Efficacy of an Intranasal Immunization with gEgC and gEgI Double-deletion Mutants of Aujeszky’s Disease Virus in Maternally Immune Pigs and the Effects of a Successive Intramuscular Booster with Commercial Vaccines

In this study, an intranasal immunization strategy was set up in maternally immune pigs in order to protect them not only clinically but also virologically. Two genetically engineered Aujeszky’s disease virus (ADV) strains, Kaplan gE^?gI^? and Kaplan gE^?gC^?, were used for intranasal immunization. Both strains were safe for 4-week-old pigs. A single intranasal inoculation of 10^6.0 TCID₅₀ of Kaplan gE^?gI^? and Kaplan gE^?gC^? at 4 weeks of age in the presence of moderate titres of maternally derived antibodies (SN titres: 12–16) reduced the amount of weight loss, fever and virus excretion upon challenge 6 weeks later. In a second experiment, the effect of an additional intramuscular booster with three different commercial vaccines (containing attenuated Bartha or NIA₃-783 or inactivated Phylaxia; all suspended in an oil-in-water emulsion) at 10 weeks of age was evaluated. One month after the last intramuscular booster, between five and seven pigs from each group were selected for challenge. All intranasally/intramuscularly immunized pigs showed a significantly better clinical and virological protection after challenge than the single intranasally immunized pigs. In the double immunized group, the protection was better when Kaplan gE^?gC^? was used for the intranasal priming (only two of 14 pigs excreted virus with a duration of 4 days) than when Kaplan gE^?gI^? was used (13 of 18 pigs excreted virus with a duration ranging from 1 to 4 days). The virological protection was not influenced by the type of vaccine used for booster vaccination. Because the intranasal/intramuscular immunization approach is very compatible with current pig movements on farms and pigs with moderate levels of maternally derived antibodies can effectively be immunized, it can be considered as a good alternative to intramuscular/intramuscular vaccinations especially in regions with a high ADV prevalence.     

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.     

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.     

Intranasal vaccination of pigs against Aujeszky's disease. 4. Comparison with one or two doses of an inactivated vaccine in pigs with moderate maternal antibody titres

Intranasal (IN) vaccination of pigs with low levels of maternally-derived antibody (MDA) has previously been shown to confer good protection against challenge with virulent Aujeszky's disease virus (ADV). The objective of the present study was to determine the efficacy of IN vaccination with an attenuated ADV, in comparison with that of an inactivated vaccine given parenterally, in pigs with higher MDA titres at the time of vaccination. In one experiment, vaccinations were done at 6 weeks of age, and in another experiment pigs were vaccinated at 4 and/or 9 weeks of age. Two months after (the last) vaccination pigs were challenged intranasally with a virulent ADV. Protection was evaluated on the basis of mortality, periods of growth arrest, fever and virus shedding after challenge. The presence of MDA markedly depressed the serum-neutralizing antibody response after vaccination. Sensitisation occurred after parenteral vaccination with an inactivated vaccine despite high MDA levels. Although the intranasally-vaccinated pigs had lower levels of neutralizing antibody at the time of challenge, they were significantly better protected than pigs given 1 or 2 doses of the inactivated vaccine. Comparing the present results with those of a previous study, it appears that the efficacy of parenteral as well as intranasal ADV vaccination decreases with increasing levels of MDA at the time of vaccination.     

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.     

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.     

Respiratory tract protection upon challenge of pigs vaccinated with attenuated porcine reproductive and respiratory syndrome virus vaccines

In this study, the efficacy of two attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines was assessed. The virological protection in the lungs of vaccinated pigs upon challenge was studied. Also, challenged pigs were exposed to lipopolysaccharide (LPS) to evaluate clinical protection. Six-week-old pigs were immunized intramuscularly with commercial vaccines based on either an attenuated American or an attenuated European virus strain. Non-immunized pigs and pigs intramuscularly inoculated with the virulent Lelystad strain were included as controls. Six weeks after immunization, pigs were challenged either intratracheally or intranasally with the Lelystad strain, and 3 and 6 days later intratracheally exposed to Escherichia coli LPS. After LPS administration, pigs were monitored for clinical signs. At 4 and 7 days after challenge, pigs were euthanized to determine virus quantities in broncho-alveolar lavage (BAL) fluids and in lungs. Challenge virus was recovered from three out of eight pigs that had been primo-inoculated with the Lelystad strain with titers ranging between 0.3 and 3.1 log(10). Fifteen out of sixteen pigs vaccinated with the attenuated American strain were positive for challenge virus and their mean virus titers were similar to those of non-immunized challenge controls. Eleven out of 16 pigs vaccinated with the attenuated European strain were positive for challenge virus and their mean virus titers were 2.0-2.5 log(10) lower than those of non-immunized challenge controls. Thus, the virological protection in the lungs of vaccinated pigs upon challenge was incomplete, but was more pronounced in the homologous situation. Clinical signs upon LPS exposure in both vaccinated groups were not reproducible in two experiments.     

Mucosal vaccination against influenza: protection of pigs immunized with inactivated virus and ether-split vaccine

Effective vaccinations against swine influenza reduce the economic loss of pig industries, and also may minimize the possibility of emergence of new pandemic viruses, since pigs are intermediate hosts to generate reassortant viruses among avian and mammalian influenza viruses. In this study, we showed that intranasal immunization of pigs with formalin-inactivated or ether-split influenza vaccine (A/Aichi/2/68) induced virus-specific IgG, IgM, and IgA antibodies in their nasal secretions and sera, resulting in complete protection from virus challenge. Antibody response to the challenge virus was not observed in the immunized pigs, suggesting that the replication of the virus in the primary targets, respiratory epithelial cells, was inhibited. The present results indicate that intranasal immunization of pigs with inactivated vaccines is effective to control swine influenza, and also provide a good model, as well as a mouse model, to evaluate an intranasal application of influenza vaccine for humans.     

Studies on immunisation of pigs with the Bartha strain of Aujeszky's disease virus.

The K strain of Aujeszky's disease virus (ADV) grown in Vero cells was used to vaccinate pigs. Following intramuscular inoculation, the pigs remained healthy, no vaccine virus was excreted and virus could be detected only at the inoculation site. One inoculation gave good protection against challenge with a virulent strain of ADV, and the amount of virulent ADV excreted was geatly curtailed. Following vaccination only low leads of serum neutralizing antibody were detected (geometric mean titre 1/2), but three weeks after challenge very high levels were found (GMT 1/1773). Intranasal vaccination gave similar results. There was minimal excretion of vaccine virus. The clinical reaction on challenge was less severe than in the intramuscularly challenged group, although lower antibody levels were detected three wekks following challenge (GMT 1/483). A field trial, using this strain given subcutaneously, indicated that one inoculation of this vaccine is effective.     

The protective value of vaccine-induced neutralising antibody titres in swine fever

The relationship between vaccine-induced antibody titres against swine fever virus (SFV), as measured by the neutralisation peroxidase-linked assay (NPLA), and protection against virus multiplication, excretion and transmission, disease and death was studied in 46 pigs. The pigs were housed individually and challenged intranasally with 100 pig ID50 of the virulent Brescia strain of SFV. In order to detect virus transmission, a swine fever (SF)-susceptible sentinel pig was placed in contact with the vaccinated animal 2 days after challenge. All 11 pigs with pre-challenge NPLA titres less than 12.5 responded to the challenge with fever, 8 out of 10 showed leucopenia, 7 transmitted virus to their contact and 3 died. Of the 9 animals with titres greater than or equal to 12.5 and less than 25, 8 developed fever, 6 out of 7 had leucopenia, 2 excreted and/or transmitted virus and all survived. Of the 12 pigs with pre-challenge titres greater than or equal to 25 and less than 50, 5 responded with fever, 6 out of 10 had leucopenia, 4 excreted virus and none died. Although all pigs with prechallenge titres greater than or equal to 50 showed a booster response, virus transmission was not observed, indicating that in the case of exposure such animals would not contribute towards the spread of field virus. From an epidemiological point of view, titres less than 32 were found inadequate.     

Multiplication and immunogenicity of a temperature-sensitive and thymidine kinase-deficient strain of Aujeszky's disease virus in pigs.

Hysterectomy-produced colostrum-deprived 5- and 27-day-old pigs were inoculated intramuscularly (IM) or intranasally (IN) with the temperature-sensitive and thymidine kinase-deficient ZHtsTK- strain of Aujeszky's disease virus (ADV), and the nasal swabs and organs of the pigs were periodically collected for virus isolation. No abnormal clinical signs were observed in these pigs, except for a mild febrile response. Viral shedding in the nasal swabs with low titers was detected in the pigs inoculated IN between postinoculation day (PID) 1 and 5, but not in those of the pigs inoculated IM. No contact infection, however, occurred in the cohabiting pigs. Viruses with low titers were isolated only from the muscles and lymph nodes at the site of inoculation in the pigs inoculated IM on PID 2 and 4, but not from any organs of the pigs inoculated IN. To investigate the ability of the ZHtsTK- strain to establish a latent infection in pigs, the pigs inoculated IM or IN with the ZHtsTK- strain were treated with prednisolone. No virus was detected in the trigeminal ganglia or the nasal swabs collected after prednisolone treatment by the cocultivation method. The immunological evaluation demonstrated that immunization of pigs with this strain was effective in preventing clinical signs caused by ADV infection. The duration of virus shedding was markedly shortened in immunized pigs, particularly in those immunized twice and the total quantity of virus recovered from immunized pigs was reduced in comparison with unimmunized pigs.     

The use of different vaccination schedules for sows to protect piglets against Aujeszky's disease

Several Aujeszky's disease virus (ADV) vaccination protocols of sows were evaluated with regard to the passive protection conferred on piglets in a recently built commercial farm. Three different groups of sows were vaccinated using a Bartha K-61 strain. One group received an inactivated vaccine during pregnancy and the other two groups received attenuated vaccines, either during pregnancy (day 65) or on the seventh day of lactation. At farrowing, sows vaccinated during lactation had lower seroneutralization titres than those vaccinated during pregnancy either with inactivated or attenuated vaccines. Accordingly, their piglets were the ones with lower levels of maternally transferred neutralizing antibodies. At 4 weeks of age, five piglets born of each group of sows were challenged intranasally with a neurotropic strain of ADV. Piglets born of sows vaccinated during pregnancy with inactivated and attenuated vaccines gained 1.50 kg bodyweight and 2.50 kg bodyweight during 7 days, respectively, and did not show clinical signs, while piglets from sows vaccinated during the previous lactation lost 0.60 kg and presented moderate to severe clinical signs of ADV. Vaccination of sows during pregnancy provided more protection against ADV for piglets than sow vaccination before mating. Piglets born from sows vaccinated with attenuated or inactivated vaccines did not present remarkable differences on protection.     

Immunization of pigs with a purified Shiga-like toxin II variant toxoid.

Passive transfer of neutralizing antibodies and active immunization with a toxoid of purified Shiga-like toxin II variant (SLT-IIv, edema disease toxin) were used to protect pigs against challenge with SLT-IIv. Six 6-week-old pigs were passively immunized by intraperitoneal administration of an immunoglobulin preparation from porcine antiserum against purified SLT-IIv. Six 6-week-old pigs and twelve 2-week-old pigs were actively immunized by two intramuscular injections of 25 micrograms of SLT-IIv toxoid given 2 weeks apart. The 24 immunized pigs and an equal number of age-matched unimmunized control pigs were all challenged by intravenous injection of purified SLT-IIv (6 ng/kg body weight). The six passively immunized pigs acquired neutralizing SLT-IIv antibody titers of 1280 or 2560 and the 18 actively immunized pigs mounted a serum immune response with toxin neutralizing titers ranging from 128 to 10,240. All 24 immunized pigs survived the challenge but nine pigs with the lowest titers showed mild clinical signs of edema disease. All 24 control pigs died within 30 hours of challenge.     

Newcastle disease virus pathogenesis in the respiratory tract of local or systemic immunized chickens.

Establishment of selective immunity, local or systemic, made it possible to evaluate the pathogenesis of Newcastle disease virus (NDV) in the respiratory tract of chickens that were previously immunized with beta-propiolactone-inactivated antigen. NDV was inoculated intranasally or intramuscularly to chickens in different states of immunity (local or systemic). Humoral antibodies protected chickens against intranasal as well as intramuscular infection. Local antibodies, on the other hand, conferred immunity only against intranasal challenge. The respiratory tract supported multiplication of the virus, producing a self-limited subclinical infection. Replication of the virus in this system was negligible, playing only a minor role in the pathogenesis of the disease.     

The use of a systemic prime/mucosal boost strategy with an equine influenza ISCOM vaccine to induce protective immunity in horses

In horses, natural infection confers long lasting protective immunity characterised by mucosal IgA and humoral IgGa and IgGb responses. In order to investigate the potential of locally administered vaccine to induce a protective IgA response, responses generated by vaccination with an immunostimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F) containing A/eq/Newmarket/77 (H7N7), A/eq/Borl?nge/91 (H3N8) and A/eq/Kentucky/98 (H3N8) using a systemic prime/mucosal boost strategy were studied. Seven ponies in the vaccine group received EQUIP F vaccine intranasally 6 weeks after an initial intramuscular immunisation. Following intranasal boosting a transient increase in virus-specific IgA was detected in nasal wash secretions. Aerosol challenge with the A/eq/Newmarket/1/93 reference strain 4 weeks after the intranasal booster resulted in clinical signs of infection and viral shedding in seven of seven influenza-naive control animals whereas the seven vaccinated ponies had statistically significantly reduced clinical signs and duration of virus excretion. Furthermore, following this challenge, significantly enhanced levels of virus-specific IgA were detected in the nasal washes from vaccinated ponies compared with the unvaccinated control animals. These data indicate that the intranasal administration of EQUIP F vaccine primes the mucosal system for an enhanced IgA response following exposure to live influenza virus.     

Vaccines against Aujeszky's disease: Evaluation of their efficacy under standardized laboratory conditions

Summary

A standardized test was developed to compare the efficacy of Aujeszky's disease virus (ADV) vaccines under laboratory conditions. Per test 3 groups of 6 to 8 sero‐negative pigs were used. The first vaccination was done at 10 weeks of age. One group was vaccinated once, another was vaccinated twice and the 3rd served as control. Pigs were challenge exposed to the virulent NIA‐3 strain of ADV 12 weeks after the first vaccination. Apart from mortality, average periods of growth arrest, fever and virus shedding after challenge were used as parameters to evaluate vaccine efficacy.

Two inactivated and 4 attenuated vaccines were tested. Two attenuated vaccine viruses were excreted after vaccination. Despite maximal standardization, a considerable variation still existed between the experiments in mortality and growth arrest periods of control pigs after challenge. However, the controls were always more severely affected than the vaccinated pigs. All vaccines except one were effective in preventing death after challenge, but none conferred complete protection. Most vaccinated pigs still lost weight, developed fever and shed virus after challenge. Revaccination after 3 or 4 weeks had little effect, particularly with the attenuated vaccines. The results of the present study indicate that 2 of the attenuated vaccines conferred the best protection, I attenuated vaccine appeared to be as effective as the 2 inactivated ones, and the 4th attenuated vaccine was least effective.     

The effect of dose, route and virulence of bovine herpesvirus 1 vaccine on experimental respiratory disease in cattle.

Three experiments were conducted with calves in which, following intramuscular or intranasal vaccination with virulent or attenuated bovine herpesvirus 1, calves were protected against bovine herpesvirus 1 -- Pasteurella haemolytica challenge. Calves receiving low doses of vaccine had lower levels of antibody and greater evidence of virus replication upon challenge than those receiving higher doses. In contrast 11/13 unvaccinated controls had fibrino-purulent pneumonia following challenge. The immune response developed later in younger calves and those given low doses of vaccine. Neutralizing antibodies to bovine herpes-virus 1 were not found in nasal secretions, but were present in serum seven days after vaccination. Bovine herpesvirus 1 was isolated before challenge from nasal secretions of calves vaccinated intranasally or intramuscularly with virulent virus but not those vaccinated intramuscularly with vaccine virus. It was concluded that both routes of vaccination with either virulent or attenuated bovine herpesvirus 1 provided protection from challenge with homologous or heterologous bovine herpesvirus 1 and that live vaccines should contain at least 10(3) plaque forming units/dose for effective immunization.     

猪流行性腹泻病毒实验感染猪血清抗体的消长规律

应用微量中和试验(MT)、间接血凝试验(IHA)和间接酶联免疫吸附试验(间接ELISA)对三种不同途径即滴鼻、口服和肌肉注射人工感染猪流行性腹泻病毒的12头仔猪的血清抗体进行了检测。感染猪在第1～2周时即可测出抗体,3～5周后抗体滴度达高峰,抗体持续时间最短为18周,最长可达22周以上。其中以肌注和滴鼻组抗体产生较早,而口服组稍晚。肌注组抗体上升较快,抗体持续时间较短,而滴鼻和口服组抗体上升稍慢,抗体持续时间较长。     

Pathogenesis and Subsequent Cross‐Protection of Influenza Virus Infection in Pigs Sustained by an H1N2 Strain

The H1N1, H3N2 and, more recently, H1N2 subtypes of influenza A virus are presently co‐circulating in swine herds in several countries. The objectives of this study were to investigate the pathogenesis of Sw/Italy/1521/98 (H1N2) influenza virus, isolated from respiratory tissues of pigs from herds in Northern Italy, and to evaluate its potential cross‐protection against the Sw/Fin/2899/82 (H1N1) strain. In the pathogenesis test, eight pigs were intranasally infected with H1N2 virus; at pre‐determined intervals, these animals were killed and necropsied, along with eight uninfected animals. In the cross‐protection test, sixteen pigs were infected by intranasal (i.n.) and intratracheal (i.t.) routes with either H1N2 or H1N1 virus. Twenty days later, all pigs were challenged (by the same route), with either the homologous H1N2 or heterologous H1N1 virus strains. Control group was inoculated with culture medium alone. On post‐challenge days (PCD) 1 and 3, two pigs from each infected group, along with one control pig, were killed. Clinical, virological, serological and histopathological investigations were performed in both the pathogenicity and cross‐protection tests. In the pathogenicity test, mild clinical signs were observed in two pigs during 3 and 4 days, respectively. Virus was isolated from two pigs over 6 days and from lung samples of pigs killed on post‐infection days 2 and 4. Seroconversion was detected in the two infected animals killed 15 days after infection. In the cross‐protection study, mild clinical respiratory signs were detected in all pigs infected with either the H1N2 or H1N1 virus. The virus was isolated from nasal swabs of almost all pigs till 6 days. After the challenge infection, the pigs remained clinically healthy and virus isolation from the nasal secretions or lung samples was sporadic. Antibody titres in H1N1 or H1N2 infected groups were similar, whereas the H1N2 sub‐type induced less protection against re‐infection by homologous and heterologous virus than H1N1 sub‐type. The controls had no signs of the disease. In the H1N2 infected pigs, a reduced number of goblet cells in nasal and tracheal mucosa and small foci of lymphomononuclear cell infiltrates in the submucosa were detected. Furthermore, the goblet cell reduction was related to the time of infection. Diffuse mild interstitial pneumonia was also recorded in pigs infected with the H1N2 virus and challenged with either H1N1or H1N2 pigs. These studies showed the moderate virulence of the H1N2 virus and a partial cross‐protection against heterologous infection.