Safety of an Aujeszky's disease vaccine based on deletion mutant strain 783 which does not express thymidine kinase and glycoprotein I

The safety of an Aujeszky's disease virus vaccine based on strain 783, a deletion mutant which does not express glycoprotein I and thymidine kinase, was assessed in pigs, calves and sheep. Four-day-old piglets which were inoculated intranasally and intramuscularly with 10(7) plaque forming units (PFU) developed only slight depression and fever. The virus was transmitted to a sentinel piglet. Six weeks after inoculation, the pigs were injected with high doses of corticosteroids in an attempt to reactivate the vaccine virus. The pigs did not shed Aujeszky's disease virus, did not develop a rise in virus neutralising antibody titres and sentinel pigs remained seronegative to Aujeszky's disease virus. Strain 783 was passaged in two series of three- to five-day old piglets, but after the third and fourth passages virus could no longer be recovered. Pregnant sows were inoculated with 10(7) PFU of virus strain 783 around day 35 or on day 85 of pregnancy, and their fetuses and piglets were assayed for Aujeszky's disease virus and antibodies against Aujeszky's disease virus. No evidence was found for transplacental transmission of the virus. Calves and sheep were given 10(7) PFU of virus strain 783 intranasally or intramuscularly; they survived and did not develop clinical signs of Aujeszky's disease. All the sheep and the calves inoculated intramuscularly developed neutralising antibodies to Aujeszky's disease virus.     

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.     

Intranasal vaccination of pigs against Aujeszky's disease: protective immunity at 2 weeks, 2 months and 4 months after vaccination in pigs with maternal antibodies.

The purpose of the study was to evaluate the short- and long-term immunity after intranasal vaccination in pigs with maternally derived antibodies (MDA). In two experiments, 10-week-old pigs with moderate MDA titres against Aujeszky's disease virus (ADV) were vaccinated intranasally with the Bartha strain of ADV to evaluate the protective immunity conferred at 2 weeks, 2 months and 4 months after vaccination. Protection was evaluated on the basis of severity of clinical signs, periods of fever and growth arrest, and duration and amount of virus excreted after challenge with a virulent ADV. During the first 2-3 weeks after vaccination, antibodies to ADV continued to decline as in unvaccinated control pigs. After that, antibody titres stabilized or gradually increased. At 2 weeks, 2 months and 4 months after vaccination, vaccinated pigs were significantly better protected than unvaccinated controls. The vaccinated pigs challenged 2 weeks after vaccination hardly developed any sign of disease. Mild signs of Aujeszky's disease and a growth arrest period of 5 days were observed in vaccinated pigs challenged 2 months after vaccination, whereas vaccinated pigs challenged 4 months after vaccination developed severe signs of disease and a growth arrest period of 13 days. Vaccinated pigs challenged 2 weeks after vaccination did not excrete challenge virus, and pigs challenged 2 or 4 months after vaccination excreted far less virus than unvaccinated controls. The results demonstrate that intranasal ADV vaccination of pigs with moderate MDA titres protected them from 2 weeks to at least 4 months after vaccination. Immunity steadily declined, however, after vaccination.     

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.     

Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky's disease vaccines

Different deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion optimally upon infection. Groups of pigs were vaccinated once with attenuated deleted Aujeszky's disease vaccine (gI, gX or gp63 negative), suspended in phosphate buffered saline. Two additional groups were vaccinated with a gI deleted vaccine virus suspended in an oil-in-water emulsion. Other groups were vaccinated twice with gI deleted inactivated vaccines. The three control groups included were: pigs immune after infection, unvaccinated pigs and pigs receiving vaccine without known deletion in the envelope. Experimental challenge took place 3 or 4 weeks after the only or the last vaccination. The number of excreting pigs, the duration of excretion and the virus titers excreted, were determined for all the groups. All the pigs vaccinated with glycoprotein deletion vaccines suspended in phosphate buffered saline, excreted virus for 2 to 6 days after challenge. A 100 to 1000 fold reduction in excreted virus titers was obtained in vaccinated pigs compared to unvaccinated ones. Some vaccines suppressed virus excretion better than others, but no correlation could be made between the type of deletion (gI, gX or gp63) and the degree of reduction in virus excretion. Similar results were obtained with two applications of inactivated vaccines. The lowest number of excreting pigs, the lowest duration of excretion and the lowest titers were obtained in groups vaccinated with the attenuated vaccine suspended in an oil-in-water emulsion. No vaccine suppressed virus excretion totally.     

A novel concept for the control of Aujeszky's disease: experiences in two vaccinated pig herds

A study was conducted to examine the usefulness of a glycoprotein I (gI)-ELISA to monitor Aujeszky's disease virus infection in two vaccinated pig herds; the gI-ELISA can differentiate between pigs infected with Aujeszky's disease virus and pigs vaccinated against Aujeszky's disease with gI-negative vaccines. The two herds had been vaccinated with gI-negative vaccines for several years. The first survey, in September 1986, revealed that approximately 10 per cent of the breeding pigs in a large multiplier herd were seropositive for antibodies to gI of Aujeszky's disease virus, and it was decided to try to eliminate the virus from the herd by gI-ELISA testing and culling of gI-seropositive pigs. A one month quarantine period for incoming stock was established, and only gI-seronegative pigs were admitted to the herd. After two rounds of testing and culling the herd appeared to be free of wild-type Aujeszky's disease virus, and neither Aujeszky's disease virus nor antibodies could be detected either in 21 sentinel pigs placed on the farm or in 347 stillborn piglets or piglets that died shortly after birth. The herd probably remained free of Aujeszky's disease virus until the end of the 27-month period of monitoring except for two of 639 breeding pigs that were unexpectedly found to be positive in the gI-ELISA in November 1987. These sows were culled. A second breeding herd was monitored for antibodies to gI of Aujeszky's disease virus for two years. The gI-seropositive sows constituted approximately 30 per cent of the herd's breeding pigs, but they were not culled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody response to glycoprotein I in maternally immune pigs exposed to a mildly virulent strain of pseudorabies virus.

To study the antibody response to glycoprotein I (gI) of pseudorabies virus (PRV) in maternally immune pigs, 3 groups of 6 pigs were given low doses of the mildly virulent Sterksel strain of PRV at 3 and 11 weeks of age. Group A consisted of seronegative pigs; groups B and C consisted of pigs with maternal antibodies deficient of antibodies to gI. At 3 weeks of age, 3 pigs of each group were inoculated intranasally with 10(2.5) plaque-forming units (groups A and B), or with 10(3.5) plaque-forming units (group C) of PRV. The 3 other pigs in each group were contact-exposed to the inoculated pigs. In group A, 4 of 6 pigs shed virus and all developed antibodies to gI of PRV and produced PRV-specific IgM and virus-neutralizing antibodies. In groups B and C, 10 pigs shed virus and all developed low and inconsistent titers of gI antibodies, whereas only 3 pigs produced PRV-IgM antibodies with low titers. Thus, after PRV infection of pigs with high concentrations of maternal antibodies deficient of gI antibodies, the antibody responses to PRV were severely inhibited. The pigs were reinoculated with 10(3) plaque-forming units of the same virus 8 weeks after the first inoculation. The pigs in group A did not respond at all, as they were immune. The pigs in groups B and C shed considerable amounts of virus. Three pigs had a clear secondary antibody response to gI, whereas the others developed an early to normal antibody response to gI. None of the pigs mounted a secondary neutralizing antibody response to PRV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intranasal vaccination of pigs against Aujeszky's disease: comparison with one or two doses of attenuated vaccines in pigs with high maternal antibody titres

Ten-week-old pigs with high levels of maternally derived antibody (MDA) against Aujeszky's disease virus (ADV) were given either a single intranasal vaccination or one or two doses (with an interval of three weeks) of commercially available attenuated ADV vaccines intramuscularly. The pigs did not produce a clear neutralising antibody response to ADV. However, pigs vaccinated intranasally and pigs given two doses of attenuated ADV vaccines were protected against intranasal challenge with virulent ADV two months after the first vaccination. Pigs given one parenteral dose of attenuated ADV vaccine were insufficiently protected. Protection was shown by shorter periods of growth arrest and fever and a greater reduction of virulent virus shedding after challenge in vaccinated pigs than in unvaccinated control pigs. Although intranasal vaccination conferred protection comparable to two parenteral doses of attenuated vaccines, it reduced shedding of virulent virus much more effectively. These results, together with those of other studies, show that intranasal vaccination confers better protection against Aujeszky's disease in pigs with MDA than parenteral vaccination. However, the efficacy of intranasal vaccination also decreases with increasing levels of MDA at the time of vaccination.     

The porcine humoral response to detergent extracted Aujeszky's disease (pseudorabies) virus antigens

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.     

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.     

A comparison of the efficacy of two commercial Aujeszky's disease vaccines with glycoprotein-I deletion in pigs

Two commercial Aujeszky's disease vaccines, a modified killed vaccine and a sub-unit vaccine, both carrying a deletion of glycoprotein-I, were evaluated in pigs. Each vaccine was administered to two groups of four pigs, twice at 4-week intervals, with two pigs held as unvaccinated controls. All pigs were challenged with a New Zealand field isolate of Aujeszky's disease virus 3 weeks after the second vaccination. The results indicate that the sub-unit vaccine was able to protect pigs against clinical Aujeszky's disease much better than the pigs vaccinated with the modified killed vaccine when challenged with a virulent virus. However, the amount and the duration of virulent virus excretion following challenge was greater with the sub-unit vaccine than the modified killed vaccine. Pigs vaccinated with the sub-unit vaccine were shown to be latently infected following challenge. Latent infection was demonstrated by excretion of Aujeszky's disease virus from the nasal cavity after dexamethasone treatment and seroconversion of a sentinel in contact pigs to Aujeszky's disease virus.     

Aujeszky's disease in a cow

A non-suppurative encephalitis accompanied by intraneuronal intranuclear inclusions were observed in the brain from a cow that died within 10 hours of developing nervous signs. Immunogold-silver staining located Aujeszky's disease virus antigen in neuronal cytoplasm and the virus was isolated from large volumes of suspensions of nervous tissues and tonsils. Fattening pigs in adjacent buildings had high antibody titres to Aujeszky's disease virus. The methods by which the cow could have acquired infection are considered, and the significance of transient low titres of antibodies to Aujeszky's disease virus in in-contact cows is discussed.     

Marker vaccines, virus protein-specific antibody assays and the control of Aujeszky's disease

Vaccination of pigs is widely practised to control Aujeszky's disease (AD). Molecular biological research revealed that several conventionally attenuated virus vaccines harbour deletions in their genomes. The deleted genes are nonessential for virus replication and can be involved in the expression of virulence. These findings have prompted several groups to construct well-characterized deletion mutants of AD virus that do not express either glycoprotein gI, gX or gIII. These mutants have also been rendered thymidine kinase negative. Although data on vaccine efficacy and safety have been published, widely varying test conditions have made it impossible to identify the most efficacious deletion mutant vaccine(s). Vaccination enhances the amount of virus required for infection and reduces, but does not prevent, the shedding of virulent virus and the establishment of latency in pigs infected with virulent AD virus. Therefore, while a vaccination programme will reduce the circulation of virus in the field, it will not eliminate AD virus from pig populations. To eradicate AD, the ability to differentiate infected from vaccinated pigs is crucial. The use of marker vaccines enables us to identify infected pigs in vaccinated populations by detecting antibodies against the protein whose gene is deleted from vaccine strains. The antibody response to gI appears to persist for more than 2 years, and all of about 300 field strains tested so far express gI. The use of vaccines lacking gI in combination with an enzyme linked immunosorbent assay to detect antibodies to gI and culling of gI-seropositive pigs, may help to eradicate AD in countries where vaccination is widely practised.     

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.     

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.     

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.     

伪狂犬病基因缺失疫苗株(SA215)某些生物学特性研究

本试验测定了伪狂犬病ｇＥ－／ｇＩ－／ ＴＫ－／ ＬａｃＺ＋基因缺失疫苗株（ＳＡ２１５）的致细胞病变效应、安全性、免疫原性和免疫期等生物学特性。试验结果显示,该疫苗株能在Ｖｅｒｏ细胞上适应生长,并形成典型的蚀斑。其对１日龄仔猪、怀孕母猪、牛、羊以及家兔安全,无不良接种反应,接种动物不向体外散毒。ＳＡ２１５疫苗接种猪能抵御高剂量（１０７ＰＦＵ）Ｆａ株强毒感染,攻毒后试验猪的发热期、增重受阻天数、散毒滴度均低于Ｂａｒｔｈａ株疫苗接种猪,远远低于对照组猪。ＳＡ２１５接种猪能维持长时间的高水平中和抗体滴度,免疫期可达半年以上。试验结果表明,ＳＡ２１５株是一株安全、免疫原性好、免疫期长的疫苗株。     

Detection of Aujeszky's disease virus in nasal cells of fattening pigs by immunoassay

Both conventional and specific pathogen free pigs were inoculated intranasally with a strain of Aujeszky's disease virus (ADV). Nasal cells were collected daily by swab, aspiration or wash. The nasal cells were examined for ADV by isolation on cell culture, direct or indirect immunofluorescence and immunoperoxidase staining by monoclonal antibodies. The infected pigs were studied for nasal shedding of infected cells until 30 days after infection. The study was also extended to naturally infected farm pigs. Swabbing, washing and aspiration proved effective methods of collecting between 10(5) and 10(8) pavement or columnar epithelial cells and non-epithelial cells. Macrophages and polymorphonuclear leucocytes were also identified. Infected nasal cells were detected by immunofluorescence and immunoperoxidase from one to 21 days after infection. The viral antigen was detected in both epithelial and non-epithelial cells, the fluorescence was nuclear and, or, 'cytoplasmic', in the latter case only the cell membrane was stained. ADV antigens were detected in nasal cavity cells in pigs infected with a virulent and a hypovirulent strain. Nasal swabs proved effective in confirming infection both by virus isolation and immunological assay, and the latter was shown to be a useful experimental tool for the rapid diagnosis of Aujeszky's disease virus infection in fattening pigs suffering from acute respiratory distress.     

Protection of fattening pigs against challenge with Aujeszky's disease virus after a successive intranasal/intramuscular vaccination.

In this study, the efficacy of successive intranasal (i.n.)/intramuscular (i.m.) vaccination against Aujeszky's disease virus (ADV) was assessed in experimental pigs. The double deletion-mutant Kaplan gE-gl- was used for i.n. vaccination at 4 weeks of age and the commercially available Bartha strain, suspended in an oil-in-water emulsion, was used for the i.m. booster vaccination at 10 weeks of age. Efficacy was compared with that of the traditional double i.m. vaccination with the commercially available Bartha strain at 10 and 14 weeks of age by challenging the pigs at the end of the fattening period. There were significant differences in clinical signs, mean daily weight gain, and virus excretion between the vaccinated groups and the unvaccinated controls; however, the differences between the vaccinated groups were not statistically significant.     

Studies on the ability of a 98-kilodalton pseudorabies virus diagnostic antigen to detect latent infections induced by low-dose exposure to the virus

The effect of low-dose challenge of immunity with pseudorabies virus (PRV) on subunit-vaccinated pigs was studied in 2 experiments. In the first experiment, we studied the effect of challenge dose on the antibody response to an early excreted 98-kilodalton PRV-glycoprotein that was used as a diagnostic antigen in the ELISA. In the second experiment, we studied the effect of low doses of virus on the establishment of latent infections in subunit-vaccinated pigs. The relationship of virus exposure dose and vaccine dose to the response of pigs to diagnostic antigen was studied in 18 pigs. Two groups of 3 pigs were vaccinated with a total of 200 micrograms of a lectin-derived PRV subunit vaccine over a 5-week period. Two groups of 3 pigs were similarly vaccinated with a total of 100 micrograms. Two groups of 3 pigs served as nonvaccinated controls. One group of pigs from each of the preceding categories was intranasally exposed to 10(6.0) and 10(2.7) plaque-forming units (PFU) of virus. Antibody to diagnostic antigen was detected by the ELISA and radioimmunoprecipitation 3 to 7 days earlier in pigs exposed to 10(6.0) PFU, demonstrating that the size of the virus challenge dose affects the antibody response to diagnostic antigen. The establishment of latent infections by low PRV doses and the ability to detect these infections was studied in 10 subunit-vaccinated pigs. Each pig was intranasally exposed to 10(2.3) PFU of virus (day 0).(ABSTRACT TRUNCATED AT 250 WORDS)