Toxigenic Pasteurella Multocida Research Articles

Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran

Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular–LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants.

Pasteurella multocida Toxin Triggers RANKL-Independent Osteoclastogenesis.

Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-α, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.

Comparison of Two Challenge Models of Atrophic Rhinitis in Piglets

Background: Swine atrophic rhinitis (AR) is a multiple chronic respiratory diseasemainly caused by porcine Bordetella bronchiseptica (Bb) and Toxigenic Pasteurella multocida (T+Pm). There are two challenge models using Bordetella bronchiseptica and Pasteurella multocida. The first model is to treat pigs with Bordetella bronchiseptica followed by Toxigenic Pasteurella multocida infection. Another model is to inoculate pigs with Bordetella bronchiseptica and Pasteurella multocida simultaneously. So far, there is no report about comparison of these two challenge models as for their effectiveness to establish infection. Methods: Thirteen 42 day-old piglets were divided into three groups. Pigs in the first group were challenged with Bordetella bronchiseptica followed by Toxigenic Pasteurella multocida infection (model 1). Pigs in the second group were challenged with Bordetella bronchiseptica and Pasteurella multocida at the same time (model 2). Pigs in the third group worked as sterile controls. Clinical symptoms, turbinate lesions, lung lesions and daily bodyweight gain were used as parameters to evaluate the effectiveness of above challenge models. Results: All challenged piglets showed different degrees of clinical symptoms, turbinate lesions, lung lesions and loss of average daily bodyweight gain. There was no significant difference in clinical symptoms and lung lesions between two challenge models. However, significant differences in turbinate lesion score and average daily gain were observed between these two challenge models. Discussion and Conclusion: Turbinate lesions score of piglets in first group ranged from 4 to 10 and only 1/5 of piglets had a total turbinate score of 10. By contrast, turbinate lesions score of piglets in the second group ranged from 8 to 16 and 4/5 of piglets had a total turbinate score of equal or above to 10. Therefore, all above data indicated that co-infection of Bordetella bronchiseptica and Pasteurella multocida was more suitable for to establish AR infection model.

Evaluation of an Experimental Vaccine for Atrophic Rhinitis in Piglets

Background: Commercial swine atrophic rhinitis (AR) vaccine contains killed Bordetella bronchiseptica and P. multocida toxin (PMT) from Toxigenic Pasteurella multocida. The recombinant PMT proteins used as vaccine candidates were seldom reported. Methods: In this study, we developed recombinant fragments of PMT to replace P. multocida toxin in AR vaccine to compare the efficacy of this vaccine candidate with the commercial vaccine. Results: The experimental AR vaccine showed similar efficacy after bacterial challenges since there were no significant differences in clinical symptoms, turbinate lesions, lung lesions and daily body gain between the two vaccinated groups. Discussion and conclusion: The replacement of recombinant fragments of PMT to P. multocida toxin in AR vaccine provided same protection as commercial vaccine after bacterial challenges. However, high yield and simple processing of recombinant fragments of PMT from E.coli cells to replace purified P. multocida toxin in AR vaccine will dramatically reduce the cost of vaccines and simplify the antigen preparation.

A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

BackgroundProgressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs.ResultsIn comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR.ConclusionsThe approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13028-016-0267-7) contains supplementary material, which is available to authorized users.

Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation.

BackgroundPasteurella multocida toxin (PMT) is a potent inducer of osteoclast formation. Pigs suffering from an infection with toxigenic Pasteurella multocida strains develop atrophic rhinitis characterised by a loss of turbinate bones and conchae. However, on the molecular level the process of bone loss remains largely uncharacterised.ResultsRecently it was found that PMT activates the serine/threonine kinase mammalian target of rapamycin (mTOR) in fibroblasts. Using RAW264.7 macrophages, we investigated the role of the mTOR complex 1 (mTORC1) in PMT-mediated osteoclast formation. PMT induces the differentiation of RAW264.7 macrophages into multinucleated, tartrate resistant acid phosphatase (TRAP) positive osteoclasts that are capable to resorb bone. In the presence of the mTORC1 inhibitor rapamycin, PMT was significantly less able to induce the formation of TRAP-positive osteoclasts. Accordingly, the resulting resorption of bone was strongly reduced. A major target of mTOR is the 70 kDa ribosomal protein S6 kinase 1 (p70 S6K1). Activated p70 S6K1 decreases the expression of programmed cell death protein 4 (PDCD4), a negative transcriptional regulator of osteoclastogenesis, at the protein and gene level. Ultimately this results in the activation of c-Jun, a component of the activator protein 1 (AP-1) complex, which is a major transcription factor for the induction of osteoclast-specific genes. We now demonstrate that c-Jun and its downstream target, the osteoclast-specific bone degrading protease cathepsin K, are upregulated upon PMT treatment in an mTOR-dependent manner.ConclusionsActivation of mTOR signalling plays a central role in the formation of osteoclasts through the bacterial toxin PMT. On the molecular level, PMT-induced activation of mTOR leads to down regulation of PDCD4, a known repressor of AP-1 complex, culminating in the activation of c-Jun, an essential transcription factor for triggering osteoclastogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0117-7) contains supplementary material, which is available to authorized users.

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

BackgroundAtrophic 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.ResultsB. 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.ConclusionsB. 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.

Kinetics of the response of four positive acute phase proteins in pigs experimentally infected with toxigenic Pasteurella multocida

In the present study, acute phase proteins (APP) responses in pigs after infection with toxigenic strain of Pasteurella multocida (Pm) were evaluated. Twelve piglets from a herd free from toxigenic Pm were used. Six of them were infected intranasally with Pm. CRP, Hp, SAA and Pig-MAP concentrations were measured using commercial ELISA tests. CRP and Hp were significantly induced from 2 days post inoculation (dpi). The concentration of Hp in inoculated pigs remained elevated until the end of the study. The concentrations of SAA and Pig-MAP increased significantly from 3dpi, and remained elevated to 5 or 7dpi, respectively. Strong correlations were observed between concentration of Hp or SAA and changes in the lungs. No correlations were found between levels of APP in serum and changes observed in the turbinates. On the basis of our investigation, we cannot state that evaluation of APP concentrations in serum may provide useful information about severity of atrophic rhinitis. However, the concentration of all investigated APP increased after inoculation. Thus, APP-measurements may reveal ongoing infection. Monitoring of APP concentrations in the pig herds may help to pinpoint infected animals even before clinical signs are present. If found, these “APP-high” animals may be chosen next for pathogen-specific diagnostics. Early diagnosis and therapy may prevent the infection from spreading in the herd. Moreover, monitoring of APP concentrations in serum may be useful for selecting clinically healthy pigs before integration into an uninfected herd. Future studies should focus on the possibility of distinguishing infected and non-infected pigs under field conditions.

The occurrence of Bordetella bronchiseptica in pigs with clinical respiratory disease

Between January 2003 and September 2008, 652 Bordetella bronchiseptica isolates were cultured from 3506 lung samples collected from pigs with respiratory disease. Over the 6-year period, the average isolation rate was 18.6%, making B. bronchiseptica the fourth most frequently isolated pathogenic bacterium from those lung samples. The isolation rates in different years and provinces ranged from 15.2% to 25.7% and 17.3% to 20.7%, respectively. There were significant influences of sampling month and pig age on bacterial isolation (P<0.05). Streptococcus suis (29.9%), Haemophilus parasuis (26.7%) and Escherichia coli (21.6%) were isolated most frequently in association with B. bronchiseptica. All 12 toxigenic Pasteurella multocida strains co-isolated with B. bronchiseptica from 63 cases of atrophic rhinitis were classified into serogroup D. The results suggest that B. bronchiseptica infection is highly prevalent in pig farms in China, and is often accompanied by co-infection with other bacteria.

Pasteurella multocida toxin is a potent activator of anti-apoptotic signalling pathways

Toxigenic Pasteurella multocida strains produce a 146 kDa protein toxin (PMT) that due to its high mitogenic activity is thought to possess carcinogenic properties. PMT affects several signal transduction pathways related to cancer by constitutively stimulating heterotrimeric G proteins. Downstream of Galpha(q), Galpha(13) and Galpha(i), the toxin activates the small GTPase RhoA, MAP kinases and signal transducer and activator of transcription (STAT) proteins. PMT also stimulates Gbetagamma signalling and activates phosphoinositide 3-kinase (PI3K)-related pathways, which play a crucial role in proliferation and apoptosis. We show that treatment of HEK293 cells with PMT inhibits staurosporine-mediated apoptosis through PI3K-dependent phosphorylation of Akt and constitutive expression of Pim-1 kinase. Simultaneous activation of these survival kinases allows the activation of pro-survival pathways, such as GSK3beta, Mcl-1, Bcl-xL and Bcl-2, as well as the downregulation of apoptotic signals by Bax or Puma. Only the combined inhibition of Akt and Pim reverses the PMT-induced protection from staurosporine-induced apoptosis. In addition, we show that apoptosis induced by tumour chemotherapeutic agents is blocked by PMT in human cancer cell lines. Our data indicate that PMT is a highly potent anti-apoptotic agent, which supports the view of a carcinogenic potential of the toxin.

Expression of the dermonecrotic toxin by Bordetella bronchiseptica is not necessary for predisposing to infection with toxigenic Pasteurella multocida

This experiment was designed to determine whether a Bordetella bronchiseptica mutant that does not produce dermonecrotic toxin (DNT) is still capable of predisposing pigs to infection with toxigenic Pasteurella multocida. Three groups of pigs were initially inoculated intranasally with a wild type B. bronchiseptica that produces DNT, an isogenic mutant of B. bronchiseptica that does not produce DNT, or PBS. All pigs were then challenged intranasally with a toxigenic strain of P. multocida 4 days later. P. multocida was recovered infrequently and in low numbers from pigs initially inoculated with PBS, and no turbinate atrophy was present in these pigs. P. multocida was isolated in similar numbers from the pigs initially inoculated with either the wild type or the DNT mutant of B. bronchiseptica, and turbinate atrophy of a similar magnitude was also seen in pigs from both of these groups. Thus, although the DNT has been shown to be responsible for much of the pathology seen during infection with B. bronchiseptica by itself, infection with non-DNT-producing strains can still predispose to secondary respiratory infections with P. multocida.

Evaluation of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) using Recombinant Toxin for Detection of Antibodies against Pasteurella multocida Toxin

To facilitate the control of progressive atrophic rhinitis (PAR) of swine caused by toxigenic Pasteurella multocida, an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (NT) have recently been developed to detect antibodies against the P. multocida dermonecrotic toxin (PmDNT). However, the NT is a cumbersome and time-consuming technique. To overcome these drawbacks, we developed an indirect ELISA, using recombinant PmDNT expressed in Escherichia coli, for the detection of antibodies to PmDNT in serum samples from pigs. The practical usefulness of this ELISA was compared with the NT using serum samples obtained from experimentally infected and naturally infected pigs. In the pigs experimentally inoculated with vaccine including PmDNT toxoid, the ELISA and neutralization antibodies were detected at almost the same time, and a good correlation was demonstrated between both tests (P<0.01, R(2)=0.807). Therefore, the ELISA can be used to evaluate the immune reaction of pigs after vaccination with P. multocida toxoid. In a survey conducted on a field herd with a history of clinical AR, the seropositivity by ELISA in pigs of age 4.5-6 months was increased even though the NT was negative, and the correlation was low between the results obtained with the two tests (P<0.01, R(2)=0.38). Therefore, the results indicated that this ELISA might be a useful alternative to the NT currently used to detect the antibody to PmDNT after vaccination or infection with P. multocida.

Clinical and pathological responses of weaned pigs to atmospheric ammonia and dust

Nine hundred and sixty weaned pigs were exposed for five weeks to controlled concentrations of atmospheric ammonia and dust in a single, multifactorial experiment. The treatments were a mean dust...

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.

Objective Measurement of Health in Pigs – Application of Acute Phase Proteins

In this presentation health is defined as absence of disease in individual animals or populations. High health status indicates that no or only mild disease is present. Low health status indicates that several or severe diseases are present. Measurement of health status. Objective measurement of health status is difficult. Serological or microbiological testing for a number of specific infections is possible, but often impractical. Clinical examination may reveal clinical disease problems but do not reveal sub-clinical disease an may suffer from lack of objectivity. Sub-clinical disease may be measured indirectly by recording of growth rate, feed consumption or reproductive performance. However, these indicators are difficult to interpret. Pathological findings at slaughter only give a prevalence estimate and data-quality may be poor. Acute phase proteins are produced as a non-specific response to inflammation, trauma and infection in animals and may be measured objectively in serum samples. Why measure health status? Objective measurement of health status or disease severity is important in several situations. It creates an important research tool when studying pathogenesis and inflammatory processes in experimental animals or when testing the efficacy of pharmaceuticals or comparing their effect in experimental or field trials. Objective measurement of health status allows comparison of health in different production. Categorisation of herds in health levels may be relevant in the future. Acute phase proteins in pigs. Acute phase proteins are an important part of the acute phase response following tissue damages. The damage may be induced by infections, toxins, trauma etc. and results in liberation of inflammatory mediators into the systemic circulation. These mediators include cytokines such as IL-1, TNF, IL-4, IL-6, IL-8, IL-10 and IL-11. The cytokines induce increased (positive) or decreased (negative) hepatic synthesis of serum acute phase proteins. Several porcine infections has been shown to mount an acute phase response e.g. Actinobacillus pleuropneumoniae, toxigenic Pasteurella multocida, Mycoplasma hyorhinis, PRRS virus etc. The major acute phase proteins in pigs include haptoglobin, major acute phase protein (MAA), serum amyloid A (SAA), alfa1 acid glycoprotein (AGP) and C-reactive protein. Serum haptoglobin in relation to clinical signs and production systems. In a case-control study in 14 pig herds including serum samples from 170 healthy and clinically diseased pig pairs. A significant increase in haptoglobin concentrations was found in pigs with clinical signs such as respiratory disease, lameness, ear and tail biting and diarrhoea. In another study 7 herds free from 5 specific pathogens (Danish SPF-herds) were compared to 4 conventional herds suffering from some or all of the infections absent in the SPF herds. In each herd serum samples were taken from young, medium or old finishing pigs. At the same time the animals were subjected to a standardised clinical examination. The study showed that finishing pigs from SPF herds had a significantly lower haptoglobin serum concentration than pigs from conventional herds. However, the variation between SPF herds was high, indicating that SPF-production is not always equivalent to high health status.

Effect of Aerial Ammonia on Porcine Infection of the Respiratory Tract with Toxigenic Pasteurella multocida

The objective of the experimental study was to examine whether aerial ammonia alone could predispose the respiratory system of pigs to infection with toxigenic Pasteurella multocida type A. Two groups of 5 pigs each were continuously exposed to 50 ppm ammonia and less than 5 ppm ammonia, respectively, for a 59-day period (from 37 kg to 90 kg bodyweight) followed by necropsy. In an aerosol chamber all pigs were exposed to an aerosol of toxigenic P. multocida type A (mean bacterial concentration in the aerosol-exposure chamber: 10(5) colony forming units/m3; exposure period: 25 min) at day 10, 21, 35 and 49 after the onset of ammonia exposure. During the experiment none of the pigs showed clinical signs of pneumonia nor did they develop visible distortion of the snout. None of the pigs had gross lesions in the lungs at necropsy and toxigenic P. multocida was not detected by culture from the lungs from any of the pigs. The chance of recovering toxigenic P. multocida from nasal swabs (collected during experiment) was 2-4 times greater in the test group compared to the control group. The average daily weight gain was lower for the ammonia exposed pigs compared to the control group. In conclusion the results from this study suggest that ammonia in concentrations of 50 ppm is unlikely to predispose growing pigs to pulmonary infection with toxigenic P. multocida.

Effect of ovalbumin aerosol exposure on colonization of the porcine upper airway by Pasteurella multocida and effect of colonization on subsequent immune function.

Seventy-three piglets were weaned at 1 week of age, randomly assigned to 10 groups (A to J), accommodated in stainless steel exposure chambers, and exposed continuously to a controlled environment containing aerosolized ovalbumin. The concentrations of ovalbumin dust were as follows (milligrams per cubic meter): A and F, 16.6; B and G, 8.4; C and H, 4.2; D and I, 2.1; E and J, 0. At weekly intervals, the pigs were bled via venipuncture and anesthetized for nasal lavage and tonsilar biopsies performed for subsequent bacteriologic analysis. At 2 weeks of age, the pigs in groups A to E were challenged with toxigenic Pasteurella multocida (10(8) CFU pig(-1)), and at 6 weeks of age, the pigs were euthanatized. At postmortem, the extent of turbinate atrophy was assessed on the snout sections by using a morphometric index. Exposure to aerial ovalbumin resulted in a dose-dependent increase in serum antiovalbumin immunoglobulin G (IgG; P < 0.001) and serum antiovalbumin IgA (P < 0.001). Exposure also caused a significant increase in the numbers of P. multocida organisms isolated from the upper respiratory tract (P < 0.001) and a corresponding increase in turbinate atrophy, as judged by the morphometric index (P < 0.001). Concurrent challenge with P. multocida and ovalbumin resulted in a significant decrease in both the IgG and IgA responses to ovalbumin (P < 0.001). These results show that ovalbumin exposure increases pig susceptibility to P. multocida colonization and that toxigenic P. multocida modifies the serum IgG and IgA responses to ovalbumin in the pig. Both of these effects may enhance the virulence of this respiratory pathogen and so influence the pathogenesis of atrophic rhinitis in pigs.

Effects of ammonia inhalation and acetic acid pretreatment on colonization kinetics of toxigenic Pasteurella multocida within upper respiratory tracts of swine.

Pigs reared in intensive production systems are continuously exposed to ammonia released by the microbial degradation of their excrement. Exposure to this gas has been shown to increase the severity of the disease progressive atrophic rhinitis by facilitating colonization of the pig's upper respiratory tract by Pasteurella multocida. The etiological mechanism responsible for this synergy was investigated by studying the colonization kinetics of P. multocida enhanced by ammonia and comparing them with those evoked by an established disease model. Three-week-old Large White piglets were weaned and allocated to five experimental groups (groups A to E). Pigs in groups A and B were exposed continuously to ammonia at 20 ppm for the first 2 weeks of the study. Pigs in group C were pretreated with 0.5 ml of 1% acetic acid per nostril on days -2 and -1 of the study. On day 0 all the pigs in groups A, C, and D were inoculated with 1.4 x 10(8) toxigenic P. multocida organisms given by the intranasal route. The kinetics of P. multocida colonization were established by testing samples obtained at weekly intervals throughout the study. The study was terminated on day 37, and the extent of turbinate atrophy was determined by using a morphometric index. The results of the study showed that exposure to aerial ammonia for a limited period had a marked effect on the colonization of toxigenic P. multocida in the nasal cavities of pigs, which resulted in the almost total exclusion of commensal flora. In contrast, ammonia had only a limited effect on P. multocida colonization at the tonsil. The exacerbation of P. multocida colonization by ammonia was restricted to the period of ammonia exposure, and the number of P. multocida organisms colonizing the upper respiratory tract declined rapidly upon the cessation of exposure to ammonia. During the exposure period, the ammonia levels in mucus recovered from the nasal cavity and tonsil were found to be 7- and 3.5-fold higher, respectively, than the levels in samples taken from unexposed controls. Acetic acid pretreatment also induced marked colonization of the nasal cavity which, in contrast to that induced by ammonia, persisted throughout the time course of the study. Furthermore, acetic acid pretreatment induced marked but transient colonization of the tonsil. These findings suggest that the synergistic effect of ammonia acts through an etiological mechanism different from that evoked by acetic acid pretreatment. A strong correlation was found between the numbers of P. multocida organisms isolated from the nasal cavity and the severity of clinical lesions, as determined by using a morphometric index. The data presented in the paper highlight the potential importance of ammonia as an exacerbating factor in respiratory disease of intensively reared livestock.

EFFECT OF CHRONIC EXPOSURE TO GASEOUS AMMONIA ON THE NASAL TURBINATES OF GNOTOBIOTIC PIGS

Pigs reared in and stock persons working in intensive production systems are continuously exposed to ammonia released by microbial degradation of animal excrement. Experimental studies have shown that exposure to this gas, at concentrations comparable to those encountered in buildings used for the intensive rearing of swine, increases the severity of the clinical disease progressive atrophic rhinitis in pigs by facilitating colonization of the upper respiratory tract by toxigenic Pasteurella multocida. During the course of these studies it was observed that mild turbinate atrophy also occurred in pigs from control groups exposed to ammonia but maintained free from P. multocida and Bordetella bronchiseptica. To determine whether exposure to ammonia is detrimental to the normal anatomical development of the mammalian nasal cavity, a study was conducted using gnotobiotic piglets. Twenty-one gnotobiotic piglets were derived from two sows by hysterectomy. Each litter was split into two groups, and the four groups were accommodated separately in sterile positive-pressure isolators supplied with high-efficiency particulate air (HEPA) filtered air. From 1 wk of age onward the air in the isolators housing one of the groups from each litter was modified by the addition of ammonia at a concentration of 9.1 and 15.7 ppm. The air supply to the isolators housing the littermate groups was not modified. At 6 wk of age all the pigs were euthanatized. The effect of ammonia exposure on the morphology of the nasal cavity of the pig was assessed by image analysis of a cross section of the pig's snout. Pigs exposed to ammonia were found to have a mild but statistically significant level of turbinate atrophy when compared to nonexposed lit termates. Histological examination revealed that prolonged ammonia exposure evoked changes to the mucous membranes lining the nasal cavity, characterized by epithelial hyperplasia with micro-abscess formation, goblet-cell hypoplasia, and inflammatory cell infiltration. Mild degenerative changes within the bony core of the ventral turbinate were also apparent, with a decline in the population of osteoblasts and simultaneous osteoclast proliferation. Group analysis revealed a correlation between the severity of turbinate atrophy and the increase in the number of osteoclasts per unit area of bony core (p<.05). These findings could explain why mild turbinate atrophy is seen on some pig units deemed free from pathogenic bacteria associated with the clinical disease atrophic rhinitis.

Direct PCR analysis for toxigenic Pasteurella multocida.

A more rapid, accurate method to detect toxigenic Pasteurella multocida is needed for improved clinical diagnosis, farm biosecurity, and epidemiological studies. Toxigenic and nontoxigenic P. multocida isolates cannot be differentiated by morphology or standard biochemical reactions. The feasibility of using PCR for accurate, rapid detection of toxigenic P. multocida from swabs was investigated. A PCR protocol which results in amplification of an 846-nucleotide segment of the toxA gene was developed. The PCR amplification protocol is specific for toxigenic P. multocida and can detect fewer than 100 bacteria. There was concordance of PCR results with (i) detection of toxA gene with colony blot hybridization, (ii) detection of ToxA protein with colony immunoblot analysis, and (iii) lethal toxicity of sonicate in mice in a test set of 40 swine diagnostic isolates. Results of an enzyme-linked immunosorbent assay for ToxA agreed with the other assays except for a negative reaction in one of the 19 isolates that the other assays identified as toxigenic. In addition to accuracy, as required for a rapid direct specimen assay, toxigenic P. multocida was recovered efficiently from inoculated swabs without inhibition of the PCR. The results show that PCR detection of toxigenic P. multocida directly from clinical swab specimens should be feasible.