A protein-based subunit vaccine with biological adjuvants provides effective protection against Pasteurella multocida in pigs.

Streptococcus suis (S. suis) and Pasteurella multocida (P. multocida) are pathogens that can cause zoonotic diseases. P. multocida toxin (PMT) is an important virulence factor that causes atrophic rhinitis in pigs. Suilysin (Sly) is an extracellular protein of S. suis and has been shown to be a potential adjuvant. Previous studies have indicated that subunit vaccines containing several fragments of PMT as antigens are safer than traditional inactivated or live-attenuated vaccines. However, protein-based vaccines need strong adjuvants to enhance their immunogenicity. In this study, recombinant PMT-NC (rPMT-NC) protein antigen was formulated with either recombinant Sly (rSly) or CpG oligodeoxynucleotides (CpG) as the adjuvant. The immune responses elicited by these vaccines and the protective efficacy after challenge with live P. multocida were evaluated in piglets. In the dose-dependent test, piglets immunized with the low dose (100 µg) of rSly had increased antigen-specific total IgG, interferon (IFN)-γ gene expression, and CD4+ and CD8+ T-cell populations. Compared to piglets in the commercial (Al-gel) adjuvant and the control groups (p < 0.05), piglets in the biological adjuvant groups showed significantly reduced turbinate atrophy, nasal distortion, and lung lesion scores after challenge with P. multocida serotype A. Vaccines containing rSly or CpG adjuvant enhanced humoral and cellular immune responses and protection against P. multocida. This combination of a protein-based antigen formulated with a biological adjuvant showed synergistic and protective effects against atrophic rhinitis and has potential to be developed as part of a bivalent vaccine.

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.

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.

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.

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.

Prevalence of clinical signs of disease in Danish finisher pigs.

Between December 1999 and February 2001, two visits, eight weeks apart, were made to 90 herds of Danish finisher pigs. The prevalence of clinical signs was recorded by three veterinary technicians from the Danish Bacon and Meat Council according to a standardised procedure; they had been trained and their observations were monitored and validated before and during the study. A total of 154,347 finisher pigs were examined and 22,136 clinical signs were recorded. Vices accounted for 43 per cent of the signs. The highest mean prevalence was observed for ear necrosis (4.44 per cent), followed by respiratory signs (2.17 per cent), lameness (1.92 per cent), other skin diseases (1.73 per cent), tail bites (1.26 per cent), umbilical hernia (0.78 per cent), flank bites (0.52 per cent), diarrhoea (0.27 per cent), respiratory distress (0.12 per cent), atrophic rhinitis (0.10 per cent), recumbency (0.09 per cent) and central nervous disease (0.05 per cent). The prevalence of atrophic rhinitis was higher in conventional herds than in specific pathogen-free herds. The prevalence of clinical signs of atrophic rhinitis was higher among finishers weighing 51 to 75 kg than among finishers weighing up to 50 kg, and the prevalence of respiratory signs was higher among finishers weighing 51 to 75 kg then among finishers weighing 76 to 100 kg.

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.

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.

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.

Analytical verification of a multiplex PCR for identification of Bordetella bronchiseptica and Pasteurella multocida from swine.

Bordetella bronchiseptica and Pasteurella multocida are etiologic agents of progressive atrophic rhinitis (PAR) and bronchopneumonia in swine. Only dermonecrotic toxin-producing strains of P. multocida play a role in atrophic rhinitis while both toxigenic and nontoxigenic strains have been associated with pneumonia. Monitoring and investigation of outbreaks involving these bacteria require sensitive and accurate identification and reliable determination of the toxigenic status of P. multocida isolates. In the present study, we report the development, optimization, and performance characteristics of a multiplex PCR assay for simultaneous amplification of up to three different targets, one common to all P. multocida strains, one found only in toxigenic P. multocida strains, and one common to B. bronchiseptica strains. Based on analysis of 94 P. multocida isolates (31 toxigenic) and 126 B. bronchiseptica isolates assay sensitivity is 100% for all amplicons. Evaluation of 22 isolates of other bacterial genera and species commonly found in the swine respiratory tract demonstrated a specificity of 100% for all gene targets. The limit of detection for simultaneous amplification of all targets is 1-10pg of DNA per target, corresponding to a few hundred genomes or less. Amplicon mobility in agarose gels and sequence analysis indicate the amplicons are highly stable. The data presented establish this multiplex PCR as a reliable method for identification of B. bronchiseptica and both toxigenic and nontoxigenic P. multocida that may greatly simplify investigations of swine PAR and bronchopneumonia.

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

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

Association of growth performance with atrophic rhinitis and pneumonia detected at slaughter in a conventional pig herd in Hungary.

The influence of atrophic rhinitis (AR) and pneumonia on growth performance was assessed in a conventional farrow-to-finish pig farm affected by both diseases. All tested pigs (n = 138) were individually weighed at birth, at weaning, at moving to the growing/finishing unit, and at slaughtering. The extent (grade) of turbinate atrophy and lung consolidation attributable to pneumonia was determined in each pig at slaughter. A negative correlation was found between birth weight and the prevalence of nasal lesions at slaughter, suggesting that pigs born with smaller weight may be more susceptible to AR. The growth performance of the pigs also showed a negative correlation with the degree of turbinate atrophy. In the nursery period, the average daily gain (ADG) of pigs with moderate/severe turbinate atrophy was 13.3% lower than that of animals with healthy snouts. In the finishing period, pigs with mild AR lesions had an ADG reduction of 6.2%, while pigs with severe AR lesions had a significant, 9.4% reduction in ADG as compared to the AR-free pigs. The extent of weight gain reduction over the whole life cycle was very similar (approximately 6%) in the pigs having either AR or pneumonia alone. In those pigs where both respiratory diseases were present, their effects seemed to be added up (11.5%); however, nasal lesion scores and percentage of lung consolidation did not show statistically apparent interactive effects on growth performance.

Pili of Pasteurella multocida of porcine origin.

Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pasteurella multocida isolated from pigs with atrophic rhinitis. Rigid pili were found on 60-80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocida and as a result frequently were difficult to see. After growth in vitro, piliated P. multocida cells produced few pili (approx. 3-5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.

Virulence of Bordetella bronchiseptica from pigs with or without atrophic rhinitis.

The virulence of 17 isolates of Bordetella bronchiseptica from 13 pig herds was compared by intranasal infection of gnotobiotic piglets and LD50 tests on mice. Of 59 piglets given 8.1-10-5 log 10 colony-forming units (cfu) of isolates from two herds with atrophic rhinitis (AR isolates) or isolates from six unaffected herds (non-AR isolates), 16 died of acute pneumonia; the survivors developed non-progressive turbinate hypoplasia and chronic pneumonia. Infection of 11 piglets with c. 3.0 log to cfu of three AR isolates or three non-AR isolates caused turbinate hypoplasia, but only slight pneumonia and no deaths. There were no significant differences between the virulence of AR and non-AR isolates in piglets. In LD50 tests in mice, there were no significant differences between the results from six AR isolates and six non-AR isolates, or from toxin prepared from two AR isolates and one non-AR isolates was fairly uniform, and that other factors must be responsible for the occurrence of progressive lesions of atrophic rhinitis in some but not all infected herds.

Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis.

One hundred and fifty-eight porcine strains of Pasteurella multocida, recovered primarily from cases of pneumonic pasteurellosis or progressive atrophic rhinitis (PAR) in England and Wales, were characterized by determination of their capsular types, presence or absence of the toxA gene and molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. Eighteen groups (clones) of strains were identified on the basis of specific combinations of capsular type, toxA status and outer-membrane protein (OMP)-type. The data provided evidence that different subpopulations of P. multocida are responsible for pneumonia and PAR in pigs. The majority (88 %) of cases of pneumonia were associated exclusively with non-toxigenic capsular type A strains of OMP-types 1.1, 2.1, 3.1 and 5.1 and capsular type D isolates of OMP-type 6.1. These strains were recovered from widespread geographical locations within England and Wales over a 12-year period and represented mostly single sporadic cases. The association of a small number of P. multocida variants with the majority of cases of porcine pneumonia suggests that these strains are not opportunistic pathogens of low virulence but represent primary pathogens with a relatively high degree of virulence. In contrast, the majority (76 %) of cases of PAR were associated with toxA-containing capsular type D strains of OMP-type 4.1 and capsular type A and D strains of OMP-type 6.1. Toxigenic capsular type A strains associated with PAR and non-toxigenic capsular type A strains associated with pneumonia represent distinct subpopulations of P. multocida that can be differentiated by their OMP-types. The association of capsular types A and D with strains of the same OMP-types, and the absence and presence of the toxA gene in strains of the same OMP-types, suggest that horizontal transfer of capsular biosynthesis and toxA genes has occurred between strains representing certain subpopulations of P. multocida.

Demonstration that Australian Pasteurella multocida isolates from sporadic outbreaks of porcine pneumonia are non-toxigenic (toxA-) and display heterogeneous DNA restriction endonuclease profiles compared with toxigenic isolates from herds with progressive atrophic rhinitis.

Capsular types A and D of Pasteurella multocida cause economic losses in swine because of their association with progressive atrophic rhinitis (PAR) and enzootic pneumonia. There have been no studies comparing whole-cell DNA profiles of isolates associated with these two porcine respiratory diseases. Twenty-two isolates of P. multocida from diseased pigs in different geographic localities within Australia were characterised genotypically by restriction endonuclease analysis (REA) with the enzyme CfoI. Seven of 12 P. multocida isolates from nasal swabs from pigs in herds where PAR was either present or suspected displayed a capsular type D phenotype. These were shown to possess the toxA gene by polymerase chain reaction (PCR) and Southern hybridisation, and further substantiated by production of cytotoxin in vitro. The CfoI profile of one of these seven isolates, which was from the initial outbreak of PAR in Australia (in Western Australia, WA), was identical with profiles of all six other toxigenic isolates from sporadic episodes in New South Wales (NSW). The evidence suggests that the strain involved in the initial outbreak was responsible for the spread of PAR to the eastern states of Australia. Another 10 isolates, representing both capsular types A and D, were isolated exclusively from porcine lung lesions after sporadic outbreaks of enzootic pneumonia in NSW and WA. CfoI restriction endonuclease profiles of these isolates revealed considerable genomic heterogeneity. Furthermore, none of these possessed the toxA gene. This suggests that P. multocida strains with the toxA gene do not have a competitive survival advantage in the lower respiratory tract or that toxin production does not play a role in the pathology of pneumonic lesions, or both. REA with polyacrylamide gel electrophoresis and silver staining was found to be a practical and discriminatory tool for epidemiological tracing of P. multocida outbreaks associated with PAR or pneumonia in pigs.

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.

Protection of Pasteurella multocida dermonecrotic toxin-challenged rats by toxoid-induced antibody.

Two different doses of glutaraldehyde-treated Pasteurella multocida dermonecrotic toxin (PMDT) were used to immunize rats. Rats developed serum IgG antibodies specific for native PMDT, and IgG titers increased with dose and number of toxoid immunizations. Survival rates in both active immunization and passive serum neutralization experiments were dependent on dose of toxoid vaccination and serum levels of anti-PMDT IgG. Vaccination with toxoid prevented weight loss but not leukocytosis and increased complement titers in toxin-challenged rats. Toxoid, itself, induced minimal leukocytosis but no alterations in complement titers or weight gain.

Comparison of three biological assays for the detection of toxigenic properties of Bordetella bronchiseptica associated with atrophic rhinitis.

The toxigenic properties of Bordetella bronchiseptica strains associated with clinical atrophic rhinitis were compared using three biological assays: the guinea pig skin test, the mouse spleen atrophy and the suckling mice mortality assay. Forty one sick pigs were cultured and, in 20 of them B. bronchiseptica strains were isolated. Eighteen of the strains were toxigenic by at least one test. However, positivity did not always coincide amongst assays. This discrepancy raises the possibility of either differences in sensitivity between assays or the existence of more than one toxin.