Rapid diagnosis of Aujeszky's disease in pigs by immunofluorescence

Direct immunofluorescence on impression smears of brain and pharynx was compared with virus isolation in cell culture for the diagnosis of Aujeszky's disease in experimentally and naturally infected pigs. Pharyngeal impression smears were more sensitive than virus isolation in two pigs killed 10 and 12 days after experimental infection. Both methods were of similar sensitivity in the detection of virus from field cases of disease. Smears of brain and pharynx were more sensitive than virus isolation for tissue which had been stored at room temperature (approximately 20 degrees C) for up to 48 hours. Some reduction in the amounts of virus recovered from tissues and the intensity of fluorescent staining occurred in these samples.     

Serological response of pigs infected with Aujeszky's disease virus

The temporal development of antibody in four groups of pigs infected with different Aujeszky's disease virus isolates was examined. The enzyme-linked immunosorbent assay detected antibody by five to six days after infection and the antibody-dependent cell-mediated cytotoxicity assay detected antibody seven to nine days after infection. Neutralising antibody was first detected nine to 10 days after infection, whereas assays measuring complement mediated antibody lysis did not detect antibody until 10 days after infection. These results are discussed in terms of their importance to the diagnosis of and recovery from Aujeszky's disease.     

Potency of an experimental DNA vaccine against Aujeszky's disease in pigs

Intradermal vaccination with plasmid DNA encoding envelope glycoprotein C (gC) of pseudorabies virus (PrV) conferred protection of pigs against Aujeszky's disease when challenged with strain 75V19, but proved to be inadequate for protection against the highly virulent strain NIA-3. To improve the performance of the DNA vaccine, animals were vaccinated intradermally with a combination of plasmids expressing PrV glycoproteins gB, gC, gD, or gE under control of the major immediate-early promotor/enhancer of human cytomegalovirus. 12.5 microg per plasmid were used per immunization of 5-week old piglets which were injected three times at biweekly intervals. Five out of six animals survived a lethal challenge with strain NIA-3 without exhibiting central nervous signs, whereas all the control animals succumbed to the disease. This result shows the increased protection afforded by administration of the plasmid mixture over vaccination with a gC expressing plasmid alone. A comparative trial was performed using commercially available inactivated and modified-live vaccines and a mixture of plasmids expressing gB, gC, and gD. gE was omitted to conform with current eradication strategies based on gE-deleted vaccines. All six animals vaccinated with the live vaccine survived the lethal NIA-3 challenge without showing severe clinical signs. In contrast, five of six animals immunized with the inactivated vaccine died, as did two non-vaccinated controls. In this test, three of six animals vaccinated with the DNA vaccine survived without severe clinical signs, whereas three succumbed to the disease. Comparing weight reduction and virus excretion, the DNA vaccine also ranged between the inactivated and modified-live vaccines. Thus, administration of DNA constructs expressing different PrV glycoproteins was superior to an adjuvanted inactivated vaccine but less effective than an attenuated live vaccine in protection of pigs against PrV infection. Our data suggest a potential use of DNA vaccination in circumstances which do not allow administration of live attenuated vaccines.     

Horizontal transmission of Aujeszky's disease virus from sheep to pigs

Eight 2-month-old merino lambs were inoculated intranasally with different (10(2.0)-10(5.0)TCID50) amounts of Aujeszky's disease virus (ADV). Electron microscopic studies indicated that ADV replicated in extra-neural sites, in the epithelial cells of the mucosa of the upper and lower respiratory tract. Although the virus was excreted continuously in nasal discharges, horizontal transmission to contact lambs failed. The surviving exposed and contact lambs had no demonstrable antibodies against ADV and they were susceptible when challenged by ADV. However, the virus was transmitted to susceptible pigs in contact with the exposed lambs. One of the five contact pigs showed characteristic clinical signs of Aujeszky's disease, developed a nonsuppurative meningoencephalomyelitis and ADV was recovered from the brain, nasal discharge and other organs. Restriction enzyme analysis of DNA from this virus confirmed the sheep origin of the isolate. The other 4 pigs seroconverted. ADV infection in sheep is therefore a possible source of infection for pigs, but the lack of horizontal transmission in sheep was confirmed.     

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.     

Infection of pigs by Aujeszky's disease virus via the breath of intranasally inoculated pigs

Aujeszky's disease is a worldwide problem in the pig industry. In this experiment, four pigs chosen to act as shedder pigs were intranasally infected with Aujeszky's disease virus. Next, on three consecutive days, eight recipient pigs were exposed to the breath of a pair of shedder pigs via a mask-to-mask module. Except for the virtual absence of CNS signs, shedder pigs expressed clinical signs that were similar to pigs infected naturally or experimentally. Only mild respiratory signs occurred in recipient pigs, but all were infected by aerosols of Aujeszky's disease virus as evidenced by seroconversion. The pig is a much more sensitive indicator of airborne virions than our aerosol collection methods. We conclude that the mild respiratory disease acquired by the aerogenous route in recipient pigs is an easily managed model for studying the transmission of airborne respiratory infections and the immune responses to this type of infection.     

Local humoral and cellular responses in Aujeszky's disease virus infection in pigs

Mucosal and tracheal washings from pigs vaccinated parenterally and intranasally with Aujeszky's disease virus were tested for specific anti-Aujeszky's disease virus responses. Antibody tests included complement dependent antibody lysis, antibody dependent cellular cytotoxicity, virus neutralisation, and anti-Aujeszky's disease virus IgA and IgG levels. There was no correlation between the levels of these antibodies and protection from subsequent challenge. Direct lymphocyte cytotoxicity against cells infected with Aujeszky's disease virus was found in lymph nodes draining the tonsillar area.     

Enzyme-linked immunosorbent assay for detecting antibodies to Aujeszky's disease virus in pigs

An indirect micro enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Aujeszky's disease virus in pigs is described. A control antigen prepared from infected cells was included for each serum tested. Of 243 sera from serologically positive farms, 175 (72 per cent) and 147 (60 per cent) were positive by the ELISA test and microtitre serum neutralisation test, respectively. Failure to include a control antigen for each serum would have resulted in 14 sera (6 per cent) being differently recorded. Results for sera from experimental and field infections indicated that seroconversion was more quickly detected by the ELISA test than the microtitre serum neutralisation test. In addition to greater sensitivity the ELISA test has other advantages over the serum neutralisation test. ELISA is a rapid, cheap test which is not dependent on a continuous supply of cell cultures and which can be readily automated.     

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.