Vaccine potential of an attenuated Pasteurella multocida that expresses only the N-terminal truncated fragment of P. multocida toxin in pigs

Previously we described the development of an attenuated Pasteurella multocida mutant that expresses only the N-terminal truncated fragment of P. multocida toxin (N-PMT) and its protective effects in a mouse model. This paper details our evaluation of the vaccine potential of this mutant strain in pigs. Pigs vaccinated with the mutant showed significantly higher rates of antibody induction and lower nasal conchal (turbinate) scores for atrophic rhinitis than controls, which suggests that this mutant strain may be a good candidate for a live attenuated vaccine.     

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 80kDa 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 72h 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.     

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.     

Atypical Pasteurella strains producing a toxin similar to the dermonecrotic toxin of Pasteurella multocida subspecies multocida

This paper is the first report of the production of a dermonecrotic toxin by pasteurella strains that do not belong to the species Pasteurella multocida subspecies multocida. Four strains, isolated from cattle with atrophic rhinitis, were characterised phenotypically. The strains were related to pasteurellaceae, but their taxonomic position remained unclear. The strains produced a toxin that caused a haemorrhagic dermonecrosis in guinea pigs and was lethal to mice. Both effects were neutralised by an antiserum against the purified dermonecrotic toxin of P multocida subspecies multocida. Western blot analysis of culture filtrates of the bovine strains revealed a protein, with the same molecular weight as dermonecrotic toxin, which reacted with both polyclonal and monoclonal antibodies against the toxin. In an immunodiffusion test, anti-dermonecrotic toxin serum did not discriminate between the toxin of the bovine strains and the toxin of P multocida subspecies multocida. It is concluded that these atypical pasteurella strains produce a toxin that is closely related to the dermonecrotic toxin of P multocida subspecies multocida.     

Characterization of the immunogenicity of formaldehyde detoxified Pasteurella multocida toxin.

The immunogenicity of the Pasteurella multocida toxin (PMT) was studied in murine model systems. Mice were vaccinated with either formaldehyde treated pure PMT (pure toxoid) or formaldehyde treated crude extract of toxigenic P. multocida (crude toxoid). The corresponding mean anti-PMT titres, sero-conversion rates and survival rates after challenge with affinity purified PMT were compared. When assessed both by anti-PMT titres and seroconversion and challenge, pure toxoid was a more potent immunogen than crude toxoid. This greater immunogenic potency was unaffected by the addition of killed cell preparations of Bordetella bronchiseptica, non-toxigenic P. multocida and B. pertussis. Increasing anti-PMT titres and seroconversion rates were induced by increasing doses of formaldehyde treated PMT (fPMT) in the pure toxoid vaccines, but not in the vaccines containing crude toxoid. However, improved survival rates were observed for both types of vaccine, when the fPMT content was raised. Immunization of pregnant mice with vaccines containing fPMT induced protection of the offspring against challenge with PMT; the protection of the offspring corresponded to that of the mother.     

猪多杀性巴氏杆菌的分离鉴定及毒素检测

多杀性巴氏杆菌(pasteurella multocida，Pm)在世界各地分布非常广泛，在畜禽中带菌率较高，有资料指出，猪的鼻道深处和喉头内，带菌率为30．9％。多杀性巴氏杆菌按荚膜的不同可分为A、B、C、D和E5个血清型，其中A、B和D型已在猪中发现；多杀性巴氏杆菌还有16个体细胞血清型，在猪中检测到的有血清3型与5型，     

Purification of three fragments of the dermonecrotic toxin from Pasteurella multocida

Dermonecrotic toxin purified from sonicates of Pasteurella multocida was mildly trypsinized. The trypsinized preparations were reversibly dissociated into three polypeptides, with molecular weights of about 23,000 (fragment a), about 64,000 (fragment b), and about 74,000 (fragment c) by treatment with 100 mM dithiothreitol and 6 M urea. Upon removal of dithiothreitol and urea from the dissociated toxin by dialysis, the fragments reassociated and formed dermonecrotic toxin indistinguishable from the native toxin. The three fragments were separated from the dissociated toxin by gel filtration on a Sephadex G-200 column equilibrated with buffer containing 4 M urea and 1 mM dithiothreitol. The purified fragments a, b, and c did not show dermonecrotic activity for guinea pigs. Immunodiffusion and immunoelectrophoretic analysis with rabbit anti-dermonecrotic antiserum showed that the three purified fragments were antigenically distinct but had partial identity with the native toxin.     

Induction of nasal turbinate atrophy in germ-free pigs, using Pasteurella multocida as well as bacterium-free crude and purified dermonecrotic toxin of P multocida

To establish the role of the dermonecrotic toxin (DNT) of Pasteurella multocida in the cause and pathogenesis of atrophic rhinitis, germ-free pigs were inoculated with several strains of P multocida, crude DNT, or purified DNT. In some experiments, the aforementioned inocula were combined with Bordetella bronchiseptica. All DNT-producing P multocida strains induced severe turbinate atrophy. Histologic examination of the remnants of the nasal turbinates revealed intact, but undulated, ciliated epithelium and numerous osteoclasts. Inflammation was minimal or absent. A DNT-producing B bronchiseptica strain induced only mild turbinate atrophy. The lesions were characterized histologically by loss of cilia and ciliated cells and by an infiltration of predominantly mononuclear cells. Bone formation seemed impaired. Turbinate lesions were most severe in pigs infected with a combination of B bronchiseptica and a DNT-producing P multocida strain. Intranasal administration of sterile DNT-containing culture filtrate of P multocida or purified DNT of P multocida did not result in turbinate atrophy. In contrast, turbinate atrophy developed when these preparations were injected IM or when intranasal administration of DNT was preceded by inoculation of B bronchiseptica.     

Atrophic rhinitis of swine: neutralization of the toxin of Pasteurella multocida

The neutralization of the lethal as well as the growth-retarding action of the toxin on mice by an antitoxic serum is described.     

Infection of duck plague carriers with Pasteurella multocida and P. anatipestifer

Mallards (Anas platyrhynchos platyrhynchos) and white pekin ducks (Anas platyrhynchos domesticus) were infected with duck plague virus and challenged with LD20's of Pasteurella multocida and P. anatipestifer. There was no difference between mortality rates of duck plague-infected ducks and controls, suggesting that these organisms do not act synergistically under the conditions of our experiments. There was a difference of about 500-fold between the LD20 of P. multocida for mallards and that for white pekin ducks, indicating that mallards are much more susceptible to avian cholera than white pekin ducks.     

Investigation into the pathogenesis of atrophic rhinitis in pigs. I. Atrophic rhinitis caused by Bordetella bronchiseptica and Pasteurella multocida and the meaning of a thermolabile toxin of P. multocida

In two groups of swine herds, herds with and without clinical AR the presence of Atrophic Rhinitis (AR) correlated with the presence of toxinogenic Pasteurella multocida (PM) and not with the Bordetella bronchiseptica (BB) infection. Six BB- and eighteen PM-strains have been investigated for AR pathogenicity. Broth cultures were injected intradermally in guinea-pigs (GPST) or intranasally in 3-week-old colostrum deprived specific pathogen free (SPF) piglets. The average atrophy of the ventral conchae (AVC) correlated with the GPST in 4 BB-and 7 PM-strains. One BB- and 2 PM-strains were qualified as doubtful, the others as non-AR pathogenic. With AR pathogenic BB-and PM-strains clinical AR could be induced in 3-and 6-week-old piglets. AVC lesions (gradation greater than 1) could be induced with BB in piglets of 6 and with pathogenic PM in 16-week-old piglets. Six of seven AR pathogenic PM-strains resembled Carter-type D and one resembled type A. No significance was found between AR pathogenicity and somatic serotypes. Intranasal instillations of cell-free broth culture filtrates of AR pathogenic PM-strains also caused AR in piglets. These filtrates also caused lethality in piglets and in mice lethalitytest (MLT) and induced a positive GPST. After heating the pathogenic effects of the filtrates disappeared. The name AR toxin has been introduced for this thermolabile, haemorrhagic dermonecrotic (HDNT) fraction of the AR inducing filtrates. The severity of the AR lesions depended on the amount of the AR toxin intranasally instilled in pigs. Cross protecting antibodies obtained in rabbits against the AR toxins of two PM strains could be demonstrated by a toxin neutralisation test in the MLT and the GPST. Broth cultures were injected intradermally in guinea-pigs (GPST) or intranasally in 3-week-old colostrum deprived specific pathogen free (SPF) piglets.     

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.     

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

Heat-labile toxin from a cell sonicate of a virulent type-D 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.     

Pasteurella multocida toxin induces IL-6, but not IL-1 alpha or TNF alpha in fibroblasts.

Pasteurella multocida toxin (PMT) is the major virulence factor in Progressive Atrophic Rhinitis of swine. Other workers' previous findings that PMT was mitogenic for 3T3 fibroblasts, were confirmed in the present study. In addition, PMT stimulated 3T3 cells to release IL-6, but IL-1 alpha or TNF alpha were not detected in fibroblast supernatants sampled 24, 48, or 72 h after stimulation. In view of the role of IL-6 in osteoclastic bone resorption, these findings provide a new working hypothesis for investigations into the molecular pathogenesis of this important disease.     

禽多杀性巴氏杆菌强毒株与弱毒株制备的蜂胶灭活疫苗免疫原性的比较试验

用禽多杀性巴氏杆菌强毒株(C48-1)与弱毒株(G190E40和B26-T1200)制备的蜂胶灭活疫苗的免疫原性对比试验结果表明,强毒株C48-1制备的禽霍乱蜂胶灭活疫苗的免疫效果显著高于弱毒株G190E40和B26-T1200,近期保护率相差20%～40%,3个月后相差40%～80%.强毒株C48-1的免疫原性显著高于弱毒株G190E40和B26-T1200,说明禽多杀性巴氏杆菌荚膜上的毒力蛋白可能具有免疫原性.     

Protection by toxoid-induced antibody of gnotobiotic piglets challenged with the dermonecrotic toxin of Pasteurella multocida

A crude dermonecrotic toxin (DNT) of Pasteurella multocida (P.m.) type D was prepared by repeated sonication and freezing. It was sterilized by filtration. A toxoid was then made and pigs were hyperimmunized with it to get an antiserum. A control serum was obtained by hyperimmunization of pigs with a preparation derived from nontoxigenic P.m. type D in the same manner as the toxoid. Three gnotobiotic piglets were injected with the antiserum. This resulted in neutralization indices (NI) of 25 in their sera, as tested on mice. Three litter-mated controls were given the control serum. Their NI remained 1. All piglets were challenged intramuscularly 4 times, every third day, with 30 mouse LD50 of the DNT. When euthanized 15 days after the last DNT administration no snout lesions were found in passively immunized piglets, whereas control animals showed severe turbinate atrophy and other changes typical for atrophic rhinitis. The next experiment was identical to the previous one except for the challenge, which was given intranasally (4 times 300 mouse LD50). Also in this case circulating antitoxin protected the piglets from damage of the nasal turbinates caused by the DNT.     

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.