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1.
An indirect ELISA, based on the specific and strongly antigenic recombinant peptide of the N'-terminal half of the lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides small colony type (SC) was developed for the detection of antibodies to M. mycoides subsp. mycoides SC. It was evaluated for its suitability for serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP). The recombinant peptide containing poly-histidine residue tails was expressed in Escherichia coli and subsequently purified by Ni(2+) chelate affinity chromatography to be used as antigen to coat microtiter ELISA plates. The specificity of the antigen was tested against rabbit hyperimmune sera directed against related Mycoplasmas of the M. mycoides cluster and with sera from cattle that were either free of CBPP, but suffered from other mycoplasmal infections such as M. bovis, or showed cross-reactions in the complement fixation test. The sensitivity of the ELISA was assessed with sera from artificially infected animals and with sera from cattle originating from areas where CBPP was endemic at the time of blood sampling. The study revealed that the ELISA was both specific and sensitive for CBPP positive bovine sera and was shown also to be robust to harsh climatic conditions.  相似文献   

2.
The complement fixation test (CFT), the c-ELISA and an indirect LppQ ELISA were compared to post-mortem (PM) inspection for the diagnosis of contagious bovine pleuropneumonia (CBPP). Sera from 797 cattle in the CBPP affected area of Kazungula, Zambia and 202 sera from Lusaka, Zambia, a CBPP-free area were used. The clinical history of CBPP was recorded and all the cattle from Kazungula were slaughtered and PM inspections conducted. The prevalence of CBPP in Kazungula was 67.5% (95%CI 67.2%, 70.8%), 52.6% (95%CI 49.2%, 56.2%), 59.0% (95%CI 55.5%, 62.4%) and 44.4% (95%CI 41.0%, 47.9%) using PM inspection, CFT, c-ELISA and LppQ ELISA, respectively. Three of the 202 negative control animals tested positive on the c-ELISA although they were from a known CBPP negative zone. In this study, the c-ELISA was more sensitive in detecting cattle with lesions in the chronic stage than any other test whilst the CFT detected more during the onset stage. No single serological test could detect all stages of CBPP infection, therefore the use of more than one test is advised.  相似文献   

3.
A competitive ELISA, using a specific monoclonal antibody, was designed to detect antibodies to Mycoplasma mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia. One monoclonal antibody was found suitable for such a test, ‘117/5', it does not cross-react with any of the other mycoplasma species tested, furthermore, its binding is inhibited by positive sera. The cutoff, 50% of inhibition, was determined using a set of negative sera from CBPP-free areas. The sensitivity was controlled with sera from artificially infected animals as well as from sera from areas where CBPP is enzootic. In both cases, cELISA compared favorably with CFT. The precocity of detection was similar but cELISA detected more positives and the positive titers seemed to persist longer than in the case of CFT. Lysis of the antigen used to coat the ELISA plates reduced the variability of fixation and improved the repeatability of the test. A field evaluation is now in progress which will determine the true sensitivity and specificity of the test and also check if antibodies are detected after vaccination.  相似文献   

4.
Diagnosis of bovine brucellosis by enzyme immunoassay of milk   总被引：1，自引：0，他引：1  
Enzyme-immunoassays using lipopolysaccharide as antigen were developed for the detection of bovine IgG1, IgG2 or IgA Brucella antibodies (Ab) in milk. The results of these tests were compared with those of the milk ring test (MRT) by analyzing 3212 bulk milk samples from farms located in regions where brucellosis is prevalent. Among the 105 herds detected by ELISA and/or MRT, 29 infected herds were detected by ELISA only. The 40 MRT-positive herds were also ELISA positive. Five herds became infected during the study and were detected by ELISA 15 days to 6 months prior to detection by MRT. The ELISA IgG1 titration (IgG1 ELISA) detected 92.8% of the herds found positive in the three ELISA assays. The concomitant use of IgA ELISA raised the sensitivity to 100% but slightly decreased the specificity. The IgG2 ELISA did not improve the diagnosis. The sensitivity of MRT and IgG1 ELISA was compared by testing successive dilutions in negative milk of 110 individual MRT positive milks samples. On average, IgG1 ELISA was 22 times more sensitive than MRT.  相似文献   

5.
Cattle from Northern Portugal, many with pulmonary lesions typical of contagious bovine pleuropneumonia, were investigated for the presence of Mycoplasma mycoides subspecies mycoides small colony (MmmSC), which is the causative agent of CBPP, with several detection tests. Sandwich ELISA that included a culture enrichment stage, and 2 different PCR diagnostic systems were used to detect MmmSC in lung and mediastinal lymph node tissues from these animals. The comparison of typical CBPP pathology with the results of detection revealed that no single one of these methods provided a perfect match to the pathological data. Best performing tests were the PCR with laser induced fluorescence and PCR with pleuroTRAP kit (Chemicon, Australia), which are diagnostic systems based on amplification of genomic MmmSC DNA followed by sensitive detection of the amplified products. These were followed by the broth-enriched sandwich ELISA, which uses a monoclonal antibody specific to the M. mycoides cluster, to capture the antigen.  相似文献   

6.
A cross-sectional study was designed to investigate the prevalence of bovine brucellosis and contagious bovine pleuropneumnia (CBPP) in Kongor Rural Council, southern Sudan and to assess associations between the presence of antibodies for these diseases with age, sex and breed. The detection of Brucella antibodies using the Rose-Bengal plate test was significantly associated with increasing age. The difficulty of estimating the true prevalence of brucellosis from the apparent prevalence based on test results is disscussed. Although no linear association between age and CBPP complement fixation test results were found, cattle of 2–4 years of age appeared to be at higher risk. Local factors important in CBPP disease control planning are discussed.  相似文献   

7.
Mycoplasma bovis and Mycoplasma mycoides subspecies mycoides small colony (MmmSC) are causes of bovine mycoplasmosis and contagious bovine pleuropneumonia (CBPP), respectively, and are responsible for serious economic losses in cattle around the world. CBPP was last reported in Poland in 1939 but bovine mycoplasmosis is believed to be endemic. A survey of 3670 serum samples for antibodies to M bovis and MmmSC from 361 herds in 16 Polish provinces Poland between 2007 and 2010 found no evidence of CBPP. The seroprevalence of M bovis, however, appeared high with 76.7 per cent of samples giving a positive reaction in the ELISA test, which did not appear to reflect the clinical disease status of the cattle. Adjusting the sensitivity of the test reduced the prevalence to 28.2 per cent and reflects the levels reported in other European countries.  相似文献   

8.
The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen-antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.  相似文献   

9.
Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies to Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (CLA). However, none are commercially available in the UK. It was therefore necessary to develop a new, economic ELISA for use in a research project studying the epidemiology of CLA in UK sheep. The ELISA with its diagnostic qualities is presented. The ELISA was developed using sonicated C. pseudotuberculosis and optimised to detect total antibody or IgG class antibody in serum. Receiver operating characteristic (ROC) curves were obtained and the area under the ROC curve was used to compare the sensitivity and specificity of the two ELISAs. Both versions of the ELISA were evaluated on a panel of 150 positive reference sera and 103 negative reference sera. Using the test at 100% specificity, the sensitivity of detection of total antibody was 71% (95% confidence interval 63-78%), and the sensitivity of detection of IgG antibody to C. pseudotuberculosis was 83% (76-89%), which compares favourably with other reported ELISA tests for CLA in sheep. The sensitivity of the IgG antibody assay may be higher because of the greater affinity of IgG class antibodies compared with the IgM antibodies also detected by the total antibody ELISA. The results of ROC analysis indicated that the IgG isotype ELISA was more accurate than the total antibody ELISA. The efficiency of the test was greatest when serum samples were run in a dilution series than when any single serum dilution was used. The ELISA is considered to be suitable for application in field studies of CLA in UK sheep.  相似文献   

10.
A blocking enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against bovine leukemia virus (BLV) is described. The test is based on the biotin-streptavidin system using unlabelled polyclonal bovine IgG against BLV as catching antibody and biotinylated bovine anti-BLV IgG as detecting antibody. The sensitivity was found to be 50-100 times higher than the agar gel immunodiffusion test, with a specificity of practically 100%. The blocking ELISA proved to be suitable for detection of antibodies against BLV in serum and milk. In 34 paired milk/serum samples, the average ratio of BLV antibody titres was 1:26. So far, more than 700,000 sera have been screened by blocking ELISA for BLV antibodies in the course of the Danish surveillance programme for BLV infection.  相似文献   

11.
Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease of cattle and buffalo caused by Mycoplasma mycoides subsp. mycoides "Small Colony" (MmmSC). The agent of CBPP has been isolated from goats in different countries including CBPP-free areas. Goats can therefore be regarded as a putative MmmSC reservoir. No diagnostic test for CBPP surveillance in goats has been proposed as yet. Furthermore, serological tests could be seriously hampered by a widespread caprine infection due to the subspecies M. mycoides subsp. capri (Mmc), which is antigenically very close to MmmSC and displays high levels of genetic variability. A competition ELISA (cELISA) is currently used to screen for CBPP in cattle at the herd level in infected areas. The aim of this study was to see if the same cELISA would be specific enough to be used to screen goats despite the potential concomitant infection with Mmc. The cELISA titers of goats from Mmc-infected and non-infected herds were comparable and negative using the accepted cutoff for bovine sera. In contrast, seroconversion was observed in goats experimentally inoculated with an Mmc strain that cross-reacted with a monoclonal antibody targeting the same epitope as that used in cELISA. The probability of such false positivity occurring under field conditions is very low since Mmc strains with such an atypical antigenic profile emerge only rarely as a result of random nucleotide variation of the epitope-coding region. In conclusion, the commercially available cELISA can be considered specific enough to be used as a primary test to monitor passage of the CBPP agent in goats, but its sensitivity in goats requires further investigation.  相似文献   

12.
Using radial immunodiffusion as a standard, 4 screening techniques for detection of failure of passive transfer in equine neonates were compared for sensitivity, specificity, positive and negative predictive values, efficiency, and cost. The techniques compared were latex agglutination test, membrane filter ELISA, dipstick ELISA, and glutaraldehyde coagulation (GC) test. Test results of 50 serum samples from foals 24 to 60 hours old revealed consistently highest accuracy in the GC test at IgG concentrations of 400 and 800 mg/dl, and lowest cost per test, using the GC test. Two hundred fifty-three serum samples from foals 24 to 60 hours old were evaluated for comparison of results of GC and radial immunodiffusion tests. Overall efficiency was 92 and 91% at serum IgG concentrations of 400 and 800 mg/dl, respectively. Under most field circumstances, the GC test would be the preferred screening test for detection of failure of passive transfer in equine neonates.  相似文献   

13.
旨在制备抗丝状支原体丝状亚种(Mmm)的特异性单克隆抗体(MAb),为牛传染性胸膜肺炎(CBPP)病原诊断的免疫学方法提供特异性抗体。本研究利用生物信息学技术分析了Mmm国内分离株Ben-1不同传代株的全基因组序列,选取M0071蛋白作为研究对象。将原核表达的可溶性重组蛋白M0071(rM0071)作为免疫原免疫BALB/c小鼠,通过有限稀释法和间接ELISA方法筛选得到能稳定分泌抗rM0071蛋白的单克隆抗体的杂交瘤细胞株。进一步制备单抗腹水并纯化,利用Western blot方法对该单抗进行特异性鉴定,同时测定其抗体效价和抗体亚类。随后利用间接免疫荧光试验(IFA)评价该单抗对细胞感染Mmm的检测能力。结果表明成功获得1株单克隆细胞株3C4A1,将其分泌抗体命名为MAb 3C4A1。特异性结果表明,MAb 3C4A1能与Mmm的分离株和标准株发生特异性反应,而不与山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、牛鼻支原体、无乳支原体、牛支原体、leachii支原体和牛A型巴氏杆菌等发生反应。抗体亚类鉴定MAb 3C4A1属于IgG1亚类、轻链为κ链。经间接ELISA测定其抗体效价为1∶256 000。IFA试验结果表明,MAb 3C4A1仅与感染EBL细胞的Mmm发生绿色荧光反应,而与牛鼻支原体、无乳支原体、牛支原体感染的细胞不发生荧光反应,特异性良好。本研究制备的MAb 3C4A1具有良好的特异性和免疫反应性,可作为CBPP病原免疫学诊断的工具,为进一步研制CBPP病原鉴别诊断试剂盒提供了基础材料。  相似文献   

14.
Postmortem observations of 37 cattle from an outbreak of contagious bovine pleuropneumonia (CBPP) in north Italy in 1993 were made at the abattoir, where samples of lung and tracheobronchial lymph node tissues were taken for culture and identification of Mycoplasma mycoides subspecies mycoides (MmmSC), immunohistochemistry with the peroxidase anti-peroxidase (PAP) system, and molecular detection by the polymerase chain reaction (PCR) amplification of specific DNA from MmmSC. Nasal swabs were also taken for testing by PCR Lung pathology typical of CBPP was observed in 38 per cent of the animals, and MmmSC was isolated from 19 per cent DNA of MmmSC was detected by PCR in 64 per cent of lung samples and 35 per cent of the nasal swabs. Staining of lung tissue and lymph node tissue by PAP was positive in 27 per cent and 30 per cent of cases, respectively, and was a useful back-up test. These results suggest that PCR amplification from lung tissue may be used as a rapid and accurate confirmatory test for cases with pathology resembling CBPP.  相似文献   

15.
The dot enzyme-linked immunosorbent assay (Dot-ELISA) and the enzyme-linked immunosorbent assay (ELISA) were compared with the immunofluorescent antibody test (IFA) for detection of IgM- and IgG-specific antibodies to human toxoplasmosis. Reciprocal titers were determined in all three assays using sera from 56 patients with suspected toxoplasmosis or with symptoms and diseases requiring exclusion of toxoplasmosis and control sera from 56 healthy persons. Using the Dot-ELISA, six patient sera (10.7%) were positive at titers of greater than equal to 1024 for IgM antibodies (titer range 1024-16 384) and 47 sera (84%) were positive for IgG antibodies (titer range 16-262 144) at a titer of greater than or equal to 16. One control serum was reactive for IgM (titer 1024) and 10 control sera (18%) were positive for IgG in the Dot-ELISA. In the ELISA, at titers of greater than or equal to 128, five sera (9%) were reactive for IgM (titer range 128-512) and 52 sera (92.8%) were reactive for IgG (titer range 32-8192) at a titer of greater than or equal to 32; no control sera gave positive reactions for IgM while 10 sera (18%) were positive for IgG in the ELISA. Using the IFA test at reciprocal titers of greater than or equal to 16, four sera (7.1%) were positive for IgM (titer range 32-512), and 51 sera (91%) were positive for IgG (titer range 16-8192). None was reactive for IgM, and eight sera (14%) were positive for IgG (titer range 32-128) in the IFA test. The Dot-ELISA correlated well with the IFA test (correlation coefficient = 0.895) and the ELISA correlated slightly higher with the IFA test (correlation coefficient = 0.910) for detection of IgG antibodies to Toxoplasma gondii.  相似文献   

16.
A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.  相似文献   

17.
The "gold standard" for the detection of antibodies to Ehrlichia canis, the cause of canine monocytic ehrlichiosis (CME), is the indirect immunofluorescence antibody (IFA) test. The IFA test however is generally available only in selected laboratories and requires extensive equipment and trained personnel. A double-blind study was conducted to compare the ability of an in-clinic standardized enzyme-linked immunosorbent assay (ELISA) test kit to measure E. canis IgG antibodies in dogs compared with the standard IFA technique. A good correlation was found between the 2 techniques (r2 = 0.8793; P < 0.0001). Evidence for the sensitivity of the ELISA technique for the early detection of E. canis IgG antibodies was demonstrated by comparing the appearance of E. canis antibody titers by the IFA and ELISA techniques after artificial infection of 2 sets of dogs. In both experimental infections, both tests were equally sensitive for the early detection of IgG antibodies against E. canis, and the results correlated well with the appearance of fever and clinical signs. Proposed application of the in-clinic ELISA test is to aid in the diagnosis of CME.  相似文献   

18.
旨在建立一种检测牛传染性胸膜肺炎（CBPP）血清抗体的间接ELISA方法,用于该病的监测。利用生物信息学软件对CBPP的病原丝状支原体丝状亚种国内分离株Ben-1株的全基因组进行分析,选取脂蛋白rP0308作为包被抗原,通过一系列反应条件的优化,建立了基于rP0308蛋白的间接ELISA抗体检测方法,并对其性能进行评价。结果显示该方法的敏感性为92%,特异性为96%,与牛支原体、牛鼻支原体、无乳支原体、口蹄疫、牛病毒性腹泻、牛传染性鼻气管炎和牛结核分枝杆菌等阳性血清均不发生交叉反应,批内变异系数在2.41%~6.03%,批间变异系数在2.94%~6.59%,重复性良好。利用本方法和商品化的cELISA试剂盒分别对实验室保存的1 648份临床样品进行检测,该方法与商品化试剂盒的阴性符合率为89.1%,阳性符合率为79.2%,总符合率为88.7%。以上研究结果表明,本研究建立的间接ELISA方法具有良好的敏感性、特异性和重复性,具有良好的临床应用前景。  相似文献   

19.
Serology is currently used for the diagnosis of canine sino-nasal aspergillosis (SNA). However, the accuracy of serological testing using commercially available, standardized purified antigen preparations of Aspergillus (CAPurAspAg) has only been poorly documented. The aim of the present study was to assess the diagnostic value of an agar-gel double immunodiffusion (AGDD) test and an anti-Aspergillus IgG ELISA, using CAPurAspAg and the commercially available Platelia test for the detection of serum galactomannan. Sera from 17 dogs with SNA, 18 dogs with a nasal tumour (NT), 11 dogs with lymphoplasmacytic rhinitis (LPR) and 33 control dogs were tested with the 3 methods. AGDD result was positive in 76.5% of dogs with SNA, whereas all sera from dogs with non-fungal nasal disease and control dogs were negative. A positive IgG ELISA result was obtained in 88% of dogs with SNA and in 18% of dogs with LPR. All patients with NT and control dogs had a negative IgG ELISA result. The Platelia test was positive in 24% of dogs with SNA, 11% of dogs with NT, 9% of dogs with LPR and 24% of control dogs. The results of this study suggest that (1) the detection of serum Aspergillus-specific antibodies with AGDD or ELISA, using CAPurAspAg, provides excellent specificity and good sensitivity, (2) the specificity is higher for AGDD (100%) than for ELISA (96.8%) while sensitivity is higher for ELISA (88.2%) than for AGDD (76.5%) and (3) serum galactomannan quantification with the Plateliat test is unreliable for the diagnosis of canine SNA.  相似文献   

20.
An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.  相似文献   

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