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1.
本试验以超声破碎和高速离心方法提取禽波氏杆菌外膜蛋白,作为抗原免疫家兔,制备并纯化兔抗禽波氏杆菌外膜蛋白IgG,建立了能进行禽波氏杆菌抗原定位和检测的间接免疫酶组化法,并对试验条件进行了优化。对禽波氏杆菌人工感染海兰褐雏鸡的检测结果表明,该法能特异性地对气管、肺、肝、心、脾、肾中的禽波氏杆菌进行抗原定位,同细菌分离鉴定的结果符合率为100％。同时对感染鸡胚肝脏和心脏中禽波氏杆菌的检测结果均为阳性。该法具有高度敏感性和特异性特点,可用于禽波氏杆菌临床和试验感染条件下的诊断和定位检测。  相似文献   

2.
采用改良的Wooldridge法提取了禽波氏杆菌外膜蛋白（0MP）,通过Bradford方法测定禽波氏杆菌OMP含量为320μg／mL,SDS-PAGE电泳检测发现禽波氏杆菌P5株OMP含有5种成分,相对分子质量分别为58、47、41、36、24ku;P8株OMP含有6种成分,相对分子质量分别为58、47、41、38、21、16ku。将提取的禽波氏杆菌OMP免疫健康青紫兰兔,每周免疫1次,共免疫4次,获得了兔抗禽波氏杆菌OMP高免血清,以此高免血清建立了检测禽波氏杆菌的间接ELISA和间接免疫荧光（IFA）方法,并对其工作条件进行了优化和筛选,试验结果表明间接ELISA抗原、抗血清最佳工作浓度分别为10μg／mL、1：12800,酶标二抗工作浓度为1：5000,最佳包被条件为4℃、24h,最佳封闭条件为37℃、1h;间接免疫荧光（IFA）兔高免血清释释度为1：20,FITC标记羊抗兔IgG稀释度为1：10,感作时间为45min时,禽波氏杆菌特异性黄绿色荧光最清晰,非特异性荧光最弱。经临床检测证明该两种方法均具有简便、快速、特异性强的特点,为禽波氏杆菌病的流行病学、血清学调查和临床快速诊断奠定了基础。  相似文献   

3.
差速离心和蔗糖密度梯度离心纯化鸭病毒性肠炎病毒(DEV)免疫兔制备兔抗DEV高免血清后,用DEAE-Sephadex A-50柱层析纯化的兔抗DEVIgG建立了间接免疫荧光染色法(IFA)检测石蜡切片中DEV的方法。IFA的最佳条件为:石蜡切片用10mmol/L pH6.0的柠檬酸缓冲液为微波修复液微波修复10min,胰酶修复20min;10%小牛血清室温封闭30min;加入1∶25的兔抗DEVIgG于4℃孵育过夜;再加入FITC标记的羊抗兔IgG于37℃孵育45min。以建立的IFA对DEV人工皮下感染死亡鸭的各组织器官进行检测,在死亡鸭的脾脏、胸腺、法氏囊、肝脏、食道、十二指肠、直肠及肺脏中检测到DEV抗原。研究表明,IFA检测石蜡切片中的DEV具有直观、特异性强的优点,是对DEV进行检测和抗原定位的良好方法。  相似文献   

4.
以鸽抗鸡劳氏肉瘤病毒(RSV)血清IgG作为包被抗体及酶标抗体的双抗体夹心法Dot-ELISA,检测禽白血病病毒抗原,其特异性及敏感性与澳大利亚ELISA(用兔抗AMV-P_(27)血清IgG作为包被抗体及酶标抗体)相同。可用以检测卵清中禽白血病病毒抗原及用鸡胚或鸡胚细胞生产的各种疫苗中污染的禽白血病病毒抗原。  相似文献   

5.
直接荧光抗体法检测禽波氏杆菌   总被引：1，自引：0，他引：1  
制备了兔抗禽波氏杆菌特异性荧光抗体并建立了直接荧光抗体检测方法 ,对禽波氏杆菌在人工感染雏鸡内的致病机理作了初步研究。结果 ,该荧光抗体只与其相应菌株发生特异性荧光反应 ,而不与其他病原菌株发生反应。对人工感染发病雏鸡的检测结果表明 ,禽波氏杆菌主要定植于上呼吸道黏膜 ,并造成损害。该技术具有简便、快速、敏感和特异性强等优点  相似文献   

6.
J亚群禽白血病病毒的免疫荧光检测效果   总被引：14，自引：0，他引：14  
应用特异性抗J亚群禽白血病病毒（ALV－J）的单克隆抗体JE9建立了免疫荧光检测ALV－J抗原和抗体的方法。结果表明，在ALV－JADOL－Hcl感染鸡胚成纤维细胞（CFF）24小时后，可以用1FA检测出阳性细胞，感染72小时后，可以检测出最小病毒感染量≥1．25TCID50／孔。对人工感染ALV－J鸡肾脏冰冻切片检测结果证明，免疫荧光法可以检测出组织中的ALV－J抗原，表现强阳性反应。用重组病毒rBac4817－env感染的Sf9细胞作抗原，可以检测出鸡血清中抗ALV－J的特异性抗体。该方法在ALV－J的控制中必将产生重要作用。  相似文献   

7.
提取A型鼻气管鸟杆菌(ORT)外膜蛋白,经纯化后包被反应板,通过方阵滴定确定最佳抗原包被浓度、血清稀释倍数、兔抗鸡酶标二抗稀释倍数,建立了检测ORT抗体的间接ELISA方法.经方阵滴定确定最佳抗原包被质量浓度为11.85 mg/L,血清样品稀释倍数为1∶20,兔抗鸡IgG辣根过氧化物酶结合物最佳稀释倍数为1∶1 000,抗原抗体最佳结合时间为1 h,血清与二抗最适反应时间为45 min.试验结果确定的判定标准为:样品D490 nm≥0.639判定为阳性,D490 nm≤0.350判定为阴性,介于两者之间的为可疑.试验证明该方法具有良好的稳定性、重复性和特异性,将205份疑似ORT的鸡血清用本方法进行检测,其阳性检出率为11.7%(24/205).  相似文献   

8.
以抗新城疫病毒F蛋白的单克隆抗体为一抗,建立了间接免疫荧光染色法(IFA)检测石蜡切片中鸭副黏病毒(DPMV)的方法。以建立的IFA对DPMV人工感染鸭的各组织器官进行检测,结果显示:各组织器官均能检测到DPMV,但不同时间取样,阳性信号分布的器官不同。脾脏、胸腺、法氏囊、肠道、肝脏、肺脏DPMV的阳性检出率较高,表明这些器官为DPMV的主要靶器官。在所检测的阳性组织中,病毒抗原分布在细胞浆中。IFA检测石蜡切片中的DPMV具有直观、特异性强的优点,是对DPMV进行检测和抗原定位的良好方法。  相似文献   

9.
为建立检测细胞培养物和组织切片中羊口疮病毒(Orf virus,ORFV)的间接免疫荧光(IFA)方法,用已知滴度的ORFV感染牛睾丸上皮细胞,羊口疮病毒单克隆抗体(anti-ORFV mAb)为一抗,荧光素标记羊抗小鼠IgG为二抗,通过反应条件的优化,建立细胞培养物ORFV的IFA检测方法,并制备ORFV感染的山羊唇部组织冰冻切片,对组织中病毒进行检测。结果显示,用牛睾丸上皮细胞增殖的羊口疮病毒TCID_(50)达到10~(-6.29)/0.1 mL,ORFV适宜的接种滴度和培养条件为10~(3.70) TCID_(50) ORFV接种细胞,37℃、体积分数为5%的CO_2恒温箱培养42 h,荧光素标记羊抗小鼠二抗适宜工作浓度为1∶400稀释,此时羊口疮病毒单抗应用于IFA检测的特异性和灵敏度最佳,且可应用于病料组织中ORFV的定位检测。表明成功建立了羊口疮病毒单抗检测牛睾丸上皮细胞和组织切片中ORFV的间接免疫荧光方法,有助于ORFV感染的实验室诊断及其在感染细胞中的定位和动态分布研究。  相似文献   

10.
本文旨在克隆禽波氏杆菌外膜蛋白OmpA的编码基因,并预测OmpA蛋白二级结构和B细胞抗原表位,从而探讨禽波氏杆菌外膜蛋白OmpA在免疫保护中所起的作用。作者对禽波氏杆菌的外膜蛋白ompA基因进行PCR扩增、克隆及序列测定。应用生物信息学相关软件和方法,对禽波氏杆菌OmpA蛋白的二级结构和B细胞抗原表位进行预测。禽波氏杆菌ompA基因全长597bp,编码199个氨基酸。二级结构以无规卷曲为主,有少量的α-螺旋和β-片层,少见β-转角;推测OmpA蛋白有5个B细胞优势抗原表位区域、2个糖基化位点。本研究为进一步分析禽波氏杆菌免疫机理、制备单克隆抗体和设计表位疫苗等奠定理论基础。  相似文献   

11.
When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.

A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.  相似文献   


12.
Fowl adenovirus-1 (FAV-1), isolated from field outbreaks of inclusion body hepatitis (IBH), was administered orally to 3-week-old disease-free broiler chicks. Humoral immune competency was evaluated by determining the antibody response of infected chicks to sheep red blood cells (SRBC) and Brucella abortus. FAV-1 infection significantly decreased the antibody response of chicks to B. abortus (T-cell-independent antigen) by decreasing IgM responses, however, the decreased antibody response to SRBC (T-cell-dependent antigen) was statistically non-significant. Bursal index was also found lowered in infected chicks as compared to the control chicks. A significant decrease was seen in blastogenesis response of peripheral blood lymphocytes to phytohaemagglutinin (PHA-P) in FAV-1-infected chicks on 2 and 3 weeks post-infection (WPI). These results indicated that FAV-1 affects humoral as well as cellular immune competency of infected chicks.  相似文献   

13.
Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA’s, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-γ test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals.

Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.  相似文献   


14.
Epidemiological aspects of Babesia vogeli infection were studied in the canine population of a rural town located in the Brazilian “Drought Polygon” of the state of Minas Gerais, Brazil. The survey was carried out in March 2003, when 505 dogs were identified and their characteristics registered on appropriate forms. Blood samples were collected at this time and again in June, September and December 2003. Serum samples were tested by the indirect fluorescent antibody test (IFAT) to detect antibodies against B. vogeli. The prevalence of anti-B. vogeli antibodies was 18.8%; however, no correlations were found between prevalence of infection and the age or gender of the animals. Cross-bred dogs presented a higher chance of acquiring infection in comparison to pure-bred dogs. Significant differences concerning the incidence of the disease were found during the period April–June in comparison to other months, demonstrating that transmission of B. vogeli is related to seasonal variations of tick infestations. The results indicate that climatic factors within the semiarid area interfere directly in the epidemiology of canine babesiosis.  相似文献   

15.
以马立克氏病病毒（ＭＤＶ）感染１日龄肉用雏鸡，在感染后５、２５、４５ｄ采取法氏囊、脾脏、盲肠扁桃体和哈德尔腺，用彩色免疫金银染色法检查免疫器官组织中ＩｇＧ、ＩｇＭ和ＩｇＡ抗体生成细胞数量的动态变化。结果：ＭＤＶ感染雏鸡的法氏囊、脾脏和哈德尔腺中以ＩｇＧ抗体生成细胞居多，ＩｇＧ、ＩｇＭ和ＩｇＡ抗体生成细胞均较正常对照鸡显著减少；盲肠扁桃体中以ＩｇＡ抗体生成细胞居多，ＩｇＡ、ＩｇＧ和ＩｇＭ抗体生成细胞数量显著低于正常对照鸡。由此表明，ＭＤＶ感染鸡全身免疫器官和消化道、呼吸道局部粘膜体液免疫机能明显抑制。  相似文献   

16.
窄竹叶柴胡(Bupleurum marginatum var.stenophyllum)为柴胡属多年生高大型草本植物，以根入药称为藏柴胡，具有较高的药用价值。本试验采用实时荧光定量PCR技术，利用geNorm、NormFinder及BestKeeper3种不同数据分析软件，从5个常用的候选基因(Actin,α-tubulin,β-tubulin,Cyclophilin,EF-1α)筛选出可在窄竹叶柴胡不同器官稳定表达的基因作为内参基因，并利用筛选得到的内参基因分析皂苷合成关键酶基因(HMGR,IPPI,FPS,SS,β-AS)在窄竹叶柴胡不同器官的相对表达量。结果表明β-tubulin基因可作为窄竹叶柴胡不同器官稳定表达的内参基因；皂苷合成关键酶基因在窄竹叶柴胡的不同器官中表达量有显著差异，IPPI基因在种子中表达量最高，其他基因在根中的表达量均高于在其他器官中的表达量，其中HMGR,FPS,SS在侧根的表达量高于主根。因此，窄竹叶柴胡各器官皂苷合成关键酶基因的表达量不同，最适内参基因为β-tubulin基因，本研究为窄竹叶柴胡分子生物学研究提供一定依据，促进其合理开发和利用。  相似文献   

17.
All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.  相似文献   

18.
The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.  相似文献   

19.
Prevalence of intestinal pathogens in Danish finishing pig herds   总被引：5，自引：0，他引：5  
Our aim was to determine the prevalence of the intestinal bacteria: Lawsonia intracellularis, Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens, Brachyspira pilosicoli, pathogenic Escherichia coli (serogroups O138, O139, O141 and O149) and Salmonella enterica in Danish finishing pig herds. A total of 79 herds was randomly selected and visited during 1998. From each herd, 20 faecal samples were collected from individual pigs weighing 30–50 kg. Furthermore, 10 pooled pen samples were collected and examined for S. enterica. In total, 1580 faecal samples and 790 pen samples were collected and examined by polymerase chain reaction (PCR) or culture. L. intracellularis was found in 74 herds (93.7%), B. hyodysenteriae in two herds (2.5%), S. intermedia in 10 herds (12.7%), B. innocens in 27 herds (34.2%), B. pilosicoli in 15 herds (19.0%), pathogenic E. coli in 19 herds (24.1%) and S. enterica in eight herds (10.1%). The within-herd prevalences of L. intracellularis and B. hyodysenteriae were 25–30%; the within-herd prevalences of the other agents were 5–10%. Three herds (4%) were not infected with any of the bacteria and 25 herds (32%) were only infected with L. intracellularis.  相似文献   

20.
鸡传染性贫血疫苗免疫母鸡后子代雏鸡的免疫学变化   总被引：1，自引：0，他引：1  
应用免疫学新技术对鸡传染性贫血（CIA）疫苗免疫母鸡后，其子代雏鸡外周血液和免疫器官组织及局部体液的免疫学变化进行了动态研究。结果表明：CIA疫苗免疫母鸡后，其子代雏鸡外周血液T、B细胞数量和IgG、IgM、IgA含量及免疫器官组织的T细胞和IgG、IgM、IgA抗体生成细胞以及泪液、气管液、胆汁、肠液的IgA、IgM、IgG含量均不同程度地高于未免疫的相应对照雏鸡，表明CIA疫苗免疫母鸡后，其子代雏鸡全身的体液免疫和细胞免疫功能明显增强。而CIAV强毒攻击后，未免疫的子代雏鸡，其外周血液和免疫器官组织及局部体液的上述各项指标均明显低于疫苗免疫的子代雏鸡。  相似文献   

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