Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 209 - 23 Emended description of porcine {Pasteurella} aerogenes, {Pasteurella} mairii and {Actinobacillus} rossii; Christensen H et al.; The aim of this study was to improve the definition and identification of a group of veterinarily important bacteria referred to as the {Pasteurella} aerogenes-{Pasteurella} mairii-{Actinobacillus} rossii complex . These organisms have mainly been isolated from the reproductive and intestinal tracts of pigs and in most cases have been considered as opportunistic pathogens . A collection of 87 strains were characterized by phenotypic analysis from which 41 strains were selected for 16S rRNA gene sequence comparison, out of which 23 have been sequenced in the present study . One group of 21 strains phenotyped as biovars 1, 3-5, 9-11, 19 and 25-27, including the type strain of {P.} aerogenes, showed 16S rRNA gene sequence similarities of 99.6 % or higher; another group of 18 strains including biovars 2, 6-8, 12-15, 21, 23, 24 and 26A and the type strain of {A.} rossii showed 97.8 % or higher 16S rRNA gene sequence similarity . Between the two groups, 93.8-95.7 % 16S rRNA gene sequence similarity was observed . Strains of {P.} mairii showed 99.5 % similarity, with 95.5-97.2 and 93.8-95.5 % similarity to strains of {P.} aerogenes and {A.} rossii, respectively . Four strains could not be classified with any of these groups and belonged to other members of Pasteurellaceae . Comparisons were also made to DNA-DNA hybridization data . Biovars 1, 9, 10, 11 and 19, including the type strain of {P.} aerogenes, linked at 70 % DNA reassociation, whereas strains identified as biovars 2, 6, 7, 8, 12, 15 and 21 of {P.} aerogenes linked at 81 % . The latter group most likely represents {A.} rossii based on the 16S rRNA gene sequence comparisons . DNA reassociation between the {P.} aerogenes and {A.} rossii groups was at most 37 %, whereas 47 % was the highest DNA reassociation found between {P.} aerogenes and {P.} mairii . The study showed that {P.} aerogenes, {P.} mairii and {A.} rossii can not be easily separated and may consequently be misidentified based on current knowledge of their phenotypic characteristics . In addition, these taxa are difficult to separate from other taxa of the Pasteurellaceae . A revised scheme for separation based upon phenotypic characters is suggested for the three species {P.} aerogenes emend., {P.} mairii emend . and {A.} rossii emend., with the respective type strains ATCC 27883(T), NCTC 10699(T) and ATCC 27072(T). J Vet Med Sci, 2004 Dec, 66(12), 1603 - 4 Protectivity of an Immunoaffinity-Purified 39 kDa Capsular Protein of Avian Pasteurella multocida in Mice; Ali HA et al.; A 39 kDa protein of avian Pasteurella multocida strain P-1059 (serovar A:3) was purified from a crude capsular extract by immunoaffinity chromatography by using a ligand of purified mouse monoclonal antibody to the 39 kDa capsular protein of the strain . Protective activity of the purified 39 kDa protein antigen was determined by inoculation of ddY mice twice with 25 or 125 mug of the protein and challenge-exposure with 10 or 50 LD(50) of strains P-1059 or X-73 (serovar A:1) . The results showed that the antigen gave high protection (60 to 100%) . These results indicated that the 39 kDa protein of avian P . multocida is a cross-protective antigen over serovars A:1 and A:3. Trop Anim Health Prod, 2004 Nov, 36(8), 743 - 50 Antibiotic sensitivity patterns among Indian strains of avian Pasteurella multocida; Shivachandra SB et al.; An investigation was carried out to study the antibiotic sensitivity of avian strains of Pasteurella multocida and to select an effective antimicrobial agent for control of avian pasteurellosis in India . A total of 123 strains of P . multocida recently isolated from different avian species (chicken, duck, turkey, quail, and goose), from different regions of India were subjected to antibiotic sensitivity tests using 20 different antibiotics . Absolute resistance was observed against sulfadiazine . The studies indicated that the strains were most sensitive to chloramphenicol (73.98%), followed by enrofloxacin (71.54%), lincomycin (64.23%), norfloxacin (61.79%) and doxycycline-HCl (56.91%) . The majority of the strains were found to exhibit intermediate sensitivity . Chloramphenicol was selected and suggested for treatment . Antibiogram studies also revealed the emergence of multidrug-resistant strains of P . multocida among Indian poultry. J Clin Microbiol, 2005 Jan, 43(1), 259 - 70 Characterization of sucrose-negative Pasteurella multocida variants, including isolates from large-cat bite wounds; Christensen H et al.; To validate the identification of Pasteurella multocida-like bacteria negative for acid formation from sucrose, including isolates from bite wounds caused by large cats, 17 strains were phenotypically and genotypically characterized . Phylogenetic analysis of partially sequenced rpoB and infB genes showed the monophyly of the strains characterized and the reference strains of P . multocida . The sucrose-negative strains formed two groups, one related to reference strains of P . multocida and the other related to a separate species-like group (taxon 45 of Bisgaard) . DNA-DNA hybridization further documented the species-like nature of this group . Ribotyping showed the heterogeneity of all strains except four strains that shared the same ribotype and that were isolated from bovine lungs . Phylogenetic analysis by 16S rRNA sequence comparison showed the monophyly of the strains characterized and the reference strains of P . multocida . Two strains isolated from leopard bite wounds were related to the type strain of P . dagmatis; however, they represented a new taxon (taxon 46 of Bisgaard), in accordance with their distinct phenotypic and genotypic identifications . The present study documents that sucrose-negative strains of P . multocida-like bacteria belong to two genotypically distinct groups . The study further confirms the phenotypic heterogeneity of P . multocida strains and documents two new species-like taxa of Pasteurella related to P . multocida . Until diagnostic tools have been further elaborated, special care should be taken in the identification of Pasteurella-like bacteria isolated from bite wounds caused by large cats . The evidence of phenotypic and genotypic divergence calls for the further development of PCR tests and DNA sequencing to document doubtful isolates. Vet Immunol Immunopathol, 2005 Feb 10, 103(3-4), 187 - 93 Incubation of bovine PMNs with conditioned medium from BHV-1 infected peripheral blood mononuclear cells increases their susceptibility to Mannheimia haemolytica leukotoxin; Leite F et al.; Active infection with bovine herpesvirus-1 (BHV-1) increases the susceptibility of cattle to secondary bacterial pneumonia with Mannheimia (Pasteurella) haemolytica A1 . In the present study we found that bovine PMNs incubated with conditioned media from BHV-1 infected peripheral blood mononuclear cells (PBMCs) exhibited increased LFA-1 expression, enhanced LKT binding and increased LKT cytotoxicity . These effects were abrogated when the conditioned medium was pre-incubated with an anti-IL-1beta Mab before being added to the PMNs . These findings suggest that BHV-1 infection, and the resulting release of IL-1beta and perhaps other inflammatory cytokines, can stimulate activation of LFA-1 in bystander bovine PMNs, thus enhancing the binding and biological effects of LKT. Carbohydr Res, 2005 Jan 17, 340(1), 59 - 68 Structural analysis of the lipopolysaccharide of Pasteurella multocida strain VP161: identification of both Kdo-P and Kdo-Kdo species in the lipopolysaccharide; St Michael F et al.; The structure of the lipopolysaccharide from the Pasteurella multocida strain VP161 was elucidated . The lipopolysaccharide 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 following structures for the lipopolysaccharides were determined on the basis of the combined data from these experiments . Based on the NMR data, all sugars were found in pyranose ring forms, and Kdo is 2-keto-3-deoxy-octulosonic acid, l-alpha-d-Hep is l-glycero-d-manno-heptose, PPEtn is pyrophosphoethanolamine and PCho is phosphocholine . Intriguingly, when the O- and fully deacylated LPS was examined, it was evident that there was variability in the arrangement of the Kdo region of the molecule . Glycoforms were found with a Kdo-P moiety, as well as glycoforms elaborating a Kdo-Kdo group . Furthermore the Glc II residue was not attached to Hep I when two Kdo residues were present, but it was attached when the Kdo-P arrangement was elaborated, suggesting a biosynthetic incompatibility due to either steric hindrance or an inappropriate acceptor conformation . This variation in the Kdo region of the LPS was also observed in several other Pasteurella multocida strains investigated including the genome strain Pm70. Infect Immun, 2005 Jan, 73(1), 413 - 21 Pasteurella multocida toxin activates human monocyte-derived and murine bone marrow-derived dendritic cells in vitro but suppresses antibody production in vivo; Bagley KC et al.; Pasteurella multocida toxin (PMT) is a potent mitogen for fibroblasts and osteoblastic cells . PMT activates phospholipase C-beta through G(q)alpha, and the activation of this pathway is responsible for its mitogenic activity . Here, we investigated the effects of PMT on human monocyte-derived dendritic cells (MDDC) in vitro and show a novel activity for PMT . In this regard, PMT activates MDDC to mature in a dose-dependent manner through the activation of phospholipase C and subsequent mobilization of calcium . This activation was accompanied by enhanced stimulation of naive alloreactive T cells and dominant inhibition of interleukin-12 production in the presence of saturating concentrations of lipopolysaccharide . Surprisingly, although PMT mimics the activating effects of cholera toxin on human MDDC and mouse bone marrow-derived dendritic cells, we found that PMT is not a mucosal adjuvant and that it suppresses the adjuvant effects of cholera toxin in mice . Together, these results indicate discordant effects for PMT in vitro compared to those in vivo. Antimicrob Agents Chemother, 2005 Jan, 49(1), 414 - 7 dfrA20, A novel trimethoprim resistance gene from Pasteurella multocida; Kehrenberg C et al.; A novel trimethoprim resistance gene, designated dfrA20, was detected on the 11-kb plasmid pCCK154 from Pasteurella multocida . The dfrA20 gene codes for a dihydrofolate reductase of 169 amino acids . Sequence comparisons revealed that the DfrA20 protein differed distinctly from all dihydrofolate reductases known so far. Vet Res Commun, 2004 Nov, 28(8), 657 - 67 Prevalent serotypes of Pasteurella multocida isolated from different animal and avian species in India; Kumar AA et al.; Identification and estimation of the prevalence of Pasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out . Out of 418 samples collected from different outbreaks suspected to be caused by P . multocida, a total of 206 bacterial cultures were identified as P . multocida on the basis of cultural, morphological and biochemical characteristics . All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer . Serotyping of P . multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1 and -:1) among the livestock population . P . multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of approximately 460 bp specific for P . multocida . The results of PCR assay correlated well with conventional methods of identification . The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species. Tidsskr Nor Laegeforen, 2004 Dec 16, 124(24), 3194 - 6 {Bite wound infections}; Yaqub S et al.; BACKGROUND: The lifetime risk of experiencing a bite wound, human or animal, is approximately 50%, and bite wounds account for approximately 1% of all visits to emergency departments . The majority of bite wounds are inflicted by dogs and cats . MATERIAL AND METHODS: A review of the literature on the diagnosis and treatment of bite wound infections is presented . RESULTS: The most common pathogens associated with bite wounds are Streptococcus species, Staphylococcus species, Pasteurella multocida, Capnocytophaga canimorsus and anaerobic bacteria . Sporadically other pathogens are isolated from bite wounds . Human bites differ from animal bites by higher prevalence of Staphylococcus aureus and Eikenella corrodens . INTERPRETATION: It is important to be aware of the possibility of complicating infections following bite wounds, particularly after cat bites . Phenoxymethyl penicillin should be the drug of choice in treatment of infections associated with cat and dog bites . However, in case of slow recovery or no improvement, simultaneous lymphadenopathy or pneumonia, S . aureus or Francisella tularensis should be suspected; ciprofloxacin is recommended . For human bite infections the recommend treatment is phenoxymethyl penicillin in combination with penicillinase-stable penicillin. J Vet Med B Infect Dis Vet Public Health, 2004 Dec, 51(10), 467 - 9 Identification of Pasteurella multocida Serogroup F Isolates in Rabbits; Jaglic Z et al.; Summary A total of 24 Pasteurella multocida rabbit isolates obtained from 24 rabbit flocks in the Czech Republic during the period of between 2001 and 2004 were analysed by capsular PCR typing . Apart from isolates identified as serogroups A (n = 14, 58.4%) and D (n = 2, 8.3%), eight isolates (33.3%) were identified as members of serogroup F . This serogroup had been predominantly associated with poultry infections so far . The rabbit serogroup F isolates were characterized in detail by ribotyping with restriction to endonuclease MspI revealing two distinct ribotypes . Seven serogroup F isolates were assigned to ribotype 1 and one isolate was assigned to ribotype 2. South Med J, 2004 Nov, 97(11), 1113 - 5 Spontaneous bacterial peritonitis with Pasteurella multocida in cirrhosis: case report and review of literature; Tamaskar I et al.; Most Pasteurella multocida human infections involve skin and soft tissues and invariably develop after a bite or a scratch from a dog or a cat . However, other infections with this organism occur infrequently . Enteric microorganisms are the common cause of spontaneous bacterial peritonitis (SBP) . We report a case of SBP in a cirrhotic patient from P . multocida . English literature (Pubmed) review revealed 12 adult cases of SBP in cirrhotic patients with P multocida . Nine patients were exposed to animals, though a break in the skin or a bite was not reported in each case . The SBP was fatal in four of these patients. Berl Munch Tierarztl Wochenschr, 2004 Nov-Dec, 117(11-12), 480 - 92 {Data on the prevalence of antimicrobial susceptibility of veterinary pathogens from cattle and pigs: national antibiotic resistance monitoring 2002/2003 of the BVL}; Wallmann J et al.; The national antimicrobial resistance monitoring determines the current quantitative resistance level of life-stock pathogens, in order to permit the evaluation and surveillance of the distribution of resistances on a valid basis . During the examination period from June 2002 to July 2003, a total of 1849 pathogens was collected, following a representative German-wide pattern in collaboration with 29 laboratories . The selection of examined bacterial strains included different specimen causing respiratory diseases in fattening pigs (Pasteurella multocida, Bordetella bronchiseptica) and cattle (Pasteurella multocida, Mannheimia haemolytica), respectively, as well as strains causing mastitis in dairy cows (Staphylococcus spp., Streptococcus spp., E . coli) . Determination of the in-vitro susceptibility (minimal inhibitory concentration) to at least 17 antimicrobial agents was performed centrally by the BVL using the microdilution broth method . The findings approximately match results of an analogous study conducted in 2001, and correspondingly revealed significantly lower resistance-values in comparison to data published for Germany so far . No correlation could be established between the incidence of resistance and differing stock densities . By means of the resistance monitoring data gathered by the BVL, the risk potential of antimicrobials applied in Germany can be reliably defined . This valuable information about the epidemiological situation of resistance in Germany can be helpful to veterinarians as a decision guidance when choosing appropriate means of therapy . Accordingly, the results stated by the BVL are apt to provide an important contribution to improving the safety of food-animal products . The experiences obtained from the monitoring also show that valid data about the antimicrobial susceptibility can only be raised on an interdisciplinary approach (federal agencies, county diagnostic laboratories, universities, industry) . Currently, the BVL put into practice a study with an extended spectrum of bacterial species and indications. J Clin Microbiol, 2004 Dec, 42(12), 5542 - 8 Nicoletella semolina gen . nov., sp . nov., a new member of Pasteurellaceae isolated from horses with airway disease; Kuhnert P et al.; Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease . On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen . nov, sp . nov., a new member of the family Pasteurellaceae . N . semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars . Growth is fastidious, and the isolates have a distinctive colony morphology, with the colonies being dry and waxy and looking like a semolina particle that can be moved around on an agar plate without losing their shape . DNA-DNA hybridization data and multilocus phylogenetic analysis, including 16S rRNA gene (rDNA), rpoB, and infB sequencing, clearly placed N . semolina as a new genus in the family Pasteurellaceae . In all the phylogenetic trees constructed, N . semolina is on a distinct branch displaying approximately 5% 16S rDNA, approximately 16% rpoB, and approximately 20% infB sequence divergence from its nearest relative within the family Pasteurellaceae . High degrees of conservation of the 16S rDNA (99.8%), rpoB (99.6%), and infB (99.7%) sequences exist within the species, indicating that N . semolina isolates not only are phenotypically homogeneous but also are genetically homogeneous . The type strain of N . semolina is CCUG43639(T) (DSM16380(T)). Microbiology, 2004 Dec, 150(Pt 12), 4199 - 210 Genetic diversity among Pasteurella multocida strains of avian, bovine, ovine and porcine origin from England and Wales by comparative sequence analysis of the 16S rRNA gene; Davies RL; Genetic diversity among 86 Pasteurella multocida isolates was investigated by comparative sequence analysis of a 1468 bp fragment of the 16S rRNA gene . The strains included 79 field isolates recovered from birds (poultry) (22), cattle (21), pigs (26) and sheep (10) within England and Wales, four Asian isolates associated with bovine haemorrhagic septicaemia, and the type strains of the three subspecies of P . multocida . Dulcitol and sorbitol fermentation patterns were also determined to establish correlations between subspecies status and phylogenetic relatedness . Nineteen 16S rRNA types were identified, but these were clustered into two distinct phylogenetic lineages, A and B . Sequences within lineages A and B had a mean number of nucleotide differences of 21.12+/-3.90 . Isolates within lineage A were associated with birds, cattle, pigs and sheep, whereas those belonging to lineage B were recovered from birds and a cat . Eighty-seven per cent of the isolates were classified as P . multocida subsp . multocida by dulcitol and sorbitol fermentation patterns, but these have diverse 16S rRNA gene sequences that were represented in both lineages A and B . Avian P . multocida subsp . septica isolates were associated exclusively with lineage B, but bovine P . multocida subsp . septica isolates were present in lineage A . P . multocida subsp . gallicida isolates of avian, bovine and porcine origin represent a homogeneous group within lineage A, but they have the same 16S rRNA type as certain P . multocida subsp . multocida isolates . These findings provide strong support for the view that dulcitol and sorbitol fermentation patterns are inaccurate indicators of genetic relatedness among P . multocida strains . Avian capsular type B isolates and capsular type B and E isolates associated with haemorrhagic septicaemia of cattle and water buffaloes are closely related and form a distinct cluster within lineage A . The current subspecies nomenclature of P . multocida neither accurately reflects the 16S rRNA-based phylogenetic relationships among isolates nor does it adequately encompass the full range of diversity within the species . The study provides a 16S rRNA-based evolutionary framework that will form the basis of further studies into the genetic diversity of P . multocida and will also help in the reclassification of the species. J Bacteriol, 2004 Dec, 186(24), 8529 - 32 Identification of a distinct, cryptic heparosan synthase from Pasteurella multocida types A, D, and F; Deangelis PL et al.; The extracellular polysaccharide capsules of Pasteurella multocida types A, D, and F are composed of hyaluronan, N-acetylheparosan (heparosan or unsulfated, unepimerized heparin), and unsulfated chondroitin, respectively . Previously, a type D heparosan synthase, a glycosyltransferase that forms the repeating disaccharide heparosan backbone, was identified . Here, a approximately 73% identical gene product that is encoded outside of the capsule biosynthesis locus was also shown to be a functional heparosan synthase . Unlike PmHS1, the PmHS2 enzyme was not stimulated greatly by the addition of an exogenous polymer acceptor and yielded smaller- molecular-weight-product size distributions . Virtually identical hssB genes are found in most type A, D, and F isolates . The occurrence of multiple polysaccharide synthases in a single strain invokes the potential for capsular variation. Respir Care, 2004 Dec, 49(12), 1528 - 9 Community-acquired pneumonia due to Pasteurella multocida; Marinella MA; Most cases of community-acquired pneumonia result from infection with predictable common pathogens . However, rare patients develop pneumonia from unusual bacterial species such as Pasteurella multocida, a Gram-negative oral commensal of most dogs and cats . The majority of P . multocida infections involve skin and soft tissue and complicate a bite or scratch . I report the case of an elderly man who owned 16 cats and developed bacteremic pneumonia with P . multocida . . Int J Antimicrob Agents, 2004 Dec, 24(6), 592 - 8 A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets; Meunier D et al.; This study, Vetoquinol S.A . epidemiosurveillance, was conducted from 1994 to 2001 in order to determine the susceptibility (by MIC determination) to marbofloxacin (a third generation fluoroquinolone used only in individual administration for animals) . Strains from infected pets originated from six European countries . Isolates were collected from urinary infections (Escherichia coli), respiratory infections (Pasteurella multocida), dermatological infections (Staphylococcus intermedius, Pseudomonas aeruginosa) and otitis (S . intermedius, P . aeruginosa) . The MIC distribution for each species was the same both before and after the launch of marbofloxacin in 1995 . In E . coli, a resistant population was present before the use of marbofloxacin; this resistance was induced by co- or cross-resistance to other antibiotics used previously . Over this period, there was no significant evolution of MIC(90) for any bacterial species studied and no development of resistance was observed . Marbofloxacin was the most active antibiotic against P . multocida isolates and had the lowest MIC . No difference in MIC distribution was seen between the S . intermedius (unimodal distribution) isolated from dermatological infections and those from otitis . This was also true for P . aeruginosa . The use of marbofloxacin was not found to have induced a significant increase or spread of resistant bacteria. Pediatr Infect Dis J, 2004 Nov, 23(11), 1063 - 5 Pasteurella multocida meningitis and cervical spine osteomyelitis in a neonate; Hirsh D et al.; A 20-day-old male infant presented with fever, decreased alertness and quadriparesis as a result of Pasteurella multocida meningitis and C1-2 vertebral osteomyelitis . Although his household contained 2 pet cats, there was no history of bites, scratches or licks . We speculate that colonization of the nasopharynx was followed by contiguous spread to the retropharyngeal soft tissue, cervical vertebrae and meninges. Chest, 2004 Nov, 126(5), 1636 - 44 Intrapleural injection of transforming growth factor-beta antibody inhibits pleural fibrosis in empyema; Kunz CR et al.; STUDY OBJECTIVES: Transforming growth factor (TGF)-beta is a cytokine that has been demonstrated to be an important modulator of inflammation and angiogenesis, as well as a potent stimulator of pleural fluid production and fibrosis . We previously demonstrated that rising levels of pleural fluid TGF-beta(1) correlate with pleural fibrosis in experimental empyema in rabbits . In this study, our hypothesis is that neutralization of TGF-beta with an intrapleural injection of a monoclonal antibody to TGF-beta will decrease pleural fibrosis in empyema . DESIGN: Prospective, randomized, blinded study . SETTING: Animal research laboratory . SUBJECTS: Nineteen rabbits . INTERVENTIONS: An empyema was induced in 19 rabbits by intrapleural injection of Pasteurella multocida . A panspecific monoclonal antibody to TGF-beta was injected into the pleural space on 2 subsequent concurrent days in nine rabbits . Ten rabbits received intrapleural injections of bacteria alone and served as controls . All animals were then killed on day 6 . Immunohistochemistry, using the antibody to TGF-beta, was performed on pleural tissue specimens from the control rabbits . MEASUREMENTS AND RESULTS: Immunohistochemistry revealed localization of TGF-beta to macrophages in the exudative material and the visceral pleura . After injection of the antibody to TGF-beta, the amount of purulent, exudative material in the pleural space of the nine experimental animals was markedly decreased at autopsy on day 6, relative to control animals . All markers of empyema and pleural fibrosis were also significantly decreased in the rabbits receiving intrapleural anti-TGF-beta . CONCLUSIONS: TGF-beta localizes to macrophages in experimental empyema . Early intrapleural injection of an antibody to TGF-beta inhibits empyema formation and significantly decreases pleural fibrosis in experimental empyema. Glycobiology . 2004 Nov 10; {Epub ahead of print} Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identification of the putative lipopolysaccharide glycosyltransferases; St Michael F et al.; Pasteurella multocida is an important multi-species 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 the LPS 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 following structure for the core oligosaccharide was determined on the basis of the combined data from these experiments, where based on the NMR data all sugars were found in pyranose ring forms . Glucose, galactose and N-acetyl-galactosamine residues were all present as D-isomers . Kdo is 2-keto-3-deoxy-octulosonic acid, L-alpha-D-Hep is L-glycero-D-manno-heptose, and PEtn is phosphoethanolamine . Identification of the core oligosaccharide structure enabled a search for glycosyltransferase homologues in the Pm70 genome, and revealed a clustering of the genes putatively responsible for outer core oligosaccharide biosynthesis. Vet Microbiol, 2004 Nov 30, 104(1-2), 55 - 62 Invasion of chicken embryo fibroblast cells by avian Pasteurella multocida; Al-haj Ali H et al.; Invasion of chicken embryo fibroblast (CEF) cells by the virulent encapsulated Pasteurella multocida strains P-1059 (serovar A:3) and X-73 (serovar A:1) and an avirulent noncapsulated derivative P-1059B (serovar -:3) was investigated . The number of intracellular bacteria increased for all the strains after 2, 4 and 6 h post-inoculation to CEF cells . By 6 h post-inoculation, the number of invaded bacteria of encapsulated strains was significantly higher than noncapsulated strain and reached 150- and 112-fold for strains P-1059 and X-73, respectively, while it was 9-fold for strain P-1059B as compared to the number of invaded bacteria recovered after 2 h post-inoculation . Electron microscopy of invasion by encapsulated strains showed that the bacteria were adhering to CEF cells membrane after 1 h of inoculation . By 4-h, one or two bacteria were detected within membrane-bound vacuoles of the intracellular space . The number of intracellular bacteria markedly increased at 14 h post-inoculation . Invasion of all strains was inhibited significantly when the monolayers were treated with periodic acid (P<0.001) or trypsin (P<0.05) . The treatment of bacteria with hyaluronidase did not affect invasion . The present results indicate that avian P . multocida capsular type A strains are invasive and that the receptor on CEF cell surface might be glycoprotein. Avian Dis, 2004 Sep, 48(3), 463 - 70 Pasteurella multocida infection in heterophil-depleted chickens; Bojesen AM et al.; The present study was aimed at elucidating the role of heterophil granulocytes during the initial infection with Pasteurella multocida subsp . multocida in chickens . Chickens (17 and 19 wk old) were depleted of their heterophil granulocytes by 5-fluorouracil treatment . When the heterophil blood counts were significantly reduced, the birds were inoculated intratracheally with 1.8-4.3 x 10(4) colony-forming units of P . multocida . Twelve, 24, or 48 hr postinoculation, the birds were euthanatized and examined for macroscopic and histologic lesions in the lungs . Bacterial invasion was determined by culture of P . multocida from the spleen . Recruitment of heterophils into the respiratory tract during infection was found to contribute considerably to the lung lesions in chickens and was found to mediate tissue damage, possibly allowing a more rapid systemic spread of P . multocida . However, during progression of the infection, the heterophil-mediated necrosis in chickens seemed to stimulate giant cell demarcation of infected lung tissue, which coincided with the clearance of P . multocida from the spleen, thus hampering further invasion . Consequently, heterophil activation plays a dual role for the outcome of a P . multocida infection in chickens, where it initially seems to promote invasion and systemic spread but subsequently helps limit the infection by giant cell formation and bacterial clearance. Vet Microbiol, 2004 Nov 15, 103(3-4), 201 - 7 An experimental mouse model of progressive atrophic rhinitis of swine; Jordan RW et al.; Pasteurella multocida is responsible for a variety of diseases of veterinary importance, including the pig disease progressive atrophic rhinitis (PAR) . The feasibility of using the mouse as an experimental model of PAR was evaluated . We experimentally infected the upper respiratory tract of immature mice with a pig isolate of P . multocida that produces the toxin responsible for causing the nasal lesions characteristic of PAR . We tracked the health status and weight gain of these mice for one month following infection, after which the mice were killed and the integrity of the nasal turbinates was examined . Mice infected with P . multocida appeared healthy throughout the study, although the growth rate of these mice was reduced significantly compared with non-infected control animals . Infected animals also demonstrated marked nasal atrophy analogous to that seen in naturally occurring PAR of swine, with shortening and thinning of the turbinate scrolls and inflammatory cell involvement . The mouse therefore provides a convenient model for the further investigation of PAR of swine. Berl Munch Tierarztl Wochenschr, 2004 Sep-Oct, 117(9-10), 367 - 86 {Pasteurella: insights into the virulence determinants of a heterogenous bacterium}; Ewers C et al.; Pasteurella (P.) multocida is the causative agent of numerous economically relevant diseases worldwide . These are enzootic bronchopneumonia in cattle and sheep and hemorraghic septicemia in cattle and buffaloes, Rhinitis atrophicans in swine, snuffles in rabbit, and fowl cholera . All disease complexes are associated with certain capsular and somatic antigens . Even as human pathogen P . multocida is of increasing importance, causing wound infections, and even septicemia, meningitis, and endocarditis . Despite extensive research activities including the genome analysis of one fowl cholera isolate in the year 2001 there are a lot of open questions concerning the molecular pathogenic mechanisms . Problems encountered are the high antigenic variability and the wide host spectrum of P . multocida as well as different courses of infection . In consequence there are enormous difficulties in producing vaccines . Transcriptomics and proteomics hopefully will give new insight into the pathogenesis of P . multocida infections in different hosts . A frequent problem particular in classical diagnostic laboratories is the diagnosis of P . multocida and its differentiation from other P . species and Mannheimia (M.) haemolytica . The biochemical identification of P . multocida is not reliable due to variable phenotypical characteristics often caused by different culture conditions, and it is time consuming and cost-intensive . Extensive molecular biologic studies concerning the prevalence and distribution of virulence associated genes known so far in P . species, which will be described in detail in this paper, could contribute to the establishment of a diagnostic tool, such as a multiplex polymerase chain reaction, that would provide a cheap and time-saving identification and characterization of wildtype strains. Schweiz Arch Tierheilkd, 2004 Sep, 146(9), 417 - 22 {Comparison of antimicrobial resistance pattern of selected respiratory tract pathogens isolated from different animal species}; Wettstein K et al.; The antibiotic resistance pattern of respiratory tract pathogens isolated of different animal species suffering from respiratory tract diseases has been investigated by antibiograms performed by agar diffusion test . The results show that the resistance situation in Switzerland is favourable compared with studies from other countries . However, high resistance rates were found in certain species: 61% of Streptococcus spp . were resistant to erythromycin and 44% to tetracycline, 59% of Bordetella bronchiseptica were resistant to ampicillin and 50% of Mannheimia (Pasteurella) haemolytica were multiresistant to tetracycline, ampicillin and streptomycine . The gram negative isolates were widely resistant to streptomycine. J Wildl Dis, 2004 Jul, 40(3), 377 - 82 Are wetlands the reservoir for avian cholera? Samuel MD, Shadduck DJ, Goldberg DR. 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. J Vet Diagn Invest, 2004 Sep, 16(5), 458 - 60 Molecular epidemiology of two fowl cholera outbreaks on a free-range chicken layer farm; Zhang P et al.; Two outbreaks of fowl cholera on a multiage free-range egg farm were investigated . The outbreaks occurred in 1994 and 2002 . A total of 22 strains of Pasteurella multocida were available for study, 11 from the 1994 outbreak and 11 from the 2002 outbreak . Lesions typical of acute fowl cholera were seen in the 1994 outbreak, whereas both acute and chronic fowl cholera occurred in the 2002 outbreak . The isolates were examined in an extended phenotypic typing methodology, by a P . multocida-specific polymerase chain reaction (PCR), by the Heddleston somatic serotyping scheme, and by restriction endonuclease analysis (REA) typing using the enzyme HpaII . All 22 strains had the same phenotypic properties, all were confirmed as P . multocida by PCR, all were Heddleston serovar 4, and all had the same REA pattern . The results indicate that these 2 outbreaks were caused by the same clone of P . multocida--despite the 8-year time period between the outbreaks. J Vet Diagn Invest, 2004 Sep, 16(5), 426 - 31 Isolation and antimicrobial susceptibilities of bacterial pathogens from bovine pneumonia: 1994--2002; Welsh RD et al.; Between 1994 and 2002, a total of 390 (46.3%) Mannheimia haemolytica, 292 (34.7%) Pasteurella multocida, and 160 (19.0%) Histophilus somni were isolated at the Oklahoma Animal Disease Diagnostic Laboratory from lungs from 6-18-month-old beef cattle with pneumonia . The ratio of M . haemolytica isolations to P . multocida isolations decreased from 3.1 in 1994 to 0.8 in 2000 while increasing to 1.5 in 2002 . Mannheimia haemolytica isolations significantly (P < 0.05) decreased from 62.5% in 1994 to between 30.6% and 50.4% in 1998--2002 . Pasteurella multocida isolations significantly (P < 0.05) increased from 20.0% in 1994 to between 28.6% and 47.4% in 1998--2002 . Histophilus somni isolations were <19% except in 1998 (40.8%) and 1999 (23%) . Antimicrobial susceptibilities for M . haemolytica significantly declined for erythromycin (P = 0.0001), florfenicol (P = 0.0004), spectinomycin (P = 0.0001), and tilmicosin (P = 0.03) . For P . multocida, antimicrobial susceptibilities significantly declined for erythromycin (P = 0.0001), florfenicol (P = 0.004), spectinomycin (P = 0.03), sulfachloropyridizine (P = 0.028), tetracycline (P = 0.017), tilmicosin (P = 0.0001), and trimethoprim/sulfamethoxazole (P = 0.0003) . Antimicrobial susceptibilities for H . somni were variable for spectinomycin and sulfachloropyridizine, whereas susceptibilities to other antibiotics remained consistently high. Mol Microbiol, 2004 Oct, 54(1), 239 - 50 Identification and characterization of the Pasteurella multocida toxin translocation domain; Baldwin MR et al.; The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event . In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 - residues 402-423, H2 - 437-457) linked by a hydrophilic loop (PMT-TL - 424-436) . The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1 . To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine . Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity . Interestingly, mutation of D401 located immediately outside the predicted helix-loop-helix motif completely abolished toxin activity . Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse . Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion. Rev Physiol Biochem Pharmacol, 2004, 152, 93 - 109 Epub 2004. Pasteurella multocida toxin as a tool for studying Gq signal transduction; Wilson BA et al.; Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C (PLC) signal transduction through its selective action on the Galphaq subunit . This review summarizes what is currently known about the molecular action of PMT on Gq and the resulting cellular effects . Examples are presented illustrating the use of PMT as a powerful tool for dissecting the molecular mechanisms involving pertussis toxin (PT)-insensitive heterotrimeric G proteins. FEMS Microbiol Lett, 2004 Oct 1, 239(1), 125 - 30 Isolation and characterization of a cytotoxin produced by Plesiomonas shigelloides P-1 strain; Okawa Y et al.; In order to clarify the enteropathogenicity of Plesiomonas shigelloides, we investigated a cytotoxin produced by the P-1 strain isolated from patients suffering from diarrhea . The cytotoxicity of the culture filtrate of the strain reached a maximum in culture at 37 degrees C after 12 h shaken in BHI medium . The cytotoxin in the cultures was purified by (NH4)2SO4 precipitation, and Sephacryl S-100, Mono Q HR, and Superdex 200 HR column chromatographies . An approximate 340-fold purification was achieved, with a recovery of about 1.4%, from the culture supernatant . The cytotoxin is heat-stable, and is a complex of three major proteins (LPS-binding proteins with molecular weights of 32, 40, and 48 kDa), with lipopolysaccharide (LPS) giving a total a molecular weight of more than 600 kDa . The ratio of protein to LPS in the cytotoxin was 6-5 . The cytotoxic activity was reduced by about 80% by proteinase K treatment or when incubated with anti-cholera toxin antibody (Anti-CT) . Western blotting of the cytotoxin with Anti-CT demonstrated the presence of two anti-cholera toxin-reactive protein (ACRP) bands with molecular weights of 40 kDa (a major single protein band) and 48 kDa . The N-terminal amino acid sequence (20 residues) of the 40 kDa protein was 75% identical to Pasteurella multocida cell membrane proteins . The cytotoxin gave a positive reaction in the suckling mouse assay whereas LPS alone hardly exhibited any cytotoxic or enterotoxigenic activity . In conclusion, P . shigelloides produces a cytotoxin that consists of a complex of protein and LPS with the former component exhibiting both cytotoxicity and enteropathogenicity . This cytotoxin has the potential to have an important role in the enteropathogenicity of P . shigelloides. J Infect Chemother, 2004 Aug, 10(4), 250 - 2 Pasteurella multocida septicemia caused by close contact with a domestic cat: case report and literature review; Kimura R et al.; We report here a case of Pasteurella multocida infection caused by cat exposure presenting with septic shock, sinusitis, and pneumonia . The patient was a febrile 20-year-old woman who had been experiencing disturbed consciousness progressively . She had close contact with a domestic cat and had received some scratches on both arms . A magnetic resonance imaging (MRI) scan of the head showed a high intensity in the paranasal cavity, and a computed tomographic (CT) scan of the chest showed bilateral lung consolidations . The pathogen was identified as P . multocida by the cultures from blood and nasal discharge . She was given intensive antibiotic therapy with ceftriaxone and piperacillin, continuous hemodiafiltration (CHDF) therapy, and anticoagulation therapy . Owing to these therapeutic regimens, the septic shock was successfully treated without complications . We also review the literature on P . multocida septicemia. Clin Diagn Lab Immunol, 2004 Sep, 11(5), 825 - 34 Serological response to Pasteurella multocida NanH sialidase in persistently colonized rabbits; Sanchez S et al.; Pasteurella multocida is a mucosal pathogen that colonizes the upper respiratory system of rabbits . Respiratory infections can result, but the bacteria can also invade the circulatory system, producing abscesses or septicemia . P . multocida produces extracellular sialidase activity, which is believed to augment colonization of the respiratory tract and the production of lesions in an active infection . Previously, it was demonstrated that some isolates of P . multocida contain two unique sialidase genes, nanH and nanB, that encode enzymes with different substrate specificities (S . Mizan, A . D . Henk, A . Stallings, M . Meier, J . J . Maurer, and M . D . Lee, J . Bacteriol . 182:6874-6883, 2000) . We developed a recombinant antigen enzyme-linked immunosorbent assay (ELISA) based on the NanH sialidase of P . multocida and demonstrated that rabbits that were experimentally colonized with P . multocida produce detectable anti-NanH immunoglobulin M (IgM) and IgG in serum, although they demonstrated no clinical signs of pasteurellosis . In addition, clinically ill pet rabbits infected with P . multocida possessed IgM and/or IgG antibody against NanH . The NanH ELISA may be useful for the diagnosis of P . multocida infections in sick rabbits as well as for screening for carriers in research rabbit colonies. Can J Vet Res, 2004 Jul, 68(3), 188 - 92 Bacteriology and somatic cell counts in milk samples from ewes on a Scottish farm; Hariharan H et al.; Milk samples from 50 sheep on a single Scottish research farm were collected weekly for 10 wk postpartum . Samples were analyzed for somatic cell counts (SCC) each week and bacteriologic culture was done for 7 of the 10 wk . A total of 492 udder half samples were cultured, of which 467 had corresponding cell count data . Statistical analysis on complete SCC and culture data showed no association between SCC and bacterial isolation, even when more than 10 colonies of a single bacterial species were present . Only 3.6% of the samples were simultaneously positive for high count (> 10 colonies from 0.01 mL of milk) of any one bacterial species and high SCC (> 1 x 10(6)/mL) . The bacteria recovered were: Staphylococcus equorum (19 times), S . xylosus (7 times), S . simulans (6 times), Streptococcus uberis (3 times) and other streptococci (4 times), Mannheimia (Pasteurella) haemolytica (2 times), Staphylococcus aureus (1 time), S . capitis (1 time), and Enterococcus faecium (1 time) . There was an association between the test day and SCC, with higher SCC values in the first 2 wk . In addition, significantly higher SCC values were found in the oldest animals compared to the other age groups. J Bacteriol, 2004 Sep, 186(17), 5741 - 52 Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi; Davies RL et al.; The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria . The protein contains eight membrane-traversing beta-strands and four surface-exposed loops . The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis . The OmpA proteins of M . haemolytica and M . glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops . The hypervariable domains of OmpA proteins from bovine and ovine M . haemolytica isolates are very different but are highly conserved among strains from each of these two host species . Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M . haemolytica and M . glucosida . Class I, II, and IV alleles were associated with bovine M . haemolytica, ovine M . haemolytica, and M . glucosida strains, respectively, whereas class III alleles were present in certain M . haemolytica and M . glucosida isolates . Class I and II alleles were associated with divergent lineages of bovine and ovine M . haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination . Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination . Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation . It is proposed that the OmpA protein of M . haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep . P . trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes . The P . trehalosi ompA genes are highly diverged from those of M . haemolytica and M . glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer. J Biol Chem, 2004 Oct 1, 279(40), 42345 - 9 Epub 2004 Aug 05. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers; Jing W et al.; The length of the hyaluronan (HA) polysaccharide chain dictates its biological effects in many cellular and tissue systems . Long and short HA polymers often appear to have antagonistic or inverse effects . However, no source of very defined, uniform HA polymers with sizes greater than 10 kDa is currently available . We present a method to produce synthetic HA with very narrow size distributions in the range of approximately 16 kDa to approximately 2 MDa . The Pasteurella HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer utilizing monosaccharides from UDP-sugar precursors . Recombinant pmHAS will also elongate exogenously supplied HA oligosaccharide acceptors in vitro in a nonprocessive fashion . As a result of bypassing the slow initiation step in vitro, the elongation process is synchronized in the presence of acceptor; thus all of polymer products are very similar in length . In contrast, without the use of an acceptor, the final polymer size range is difficult to predict and the products are more polydisperse . HA polymers of a desired size are constructed by controlling the reaction stoichiometry (i.e . molar ratio of precursors and acceptor molecules) . The use of modified acceptors allows the synthesis of HA polymers containing tags (e.g . fluorescent, radioactive) . In this scheme, each molecule has a single foreign moiety at the reducing terminus . Alternatively, the use of radioactive UDP-sugar precursors allows the synthesis of uniformly labeled native HA polymers . Overall, synthetic HA reagents with monodisperse size distributions and defined structures should assist in the elucidation of the numerous roles of HA in health and disease. J Rheumatol, 2004 Aug, 31(8), 1663 - 5 Septic arthritis caused by Actinobacillus ureae in a patient with rheumatoid arthritis receiving anti-tumor necrosis factor-alpha therapy; Kaur PP et al.; Actinobacillus ureae, formerly known as Pasteurella ureae, is a rare human pathogen . We describe a case of septic arthritis and abscess formation caused by this unusual organism in a patient with rheumatoid arthritis, who was being treated with tumor necrosis factor-alpha inhibitors. Vet Microbiol, 2004 Aug 19, 102(1-2), 117 - 22 Differentiation of Pasteurella multocida isolates from cases of atrophic rhinitis in pigs from Zimbabwe by RAPD and ribotyping; Dziva F et al.; Atrophic rhinitis in pigs is rarely reported in Southern Africa . To determine the relationship between Pasteurella multocida clones from clinical cases of atrophic rhinitis, twenty-one strains were characterised by selected phenotypic and genotypic methods . Biochemical analysis classified 18 strains as P . multocida subspecies multocida, whilst the remainder were grouped into separate unassigned biotypes . Capsular groups A (16/21) and D (l/21) were found among the isolates by PCR . Four ribotype patterns were obtained following HpaII ribotyping, whilst random amplification of polymorphic DNA (RAPD) revealed three main clusters . However, subclusters were also noted for each RAPD cluster . Our results indicate that RAPD offers a better discrimination of strains than ribotyping and that none of the phenotypic characters were directly related to the genotypic clusters. Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1393 - 9 Phylogeny of the family Pasteurellaceae based on rpoB sequences; Korczak B et al.; Sequences of the gene encoding the beta-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae . A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study . Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene . In parallel, 16S rDNA was sequenced from all strains . The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level . Only a few discrepancies between the trees were observed . In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA . The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae . Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family. Colloids Surf B Biointerfaces, 2004 Aug 1, 36(3-4), 127 - 37 Lipid composition-dependent incorporation of multiple membrane proteins into liposomes; Daghastanli KR et al.; Membrane proteins from bacteria Pasteurella multocida were used as a model for studying its incorporation into liposomes . An important step to achieve efficient high yield protein incorporation in proteoliposomes is the study of the more suitable lipid composition . To this end, we compared the amount of total protein, reconstituted by co-solubilization methods, into liposomes of phospholipids with different polar head groups and acyl chain lengths . The liposomes and proteoliposomes were characterised by isopycnic centrifugation in sucrose gradient and by dynamic light scattering . Experimental and theoretical results were compared considering the effects exerted through the hydrocarbon chain length, volume, and optimal cross-sectional area of the phospholipid (combined in the geometrical critical packing parameter, lipid-protein matching), critical spontaneous radius of curvature of the bilayer vesicle, phase transition temperature of the lipid and ratio of lipid-protein molecules present in the vesicles . The highest incorporation of multiple proteins was found with dipalmitoylphosphatidylcholine (DPPC), reaching a yield of 93% compared to the lower relative amounts incorporated in proteoliposomes of the other lipids . The incorporation of multiple proteins induces a proportional enhancement of vesicular dimension, since DPPC-proteoliposomes have an average diameter of 1850A, compared to the 1430A for pure DPPC vesicles. Glycobiology, 2004 Dec, 14(12), 1217 - 28 Epub 2004 Jul 14. Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1); Saribas AS et al.; Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis . This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan . N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis . We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein . The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities . N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1 . However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate . The rNDST-1 was partially purified on heparin Sepharose CL-6B . Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E . coli K5 polysaccharide or Pasteurella multocida polysaccharide. J Microbiol Methods, 2004 Aug, 58(2), 263 - 7 Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes; Liu D et al.; Pasteurella multocida is an important animal pathogen that may also infect humans through animal bites and scratches . After comparison of transcriptional regulator gene sequences from the P . multocida genome with other DNA sequences at GenBank, we identified two genes (i.e., Pm0762 and Pm1231) uniquely present in P . multocida . By using oligonucleotide primers (Pm0762F/R and Pm1231F/R) designed from these genes in PCR, it was found that specific DNA products of expected sizes were obtained with genomic DNA from P . multocida only, but not from other bacteria . These results indicated that the putative transcriptional regulator genes Pm0762 and Pm1231 are species-specific, and that the PCR methods targeting these genes provide a useful means of rapidly and precisely identifying P . multocida from other bacteria . Further elucidation of the roles and functions of these putative transcriptional regulator genes (Pm0762 and Pm1231) and their protein products may help provide valuable insight into the molecular mechanism of P . multocida virulence and pathogenicity. Vet Res, 2004 May-Jun, 35(3), 309 - 24 Pathophysiological changes occurring during Escherichia coli endotoxin and Pasteurella multocida challenge in piglets: relationship with cough and temperature and predicitive value for intensity of lesions; Halloy DJ et al.; The aims of this study were (1) to correlate cough and body temperature (BT) with the severity of bronchopneumonia in pigs, (2) to determine whether these clinical signs can be used to early diagnose bronchopneumonia and (3) to assess the predictive values of cough and BT regarding lung lesions . Bronchopneumonia was induced by administering E . coli endotoxin (LPS) combined with Pasteurella multocida type A (PmA) in the trachea of 13 piglets . Saline-instilled negative controls (n = 8), PmA inoculated (n = 6) and LPS instilled (n = 5) groups were also constituted . Cough and BT were recorded daily while the bronchopneumonia severity was assessed using bronchoalveolar lavage fluid (BALF) cytology, cytokines and measurement of lung lesion volume . Changes in expiratory breathing pattern were also measured (Penh) . The combination of LPS and PmA induced a subacute bronchopneumonia characterised by macrophage, neutrophil, and lymphocyte infiltration, changes in Penh and an increase in the mRNA level of IFN-gamma while IL8, IL-18 and TNF-alpha mRNA levels remained unchanged . The daily body weight gain of infected animals was significantly reduced . Cough and BT changes were proportional to the intensity of the lung inflammatory process, functional respiratory changes and to the extent of macroscopic lesions . When comparing the individual values of cough and BT to thresholds defined for both parameters, an early diagnosis of pneumonia was possible . Considering the pooled data of each group, it was possible to define thresholds allowing an early segregation between the groups of diseased and healthy piglets . The daily values of cough and BT were predictive for the volume of lung lesions recorded at the end of the trial . In conclusion, cough and BT appear as potential indicators for the intensity and the evolution of the respiratory disease . They also seem to be good predictors for the magnitude of lung lesions and weight gain recorded at the study endpoint . Vet Immunol Immunopathol, 2004 Aug, 100(3-4), 197 - 207 Pathophysiological and immune cell responses in calves prior to and following lung challenge with formalin-killed Pasteurella multocida biotype A:3 and protection studies involving subsequent homologous live challenge; Dowling A et al.; Pneumonic pasteurellosis is a common respiratory infection in cattle that has major economic and welfare implications world-wide and the incidence in the UK due to Pasteurella multocida, currently the same as that associated with Mannheimia haemolytica, is increasing . Whereas much is known regarding the pathogenesis of M . haemolytica infections little information is available on the pathogenic process of pasteurellosis initiated by P . multocida . In the present work calf systemic and innate immune responses to intratracheal challenge with formalin-killed P . multocida biotype A:3 and to subsequent experimental lung infection with live P . multocida were investigated . Eight-week-old calves were challenged intratracheally on day 0 with either 10(9) colony forming units (cfu) of formalin-killed P . multocida biotype A:3 in 300 ml saline (n = 10) or 300 ml saline alone (n = 10), followed, at day 21, by challenge with 10(9) cfu live P . multocida . Pathophysiological and lung phagocyte responses were assessed by clinical monitoring, sequential lung lavage and blood sampling . Results for samples obtained before, during and after challenge showed clinical and acute phase protein responses to both bacterial culture and saline control treatments, although higher responses were associated with bacterial challenge . Phagocytosis of P . multocida during 1h incubation periods with lavaged cells in vitro was unaffected by exposure in vivo to killed P . multocida and there was evidence that P . multocida was able to survive intracellularly during this assay . There was no indication that lung exposure to formalin-killed P . multocida conferred protection against subsequent homologous live challenge. J Zoo Wildl Med, 2004 Mar, 35(1), 88 - 93 A Pasteurella-like bacterium associated with pneumonia in captive megachiropterans; Helmick KE et al.; A novel Pasteurella-like organism was recovered postmortem from lung tissue of two captive Wahlberg's epauleted fruit bats (Epomophorus wahlbergi), with severe, unilateral pneumonia . The bats had been recently shipped and died shortly after release from a 30-day quarantine . One presented with clinical signs of anorexia and lethargy before death; the other died without prior clinical symptoms . The same Pasteurella-like organism was recovered antemortem from subcutaneous abscesses in two captive little golden mantled flying foxes (Pteropus pumilus) housed with additional E . wahlbergi . The organism was also cultured on tracheal wash from one Malaysian flying fox (Pteropus vampyrus) and another E . wahlbergi, both demonstrating clinical signs of pneumonia . All recovered isolates appeared morphologically and biochemically similar to the initial isolates and were further characterized as either a Pasteurella or Actinobacillus organism on the basis of biochemical and cellular fatty acid profiles . Screening of the current collection using pharyngeal swabs isolated this organism from 12 of 15 E . wahlbergi, two of three P . vampyrus, one of 26 island flying foxes (Pteropus hypomelanus), and one of nine Rodrigues fruit bats (Pteropus rodricensis) . The organism was not identified in pharyngeal culture from eight Indian flying foxes (Pteropus giganteus), nine Egyptian fruit bats (Rousettus aegypticus), or an additional 16 P . pumilus. J Biol Chem, 2004 Aug 13, 279(33), 34150 - 5 Epub 2004 Jun 10. Action of Pasteurella multocida toxin depends on the helical domain of Galphaq; Orth JH et al.; Pasteurella multocida produces a 146-kDa protein toxin (PMT), which activates multiple cellular signal transduction pathways, resulting in the activation of phospholipase Cbeta, RhoA, Jun kinase, and extracellular signal-regulated kinase . Using Galpha(q)/Galpha(11) -deficient cells, it was shown that the PMT-induced pleiotropic effects are mediated by Galpha(q) but not by the highly related Galpha(11) protein (Zywietz, A., Gohla, A., Schmelz, M., Schultz, G., and Offermanns, S . (2001) J . Biol . Chem . 276, 3840-3845) . Here we studied the molecular basis of the unique specificity of PMT to distinguish between Galpha(q) and/or Galpha(11) . Infection of Galpha(q) -deficient cells with retrovirus-encoding Galpha(q) caused reconstitution of PMT-induced activation of phospholipase Cbeta, whereas Galpha(11) -encoding virus did not reconstitute PMT activity . Chimeras between Galpha(q) and/or Galpha(11) revealed that a peptide region of Galpha(q), covering amino acid residues 105-113, is essential for the action of PMT to activate phospholipase Cbeta . Exchange of glutamine 105 or asparagine 109 of Galpha(11), which are located in the all-helical domain of the Galpha subunit, with the equally positioned histidines of Galpha(q), renders Galpha(11) capable of transmission PMT-induced phospholipase Cbeta activation . The data indicate that the all-helical domain of Galpha(q) is essential for the action of PMT and suggest an essential functional role of this domain in signal transduction via G(q) proteins. Can J Vet Res, 2004 Apr, 68(2), 118 - 27 Experimental infectious respiratory disease in groups of calves: lobar distribution, variance, and sample-size requirements for vaccine evaluation; Jericho KW et al.; The distribution and variance of respiratory disease produced with aerosols of bovine herpesvirus 1 (BHV-1) and Mannheimia haemolytica in control (183 calves in 44 experiments) and vaccinated calves were studied in experiments conducted at the Animal Diseases Research Institute, Lethbridge, Alberta, from 1975 to 1989 . All calves had been born and raised at this institute and exposed similarly for 5 min by means of a face mask to viral and bacterial aerosols produced by a Collison atomizer (particles < 3 microm in diameter) . We summarized the macroscopic pathological responses of pneumonia (main end point), tonsillitis, tracheitis, and other microbiologic and experimental variables . We also summarized the lobar distribution of pneumonia in 202 control and 192 vaccinated calves with this disease model and in calves similarly exposed to parainfluenza 3 virus/M . haemolytica or BHV-1/Pasteurella multocida . Pneumonia in control calves began in ventral tissues of all lobes, with lobar preferences, and progressed dorsally, the dorsal parts of both large caudal lobes being least affected . A high variance of pneumonia was evident within and among experiments . From the magnitude of variance observed in the control groups, the number of calves per group required in vaccine-challenge studies using this BHV-1/M . haemolytica disease model was estimated . Such estimates are required for any disease model used in vaccine-challenge studies. Microbiology, 2004 Jun, 150(Pt 6), 1757 - 67 Revised description and classification of atypical isolates of Pasteurella multocida from bovine lungs based on genotypic characterization to include variants previously classified as biovar 2 of Pasteurella canis and Pasteurella avium; Christensen H et al.; Strains deviating in key phenotypic characters, mainly isolated from cases of bovine pneumonia in five European countries, were genotyped in order to examine their genotypic relationship with Pasteurella multocida . Twenty-two strains of Pasteurella avium biovar 2, including variants in indole, xylose and mannitol, 18 strains of Pasteurella canis biovar 2 and variants of this taxon, five strains of P . multocida subsp . septica showing variations in indole and ornithine decarboxylase, nine strains of P . multocida subsp . multocida showing variation in ornithine decarboxylase and mannitol, and type strains of the subspecies of P . multocida were included . Ribotyping was used to examine the relationship of the strains, and 13 types, each containing between one and 20 isolates, were observed . Identical ribotypes were observed in some cases for P . avium biovar 2 and either P . canis biovar 2 or P . multocida subsp . septica . ITS (16S-23S rRNA internal transcribed spacer) fragment-length profiling showed identity of the majority of strains (47 of 52), representing all four taxa, with only five divergent strains . A 16S rRNA sequence comparison of 11 strains representing the main ribotype clusters showed 99.9 % similarity to the type strain of P . multocida subsp . multocida, but only 97.4 % similarity was obtained to P . canis (biovar 1) and 93.7 % to P . avium (biovar 1) . A species-specific PCR test for P . multocida gave a positive result with biovar 2 variants of P . avium and P . canis . DNA-DNA hybridizations between strains of P . multocida, biovar 2 variants of P . avium and P . canis, and P . multocida subsp . septica confirmed similarity at the species level . It is proposed, on the basis of genotypic similarity, that P . multocida be reclassified to include the biovar 2 variants of P . avium and P . canis and that the existence of the biovar 2 variants of P . avium and P . canis is highly questionable . It is concluded that the redefined P . multocida is genotypically homogeneous, although phenotypically diverse lineages exist with respect to ornithine decarboxylase, indole and mannitol, characters that have been regarded as essential for identification to the species level . A formal reclassification of the species is not possible, however, since too few strains have been found to vary in these key characters . Considering the phenotypic diversity of P . multocida, identification will have to depend partly on genotypic methods and the source host also seems important for safe diagnosis. Microbiology, 2004 Jun, 150(Pt 6), 1723 - 34 Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae; Srikumar R et al.; From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA . Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida . Upon screening two genomic libraries of A . pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp . HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box . The promoter region of hgbA from A . pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site . RT-PCR confirmed that hgbA of A . pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium . While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb . HgbA is required for growth of A . pleuropneumoniae in the presence of Hb as sole iron source. Infect Immun, 2004 Jun, 72(6), 3436 - 43 A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence; Harper M et al.; Pasteurella multocida is the causative agent of fowl cholera in birds . In a previous study using signature-tagged mutagenesis, we identified a mutant, AL251, which was attenuated for virulence in mice and in the natural chicken host . Sequence analysis indicated that AL251 had an insertional inactivation of the gene waaQ(PM), encoding a putative heptosyl transferase, required for the addition of heptose to lipopolysaccharide (LPS) (M . Harper, J . D . Boyce, I . W . Wilkie, and B . Adler, Infect . Immun . 71:5440-5446, 2003) . In the present study, using mass spectrometry and nuclear magnetic resonance, we have confirmed the identity of the enzyme encoded by waaQ(PM) as a heptosyl transferase III and demonstrated that the predominant LPS glycoforms isolated from this mutant are severely truncated . Complementation experiments demonstrated that providing a functional waaQ(PM) gene in trans can restore both the LPS to its full length and growth in mice to wild-type levels . Furthermore, we have shown that mutant AL251 is unable to cause fowl cholera in chickens and that the attenuation observed is not due to increased serum sensitivity. Int J Med Microbiol, 2004 Apr, 293(7-8), 505 - 12 The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation; Lax AJ et al.; Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture . It is an intracellularly acting toxin that stimulates several signal transduction pathways . The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates . The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements . Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains . The location of all three domains has been confirmed directly . Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain . Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain . Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin . Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process . We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein . Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation . Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling . Natural or experimental infection of animals with toxigenic P . multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone . The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone . PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin . These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation . Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro . In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells. Harefuah, 2004 Feb, 143(2), 92 - 6, 168 {Pasteurella multocida infections--10 years' experience}; Paz A et al.; Dog and cat bites are commonly seen at emergency rooms, but have been inadequately characterized . This study attempted to characterize the clinical features of 10 patients from whom P . multocida was cultured . During the past 10 years, 108 patients have been hospitalized for pet bites, at a rate of 3.4/10,000 hospitalizations . Five patients had a documented exposure to cats, 3 to dogs, and two had an unknown exposure . The mean age was 50.8 years (+/- 20.5) and 80% were men . An average delay of 5.7 days was noted from exposure to hospitalization, and additional 4.4 days until P . multocida was characterized . P . multocida was cultured from wounds in six patients, and three patients had bacteremia; another patient had septic arthritis . Six patients needed debridement and the average hospital stay was 11.7 days (3 times our hospital's average) . Animal bites may take a complicated course . Our findings call for reassessment of the need for prophylaxis in animal bites. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 813 - 8 Reclassification of Bisgaard taxon 33, with proposal of Volucribacter psittacicida gen . nov., sp . nov . and Volucribacter amazonae sp . nov . as new members of the Pasteurellaceae; Christensen H et al.; A total of 25 strains isolated from parrots, budgerigars, parakeets (Psittaciformes) and a chicken, mostly associated with respiratory disease or septicaemia, were classified as a new genus, Volucribacter gen . nov., within the family Pasteurellaceae, on the basis on unique phenotypic characteristics and clear monophyly as determined by 16S rRNA gene sequence comparison . Comparison of 16S rRNA gene sequences from six strains showed at least 98.8 % similarity and the closest similarity outside the genus was found to Bisgaard taxon 34 and to Pasteurella avium, at 94.6 and 94.5 %, respectively . Phenotypes that separate the new genus from other genera of the Pasteurellaceae included at least two characters . The genus includes two species, Volucribacter psittacicida sp . nov . and Volucribacter amazonae sp . nov., corresponding to the two biovars previously outlined, underlining that most isolates have been obtained from psittacine birds . The two species can be separated by fermentation of meso-inositol, (-)-L-fucose, maltose and dextrin and a positive ONPG test . The type strains for Volucribacter psittacicida and Volucribacter amazonae are respectively Gerl . 236/81(T) (=CCUG 47536(T)=DSM 15534(T)) and 146/S8/89(T) (=CCUG 47537(T)=DSM 15535(T)). Vet Immunol Immunopathol, 2004 Jun, 99(3-4), 193 - 202 BHV-1 infection and inflammatory cytokines amplify the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood mononuclear cells in vitro; Leite F et al.; Bovine herpesvirus-1 (BHV-1) has been reported to increase the susceptibility of cattle to respiratory disease caused by Mannheimia (Pasteurella) haemolytica A1 . The principal virulence factor of M . haemolytica is a leukotoxin (LKT) that can specifically kill ruminant leukocytes following its binding to the beta2-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 (LFA-1)) . In this study, we investigated the effects of experimental infection of bovine peripheral blood mononuclear cells (MNCs) with BHV-1 in vitro, on the subsequent interaction of these cells with the M . haemolytica LKT . We found that BHV-1 infection increased LFA-1 expression (as assessed by flow cytometry), and subsequently enhanced LKT binding and cytotoxicity to bovine MNCs . We also found that BHV-1 infection increased CD18, IL-1beta, and IFN-gamma mRNA expression by MNCs . As previously reported for bovine polymorphonuclear neutrophils (PMNs), MNCs increased their expression of LFA-1, and their LKT binding and cytotoxicity, following exposure to IL-1beta, TNF-alpha, and IFN-gamma . These findings suggest that BHV-1 infection, and the resulting release of inflammatory cytokines, can stimulate expression of LFA-1 in bovine MNCs, thus enhancing the binding and biological effects of LKT . If such a mechanism occurs in vivo it might explain, in part, the increased susceptibility of BHV-1 infected cattle to bovine pasteurellosis . Vet Microbiol, 2004 May 20, 100(1-2), 43 - 53 Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies; Ali HA et al.; The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs) . Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P . multocida strain P-1059 (serovar A:3) . Totally eight hybridomas producing Mab were obtained . Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE . Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous . The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein . Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule . The Mabs significantly inhibited the adherence of encapsulated P . multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains . Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1) . Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P . multocida type A strains. Int J Infect Dis, 2004 May, 8(3), 171 - 4 A case of Pasteurella multocida peritoneal dialysis-associated peritonitis and review of the literature; Cooke FJ et al.; OBJECTIVES: Two episodes of peritoneal dialysis-associated peritonitis, which occurred four months apart and were both due to Pasteurella multocida, were noted in a 73 year old woman . This report aims to describe the clinical history of these episodes and the microbiological investigations that were undertaken . The relevant literature will also be discussed . METHODS AND RESULTS: Basic microbiological tests identified the organism as Pasteurella multocida, and further work at a specialist laboratory classified it as Pasteurella multocida subsp . multocida . Pulsed field gel electrophoresis confirmed that the strains isolated from the two clinical episodes originated from the same clone . A literature search was performed, looking particularly for patients who experienced more than one episode of peritonitis caused by Pasteurella spp, whether due to recurrence or re-infection . CONCLUSIONS: It is likely that the infection resulted from a domestic cat, as there was evidence of a cat bite to the dialysis tubing in the period between the two episodes . Re-infection with two identical strains of pasteurella is more probable than relapse, for reasons discussed . Strict hygiene and avoiding contact between dialysis tubing and domestic animals must be emphasised to try to prevent pasteurella and other animal-associated infections in this already vulnerable population. Pediatr Infect Dis J, 2004 Apr, 23(4), 368 - 70 Pasteurella aerogenes hamster bite peritonitis; Freeman AF et al.; Pasteurella multocida has been reported to cause peritoneal dialysis-associated peritonitis after a cat bite or scratch to the catheter . We report a teenager with hamster bite peritonitis caused by P . aerogenes, an organism predominantly isolated from swine. Curr Microbiol, 2004 Mar, 48(3), 189 - 95 Evaluation of PCR based on gene apxIVA associated with 16S rDNA sequencing for the identification of Actinobacillus pleuropneumoniae and related species; da Costa MM et al.; The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases . Biochemical and serological tests are widely applied for App diagnosis and characterization . However, in some isolates, conflicting results are found . The present work focus on the characterization of 29 isolates biochemically classified as A . pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia . Sixteen isolates were from healthy swine, initially classified as nonserotypable A . pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III) . Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing . All 29 isolates were analyzed by PCR for the presence of the apxIVA gene . Thirteen isolates (45%) were confirmed to be A . pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping . The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A . pleuropneumoniae, resulting in eleven A . minor, three A . porcinus, and two Pasteurella sp . Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data . Biochemical characterization proved to be efficient for the majority of the A . pleuropneumoniae isolates . Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A . pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification . We also observed a great variety in rDNA 16S sequences from different A . minor isolates. J Vet Diagn Invest, 2004 Mar, 16(2), 121 - 5 Evaluation of Pasteurella multocida isolated from rabbits by capsular typing, somatic serotyping, and restriction endonuclease analysis; El Tayeb AB et al.; The goal of this study was to characterize Pasteurella multocida isolated from rabbits . Five hundred and fifty-three apparently healthy rabbits were sampled for this study . Nasal swabs were collected from each rabbit for P . multocida isolation and identification . Isolates were further characterized by capsular and somatic antigens and genomic DNA fingerprinting . Thirty-nine P . multocida isolates were recovered from 553 rabbits (7%) . Capsular typing was done by depolymerization of P . multocida capsule by Staphylococcus aureus hyaluronidase and by disc diffusion with mucopolysaccharidase enzymes (heparinase III, chondroitinase AC, and hyaluronidase) . Thirty-one (79%) of the isolates were capsular type A, and 8 isolates (21%) had untypable (UT) capsules . The gel-diffusion precipitin test was used to determine the somatic type of P . multocida isolates . Nineteen isolates were somatic serotype 3 (49%), 12 were serotype 1 (31%), 1 was serotype 2, 2 were serotype 5, 2 were serotype 12 with a weak reaction to antiserum raised against serotype 7 (5%), and 1 was serotype 4 . Two of the isolates (5%) were UT . Restriction endonuclease analysis of the DNA of the isolates revealed 7 distinct profiles by digestion with HindIII, and 12 profiles were obtained with HpaII, whereas digestion with EcoRI did not differentiate between any of the P . multocida DNA isolates studied . The DNA restriction endonuclease enzyme HpaII was found more useful for differentiating between DNA fingerprints of P . multocida rabbit isolates . However, no correlation between capsular type, somatic serotypes, and DNA fingerprints was seen in this study. Res Vet Sci, 2004 Jun, 76(3), 179 - 85 Specific identification of Pasteurella multocida serogroup-A isolates by PCR assay; Gautam R et al.; A polymerase chain reaction (PCR) assay targeting the hyaC-hyaD gene was developed and used to identify strains of Pasteurella multocida belonging to serogroup-A . A set of serogroup-specific-PCR primers amplified a 564 bp product from genomic DNA prepared from bacterial cells or directly from bacterial colonies . This method detected as low as 10 ng of bacterial DNA and had a specificity of 100% for P . multocida serogroup-A . A nested PCR method yielded a single 374 bp product . All fifty isolates were also shown to be identical by restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with BglII. Berl Munch Tierarztl Wochenschr, 2004 Mar-Apr, 117(3-4), 97 - 115 {Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia}; Ewers C et al.; Mannheimia (M.) haemolytica (formerly Pasteurella {P.} haemolytica) is the primary aetiological agent of pneumonic pasteurellosis--one of the most important respiratory diseases in cattle and sheep . While bovine pneumonic pasteurellosis is regarded to be mainly caused by M . haemolytica serotype A1, and in Germany during the last years also by serotype A6, sheep can be infected by all serotypes although there is an increased prevalence of serotypes A2 and A5-7 . The obligate pathogenicity of M . haemolytica is proven by isolation of pure cultures from pneumonic lungs as well as by infection studies . Knowledge about the virulence mechanisms of M . haemolytica and their molecular basis are fragmentary, most probably due to the complex gene regulation of virulence associated factors in lung tissues . This review summarizes the current literature covering virulence factors to substantiate a model of pathogenesis . After serotype A1 strains have colonized the bovine upper respiratory tract they replace other serotypes by mechanisms unknown to date . After fulminant proliferation in the upper respiratory tract the microorganisms colonize the lower respiratory tract, finally entering alveolar spaces . An inflammatory cascade is initiated by M . haemolytica LPS and Leukotoxin, causing activation of the complement system and release of cytokines . Pathognomonic for bovine pneumonic pasteurellosis is the strong influx of neutrophiles accompanied by accumulation of fibrin, finally causing necrosis of alveolar spaces . Depending on lesion size this fibronecrotizing pneumonia can result in death of the animals . In addition, possible protective antigens are discussed . There is still a great effort in the development of efficacious vaccines against pneumonic pasteurellosis in cattle and sheep caused by various M . haemolytica serotypes worldwide . The scarce knowledge concerning presence and distribution of virulence associated factors in M . haemolytica strains and their role in pathogenesis made it difficult to determine a suitable vaccine candidate in the past . In addition, there is lack of knowledge concerning the variability of virulence factors in individual isolates . Genome sequence analysis of M . haemolytica, enabling proteomics and transciptomics, hopefully will give new insight into the pathogenesis of pneumonic pasteurellosis. J S Afr Vet Assoc, 2003 Dec, 74(4), 135 - 6 Pasteurella testudinis associated with respiratory disease and septicaemia in leopard (Geochelone pardalis) and other tortoises in South Africa; Henton MM; The first recorded isolates of Pasteurella testudinis from South African tortoises kept in captivity is presented . P . testudinis was found in association with respiratory disease in affected animals. Vet Microbiol, 2004 Mar 5, 98(3-4), 251 - 60 tRNA-intergenic spacer PCR for the identification of Pasteurella and Mannheimia spp; Catry B et al.; tRNA-intergenic spacer PCR (tDNA-PCR) was evaluated for its effectiveness in differentiating Pasteurella and Mannheimia (sub)species predominantly of ruminant origin . For this purpose, 38 reference strains and 13 field isolates belonging to both genera were investigated . tDNA-PCR enabled discrimination of all Pasteurella species tested (Pasteurella (P.) aerogenes, P . avium, P . canis, P . lymphangitidis, P . multocida, P . trehalosi) . For the differentiation of the subspecies of P . multocida, an additional dulcitol reaction was required . Two of the five so far-defined Mannheimia species, M . granulomatis and M . varigena, had a distinct fingerprinting profile . The remaining three phylogenetically highly related species (M . haemolytica, M . glucosida, and M . ruminalis) clustered together . Nevertheless, M . ruminalis is non-haemolytic, and M . haemolytica and M . glucosida can be differentiated on the basis of two additional phenotypic characteristics (beta-glucosidase and aesculin hydrolysis) . In conclusion, tDNA-PCR is a useful tool in differentiating organisms belonging to the genera Pasteurella and Mannheimia. Microbes Infect, 2004 Mar, 6(3), 290 - 8 Genomic-scale analysis of Pasteurella multocida gene expression during growth within liver tissue of chickens with fowl cholera; Boyce JD et al.; We have recently reported the gene expression profile of Pasteurella multocida during growth in the blood of chickens with fowl cholera . Here we report the gene expression profile of P . multocida during growth in the livers of similarly infected chickens . We compared expression profiles of bacteria harvested from the livers of infected chickens with late-stage fowl cholera with those of bacteria grown in rich medium . Independent analysis of bacterial expression profiles from three individual chickens indicated that 93 P . multocida genes were always differentially expressed during growth in liver tissue . Of these 93 genes, 49 were upregulated and 44 downregulated in the host . Many of the upregulated genes were involved in energy production and conversion (9/49) and carbohydrate transport and metabolism (8/49), and a number of these have been shown to be induced under anaerobic conditions in other species . The downregulated genes were generally of unknown or poorly characterised functions (14/44) . Comparison of the differentially regulated gene sets identified for growth in liver with those identified previously for growth in blood allowed the identification of a core set of 13 upregulated and 16 downregulated genes that were differentially regulated in at least five of the six infections studied. Vet Microbiol, 2004 Apr 5, 99(2), 145 - 58 Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin; Davies RL et al.; One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis . All of the strains were of capsular type A with the exception of a single capsular type F isolate . Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins . However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type . Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P . multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones . Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds . The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones . Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions . Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P . multocida . However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P . multocida. Vet Microbiol, 2004 Apr 5, 99(2), 103 - 12 Pasteurella multocida contains multiple immunogenic haemin- and haemoglobin-binding proteins; Bosch M et al.; Iron-dependent outer membrane proteins (IROMPs) play an important role in bacterial pathogenesis and present several attributes of potential vaccine candidates . TBLASTN analysis of the Pasteurella multocida Pm70 genome using the same molecules of other bacterial pathogens as a query identified eight putative haemin and haemoglobin receptors for this organism . Quantitative binding assays have demonstrated that the proteins PM0040, PM0236, PM0741, PM1081, PM1428, PM0592 and HgbA bind both haemin and haemoglobin, whereas PM0576 and PM1282 ORFs only bind either haemoglobin or haemin, respectively . Furthermore, Western blot analysis showed that P . multocida-infected mice generate specific antibodies against PM0040, PM0236, PM0741, PM1081, PM1428, PM0592 and HgbA proteins . Nevertheless, inoculation of mice with any single one of these receptors alone did not protect against P . multocida infection. Arthroscopy, 2004 Mar, 20(3), 311 - 3 Arthroscopic treatment of septic arthritis in a patient with posterior stabilized total knee arthroplasty; Polzhofer GK et al.; We report on a case of arthroscopic treatment of septic arthritis of the knee in a 73-year-old woman with a posterior stabilized knee endoprosthesis . Six months after arthroplasty of the right knee joint because of osteoarthritis, the patient experienced an erysipelas of the right lower leg after a cat bite . Although given intravenous antibiotic therapy, the patient developed septic arthritis of the right knee . Pasteurella multocida could be identified as the causative organism . The joint infection was classified as stage I according to Gachter . Via arthroscopic joint debridement, partial synovialectomy, the use of continuous irrigation-suction drains, and intravenous antibiotic therapy, the empyema could be cured without removal of the total endoprosthesis of the right knee. J Bone Miner Res, 2004 Apr, 19(4), 661 - 70 Epub 2004 Jan 05. Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation; Harmey D et al.; The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells . The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation . These effects correlated with altered BMP-2 and -4 expression . These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation . INTRODUCTION: The signal transduction pathways controlling osteoblast differentiation are not well understood . In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro . MATERIALS AND METHODS: Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation . Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy . RESULTS: PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression . There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation . PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK . Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation . Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and -4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation . CONCLUSIONS: These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation . Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone. J Comp Pathol, 2004 Feb-Apr, 130(2-3), 137 - 42 Characterization of Pasteurella spp . strains isolated from human infections; Donnio PY et al.; This report describes the distribution of species and capsular groups in a collection of 143 strains of Pasteurella recovered from human patients . The organism isolated most frequently was Pasteurella multocida subsp . multocida . As in animals, most of the group A strains were recovered from the respiratory tract . The distribution of species in relation to the animal source suggests that P . multocida subsp . multocida is more infective than other Pasteurella species or subspecies for man. Vet Res Commun, 2004 Jan, 28(1), 17 - 25 Cloning and sequencing of a 16 kDa outer membrane protein gene of Pasteurella multocida P52; Goswami PP et al.; The outer membrane proteins (OMPs) of Pasteurella multocida are potential immunogens . The 16 kDa OMP of P . multocida P52, serotype B:2, was identified as one of the major immunodominant antigens . The gene omp16, encoding a 16 kDa outer membrane protein, was amplified, cloned into a pBluescript SK(-) vector and sequenced . Complete sequence homology was observed between the 16 kDa OMP gene of P . multocida P52 (serotypes B:2) and P . multocida T16 (somatic serotype 3,4) . This gene was distributed among different serotypes of P . multocida and found to localize in a 6.0 kb HindII fragment of the P . multocida genome. Can J Vet Res, 2004 Jan, 68(1), 66 - 70 Antibody response in sows and piglets following vaccination against Mycoplasma hyopneumoniae, toxigenic Pasteurella multocida, and Actinobacillus pleuropneumoniae; Kristensen CS et al.; The aim of the experimental study was to compare the humoral immune response and occurrence of adverse effects following single or multiple simultaneous vaccination of sows against Mycoplasma hyopneumonia, toxigenic Pasteurella multocida, and Actinobacillus pleuropneumoniae . In addition, passively transferred antibodies to piglets were studied until weaning at 3 weeks of age . Fever was seen in a few sows within the first 12 hours after the 1st and 2nd vaccination . No difference in the occurrence of other adverse effects was observed between groups . Antibody levels were significantly higher in vaccinated sows and their offspring compared with the control group . This was found to be independent of single or simultaneous vaccinations with the 3 vaccines . In conclusion, applying multiple vaccines simultaneously to sows appeared not to influence the occurrence of adverse effects or the sow's serum levels of antibodies at the time of farrowing, nor the offspring's serum levels up to 3 weeks of age. J Arthroplasty, 2004 Feb, 19(2), 244 - 7 Acute pasteurella multocida in total knee arthroplasty; Stiehl JB et al.; Pasturella multocida is a rare cause of joint sepsis in total joint arthroplasty, and all case reports have identified a distant source of infection from an animal bite that has caused potential hematogenous seeding of the prosthesis . We report a case in which no potential distal wound source was found and the only likely etiology was local wound seeding from an old injury . In that injury, a saddle stirrup had caused a severe traumatic soft tissue injury as a horse had rolled over the patient . We draw attention to the fact that this particular bacteria is virulent in producing septic contamination of a total joint prosthesis, and aggressive treatment is indicated when such infection is identified. JAAPA, 2003 Apr, 16(4), 28 - 32, 34, 37 Managing dog, cat, and human bite wounds; Correira K; All bite wounds require the same general approach regarding history and physical exam, selective ancillary testing, wound cleaning, and immobilization . Primary closure should be considered only for bites in which the concerns about cosmetic outcome outweigh the risk of infection . Antibiotic prophylaxis should be initiated for patients with high-risk bite wounds and for those who are at risk for serious wound infection complications . The chosen antibiotic should cover beta-lactamase-producing aerobic and anaerobic organisms, including Pasteurella species in animal bites and E corrodens in human bites. J Clin Pathol, 2004 Feb, 57(2), 210 - 2 Fatal Pasteurella dagmatis peritonitis and septicaemia in a patient with cirrhosis: a case report and review of the literature; Ashley BD et al.; Pasteurella species cause zoonotic infections in humans . Human pasteurella infections usually manifest as local skin or soft tissue infection following an animal bite or scratch . Systemic infections are less common and are limited to patients at the extremes of age or those who have serious underlying disorders, including cirrhosis . Most human pasteurella infections are caused by the multocida species . We report a case of Pasteurella dagmatis peritonitis and septicaemia in a patient with cirrhosis . The infection followed a scratch inflicted by a pet dog . Despite appropriate antibiotic treatment the infection proved fatal . Spontaneous bacterial peritonitis caused by P dagmatis has not been reported previously . Pasteurella dagmatis is a relatively recently described species, which is rarely reported as a human pathogen . This species may be misidentified unless commercial identification systems are supplemented by additional biochemical tests. Infect Immun, 2004 Feb, 72(2), 1195 - 8 Identification of five outer membrane-associated proteins among cross-protective factor proteins of Pasteurella multocida; Tabatabai LB et al.; Fowl cholera is caused by Pasteurella multocida serovars A:1, A:3, and A:4 . The 39-kDa cross-protective factor protein and four other membrane proteins of the membrane proteome of P . multocida were identified . We determined that the 39-kDa cross-protective protein was Pasteurella lipoprotein B, or PlpB. Infect Immun, 2004 Feb, 72(2), 701 - 8 Two TonB systems in Actinobacillus pleuropneumoniae: their roles in iron acquisition and virulence; Beddek AJ et al.; Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system . Tonpitak et al . (W . Tonpitak, S . Thiede, W . Oswald, N . Baltes, and G.-F . Gerlach, Infect . Immun . 68:1164-1170, 2000) have described one such system, associated with tbpBA encoding the transferrin receptor, and here we report a second, termed tonB2 . This gene cluster (exbB2-exbD2-tonB2) is highly homologous to those in other Pasteurellaceae, unlike the earlier system described (now termed tonB1), suggesting that it is the indigenous system for this organism . Both tonB2 and tonB1 are upregulated upon iron restriction . TonB2, but not TonB1, was found to be essential for growth in vitro when the sole source of iron was hemin, porcine hemoglobin, or ferrichrome . In the case of iron provided as iron-loaded porcine transferrin, neither tonB mutant was viable . The tonB1 phenotype could be explained by a polar effect of the mutation on transcription of downstream tbp genes . We propose that TonB2 is crucial for the acquisition of iron provided in this form, interacting with accessory proteins of the TonB1 system that have been demonstrated to be necessary by Tonpitak et al . TonB2 appears to play a much more important role in A . pleuropneumoniae virulence than TonB1 . In an acute porcine infection model, the tonB2 mutant was found to be highly attenuated, while the tonB1 mutant was not . We hypothesize that acquisition of the tonB1-tbp gene cluster confers a biological advantage through its capacity to utilize transferrin-iron but that TonB1 itself plays little or no part in this process. Vaccine, 2004 Jan 26, 22(5-6), 643 - 9 Maternally derived humoral immunity to bovine viral diarrhea virus (BVDV) 1a, BVDV1b, BVDV2, bovine herpesvirus-1, parainfluenza-3 virus bovine respiratory syncytial virus, Mannheimia haemolytica and Pasteurella multocida in beef calves, antibody decline by half-life studies and effect on response to vaccination; Fulton RW et al.; The passive immunity transferred to calves from their dams was investigated in a beef herd to determine half-life of antibody, estimated time to seronegative status and effect on immunization . One hundred two beef calves in a commercial ranch under standard management conditions were utilized . Samples were collected at branding (day 0) . This was the first possible date to collect samples postcalving . This was approximately 2 months postcalving, and days 95 and 116 . The calves were divided into two groups: vaccinates (51) and nonvaccinates (51) . The calves were vaccinated with a commercial inactivated viral vaccine containing bovine viral diarrhea virus (BVDV)1a, BVDV2, bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PI-3V), and bovine respiratory syncytial virus (BRSV) on days 0 and 95 . Half of the vaccinated and unvaccinated calves also received one dose of an experimental Mannheimia haemolytica and Pasteurella multocida vaccine at day 95 . Serums were tested for neutralizing antibody titers to BVDV1a, BVDV1b, BVDV2, BHV-1, PI-3V, and BRSV . Antibodies were detected by ELISA to M . haemolytica whole cell, M . haemolytica leukotoxin, and P . multocida outer membrane