in dealing with wildlife reservoirs of disease is to interrupt inter‐species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white‐tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 10⁷ CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 10⁷ CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid‐fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white‐tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.     

Farm and slaughter survey of bovine tuberculosis in captive deer in Switzerland

In 1998, a survey was conducted by postal questionnaire to gather basic knowledge about the management, health and productivity of captive deer in Switzerland. In addition, lymph nodes were collected from slaughtered deer from 124 of the 262 holdings surveyed, and tested for Mycobacterium bovis and Mycobacterium tuberculosis. The total farmed deer population was 8389 animals kept on 485 holdings; 87 per cent were fallow deer, 8 per cent red deer, 4 per cent sika deer, and there were small numbers of other species. The median herd sizes were 12 for fallow deer and eight for red deer. Few owners had handling facilities or crushes. In none of the lymph nodes examined were lesions typical of bovine tuberculosis observed, and neither M bovis nor M tuberculosis was cultivated from any of the samples.     

Improving protective efficacy of BCG vaccination for wildlife against bovine tuberculosis

Possums are a wildlife vector of bovine tuberculosis in New Zealand. Vaccination of possums with BCG is being considered as a measure to control the spread of bovine tuberculosis to cattle and deer. Delivery via oral bait is feasible but BCG is degraded in the stomach. The aim was to determine whether ranitidine (Zantac) would reduce gastric acidity and enhance the efficacy of intragastrically administered BCG. A dose of 75 mg reduced gastric acidity for at least 4 h. Thus, possums were vaccinated intragastrically with BCG after receiving 75 mg ranitidine or ranitidine or BCG alone, as controls, before challenge with virulent Mycobacterium bovis. Proliferative responses of blood lymphocytes to M. bovis antigens after vaccination were significantly higher in possums given ranitidine/BCG compared to controls and seven weeks after challenge they had significantly lower lung weights and spleen bacterial counts than ranitidine alone controls. Vaccination with BCG alone only gave a reduction in loss in body weight. Agents that reduce gastric acidity may be useful in formulating BCG for oral bait delivery to wildlife for vaccination against bovine tuberculosis.