Use of a combination of an effective gE gene-deleted pseudorabies virus (PRV) vaccine with a companion diagnostic kit for PRV glycoprotein gE has proven successful in several pseudorabies-eradication programs. To produce a large quantity of functional gE protein for development of a PRV-gE diagnostic kit, an Escherichia coli expression system containing the distal region of the PRV-gE gene of a PRV strain CF was constructed. The expressed protein contained 134 amino acids of gE protein (amino acids 77-210) fused to a 19-amino acids tag containing 6 histidine residues. After induction, a truncated PRV-gE polypeptide of 18-kd was expressed to about 20% of the total E coli proteins. Results of immunoblot analysis indicated that this E coli-produced PRV-gE protein reacted specifically with serum from PRv-hyperimmunized pigs and from field PRv-infected pigs, but not with serum samples from specific-pathogen-free pigs or pigs inoculated with gE-deleted PRV vaccine. These data indicate that, although the recombinant gE protein is produced in E coli, it still retains the antigenicity of the viral gE glycoprotein. Comparison between the recombinant gE protein, using immunoblot analysis with a commercial gE ELISA containing natural PRV-gE protein, revealed comparable test performance. This finding indicated that recombinant gE protein produced by E coli can be used for development of a companion serologic assay for a PRV-gE gene-deleted vaccine.

Pregnant sows, immune against pseudorabies after vaccination, were inoculated at 70 days of gestation either with autologous blood mononuclear cells that had been infected in vitro with pseudorabies virus (PRV) or with cell-free PRV. The infected cells or cell-free PRV were inoculated surgically into the arteria uterina. Eight sows (A to H) had been vaccinated with an inactivated vaccine. The titer of seroneutralizing antibodies in their serum varied between 12 and 48. Five sows (A to E) were inoculated with autologous mononuclear cells, infected either with a Belgian PRV field strain or with the Northern Ireland PRV strain NIA. These 5 sows aborted their fetuses: 2 of them (B and C) 3 days after inoculation, and the other 3 (A, D, and E) 10, 11, and 12 days after inoculation, respectively. Sows F, G, and H were inoculated with a cell-free PRV field strain. They farrowed healthy Utters after normal gestation. Neutralizing antibodies were absent against PRV in the sera of the newborn pigs, which were obtained prior to the uptake of colostrum. The 23 fetuses that were aborted in sows B and C 3 days after the inoculation were homogeneous in appearance and size. Foci of necrosis were not detected in the liver. Viral antigens were located by immunofluorescence in individual cells in lungs, liver, and spleen of 15 fetuses. Virus was isolated from the liver, lungs, or body fluids of 12 fetuses. The 39 fetuses that were aborted in sows A, D, and E between 10 and 12 days after inoculation were of 2 types: 17 were mummified and 22 were normal-appearing. Foci of necrosis were found in the liver of all mummified fetuses and 13 of the normal-appearing fetuses. In fetuses with foci of necrosis in the liver, viral antigens were located in groups of cells in the liver, lungs, and spleen. Virus was isolated from 16 normal-appearing fetuses and from 11 mummified fetuses. Pseudorabies virus was isolated from vaginal excretions of sows A and D until 1 and 2 days after abortion, respectively, and of sows B and C until 4 and 5 days after abortion, respectively. Virus was not isolated from sow E. It was concluded that PRV can reach the uterine and fetal tissues, via infected mononuclear cells, in the presence of circulating antibodies induced on vaccination. This cell-associated spread led to abortion. Cell-free virus did not induce abortion under similar circumstances.

A fully age-structured deterministic model of the population biology of a pseudorabies epizootic in a farrow-to-finish operation was used to examine the disease-related change in productivity following the initial disease episode. A strategy involving continual sow vaccination was compared with various strategies involving the vaccination of growing pigs, as well as sows. The model suggests that vaccinating growing pigs, in addition to the breeding herd, results in only a relatively small improvement in long-term productivity following a pseudorabies epizootic.

A deterministic mathematical model of the population biology of pseudorabies in swine was used to clarify some of the basic features of the host-virus relationship and to inquire into the circumstances that promote or impede virus persistence in a single herd. When the basic reproductive rate of the infection (ie, the number of secondary infections resulting from the introduction of a single infective animal into a wholly susceptible herd) is greater than unity, the model suggests that the number of infective individuals in the herd will undergo highly damped oscillations to a final equilibrium level. The most important determinants of virus persistence are herd size and the density at which sows are maintained. There is a threshold density of susceptible individuals below which the virus will eventually be eliminated from the herd, even when specific control measures are lacking. Test and removal strategies hasten virus elimination when herd density is already below threshold, but are otherwise likely to succeed only when the removal of latent infections reduces the basic reproductive rate of the infection below unity. Vaccination strategies may also result in virus elimination, but only in relatively small herds.

Pseudorabies is a porcine herpesvirus of major importance in the swine industry. Isoprinosine is an immunomodulating drug that has been shown to be beneficial in treating herpesvirus infections. Twenty-four 7-week-old pigs were allotted within litters to 1 of 4 groups: control, isoprinosine (ISO), pseudorabies virus (PRV), or isoprinosine and pseudorabies virus (ISO-PRV). Isoprinosine was administered daily for 16 days to the ISO and ISO-PRV groups (75 mg/kg of body weight/day, PO). Immunity in pigs in the PRV and ISO-PRV groups was challenged with pseudorabies virus (10(5) TCID50 units) on day 4. Rectal temperatures and viral excretion were monitored daily; total and differential leukocyte counts, lymphocyte response to mitogens, and interleukin-2 production were monitored every 4 days. Pigs challenge-inoculated with pseudorabies virus became ill, with the ISO-PRV group most severely affected. Rectal temperatures were high (P less than 0.05) in virally challenged pigs on days 5 to 12 and 14 to 16; isoprinosine did not alter this effect. Pseudorabies virus-infected pigs had leukocytosis (P less than 0.05) on days 12 and 16, primarily caused by neutrophilia. Concanavalin A-stimulated lymphocyte proliferation was decreased (P less than 0.06) in both PRV and ISO-PRV groups on day 12, compared with control pigs, but only in the PRV group on day 16. Pokeweed mitogen-stimulated lymphocyte proliferation was decreased (P less than 0.02) in ISO-PRV pigs on day 8 of the experiment. Interleukin-2 concentrations, pooled over all sampling days, were decreased (P less than 0.03) in pseudorabies virus-infected pigs. Viral excretion was not altered by isoprinosine treatment. These data suggest that pseudorabies virus infection decreased lymphocyte proliferative responses and interleukin-2 prodcution in pigs, and that isoprinosine did not mitigate these effects.

To determine the sites of replication and the evolution of pseudorabies virus infection in boar genital organs, 5 Belgian Landrace boars were inoculated with pseudorabies virus unilaterally in the cavum vaginale of the testis. Virus replication took place only in cells of the tunica vaginalis of both cava vaginalia. Infection of the serosa led to exudative periorchitis and increased scrotal fluid, resulting in a severely swollen scrotal region. These experimental findings were similar to findings in boars with naturally aquired pseudorabies virus infection. Scrotal fluid contained large amounts of virus, making it a useful specimen for diagnosis of the disease in affected boars.

The antibody response to pseudorabies virus nucleocapsid proteins (NCP) was evaluated by the western immunoblot analysis before and after challenge of immunity by nasal inoculation of 10(2.3) plaque-forming units of virus in 10 pigs that had been vaccinated with pseudorabies virus envelope glycoproteins. Antibody to 5 NCP with molecular mass of 140, 63, 41, 34, and 23 kD was first detected in vaccinated and nonvaccinated pigs on day 14 after challenge of immunity. Antibody to 2 of the 5 NCP continued to be detected through day 113 in 9 of 10 vaccinated pigs. Beyond day 32, antibody to NCP was not detected in 1 vaccinated pig. The 23-, 34-, and 41-kD proteins were the most immunogenic. Antibody to each of these proteins was first detected on day 14 in 10, 10, and 8 pigs, respectively. Seven, 6, and 8 pigs, respectively, were antibody-positive for these proteins on day 113. The 140- and 63-kD proteins were the least immunogenic. Antibody to these proteins was detected in 8 and 9 pigs, respectively, on day 14, and in 4 and 5 pigs, respectively, on day 113. Chi-square analysis for dependency indicated that the antibody response to the 140- and 63-kD proteins was interdependent. These results suggested that combinations of NCP may be useful as non-vaccine diagnostic antigens.

Hybridomas were selected for secretion of monoclonal antibodies directed against pseudorabies virus (PRV) glycoproteins. Each monoclonal antibody was capable of neutralizing PRV in vitro in the presence of complement. This panel of antibodies was used in passive immunization studies to protect mice and swine from PRV-induced mortality. The most protective antibody in mice was 3A4, specific for PRV glycoprotein gp50, which afforded as high as 100% protection. Although antibody 3A4 was partially protective in swine, antibody 3D11, which is specific for PRV glycoprotein gIII, afforded greater protection-83% protection when ascitic fluid was used and 100% protection when immunoglobulin concentrated from cell cultures was used at a dose of 150 mg/pig. These studies demonstrated that monoclonal antibodies may be useful for short-term prophylaxis against PRV-induced disease and that antibody directed against either PRV gylcoprotein gIII or gp50 is sufficient to protect animals from PRV-induced mortality.

A field trial was conducted on a commercial swine farm quarantined because of infection with pseudorabies virus. The purpose was to investigate, in growing pigs born to hyperimmunized sows, the immunogenicity of a vaccine with a glycoprotein I (gE) deletion. One hundred twenty pigs were assigned at random to 1 of 3 vaccination schedules at ages: 8 and 12 weeks; 8, 12, and 14 weeks; and 8, 12, and 16 weeks. Immune response was measured at 8, 12, 14, 16, and 18 weeks, using the serum neutralization test, a screening ELISA, and assays of IgG and IgA in serum and nasal secretions. Results of the serum neutralization test and the screening ELISA indicated that, for pigs vaccinated only at 8 and 12 weeks, the percentage of pigs with pseudorabies virus serum antibodies decreased substantially by 18 weeks; for pigs given a booster at 14 or 16 weeks, the prevalence of serum antibodies at 18 weeks was higher, with 16-week booster vaccination eliciting the best response. At each age, nasal IgA and IgG values were highly correlated (r greater than or equal to 0.70), as were serum IgA and IgG values; correlations of serum with nasal IgA and IgG values were somewhat lower (approx range, r = 0.40 to 0.70). Nevertheless, an increase in serum IgA or IgG values on vaccination was no guarantee of an increase in nasal IgA or IgG values. For serum and nasal mucosal antibodies, a poor immune response was associated with high quantities of maternally derived antibodies. Vaccination at 16 weeks was necessary to ensure eliciting of an immune response in almost all pigs.

Whereas the clinical efficacy of vaccination against pseudorabies has been studied extensively, methods to evaluate the influence of vaccination on pseudorabies virus (PRV) transmission have only recently become available. In this study, PRV transmission and growth performance in finishing pigs vaccinated either once or twice were compared. The incidence of PRV infections was significantly (P = 0.039) higher in the group vaccinated once (38%) than in the group vaccinated twice (10%). The reproduction ratio R, which is defined as the average number of new infections caused by 1 infectious individual, was estimated in both groups. This ratio was also significantly (P = 0.025) higher among single vaccinated pigs (R = 3.4) than among pigs that had received double vaccination (R = 1.5). In compartments where serologic evidence of PRV introduction was observed, the mean daily weight gain was significantly (P = 0.029) lower in pigs vaccinated once (698 g/d) than in pigs vaccinated twice (721 g/d). Results of this study document the possibility to objectively evaluate the effect of vaccination on PRV transmission under field conditions. From the results, we concluded that double vaccination is advantageous in populations of finishing pigs at risk for PRV introduction. However, even among pigs vaccinated twice, extensive spread of PRV can occur.