Detection of pseudorabies virus antibodies: time of appearance by two serotests

This report describes a time-course comparison of detection of pseudorabies virus antibodies in experimentally infected swine by the virus-neutralization (VN) and indirect hemagglutination tests. Specific antibody titers were observed by the IHA test at 5 days after swine were inoculated, but not until 12 days by the VN test. The predominant immunoglobulin (Ig) class present in the serums of the swine at 5 and 7 days after inoculation was IgM, as determined by sulfhydryl reductions. The VN test lacked sensitivity to early Ig levels (IgM) in these experimentally infected swine, while the indirect hemagglutination test was highly sensitive to these same levels. On the basis of these results, it is possible that the VN test may read early infections as pseudorabies virus negative, due to the low presence of IgG in these samples.     

Virus isolation and immune responses in susceptible swine exposed with pseudorabies virus (Shope strain).

Eighteen seronegative swine weighing from 9 to 11 kg were exposed intranasally with the Shope strain of pseudorabies virus (PRV) and were observed for 21 days in an experiment to detect virus shedding and immune responses. All swine had PRV in their nasal passages at 7 days after exposure; they also had precipitating antibodies to PRV as determined by the microimmunodiffusion test (MIDT) and very low levels of virus-neutralizing (VN) antibodies. The PRV was isolated from only 2 swine at postexposure day 14; all swine were MIDT positive, and VN titers ranged from 4 to 128. Virus was not isolated from the swine at 21 days after exposure, but all were MIDT positive; VN titers ranged between 8 and greater than or equal to 256.     

Sensitivity of the standardized pseudorabies virus neutralization test varies with the test strain used.

The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.     

Evaluation of the sensitivity and specificity of two diagnostic tests for antibodies to pseudorabies virus glycoprotein X.

The diagnostic performance of 2 enzyme-linked immunosorbent assays (gX-T, gX-H) for antibodies to pseudorabies virus (PRV) glycoprotein X (gX) were evaluated using 311 serum samples from a nonvaccinated quarantined herd. When the standardized virus neutralization (VN) test, which uses the Shope strain (VN Shope), was used as the comparative diagnostic standard, the gX-T test had a 7% false-negative rate and a 52% false-positive rate, and the gX-H test had a 19% false-negative rate and a 19% false-positive rate. When the VN test with a Bartha recombinant strain (VN Bartha gIIIKa) was used as the diagnostic standard, the gX-T test had a 9% false-negative rate and a 26% false-positive rate, and the gX-H test had a 24% false-negative rate and a 11% false-positive rate. Thus, the gX-T test was more sensitive and the gX-H test was more specific. Additional diagnostic tests on 79 serum samples from a noninfected herd did not produce false positives for the gX-H test, but there was an 8% false-positive rate for the gX-T test. Previous studies from our laboratory have demonstrated that VN Bartha gIIIKa has higher sensitivity than VN Shope, without losing specificity, and thus is a better comparative diagnostic standard. When adding a suspect range to the gX-T test, using the same criteria as the suspect range for the gX-H test, the false-positive rate of the gX-T test was reduced to 5% when evaluated versus VN Bartha gIIIKa in the infected herd and to 1% for the PRV-negative herd.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and evaluation of a microimmunodiffusion test for detection of antibodies to pseudorabies virus in swine serum.

A microimmunodiffusion test (MIDT) was developed for the detection of pseudorabies virus (PRV) antibodies in swine serum. The optimal medium for the MIDT was determined to contain 0.69% agarose in 0.05 M tris buffer (pH 7.2) with 0.025% sodium azide and no NaCl. The PRV antigen prepared by (NH4)2SO4 precipitation of viral fluids (42.5 g/100 ml), dialyzed against distilled water, and concentrated to approximately 100-fold of the original volume with polyethylene glycol (mol wt 20,000) provided a good reproducible antigen. The sensitivity of the MIDT was compared with the microtitration procedure of the virus-neutralization (VN) test by assaying 2,203 swine serums for PRV antibodies. An equal percentage of serums was positive in both tests; 419 had VN titers of greater than or equal to 4, and 421 were MIDT positive. Serums (314) that had VN titers of greater than or equal to 16 were all positive by the MIDT. Of serum samples with a VN titer of 8 (53), 50 were MIDT positive, a 94% correlation, and of 52 serums that had VN titers of 4, 36 were MIDT positive, a 69% correlation. In addition, 8 serums that had titer of less than 4 by VN test were positive by MIDT. Seventy-one serum samples were too cytotoxic, markedly hemolyzed, or contaminated to evaluate properly in the VN test; of these serums, 13 were MIDT positive. The MIDT is an accurate, rapid, economical, and sensitive diagnostic test for the detection of PRV antibodies in swine serums.     

Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus

Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.     

Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections

An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.     

Single dilution ELISA for detection of serum antibody to foot-and-mouth disease virus in cattle

A single dilution blocking ELISA was developed and evaluated for measuring serum antibody to foot-and-mouth disease virus (FMDV). Basic parameters of the assay were established and a positive-negative threshold determined from testing 176 specific antibody negative sera from Australian cattle. Sera collected from immunised animals in Thailand were tested by ELISA and virus-neutralisation (VN) tests and the results compared. A positive correlation between ELISA and VN titres was recorded for each of the 3 FMDV serotypes endemic in Thailand, with the overall correlation coefficient being r = 0.8990. A positive correlation for each of the serotypes was also found between ELISA titre and the degree of blocking (percentage inhibition) of each test serum at a dilution of 1:16, with the overall correlation being r = 0.8704. This simplified ELISA was sensitive, specific and gave reproducible results, and had the potential to test quickly and efficiently a considerable number of sera.     

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.     

Pseudorabies virus infection in wild boars in Japan

In Japan, most pig populations are now free from pseudorabies virus (PRV) due to the recent success of an extensive eradication program. However, PRV infection persists in Japanese wild boars (Sus scrofa leucomystax), representing another potential reservoir for the virus in Japan. In this study, the seroprevalence of PRV in wild boars captured in three different prefectures was ascertained. A virus neutralization (VN) test showed that 6 of 173 serum samples (3%) were positive for VN antibody; glycoprotein E-ELISA revealed infection with the wild-type, but not the available vaccine strain, PRV. These results indicate that PRV has continued to spread among wild boars in Japan.     

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.     

Blocking ELISA to distinguish pseudorabies virus-infected pigs from those vaccinated with a glycoprotein gIII deletion mutant

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV)-infected pigs from those immunized with a glycoprotein g92(gIII) deletion mutant, PRV(dlg92dltk). The blocking ELISA utilizes 96-well microtiter test plates coated with a cloned PRV g92(gIII) antigen, a mouse monoclonal antibody against gIII antigen (moMCAgIII): horseradish peroxidase (HRPO) conjugate, and undiluted test sera. Analyses can be completed in less than 3 hours with results printed out by an automated plate reader. Analyses on over 300 pig sera from PRV-free farms, on sera from other species, and on control sera containing antibodies to microorganisms other than PRV showed that the ratio of the optical density at 405 nm for the test sample to the optical density at 405 nm for the negative control (S/N value) was greater than 0.7 for all sera. No false positives were identified. Likewise, the S/N values were greater than 0.7 for over 400 sera obtained from pigs vaccinated twice with more than 1,000 times the standard PRV (dlg92dltk) dose or 1-4 times with the standard dose (2 x 10(5) TCID50/pig). Following challenge exposure to virulent PRV, the S/N values of the vaccinates were 0.1, showing that g92(gIII) antibodies in the sera of experimentally challenged pigs strongly blocked the binding of the moMCAgIII:HRPO conjugate to the antigen-coated wells. Sera of 233 pigs from PRV-infected herds with virus neutralization (VN) titers of 1:4 or greater were tested. All except 2 of these sera had S/N values less than 0.7 and more than 175 had S/N values less than 0.1. Sixteen sera from fetal pigs with VN titers of 1:4 or greater had S/N values of 0.24 or less, but 2 sera with VN titers of 1:4 when tested 5 years prior to the PRV g92(gIII) blocking ELISA test gave false negative S/N values. Twenty-four of 29 pig sera from PRV-infected herds with VN titers less than 1:4 were positive for g92(gIII) antibodies, illustrating the sensitivity of the PRV g92(gIII) blocking ELISA test. Analyses on 7 sera with VN titers of 1:4-1:64 showed that titers obtained with the PRV g92(gIII) blocking ELISA test were from 2- to 16-fold greater than the VN titers. The accuracy and sensitivity of the PRV g92(gIII) blocking ELISA test was further demonstrated by analyses of 40 unknown sera supplied in the National Veterinary Services Laboratories 1988 PRV check test kit.     

Comparison of precipitin antibodies and virus-neutralizing antibodies to infectious bursal disease virus

Three hundred twenty-two serum samples from commercial pullets and multiplier breeders were analyzed for agar-gel precipitin (AGP) antibodies and virus-neutralizing (VN) antibodies to infectious bursal disease virus. Two hundred thirty-four of these sera were AGP-positive, and 88 were AGP-negative. The geometric mean of the reciprocal of the VN titers for the AGP-positive sera was 208.7, and 232 (99.1%) had a VN titer of 1:16 or greater. In contrast, the geometric mean of the reciprocal of the VN titers for the AGP-negative sera was 6.1, but 53 (60.2%) had a VN titer ranging from 1:4 to 1:256. When the AGP test was compared with the VN test, the sensitivity and specificity, respectively, of the AGP test were 81.5% and 100%.     

A serodiagnostic ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein.

A serodiagnostic ELISA utilizing the recombinant nucleoprotein (rN protein) of transmissible gastroenteritis virus (TGEV) was developed, and evaluated by examining a panel of 141 virus neutralization (VN) positive and 101 negative sera. The rN protein-based ELISA (rnELISA) appeared to be highly sensitive and specific (98.6% and 98.0%, respectively) when it was compared to the VN test. The result was similar to that of an ELISA based on purified viral antigens with showing good correlation (R=0.829). No cross-reaction was detected with antisera against porcine epidemic diarrhea virus, hog cholera virus, type A rotavirus, pseudorabies virus and swine vesicular disease virus in this ELISA. The rnELISA can be an alternative for the diagnosis of TGE with a great advantage in antigen preparation.     

山羊痘病毒p32基因原核表达及间接ELISA抗体检测方法的建立

为了建立山羊痘病毒(GPV)抗体快速检测方法,本研究通过人工合成密码子优化的GPV p32基因,并在大肠杆菌中进行截短表达(p32-opti).表达的重组p32-opti蛋白主要以包涵体形式存在,Western blot分析表明,p32-opti与GPV标准阳性血清具有良好的抗原性.以纯化的重组p32-opti蛋白作为ELISA包被抗原,建立间接ELISA抗体检测方法.该方法检测小反刍兽疫、蓝舌病和口蹄疫阳性血清均无交叉反应;与中和试验(VNT)比较,两者的符合率为95.7%;ELISA批内和批间重复性试验显示,OD值的变异系数小于10%.上述结果表明.该ELISA检测方法具有良好的特异性、敏感性和重复性.对来自黑龙江、内蒙古和新疆自治区的390份山羊和绵羊血清进行检测,抗体阳性率为80.2%,表明这些地区的山羊和绵羊群普遍存在羊痘病毒抗体.本研究建立的间接ELISA方法可用于GPV感染的流行病学调查以及疫苗接种动物的抗体水平监测.     

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.     

Use of a recombinant maedi-visna virus protein ELISA for the serologic diagnosis of lentivirus infections in small ruminants.

Highly purified recombinant gag and env proteins derived from Icelandic strain 1514 of maedi-visna virus were used in an indirect enzyme immunoassay (ELISA) to detect antibodies to small ruminant lentiviruses in sheep and goat sera. The recombinant protein-based ELISA performed very well relative to whole maedi-visna virus and whole caprine arthritis-encephalitis-virus-based ELISAs in its ability to detect anti-maedi visna virus and anti-caprine arthritis-encephalitis virus antibodies, despite the antigenic and genomic variability that is known to exist within and between these two small ruminant lentiviruses. The data suggest that these recombinant maedi-visna virus proteins can be reliably used in an ELISA for the routine serodiagnosis of lentiviral infections in sheep and goats.     

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4)

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.