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1.
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。 相似文献
2.
Tuberculosis in domesticated red deer: Comparison of purified protein derivative and the specific protein MPB70 for in vitro diagnosis 总被引：7，自引：0，他引：7
The use of a Mycobacterium bovis-specific protein, mycobacterial protein bovis 70 (MPB70), was compared with complex, M bovis-derived purified protein derivative (bovine PPD), for its ability to improve the diagnostic precision of in vitro assays for tuberculosis in farmed deer. A combination of lymphocyte transformation and enzyme-linked immunosorbent assay (ELISA) was used to differentiate between specific M bovis reactivity and crossreactivity due to sensitisation with saprophytic mycobacteria such as Mycobacterium avium. In the lymphocyte transformation assay the response of mononuclear cells, from red deer, to MPB70 was found to be more specific, but less sensitive, as an indicator of infection by M bovis when compared with the complex antigen bovine PPD. When used in conjunction with bovine PPD alone, MPB70 was found to increase the specificity of the ELISA in diagnosing animals with disease. 相似文献
3.
牛型分枝杆菌MPB70蛋白胶乳凝集方法的建立及应用 总被引：9，自引：0，他引：9
为了研究牛结核病新型诊断试剂，提高诊断的特异性和敏感性，我们用大肠杆菌工程菌表达了牛型分枝杆菌特异性抗原MPB70并提纯蛋白建立了牛结核病乳胶凝集试验（LAT）诊断方法。MPB70是一种牛型分枝杆菌特异性分泌而卡介苗BCG缺失的蛋白质，热稳定性好，用此蛋白建立的乳胶凝集方法具有良好的敏感性、特异性以及较长的保存期，检测70份临床奶牛血清，与皮内变态反应和间接血凝方法相比较分别具有71．4％和88．6％的符合率。该方法还可鉴别诊断自然感染和疫苗免疫接种。我们建立的牛型分枝杆菌MPB70蛋白乳胶凝集试验诊断方法将分子生物学手段和经典试验方法有机结合，为临床快速检测牛结核病血清特异性抗体水平提供了行之有效的方法。 相似文献
4.
AIMS: Recently the first case of natural infection of deer with Brucella ovis was discovered. The aim of this study was to develop and evaluate an electrophoretic immunoblotting method for testing deer serum for specific B. ovis antibodies. METHODS: An existing immunoblotting method for sheep serum was altered by using a recombinant protein G-alkaline phosphatase conjugate and Tris-buffered saline containing 3% non-fat dry milk powder for the blocking step and the serum and conjugate dilutions. The method was evaluated using 106 sheep sera from B. ovis - negative, accredited flocks, 69 sera from chronically infected rams shedding B. ovis in their semen, 110 sera from a B. ovis-infected flock, 18 sera from stags from which B. ovis was isolated, and 48 sera from deer flocks free from B. ovis infections. The immunoblotting method was applied to another 85 deer sera. RESULTS: The sensitivity of the new immunoblotting method was 98.6% for sheep and 94.4% for deer, and the specificity 99.1% for sheep and 100% for deer. Sixty-nine out of 97 deer sera, originating from the property from which the first B. ovis deer case had been reported, tested positive or suspicious in the complement fixation test. Of these, 53 sera exhibited staining patterns in blots typical for B. ovis infections and also one serum which was negative in the CFT. Only six out of 1498 deer sera. from throughout New Zealand had positive or suspicious reactions in the B. ovis complement fixation test. Of these, one exhibited a staining pattern in the blot suggestive of a B. ovis infection, while four showed patterns of suspicious reactions. CONCLUSION: The new immunoblotting technique is useful as a confirmatory serological test method for B. ovis infections in deer. 相似文献
5.
6.
塔里木马鹿结核病的诊治 总被引：1，自引：0，他引：1
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。 相似文献
7.
Falconi C López-Olvera JR Boadella M Camarena J Rosell R Alcaide V Vicente J Sánchez-Vizcaíno JM Pujols J Gortázar C 《Veterinary microbiology》2012,159(1-2):40-46
Bluetongue (BT) is an infectious disease of wild and domestic ruminants caused by bluetongue virus (BTV). BTV-4 spread through southern Spain from 2004 to 2006, whereas a BTV-1 outbreak that started in southern Spain in 2007 is still ongoing. Vaccination and movement restriction regulations are applied to domestic ruminants to control BT, but the potential reservoir role of wild European ungulates has not been clarified so far. The aim of this study was to describe the epidemiology of BTV in the wild free-ranging red deer (Cervus elaphus) population of Caba?eros National Park (CNP) in central Spain during the BTV-4 and BTV-1 epizootics, assessing the potential role of this deer population as a BTV reservoir. Blood samples from 2885 (2542 adults, 208 calves and 135 undetermined) wild red deer were collected from 2005 to 2010 in CNP and surrounding hunting estates. All sera were tested for antibodies against the BTV VP7 protein by ELISA. Ninety-four of the ELISA-positive samples were analysed by serum neutralization to detect BTV-4 and BTV-1 specific antibodies, and 142 blood samples were analysed by RT-PCR for BTV RNA. A total of 371 (12.9%) out of the 2,885 deer (35/208 calves, 307/2,542 adults, and 29/135 undetermined) were positive for antibodies against BTV. Prevalence increased in adult deer from 2005-2006 to 2008-2009, declining thereafter. No positive samples for BTV-1 were found by serum neutralization, whereas 43 deer (38 adults, four calves and one undetermined) were positive for BTV-4 specific antibodies. No BTV RNA positive deer were found by RT-PCR. Antibody detection throughout the study period suggests a maintained circulation of BTV in red deer. However, the lack of BTV RNA detection suggests a minor transmission risk to livestock. 相似文献
8.
S D Fitzgerald J B Kaneene K L Butler K R Clarke J S Fierke S M Schmitt C S Bruning-Fann R R Mitchell D E Berry J B Payeur 《Journal of veterinary diagnostic investigation》2000,12(4):322-327
A retrospective study of various diagnostic postmortem techniques used in a 4-year surveillance program for detection of Mycobacterium bovis infection in wild white-tailed deer (Odocoileus virginianus) was conducted. The tests evaluated were routine histopathology, acid-fast staining, detection of acid-fast bacilli in culture, and an M. tuberculosis group-specific genetic probe applied to pure cultures. Each of these techniques were compared with a reference or "gold standard" of mycobacterial culture and identification. Histopathology, the most rapid form of testing for M. bovis infection in white-tailed deer samples, had a sensitivity of 98% and a specificity of 87%, resulting in a positive predictive value of 94%. The detection of acid-fast bacilli by staining was less sensitive than histopathology (90%), but its higher specificity (97%) resulted in a positive predictive value of 99%. The detection of acid-fast bacilli on culture was both highly specific (93%) and sensitive (100%). The group-specific genetic probe had the highest sensitivity and specificity and produced results in complete agreement with those of mycobacterial culture, suggesting that this technique could be used as the new "gold standard" for this particular wildlife tuberculosis surveillance program. 相似文献
9.
Kim TJ Cho HS Park NY Lee JI 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2006,53(2):87-90
A protein chip based on surface plasmon resonance (SPR) was developed for measuring the Mycoplasma hyopneumoniae antibody titres using a recombinant 30-kDa fragment of P97 adhesin as an antigen. The diagnostic potential of this SPR assay, for detecting the antibody titres to the M. hyopneumoniae 30-kDa protein, was compared with that of conventional ELISA using 70 pig serum samples taken from six pig farms. The SPR assay was found to be highly specific and sensitive. Moreover, there was a strong positive correlation between the SPR and ELISA titres (n = 70, r = 0.898, P < 0.01). Therefore, this recombinant 30-kDa protein can be used as an antigen for serological studies, and the SPR, which is a label-free method, is expected to be a valuable and reproducible tool in the serodiagnosis of M. hyopneumoniae infection. 相似文献
10.
采集吉林省具有代表性的4个地区的8个梅花鹿场（四平2个梅花鹿场、通化2个梅花鹿场、长春市2个梅花鹿场、辽源2个梅花鹿场）中630份血清样本作为研究对象,进行血清学（ELISA_方法）检测,了解吉林省梅花鹿副结核病、结核病及布鲁菌病感染现状。上述4个地区均存在梅花鹿副结核病,结核病及布鲁菌病发生和流行,其中副结核病的整体阳性率为18．73％、结核病的整体阳性率为19．21％、布鲁病的整体阳性率为28．42％。吉林省梅花鹿副结核病、结核病及布鲁菌病流行不容乐观,提示广大饲养者及相关部门应该高度重视梅花鹿副结核病的防治工作。 相似文献
11.
AIMS: This study was initiated to investigate aspects of the epidemiology, pathogenesis and transmission of tuberculosis in wild red deer, with the aim of determining whether this species may be considered a reservoir host of Mycobacterium bovis in New Zealand. METHOD: One hundred and six wild red deer (Cervus elaphus) carcasses from the Castlepoint and Hauhungaroa Range areas, which are endemic for bovine tuberculosis, were examined for the presence of M. bovis infection. Samples were also examined from 46 skin test-positive farmed deer killed at two deer slaughter premises. Where possible, a standard set of tissues and excretion site samples was collected for mycobacteriological examination. RESULTS: Fifty-eight infected deer were identified, and of these 28% showed no gross lesions. The prevalence of tuberculosis confirmed by culture in the wild deer was 32%. Only one of 18 deer younger than 1 year was infected. Mature deer (>2 years) were 12 times more likely to be infected than those under 1 year of age. Infected older deer were less likely to show typical gross lesions than younger animals. Mycobacterium bovis was isolated from the oropharyngeal tonsil of 34 of 56 (61%) of the infected deer, and this was the most commonly infected site. Gross lesions were found in 18 of the 34 infected tonsils and only one of these showed a purulent tonsillitis. Mycobacterium bovis was recovered from four of 53 nasopharyngeal tonsils, four of 53 oropharyngeal swabs, one of 53 tracheal and nasal swabs, and one of 46 faecal samples, but not from any urine specimens. CONCLUSION: These findings suggest that significant bacillary excretion from infected deer was uncommon, and is more likely to occur in severely affected animals. This study has confirmed the importance of mucosa-associated lymphoid tissues (MALT), particularly the oropharyngeal tonsil, in the pathogenesis of tuberculosis in deer. The findings justify investigation of the hypotheses that the prevalence of tuberculosis in wild deer in New Zealand is high due to transmission of infection from possums, and that in the absence of an infected possum population, the prevalence of tuberculosis in deer is likely to be low, and spatially patchy. CLINICAL RELEVANCE: The results suggest that about one quarter of infected deer show no detectable gross lesions. This implies that many infected carcasses may enter the food chain unrecognised and that the estimated sensitivity and specificity of diagnostic tests may be erroneous if there is a difference in test performance between those conducted on deer with or without gross lesions. Diagnostic sensitivity following slaughter may be improved by routine culture of oropharyngeal tonsils and careful examination of lungs for adhesions and small subpleural tubercles. 相似文献
12.
本研究以鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)S1基因为研究对象,设计特异性引物扩增S1抗原集中区目的基因,长度为198 bp,亚克隆至原核表达载体pGEX-6P-1,构建重组原核表达质粒pGEX-6P-1-S1,转化至宿主菌Rosetta(DE3)后进行诱导表达。SDS-PAGE分析结果显示,表达获得1条特异性蛋白条带,其分子质量大小为33.0 ku,与预期蛋白大小一致。可溶性分析结果表明目的蛋白以包涵体形式存在。Western blotting结果显示,重组蛋白能与IBV鸡阳性高免血清反应,被GST标签单抗识别,结果表明该融合蛋白正确表达并具有良好的抗原性。本研究为制备IBV单克隆抗体奠定了抗原基础。 相似文献
13.
The single cervical intradermal tuberculin test was used on 34 farmed fallow deer in Tasmania, to determine the specificity of this test when increases in skin thickness of 1 and 2 mm were used as arbitrary significant responses. This was to assess the response that could be expected if this test was used in monitoring or export testing programs of deer in Tasmania. Tasmania is free from bovine tuberculosis, thus providing a unique opportunity to undertake such a study. All deer were slaughtered following testing, and thoroughly inspected post-mortem. Deer that reacted to the skin test had lymph nodes cultured for the presence of Mycobacteria sp. With the reactor response set at 1 mm or more, 25 out of 34 deer tested did not react to the skin test, giving a specificity of 73.5% (95% CI 57.5–89.5%). With the reactor response set at 2 mm or greater, the specificity of the test was 100%.
One lymph node each from two reactors contained mycobacteria identified as Mycoplasma avium and Mycoplasma scrofulaceum. This trial indicates that despite being free of bovine tuberculosis for 20 years, Tasmania also experiences the same difficulties as other countries in the use of the single cervical skin test for certifying deer free from bovine tuberculosis. 相似文献
14.
Mitchell V Palmer W Ray Waters Diana L Whipple Ralph E Slaughter Stephen L Jones 《Journal of veterinary diagnostic investigation》2004,16(1):17-21
Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6-9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-gamma (IFN-gamma) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-gamma in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis-infected deer as compared with uninfected control deer, whereas IFN-gamma production to PWM did not differ significantly between infected and control deer. Measurement of IFN-gamma production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae. 相似文献
15.
低分子质量的蛋白抗原CFP-10是一种重要的牛分支杆菌早期分泌蛋白.为了检测该蛋白和其他几种抗原在牛结核病诊断中的临床应用,对CFP-10的基因进行克隆,鉴定,并在原核系统中表达.PCR法扩增cfp-10基因片段,连接到pET-22b( )原核表达载体中,再转入表达宿主E.coli BL21(DE3)PlysS菌株内,用IPTG诱导,进行蛋白表达、纯化.分别以CFP-10、ESAT-6、MPT83、MPT70、牛PPD、CFP-10与ESAT-6混合蛋白,MPT83与MPT70混合蛋白为抗原,用间接ELISA法诊断牛结核病.结果表明,以CFP-10与ESAT-6混合蛋白作为抗原检测牛结核病的特异性和敏感性分别达到了100%和63.6%,均超过了其他单抗原或抗原组合,为以筛选合适抗原为基础的血清学诊断技术提供了有力的支持并创造了良好的应用前景. 相似文献
16.
狍Sry基因PCR扩增的初步研究 总被引：3，自引：2，他引：3
为研究狍性别决定机制,采用聚合酶链式反应(PCR)技术对野生狍(Capreoluscapreolus)(n♀=2,n♂=2)Sry基因(哺乳动物Y染色体DNA雄性特异区)进行扩增。根据人的SRY基因核心序列设计合成了1对引物1,2。结果在野生狍雄性个体中扩增出1条带,大小约为220bp,而在雌性个体中未见扩增带,表明了Sry基因的性别特异性,为探讨野生狍的性别决定机制提供了分子资料。 相似文献
17.
Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay 总被引：1，自引：0，他引：1
Waters WR Palmer MV Thacker TC Orloski K Nol P Harrington NP Olsen SC Nonnecke BJ 《The Veterinary record》2008,162(7):203-208
Mitogen- and antigen-induced interferon-gamma (IFN-gamma) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer, M bovis BCG-vaccinated white-tailed deer and elk, and unvaccinated, uninfected white-tailed deer, fallow deer, elk and reindeer. The responses of the M bovis-infected white-tailed deer to pokeweed mitogen (PWM) varied with time and between individuals. The responses of the M bovis-infected reindeer to PWM and M bovis purified protein derivative (PPD) were positively associated. Samples from tuberculosis-free captive herds in various parts of the USA were also evaluated. Four per cent of fallow deer, 20 per cent of elk, 44 per cent of white-tailed deer, and 91 per cent of reindeer had responses to PWM exceeding 0.25 Delta optical density, that is, PWM stimulation minus no stimulation. The specificity of the responses to M bovis PPD and a Mycobacterium tuberculosis complex-specific antigen rESAT-6:CFP-10, excluding animals not responding to PWM, ranged from 78 per cent to 100 per cent and was dependent upon the species and the positive response cut-off value. The results show that the commercial assay is valid for the detection of TB in reindeer; however, further development of the assay will be required before it is used in surveillance programmes for white-tailed deer, fallow deer, and elk. 相似文献
18.
根据GenBank公布的肺炎支原体(Mycoplasma pneumoniae,Mp)P1蛋白羧基端序列(AF290001.1),设计合成2对特异性引物,采取套式PCR方法,以肺炎支原体培养物中提取的DNA为模板,扩增肺炎支原体P1蛋白羧基端基因序列,将其克隆至pET32a表达载体,构建重组质粒pET32a/P1(3 802～4 695),转化大肠埃希菌BL21-gold,测序验证后IPTG诱导表达,经SDS-PAGE表明重组蛋白表观分子质量与预期一致,可溶性表达产物经Western blot证实重组蛋白具有免疫反应性。 相似文献
19.
A comparative cervical skin test using 1.0 mg/ml bovine purified protein derivative and 0.5 mg/ml avian purified protein derivative was evaluated as a method for detecting tuberculosis in farmed deer. A positive comparative cervical skin test reaction was defined as a bovine response with a 2 mm or greater increase in skin thickness which was greater or equal to the avian response. Estimates of the sensitivity of the comparative cervical skin test were obtained from a series of experiments conducted on 60 deer intratracheally inoculated with Mycobacterium bovis. Prior tuberculin skin testing was found to suppress the skin reactivity to a subsequent comparative cervical skin test. This effect was most pronounced at short intervals of 3-7 days, but could still be measured 60 days after the previous test. When the test interval was greater than 60 days, the sensitivity of the comparative cervical skin test was 91.4%. The specificity of the comparative cervical skin test was 98.7% when 1157 deer from 17 uninfected herds with a history of nonspecific skin test reactions were examined. There was no statistical difference in the mean skin thickness increases of three groups of infected animals tested with 2 mg/ml, 0.2 mg/ml and 0.02 mg/ml of bovine purified protein derivative respectively. 相似文献
20.
Lyashchenko KP Greenwald R Esfandiari J Chambers MA Vicente J Gortazar C Santos N Correia-Neves M Buddle BM Jackson R O'Brien DJ Schmitt S Palmer MV Delahay RJ Waters WR 《Veterinary microbiology》2008,132(3-4):283-292
Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock. 相似文献