Atrophic Rhinitis In Pigs Research Articles

Differentiation of Pasteurella multocida isolates from cases of atrophic rhinitis in pigs from Zimbabwe by RAPD and ribotyping

Atrophic rhinitis in pigs is rarely reported in Southern Africa. To determine the relationship between Pasteurella multocida clones from clinical cases of atrophic rhinitis, twenty-one strains were characterised by selected phenotypic and genotypic methods. Biochemical analysis classified 18 strains as P. multocida subspecies multocida, whilst the remainder were grouped into separate unassigned biotypes. Capsular groups A (16/21) and D (l/21) were found among the isolates by PCR. Four ribotype patterns were obtained following HpaII ribotyping, whilst random amplification of polymorphic DNA (RAPD) revealed three main clusters. However, subclusters were also noted for each RAPD cluster. Our results indicate that RAPD offers a better discrimination of strains than ribotyping and that none of the phenotypic characters were directly related to the genotypic clusters.

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 80 kDa 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 72 h 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.

Protection of piglets against atrophic rhinitis by vaccinating the sow with a vaccine against Pasteurella muftocida and Bordetelia bronchiseptica

The efficacy of a new vaccine against atrophic rhinitis in pigs was tested in the Netherlands and Denmark. The vaccine contained protein dO (a truncated Pasteurella multocida toxin which is...

The capsule biosynthetic locus of Pasteurella multocida A:1.

Pasteurella multocida is the aetiological agent of fowl cholera, bovine haemorrhagic septicaemia and atrophic rhinitis in pigs. Many strains of P. multocida express a capsule on their surface. However, nothing is known about the capsule biosynthetic locus in P. multocida although the capsule has been implicated as a virulence factor. The entire capsule locus of P. multocida A:1 was cloned and sequenced. The locus is divided into three regions. Region 1 comprises four ORFs which are involved in the transport of the capsule polysaccharide to the surface. Region 2 comprises five ORFs whose postulated protein products are involved in the biosynthesis of the polysaccharide capsule. Region 3 comprises two ORFs whose postulated products show similarity to proteins that are involved in the phospholipid substitution of the polysaccharide capsule.

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.

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.

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.

Intranasal administration of Pasteurella multocida toxin in a challenge-exposure model used to induce subclinical signs of atrophic rhinitis in pigs

Summary A challenge-exposure model was developed for dose-dependent induction of subclinical (moderate) atrophic rhinitis (ar) in conventionally raised Dutch Landrace and Large White pigs, about 4 weeks old. Under favorable climatic and housing conditions, pigs were intranasally challenge-exposed with Pasteurella multocida-derived toxin (Pm-T) 3 days after pretreatment by inoculation with 1% acetic acid. Pigs were challenge-exposed with 1 of the following Pm-T doses: 0 (control), 5, 13, 20, or 40 μg of Pm-T/ml of phosphate-buffered saline solution (pbss), 0.5 ml/ nostril/d on 3 consecutive days. Five weeks after challenge exposure, subclinical (moderate) ar status was defined as intermediate conchal atrophy (grade 2 for ventral conchae on a 0 to 4 scale and grade 1 or 2 for dorsal conchae on a 0 to 3 scale, respectively) and perceptible difference in change in brachygnathia superior (cbs) between control and challenge-exposed pigs between the beginning and end of the study. All Pm-T-exposed pigs had nasal damage that was dose-dependent. The higher Pm-T doses resulted in higher ventral conchae atrophy and dorsal conchae atrophy scores. The CBS increased with applied Pm-T dose, resulting in significant (P &lt; 0.05) differences between controls (3.88 mm) and the 13-, 20-, and 40-μg Pm-T-treated groups (7.77, 6.58, and 7.98 mm, respectively). In response to the applied dose, weight gain per week for Pm-T-exposed pigs was lower than that of controls after week 3 (P &lt; 0.01). Difference from controls was 32, 54, 52, and 96 g/d/pig for 5-, 13-, 20-, and 40-μg Pm-T-treated groups respectively, in the last 2 weeks. For Dutch Landrace and Large White pigs, intranasally administered Pm-T mimicked the pathogenic effect of in vivo infection with toxigenic Pm strains. The optimal model to induce subclinical ar appeared to be 13 μg of Pm-T/ml (0.5 ml/nostril/d) on 3 consecutive days. Our model should enable studies of exogenous and endogenous factors involved in development of ar, independent of the colonizing ability of the Pm strain used.

Nasal epithelial changes induced in piglets by acetic acid and by Bordetella bronchiseptica

Research on atrophic rhinitis of pigs has shown that both Bordetella bronchiseptica infection and experimental treatment with acetic acid predispose the nasal mucosa to colonization with Pasteurella multocida. Gnotobiotic piglets aged 3 days were dosed intranasally with either B. bronchiseptica (n = 6) or acetic acid 1 per cent (n = 10) and killed at intervals up to the 4th day after treatment. Samples of the ventral turbinates were examined by light microscopy and scanning and transmission electron microscopy. Within 12 h acetic acid induced loss of cilia, oedema, focal cell exfoliations, mitochondrial swelling and inflammatory cell infiltration. Bordetella bronchiseptica induced only a limited oedema and loss of cilia. Colonization of cilia by the bacteria was observed 96 h after infection. We conclude that, although acetic acid and B. bronchiseptica do not induce the same modifications of the nasal respiratory epithelium, their action causes stagnation of nasal mucus, which results in a nasal environment favourable to colonization by Pasteurella multocida.

Effect of Pasteurella multocida toxin on bone resorption in vitro.

Pasteurella multocida toxin (PMT), which is the primary etiologic factor in the pathogenesis of progressive atrophic rhinitis in pigs, was found to stimulate bone resorption in vitro. This stimulation was observed both in cultures of murine calvaria by measuring the release of calcium and of the lysosomal enzyme beta-glucuronidase and in murine long bone cultures by measuring the release of calcium. Both systems showed the same dose response curve, with the maximal effect at a concentration of 5 ng/ml. The effect on calvaria was studied in more detail. PMT increased bone resorption 24 h after its addition and always had to be present to express an effect. Calcitonin was able to inhibit this increase of resorption completely, and inhibitors of prostaglandin synthesis suppressed it partially. Although the data show an effect of PMT on bone tissue, the results do not exclude an action on cells in the nasal cavity, which could indirectly stimulate bone resorption.

Detection of stable epitopes on formaldehyde-detoxified Pasteurella multocida toxin by monoclonal antibodies

Progressive atrophic rhinitis in pigs can be prevented by vaccination of pregnant sows with formaldehyde-detoxified preparations of either crude extract of toxigenic Pasteurella multocida or purified P. multocida toxin (PMT). The protective value of a vaccine is expected to be related to its content of detoxified immunogenic PMT, but previously described methods for detection of PMT cannot be used for the quantification of formaldehyde-detoxified PMT. In contrast to the epitopes on PMT recognized by previously produced anti-PMT monoclonal antibodies (mAbs), two of the epitopes recognized by mAbs developed in this study showed a significant stability after treatment with formaldehyde under conditions relevant for production of vaccines. When analysed in a sandwich ELISA based on these two mAbs, the titre of a preparation of PMT, detoxified by treatment with 1% (w/v) formaldehyde for 48 h at 20°C, decreased by <30% when compared to the titre of native PMT. A close relationship between the amount of formaldehyde-treated PMT in a vaccine determined by the sandwich ELISA and its immunogenic properties in mice was observed.

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

Recombinant derivatives of Pasteurella multocida toxin: candidates for a vaccine against progressive atrophic rhinitis

Potential vaccine components for protection against atrophic rhinitis in pigs were developed. This was achieved by deletion mutagenesis of the gene encoding the Pasteurella multocida toxin. Four purified toxin derivatives lacking different and widely separated regions in the amino acid sequence were characterized by a lack of toxic activity. One such component was shown to induce efficient protection of vaccinated female mice and their offspring against challenge with purified P. multocida toxin.

A Survey of the concentration of ammonia recorded in pig housing in England

In animal housing ammonia is an aerial pollutant resulting from the microbial conversion of nitrogenous materials excreted in faeces and urine. Ammonia is an irritant to cells lining the respiratory tract and an increased incidence of pneumonia in pigs and humans and atrophic rhinitis in pigs have been correlated with elevated ammonia levels. In a survey of 12 farms and 49 pig buildings concentration of ammonia was identified specifically as a causative agent in the aetiology of atrophic rhinitis (Robertson, Wilson and Smith 1990). Furthermore, high levels of ammonia can delay the onset of puberty in gilts perhaps as a result of the masking of boar odour (Malayer, Brandt, Green, Kelly, Sutton and Diekman 1988).Regulations have been introduced (Control of Substances Hazardous to Health (COSHH)) limiting the exposure of humans to atmospheric ammonia to a mean level of 25 ppm over an 8h work shift and 35 ppm for 10 minute exposure. As part of a study on the effectiveness of a feed additive formulated to control atmospheric ammonia, levels of ammonia in pig housing were monitored.

Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida

SUMMARY Heat-labile toxin from a cell sonicate of a virulent typeD strain of Pasteurella multocida was purified by ammonium sulfate precipitation followed by ion exchange chromatography, gel filtration chromatography, and Polyacrylamide gel electrophoresis. Toxic activity was assayed during toxin purification by cytopathic effect in Vero or bovine embryonic lung cell cultures. Toxicity for cells correlated with dermonecrosis in guinea pig skin. Toxicity was accounted for by a single protein with a molecular weight of 149,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbits were inoculated intranasally with purified toxin to determine whether toxin had a role in the induction of pneumonia in rabbits infected with P multocida. Pneumonia, pleuritis, acute hepatic necrosis, and splenic lymphoid atrophy were found in 4 of 5 rabbits. One of 5 rabbits had bilateral turbinate atrophy. Western blotting with monoclonal antibodies to toxin from a P multocida isolate causing atrophic rhinitis in pigs revealed the toxin that induces pleuritis and pneumonia in rabbits to be the same or a closely related toxin.

Colonisation by Pasteurella multocida in atrophic rhinitis of pigs and immunity to the osteolytic toxin

Gnotobiotic pig antisera to purified toxoid from a capsule type A or D strain of Pasteurella multocida contained large quantities of antitoxin but comparatively little antibody to a crude lysate of P. multocida. These sera given intraperitoneally to further pigs were almost completely protective against turbinate atrophy after intranasal inoculation of dilute acetic acid and infection with type D toxigenic P. multocida. In contrast, antisera to a crude lysate or bacterin of toxigenic P. multocida which contained large titres of antibody to P. multocida lysate, but no detectable antitoxin, were not protective. Colonisation by toxigenic P. multocida was significantly reduced in protected pigs and was similar to colonisation by nontoxigenic P. multocida in pigs untreated or treated with dilute acetic acid. These results indicated (1) that antitoxin was protective and cross protective between toxins from different capsule types; and (2) that the toxin was the main colonisation factor produced by toxigenic bacteria in the acetic acid model of infection and that immunity to it did not eliminate infection.

Diagnosis of atrophic rhinitis by computerised tomography: a preliminary report.

Computerised tomography, used as a diagnostic tool for atrophic rhinitis in pigs, facilitated the macroscopic grading of the nasal structures in live pigs of any age. The results of sequential scans in normal and affected pigs are described; transient atrophy of the ventral conchae was observed in one pig.

A comparison of different challenge methods for induction of atrophic rhinitis in pigs

Transmission and development of atrophic rhinitis (AR) was studied in 5- to 15-week-old pigs (Groups 2-7) originating from a herd free of AR, and compared to unexposed healthy pigs (Group 1), and pigs from a herd with endemic AR (Group 8). At the start of the trial, pigs in Groups 2-5 were challenged intranasally twice a week for 3 weeks with pure cultures of bacteria originating from the endemic AR herd: Nontoxigenic Pasteurella multocida type A (PmA) plus Bordetella bronchiseptica phase I (Bb) (Group 2); PmA + toxigenic Pm type D (PmD) (Group 3); PmD only (Group 4); and PmD + Bb (Group 5). Group 6 pigs were challenged with nasal wash of pigs from the endemic AR herd, and Group 7 pigs were challenged by being housed together in the same pen with Group 8 pigs throughout the study. Nasal swabs of all pigs were cultured 5 times during the study. Serum was collected at 6 weeks post challenge. Average daily gain (ADG) and turbinate lesions (turbinate gross lesions by visual scoring and by Turbinate Perimeter Ratio, TPR, scoring, and histopathological lesions) were measured at the time of slaughter at 15 weeks of age. Mean TPR value for the Group 1 pigs was 1.64, which was significantly (P less than 0.05) different from the mean TPR value of 0.58 for the pigs from the endemic AR herd (Group 8), the 0.79 value for Group 6 pigs, and 1.03 value for Group 7 pigs. Of pigs challenged with pure bacterial cultures, only Group 5 (PmD + Bb) developed significant AR (mean TPR = 1.24). Only one pig in each of Groups 2 and 3, and two pigs in Group 4 showed TPR values indicative of AR (TPR less than 1.30). However, histopathological examination showed that those pigs were recovering from the infection 7 weeks post challenge. Constant exposure to certain bacteria or other factors in nasal washings, stress of crowding or poor environmental conditions might be required to experimentally produce AR in 5-week and older pigs similar to that in naturally infected pigs. There was no relationship between turbinate lesions and the isolation frequency or quantity of PmA, PmD, or Bb. Antibody levels against PmA or PmD had moderate to high correlation with TPR values (r = -0.694 and -0.503 respectively). ELISA values also corresponded well with the type of bacteria inoculated in each group of pigs and appeared to be a sensitive test for PmA, PmD, and Bb infections in pigs.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and expression of the Pasteurella multocida toxin gene, toxA, in Escherichia coli.

A chromosomal DNA library of a toxigenic type D strain of Pasteurella multocida subsp. multocida was established in Escherichia coli. From this library two clones, SPE308 and SPE312, were identified by using a monoclonal antibody against the osteoclast-stimulating P. multocida toxin (PMT). Extracts of these clones showed cytopathic activity identical to that of extracts of toxigenic P. multocida. The recombinant plasmids, pSPE308 and pSPE312, directed the synthesis of a protein with an apparent molecular weight of 143,000 which could be specifically detected by anti-PMT antibody. The recombinant toxin, which was located in the cytoplasm of E. coli, was purified by affinity chromatography with immobilized monoclonal antibody and was shown to react in a manner identical to that of PMT in a quantitative structural test using a series of monoclonal antibodies as well as in all quantitative functional tests used, i.e., tests for dermonecrotic activity and mouse lethality and the embryonic bovine lung cell test for cytopathic activity. The gene encoding this toxic activity was named toxA and was found to be present in the chromosome of toxigenic strains only of P. multocida. A probe spanning the toxA gene therefore has potential in the diagnosis and surveillance of progressive atrophic rhinitis in pigs.

Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs

Three strains of Bordetella bronchiseptica were compared for their ability to assist colonisation of the nasal cavity of gnotobiotic pigs by toxigenic Pasteurella multocida. Toxigenic P multocida (counted in nasal washings) colonised the cavity in large numbers in pigs previously infected with a cytotoxic phase I strain of B bronchiseptica (B58), whereas it colonised only in small numbers in those previously infected with B65, a phenotypic phase III variant of B58. Toxigenic P multocida colonised pigs infected with a non-cytotoxic phase I strain of B bronchiseptica (PV6) in fewer numbers than were seen in pigs infected with the cytotoxic phase I strain but in greater numbers than in pigs infected with the phase III strain. The turbinates of pigs infected with the cytotoxic phase I strain of B bronchiseptica and toxigenic P multocida were most severely affected and those in pigs infected with the non-cytotoxic phase I strain and toxigenic P multocida were moderately reduced in size. The turbinates of pigs infected with the phase III strain and toxigenic P multocida were slightly reduced in size except for one piglet whose turbinates were severely affected. Pigs infected with the non-cytotoxic phase I strain of B bronchiseptica alone showed no signs of atrophy and their turbinates were used to calculate reductions (per cent) in those infected with P multocida. The reduction (per cent) in size of turbinates and total numbers of P multocida isolated from the nasal washings of each pig were linearly related.(ABSTRACT TRUNCATED AT 250 WORDS)