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 the immunobiological effects of a regenerative far-infrared heating system in pigs.

Thermal conditions are an important environmental factor in maintaining healthy pigs because they affect feed intake, growth efficiency, reproduction and immune responses in pigs. RAVI, a regenerative far-infrared heating system, can effect pig production by emitting an optimal far-infrared wavelength. Far-infrared radiation has been reported to increase microvascular dilation and vascular flow volume. The purpose of this study was to evaluate the immunobiological differences between pigs raised with the RAVI system and the gasoline heater system. Twenty-six-week-old weaned pigs were raised in two rooms that were equipped with a RAVI system or a gasoline heater for 8 weeks. A porcine atrophic rhinitis vaccine was administered after two weeks and transcriptome analysis in whole blood were analyzed at 2-week intervals. Signaling pathway analyses of the RAVI group at 8 weeks showed the activation of pathways related to nitric oxide (NO) production. This suggests that the application of RAVI might induce the production of NO and iNOS, which are important for increasing the immune activity. Similar to the result of microarray, phenotypic changes were also observed at a later period of the experiment. The increase in body weight in the RAVI group was significantly higher than the gasoline heater group at 8 weeks. The antibody titer against the vaccine in the RAVI group was also higher than that the gasoline heater group at 4 weeks and 8 weeks. This evaluation of the use of a far-infrared heating system with pigs will be helpful for applications in the pig farm industry and pig welfare.

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.

Influence of Pasteurella multocida Toxin on the differentiation of dendritic cells into osteoclasts.

Dendritic cells (DC) are antigen-presenting cells that connect the innate and adaptive immune system to ensure an efficient immune response during the course of an infection. Recently, DC came into the spotlight as a potential source of osteoclast progenitors, especially under (auto)inflammatory conditions. The virulence factor Pasteurella multocida Toxin (PMT) causes atrophic rhinitis in pigs, a disease characterised by a severe reduction of nasal bone. Our group and others have shown the potential of PMT in mediating differentiation of monocytes/macrophages into bone-resorbing osteoclasts. However, whether DC are target cells for PMT-induced osteoclast differentiation, is currently unknown. Using different murine DC model systems, we investigated the ability of PMT to induce osteoclast formation in DC. Similar to our previous observations in macrophages, PMT was endocytosed by DC and triggered intracellular deamidation of residue Q209 of the Gq alpha subunit. Still, PMT failed to induce prolonged secretion of osteoclastogenic cytokines and osteoclast formation; instead PMT-treated DC secreted interleukin-12 (IL-12), an inhibitor of osteoclastogenesis. In this study, we show that in comparison to bone marrow-derived macrophages, PMT induces maturation of DC through increased expression of the activation markers CD80 and CD86. As maturation of DC prevents their transdifferentiation into osteoclasts, we hypothesize that PMT, a potent osteoclastogenic toxin, fails to trigger osteoclastogenesis in DC due to its effect on DC maturation and IL-12 production.

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.

Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor.

The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs.

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.

Pasteurella multocida: diseases and pathogenesis.

Pasteurella multocida is an enigmatic pathogen. It is remarkable both for the number and range of specific disease syndromes with which it is associated, and the wide range of host species affected. The pathogenic mechanisms involved in causing the different syndromes are, for the most part, poorly understood or completely unknown. The biochemical and serological properties of some organisms responsible for quite different syndromes appear to be similar. Thus, the molecular basis for host predilection remains unknown. The recent development of genetic manipulation systems together with the availability of multiple genome sequences should help to explain the association of particular pathological conditions with particular hosts as well as helping to elucidate pathogenic mechanisms.

A genome-wide scan for quantitative trait loci affecting respiratory disease and immune capacity in Landrace pigs.

Respiratory disease is the most important health concern for the swine industry. Genetic improvement for disease resistance is challenging because of the difficulty in obtaining good phenotypes related with disease resistance; however, identification of genes or markers associated with disease resistance can help in the genetic improvement of pig health. The purpose of our study was to investigate whether quantitative trait loci (QTL) associated with disease resistance were segregated in a purebred population of Landrace pigs that had been selected for meat production traits and mycoplasmal pneumonia of swine (MPS) scores over five generations. We analysed 1395 pigs from the base to the fifth generation of this population. Two respiratory disease traits [MPS scores and atrophic rhinitis (AR) scores] and 11 immune-capacity traits were measured in 630-1332 animals at 7 weeks of age and when the animal's body weight reached 105 kg. Each of the pigs, except sires in the base population, was genotyped using 109 microsatellite markers, and then, QTL analysis of the full-sib family population with a multi-generational pedigree structure was performed. Variance component analysis was used to detect QTL associated with MPS or AR scores, and the logarithm of odds (LOD) score and genotypic heritability of the QTL were estimated. Five significant (LOD > 2.51) and 18 suggestive (LOD > 1.35) QTL for respiratory disease traits and immune-capacity traits were detected. The significant QTL for Log-MPS score, located on S. scrofa chromosome 2, could explain 87% of the genetic variance of this score in this analysis. This is the first report of QTL associated with respiratory disease lesions.

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.

Alterations in epithelial barrier function and host defense responses in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is characterized by a chronic symptomatic inflammation of the nasal and paranasal sinus mucosae and is one of the most frequently reported chronic diseases in the United States, with an estimated prevalence of greater than 10% of the general population. Although the pathogenesis of CRS remains poorly understood, there is evidence for a role of bacteria and fungi, as well as the presence of a robust adaptive immune response in the upper airways and sinuses. Recent studies of CRS, as well as several other diseases in the skin and respiratory epithelium, have uncovered evidence that deficiencies in epithelial immune barrier function might compromise the interaction between the host and external immune stimuli. Recent studies suggest the hypothesis that reduced expression of antimicrobial S100 proteins, particularly psoriasin and calprotectin, might lead to increased susceptibility to bacterial and fungal colonization in patients with CRS. The main emphasis of this review will be to highlight the current literature that suggests that a defect in the expression of a broad set of epithelially derived genes might lead to barrier compromise and subsequently a dysfunctional host immune response to environmental agents in patients with CRS.

Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs.

The immune-stimulating activities of Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) loaded in chitosan microspheres (CMs) have already been reported in vitro and in vivo with a mouse alveolar macrophage cell line (RAW264.7) and mice. Therefore, this study attempted to demonstrate the successful induction of mucosal immune responses after the intranasal administration of BBD loaded in CMs (BBD-CMs) in colostrum-deprived pigs. The BBD was introduced to the CMs using an ionic gelation process involving tripolyphosphate (TPP). Colostrum-deprived pigs were then directly immunized through intranasal administration of the BBD-CMs. A challenge with a field isolate of B. bronchiseptica was performed ten days following the final immunization. The BBD-specific IgG and IgA titers, evident in the nasal wash and serum from the vaccinated pigs, increased with time (p<0.05). Following the challenge, the clinical signs of infection were about 6-fold lower in the vaccinated pigs compared with the nonvaccinated pigs. The grades for gross morphological changes in the turbinate bones from the vaccinated pigs were also significantly lower than the grades recorded for the nonvaccinated pigs (p<0.001). Therefore, the mucosal and systemic immune responses induced in the current study would seem to indicate that the intranasal administration of BBD-CMs may be an effective vaccine against atrophic rhinitis in pigs.

Atrophic rhinitis vaccine composition triggers different serological profiles that do not correlate with protection.

Atrophic rhinitis (AR) is a widespread and economically important disease of swine caused by Bordetella bronchiseptica and Pasteurella multocida. It can be controlled by vaccination. This study investigates the effect of altering the composition (adjuvants and/or addition of formalin-inactivated P. multocida toxin, fPMT) of conventional vaccines on the serological profile and on protection against AR in swine. A significantly higher B. bronchiseptica specific antibody titre was detected for vaccines with novel immunostimulants, the best being Montanide IMS 1313 (1:630 compared to 1:274 obtained with alum). The highest B. bronchiseptica antibody titre was demonstrated for a combination of B. bronchiseptica--fPMT, while PMT antibody titre was highest for monovalent fPMT (both adjuvanted with IMS 1313). The AR-specific antibodies were transmitted from dams to their offspring in similar titres and with the same hierarchy of effectiveness. After a B. bronchiseptica--P. multocida bacterial challenge, piglets from dams vaccinated with fPMT combined with B. bronchiseptica or B. bronchiseptica--P. multocida bacterins showed the lowest nasal lesions scores (4.5 and 3.2, respectively, out of a possible maximum score of 18). These combinations, both of which were adjuvanted with IMS 1313, gave the best protection against experimentally induced AR. Our results show that the adjuvant and the antigen composition of the vaccine strongly affect seroconversion, and that the AR-specific antibody titre does not necessarily correlate with the degree of protection.

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.

Modulation of the humoral immune response of swine and mice mediated by toxigenic Pasteurella multocida.

Progressive atrophic rhinitis is an upper respiratory tract disease of pigs caused by toxigenic strains of the bacterium Pasteurella multocida. In this study the effect of P. multocida on the humoral immune response of pigs and mice was investigated. Pigs were given live intranasal challenge with either a toxigenic strain or a non-toxigenic strain of P. multocida, or were given daily intranasal instillation of a cell-free lysate of the toxigenic strain. Mice were given a live intranasal challenge of either a toxigenic or a non-toxigenic strain of P. multocida. All of the animals were immunised with ovalbumin and serum concentrations of anti-ovalbumin antibodies were quantified and compared between different treatment groups and control animals. Intranasal challenge with toxigenic P. multocida caused a significant reduction in the levels of anti-ovalbumin IgG in both species. A similar effect was seen in pigs given a cell-free extract of toxigenic P. multocida. Whilst the mechanism of this suppression is unclear, we surmise that immunomodulation of the host is an important virulence factor for toxigenic P. multocida, and could be an important function of the toxin. This immunomodulatory effect may enhance colonisation of P. multocida aiding horizontal transmission and may predispose to concurrent infection with other potential pathogens.

Clinical, genetic and immunologic analysis of a family affected by ozena.

Primary atrophic rhinitis is a chronic inflammation of the nasal mucosa characterized by atrophy of the mucous and bony tissue of the turbinates and by a thick, dense secretion, which quickly forms a characteristically fetid-smelling, greenish crust. We report the results of the clinical, genetic and immunologic investigations performed on eight subjects (three with ozena and five asymptomatic), members of the same familial group. The presence of the disease in the family fits well with dominant inheritance. All the culture specimens from the patients affected by ozena were positive for Klebsiella ozaenae, and one of them was also positive for Pseudomonas aeruginosa. All the three patients with ozena and two of the five apparently unaffected family members were positive for antinuclear antibodies. Immunoblotting showed a reactivity to a 50-kD protein, which was not identified by the common, recognized nuclear autoantigens. This was present in one of the three patients and three of the five other family members. Positivity for IgG-class anticardiolipins was correlated with disease manifestation in that it was found in two of the three patients and only in one of the five asymptomatic family members. The hypothesis of a genetic factor that could drive the chronicity of the inflammatory pattern of a pre-existing infectious nasal disease is suggested.