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.     

Second-generation pseudorabies virus vaccine with deletions in thymidine kinase and glycoprotein genes

A modified-live pseudorabies virus (PRV) vaccine, designated PRV(dlg92/d1tk), with deletions in the thymidine kinase (tk) and glycoprotein-gIII (g92) genes, was derived from the PRV (Bucharest [BUK]-d13) vaccine strain. The vaccine virus also contained a deletion in glycoprotein gI. Despite 3 deletions, PRV(dlg92/d1tk) replicated to high titers in cell culture from 30 C to 39.1 C. Enzyme assays and autoradiography revealed that PRV(dlg92/d1tk) did not induce a functional tk activity in infected tk- RAB(BU) cells (rabbit skin). Rabbit skin cells were infected with PRV(dlg92/d1tk), with vaccine strains derived from BUK or Bartha K strains of PRV or with the virulent Illinois (ILL), Indiana-Funkhauser (IND-F), and Aujeszky (Auj) strains of PRV and were labeled with [3H]mannose from 4 or 5 to 24 hours after infection to investigate whether these viruses induced the synthesis of glycoprotein gIII. Nonionic detergent extracts were prepared and immunoprecipitated with antisera from pigs vaccinated with tk(-)-PRV(BUK-d13) or tk+-Bartha K, pigs vaccinated with tk+-PRV(BUK) strains and then challenge exposed to tk+-PRV(IND-F), naturally infected domestic or feral pigs, and pigs vaccinated with tk-)-PRV(dlg92/d1tk). Mouse monoclonal antibodies against PRV glycoproteins gIII, gp50, and gII were also studied. After immunoprecipitation, labeled PRV-specific proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. The PRV glycoprotein-gII complex, but not glycoprotein gIII, was synthesized in PRV(dlg92/d1tk)-infected cells. Glycoprotein gII and gIII were made in cells infected with PRV vaccine strains BUK, Bartha K, and BUK-d13 and with virulent PRV strains ILL, IND-F, and Auj. Cells infected with PRV(dlg92/d1tk) and with PRV strains ILL, IND-F, Auj, Bartha K, BUK, and BUK-d13, excreted into the cell culture medium a highly sulfated glycoprotein gX of about 90 kilodaltons. Antibodies to glycoprotein gIII were not detected in the sera of pigs inoculated with PRV(dlg92/d1tk), but were found in all other swine sera.     

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.     

伪狂犬病弱毒株的分离鉴定及生物学特性的研究

在流行病学调查中分离到1株病毒,经鉴定为伪狂犬病弱毒株,定名为F971株。分离病毒经克隆纯化后测得其毒价为10^7.59TCID50/ml,通过细胞中和试验表明分离病毒能也有效地被猪伪狂犬病毒闽A株阳性血清中和。病毒在电镜下可以清楚地观察到囊膜及外周纤突。分离株对3日龄乳鼠有一定的致病力,但对家兔、3日龄乳猪及妊娠母猪都有很高的安全性。用不同的剂量10^0、10^-1、10^-2肌肉注射3日龄乳猪后14天用10^5.7TCID50伪狂犬病强毒攻击,所有试验仔猪均得到保护。用分离株免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵御10^5.7TCID50强毒的攻击。用ELISA普查试剂盒测定免疫猪抗体,结果均为阳性,而用g^1-ELISA试剂盒测定抗体时,结果均为阴性。证明分离株具有缺损g^1糖蛋白的特性。综合上述特性,确定F971为1株g^1糖蛋白缺损的猪伪狂犬病弱毒株。     

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.     

Immune response of pigs inoculated with virulent pseudorabies virus and pigs inoculated with attenuated or inactivated pseudorabies virus vaccine before and after challenge exposure

Pseudorabies virus (PRV) antibodies, detectable by indirect radioimmunoassay (IRIA), serum-virus neutralization test (NT), or microimmunodiffusion test (MIDT) were developed within 8 days after pigs were inoculated with virulent PRV or attenuated PRV vaccine. Indirect radioimmunoassay and NT titers in pigs inoculated with virulent PRV were developed at the same rate, with IRIA titers being higher than NT titers. Pigs inoculated with attenuated or inactivated PRV vaccine developed peak mean prechallenge NT antibody titers of 4 and 1 (reciprocals of serum dilutions), respectively. Pigs inoculated with attenuated PRV vaccine had peak mean prechallenge IRIA antibody titers of 6, whereas pigs inoculated with inactivated PRV vaccine had mean IRIA antibody titers of 64. Challenge exposure of swine inoculated with attenuated or inactivated PRV vaccine elicited quantitatively equivalent responses, as measured by IRIA or NT, which were higher than prechallenge titers. There were no false-positive IRIA, NT, or MIDT results obtained when sera from nonvaccinated, nonchallenge-exposed pigs were tested. It appears that the PRV infection status of a seropositive swine herd could be ascertained by serologically monitoring several representative animals from a herd, using the NT. If 2 or more tests of representative animals at 14-day intervals were done and the mean NT titer was 4 or less, it could be concluded that the herd was vaccinated against, but not infected with, virulent virus.     

Intranasal vaccination of pigs against pseudorabies: absence of vaccinal virus latency and failure to prevent latency of virulent virus

The avirulent Bartha's K strain of pseudorabies virus (PRV) was used to vaccinate 8 pigs at 10 weeks of age by the intransal route (experiment 1). On postvaccination days (PVD) 63 and 91, pigs were treated with corticosteroids. Viral shedding could not be detected. Explant cultures of trigeminal ganglia and tonsils did not produce virus. Four pigs with maternal antibody were vaccinated intranasally with Bartha's (attenuated) K strain of PRV at 10 weeks of age and were challenge exposed with a virulent strain of PRV on PVD 63 (experiment 2). Corticosteroid treatment, starting on postchallenge exposure day 70 (PVD 133) resulted in viral shedding in 1 of 4 pigs. In another pig of these 4, a 2nd corticosteroid treatment was required to trigger reactivation. In both pigs, sufficient reactivated virus was excreted to infect susceptible sentinel pigs. Restriction endonuclease analysis indicated that viruses isolated from the 2 pigs after challenge exposure and corticosteroid treatment were indistinguishable from the virulent virus. Evidence was not obtained for simultaneous excretion of vaccinal and virulent virus. Of 4 pigs without maternal antibody vaccinated twice with 1 of 2 inactivated PRV vaccines, challenge exposed on PVD 84, and treated with corticosteroids on postchallenge exposure day 63 (PVD 147), 1 was latently infected, as evidenced by the shedding of PRV (experiment 3). However, its sentinel pig remained noninfected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunogenicity in Aujeszky's disease virus structural glycoprotein gVI (gp50) in swine.

Aujeszky's disease virus (ADV) envelope glycoprotein gVI (gp50) was purified from virus-infected Vero cells by ion-exchange and immunoaffinity chromatography and its usefulness as a subunit vaccine was evaluated in active and passive immunization studies. Four-week-old piglets were immunized intramuscularly (IM) with purified gVI twice two weeks apart and challenged intranasally (IN) 10 days after the second immunization with 30 LD50 (10(8)PFU) of a virulent strain of ADV. Pigs, vaccinated with 100 micrograms of purified gVI, produced virus neutralizing antibodies and did not develop clinical signs after challenge exposure. The challenge virus was not isolated from nasal swabs and tonsils of gVI-vaccinated pigs, whereas non-vaccinated control pigs developed illness after challenge exposure with the same virulent ADV strain which was later recovered from their nasal swabs and tonsils. Pregnant sows vaccinated twice with purified gVI (IM) at a three week interval produced virus neutralizing antibodies in colostrum. Four-day-old sucking piglets born of vaccinated sows were passively protected by colostral antibodies against intranasal challenge with a lethal dose of virulent ADV. Sera from gVI-vaccinated pigs were distinguished from experimentally infected swine sera by their differential reactivity in enzyme-linked immunosorbent assay (ELISA) using four major viral glycoproteins (excluding gVI) as antigen purified by the use of lentil-lectin.     

Evaluation in swine of a subunit vaccine against pseudorabies

A subunit vaccine against pseudorabies virus (PRV) was prepared by treating a mixture of pelleted virions and infected cells with the nonionic detergent Nonidet P-40 and emulsifying the extracted proteins incomplete Freund's adjuvant. Three 7-week-old pigs without antibodies against PRV were given 2 IM doses of this vaccine 3 weeks apart. Thirty days after the 2nd vaccination, 10(6) median tissue culture infective doses (TCID50) of a virulent strain of PRV were administered intranasally. Tonsillar and nasal swabs were collected daily between 2 and 10 days after challenge exposure. The pigs vaccinated with the subunit vaccine were not found to shed virulent PRV. Two groups of five 7-week-old pigs vaccinated with commercially available vaccines, either live-modified or inactivated virus, and subsequently exposed to 10(6) TCID50 of virulent PRV, shed virulent virus for up to 8 days. The subunit vaccine induced significantly higher virus-neutralizing antibody titers than either the live-modified or inactivated virus vaccine.     

The Recombinant Nonstructural Polyprotein NS1 of Porcine Parvovirus (PPV) as Diagnostic Antigen in ELISA to Differentiate Infected from Vaccinated Pigs

To differentiate pigs infected with porcine parvovirus (PPV) from those vaccinated with inactivated whole-virus vaccine, an enzyme-linked immunosorbent assay (ELISA) based on detection of a nonstructural polyprotein 1 (NS1) was developed. A threshold of 0.23 optical density units was established and the assayhad high specificity (100), sensitivity (88), accuracy (90) and positive predictive value (100) using haemagglutination inhibition as the standard method. A reproducibility test revealed that the coefficients of variation of sera within-plates and between-run were less than 10%. The assayshowed no cross-reactivitywith antibodies to porcine reproductive respiratorysyndrome virus, pseudorabies virus, foot and mouth disease virus, Actinobacillus pleuropneumoniae, Toxoplasma or Chlamydia. Sera obtained from pigs infected with PPV reacted with recombinant NS1 protein in the ELISA. Sera from pigs vaccinated with inactivated whole virus did not recognize this protein in the ELISA. In contrast, antibodies against PPV whole virus were present in both PPV-infected and vaccinated animals. Serum conversion against NS1 was first detected 10 days after infection and NS1-specific antibodies were detectable up to half a year post infection. In conclusion, the PPV-NS1 ELISA can differentiate PPV-infected versus inactivated PPV-vaccinated pigs and could be applied in disease diagnosis and surveillance.     

A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds.

A commercially available blocking ELISA was analyzed for its ability to identify antibodies to porcine coronaviruses (transmissible gastroenteritis virus [TGEV] or porcine respiratory coronavirus [PRCV]), to differentiate antibodies to TGEV and PRCV, and to identify TGEV-infected herds. Nine sera from uninfected pigs, 34 sera from 16 pigs experimentally infected with TGEV, and sera from 10 pigs experimentally infected with PRCV were evaluated using both the TGEV/PRCV blocking ELISA and a virus neutralization (VN) assay. The ELISA was not consistently effective in identifying pigs experimentally infected with TGEV until 21 days postinfection. Sera from 100 commercial swine herds (1,783 sera; median 15 per herd) were similarly evaluated using both tests. Thirty of these commercial herds had a clinical history of TGEV infection and a positive TGEV fluorescent antibody test recorded at necropsy within the last 35 months, while 70 herds had no history of clinical TGEV infection. The blocking ELISA and the VN showed good agreement (kappa 0.84) for the detection of porcine coronavirus antibody (TGEV or PRCV). The sensitivity (0.933) of the ELISA to identify TGEV-infected herds was good when considered on a herd basis. The ELISA was also highly specific (0.943) for the detection of TGEV-infected herds when the test results were evaluated on a herd basis. When sera from specific age groups were compared, the ELISA identified a greater proportion (0.83) of pigs in herds with TGEV antibody when suckling piglets were used. In repeatability experiments, the ELISA gave consistent results when the same sera were evaluated on different days (kappa 0.889) and when sera were evaluated before and after heating (kappa 0.888). The blocking ELISA was determined to be useful for herd monitoring programs and could be used alone without parallel use of the VN assay for the assessment of large swine populations for the detection of TGEV-infected herds.     

三种检测猪伪狂犬病抗体的方法比较

应用血清中和试验(SNT)、乳胶凝集试验(LAT)和PRVgpI抗体鉴别ELISA三种方法检测了42份猪血清中的伪狂犬病抗体效价并进行了比较。结果表明SNT与LAT二种方法的阳性符合率为87.1%(27/31),阴性符合率为90.1%(10/11),总符合率为88.1%(37/42),且SNT的敏感性高于LAT,但PRVgpI抗体与SN和LA抗体无相关性。     

3种非猪瘟病毒单独或混合感染对猪瘟弱毒疫苗免疫效力的影响

将 2 0头 9月龄左右猪瘟、伪狂犬、猪繁殖与呼吸障碍综合征抗原、抗体阴性猪分成 6组 ,分别利用猪细小病毒(PPV)、猪伪狂犬病毒 (PRV)和猪繁殖与呼吸障碍综合征病毒 (PRRSV)单独或混合感染。 7d后连同对照猪 4头 ,免疫接种猪瘟兔化弱毒疫苗 (HCL V) ,13d后连同 4头阴性对照猪一起攻击猪瘟石门强毒。整个试验期间分别每天测温 ,观察临床症状 ,每周采集扁桃体和血样做各种病毒抗原及抗体检测。结果表明 ,非猪瘟病毒感染 7d后 ,所有各组猪均从体内检测到了相应感染的病原 ,表明 3种非猪瘟病毒感染成功。在攻击猪瘟石门强毒后 2周 ,感染了非猪瘟病毒后接种 HCL V疫苗的 4个免疫组 12头猪除 1头外 ,11头全为猪瘟病毒 (HCV)抗原检测阳性 ,且多呈强阳性 ;而单一 HCL V疫苗免疫组在猪瘟强毒攻击后检测不到 HCV;所有 HCL V疫苗免疫猪均存活 ,而非免疫对照组 4头猪全部在攻毒 16 d内死亡。     

猪瘟不同顺序零天免疫的免疫效果比较

以猪瘟兔化弱毒细胞苗分别免疫3组初生仔猪，每头1．5头剂。第1组20头先免疫，1小时后吸初乳。第2组15头，吸初乳后1小时免疫。每3组13头，吸初乳后半小时免疫。75日龄，以猪瘟弱毒单抗酶联方法测定3组动物的平均抗体（OD值）分别为0．25、0．23及0．21。80日龄，每组各随机抽出4头猪，连同非免疫对照猪6头以猪瘟石门系强毒攻击后，对照猪全死，第1组100％保护，第2组75％保护，第3组50％保护。第1组的免疫效果明显优于其他两组。     

A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus (PRV) mutant inoculated by different routes to protect pigs against PRV infection

The capacity of a TK-negative (TK-) and gI/gE-negative (gI/gE-) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)-intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)-intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild-type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.     

伪狂犬病病毒弱毒株LY株的分离鉴定

从辽阳某猪场的10日龄仔猪中分离到1株病毒,经纯化后测得其毒价为107.29TCID50/mL.细胞中和试验表明,该病毒能被猪伪狂犬病病毒标准阳性血清所中和.电镜下可见到典型的疱疹病毒粒子,具有囊膜及外周纤突.所分离的病毒对氯仿、胰蛋白酶、乙醚敏感,在pH5.0～9.0下稳定,56℃ 30 min可以灭活.应用特异性引物,通过PCR能扩增出伪狂犬病病毒1 240 bp的gD基因.分离病毒对3日龄乳鼠有一定的致病力,但对家兔、3～5日龄仔猪及妊娠母猪都有很高的安全性.用不同剂量的病毒培养液肌肉注射于3～5日龄仔猪,14 d后用105.7TCID50伪狂犬病病毒强毒攻击,所有试验仔猪均可得到有效保护.用分离毒免疫母猪,其后代可获高滴度的母源抗体,15日龄的仔猪能抵抗105.7TCID50强毒的攻击.试验的结果初步说明,所分离的病毒为伪狂犬病病毒(命名为PRV LY株),并可能是一株弱毒株,而且具有很好的免疫保护作用.     

Anamnestic immune response of pigs to pseudorabies virus: latent virus reactivation versus direct oronasal and parenteral exposure to virus.

The humoral antibody response of pseudorabies-immune pigs to reactivation of latent pseudorabies virus (PRV) was compared with the response following direct exposure to virulent PRV. Nine pigs that had been vaccinated for pseudorabies and later exposed to virulent virus to establish latent infection were given dexamethasone to reactivate latent virus (3 pigs), were exposed oronasally and parenterally to virulent virus (3 pigs), or were kept as nontreated controls (3 pigs). Sera collected from all 9 pigs just before and 3 weeks after treatment were tested by virus neutralization and radioimmunoprecipitation. The 3 pigs exposed directly to virulent virus and 2 of the 3 pigs given dexamethasone had a 4-fold or greater increase in neutralizing antibody titer. All 6 of these pigs had an increase in precipitating antibody activity. Precipitation patterns changed both quantitatively and qualitatively, especially for virus-coded proteins of relatively low molecular weight (less than 46 K). There were some differences in the precipitation patterns associated with sera of individual pigs. However, there was no clear indication of any difference between the 2 treatment groups and therefore no evidence that reactivation of latent virus is associated with any unique immunologic response that could be detected by radioimmunoprecipitation and used diagnostically. Clinical signs were limited to the 3 pigs that were exposed oronasally and parenterally to virulent virus even though the dexamethasone-treated pigs shed more virus for much longer than did those exposed directly to virus.     

Detection of pseudorabies virus infection in subunit-vaccinated and nonvaccinated pigs using a nucleocapsid-based enzyme-linked immunosorbent assay.

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 10(4) PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid-specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (10(2.3) PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs.     

gIII antibody responses in pigs following vaccination and challenge to Aujeszky's disease.

Serological responses to a genetically engineered Aujeszky's disease "marker" vaccine (dl gIII + dl tk) were monitored using a blocking-ELISA (B-ELISA), a serum neutralisation test (SNT) and an indirect ELISA (I-ELISA). The B-ELISA is capable of differentiating pigs vaccinated with the above vaccine from natural infection. The SNT and the I-ELISA indicated that the pigs responded to vaccination and challenge. All three tests showed that the controls and the in-contact pigs always reacted negative for antibodies. The B-ELISA was able to detect pigs challenged with a field isolate 24 days post-challenge. These pigs remained positive until 110 days post-challenge when last tested. These findings indicate that the B-ELISA could be used successfully with this vaccine in a control eradication programme. This trial also shows that the vaccine virus did not spread to the in-contact pigs and also the vaccinated and challenged pigs did not transmit the disease to other susceptible pigs when they were introduced 14 days after challenge.