Tonsillar changes in pigs given pseudorabies (Aujeszky's disease) virus

All of the eight 5-day-old pigs orally given pseudorabies (Aujeszky's disease) virus developed tonsillitis. The initial changes occurred in the subepithelial area between the lymphoid nodule and the crypt epithelium, showing a characteristic pattern of necrosis. The necrosis became more severe and gained access into the lymphoid nodule and crypt epithelium. Coincident with the histopathologic changes, numerous specific immunofluorescences were detected, first in the nucleus and in some parts of the cytoplasm of cells distributed in the subepithelial area. The fluorescence subsequently spread into adjacent lymphoid nodules and crypt epithelial cells. Ultrastructurally, many enveloped virus particles were detected in the center of the necrosis. Thereafter, the crypt epithelial cells also underwent degeneration, and a small number of virus particles were detected in the nucleus of the degenerating epithelial cells. In the more advanced stage, the enveloped virus particles were discharged into the crypt lumen.     

Air sampling procedure for evaluation of viral excretion level by vaccinated pigs infected with Aujeszky's disease (pseudorabies) virus.

Five groups of eight fattening pigs were vaccinated and then infected with Aujeszky's disease virus. Viral excretion was evaluated by two means: deep nasal swabbing and air sampling. It appeared that infectious airborne virus could be recovered from day 1 to day 6 after infection in the isolated units where control animals were raised. In vaccinated animals, airborne particles were also detected but the amount and duration varied in relation to their immune status at the day of virulent challenge: viral excretion was significantly lower in pigs presenting a high antibody level (1/16 to 1/64) just before infection. Results obtained with nasal swabs and with air samples were closely related. Despite its low sensitivity, the air sampling procedure could be considered as an efficient tool for reflecting infectious viral pressure in a confined atmosphere.     

Reactivation of latent pseudorabies virus infection in vaccinated commercial sows

Pseudorabies virus (PRV) was isolated from 9 of 44 PRV-vaccinated seropositive sows on 5 of 11 farms. Although serum-neutralization antibody titers were 1:16 to 1:256, 28 virus isolates were obtained from tonsil, nasal, or buccal swab samples from 9 sows given 2 ml of dexamethasone/kg of body weight IM for 5 days. Pseudorabies virus was isolated from 6 of 20 sows (3 of 5 farms) given a killed-virus vaccination. Virus was obtained from 3 of 24 sows (2 of 6 farms) given modified-live virus and killed-virus vaccination. Evaluation of the 9 PRV with 5 restriction endonucleases revealed 4 PRV existing genotypes. The 9 isolated types of PRV appeared to be indistinguishable by Kpn I and BamHI restriction endonuclease analysis; however, when analyzed with Sal I, HinfI, and Pst I, isolates 7 (farm D), 8 (farm C), and 9 (farm B) had numerous differences. Isolates 1, 2, 3, and 4 (farm F) and 5 and 6 (farm G) appeared to be the same genotype when further analyzed with Pst I, HinfI, and Sal I.     

Molecular biology of pseudorabies (Aujeszky's disease) virus.

In this review, some of the aspects concerning the molecular biology of pseudorabies virus (PrV), the causative agent of Aujeszky's disease, will be discussed. It will mainly focus on new findings concerning viral glycoproteins, factors determining PrV virulence, the problem of PrV latency and the development regarding genetically engineered vaccines.     

Experimental infection of fattening pigs with pseudorabies (Aujeszky's disease) virus: efficacy of attenuated live- and inactivated-virus vaccines in pigs with or without passive immunity

The efficacies of attenuated live- and inactivated-virus vaccines against pseudorabies (PR) in fattening pigs were compared. Pigs born from vaccinated or nonvaccinated sows were vaccinated with one or the other vaccine and were challenge exposed at the end of the fattening period. The particular form of PR observed in fattening units in the field could be reproduced. A marked difference was seen between the control lot and the lots of the pigs vaccinated with the attenuated live- and inactivated-virus vaccines. The protection was real, but not absolute, in the vaccinated pigs. The inactivated-virus vaccine conferred a strong passive immunity to the young pigs of vaccinated dams which interfered with the development of an active immunity. The titer of the colostral antibodies in the sera of pigs born from the sows vaccinated with the attenuated-live virus vaccine was low and decreased rapidly. The active protection obtained with this vaccine was similar to that observed with the inactivated-virus vaccine. Thermal curves, weight losses, and time necessary for recovering the weight of pigs at the time of challenge exposure seemed to be good criteria for measuring the protection of fattening pigs against this particular form of PR. The conditions of the outcome of respiratory tract disorders in these pigs are discussed.     

Sensitive glycoprotein gIII blocking ELISA to distinguish between pseudorabies (Aujeszky's disease)-infected and vaccinated pigs.

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an anti-PRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of greater than 0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera). Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII+ vaccines were positive for gIII antibodies (S/N less than 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII+ antibodies 7-10 days postinfection. Sera from pigs vaccinated with gpX- and gI- vaccines seroconverted to gIII+ antibodies 7-8 days after vaccination. The gIII antibodies persisted after gIII+ vaccinated for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII+ antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on the United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays.     

Aujeszky's disease (pseudorabies) virus detection in cerebrospinal fluid in experimentally infected pigs

The presence of Aujeszky's disease virus in cerebrospinal fluid of experimentally infected pigs was studied using the techniques of virus isolation and PCR. Pigs, some of which were previously vaccinated against Aujeszky's disease, were inoculated with different doses of the Aujeszky's disease NIA-3 strain. At the time of death or sacrifice, a sample of cerebrospinal fluid was taken and tested for the presence of virus using the mentioned techniques. Virus was isolated only from one sample, while it was detected by PCR in most of them. The higher sensitivity of the PCR technique and the possible presence of antiviral antibodies in the cerebrospinal fluid are reasons that can be argued to explain this fact. By PCR, the virus was detected more efficiently when digested cerebrospinal fluid cells were used as DNA source than when using whole cerebrospinal fluid, suggesting that the virus could be cell-associated. Aujeszky's disease virus could not be detected by PCR in pigs which survived the acute phase of the infection and were euthanased at 8 weeks post-inoculation, when they were latently infected. This indicated that the cerebrospinal fluid is not an adequate sample for the diagnosis of latency. Since Aujeszky's disease virus was detected from most of the tested samples, we believe that this could be an adequate procedure for the quick diagnosis of Aujeszky's disease.     

Abortion induced by cell-associated pseudorabies virus in vaccinated sows.

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 NIA3. 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 litters 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional antibody responses in pigs vaccinated with live and inactivated Aujeszky's disease virus

Functional antibody tests, including virus neutralising activity of serum, antibody dependent cellular cytotoxicity and complement mediated lysis, were used to measure the response of pigs given either live or inactivated Aujeszky's disease virus vaccines. Pigs were then challenged with virulent Aujeszky's disease virus and antibody responses were analysed and found not to correlate with protection. Reasons for this lack of correlation are discussed and it is suggested that these results indicate that more emphasis should be placed on measuring the local immune response.     

Prevalence of herds with young sows seropositive to pseudorabies (Aujeszky's disease) in northern Belgium

In Belgium, pseudorabies in swine has been the subject of a mandatory eradication programme since 1993. From December 1995 to February 1996, a survey was conducted in the five provinces of northern Belgium to estimate the provincial pseudorabies virus (PRV) herd seroprevalence. Seven hundred and twenty randomly selected herds were included in this survey. To detect recently infected animals, only young sows were sampled. The results show that 44% of these herds had an important number of PRV-seropositive young sows. The highest herd seroprevalence was observed in West Flanders (68%), followed by Antwerp (60%), East Flanders (43%), Limburg (18%), and Flemish Brabant (8%). Assuming a diagnostic test sensitivity and specificity of 95% and 99%, respectively, and a true PRV within-herd prevalence of 43%, the overall true PRV herd prevalence was estimated to be 35%. A logistic multiple-regression revealed that the presence of finishing pigs was associated with a two-fold increase in odds of a herd being seropositive (odds ratio (OR)=2.07, 95% confidence interval (CI)=1.31–3.26); a breeding herd size ≥70 sows was associated with a four-fold increase in odds of a herd being seropositive (OR=4.09, 95% CI=2.18–7.67); a pig density in the municipality of ≥455 pigs/km² was associated with a 10-fold increase in odds of a herd being seropositive (OR=9.68, 95% CI=5.17–18.12). No association was detected between the PRV herd seroprevalence and purchase policy of breeding pigs (purchased gilts, or use of homebred gilts only).     

Rapid detection of Aujeszky's disease (pseudorabies) virus infection of pigs by direct filter hybridisation of nasal and tonsillar specimens

A direct filter hybridisation method has been developed to diagnose acute Aujeszky's disease in live pigs. The advantages of the method are easy, fast sample processing; no DNA-purification is needed, and the hybridisation itself is simplified. The direct filter hybridisation method has been tested on pseudorabies virus infected cultured cells, experimentally infected pigs and on specimens from an outbreak of Aujeszky's disease. Virus isolation and filter hybridisation gave comparable results, indicating that the direct filter hybridisation method is a good tool for rapid diagnosis. It is independent of cell culture facilities and the disease can be diagnosed in live animals within 15 hours.     

Antigens involved in vaccination of swine against Aujeszky's disease (pseudorabies) virus.

The polypeptide and glycopolypeptide composition of a local virulent Aujeszky's disease virus (suid herpesvirus 1, SHV-1) strain (E-974) was determined in order to characterize the individual SHV-1 antigens inducing the serological responses in immunized and non-immunized animals. A commercially available inactivated vaccine of known efficacy and three experimental immunogen preparations (whole inactivated SHV-1 particles, lectin-purified glycoproteins from SHV-1 culture, and a combination of both) were used for immunization. Sera of two-month old immunized and non-immunized animals were analyzed by ELISA, seroneutralization and Western immunoblotting prior to and following challenge with E-974. Sera of 7- to 30-day-old piglets littered by immunized and non-immunized sows were likewise analyzed by immunoblotting. The following variables were determined: the total level of anti-SHV-1 antibodies, the level of neutralizing antibodies, the IgG responses to individual SHV-1 antigens, and the clinical parameters and degree of protection of the animals. The whole-particle experimental immunogen conferred greatest protection, but correlation between antibody levels and the degree of protection was imperfect. Serological responses seemed to be directed against certain structural polypeptides and viral envelope glycoproteins. The glycoprotein immunogen caused a selective response to bands which closely resemble the glycopolypeptides gII and gIII. A 71 kDa component of uncertain location within the viral structure appeared to be one of the main antigens involved in porcine serological response to SHV-1 and colostral protection of piglets.     

In vivo and in vitro reactivation of latent pseudorabies virus in pigs born to vaccinated sows

Latency of pseudorabies virus (PRV) was established in 8 of 9 pigs born to 2 vaccinated sows. Pigs had high, low, or no maternal antibody titers at the time of the initial inoculation. At postinoculation months 3 to 4, latent PRV could be reactivated in vivo by the administration of large doses of corticosteroids. In most pigs, the stress-simulating treatment resulted in recrudescence of virus shedding after lag periods of 4 to 11 days. In 3 pigs, virus shedding was without clinical signs of disease, whereas clinical signs that developed in 4 pigs appeared to be due to the corticosteroid treatment, rather than to the reactivation of PRV. Pigs with a log10 neutralizing antibody titer of less than or equal to 2.55 at the onset of corticosteroid treatment had a booster response. Reactivated PRV spread to sentinel pigs housed with the inoculated pigs. Reactivation of PRV was also demonstrated in vitro. Explant cultures of trigeminal ganglia from pigs killed between postinoculation months 4 to 5 produced infectious virus. Restriction endonuclease analysis indicated that the reactivated PRV was indistinguishable from virus isolated shortly after the primary infection. Seemingly, pigs with maternal antibodies can become latently infected and therefore may be regarded as potential sources of dissemination of PRV.     

Studies of transplacental and perinatal infection with two clones of a single Aujeszky's disease (pseudorabies) virus isolate

The effect of Aujeszky's disease virus (ADV) infection in the last third of gestation was studied using two clones from an ADV isolate. Twelve sows were infected with one or other clone at 85 +/- 1 days of gestation. The dose of infection was 5 X 10(6) TCID50 per sow. The clinical and serological responses to the infection were different. One clone, Ls-1, produces a severe and acute illness. The course of the disease went from 9 to 14 days while the other clone, Ls-2, caused a mild or silent infection and for a shorter time. Transplacental infection occurred in only one sow which had been infected with Ls-2. The litter consisted of 8 mummified fetuses. Sows infected with Ls-1 produced piglets or mummified fetuses that were virus-negative. Perinatal infection was found in litters from both groups. Colostrum-deprived piglets that did not have postpartum contact with the sow, produced specific antibodies, 2 out of 6 born to sows infected with Ls-1 and 1 out of 4 born to sows infected with Ls-2. The antibody titers in colostrum-fed piglets were related to the extent of clinical response of the sows to the infection. Litters born to sows infected with Ls-1 had mean SNT titers from 1/12 to 1/112 while litters born to sows infected with Ls-2 showed titers of 1/2 or less than 2.     

Evidence of long distance airborne transmission of Aujeszky's disease (pseudorabies) virus

Aujeszky's disease has been the subject of an eradication campaign in Denmark since 1980. A detailed knowledge of the virus strains present in the country was provided by restriction fragment analyses of older clinical isolates, and of isolates from all the virologically confirmed outbreaks since 1985. The introduction of foreign strains into southern border areas was demonstrated during the winters of 1984/85, 1986/87 and 1987/88. An epizootic during the winter of 1987/88 was shown to correlate with an unusual predominance of southerly winds. Both conventional and specific pathogen free herds became infected. A herd level case-control analysis of the outbreaks during the winter of 1987/88 revealed that there was a positive correlation between the risk of infection and the size of the herd. The observations support the hypothesis of airborne transmission of the disease.     

Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's disease vaccines.

Seven deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion. For each vaccine, the levels of clinical protection and viral excretion were compared. Groups of eight pigs were vaccinated twice with attenuated deleted Aujeszky's disease vaccines (which do not express certain glycoproteins: gI, gX or gp63). Pigs were vaccinated at the beginning of the fattening period and challenge took place at the end of it when the pigs were 18-19 weeks old. Live virus vaccines were suspended in water or in an oil-in-water emulsion. The experiment was performed in three successive assays of two groups of eight pigs (except three groups for the first assay). At each assay, a control unvaccinated group of eight pigs was added to compare the effects of challenge between vaccinated and unvaccinated animals. In total, 80 pigs were involved in this experiment. All the vaccinated pigs excreted virus from 3 to 9 d after challenge. However the level of viral excretion and the duration of the period of excretion were reduced after vaccination and especially, when oil-in-water emulsion was used. There were obvious differences between vaccines. With some vaccines, when the level of viral excretion was low, the level of clinical protection was high. However, in other cases, the level of clinical protection could be good despite a higher level of viral excretion. The seroneutralizing titres were significantly and inversely related to a low level of viral excretion but not to the level of clinical protection.