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.     

Development of a toxA gene knock-out mutant of Pasteurella multocida and evaluation of its protective effects

Pasteurella multocida is an important veterinary and opportunistic human pathogen. In particular, strains of P. multocida serogroup D cause progressive atrophic rhinitis, and produce a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. To further investigate the toxigenic and pathogenic effects of PMT, a toxA-deleted mutant was developed by homologous gene recombination. When administrated to mice, the toxigenicity of the toxA mutant P. multocida was drastically reduced, suggesting that the PMT contributes the major part of the toxigenicity of P. multocida. Similar results were obtained in a subsequent experiment, while high mortalities were observed when toxA(+) P. multocida bacterial culture or culture lysate were administrated. Mice immunized with toxA(-) P. multocida were not protected (none survived) following challenge with toxA(+) P. multocida or bacterial culture lysate (toxin). These results suggest that the toxigenicity of P. multocida is mainly derived from PMT.     

Characterization of Pasteurella multocida associated with pneumonia in bighorn sheep

Pasteurella multocida is a highly diverse group of bacteria recognized as important pathogens. Although P. multocida is not ordinarily associated with disease in Rocky Mountain bighorn sheep (Ovis canadensis canadensis), numerous isolates were cultured in high numbers from free-ranging bighorn sheep in the Hells Canyon area of Idaho, Washington, and Oregon (USA) during the winter of 1995-96. Animals captured in Hells Canyon and held in captivity, and their offspring, also harbored P. multocida. Biochemical utilization tests on 90 isolates identified three subspecies: P. multocida multocida a (n = 54); P. multocida multocida b (n = 13); and P. multocida gallicida (n = 15); and a non-speciated biotype, U6 (n = 8). Genomic DNA digestion with restriction endonuclease Hha I separated the isolates into 62 unique restriction fragment length polymorphism profiles. Capsular type A was predominant (72% of isolates). Only one isolate type, which may have been transmitted from a feral goat, was capsular type D, possessed the structural gene, toxA, for dermonecrotoxin detected by polymerase chain reaction, and produced toxin as determined by monoclonal antibody immunoblot. In conclusion, bighorn sheep in this study carried diverse types of generally non-toxigenic P. multocida associated with epizootic pneumonia.     

Decoration of Pasteurella multocida lipopolysaccharide with phosphocholine is important for virulence

Phosphocholine (PCho) is an important substituent of surface structures expressed by a number of bacterial pathogens. Its role in virulence has been investigated in several species, in which it has been shown to play a role in bacterial adhesion to mucosal surfaces, in resistance to antimicrobial peptides, or in sensitivity to complement-mediated killing. The lipopolysaccharide (LPS) structure of Pasteurella multocida strain Pm70, whose genome sequence is known, has recently been determined and does not contain PCho. However, LPS structures from the closely related, virulent P. multocida strains VP161 and X-73 were shown to contain PCho on their terminal galactose sugar residues. To determine if PCho was involved in the virulence of P. multocida, we used subtractive hybridization of the VP161 genome against the Pm70 genome to identify a four-gene locus (designated pcgDABC) which we show is required for the addition of the PCho residues to LPS. The proteins predicted to be encoded by pcgABC showed identity to proteins involved in choline uptake, phosphorylation, and nucleotide sugar activation of PCho. We constructed a P. multocida VP161 pcgC mutant and demonstrated that this strain produces LPS that lacks PCho on the terminal galactose residues. This pcgC mutant displayed reduced in vivo growth in a chicken infection model and was more sensitive to the chicken antimicrobial peptide fowlicidin-1 than the wild-type P. multocida strain.     

Characteristics of Pasteurella multocida isolated from waterfowl and associated avian species in California

Characteristics of Pasteurella multocida isolated from tissues of dead waterfowl and associated avian species found at 23 sites located in northern and central California, from January 1986 through January 1988 are reported. Two hundred ninety five isolates of P. multocida were obtained from 23 avian species. Most of the isolates belonged to the subspecies P. multocida multocida (63%), followed by P. multocida gallicida (37%), and by P. multocida septica (less than 1%). There appeared to be a higher prevalence of P. multocida multocida in Ross' geese (Chen rossi) and Snow geese (Chen coeruleus). All of the isolates belonged to somatic serotype 1, possessed the A capsule type and were susceptible to the 8 antimicrobial agents tested. None contained plasmid DNA.     

Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant

The aroA gene from Pasteurella multocida was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The nucleotide sequence of the P. multocida aroA gene indicated an open reading frame encoding a protein of 441 amino acids, which showed a high degree of homology with the amino acid sequences of various other bacterial AroA proteins. The cloned P. multocida aroA gene was inactivated by insertion of a kanamycin-resistance gene and reintroduced by allelic exchange into the chromosome of P. multocida using the suicide vector pJM703.1. The P. multocida aroA mutant was highly attenuated in a mouse model. Mice immunized intraperitoneally with two doses of live P. multocida aroA mutant were completely protected against a lethal parental strain challenge.     

Are wetlands the reservoir for avian cholera?

Wetlands have long been suspected to be an important reservoir for Pasteurella multocida and therefore the likely source of avian cholera outbreaks. During the fall of 1995-98 we collected sediment and water samples from 44 wetlands where avian cholera epizootics occurred the previous winter or spring. We attempted to isolate P. multocida in sediment and surface water samples from 10 locations distributed throughout each wetland. We were not able to isolate P. multocida from any of the 440 water and 440 sediment samples collected from these wetlands. In contrast, during other investigations of avian cholera we isolated P. multocida from 20 of 44 wetlands, including 7% of the water and 4.5% of the sediment samples collected during or shortly following epizootic events. Our results indicate that wetlands are an unlikely reservoir for the bacteria that causes avian cholera.     

Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identification of the putative lipopolysaccharide glycosyltransferases

Pasteurella multocida is an important multispecies veterinary pathogen. The cell surface lipopolysaccharide (LPS) is an important virulence factor and forms the basis of the serotyping scheme, although little structural information about it is known. The structure of the LPS from the Pasteurella multocida genome strain Pm70 was elucidated in this study. The LPS was subjected to a variety of degradative procedures. The structures of the purified products were established by monosaccharide and methylation analyses, NMR spectroscopy, and mass spectrometry. The structure of the core oligosaccharide was determined on the basis of the combined data from these experiments. Identification of the core oligosaccharide structure enabled a search for glycosyltransferase homologs in the Pm70 genome and revealed a clustering of the genes putatively responsible for outer core oligosaccharide biosynthesis.     

Interaction of Pasteurella multocida with free-living amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

多杀性巴氏杆菌aroA基因缺失突变株的构建及鉴定

【目的】构建多杀性巴氏杆菌aroA基因缺失突变株,并验证其致病性。【方法】采用正向筛选同源重组技术构建多杀性巴氏杆菌aroA基因缺失突变株,利用PCR对突变株进行鉴定,分析其遗传稳定性、生长特性和致病性。【结果】成功构建多杀性巴氏杆菌aroA基因缺失突变株,连续传代20代,遗传稳定;突变株体外生长曲线表明,在前6h生长速度稍慢于亲本菌,随后两者生长速度一致。对小鼠的致病性试验表明:经腹腔注射aroA基因缺失突变株在1.0×106 CFU对小鼠无致死性,而亲本菌株在1.0×102 CFU对小鼠是致死性的。【结论】本研究获得多杀性巴氏杆菌aroA基因缺失突变株,对小鼠的致病性是减弱的。多杀性巴氏杆菌突变株的构建有助于研究其致病机理。     

Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.     

Isolation and sequencing of a temperate transducing phage for Pasteurella multocida

A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae, presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA(Leu). By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation.     

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.     

Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates

Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.     

Effects of antibiotics on the growth and morphology of Pasteurella multocida.

The effects of subminimal inhibitory concentrations (subMICs) of certain antibiotics, namely penicillin G, tetracycline and trimethoprim/sulphamethoxazole, on the growth and morphology of Pasteurella multocida were evaluated. SubMICs of penicillin markedly reduced the growth of P. multocida. Tetracycline and trimethoprim/sulphamethoxazole had no effect on its growth. SubMICs of penicillin greatly affected the morphology of P. multocida. At the highest concentrations tested (1/2 and 1/4 MIC) cells were acapsulate, and long filamentous cells (4-6 microns) were observed with some isolates. There was no correlation between the observed differences in the penicillin-binding proteins of the P. multocida isolates, and the extent of cell filamentation induced by penicillin G. SubMICs of tetracycline and trimethoprim/sulphamethoxazole did not seem to affect capsule production although filamentation was observed. Our results indicate that subMICs of penicillin can reduce growth of P. multocida. Furthermore, results also indicate that subMICs of antibiotics can affect the production of capsular material and the morphology of P. multocida.     

Survival of toxigenic Pasteurella multocida in aerosols and aqueous liquids.

The survival of toxigenic Pasteurella multocida in air and liquids was studied to identify possible risk factors in the etiology of atrophic rhinitis. In aerosols, at low relative humidity (28%), the viability of toxigenic P. multocida 5 min after aerosolization was at least 22% of its initial value. Viability at low relative humidity declined to 8% after 45 min. Viability at high relative humidity (79%) was 69% after 5 min and declined to 2% after 45 min. Survival of toxigenic P. multocida in liquids depended on storage and constituents in the liquid. Toxigenic P. multocida became nonculturable 1 to 14 days after inoculation in water and artificial seawater, depending on the storage temperature. Toxigenic P. multocida stored at 37 degrees C could be detected for up to 6 days in pig slurry and more than 36 days in Bacto Tryptose broth and nasal lavages. However, in Bacto Tryptose broth and nasal lavages stored at 4 degrees C, P. multocida was detected for up to 14 days whereas at 15 and 37 degrees C it was detected for more than 49 days. These results suggest that aerosols and fomites can play a role in the transmission of atrophic rhinitis.     

Cloning and expression of the dermonecrotic toxin gene of Pasteurella multocida ssp. multocida in Echerichia coli

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.     

Transfer and properties of some natural and suicide replicons in Pasteurella multocida

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013--a narrow-host-range plasmid with the broad-host-range IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1011, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.     

The capsule biosynthetic locus of Pasteurella multocida A:1

Pasteurella multocida is the aetiological agent of fowl cholera, bovine haemorrhagic septicaemia and atrophie 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.