Protective effect of Pasteurella multocida cell-free antigen and toxoid against challenge with toxigenic strains of pasteurella multocida in mice

The cell-free antigen (CFA) obtained from the culture supernatant of Pasteurella multocida (P. multocida) and the toxin (PMT) purified from CFA were inactivated and mixed with oil adjuvant to prepare a trial vaccine. Both of the mice immunized with CFA and PMT toxoid vaccine were noticeably protected against intratracheal challenge with toxigenic strains of P. multocida. Nevertheless, the protective indices of the mice immunized with CFA vaccine indicate that it is more protective and clears away the bacteria more promptly than in the mice immunized with PMT vaccine. The results suggested that CFA would possibly be good as an effective antigen to toxigenic strains of P. multocida infection.     

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam. The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P. multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry.     

Study of the toxin-producing ability of Pasteurella multocida in mice

Cell-free sonicated extracts and broth cultures of Pasteurella multocida strains of pig origin were examined for their lienotoxicity in mice. P. multocida strains represented capsular types A and D with or without dermonecrotoxic (DNT) activity in the guinea pig skin test. Mouse lienotoxicity test was suitable for determining the toxigenicity of P. multocida strains only when bacterium-free extracts were tested. In that case both toxigenic type A and D strains were lethal to intravenously inoculated mice and caused a remarkable reduction in spleen mass when sublethal doses were used. The extracts of atoxic strains were not lethal and induced splenic hyperplasia. By testing viable cells no correlation was demonstrable between toxin production and virulence of P. multocida to mice. In one experiment the concentrated sterile culture fluids of a toxigenic type D P. multocida and a toxigenic B. bronchiseptica strain were compared. The former caused deaths and splenic atrophy among mice, while the latter was nontoxic and induced slight hyperplasia of the spleen. This fact indicates that P. multocida secretes its toxin into the culture fluid.     

Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of pigs

A toxin produced by strains of Pasteurella multocida isolated from pigs with atrophic rhinitis caused a cytopathic effect in cell cultures derived from embryonic bovine lung. The toxin was produced during the late logarithmic phase of bacterial growth and inactivated by heating for 30 minutes at 56 degrees C. The cell culture assay was reproducible and 10(3) to 10(4) times more sensitive than a lethal assay in BALB/c mice. There was complete agreement between results in the two tests with 76 isolates of P multocida. Neutralising activity was demonstrated in both assays with sera from infected gnotobiotic piglets. It was concluded that embryonic bovine lung cell cultures provided a sensitive in vitro test for the differentiation of toxigenic from non toxigenic isolates of P multocida. The assay could be used in diagnostic laboratories and for characterisation of the toxin.     

Genetic diversity of Pasteurella species isolated from European vespertilionid bats

Pasteurella are an important cause of fatal infections in free-ranging bats, but the genetic diversity of bat-derived strains is unclear. In the current study, 81 Pasteurella strains associated with pneumonia, severe organ necroses and systemic infection in free-ranging European vespertilionid bats were characterized by biochemical and molecular typing methods. Genetic relationships and subspecies status of Pasteurella multocida strains were determined by comparative 16S rDNA and rpoB gene sequence analysis. In addition, 30 representatives of the bat-derived P. multocida strains were selected based on phenotypic and genotypic tests to be compared by pulsed-field gel electrophoresis using SmaI. Most (85%) of the Pasteurella strains obtained from free-ranging bats in this study represented P. multocida ssp. septica. P. multocida ssp. multocida and Pasteurella species B were also identified in a small number of isolates. PFGE analysis correlated well with the sequencing results and revealed a high genetic diversity among bat-derived strains of P. multocida ssp. septica. Strains sharing identical or closely related SmaI fragment patterns were cultured from bats of different species, geographic origins, and years of isolation. The presence of numerous different P. multocida strains allows the assumption that Pasteurella infections in vespertilionid bats are not solely based on intra- but also on inter-species transmission. And indeed, our results present evidence of P. multocida infections in bats following cat predation.     

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.     

Occurrence of Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania

Investigation was done to determine the presence of Pasteurella multocida and related species in free ranging chickens and ducks, dogs, cats and pigs in three climatic zones (cool, warm and hot) of rural Morogoro, Tanzania. A total of 153 isolates of P. multocida ssp. multocida and related species were obtained by direct culture on blood agar, selective medium and mouse inoculation. P. multocida ssp. multocida was isolated from 0.7% of chickens and 7% of ducks. In dogs and cats, P. multocida ssp. multocida was isolated from 1 and 68%, respectively. One isolate of Pasteurella gallinarum was isolated from a duck. Other species obtained were; P. multocida ssp. septica, Pasteurella stomatis and taxon 16 from dogs and cats, while Pasteurella dagmatis and Pasteurella canis were found in dogs only. Prevalence of P. multocida ssp. multocida was significantly higher (P<0.01) in ducks of the warm zone (22%) than in ducks of other zones (0%). No significant difference was observed between the prevalence of P. multocida ssp. multocida in chickens of the warm zone (2%) and chickens of the cool and hot zones (0%). Extended phenotypic characterization revealed phenotypic similarities between two isolates from chickens and the duck strains. Mouse inoculation appeared to be more sensitive in detecting P. multocida ssp. multocida than blood agar and selective medium. Direct culture on blood agar recovered most of the isolates from dogs. This study has demonstrated for the first time the presence of P. multocida and related species in the village free ranging chickens, ducks, dogs and cats in Tanzania. Other non-classified Pasteurella spp. were also observed in the study, but further characterization is required before the final classification can be made. This paper reports for the first time the isolation of unclassified Pasteurella from dogs and cats in Africa. The results implies that fowl cholera might be occurring in free ranging poultry, and dogs and cats kept in contact might serve as sources of P. multocida to chickens and ducks. Subsequent applications of molecular techniques to analyse the epidemiological relatedness of clones isolated from different host species is indicated.     

Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida

Three doses (75 micrograms, 25 micrograms, and 25 micrograms) of purified toxin isolated from a toxigenic strain of type D Pasteurella multocida were given (by atomizer) into the right nasal cavities of each of 10 gnotobiotic pigs on the 21st, 24th, and 27th days of age, respectively. Inoculated pigs (usually 2) and 1 noninoculated control pig each were necropsied on 3, 6, 9, 12, and 15 days after inoculations were given. Severe bilateral atrophy of turbinates occurred in all toxin challenge-exposed pigs. Atrophy was more severe in the inoculated nasal cavity than that in the noninoculated side in 2 of the 10 pigs. Microscopic changes in turbinates of toxin challenge-exposed pigs were more severe in pigs killed at later dates. Dominant changes included degeneration and necrosis of osteoblasts, markedly accelerated osteoclastic osteolysis, replacement of the osseous core by a highly cellular mesenchymal stroma, and multifocal atrophy of submucosal glands. Seemingly, a protein toxin isolated from toxigenic type D strains of P multocida produced rapid atrophy of turbinates and may be a contributing factor in development of clinical progressive atrophic rhinitis in swine.     

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.     

Vaccination against progressive atrophic rhinitis with a recombinant Pasteurella multocida toxin derivative.

Vaccination against progressive atrophic rhinitis using a purified recombinant derivative of the Pasteurella multocida toxin (PMT), was carried out. Ten pregnant gilts were vaccinated twice with the nontoxic derivative (dO) which apart from a lack of 121 amino acids had an amino acid sequence identical to PMT, while seven gilts were vaccinated with a purified, formaldehyde treated, native PMT and ten gilts served as non-vaccinated controls. The resulting piglets were inoculated intranasally with Bordetella bronchiseptica and toxigenic P. multocida. Among piglets from the nonvaccinated gilts all except one developed clinical atrophic rhinitis and 90% developed severe turbinate atrophy while only a few pigs in the vaccinated groups developed clinical or pathological signs of disease. Gilt colostra from the two vaccinated groups had similar mean anti-PMT titers and the mean titers in the offspring's sera from these groups were nearly identical throughout the study. No pigs born from unvaccinated gilts were seropositive until 8 wk of age (7 wk post-challenge) but 23% became seropositive at slaughter. The infection rate with toxigenic P. multocida in piglets and the total number of P. multocida colonies cultured from nasal swabs were significantly reduced at 5 wk and 8 wk of age in the vaccinated groups, when compared to controls. There was a significantly improved weight gain (greater than 9%) from birth to slaughter in offspring from vaccinated gilts. No significant differences in feed conversion rate or % lean meat were observed among the groups. The study showed the excellent immunoprotective properties of the nontoxic derivative of the PMT molecule.     

Reclassification of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumonic calf lungs

The taxonomic relationship of 131 strains previously identified as Pasteurella multocida obtained from calf pneumonia in West Germany, United Kingdom and Netherlands was investigated by extended phenotypic and limited genotypic characterization. Twenty-four strains were classified as P. multocida ssp. multocida, 15 strains as P. avium biovar 2 and 13 strains as P. canis biovar 2. Sixty-five and five strains were tentatively classified as ornithine negative P. multocida ssp. multocida and P. multocida ssp. septica, respectively. Genetic investigations showed that ornithine negative strains of P. multocida were related on species level. Less genomic binding was found between an ornithine negative strain of P. multocida ssp. septica and the type strains of the three subspecies of P. multocida. The taxonomic position of ornithine negative strains of P. multocida is still under investigation. The taxonomic position of the remaining nine strains is uncertain underlining the need for genotypic characterization within the genus Pasteurella to aid in defining single species by phenotypic tests.     

Use of ELISA to detect toxigenic Pasteurella multocida in atrophic rhinitis in swine.

The use of an enzyme-linked immunosorbent assay (ELISA) as a means of detecting dermonecrotoxin-producing strains of Pasteurella multocida was investigated. The assay was evaluated as a means to identify toxigenic P. multocida isolates recovered from nasal secretions of swine with atrophic rhinitis. The sensitivity and specificity of the ELISA for detecting dermonecrotoxin-producing P. multocida strains were compared to those of mouse-inoculation and cytotoxicity assays. The ELISA was highly sensitive and more specific than animal inoculation or tissue culture assay and is thus a more effective method for screening swine herds for the presence of toxigenic strains of P. multocida. The ELISA is a rapid, effective, economical way to identify toxigenic P. multocida isolates.     

Toxigenic Pasteurella multocida in rabbits with naturally occurring atrophic rhinitis

In a commercial rabbitry nasal swabs were taken from 36 animals with enzootic upper respiratory disease resembling porcine atrophic rhinitis. 35 Pasteurella multocida strains were isolated from 17 rabbits. Among 30 strains tested for dermonecrotic toxin production 3, derived from 3 animals, were positive in the guinea pig skin test. 15 Bordetella bronchiseptica strains were recovered from 14 rabbits. No toxigenic strains were found among 6 isolates tested using the same method.     

Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark

The aim of the present study was to evaluate capsular-typing, plasmid-profiling, phage-typing and ribotyping for epidemiological studies of toxin-producing Pasteurella multocida ssp. multocida in Denmark. The evaluation of methods was based on 68 strains from nasal swabs and 14 strains from pneumonic lungs. Strains from lungs were all of capsular Type A, whereas strains from nasal swabs were of both capsular Types A and D. Only 9% of the strains contained plasmids, which could not be associated with antibiotic resistance. Phage-typing divided 61% of strains into 10 groups, while 39% were non-typable. CfoI ribotyping divided strains into four groups of which one type contained 94% of isolates. HindIII ribotyping divided strains into 18 types. A total of 18 strains from The Netherlands, UK and USA were subjected to HindIII ribotyping, resulting in 13 types of which six were identical to ribotypes of Danish strains. Phage-typing of isolates from an outbreak of atrophic rhinitis involving six herds in 1985 showed the existence of an epidemic strain. This type was recognised in the herd suspected of being the source of the infections and in four of the five infected herds. These findings were supported by HindIII ribotyping, as 85% of isolates from all herds were assigned to one ribotype. In conclusion, HindIII ribotyping seems to represent a useful tool for epidemiological studies of toxigenic P. multocida ssp. multocida.     

Isolation of toxigenic strains of Pasteurella multocida from lungs of pneumonic swine

Lungs from 113 pneumonic pigs were examined for Pasteurella multocida. The lungs were smeared directly onto blood agar and homogenized in brain-heart infusion broth and then inoculated intraperitoneally in mice. Pasteurella multocida isolates were typed for serotypes A (by hyaluronidase inhibition of capsule) and D (by acriflavine autoagglutination). Strains were tested for toxin production by intradermal injection of 0.2 ml of filtered 24-hour culture supernatants into guinea pigs. Most lungs (70.8%) yielded isolations. Most isolants (87.5%) were type A and 12.5% were type D. Of the type D strains, 80% were toxigenic. Of the type A isolants, 18.2% were toxigenic.     

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.     

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.     

产毒素多杀性巴氏杆菌的鉴定

采用菌落多重PCR方法对分离保存的28株多杀性巴氏杆菌进行种型和毒素基因的检测,结果表明,菌株C51-6、M-4和P-2237为产毒素多杀性巴氏杆菌,菌株C51-6和P-2237为荚膜血清D型,菌株M-4为荚膜血清A型。同时用金黄色葡萄球菌抑制试验、中性吖啶黄沉淀试验和豚鼠皮肤坏死试验对PCR方法进行了验证。基于对甘露醇、卫茅醇、山梨醇、海藻糖的发酵能力和产生鸟氨酸脱羧酶的特性,3株菌株鉴定为多杀性巴氏杆菌多杀亚种。     

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.     

Identification of toxigenic Pasteurella multocida in atrophic rhinitis of pigs by in vitro characterisation

Toxigenic strains of Pasteurella multocida were readily differentiated from non-toxigenic strains by an agarose overlay method using bovine turbinate cells or bovine lung cells. Cells which were young and densely confluent were best suited to this assay. The incubation period required to distinguish toxigenic strains was dependent on the confluence of the monolayers, which was affected by the seeding rate, cell passage level and growth time prior to overlay. The agarose overlay method correctly identified 11 of 11 reference strains of Pasteurella multocida, and visible cytotoxic changes were present in the monolayers after 48 to 65 h. Outbreaks of the enzootic form of atrophic rhinitis in 2 New South Wales piggeries were associated with the isolation of toxigenic type D strains of P. multocida.