Anti-inflammatory effects of Flos Lonicerae Japonicae Water Extract are regulated by the STAT/NF-κB pathway and HO-1 expression in Virus-infected RAW264.7 cells.

This study examined the effect of the Flos Lonicerae Japonicae water extract (FLJWE), chlorogenic acid, and luteolin on pseudorabies virus (PRV)-induced inflammation in RAW264.7 cells and elucidated related molecular mechanisms. The results revealed that FLJWE and luteolin, but not chlorogenic acid, inhibited the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines in PRV-infected RAW 264.7 cells. We found that the FLJWE and luteolin suppressed nuclear factor (NF)-κB activation by inhibiting the phosphorylation of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3, respectively). Moreover, the FLJWE significantly upregulated the expression of pNrf2 and its downstream target gene heme oxygenase-1 (HO-1). Our data indicated that FLJWE and luteolin reduced the expression of proinflammatory mediators and inflammatory cytokines, such as COX-2 and iNOS, through the suppression of the JAK/STAT1/3-dependent NF-κB pathway and the induction of HO-1 expression in PRV-infected RAW264.7 cells. The findings indicate that the FLJWE can be used as a potential antiviral agent.

Ginseng stem-leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine.

Pseudorabies, a herpesvirus infection, is mainly controlled by using attenuated live vaccines. In this study, the effect of ginseng stem and leaf saponins (GSLS) in combination with selenium (Se; in the form of sodium selenite) on vaccination against attenuated pseudorabies virus (aPrV) was evaluated. It was found that GSLS and Se have an adjuvant effect and that a combination of GSLS and Se stimulates significantly enhanced immune responses than does GSLS or Se alone. Following oral administration of GSLS, mice immunized with an attenuated PrV vaccine diluted in Se-containing physiological saline solution (PSS) provoked a significantly stronger gB-specific serum antibodies response (IgG, IgG1 and IgG2a), enhanced lymphocyte proliferation and cytolytic activity of NK cells, along with higher production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) by splenocytes. Notably, the combination of GSLS and Se conferred a much higher resistance to fPrV challenge after immunization of the mice with aPrV vaccine. This study offers convincing experimental evidence that an injection of Se with oral GSLS is a promising adjuvant combination that improves the efficacy of vaccination against PrV and deserves further study regarding improvement of responses to other animal vaccines.

Improved immune response to an attenuated pseudorabies virus vaccine by ginseng stem-leaf saponins (GSLS) in combination with thimerosal (TS).

Vaccination using attenuated vaccines remains an important method to control animal infectious diseases. The present study evaluated ginseng stem-leaf saponins (GSLS) and thimerosal (TS) for their adjuvant effect on an attenuated pseudorabies virus (aPrV) vaccine in mice. Compared to the group immunized with aPrV alone, the co-inoculation of GSLS and/or TS induced a higher antibody response. Particularly, when administered together with GSLS-TS, the aPrV vaccine provoked a higher serum gB-specific antibody, IgG1 and IgG2a levels, lymphocyte proliferative responses, as well as production of cytokines (IFN-γ, IL-12, IL-5 and IL-10) from lymphocytes, and more importantly provided an enhanced cytotoxicity of NK cells and protection against virulent field pseudorabies virus challenge. Additionally, the increased expression of miR-132, miR-146a, miR-147 and miR-155 was found in murine macrophages cultured with GSLS and/or TS. These data suggest that GSLS-TS as adjuvant improve the efficacy of aPrV vaccine in mouse model and have potential for the development of attenuated viral vaccines.

Immune response in pigs treated with therapeutic doses of enrofloxacin at the time of vaccination against Aujeszky's disease.

The effect of treatment with enrofloxacin was studied on the postvaccinal immune response in pigs. Forty pigs were used (control not vaccinated (C), control vaccinated (CV), vaccinated, received enrofloxacin (ENRO)). From day -1 to day 3 pigs from ENRO group received enrofloxacin at the recommended dose. Pigs from ENRO and CV groups were vaccinated twice against Aujeszky's disease virus (ADV). There was a significant delay in the production of humoral response of enrofloxacin dosed pigs when compared with CV group. Moreover, in ENRO group the significant decrease in IFN-γ production and significantly lower values of stimulation index after ADV restimulation was noted, as compared with CV group. The secretion of IL-6, IL-10 and TNF-α by PBMC after recall stimulation was also affected in ENRO group. The results indicate that enrofloxacin, in addition to its antimicrobial properties, possess significant immunomodulatory effects and may alter the immune response to vaccines.

Tryptophan supplements promote pregnancy success in mice challenged with pseudorabies virus (PRV) by regulating the expression of systemic cytokines, immunoglobulins, PRV-specific protein profiles, and toll-like receptors.

Tryptophan (Trp) plays an important role in regulating the maternal immune response, a key determinant of the success or failure of pregnancy, but whether Trp supplements can prevent a pseudorabies virus (PRV)-induced failure of pregnancy remains unknown. This study examined the effect of three dietary Trp levels (0.25%, 0.35%, and 0.5%) on the immunity and reproduction of PRV-challenged pregnant mice. PRV challenge resulted in decreased live embryo numbers, live litter sizes, and serum progesterone and interleukin (IL)-10 concentrations, but increased the levels of serum immunoglobulins (Igs) (PRV-specific antibody [IgG, IgA, and IgM]) and IL-1ß. Live embryo numbers, live litter sizes, serum progesterone concentration, and IgG and PRV-specific antibody levels on day 9 of pregnancy were all increased dose-dependently by Trp inclusion in the diet of PRV-challenged mice. Increased Trp levels in PRV-challenged mice promoted the up-regulation of uterine and embryonic indoleamine 2,3-dioxygenase expression, but attenuated the up-regulation of uterine and embryonic Toll-like receptor (TLR) 3 and TLR9 expression and increased serum interferon-γ concentration. Collectively, Trp supplements might improve reproductive performance of PRV-challenged pregnant mice by down-regulating TLR expression and pro-inflammatory cytokine synthesis, by up-regulating PRV-specific antibody and immunoglobulin synthesis, and by elevating the concentrations of anti-inflammatory cytokines and progesterone.

Vitamin a supplements alleviate inflammatory responses in reproductive tracts of male mice infected with pseudorabies virus.

Vitamin A is largely thought to have immune potential for mammal health; however, no conclusive mechanisms exist regarding its role in the pathogen-initiated innate immune response, or in the linkage between the innate and adaptive immune system during sperm formation in the male reproductive tract. Therefore, this study was conducted to evaluate the nutritional protective effect of vitamin A supplementation on reproductive performance and immune function of the male mouse challenged with pseudorabies virus (PRV). Sperm quality, testis toll-like receptors (TLRs) mRNA expression levels, and serum concentration of cytokines and immunoglobulins at 7 or 14 days post-injection were compared between control mice and PRV-challenged mice fed the same diet supplemented with vitamin A at 0, 4000, 10,000, 25,000 and 50,000 IU/kg, respectively. PRV- and phosphate buffered saline (PBS)-injection were performed when the mice in the unsupplemented group were marginally deficient in vitamin A. Sperm quality (sperm density and deformity ratio) of PRV-injected mice was significantly harmed by PRV, but this effect was attenuated by increased vitamin A consumption. Vitamin A supplements also attenuated PRV-challenge-induced increase in testis TLR3, TLR7, and TLR9 mRNA expression and serum pro-inflammatory cytokine (gamma interferon, IFN-gamma; and interleukin 1-beta,IL-1beta) concentration, and decrease in serum anti-inflammatory cytokine (IL-10) concentration. Higher than normal vitamin A consumption was recommended to counteract the deleterious effects of viral invasion, possibly through the downregulated expression of TLRs, and thus to improve immunity and reproductivity of male mice challenged with an invading pathogen.

Effects of sugar cane extract on pseudorabies virus challenge of pigs.

This experiment aimed to evaluate the efficacy of sugar cane extract (SCE) on the modulation of porcine immunity against pseudorabies virus (PrV) infection. Twelve-week-old experimental pigs were fed with SCE (500 mg/kg of body weight per day) for 3 days and challenged with PrV (2 x 10(5) TCID(50)) on the second day. Pigs that were only challenged with PrV and without SCE-treatment served as controls. The leukocyte functional assays were performed on the 7th and 14th day post-PrV challenge. Our results showed a significant enhancement (P<0.05) of natural killer cytotoxicity, lymphocyte proliferation, phagocytic function of monocytes, and interferon-gamma (IFN-gamma) production of CD4(+) and gammadelta T cells in the SCE-treated pigs compared with the controls. In addition, SCE administration reduced the severity of clinical signs and brain lesion in the course of disease in PrV-challenged pigs. SCE-treated pigs showed a 12% growth enhancement compared with untreated controls. SCE administration had an immunostimulating effect on porcine immunity that may subsequently enhance protective activities against PrV infection which may be extensively applied in field for the prevention of infections.

Sulfolipo-cyclodextrin in squalane-in-water as a novel and safe vaccine adjuvant.

Previously, we described synergistic adjuvanticity of combinations of synthetic sulfolipo(SL)-derivatives of polysaccharide (SL-polysaccharides) and squalane-in-water emulsions (squalane/W). In this paper, effects of type of polysaccharide and nature of oil on adjuvanticity, reactogenicity and stability are described. SL-derivatives of the following polysaccharides were synthesised: synthetic polysucroses with weight-average molecular weight (MW) of 400,000 (Ficoll400), 70,000 (Ficoll70) and 39,000 Da (Ficoll39), polyfructose of 5,000 Da (inulin), linear polyglucose of 1,200 Da (maltodextrin) and cyclic polyglucose of 1,135 Da (beta-cyclodextrin). The number of sulphate groups per monosaccharide of the different SL-polysaccharides varied between 0.15 and 0.23 and the number of lipid groups per monosaccharide between 1.15 and 1.29. Adjuvant formulations were prepared by incorporating these SL-polysaccharides into oil-in-water emulsions of either squalane, hexadecane, soya oil or mineral oil. Adjuvanticity of the formulations obtained for humoral responses to inactivated pseudorabies virus (PRV) and inactivated influenza virus strains A/Swine (A/Swine) and MRC-11 (MRC-11) in pigs and MRC-11 and ovalbumin (OVA) in mice depended on the type of oil (squalane = mineral oil > hexadecane = soya oil) but not on the type of polysaccharide backbone of the SL-derivative. Reactogenicity assessed by local swelling in mice decreased with decreasing MW (SL-Ficoll400 = Ficoll70 = Ficoll39 > SL-inulin = SL-maltodextrin > SL-cyclodextrin) when combined with squalane and decreased with the type of oil in the following order: squalane > mineral oil > hexadecane > soya oil when combined with SL-Ficoll400. Stability of the SL-polysaccharide/squalane/W emulsions at elevated temperature increased with decreasing MW of the SL-polysaccharide (SL-Ficoll400 < SL-Ficoll70 = SL-Ficoll39 < SL-inulin = SL-maltodextrin = SL-cyclodextrin). SL-cyclodextrin/squalane/W remained stable for > 2.5 years at 4 degrees C, > 18 weeks at 37 degrees C and > 10 days at 60 degrees C. We concluded that reactogenicity and stability but not adjuvanticity of SL-polysaccharide/squalane/W formulations depended on the MW of SL-polysaccharide and that SL-cyclodextrin/squalane/W is a promising non-mineral oil adjuvant as it combines strong adjuvanticity (i.e. better than the mineral oil-based adjuvant presently applied) with low reactogenicity and good stability.

Protective effect of glycoprotein gC-rich antigen against pseudorabies virus.

A trial vaccine containing pseudorabies virus (PRV) glycoprotein gC as the main component showed excellent protection against virulent virus infection in pigs. Glycoprotein gC-rich antigen was prepared by heparin affinity chromatography from PRV-infected cell lysates. The preparations were mixed with mineral oil adjuvant as a water-in-oil emulsion. Six-week-old pigs were immunized twice at two-week intervals with trial vaccines containing 128,000, 12,800 and 1,280 HA units per dose of gC antigen. They were then challenged with a virulent PRV at day 7 after the final immunization. Neutralizing (NT) antibodies were produced with increase of antibody titers after challenge. Pigs immunized with 128,000 HA units per dose of gC survived and showed no virus shedding during the 2-week experimental period after the challenge. The role of cell-mediated immunity was examined using BALB/c mice, and induction of gC-specific cytotoxic T lymphocytes (CTLs) was detected by 51Cr release assay. From these results with mice, it is inferred that cell-mediated immunity, especially CTL, may play an important role in the effectiveness of our trial vaccine in addition to humoral immunity.

Evaluation of tests for detection of antibodies to Aujeszky's disease (pseudorabies) virus glycoprotein E in the target population.

Receiver operating characteristic (ROC) curves assess the quality of tests over the entire range of test signals. We compared the ability of an ELISA to detect antibodies to Aujeszky's disease (pseudorabies) virus gE in colostrum (test A) and in a single droplet of whole blood (test B) with the results obtained in serum (gold standard) in the target population by constructing and analyzing such curves. The area under the ROC curve, which is a quantitative measure of test performance, proved to be significantly (p < 0.01) smaller in test A than in test B or the gold standard. No significant differences in the area under the ROC curve were observed between test B and the gold standard.

Vaccination with recombinant vaccinia virus vaccines expressing glycoprotein genes of pseudorabies virus in the presence of maternal immunity.

Piglets which had received colostral antibody to pseudorabie virus (PRV) were divided into four groups and inoculated with a NYVAC vaccinia recombinant expressing glycoprotein gD of PRV, a NYVAC recombinant expressing glycoprotein gB of PRV, an inactivated PRV vaccine, or no vaccine. The piglets were vaccinated twice, 3 weeks apart, beginning at approximately 2 weeks of age and later challenged with virulent PRV oronasally. All three vaccines protected similarly when no maternal antibody was present. Although all three vaccines induced some active immunity in piglets with maternal antibody, piglets receiving the NYVAC/gB vaccine were the only ones protected similarly whether or not they had maternal antibodies to PRV.

[Vaccination trial against Aujeszky's disease: development of antibody titers in sow serum, colostrum and the serum of suckling piglets and the influence of maternal antibodies on the serologic vaccination reaction of weaned piglets]. / Impfversuche gegen die Aujeszkysche Krankheit: Entwicklung der Antikörpertiter in Sauenserum, Kolostrum und Serum der Saugferkel sowie der Einfluss maternaler Antikörper auf die serologische Impfreaktion von Absatzferkeln.

In two vaccination trials the influence of different vaccination schedules of gifts on the colostral immunity of their offspring and the influence of maternally derived antibodies on the active formation of antibodies after vaccination in weanlings were tested. It could be shown, that vaccination of gifts 6 and 3 weeks prior to farrowing led to higher blood levels of maternally derived antibodies in their offspring than vaccination earlier during gestation or prior to mating. Correlation between antibody level of sow, colostrum and offspring was good, the halflife of the antibodies was 11.3 days. Low levels of maternally derived antibodies did not influence the active formation of antibodies after vaccination, higher levels reduced antibody-formation, but did not suppress it completely. Sow colostrum and blood of piglets are generally adequate substrates for the serologic control of breeding units regarding Aujeszky's disease. Existing problems are mentioned. The results of this study are discussed concerning their importance for the eradication of Aujeszky's disease with vaccination programs.

Vaccination of newborn pigs in the presence of low levels of pseudorabies colostral antibodies.

To test the hypothesis that newborn pigs with pseudorabies virus (PRV) colostral antibodies might be actively immunized with a PRV glycoprotein gIII-deleted vaccine (Omnimark-PRV), 23 piglets were obtained from four sows that had been immunized 4 weeks and 2 weeks before farrowing with this vaccine. Thirteen piglets were immunized with Omnimark-PRV when they were less than 3 days old and ten piglets served as non-vaccinated controls. Piglets were weaned at 28 days of age and challenged with virulent PRV (Shope) when they were 49 days old, at which time the vaccinated and control pigs were seronegative for PRV virus neutralizing (VN) and gIII antibodies, and all control pigs and ten vaccinees were seronegative for PRV antibodies by the latex agglutination test (LAT). Two vaccinees were LAT(+) and one was LAT(+/-). Central nervous system signs and/or respiratory disease signs were observed in six of ten control pigs with the death of one control, while two of 13 vaccinees showed only very mild and transient clinical disease signs and there were no fatalities. Non-vaccinees lost weight until postchallenge day (PCD) 6 and did not regain prechallenge weight until PCD 8. All vaccinees gained weight after challenge and at PCD 11 had mean weight gains nearly twice that of the controls. On PCD 11, the geometric mean titre for VN antibodies of non-vaccinees was 9.3, while that of vaccinees was 49.0, indicating that the vaccinated group had been immunologically primed.

Development of active immunity in newborn pigs with colostral antibodies by vaccination with gIII-deleted PRV.

Maternal antibodies interfere with active immunization of swine by gI-deleted pseudorabies virus [(PRV); Aujeszky's disease virus] vaccines. To test the hypothesis that modified-live (MLV) vaccines retaining the PRV gI and with deletions in the PRV glycoprotein gIII and thymidine kinase (TK) genes might be efficacious in circumventing colostral antibody interference, the OMNI-MARK-PRV (gI+ gIII- TK-) vaccine was administered intramuscularly to 13 newborn pigs with colostral antibodies, while 10 pigs from the same litters served as nonvaccinated controls. At 49 days of age, when PRV virus neutralization (VN) antibodies were negative and all nonvaccinated pigs as well as 10 vaccinates were latex agglutination test (LAT)-negative, the pigs were challenged intranasally with the virulent PRV(SHOPE) strain. In support of the hypothesis, it was found that several central nervous system and respiratory disease signs developed in 6 of 10 nonvaccinates, with one fatality, while 2 of 13 vaccinates showed only very mild and transient disease signs. Nonvaccinates lost weight until post challenge day (PCD) 6, did not regain prechallenge weight until PCD 8, and at PCD 11 had gained only 4.9 pounds/pig. Vaccinates gained weight after challenge and at PCD 11 showed a 9.4 pounds/pig weight gain. On PCD 11, the geometric mean titer (GMT) for VN antibodies of the nonvaccinates was 9.3, while the GMT of the vaccinates for VN antibodies was 49.0, showing that vaccinated pigs had been immunologically primed.

Intradermal application of Aujeszky's disease virus strain Begonia with tocopherol-based adjuvant and a novel design injection device.

Initially the use of intradermal application of Aujeszky's disease vaccines was shown to be very effective. However, for thus far unknown reasons the gI-deleted vaccines were much less efficacious by using this route of vaccination as compared to gI-positive vaccines. By the use of a tocopherol-based adjuvant and an improved design of the intradermal injection device it now appeared feasible to obtain the same efficacy both in specific pathogen free pigs and in pigs with material antibodies as found before when intramuscular administration was performed. With respect to safety we found a complete lack of skin lesions, no adverse systemic reactions (e.g. body temperatures) and no effect on growth rates. Last but not least, the easiness of intradermal injections is of great advantage in large-scale vaccination programs.

Use of colostrum to detect antibodies against glycoprotein I of Aujeszky's disease virus.

Serum and colostrum samples taken from 499 sows from five herds of pigs endemically infected and vaccinated against Aujeszky's disease virus, were used to investigate whether colostrum could be used to detect antibodies against glycoprotein I (gI) of the virus. Using serum as the reference, the test applied to colostrum had a sensitivity of 97 per cent and a specificity of 88 per cent. When samples were taken from 50 sows from a gI seronegative vaccinated herd, one colostrum sample was gI-positive, giving a specificity for the test of 98 per cent. The mean gI antibody titres in colostrum were about six times higher than in serum. Samples of colostrum were also taken from 132 sows from eight unvaccinated herds free of Aujeszky's disease virus. All these samples were gI-negative, giving a specificity of 100 per cent. Colostrum samples can be stored for at least six weeks at -20 degrees C without compromising the test results, and the repeatability and reproducibility of the test applied to colostrum were good.

Efficacy of various non-oily adjuvants in immunization against the Aujeszky's disease (pseudorabies) virus.

Standard oil and various non-oily adjuvants were compared for use in immunization against the Aujeszky's disease (pseudorabies) virus, both in mice and swine, and using either inactivated virions or purified glycoproteins as antigen. Mineral oil, sodium alginate, aluminium hydroxide, and saponin were assayed in mice as adjuvants for inactivated virions, saponin being the most efficient. The addition of Mab anti-CD3 did not improve either immune response or protection achieved in mice using viral particles with oil or sodium alginate. When purified glycoproteins were used as antigens, the use of ISCOM greatly enhanced specific T-cell responses and protection of mice. The incorporation of Mab anti-CD3 into ISCOM conferred 100% protection of mice. Surprisingly, when an ISCOM containing glycoproteins was assayed in swine in a single-dose trial, no improvement on the protection conferred by the oily adjuvant was observed.

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.

[The presence of antibodies against glycoprotein I (gI) of the virus of Aujeszky's disease in colostrum and milk of sows]. / De aanwezigheid van antistoffen tegen glycoproteïne I (gI) van het virus van de ziekte van Aujeszky in biest en melk van zeugen.

The possibility of using sow's milk to detect antibodies to the gI protein of Aujeszky's virus was studied by the present authors. Antibody titres in serum (collected less than 30 days ante partum) and colostrum from sixty-three sows were determined using a commercially available ELISA; twenty-four of thirty-seven animals vaccinated with gI-positive vaccines showed an antibody response in both colostrum and serum. The titre in colostrum was significantly higher than it was in serum. Antibodies could not be detected in serum or in colostrum of the twenty-six animals which were only vaccinated with gI-negative vaccines. Subsequently, anti-gI titres were determined in milk collected during lactation. Antibodies were also detectable in milk of animals showing high colostrum titres (greater than 128) for at least seventeen days. This period decreased as the initial colostrum titre became lower. Antibodies could still be detected in milk of animals vaccinated with gI-positive vaccine, which were sero-negative at the time of the experiment. Milk therefore seems to be a useful alternative to serum in detecting anti-gI antibodies in sows.

Comparative pathology of HPCD pigs infected with wild-type and ara-T-resistant strains of Aujeszky's disease virus.

Fourteen 1- to 4-week-old hysterectomy-produced and colostrum-deprived (HPCD) pigs were inoculated intranasally with wild-type and ara-T-resistant strains of Aujeszky's disease virus (ADV), and the pathological lesion induced by the two strains was compared. The wild-type strain (YS-81) led to high mortality, and the pigs developed multifocal necrosis throughout the body and encephalitis. In comparison, the ara-T-resistant strain (YS-81TR) of the virus killed only 1-week-old HPCD pigs inoculated with 10(6.0) PFU per ml of the virus and did not kill pigs more than 2-weeks of age. The latter revealed consistently severe pneumonitis on post-inoculation day (PID) 14. Results of the present study indicated that the ara-T-resistant strain of ADV was less virulent for HPCD pigs than the parental wild-type strain of ADV and that it was able to grow better in the lung than in any other tissue.