Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine.

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Cross-protection of recombinant Pasteurella multocida toxin proteins against atrophic rhinitis in mice.

Pasteurella multocida (P. multocida) infects the swine respiratory tract and mainly causes atrophic rhinitis (AR). Recently, many commercially inactivated and subunit vaccines have been used as preventive strategies. However, the best antigenic protein portion has not been selected, and the aluminum gel was used as the adjuvant, which may not induce full protection. P. multocida toxin (PMT) is the major virulence factor responsible for AR. PMT is a monomeric 146 kDa protein (approximately 1285 amino acids) encoded by the tox A gene. In this study, we expressed different fragments of recombinant PMT proteins, combined them with a water-in-oil-in-water adjuvant, and evaluated mice's immune response. The results indicated that the rPMT-C-immunized group showed significantly higher levels (p < 0.05) of IgG, IgG2a antibody and interferon-γ, IL-12 cytokine expression than other groups. Furthermore, vaccination with rPMT-C recombinant protein can provide homologous and heterologous protection against P. multocida challenge. In conclusion, our approach may be feasible for developing an effective subunit vaccine against atrophic rhinitis with a cost-down simple ingredient.

Evaluation of carbopol as an adjuvant on the effectiveness of progressive atrophic rhinitis vaccine.

The Gram-negative pathogen toxigenic P. multocida causes progressive atrophic rhinitis (PAR) in swine throughout the world. Although some vaccines are being developed against PAR, their efficacy has not been evaluated using carbopol. In our study, a mixture of killed B. bronchiseptica and P. multocida bacteria, combined with recombinant proteins containing the C- and N-termini of PMT, was emulsified using two different adjuvants (ISA-15A and carbopol 971). The efficacy of these two vaccines was evaluated in a mouse model. Balb/C mice were immunized twice at a 14-day interval. Two weeks after the secondary immunization, blood samples were collected and the mice were challenged with toxigenic P. multocida. Thirty-five days later, the mice were euthanized, blood and tissue samples were collected. Compared with mice inoculated with vaccine emulsified with ISA-15A, higher titers of SN (1:64) and significantly increased levels of TNF-α, IL-6 and IL-17A were observed in mice inoculated with vaccine emulsified with the carbopol 971P. Especially, mice immunized with vaccine emulsified with the carbopol 971P had no detectable pathological changes in snouts or organs after challenge. The results demonstrated that carbopol adjuvanted vaccine provides good protection against PAR and P. multocida infection which can induce robust humoral and cell-mediated responses. We conclude that the carbopol adjuvanted vaccine is a good candidate for PAR prevention.

Comparison of gene expression of Toll-like receptors and cytokines between Piau and Commercial line (Landrace×Large White crossbred) pigs vaccinated against Pasteurella multocida type D.

We aimed to compare Toll-like receptors (TLR) and cytokines expression in local Piau breed and a Commercial line (Landrace×Large White crossbred) pigs in response to vaccination against Pasteurella multocida type D. Seronegative gilts for Pasteurella multocida type D and Mycoplasma hyopneumoniae were used, from which peripheral blood mononuclear cells (PBMC) were collected in four time points (T0, T1, T2 and T3; before and after each vaccination dose). For bronchoalveolar lavage fluid cells (BALF), we set groups of vaccinated and unvaccinated animals for both genetic groups. Gene expression was evaluated on PBMC and BALF. In PBMC, when we analyzed time points within breeds, significant differences in expression for TLRs and cytokines, except TGFß, were observed for Commercial animals. For the Piau pigs, only TGFß showed differential expression. Comparing the expression among genetic groups, the Commercial pigs showed higher expression for TLRs after first vaccination dose, while for IL2, IL6, IL12 and IL13, higher expression was also observed in T3 and IL8 and IL10, in T1 and T3. Still comparing the breeds, the crossbred animals showed higher expression for TNFα in T1 and T2, while for TGFß only in T2. For gene expression in BALF, vaccinated Commercial pigs showed higher expression of TLR6, TLR10, IL6, IL8, IL10, TNFα and TGFß genes than vaccinated Piau pigs. The Commercial line pigs showed higher sensitivity to vaccination, while in local Piau breed lower responsiveness, which may partly explain genetic variability in immune response and will let us better understand the tolerance/susceptibility for pasteurellosis.

Immunologic study and optimization of Salmonella delivery strains expressing adhesin and toxin antigens for protection against progressive atrophic rhinitis in a murine model.

Mice were intranasally inoculated at various times to optimize the vaccination strategy with a new live candidate vaccine expressing the antigens CP39, FimA, PtfA, and ToxA of Pasteurella multocida and F1P2 of Bordetella bronchiseptica in an attenuated live Salmonella system to protect against progressive atrophic rhinitis (PAR). Sixty BALB/c mice were divided equally into 4 groups. The group A mice were vaccinated only at 12 wk of age, the group B mice received a primary vaccination at 9 wk of age and a booster at 12 wk of age, the group C mice received a primary vaccination at 6 wk of age and boosters at 9 and 12 wk of age, and the group D mice were inoculated intranasally with sterile phosphate-buffered saline as a control. The humoral and mucosal immune responses of groups A, B, and C increased significantly compared with those of the control group. Expression of the cytokines interleukin-4 and interferon-γ in splenocytes also increased significantly. In addition, the group B mice exhibited significantly fewer gross lesions in lung tissue compared with the other vaccinated groups after challenge with a virulent P. multocida strain. These results indicate that a strategy of double intranasal vaccination can optimize protection against PAR.

Des souris furent inoculées par voie intra-nasale à différents temps pour optimiser la stratégie de vaccination avec un nouveau vaccin candidat vivant exprimant les antigènes CP39, FimA, PtfA, et ToxA de Pasteurella multocida et F1P2 de Bordetella bronchiseptica dans un système vivant atténué de Salmonella afin de protéger contre la rhinite atrophique progressive (PAR). Soixante souris BALB/c ont été divisées également en quatre groupes. Les souris du groupe A furent vaccinées seulement à 12 semaines d'âge, les souris du groupe B ont reçu une première vaccination à 9 sem d'âge et un rappel à 12 sem d'âge, les souris du groupe C ont reçu une première vaccination à 6 sem d'âge et des rappels à 9 et 12 sem d'âge, et les souris du groupe D (groupe témoin négatif) furent inoculées par voie intra-nasale avec uniquement de la saline tamponnée stérile. Les réponses immunes humorales et mucosales des groupes A, B et C augmentèrent de manière significative comparativement à celles du groupe témoin. L'expression des cytokines interleukine-4 et interféron-γ dans les splénocytes augmenta également de manière significative. De plus, les souris du groupe B avaient significativement moins de lésions macroscopiques dans le tissu pulmonaire comparativement aux autres animaux des groupes vaccinés suite à une infection avec une souche virulente de P. multocida. Ces résultats indiquent qu'une stratégie de double vaccination intra-nasale peut optimiser la protection envers PAR.(Traduit par Docteur Serge Messier).

Generation of an attenuated Salmonella-delivery strains expressing adhesin and toxin antigens for progressive atrophic rhinitis, and evaluation of its immune responses in a murine model.

An expression/secretion plasmid containing genes encoding the FimA, CP39, PtfA, ToxA and F1P2 antigens associated with porcine pneumonic pasteurellosis and progressive atrophic rhinitis (PAR) was constructed and harbored in an attenuated Salmonella Typhimurium, which was used as the vaccine candidate. The immune responses induced by this delivery strain were investigated in a murine model. Each antigen secreted from the delivery strain was confirmed by Western blot analysis. Thirty BALB/c mice were divided equally into two groups; group A were intranasally inoculated with the mixture of the five delivery strains, and group B were inoculated with sterile PBS. In group A, all antigen-specific serum IgG were significantly increased compared to those of group B from the 2nd week post-inoculation (WPI) till the 8th WPI. All antigen-specific mucosal IgA in group A were also significantly greater than those of group B. In addition, the significant splenic lymphocyte proliferative responses, the elevations of CD3(+)CD4(+), CD3(+)CD8(+) and B-cell populations, and the induction of IFN-γ expression in group A were observed. In conclusion, the mixture of five delivery strains expressing specific antigen for these diseases was found to be capable of inducing significant humoral and cellular immune responses.

aroA deleted Bordetella bronchiseptica inspiring robust mucosal immune response and provide full protection against intranasal challenge.

Bordetella bronchiseptica is a Gram-negative respiratory pathogen responsible for atrophic rhinitis and bronchopneumonia in swine. Several vaccines aimed at preventing B. bronchiseptica have been used, but a safe and efficient live vaccine for use in piglets remains elusive. In this study, we constructed an aroA-deleted B. bronchiseptica strain (QH0814) and evaluated its safety and protective efficiency in piglets. Lung lesion scores in QH0814-immunized piglets post-challenge were significantly lower than those in piglets immunized with the parent strain (P<0.05). Immunization with QH0814 induced a vigorous immune response, especially at the mucosal surface of the respiratory tract. IgA titers in bronchoalveolar lavage fluid (BALF) and serum were significantly higher in the QH0814-immunized group compared to the inactivated-vaccine-immunized group. Piglets immunized with QH0814 were better protected than those in the inactivated-vaccine and negative control groups. The clinical symptoms, histopathological changes and immune responses elicited in the piglets were recorded. The results of this study suggest that QH0814 was able to confer complete protection against B. bronchiseptica infection and could thus be used as a candidate attenuated live vaccine against B. bronchiseptica in piglets.

Expression of 4 truncated fragments of Pasteurella multocida toxin and their immunogenicity.

Pasteurella multocida toxin (PMT) is a poor antigen that becomes more immunogenic after its native structure has been destroyed. In contrast, partially truncated PMT proteins, which are predicted to be good antigens when used as a vaccine, might be used to improve the control of atrophic rhinitis in pigs. In this study, 4 truncated PMT fragments were expressed in Escherichia coli, and those 4 fragments were inoculated into mice to produce the polyclonal antibodies. The results of an enzyme-linked immunosorbent assay (ELISA) revealed that #1 and #4 fragments were the most immunogenic. Immunized mice were subsequently challenged intraperitoneally with P. multocida type D. Five of the eight #1 fragment-immunized mice showed some protection against death and bacterial clearance. Pigs immunized with #1 fragment produced no or mild atrophic rhinitis (turbinate conchal score) after challenge, suggesting that this #1 fragment could be a good candidate for a subunit recombinant-type vaccine.

Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs.

Three short fragments of recombinant subunit Pasteurella multocida toxin (rsPMT) were constructed for evaluation as candidate vaccines against progressive atrophic rhinitis (PAR) of swine. PMT-specific antibody secreting cells and evidence of cellular immunity were detected in rsPMT-immunized pigs following authentic PMT challenge or homologous antigen booster. Piglets immunized with rsPMT fragments containing either the N-terminal or the C-terminal portions of PMT developed high titers of neutralizing antibodies. Pregnant sows immunized with rsPMT had higher levels of maternal antibodies in their colostrum than did those immunized with a conventional PAR-toxoid vaccine. Offspring from rsPMT vaccinated sows had better survival after challenge with a five-fold lethal dose of authentic PMT and had better growth performance after challenge with a sublethal dose of toxin. Our findings indicate these non-toxic rsPMT proteins are attractive candidates for development of a subunit vaccine against PAR in pigs.

Development of a genetically modified nontoxigenic Pasteurella multocida toxin as a candidate for use in vaccines against progressive atrophic rhinitis in pigs.

OBJECTIVE: To construct a genetically modified nontoxigenic Pasteurella multocida toxin (PMT) and examine its immunoprotective activity against challenge exposure with wild-type PMT in pigs. ANIMALS: 5 healthy pigs. PROCEDURE: A nontoxigenic PMT was created by replacing the serine at position 1164 with alanine (S1164A) and the cysteine at position 1165 with serine (C1165S). Toxic activity was determined by use of the guinea pig skin test and mouse lethality test. Three pigs were vaccinated twice with the modified PMT, and the remaining 2 pigs served as nonvaccinated control animals. Vaccinated and control pigs were challenge exposed with wild-type PMT. Pigs were euthanatized and necropsied on day 14 after challenge exposure. Turbinate atrophy was examined macroscopically and assigned a score. Serum anti-PMT antibodies were determined by use of an ELISA. RESULTS: The genetically modified PMT was characterized by a total lack of toxic activity. Pigs vaccinated with the modified PMT became seropositive; in contrast, control pigs remained seronegative. Necropsy revealed that the 2 control pigs had moderate and severe turbinate atrophy, respectively, whereas the 3 vaccinated pigs did not have any lesions in the turbinates or abnormalities in other organs. CONCLUSIONS AND CLINICAL RELEVANCE: Modification by use of S1164A and C1165S leads to a complete loss of toxic effects of PMT without impairment of the ability to induce protective immunity in pigs. Analysis of these results suggests that genetically modified PMT may represent a good candidate for use in developing a vaccine against progressive atrophic rhinitis in pigs.

In vitro study of the immune stimulating activity of an atrophic [correction of athrophic] rhinitis vaccine associated to chitosan microspheres.

Chitosan microspheres (CMs) were prepared by an ionic gelation process with tripolyphosphate and characterized. Bordetella Bronchiseptica Dermonecrotoxin (BBD), a major virulence factor of a causative agent of atrophic rhinitis (AR), was loaded on to the CMs for nasal vaccination. BBD-loaded CMs were observed as aggregated shapes although unloaded CMs were observed as relatively spherical ones. The average particle size of the BBD-loaded CMs was 4.39 microm. The lower the molecular weight of chitosan and the higher the medium pH, the greater was the release of BBD from the BBD-loaded CMs in vitro due to weaker intermolecular interaction between chitosan and BBD. Tumor necrosis factor alpha and nitric oxide from RAW264.7 cells exposed to BBD-loaded CMs were gradually secreted with time, suggesting that released BBD from CMs had immune stimulating activity of AR vaccine in vitro.

Turbinectomia parcial inferior versus cauterização submucosa para hipertrofia da concha nasal inferior / Parcial inferior turbinecomy versus submucosal cauterization os hipertrophic inferior turbinate

Obstrução nasal é a queixa crônica mais encontrada pelo otorrinolaringologista. A maioria responde ao tratamento clínico, mas em alguns casos a cirurgia é necessária. Não há consenso sobre qua técnica deve ser empregada.

Expression of a truncated Pasteurella multocida toxin antigen in Bordetella bronchiseptica.

Mild or subclinical respiratory infections caused by Bordetella bronchiseptica are widespread in pigs despite multiple control efforts. Infection with virulent B. bronchiseptica strains is a common risk factor in the establishment of toxin-producing strains of Pasteurella multocida in the nasal cavity of pigs leading to the disease, atrophic rhinitis (AR). This study was designed to explore the possibility of expressing a protective epitope of P. multocida toxin (PMT) in B. bronchiseptica to create single-component mucosal vaccine to control atrophic rhinitis in pigs. To achieve this, a P. multocida toxin fragment (PMTCE), that was non-toxic and protective against lethal challenge in mice, was cloned into a broad-host-range plasmid, PBBR1MCS2, and introduced into B. bronchiseptica by electroporation. The Pasteurella gene construct was placed under the regulatory control of a promoter region that was separately isolated from B. bronchiseptica and appears to be part of the heat shock protein gene family. B. bronchiseptica harboring the plasmid under antibiotic selection expressed the 80kDa PMTCE as determined by PAGE and Western blot with a PMT-specific monoclonal antibody. When introduced into the respiratory tracts of mice, B. bronchiseptica harboring the plasmid construct was reisolated in declining numbers for 72h post-inoculation. Antibody responses (IgM, IgA and IgG) to B. bronchiseptica were detected in serum and respiratory lavage, but PMTCE-specific antibodies were not detected. While further refinements of PMT expression in B. bronchiseptica are necessary, this study provides a basis for the development of a single-component, live-attenuated vaccine against atrophic rhinitis.

Evaluation of vaccines for atrophic rhinitis--a comparison of three challenge models.

We compared three challenge models for the assessment of atrophic rhinitis (AR) vaccines: combined infection with Bordetella bronchiseptica (Bb) and Pasteurella multocida (Pm); application of acetic acid (AA) to the nasal mucosa followed by Pm infection; and Bb infection alone. Two vaccines were tested using standardized criteria, notably nasal lesion scores. The vaccines provided different levels of protection in the Bb and the AA/Pm challenges, but were similar in the combined (Bb/Pm) challenge. It is clear that the AA/Pm model shows the protective value of only the Pm component, whereas the single Bb challenge reflects the protective value merely of the Bb component of a combined vaccine. These results suggest that the best assessment of protection is provided if the two specific challenges are performed separately.

Immunization of rabbits against Pasteurella multocida using a commercial swine vaccine.

Elaboration of heat-labile toxin (PMT) is an important virulence factor in some isolates of Pasteurella multocida from rabbits. Previously, we reported that immunization with inactivated PMT (IPMT) stimulated protective immunity to challenges from PMT. To test the hypothesis that immunization with a commercial swine vaccine containing IPMT stimulates similar protective immunity, groups of five rabbits were inoculated twice intramuscularly (i.m.), 10 days apart, with 0.5 ml of sterile saline or a commercial swine P. multocida bacterin-toxoid (BT). In addition, a group was inoculated intranasally with 5 microg of IPMT. Serum and nasal lavage samples were taken on days 0, 7, 14 and 21 after initial immunization and assayed by ELISA for anti-PMT antibody. Serum IgG and nasal lavage IgA were detectable by day 14 in BT and IPMT-immunized rabbits, but not in the saline controls. Groups of similarly inoculated rabbits were then challenged intranasally with 28 microg of PMT 21 days after initial immunization, and necropsied 7 days later, along with control challenged and non-challenged rabbits. Histological lesion severity was graded on a numerical scale. Non-immunized and saline, challenged controls developed more severe pneumonia, pleuritis, nasal turbinate atrophy and testicular atrophy than IPMT and BT-immunized rabbits. The results confirm the hypothesis that immunization with a commercial swine P. multocida BT confers protective immunity in rabbits against challenges from PMT.

Immune responses of piglets to Pasteurella multocida toxin and toxoid.

Experimental atrophic rhinitis (AR), serum antibody titres and in vitro lymphoproliferation to Pasteurella multocida derived toxin (Pm-T) were studied in piglets. Specific immune responses to Pm-T and Pm-T induced conchae atrophy were compared with AR immunity. This immunity was initiated by the Nobi-VAC AR-T vaccine administered at various times with respect to Pm-T challenge. Animals challenged with Pm-T developed conchae atrophy, but no antibodies nor cellular immune responses to Pm-T were detected. Vaccination 3 weeks before Pm-T challenge protected pigs against breakdown of nasal bony tissues. This protection was accompanied by an increase of serum antibodies and in vitro lymphoproliferation to Pm-T. Animals vaccinated 10 days before or after Pm-T challenge also had antibodies and cellular immune responses. However, these animals developed AR. In vitro, Pm-T appeared mitogenic for quiescent (non-immune) peripheral lymphocytes and Concanavalin A stimulated lymphocytes from some pigs. These in vitro lymphoproliferative responses could be partly abrogated by the addition of monomorphic anti-swine major histocompatibility complex class II DQ and DR specific monoclonal antibodies. We conclude that Pm-T is poorly immunogenic in vivo and does not initiate a protective Pm-T specific immune response. Pigs were protected from AR by vaccination, but protection was dependent on the timing of vaccine administration. We speculate that Pm-T modifies the immune response such that the response is not directed towards the toxin but to an unidentified component in the nose of piglets.

Field evaluation of thirteen regimens for the control of progressive atrophic rhinitis.

The purpose of the study was to evaluate, under field conditions, the effect of prophylactic programs for the control of progressive atrophic rhinitis (PAR)--recommended in different countries. The investigations were carried out on 280 pregnant sows divided into 14 equal groups and 50 slaughter pigs randomly selected from the litters produced by the sows of each of the groups. Efficacy of all programs was recognized on the basis of comparative evaluation of the average daily gain (ADG), morphometric examination of turbinate bones and computer conchal morphometry (TPR). An increase in ADG was noted in 12 of 13 experimental groups when compared to the control group. Usefulness of the evaluated programs differed significantly. Results of TPR relate only partially to the results of visual morphometry and results of ADG.

Field trials with a new genetically engineered vaccine for protection against progressive atrophic rhinitis in pigs.

Field trials were carried out testing a new genetically engineered vaccine against Progressive Atrophic Rhinitis. The vaccine contained a non-toxic recombinant derivative of the P. multocida toxin. The experimental vaccine was compared with a commercial vaccine in 4 farms and in 1 farm 2 different dose regimens were applied. A total of 825 sows were included. The primary efficacy variable was a comparison of post mortem evaluation of the degree of conchae atrophy in the 4585 pigs. The pigs were slaughtered at normal slaughter weight. The secondary efficacy variables were serological response and age at slaughter. In all farms the experimental vaccine provided significantly better protection of the progeny than the control vaccine. The serological response in sows was significantly higher in all farms than the response to the control vaccine. The serological response did not differ between farms.