猪伪狂犬病不同佐剂灭活疫苗对兔免疫原性初探

采用分离鉴定的猪伪狂犬病毒（HB—J株）以4种佐剂研制成4批灭活疫苗,以研究其对兔的免疫原性。疫苗分别免疫PRV抗体阴性兔后,用乳胶凝集试验法（LAT）和中和试验法（SNT）对试验兔进行血清抗体检测;于免疫后21、28d对试验兔分别进行攻毒,观察兔保护情况。试验结果表明,兔对不同佐剂的猪伪狂犬病疫苗均产生良好的免疫应答反应,且当兔免疫后血清凝集价≥1：32或血清抗体中和指数≥1479或中和价≥1：16时,可以抵抗10LD50剂量强毒的攻击;当兔免疫后血清凝集价≥1：64或血清抗体中和指数≥2187或中和价≥1：32时,可以抵抗100LD50剂量强毒的攻击;4种佐剂灭活疫苗均具有良好的免疫效果。     

猪伪狂犬病高免血清的制作与初步应用

用伪狂犬病抗体阴性健康猪,以不同剂量、间隔一定时间、多次免疫接种伪狂犬病强毒S株,制备伪狂犬病病毒高免血清,试验表明,用本方法制备的伪狂犬病病毒高免血清特异性强、抗体效价高达256倍以上,完全符合诊断用血清标准要求.并将高免血清在中和试验、ELISA、免疫胶体金等进行了对比试验.     

Comparison of a modified counterimmunoelectrophoresis test and a microimmunodiffusion test for detection of pseudorabies virus antibodies in porcine sera.

The correlation of a modified counterimmunoelectrophoresis (CIE) test and a microimmunodiffusion test for detecting pseudorabies virus antibodies in porcine sera was investigated, using as reference a standard virus neutralization test. The counterimmunoelectrophoresis test exhibited a sensitivity comparable to the microimmunodiffusion test but was not as sensitive as the virus neutralization test. The best feature of the modified counterimmunoelectrophoresis test is that it is a rapid test. It provides an alternative to currently used diagnostic tests for detection of pseudorabies virus antibodies in sera from field reared and experimentally reared swine exposed to pseudorabies virus.     

Pseudorabies virus antibodies in swine slaughtered in Iowa.

Sera from butcher swine (1,246 total) were evaluated qualitatively by the microimmunodiffusion test and quantitatively by the virus neutralization test for antibody to pseudorabies virus. Ten percent of the sera had antibody to pseudorabies virus. Follow-up contact with veterinarians whose clients included the farms from which the positive swine originated revealed that few feeder swine are vaccinated against pseudorabies and that most infections with pseudorabies virus are subclinical.     

Pseudorabies: adaptation of the countercurrent immunoelectrophoresis for the detection of antibodies in porcine serum.

A countercurrent immunoelectrophoresis test was developed for the detection of precipitating antibodies to pseudorabies virus in pig serum. The precipitation reaction occurred only between the pseudorables antigen and the homologous porcine antiserum. The sensitivity of the method was compared to that of the serum neutralization test. On the basis of its sensitivity, its specificity and the rapidity with which the results were obtained, the countercurrent immunoelectrophoresis may become a potentially valuable screening method to test large numbers of porcine serum.     

Comparison between results of virus neutralization test and those of two ELISAs when screening for antibodies to pseudorabies virus in Thailand

The virus neutralization (VN) test and two enzyme-linked immunosorbent assays (blocking and indirect ELISAs) were used to detect antibodies to pseudorabies virus on serum samples of 1,000 pigs from the central part of Thailand. The results of these tests were compared to those of VN test. Using the VN test as standard, the blocking and indirect ELISAs showed respectively 95.12% and 99.37% relative sensitivity and 92.0% and 93.5% relative specificity. The two ELISAs were considered both as practical alternatives to the VN test. However, the indirect ELISA was the more suitable test for the routine screening for antibodies to pseudorabies virus in Thailand.     

Evaluation of an enzyme immunoassay for detection of antibodies to pseudorabies virus in porcine field sera.

The performance of an indirect enzyme-linked immunosorbent assay and the standard serum neutralization test for detection of antibodies to pseudorabies virus in porcine field sera were compared, using 304 sera from pseudorabies-free pigs in Canada, and 1082 and 580 swine sera from the USA and England, respectively. The sensitivity and specificity of the ELISA relative to the serum neutralization test were in the order of 97 to 98%, with the higher agreement between the tests when a 1:40 dilution of serum samples was used for the ELISA. The indirect ELISA was considered to be a rapid and convenient procedure, offering many advantages over the serum neutralization test for routine serodiagnostic use.     

家兔效验猪伪狂犬病灭活疫苗免疫效果的初步研究

用2种猪伪狂犬病（PR）油乳剂灭活疫苗免疫PRV抗体阴性家兔,每组免疫3只,并设不免疫兔作为空白对照。定期采血,用乳胶凝集试验和中和试验测定抗体效价。乳胶凝集试验在免疫后7 d检测到抗体,中和试验在免疫后21 d检测到抗体。家兔于免疫后28 d攻毒,免疫组均无明显临床症状,空白对照组全部死亡,证明家兔免疫后乳胶凝集效价≥1∶45.3或中和试验抗体效价≥1∶8时,可以抵抗强毒的攻击。     

Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum

A radial immunodiffusion enzyme assay for the detection and quantitation of antibodies to pseudorabies virus in swine sera was developed and the methods were standardized. The assay combined the principle of radial immunodiffusion with enzyme-linked immunosorbent assay. Quantitation of pseudorabies virus antibody titers was determined by measuring the diameter of a colored circular zone after overnight incubation of antibody with antigen. The specificity and sensitivity of the radial immunodiffusion enzyme assay were compared with that of the standard virus-neutralization test, and the results were determined to be correlated highly (r = 0.694, P less than 0.0001). The assay also appeared to be highly reproducible and simple to perform.     

Rotavirus antibody assays on monkey sera: a comparison of enzyme immunoassay with neutralization and complement-fixation tests.

An enzyme immunoassay (EIA) for the detection of rotaviral antibodies was developed, using a purified, cell culture-grown SA 11 viral antigen and alkaline phosphatase as an enzyme label. This technique was evaluated by comparative testing with tube neutralization and complement-fixation assays on a collection of simian sera. There was close correlation between positive and negative results obtained by EIA and by neutralization. The EIA was as easy to perform as complement fixation testing, but showed greater sensitivity and fewer nonspecific reactions. Thus, EIA was shown to be a very suitable test for routine detection of rotaviral antibodies in serum. Results of neutralization tests suggested that the monkeys (mostly rhesus macaques) in the present study were infected with viruses varying in their antigenic relatedness to SA 11 virus and to a British isolate of calf rotavirus.     

Evaluation of a blocking ELISA using a urease conjugate for the detection of antibodies to pseudorabies virus.

A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) was evaluated for screening sera for antibodies to pseudorabies virus under field conditions. A total of 764 serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing visual assessment of results.     

A study comparing the immunologic responses of swine to pseudorabies viral antigens based on the ELISA, serum virus neutralization, and latex agglutination tests

A study of pseudorabies virus (PRV)-vaccinated pigs comparing the immune responses detected by the latex agglutination test (LAT) with responses detected by other routine tests for pseudorabies antibodies indicated that LAT was more sensitive than either the enzyme-linked immunosorbent assay (ELISA) or the serum virus neutralization test (SVNT). The LAT detected antibodies sooner than ELISA and SVNT in unvaccinated pigs after challenge with virulent PRV. The specificities of the 3 tests were found to be near 100%. The LAT is a good alternative to SVNT or ELISA for detection of PRV-specific antibodies.     

Enzyme linked immunoassay and fluorescent antibody techniques in the diagnosis of viral diseases using staphylococcal protein-A instead of anti-gamma-globulins

Staphylococcal protein-A (SpA) is known to interact with the crystallizable fragment (Fc) of IgG molecules from several species. In the present study, SpA coupled to either fluorescein isothiocyanate (FITC) or peroxidase was used in place of antisera to IgG for the fluorescent antibody (FA) techniques and the enzyme linked immunoassay (ELISA). The SpA conjugates produced low background staining when applied in these techniques, and provide a rapid, highly specific and sensitive means for the identification of viral isolates and the detection of serum antibodies. Moreover, SpA is a single reagent that replaces various preparations of anti-gamma globulin against many species. SpA-FITC conjugate was successfully applied for the identification of pseudorabies virus, hog cholera virus, swine vesicular disease virus, transmissible gastroenteritis virus, porcine parvovirus and porcine enteroviruses. Antibody titers against the mentioned viruses could be determined semi-quantitatively in the indirect FA test with SpA-FITC. In our laboratory the ELISA became a routinely practicable serological test for the detection of antibodies only after we introduced SpA-peroxidase as a marker for the IgG.     

规模化猪场猪圆环病毒2型感染的实验室诊断

为了确诊贵州省某规模化猪场保育仔猪异常死亡原因,从怀孕母猪、产房母猪、后备母猪、种公猪、哺乳仔猪、保育仔猪6个猪群采集90份血清样本采用ELISA方法分别进行血清抗体检测,并对采集的90份血清样本和1份病死猪淋巴结组织采用荧光PCR方法进行病原学检测。结果:6个猪群综合的猪瘟病毒、猪蓝耳病病毒、伪狂犬病病毒g B蛋白、伪狂犬病病毒g E蛋白血清抗体阳性率分别为87.78%、70.00%、88.89%、4.44%;猪瘟病毒、猪蓝耳病病毒、伪狂犬病病毒病原核酸检测显示阴性,猪圆环病毒2型病原核酸检测淋巴结组织样本显示阳性。试验结果表明,引起该猪场保育仔猪死亡的原因为猪圆环病毒2型感染。同时,怀孕母猪群出现了伪狂犬病病毒g E蛋白抗体阳性,提示怀孕母猪群可能存在猪伪狂犬病野毒感染。     

Indirect solid-phase microradioimmunoassay for detection of pseudorabies virus antibody in swine sera.

An indirect solid-phase microradioimmunoassay (IRIA) was developed for detection and quantitation of antibodies to pseudorabies virus (PRV) in swine serum. Qualitative results of the IRIA compared closely with results of the serum neutralization test (NT) and the microimmunodiffusion test (MIDT). The IRIA was more sensitive than the NT for detection of antibodies to PRV in swine serum. The IRIA result is expressed numerically. With the IRIA and NT, antibody to PRV was first detectable in 3 experimentally infected pigs at 9 days after inoculation. With MIDT, antibody was detected in the 3 experimentally infected pigs at 9 days after inoculation. With the MIDT, antibody was detected in the 3 experimentally infected pigs at 7, 8, and 9 days after inoculation. The IRIA results are obtainable within a few hours; the NT and MIDT require 48 hours for completion.     

A radial immunodiffusion enzyme assay for detection of antibody to equine rhinopneumonitis virus (EHV-1) in horse serum

A radial immunodiffusion enzyme assay (RIDEA) was developed for detection and quantitation of antibodies to equine herpes virus-1 (EHV-1) in horse sera. The detection and quantitation of EHV-1 antibody levels were based on the diameter of the radial diffusion zone of specific antibody in each serum sample reacting with EHV-1 antigen. The circular zone was made visible using peroxidase-conjugated rabbit anti-horse immunoglobulin G and a substrate containing hydrogen peroxide. The results of the RIDEA were compared with those of virus neutralization (VN) and enzyme-linked immunosorbent assay (ELISA) and found to be highly correlated. The relative sensitivity and specificity (percentage of agreement with VN test) were found to be 98.2 and 92.5%, respectively. Because the test procedure is relatively easy to perform, the RIDEA could be used as a field test to detect antibodies to EHV-1 in horses.     

Sensitivity and specificity of an enzyme-linked immunosorbent assay for the detection of bovine viral diarrhea virus antibody in cattle.

A reliable bovine viral diarrhea (BVD) viral antigen was prepared from BVD virus grown on Madin Darby bovine kidney (MDBK) cells by solubilizing the virus with detergent MEGA-10 (decanoyl-N-methylglucamide) followed by removal of hydrophobic proteins with Triton X-100 treatment. By these treatments, problems of high background associated with BVD viral antigen in the enzyme-linked immunosorbent assay (ELISA) were eliminated. With this new antigen, an ELISA was adapted to detect bovine serum antibody against BVD virus. The diagnostic specificity of the assay in 403 bovine sera collected from a BVD virus-free herd was 100%; in 296 bovine sera with serum neutralizing antibody titers of greater than or equal to 1:2, 289 sera were ELISA positive (relative sensitivity of 97.6%), two sera gave false negative reactions (0.7%) and five sera gave suspicious reactions (1.7%). These interpretations were based on positive/negative (P/N) ratio readings, i.e. a P/N ratio of less than 1.50, 1.50-1.99 and greater than or equal to 2.00 were interpreted as negative, suspicious and positive reactions, respectively. The ELISA results gave excellent agreement with serum neutralization in detecting both seropositive and seronegative animals (Kappa = 0.994). The ELISA assay was considered to be technically superior to the serum neutralization test for the routine detection of BVD viral antibody in bovine sera.     

检测猪脑心肌炎病毒重组非结构蛋白2C间接ELISA方法的建立

以猪脑心肌炎病毒(Encephalomyocarditis virus,EMCV)重组非结构蛋白2C作为包被抗原,确定间接ELISA反应的最佳工作条件;通过病毒阻断试验和交叉试验检验方法的特异性;通过与血清中和试验比较,确定方法的敏感性;通过对试验感染猪的血清2C抗体产生动态的检测,分析建立方法用于EMCV感染早期诊断的意义。结果显示,建立的间接ELISA方法能够特异地检出EMCV感染猪的血清2C抗体,与猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)等的抗血清没有交叉反应;与血清中和试验检测结果的符合率为95.7%(22/23),相对敏感性为90.9%(10/11);仔猪感染EMCV后第4天,应用建立的间接ELISA方法即能够检出血清中的特异性2C抗体。结果表明,建立了可以替代血清中和试验的基于EMCV重组非结构蛋白2C的间接ELISA方法。     

Sandwich enzyme-linked immunosorbent assay (ELISA) for measuring the concentration of, and detection of antibodies to, Aujeszky's disease virus

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for measuring Aujeszky's disease virus (ADV) antigen concentration and an inhibition technique based on the former was developed for detection of antibodies to ADV. The results were checked by determining the cytopathic and serum neutralization titres. The correlation was satisfactory in both cases, with correlation coefficients above 0.8. When measuring ADV antigen concentration, the lower limit of detection was 10(3) TCID 50/0.2 ml. The sensitivity of ELISA in detecting antibodies to ADV was found to be superior to that of the serum neutralization test and, thus, enabled the testing of rabbit and guinea-pig sera.     

Preparation of Polyclonal Antibodies against Swine Pseudorabies Virus and Study on the Immunological Activity

This study was aimed to obtain polyclonal antibody against swine pseudorabies virus (PRV) Min A strain,and provide a theoretical basis for the study of the treatment and detection of PRV.This study was performed on PK-15 cell and proliferation of PRV was measured as TCID₅₀ 10^-7.372,the protein concentration of PRV was measured as 3.6 mg/mL.Choosing five healthy male rabbits (2.5 kg±0.2 kg) as experimental animals and using PRV obtained as the antigen,we got polyclonal antibody against PRV.Antiserum titer was 1:32 000,antigen coating dilution was 1:40,the best coating conditions was 4 ℃ 12 h,the best blocking time was 1 h,the best working dilution of enzyme labled antibody was 1:8 000,the result of cell lesions neutralization test showed that PRV antiserum prepared in this assay at 1:16 dilution could protect 50% of PK-15 cells from being infected by PRV,and negative serum couldn't protect PK-15 cells from being infected by PRV.The study successfully prepared polyclonal antibodies against PRV.