一种鸡抗猪丁型冠状病毒阳性血清的制备方法

为研究能否用鸡制备出猪丁型冠状病毒(PDCoV)阳性血清。将PDCoV用BEI灭活制备抗原,抗原与矿物油佐剂乳化制备疫苗,疫苗多次免疫SPF鸡,采集血清,检测血清中和效价、将血清用于IFA检测PDCoV。血清中和效价随免疫次数增加逐渐增高,4免后14日的中和效价平均值为1300;中和效价1000左右的血清混合作为阳性血清,1000倍稀释可用于IFA检测PDCoV。实验说明,用鸡制备PDCoV阳性血清的方法成立,制备的阳性血清可用于中和实验、IFA检测PDCoV。     

An indirect fluorescent antibody test for the detection of antibody to swine infertility and respiratory syndrome virus in swine sera.

An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64- greater than or equal to 1:1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers greater than or equal to 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of antibody treatment of Campylobacter jejuni on the dose required to colonize chicks

This study was designed to clarify the role of antibodies in controlling chicken colonization by Campylobacter jejuni. Cecal colonization by C. jejuni was compared after the organism was exposed either to phosphate-buffered saline, normal rabbit serum, rabbit hyperimmune anti-C. jejuni serum, or anti-C. jejuni antibodies extracted from chicken bile. Antibodies from chicken bile were extracted by affinity absorption against outer-membrane proteins from the challenge organism. Sera were heated 1 hour at 56 C to destroy complement activity. Bacterial inoculum levels were enumerated after 1 hour exposure at 4 C to the various treatments. The heated sera and the bile antibodies were not bactericidal, and bacterial agglutination was not evident. Serial dilutions of the antibody-treated C. jejuni were given by gavage into 1-day-old chicks. Six days later, the ceca were removed from the chicks, and samples were cultured on Campylobacter-charcoal differential agar. The colonization dose-50% was increased by twofold to 160-fold when the organism was preincubated with hyperimmune antiserum or the bile antibodies as compared with preincubation with phosphate-buffered saline. We conclude that antibodies inhibit chicken cecal colonization by C. jejuni.     

Detection of serum antibodies against Mycoplasma gallisepticum and Mycoplasma synoviae by a dot-immunobinding technique

Both Mycoplasma gallisepticum (MG) and M. synoviae (MS) antigens prepared for the routine haemagglutination inhibition (HI) test were diluted and absorbed to the separate pieces of durapore membrane for the measurement of dot-immunobinding (DIB) titers of test sera. Besides, durapore strips bearing both antigens were employed for a DIB test with chicken sera definitely diluted 100-fold. Shortening of reaction time of chicken sera with antigens as well as with the secondary serum markedly eliminated non-specific DIB reactions exhibited at low dilutions although the same condition was not so effective on the elimination of non-specific reactions among rabbit hyperimmune sera. Rapid and specific development of DIB antibody which continued at high titer up to 1:640 for 10 weeks postinoculation was proved in the sera of SPF chickens inoculated with MG or MS, while DIB titers of sera from uninoculated chickens remained 1:20 or lower. Non-specific reactions, which occurred in the routine serum plate agglutination test with a part of sera from the inoculated chickens, were not exhibited in the DIB as well as in the HI test with the same sera. Results of the DIB test with serum samples from 287 conventionally reared chickens definitely diluted 100-fold coincided with the results of HI test at a level of 90% with MG and 89% with MS antigen. This technique seems to be useful for a rapid, simple and specific diagnosis of avian mycoplasmosis.     

Immunological relationship between Neospora caninum and Besnoitia besnoiti

Neospora caninum is a coccidian parasite identified as a major cause of abortion in cattle. A combined infection of N. caninum with another taxonomically related parasite of cattle, Besnoitia besnoiti can occur in geographical areas endemic for both species. Both infections are routinely diagnosed serologically, and incorrect diagnosis could occur if immunological cross-reactivity exists between the two parasites. To investigate the possible degree of cross-reactivity, we compared results obtained with two serological techniques, immunofluorescent antibody test (IFA), and Western blot analysis on known positive and negative sera. The test sera were derived from naturally infected cattle and from experimentally infected Mongolian gerbils. In IFA of bovine sera, no cross-reactvity was detected at the commonly used serum dilution cutoffs of 1:200 for N. caninum and 1:256 for B. besnoiti. However, at 1:64 dilution of both cattle and gerbil sera, anti-N. caninum sera reacted with B. besnoiti antigen in some individual samples. Anti-B. besnoiti serum did not react with N. caninum antigen at any dilution. This low level one directional cross-reactivity was confirmed by Western blot analysis. B. besnoiti antigen showed two immunoreactive bands when probed with anti-N. caninum serum, while no bands appeared when N. caninum antigen was probed with B. besnoiti antiserum. Immunization and challenge experiments in the highly susceptible Mongolian gerbil (Meriones unguiculatus) showed essentially no cross-protection between N. caninum and B. besnoiti.     

The use of chosen serological diagnostic methods in Lyme disease in horses. Part II. Western blot

In this investigation the Western blot test was treated as a method verifying results of the IFA, commercial ELISA and standardized ELISA tests (described in Part I). The verifying investigations were performed on 82 serum samples, which in the commercial ELISA were positive in 36 cases, dubious in 31 cases and negative in 15 cases as well as on 5 serum samples obtained from horses infected with Leptospira spp., which in the ELISA commercial were dubious (total of 87 sera samples). The antigens, against which the immunological response in horses was directed, were also established. The Milenia--Blot--Borrelia IgG test (MIDBO IgG-Kit 30 Tests: DPC Bierman GmbH) was used in the investigation. In view of species differences, rabbit anti-horse IgG (whole molecule) alkaline phosphatase conjugate, no A6063 SIGMA-ALDRICH was used interchangeably. Also the control sera were substituted with the horse control sera. It was demonstrated that the Western blot test is the most reliable in the serological diagnosis of B. burgdorferi infection in horses. The commercial ELISA and standardized ELISA tests represent a lower diagnostic value than the Western blot test, although similar to each other, while the value of the IFA is minimal. In the Western blot test antigens were established against which the immunological response in horses in mostly directed. In the sera evaluated in this test as positive the presence of antibodies, mainly against antigens with the following molecular weights: 41 kDa, 62/60 kDa, 93 kDa, 72 kDa, 34 kDa (OspB), 66 kDa was noted. At the same time, antibodies contained in the sera accepted as negative, in 55.5% cases also reacted with the antigen of 41 kDa. It points to its minimal specificity. On the basis of the results obtained it is recommended that serological examination of horses should be with the ELISA and that positive or dubious results should be verified with the Western blot test.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.     

Elimination of mycoplasmal plate agglutination cross-reactions in sera from chickens inoculated with infectious bursal disease viruses

Sera from chickens inoculated with various challenge infectious bursal disease viruses or infectious bursal disease vaccines were found to cross-react in the Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) serum plate agglutination (SPA) tests. Two-fold dilutions of these cross-reacting sera with phosphate-buffered saline before retesting eliminated all non-specific agglutination in the MG and MS SPA tests. Cross-reactions were observed in the SPA test using sera from chickens inoculated with either MG or MS. Dilutions of these sera 1:2 had little effect on the number of these cross-reactions. At 1:4 serum dilutions, however, the number of cross-reactions between MG and MS was reduced. At 1:8 dilution of test sera, cross-reactions between MG and MS were further reduced. Some reduction in specific MG and MS SPA reactions, however, also occurred at the 1:8 dilution of sera with some of the plate antigen.     

Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:16558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.     

Diagnosis of Rhodococcus equi infection in foals by the agar gel diffusion test with protein antigen

A protein antigen that reacted in the agar gel diffusion (AGD) test and which had equi factor(s) activity, was partially purified from the culture supernatant of Rhodococcus equi by successive column chromatography on diethylaminoethyl cellulose and Sepharose 4B. Employing a standard foal serum, the concentration of this antigen was adjusted for the AGD test. Optimal dilutions of the antigen reacted in the AGD test with sera from foals naturally infected with serologically different R. equi. The antigen prepared was considered suitable for use in field surveys of R. equi infection. Accordingly, four groups of sera were tested: those from 18 foals diagnosed as being infected with R. equi, those from 54 control foals with culture-negative R. equi pneumonia, arthritis or cellulitis, those from 46 diseased foals suspected of having R. equi infection and those from 51 clinically normal foals. A positive precipitation reaction was observed with sera from 100% of the first group, 69.5% of the third group and 17.7% of the fourth group. A negative reaction was obtained with sera from 100% of the second group.     

The role of specific immunoglobulins in antibody-dependent cell-mediated cytotoxicity assays during Babesia bovis infection

A stabilate prepared from Babesia bovis-infected Boophilus microplus ticks was used to infect intact adult cattle. Whole sera and immunoglobulin fractions from representative sera were tested by complement fixation (CF), indirect fluorescent antibody (IFA), and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The last test utilized 51Cr-labeled chicken erythrocytes coated with Babesia bovis antigen as targets. Mononuclear cell preparations, obtained from peripheral blood of normal donors and consisting of lymphocytes with 2--6% large monocytes, were used as the source of effector cells. Antibody activity was detected by all tests between 14 and 16 days following infection. Specific IgM and IgG1 were reactive in both CF and IFA tests, although the development of high titers was attributable to IgG, alone. The ADCC activity was restricted to IgG1 fractions and was greater in those sera or fractions with greater CF activity. No activity was demonstrated in IgG2 fractions by any test used.     

泰氏锥虫抗原制备及实验室诊断研究

应用体外培养的泰氏锥虫制备可溶性抗原Ⅰ、抗原Ⅱ和代谢抗原.经测定,其蛋白含量每毫升分别为6.5mg、7.4mg和7.1mg.薄层等电聚焦电泳测定结果表明,抗原Ⅰ出现22条区带,抗原Ⅱ21条区带,代谢抗原28条区带,对照的伊氏锥虫琼脂免疫扩散抗原14条区带.经分析,泰氏锥虫抗原和伊氏锥虫抗原有4条区带在同一迁移率上.琼脂免疫扩散反应中,3种泰氏锥虫抗原均与相应免疫兔血清发生沉淀反应,抗原Ⅰ出现1条致密沉淀线,抗原Ⅱ和代谢抗原出现2～3条沉淀线,抗原效价为1:4～16.免疫电泳显示了类似的结果,抗原Ⅰ与免疫兔血清出现1条弧形沉淀线,抗原Ⅱ和代谢抗原与免疫兔血清出现了3条弧形沉淀线.间接血凝试验结果表明,泰氏锥虫自然感染牛血清效价为1:20～40,免疫兔血清为1:1280～5120.所制泰氏锥虫抗原对伊氏锥虫和媾疫锥虫血清也能很好地发生交叉反应,3份伊氏锥虫病马血清和3份伊氏锥虫人工感染兔血清血凝效价分别为1:10～40和1:8～1024;5份媾疫马血清有4份血凝效价为1:20～320.4份环形泰勒虫病牛血清均为阴性.     

凝集试验检测兰氏D群链球菌血清抗体方法的建立

建立凝集试验,用于检测兰氏D群链球菌血清抗体.采用兰氏D群链球菌C55914株制成凝集试验抗原,制备的抗原与兰氏D群链球菌兔高免阳性血清、猪正常免疫阳性血清发生凝集反应,不与健康兔阴性血清及健康猪阴性血清发生凝集反应,与链球菌兰氏C群、兰氏E群、巴氏杆菌A群、B群阳性血清呈阴性反应.试验证明,凝集试验能够快速地检验出兰...     

Development and comparison of serologic methods for diagnosing chicken anemia virus infection.

This paper describes the development of an indirect immunoperoxidase assay (IIP) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to chicken anemia virus (VAC). The IIP assay developed used CAV-infected MDCC-MSB1 cells for detecting antibody to CAV, whereas the ELISA utilized gradient-purified immunoadsorbed CAV as the target antigen. The IIP and ELISA were compared with the standard indirect immunofluorescent antibody (IFA) assay, which is more conventionally used to screen chicken serum for antibodies against CAV. Comparative test results of 185 field samples of chicken serum by these three methods were in agreement 84% of the time. Both IFA and IIP assays yielded fewer positive tests than did the ELISA. IFA and IIP assays were in agreement 93% of the time, as compared with 91% agreement of IIP and ELISA results, or 84% agreement for comparative IFA and ELISA results.     

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.     

An agglutination test for the detection of Bordetella bronchiseptica infection in swine.

An agglutination test with the use of formalin-killed antigen of the cell carrying the capsule was developed and used for the detection of antibody in swine naturally infected with Bordetella bronchiseptica. Under optimum antigen concentration and reaction temperature 210 or 60% of 342 serum samples tested from 42 conventional swine herds were positive for Bordetella infection. In contrast, only 34 or 10% of 342 nasal swabs from the same animals were positive for Bordetella by culture technique. The test was relatively free of cross-reactivity to related organism. However, 2.7 and 13.0% of sera from growing pigs and mature hogs, respectively, reacted with antigen of Pasteurella multocida. Because of this, only agglutinin reactions in 1:20 dilutions or higher to Bordetella were considered positive. The bulk of the antibody activity of selected sera tested from various age ranges of swine was mercaptoethanol sensitive, suggesting that serum antibody in Bordetella infection may be associated with immunoglobulin IgM. Because of the high agglutinability and stability of formalin-killed antigen the test may be useful as an auxiliary aid for the diagnosis of Bordetella infection where the organism cannot be identified by culture means.     

鹦鹉热嗜性衣原体感染SPF鸡和污染相关疫苗的初步调查

为初步调查SPF鸡感染鹦鹉热嗜性衣原体状况及相关SPF鸡胚源疫苗是否出现污染,本试验通过采集不同日龄的SPF鸡血清70份、SPF种蛋卵黄膜30份,收集市场上销售的SPF鸡胚源疫苗共41支,利用国产间接血凝试剂盒检测抗体,进口免疫荧光试剂盒分别测定其抗体、抗原阳性率,以评价SPF鸡鹦鹉热嗜性衣原体的流行状况和相关疫苗的污染状况。本试验结果显示,SPF鸡血清阳性率分别为31.4%(荧光法)、5.7%(间接血凝法);SPF种蛋阳性率33.3%,SPF鸡胚源疫苗平均阳性率31.7%。SPF鸡已经感染了鹦鹉热嗜性衣原体,且发现经鸡胚卵黄膜而传播病原的新途径,进而造成SPF鸡胚源疫苗出现衣原体污染。因此,加强SPF鸡鹦鹉热嗜性衣原体监测已势在必行。     

Establishment of Indirect ELISA Method for Detecting Antibodies against Avain Infectious Bronchitis Virus

The purpose of this research was to establish a rapid detection serological method against avain infectious bronchitis virus (IBV).In this study,an indirect ELISA method was established using IBV as the detected antigen and a variety of testing conditions were optimized.The results showed that the optimal antigen coating concentration was 19.2 μg/mL and the optimal condition for coating was incubated at 37 ℃ for 1 h and then 4 ℃ overnight.The optimal dilutions of serum and enzyme labeled antibody were 1:800 and 1:7 000 incubated at 37 ℃ for 60 min,respectively.The optimal condition of chromogenic substrate was incubated at 37 ℃ for 10 min in the dark.The specificity,repeatability and sensitivity tests proved that the indirect ELISA did not cross-react with positive antiviral sera of other chicken diseases,had good repeatability and could detect IBV antibody when serum was diluted 1:12 800.We concluded that the established indirect ELISA was specific,repeatable and sensitive.     

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.