PAPS免疫微球快速诊断弓形虫病的研究

本文介绍一种新型的 PAPS 弓形虫病快速诊断试剂。PAPS(聚醛化聚苯乙烯)是我组研制成功的一种新型的载体微球,它与可溶性弓形虫腹水抗原交联制备的弓形虫病快速诊断液具有特异性强、敏感度高、重复性好,快速、简便的优点。我们应用弓形虫病快速诊断液检测了65头弓形虫阳性病猪,阳性检出率为98.4%。对200头健康猪进行检测,阴性符合率为99.1%,假阳性率为0.9%。胶乳阳性反应与血凝阳性反应的符合率为100%。对170头猪血清和血纸两种样本进行 PAPS 对比试验的阳性结果和反应强度都相吻合,两者无明显差异,故血纸可以代替血清进行试验。     

应用斑点酶标联诊盒检测牛羊“四虫病”的效果观察

应用斑点酶标“三虫病”与“四虫病”联诊盒检测牛羊血吸虫、锥虫、肝片吸血和血矛线虫病的效果比较。试验共检测牛７３８头，羊２１５只，牛锥虫病的阳性符合率９８．７％，肝片吸虫阳性符合率９８．６％，羊为９８．２％。     

检测家畜血吸虫抗体的二步金标免疫渗滤法研究

将待检家畜血纸浸出液点在硝酸纤维素膜上，以金标记的血吸虫虫卵可溶性抗原为探针（以下简称血吸虫抗原胶体金），建立了检测家畜血吸虫抗体的二步金标免疫渗滤法（Two-step Dot Immunogold Filtration Assay，T—DIGFA）。家畜血吸虫病阳性血样在50-90S形成肉眼可观察的红色斑点，可进行快速诊断。该法可以检测出人工感染血吸虫尾蚴7d和7d以上的阳性牛和兔血纸抗体，与肝片吸虫、锥虫、蛔虫病之间无交叉反应。T—DIGFA检测粪孵血吸虫毛蚴阳性牛血纸139份、阴性牛血纸130份，与粪孵法阳性符合率为100％，阴性符合率为99．2％；T—DIGFA与斑点酶联SPA法阳性符合率和阴性符合率完全相同。血吸虫抗原胶体金在4～8℃保存期可达6个月、室温保存期为3个月。试验证实，T-DIGFA有很高的敏感性、特异性、重复性和稳定性，适合于基层单位和现场进行家畜血吸虫病抗体的快速诊断、普查和检疫。     

γ-干扰素释放法检测奶牛结核病的比较试验

目的：评价γ-干扰素释放试验（IGRA）牛结核病检疫过程中的效果。方法在牛结核病检疫过程中对皮试初筛为结核病和疑似结核病的89（74/15）头奶牛同时进行r干扰素试验、单纯颈部皮试变态反应（SCT）和比较皮试变态反应（CCT）检测,结果单纯颈部皮试变态反应二次检疫中89头牛均判为阳性,单纯颈部皮试变态反应与比较皮试变态反应的阳性符合数为67头。阳性符合率为75.3％;γ-干扰素释放试验和单纯颈部皮试变态反应检测两者的阳性符合数为64头,阳性符合率为71.9％;γ-干扰素释放试验与比较皮试变态反应的阳性符合数为58头,阳性符合率为79.5％。阴性符合数为16头,阴性符合率为48.4％;结论γ-干扰素释放试验在保持较高灵敏度的同时,具有比变态反应更好的特异性。     

奶牛早孕诊断试纸条的研制及临床试验

用抗孕酮单克隆抗体S3B2D4A6和羊抗鼠IgG分别固定于硝酸纤维膜，并与胶体金标记的抗孕酮单克隆抗体S3B3E1G1及其它试剂，应用层析式双抗体夹心法原理研制奶牛早孕诊断试纸，在北京10家奶牛场进行临床检测试验，对1559头荷斯坦奶牛乳汁中孕酮含量进行临床符合验证。结果表明。该试纸条灵敏度高，最低检出量达5～8ng／mL。与无关抗原均无交叉，且批内、批间变异小；检测1559头奶牛。孕牛1316头，其中试纸检测结果阳性1274头。可疑32头。复测阳性22头，孕牛阳性临床符合率96．8％；复测后阳性总符合率达98．5％。阴性对照243头，其中试纸检测结果阴性236头。可疑6头。复测阴性2头。阴性临床符合率为97．1％。复测后阴性总符合率97．9％。结论：北京万华生物工程有限公司研制的奶牛早孕诊断试纸能够快速、准确地进行奶牛早期妊娠诊断。     

PAPS免疫微球的制备及其在寄生虫病诊断中的应用

我们研制成功一种新型的PAPS载体微球(Polyaldehyde,Polystyrone),为国内外首创。PAPS微球上的活性基团在两年之内未脱落,保存两年的PAPS和配基交联效果与新制备的一样。我们应用这种新型的PAPS载体微球与伊氏锥虫抗原、日本血吸虫虫卵抗原、弓形体的腹水S_1抗原等配基交联所制备的快速免疫诊断试剂具有特异性强,敏感性高、重复性好的特点,而且较稳定,一年内均有效。制备的诊断试剂可用血纸代替血清进行试验。一份血样本可作几种寄生虫病的诊断试验。本方法非常灵敏、快速、简便、可节省许多经费开支,是一项富有生命力的新技术。     

两种奶牛结核病检测方法与PPD检测符合率比较

对新疆某规模化奶牛场的2 106头奶牛用PPD进行普检,共检出结核病阳性牛26头;随机采集20头阴性牛和26头阳性牛抗凝血和全血,进行牛结核γ-干扰素ELISA试验和胶体金检测,然后将检测结果与PPD检测结果进行比对。结果显示:3种方法检测均为阳性的16份,均为阴性的10份,总符合率为57%。PPD和γ-干扰素ELISA相比,均为阳性的23份,阳性符合率为88%(23/26),均为阴性的11份,阴性符合率为55%(11/20);PPD和胶体金检测相比,均为阳性的19份,阳性符合率为73%(19/26),均为阴性的19份,阴性符合率为95%(19/20)。结果表明:3种检测方法的符合率较低;每种检测方法各有优缺点,γ-干扰素ELISA敏感性较高,而胶体金特异性较高。因此,在实际操作中应根据需要,合理选择相应检测方法。     

斑点酶联免疫吸附试验检测牛羊捻转血矛线虫病的研究

应用斑点酶联免疫吸附试验（Ｄｏｔ－ＥＬＩＳＡ）检测牛羊捻转血矛线虫病抗体，经对３９头剖检羊血纸检测，结果表明该法与剖检法的阳性符合率达１００％（２７／２７），阴性符合率也达１００％（１２／１２）。Ｄｏｔ＝ＥＬＩＳＡ能检出第四胃寄生１＝３３８条捻转血矛线虫羊的血清或血纸抗体；应用Ｄｏｔ－ＥＬＩＳＡ对５９９份牛、羊血纸的测定结果，与粪检地的阳性符合率达９５．５８％，阴性符合率达９１．４３％。初步认为Ｄ     

牛羊四种寄生虫病联合诊治技术的应用

本文介绍了龙游县对牛羊锥虫、肝片吸虫、捻转血矛线虫及吸虫病的诊断技术的沿革,由单虫诊断发展到二联、三联和四联法诊断。文中着重描述了１９９７年以来３年中应用四联法共检测牛羊血、粪样品１４２９８头份（牛１０７２７、羊３５２７）,检出结果与三联法比较阳（阴）性符合率,锥虫病阳性符合率９６．９２％,肝片肿虫病９８．４６％,血吸虫病阴性符合率１００％;说明四联法检测四种寄生虫病同样有效、可靠。     

应用皮内变态反应和γ-干扰素体外释放试验检测奶牛结核病的比较研究

本研究首先应用皮内变态反应对10200头奶牛进行结核病检测,然后应用γ-干扰素体外释放试验对前者检测出的阳性牛和可疑牛再次进行结核病检测,比较两者的阳性符合率。结果显示,应用皮内变态反应共检测出结核病阳性牛96头、可疑牛4头;γ-干扰素体外释放试验检测皮内变态反应呈阳性的96头奶牛,结果为阳性牛94头、阴性牛2头,而检测皮内变态反应为可疑的4头奶牛,结果全为阳性。结果表明,两种方法的阳性符合率为97．92％（94／96）,虽然皮内变态反应存在一定的假阳性,且费时、费力,但考虑到γ-干扰素试剂盒比较昂贵,建议用皮内变态反应作为初筛试验,γ-干扰素试验用于初筛阳性样品的复核,以提高结核病检测的准确性。     

Surveys in Papua New Guinea to detect the presence of Trypanosoma evansi infection

OBJECTIVE: To confirm serological evidence that Trypanosoma evansi is present in Papua New Guinea. DESIGN: Three surveys were undertaken in PNG during 1997/1998. Animals were selected for sampling on the basis of convenience. Samples of blood were examined for the presence of T evansi by the haematocrit centrifugation technique (HCT) and mouse inoculation test (MI). Sera were tested in the field using the card agglutination test for trypanosomiasis/T evansi (CATT). Bovine sera were tested at James Cook University using an antibody-detection ELISA (Ab-ELISA). Results from testing bovine sera with the Ab-ELISA and sera from wallabies with the CATT were analysed using FreeCalc to determine the probability that animals in these populations were infected with T evansi. RESULTS: A total of 545 serum samples were collected, during the three surveys of which 39 cattle, two pig and three agile wallaby samples were positive with the CATT. All bovine sera collected were negative when tested with an Ab-ELISA. T evansi was not isolated using the HCT or the MI from any of these animals. CONCLUSION: Based on the Ab-ELISA results it was concluded that T evansi infection was not present in cattle in villages around Balimo at a minimum expected prevalence of 10% (P < 0.05) and, based on the CATT results, that infection was not present in wallabies on the Bula plain at a minimum expected prevalence of 10% (P < 0.1). These results indicate that it is unlikely that T evansi is endemic in PNG.     

伊氏锥虫变异表面糖蛋白抗原双抗体夹心ELISA检测方法的建立及初步应用

A double antibody sandwich ELISA for detection of Trypanosoma evansi variant surface glucoprotein (VSG) antigen was established by using two monoclonal antibodies against Trypanosoma evansi VSG antigen.The result of sensitivity showed when positive serum was diluted to 3200 doubles, its D_{450 nm} value was still greater than the critical value of positive and negative,and specificity test result showed that there was no cross reaction with the antigens of Theileria,Toxoplasma gondii,Chlamydia and Fasciola gigantica.82 clinical sera were detected by this double antibody sandwich ELISA and its positive rate was 9.76%.The experiment results demonstrated that this double antibody sandwich ELISA for detection of Trypanosoma evansi VSG antigen had good detecting sensitivity and specificity,and could be used as an effective method for clinical detecting buffalo Trypanosoma evansi disease.     

The expression of RoTat 1.2 variable surface glycoprotein (VSG) in Trypanosoma evansi and T. equiperdum

In order to define whether the variable antigenic type RoTat 1.2 is restricted to Trypansoma evansi and could be used as antigen in serological tests to differentiate T. evansi from Trypansoma equiperdum, the appearance of RoTat 1.2-specific antibodies in rabbits, experimentally infected with T. evansi and T. equiperdum, respectively, was analyzed. Ten strains of T. evansi and 11 strains of T. equiperdum originating from Asia, Europe, Africa and Latin America were tested. Rabbit pre-infection sera and sera of days 7, 14, 25, 35 post-infection (p.i.) were analyzed for the presence of antibodies reactive with RoTat 1.2 in immune trypanolysis, ELISA/T. evansi and CATT/T. evansi. Within the duration of the infection (maximum 35 days), all T. evansi as well as 9 out of 11 T. equiperdum infected rabbits became positive in all these tests. The rabbits infected with T. equiperdum OVI (South Africa) and BoTat 1.1 (Morocco) remained negative in the immune trypanolysis test although the latter rabbit became positive in the CATT/T. evansi and ELISA/T. evansi. On the contrary, both rabbits were positive in immune trypanolysis when tested against their respective infecting population. From these data, we conclude that most T. equiperdum strains express isoVATs of RoTat 1.2. This explains, in part, why antibody tests based on T. evansi RoTat 1.2 cannot reliably distinguish between infections caused by T. evansi and those caused by T. equiperdum unless it can be proven that most described T. equiperdum are actually misclassified T. evansi.     

Comparison of serological tests for Trypanosoma evansi natural infections in water buffaloes from north Vietnam

In the present study, a collection of 415 water buffalo serum samples originating from the north of Vietnam was used for evaluation of different diagnostic antibody detection methods available to detect infections with Trypanosoma evansi. The diagnostic sensitivity and specificity of a direct card agglutination test (CATT/T. evansi), an indirect card agglutination test (LATEX/T. evansi) and a newly developed antibody detection ELISA (ELISA/T. evansi) was calculated on the basis of parasitological results, obtained by mouse inoculation, and compared for all assays. The immume trypanolysis assay with the predominant T. evansi RoTat 1.2 variable antigen type was used as reference test for antibody presence. All parasitologically confirmed animals (n=8) were positive in all tests. Diagnostic specificity was highest in CATT/T. evansi (98%) followed by the ELISA/T. evansi (95%) and the LATEX/T. evansi (82%). Concordance of the variant specific immune trypanolysis test with the other tests was calculated and revealed that few (1-8%) false positive results were actually due to a specific reactions, and that LATEX/T. evansi and ELISA/T. evansi detected more immune trypanolysis positives than the CATT/T. evansi. It was concluded that, apart from the immune trypanolysis test, which is not generally applicable, ELISA/T. evansi with a 30% positivity cut-off and LATEX/T. evansi, thanks to their superior capacity of detecting T. evansi specific antibodies, would be suitable as epidemiological tools detecting both active infections and persisting T. evansi specific antibodies. The ELISA/T. evansi with a 50% positivity cut-off and the CATT/T. evansi on the other hand, seem more appropriate to detect true infected water buffaloes.     

Evaluation of antigen and antibody rapid detection tests for Trypanosoma evansi infection in camels in Kenya

The card agglutination test for Trypanosoma evansi (CATT/T. evansi) for the detection of antibodies, and Suratex for the detection of circulating antigens were compared in a cross-sectional study involving camels in eastern and central parts of Kenya. Of the 2227 camels screened, 2038 were owned by nomadic pastoralists in T. evansi endemic areas in eastern Kenya. A herd of 86 camels were from a ranch in Mugwoni. In Athi River area, 35 camels belonged to Kenya Trypanosomiasis Research Institute, and 68 were slaughter animals. Diagnostic sensitivity estimates were obtained by testing sera from 51 camels that had been found to be parasitologically positive by the haematocrit centrifugation technique, buffy-coat technique and mouse inoculation. Diagnostic specificity was estimated by testing sera from 35 camels known to be trypanosome-free. Positive and negative predictive values (NPVs) were calculated using a range of prevalence values. The sensitivity of CATT/T. evansi (68.6%) was higher than that of Suratex (58.8%), but not significantly. Both tests had equally high specificity (100%). The overall prevalence was 2.3% (51 out of 2227) by parasite detection, 32.2% (327 out of 1017) by CATT/T. evansi and 19.6% (188 out of 961) by Suratex. Overall, there was a positive association between CATT/T. evansi and Suratex though the strength of association was low (McNemar's test=46.12, P=0.001; kappa=0.26, CI: 0.20-0.33). Parasite prevalence ranged from 0% in several herds to 27.8% in a herd in Isiolo. Prevalence was highest in Isiolo with 2.5% (51 out of 2030) by parasitological detection, 38.8% (321 out of 828) by CATT/T. evansi and 21.9% (169 out of 772) by Suratex. In Mugwoni prevalence was 7 and 18% by CATT/T. evansi and Suratex, respectively, and no parasites were detected. In Athi River Suratex detected 2.9% (3 out of 103) positive while CATT/T. evansi and parasitological methods gave negative results. At prevalence values between 10 and 100%, CATT/T. evansi as well as Suratex had infinitely high positive predictive values, whereas Suratex had a lower NPV than CATT/T. evansi. In conclusion, results of this study showed that CATT/T. evansi and Suratex were able to detect aparasitaemic infections rapidly and were more sensitive than parasitological methods in revealing the true extent of trypanosomosis in a herd. The tests effectively complemented parasitological methods in the detection of T. evansi infections in camels.     

Evaluation of mono- and polyclonal antibody-based antigen detection immunoassays for diagnosis of Trypanosoma evansi infection in the dromedary camel.

Two enzyme-linked immunosorbent assays (ELISA), one based on a mouse anti-Trypanosoma brucei group-specific monoclonal antibody and the other on rabbit anti-Trypanosoma evansi polyclonal antibodies, have been evaluated for their ability to detect circulating trypanosome antigens in camel sera as a means for the diagnosis of T. evansi infections. All 91 sera from a negative control camel herd from Kenya gave negative antigen-ELISA results in the monoclonal antibody-based ELISA and only 2 of them (2.2%) gave false positive results in the polyclonal antibody-based ELISA. In subsequent analyses of sera from infected camels (as determined by mouse inoculation), the monoclonal antibody-based ELISA detected antigens in 90 (83.3%) out of the 108 sera tested. This percentage was lower for the polyclonal antibody-based ELISA which was able to detect antigens in 67 (60.9%) out of the 110 sera tested. The two tests detected probably different antigens and when the results were combined, 99 out of 107 (92.5%) sera were shown to be ELISA positive. In a survey involving 316 camels from the Gao and Nara areas, in Mali, a high proportion of animals tested were antigen positive (43.5 and 42.9%, respectively for the mono- and polyclonal antibody-based ELISA) compared to only 22 (7.0%) diagnosed by the parasite detection techniques. Thus, these immunoassays were at least six times more sensitive than the haematocrit centrifugation technique. As a large proportion of cases may be antigen positive but parasite negative, these two of "surra" immunoassays should be used in routine diagnosis in addition to the parasite detection techniques in the dromedary camel.     

应用斑点酶联免疫吸附试验检测水牛伊氏锥虫抗体

应用斑点酶联免疫吸附试验(Dot—ELISA)检测水牛伊氏锥虫IgG抗体,并与间接血凝试验(IHA)进行了比较。检测160份伊氏锥虫疫区水牛血清160份,Dot—ELISA和IHA的阳性率分别为55.63％和53.75％;其中42份虫检阳性血清的阳性率分别为100％和92.86％,两种试验的一致率为95.63％。两种试验的滴度呈显著正相关(r=0.8842)。10份非疫区健康水牛血清两种试验均为阴性。     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

A Serological Survey of Dogs for Brucella canis in Southwestern Ontario

Sera from 2 000 dogs from southwestern Ontario were tested for antibodies to Brucella canis by a rapid slide agglutination test. The 100 positively reacting sera were tested by tube agglutination and immunoprecipitation (gel diffusion) tests. Thirty-one of these sera gave suspicious titres and one a positive titre in the tube agglutination test. Six of the rapid slide test positive sera gave positive reactions in immunoprecipitation tests. One dog was identified which was found to have a history very suggestive of B. canis infection. It was judged that 0.3% of sera tested showed serological evidence of B. canis infection. The complexities of the serological diagnosis of B. canis infection was apparent, in particular the tendency to false-positive results in the rapid slide agglutination test.     

Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.