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.

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.

Biofilm inhibitors targeting the outer membrane protein A of Pasteurella multocida in swine.

Pasteurella multocida (Pm) is the causative agent of atrophic rhinitis in swine. This study aimed to discover biofilm inhibitors against swine Pm to counteract antibiotic resistance and decrease virulence. The virulence factor outer membrane protein A (OmpA) was targeted. A library of drugs approved by the Food and Drug Administration (FDA) was used to perform virtual screening against PmOmpA. The top-scoring compounds had no effect on the growth of Pm serotype A or D. Mycophenolate mofetil showed the highest efficacy in inhibiting biofilm formation by Pm serotype A, with an IC50 of 7.3 nM. For Pm serotype D, indocyanine green showed the highest effect at an IC50 of 11.7 nM. Nevertheless, these compounds had no effect on an established biofilm of Pm. This study offers an alternative way to prevent biofilm formation by Pm that could also be applied to other pathogens.

Ozena in Immigrants of Differing Backgrounds.

Ozena, or atrophic rhinitis, is a chronic nasal process seen in Africa, India, and the Middle East. It is marked by the triad of fetid nasal discharge, crusting, and atrophy, and is often associated with Klebsiella infection. We present cases of ozena with nasal Klebsiella in three unrelated patients, including two east African children and one Saudi adult. All three patients grew Klebsiella species in culture and required prolonged treatment with multiple methodologies, including antibiotics, saline rinses, and surgical debridement. They differed greatly in time from presentation to diagnosis, and demonstrated various stages of the disease process. Ozena is rarely seen in the United States, and when it is seen, it is often misdiagnosed. Lack of prompt, adequate treatment can lead to long-term sequelae such as obliteration of nasal architecture, anosmia, sinus and skull base destruction, and social disenfranchisement due to the extreme foul smell of the nasal discharge. Clinicians should maintain a high index of suspicion for primary atrophic rhinitis when presented with its classic symptoms. Culture-directed and prolonged therapy with appropriate follow-up is a necessary component of a successful treatment plan.

Atrophic rhinitis caused by Cedecea davisae with accompanying mucocele.

Atrophic rhinitis is a chronic inflammatory disease characterized by progressive atrophy of nasal mucosa. Cedecea davisae, a rare pathogen, is a new member of Enterobacteriaceae family. In this article, we report a patient with atrophic rhinitis whose culture test revealed Cedecea davisae. The patient was operated due to accompanying posterior ethmoid mucocele. Levofloxacin and nasal irrigation were administered for two months. Significant improvement was observed in patient's complaints and nasal signs at postoperative sixth month. In conclusion, Cedecea davisae has been thought to cause atrophic rhinitis and mucocele in this patient. Patient recovered with simple treatment. These bacteria should be kept in mind as a causative agent for atrophic rhinitis.

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).

Regeneration of toxigenic Pasteurella multocida induced severe turbinate atrophy in pigs detected by computed tomography.

BACKGROUND: Atrophic rhinitis is a widely prevalent infectious disease of swine caused by Bordetella bronchiseptica and Pasteurella multocida. The course of the disease is considered to be different depending on the principal aetiological agents distinguishing B. bronchiseptica induced non-progressive and toxigenic P. multocida produced progressive forms. In order to compare the pathological events of the two forms of the disease, the development of nasal lesions has longitudinally been studied in pigs infected by either B. bronchiseptica alone or B. bronchiseptica and toxigenic P. multocida together using computed tomography to visualise the nasal structures. RESULTS: B. bronchiseptica infection alone caused moderately severe nasal turbinate atrophy and these lesions completely regenerated by the time of slaughter. Unexpectedly, complete regeneration of the bony structures of the nasal cavity was also observed in pigs infected by B. bronchiseptica and toxigenic P. multocida together in spite of seeing severe turbinate atrophy in most of the infected animals around the age of six weeks. CONCLUSIONS: B. bronchiseptica mono-infection has been confirmed to cause only mild to moderate and transient lesions, at least in high health status pigs. Even severe turbinate atrophy induced by B. bronchiseptica and toxigenic P. multocida combined infection is able to be reorganised to their normal anatomical structure. Computed tomography has further been verified to be a useful tool to examine the pathological events of atrophic rhinitis in a longitudinal manner.

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.

Swine atrophic rhinitis caused by pasteurella multocida toxin and bordetella dermonecrotic toxin.

Atrophic rhinitis is a widespread and economically important swine disease caused by Pasteurella multocida and Bordetella bronchiseptica. The disease is characterized by atrophy of the nasal turbinate bones, which results in a shortened and deformed snout in severe cases. P. multocida toxin and B. bronchiseptica dermonecrotic toxin have been considered to independently or cooperatively disturb the osteogenesis of the turbinate bone by inhibiting osteoblastic differentiation and/or stimulating bone resorption by osteoclasts. Recently, the intracellular targets and molecular actions of both toxins have been clarified, enabling speculation on the intracellular signals leading to the inhibition of osteogenesis.

Molecular biology of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is the causative agent of progressive atrophic rhinitis in swine. The 146 kDa single-chain toxin harbours discrete domains important for receptor binding, internalisation and biological activity. The molecular basis of the toxin's activity is the deamidation of a specific glutamine residue in the α-subunit of heterotrimeric G proteins. This results in an inhibition of the inherent GTPase activity leading to a constitutively active phenotype of the G protein. Due to the ability of the toxin to act on various families of heterotrimeric G proteins, a large subset of signal transduction pathways is stimulated.

Detection of urease-negative Bordetella bronchiseptica from the field.

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs™ assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.

Genetic diversity and associated pathology of Pasteurella multocida isolated from porcine pneumonia.

Pasteurella multocida is a widespread respiratory pathogen in pigs associated with atrophic rhinitis and contributing to aggravation of the pulmonary lesions. The aims of the present study were to characterize isolates of P. multocida from porcine bronchopneumonia by pulsed-field gel electrophoresis (PFGE), PCR based capsular typing and multilocus sequence typing (MLST) and to compare clonal complexes outlined with the type of histological lung lesions to investigate if a correlation between clonal lineages and lesions might exist. Isolates of P. multocida were obtained from cases of cranioventrally located porcine bronchopneumonia. All lung lesions were described and classified according to histological lesions. A total of 139 isolates, from lung (n=111), pericardial sac (n=21) and kidney (n=7) of 111 pigs were described using PFGE with ApaI as the restriction enzyme. Furthermore, 20 and 29 isolates were characterized by capsular serotyping and multilocus sequence typing, respectively. PFGE demonstrated 15 different clusters showing 50% or more similarity. All selected isolates were of capsular serotype A and only three main sequence types (ST) were detected among the isolates. Associations were not found between histopathology and clonal complexes of P. multocida. In conclusion, PFGE demonstrated a high diversity of genotypes of P. multocida associated with porcine bronchopneumonia. However, isolates obtained mainly belonged to few STs, indicating that isolates of P. multocida associated with porcine bronchopneumonia originates from a limited number of clonal lineages and therefore might have adapted to porcine hosts. No correlation was demonstrated between genotypes and types of lesions, and extra-pulmonary spreading was only rarely demonstrated.

A report on a rare case of Klebsiella ozaenae causing atrophic rhinitis in the UK.

Ozena is a chronic disease of the nasal cavity characterised by atrophy of the mucosa and bone caused by Klebsiella ozaenae. It is endemic to subtropical and temperate regions affecting the lower socio-economic group, usually the poor who live in unhygienic conditions. It is a rare disease in the UK. There is usually a delay in diagnosis due to unfamiliarity of the disease. A 25-year-old Nigerian migrant presented with nasal obstruction with purulent nasal discharge. Isolation of the bacterium was found from cultures of nasal discharge, crusting and tissue biopsies. She was treated successfully with ciprofloxacin. It is important to consider this rare condition in cases of nasal obstruction even in non-endemic areas especially with the advances of modern travel.

Association of Bordetella dermonecrotic toxin with the extracellular matrix.

BACKGROUND: Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. RESULTS: Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. CONCLUSIONS: Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

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.

Genetic diversity of porcine Pasteurella multocida strains from the respiratory tract of healthy and diseased swine.

A total of 382 porcine Pasteurella multocida strains, isolated from cases of pneumonia and progressive atrophic rhinitis (PAR) as well as from clinically healthy pigs of more than 150 German husbandries were characterized by detection of virulence-associated genes (VAGs) and ribotyping to understand the relationships between "commensal" and "pathogenic" strains, enabling a rational choice of vaccine strains. The diversity of the strains according to VAGs was low and mainly limited to capsular type genes (capA: 53.4%; capD: 45.8%; capF: 0.3%; cap-negative: 0.5%; hssB: 95.3%), dermonecrotoxin gene toxA (3.4%), as well as adhesion-related genes pfhaB (20.9%) and hgbB (84.3%). Ribotyping identified 13 patterns, but the vast majority of strains (95.8%) clustered in only three of these, namely IA-1 (45.5%), IA-7 (30.1%), and IIA-1 (20.2%). Pattern IA-1 was associated with capD(+) strains (93.6%) and harboured the majority of toxA(+) strains (84.6%). Pattern IA-7 mostly contained pfhaB(-), toxA(-)capA(+) strains (93.9%), while pattern IIA-1 was predominantly composed of pfhaB(+), toxA(-)capA(+) strains (87.0%). Clinical strains associated with pneumonia or PAR shared the above mentioned major ribotypes in comparable proportions with strains derived from healthy pigs, suggesting P. multocida to act more as an opportunistic than as an obligate pathogen in pigs. The limited number of subpopulations may either reflect a recent evolution of P. multocida in pigs or a selection by means of horizontal transfer of capsular genes, toxA or pfhaB. These data enforce further phylogenetic and epidemiological studies, examining the properties of different subpopulations of porcine P. multocida strains as well as factors of the porcine hosts themselves, which might be involved in disease susceptibility.

Efficacy of a novel Pasteurella multocida vaccine against progressive atrophic rhinitis of swine.

The efficacy of a novel vaccine composed of three short recombinant subunit Pasteurella multocida toxin (PMT) proteins in combination with a bi-valent P. multocida whole-cell bacterin (rsPMT-PM) was evaluated in field studies for prevention and control of progressive atrophic rhinitis (PAR) of swine at 15 conventional farrow-to-finish farms. Experimental piglets that were immunized twice with the rsPMT-PM vaccine developed detectable titers of neutralizing antibodies (greater than 1:8) that prevented the growth retardation and pathological lesions typically observed following challenge with authentic PMT. A total of 542 sows were vaccinated once or twice prior to parturition and serum neutralizing antibody titers were evaluated. Both single and double vaccination protocols induced neutralizing antibody titers of 1:16 or higher in 62% and 74% of sows, respectively. Notably, neither sows nor piglets at a farm experiencing a severe outbreak of PAR at the time of the vaccination trial had detectable antibody titers, but antibody titers increased significantly to 1:16 or higher in 40% of sows following double vaccination. During the year after vaccination, clinical signs of PAR decreased in fattening pigs and growth performance improved sufficiently to reduce the rearing period until marketing by 2 weeks. Collectively, these results indicate that the rsPMT-PM vaccine could be used to provide protective immunity for controlling the prevalence and severity of PAR among farm-raised swine.

Microbiological study of role of fungi in primary atrophic rhinitis.

BACKGROUND: Fungal rhinosinusitis has gained much attention in recent years. To our knowledge, no previous studies have addressed the role of fungus in primary atrophic rhinitis. STUDY DESIGN: Prospective case study. PATIENTS AND METHODS: All cases of primary atrophic rhinitis presenting to the out-patient department at El-Sahel Teaching Hospital over a five-month period were included in the study. Crusts and purulent secretions removed from patients' nasal cavities underwent microbiological analysis at the Medical Microbiology and Immunology department of the Cairo University Faculty of Medicine. Special emphasis was placed on fungal isolation. RESULTS: Fourteen consecutive cases of primary atrophic rhinitis were studied in the five-month period starting 26 November 2007. Patients comprised eight females and six males, with an age range of 12 to 65 years (mean 37 years). Microscopy of the crusts and purulent secretions showed pus cells in most of the samples. Klebsiella species were isolated from nine patients (65 per cent), and other bacterial species were isolated in most of the remainder. Fungal elements, most commonly aspergillus species, were isolated in 13 patients (93 per cent). CONCLUSION: It is proposed that the initial trigger for primary atrophic rhinitis is a virulent bacterial infection of the nasal lining, which leads to damage of the ciliated epithelium. This initiates the cascade of events leading to inflammation of the mucosa and submucosa, with secondary pyogenic osteomyelitis of the turbinate bone. The persistence of purulent secretion, within the setting of impaired mucociliary clearance, leads to saprophytic fungal colonisation which contributes greatly to the clinical picture.