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PubMed1992

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.

Actinobacillus↗
PubMed1985

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H.

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.

Escherichia coli↗
PubMed1991

Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain.

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde- and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silver-enhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

Animals↗
PubMed1975

Characterization of "Pasteurella" piscicida isolated from white perch and cultivated yellowtail.

The morphological, physiological, and biochemical characteristics of twelve "Pasteurella" piscicida strains isolated from white perch and yellowtail are described and the present uncertain taxonomic status of the organisms is discussed. The organisms isolated were gram-negative rods showing bipolar staining and pleomorphism. No spores or flagella were observed. They were non motile and viscid colonies were formed. Growth was observed in a temperature range of 20 to 30 C and the salinity range of growth was between 0.5% and 4.0%. They were aerobic and facultative anaerobic. Oxidase and catalase were produced. Nitrates were not reduced to nitrites. Lysine and ornithine decarboxylases were not produced but arginine dihydrolase was produced. The egg yolk reaction was positive. Tween 80 and tributyrin were decomposed. Phosphatase was produced. Beta hemolysis was revealed on a medium containing defibrinated blood from chickens or carp but not from mammals. Methyl red and Voges-Proskauer tests were positive, and acetoin was produced from 2,3-butanediol. Glucose was fermented without gas production. Acid was produced from fructose, mannose, galactose, sucrose, maltose, dextrin and glycerol. These organisms differed from all other members of the genus Pasteurella with respect to nitrate reduction, arginine dihydrolase, Voges-Proskauer and methyl red tests. The formation of viscid colonies and inability to grow in a medium without sodium chloride or at 37 C were additional characteristics of these organisms.

Aerobiosis↗
PubMed1998

Degradation of fluoranthene by Pasteurella sp. IFA and Mycobacterium sp. PYR-1:isolation and identification of metabolites.

The findings from a biodegradability study of fluoranthene using two pure bacterial strains, Pasteurella sp. IFA (B-2) and Mycobacterium sp. PYR-1 (AM) are reported. Of total fluoranthene, 24% (B-2) and 46% (AM) was biodegraded in an aqueous medium during 14 d of incubation at room temperature. During this period the bacteria were capable of mineralizing approximately two-thirds (B-2) and four-fifths (AM) of biodegraded fluoranthene to CO2, while one-third (B-2) and one-fifth (AM) of the original fluoranthene remained as stable metabolic products. These metabolites were isolated using liquid-liquid extraction and identified using gas chromatography-mass spectrometry (GC-MS) and derivatization techniques. Two metabolites (9-fluorenone-1-carboxylic acid and 9-fluorenone) were identified by GC-MS directly, while the metabolites 9-fluorenone-1-carboxylic acid, 9-hydroxyfluorene, 9-hydroxy-1-fluorene-carboxylic acid, 2-carboxybenzaldehyde, benzoic acid and phenylacetic acid were determined in their derivatized forms. From the identified metabolites, a fluoranthene biodegradation pathway was proposed for Pasteurella sp. IFA.

Biodegradation, Environmental↗
PubMed1990

Tn7 inserts in both orientations at a single chromosomal location and apparently forms cointegrates in Pasteurella multocida.

Pasteurella multocida transconjugants isolated after mating with Escherichia coli strains that carry one or the other of two Tn7-containing suicide plasmids, pRKTV5 and pUW964 (pRKTV5::Tn5), were analysed. These plasmids have the ColE1 replication origin and were thus expected to deliver transposons but not be maintained as free replicons in Pasteurella. Five out of six transconjugants selected for acquisition of Tn7 from E. coli (pRKTV5) had simple insertions of the transposon, in either orientation, at a single chromosomal location, while the sixth had pRKTV5 integrated at the same location. By contrast, all of 27 transconjugants selected for acquisition of either Tn7 or Tn5 from E. coli (pUW964) maintained pUW964. Of seven subsequently examined at the molecular level, all had pUW964 (in one case, a deletion derivative) integrated at the same location as the Tn7 insertions obtained with pRKTV5. A copy of Tn7 was present at each boundary between the integrated plasmids (pRKTV5 or pUW964) and the chromosome in each strain. The two copies of Tn7 at either end of an integrated plasmid were either in the same (six cases) or in opposite (two cases) orientations with respect to each other. These seem to be products of replicative transposition by Tn7 but can also derive from conservative mechanisms.

Chromosomes, Bacterial↗
PubMed1991

Characterization of toxin from different strains of Pasteurella multocida serotype A and D.

Chromosomal DNA from 13 different selected Pasteurella multocida spp. multocida strains of serotypes A and D were isolated and compared. All 10 toxigenic strains were recognized by a DNA probe which included the toxA gene coding for the Pasteurella multocida toxin (PMT). None of the three nontoxigenic strains reacted with the DNA probe. Toxin from the 10 toxigenic strains were isolated and compared. All were found to possess the biological characteristics previously described for the PMT isolated from P. multocida ssp. multocida NCTC 12178, including molecular mass of approx. 143 kDa and reactivity with a series of monoclonal antibodies. Toxin prepared from different toxigenic strains could not be differentiated immunologically by tandem crossed immunoelectrophoresis, Toxin, which was affinity purified from four of the strains and subsequently inactivated by formaldehyde, was cross-protective when used for vaccination of mice before challenge with PMT. It is concluded that the toxin from toxigenic strains of P. multocida ssp. multocida must be very similar, if not identical.

Animals↗
PubMed1998

Bacterial croup caused by Pasteurella haemolytica.

We report a case of bacterial croup caused by Pasteurella haemolytica. A nine-month-old girl was admitted to our hospital because of abrupt onset dyspnea and unconsciousness. From clinical and laboratory findings, she was diagnosed as having bacterial croup. Pasteurella haemolytica was recovered from her tracheal secretion on admission. To our knowledge, this is the first report of bacterial croup caused by P. haemolytica.

Croup↗
PubMed1981

Characteristics of 20 human Pasteurella isolates from animal bite wounds.

The bacteriological characteristics of 18 isolates of Pasteurella multocida and two isolates of Pasteurella pneumotropica from infected animal bite wounds have been studied. A high proportion (8/18) of the P. multocida isolates fermented lactose and five fermented maltose. Two failed to ferment sorbitol. Neither of the P. pneumotropica strains showed ornithine decarboxylase activity. Other biochemical characteristics were mainly in agreement with previous reports. The susceptibility of the isolates to 10 antibacterial agents has been determined by the disc diffusion method. Ampicillin, cephalothin and cotrimoxazole showed uniformly good activity whereas penicillin G and doxycycline were slightly less active. Apart from one horse bite, all bites were inflicted by cats and dogs. Two cases were serious. P. multocida seems to be a commoner occurrence in infected animal bite wounds than reports suggest. P. pneumotropica bite infections are seemingly uncommon.

Animals↗
PubMed1996

Role of Pasteurella granulomatis and Dermatobia hominis in the etiology of lechiguana in cattle.

Attempts were made to reproduce bovine lechiguana, a disease associated with Dermatobia hominis and Pasteurella granulomatis infections. Suspensions of Pasteurella granulomatis were mixed with each of the following: saponin, oil adjuvant, ground Dermatobia hominis, or 5% mucin. Each preparation was inoculated into 6 cattle. Twelve more cattle, 6 of which received dexamethasone, were inoculated with bacterial suspension alone. Abscesses but no lechiguana was produced in all 36 cattle. After abscess regression, 12 cattle were reinoculated with a suspension of mouse-passed P. granulomatis. Only abscesses were produced. The intralymphatic inoculation of P. granulomatis in 6 cattle did not produce the disease. Eleven cattle infected naturally with D. hominis had lesions containing dead larvae. These lesions were inoculated with P. granulomatis. Nine cattle were experimentally infected with larvae of D. hominis that had been contaminated with the bacteria. No lechiguana lesions were produced in these 20 cattle. Six cattle with severe natural D. hominis infection were inoculated in the larval lesions with P. granulomatis. One developed lesions indistinguishable from those of natural lechiguana. The lesions regressed after treatment with chloramphenicol. D. hominis larvae and exudate from lesions caused by the fly were collected from 7 cattle on 3 farms and examined bacteriologically. P. granulomatis was isolated from the larvae and the exudate of a healthy calf from a farm where lechiguana had never been observed. These results suggest that P. granulomatis has a causal role in lechiguana, and that D. hominis may be a carrier of the bacterium. These observations suggest that lechiguana occurs when severe D. hominis lesions are infected with P. granulomatis. The apparent long incubation period, the negative results obtained in the other experiments, and also the infrequent occurrence of the natural disease suggest that lechiguana is a disease for which Koch's postulates are not easily fulfilled.

Abscess↗
PubMed1996

Distribution of tet(H) among Pasteurella isolates from the United States and Canada.

Tetracycline-resistant isolates of Pasteurella multocida and Pasteurella haemolytica obtained from various locations in the United States and Canada were studied to determine the distribution of the tet(H) gene. Of the 31 isolates examined, 25 were found to contain the tet(H) gene. Chromosomal or plasmid DNA obtained from those that did not contain the tet(H) gene did not hybridize with probes specific for classes A through G, though chromosomal DNA from one isolate lacking tet(H) hybridized with a probe specific for class M. The tet(H) gene was found on plasmid as well as on chromosomal DNA, suggesting that it is carried on a transposable element.

Animals↗
PubMed2001

Molecular analysis of tetracycline resistance in Pasteurella aerogenes.

Tetracycline-resistant Pasteurella aerogenes isolates obtained from the intestinal tract of swine were investigated for their tet genes by PCR analysis and hybridization experiments. In contrast to Pasteurella isolates from the respiratory tract, tet(H) genes were detected in the chromosomal DNA of only 2 of the 24 isolates, one of which also carried two copies of a tet(B) gene. All other P. aerogenes isolates carried tet(B) genes, which are the predominant tet genes among Enterobacteriaceae. A single isolate harbored a tet(B) gene as part of a truncated Tn10 element on the 4.8-kb plasmid pPAT2. Comparative analysis of the pPAT2 sequence suggested that the Tn10 relic on plasmid pPAT2 is the result of several illegitimate recombination events. The remaining 21 P. aerogenes isolates carried one or two copies of the tet(B) gene in their chromosomal DNA. In the majority of the cases, these tet(B) genes were associated with copies of Tn10 as confirmed by their SfuI and BamHI hybridization patterns. No correlation between the number of tet gene copies and the MICs of tetracycline, doxycyline and minocycline was observed.

Animals↗
PubMed1989

Agarose soy casein digest medium for replacement of blood agar for potency determinations of live Pasteurella vaccines.

Blood agar, prepared with Trypticase (BBL Microbiology Systems, Cockeysville, Md.) soy agar and 5% defibrinated bovine blood, is used for testing the potency of live Pasteurella multocida and Pasteurella haemolytica vaccines, but its potential for variation makes it undesirable to use in a standard assay method. Tests done with RPMI 1640 and Trypticase soy medium indicated that the benefits obtained by adding defibrinated blood to the Trypticase soy agar medium were more likely due to neutralization of toxic components than to the presence of transferrin or iron as growth factors. Reduction of toxic components in the Trypticase soy agar medium was accomplished by replacing agar with agarose and by autoclaving glucose as a separate solution to produce the replacement medium. The replacement medium was prepared by autoclaving three separate solutions--Trypticase soy broth without glucose; glucose; and agarose--cooling to 55 degrees C, and mixing and then pouring the mixtures into petri dishes. The growth obtained with this medium as judged by determination of the number of CFU and the colony sizes of P. multocida or P. haemolytica was equal to or better than those obtained with blood agar.

Bacterial Vaccines↗
PubMed1986

Cloning of a serotype-specific antigen from Pasteurella haemolytica A1.

Recombinant plasmids coding for a soluble (or surface) antigen of Pasteurella haemolytica A1 were identified. Two plasmids, both containing the same 5.4 kilobase pairs of insert DNA, were recovered independently by screening a clone band of P. haemolytica A1 genomic DNA in Escherichia coli for the expression of P. haemolytica A1 soluble antigens (R. Y. C. Lo and L.A. Cameron, Can. J. Biochem. Cell Biol. 64:73-76, 1986). E. coli cells carrying the plasmids were found to be agglutinated by an antiserum raised against the P. haemolytica A1 soluble antigens. Analysis of the E. coli clones by electron microscopy revealed patches of amorphous material on the surface of the cells which were not present on the controls. Further characterization with protein A-colloidal gold labeled both these patches and the outer membranes of these cloned cells pretreated with the specific antiserum. These results indicated that the cloned antigen was expressed on the surface of the E. coli cells. The cloned antigen was found to be specific for serotype 1 when tested by slide agglutination against a collection of P. haemolytica typing antisera. Southern blot hybridization, using the cloned DNA as a probe, labeled the genomic DNA from P. haemolytica serotype 1 as well as the cross-agglutinating serotypes 2 and 7, but not DNA from the non-cross-agglutinating serotypes 3 and 4 and Pasteurella multocida. These results demonstrated that serotype specificity could be attributed to the particular antigenic determinants in the genome of the organism.

Antigens, Bacterial↗
PubMed1995

Pasteurella multocida produces a protein with homology to the P6 outer membrane protein of Haemophilus influenzae.

An antibody specific for a 16-kDa outer membrane protein of a rabbit strain of Pasteurella multocida was used to probe representatives of all 16 somatic serotypes of P. multocida, as well as the vaccine strains CU and M9, and all were shown to express the protein. The gene encoding this protein was cloned and sequenced and found to have extensive sequence homology with the gene encoding the P6 protein of Haemophilus influenzae. The protein in P. multocida has been designated P6-like. The gene encoding the P6-like protein was used to probe members of the family Pasteurellaceae and other gram-negative bacteria. Representatives of all 16 somatic serotypes (as well as the vaccine strains CU and M9) of P. multocida hybridized with the P6-like gene under conditions of high stringency. The DNA from H. influenzae hybridized weakly with the P6-like gene under these conditions, but Pasteurella haemolytica (representatives of A and T biotypes), Bordetella bronchiseptica, B. avium, Actinobacillus suis, A. suis-like, A. lignieresii, A. ureae, A. rossii, A. pleuropneumoniae, A. equuli, and various members of the family Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium) did not hybridize detectably. Under conditions of lower stringency, the P6-like gene also hybridized strongly with DNA from P. multocida, H. influenzae, and A. rossii but weakly with DNA from P. haemolytica and members of the genus Actinobacillus. These results suggest that the P6-like protein of P. multocida might be useful as an immunizing product to protect poultry from avian cholera. This suggestion stems from (i) our finding that the P6-like protein in P. multocida is widely distributed among all the somatic serotypes and (ii) the previous work of others demonstrating that the P6 protein of H. influenzae elicits a protective immune response in animal models of human disease.

Amino Acid Sequence↗
PubMed1995

Extracellular neuraminidase production by a Pasteurella multocida A:3 strain associated with bovine pneumonia.

The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme.

Animals↗
PubMed1997

Purification and partial characterization of the OmpA family of proteins of Pasteurella haemolytica.

This study was conducted to partially characterize and identify the purity of two major outer membrane proteins (OMPs) (with molecular weights of 32,000 and 35,000 [32K and 35K, respectively]) of Pasteurella haemolytica. The 35K and 32K major OMPs, designated Pasteurella outer membrane proteins A and B (PomA and PomB, respectively), were extracted from P. haemolytica by solubilization in N-octyl polyoxyl ethylene. The P. haemolytica strain used was a mutant serotype A1 from which the genes expressing the 30-kDa lipoproteins had been deleted. PomA and PomB were separated and partially purified by anion-exchange chromatography. PomA but not PomB was heat modifiable. The N-terminal amino acid sequences of the two proteins were determined and compared with reported sequences of other known proteins. PomA had significant N-terminal sequence homology with the OmpA protein of Escherichia coli and related proteins from other gram-negative bacteria. Moreover, polyclonal antiserum raised against the E. coli OmpA protein reacted with this protein. PomA was surface exposed, was conserved among P. haemolytica biotype A serotypes, and had porin activity in planar bilayers. No homology between the N-terminal amino acid sequence of PomB and those of other known bacterial proteins was found. Cattle vaccinated with live P. haemolytica developed a significant increase in serum antibodies to partially purified PomA, as shown by enzyme-linked immunosorbent assays, and to purified PomA and PomB, as detected on Western blots and by densitometry.

Amino Acid Sequence↗
PubMed1997

Evolutionary genetics of Pasteurella haemolytica isolates recovered from cattle and sheep.

Genetic diversity and relationships among 194 Pasteurella haemolytica isolates, which were recovered predominantly from cattle (39%) and sheep (58%) suffering from pneumonic pasteurellosis in the United Kingdom, Germany, and the United States, were estimated by examination of allelic variation at 18 enzyme-encoding loci detected by multilocus enzyme electrophoresis. The isolates formed two major divisions. One included 178 Pasteurella haemolytica sensu stricto strains representing serotypes A1, A2, A5 to A9, A12 to A14, and A16; the other was composed of 16 isolates belonging to the A11 taxon. P. haemolytica isolates were classified into 22 electrophoretic types (ETs) that formed three primary phylogenetic lineages. One lineage was represented by ovine serotype A2 isolates, a second lineage consisted of bovine serotype A2, together with serotype A7 and A13 isolates, and the third lineage included isolates representing all of the other serotypes, as well as a second group of serotype A7 strains. Electrophoretic types were nonrandomly associated with specific capsular serotypes, lipopolysaccharide (LPS) types, outer membrane protein (OMP) types, and host species. Bovine isolates were represented by only three serotypes (A1, A2, and A6) in 5 ETs, whereas ovine isolates were represented by all of the serotypes in 19 ETs. The majority (76%) of bovine isolates were of serotypes A1 or A6 and belonged to a single ET that marked a virulent, cattle-specific clonal group. Among the ovine isolates, 40% were of serotype A2 and belonged to two ETs that represented two virulent, sheep-specific clonal groups. Bovine A1 and A6 isolates and bovine A2 isolates were phylogenetically distinct from ovine isolates of the same serotypes, indicating that different subpopulations of these serotypes are associated with disease in cattle and sheep. Consistent differences in the OMP profiles of strains of the bovine and ovine lineages of these three serotypes suggest that certain OMPs are involved in host specificity and virulence. Evolutionary relationships among P. haemolytica isolates indicate that the ancestral host is the sheep and that several distinct clonal lineages have crossed the species barrier into cattle. The A11 taxon is a heterogeneous group of opportunistic pathogens of sheep that represents a separate species.

Animals↗