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PubMed2001

The effect of Pasteurella haemolytica A2 infection on phagocytosis efficiency of caprine broncho-alveolar macrophages.

An experiment was designed to study the in vivo effect of Pasteurella haemolytica A2 infection on the phagocytosis activity of caprine broncho-alveolar macrophages and the extent of pneumonic lesions. Twelve healthy local Kacang goats, about 7 months of age, were divided into two groups of six. Goats in group 1 were inoculated intratracheally with 4 ml inoculum containing 2.8 x 10(9) colony-forming units (CFU)/ml of Staphylococcus aureus. Goats in group 2 were inoculated intratracheally with 4 ml of inoculum containing 9.5 x 10(8) CFU/ml of Pasteurella haemolytica A2 isolated earlier from pneumonic lungs of goat. At intervals of 3 and 7 days post-challenge five goats from each group were killed and the lungs were washed with sterile phosphate-buffered saline. Smears were prepared from the lung washing fluid and the number of macrophages with phagocytic activity was determined. At day 3 post-infection, goats of both groups showed a similar pattern of pneumonic lesion. The lung washing fluid of goats in group 2 was found to contain numerous neutrophils and macrophages. Goats in group 2 showed significantly (P < 0.05) higher extent of lung lesions than group 1. Similarly, the average extent of lung lesions was significantly (P < 0.05) more severe in group 2 at day 7 post-infection. The lung washing fluid contained mostly macrophages. The phagocytic activity following S. aureus infection was more efficient and significantly (P < 0.01) higher compared with infection by P. haemolytica A2. There were weak correlations between the extent of pneumonic lesion and the phagocytic activity. Thus, goats with poor phagocytic activity were likely to develop more extensive lung lesions.

Administration, Intranasal↗
PubMed1990

Experimental respiratory infection of goats with caprine herpesvirus and Pasteurella haemolytica.

Groups of six male goats were inoculated intratracheally and intranasally with either caprine herpesvirus followed 6 days later by Pasteurella haemolytica, canine herpesvirus alone or P. haemolytica alone. Pneumonic lesions were observed in five of the six goats inoculated with caprine herpesvirus followed by P. haemolytica and in three of the six goats inoculated with P. haemolytica alone, but were not observed in goats inoculated with caprine herpesvirus alone or in non-infected controls. Pasteurella haemolytica was isolated from seven of eight lungs with pneumonia, but only from one of sixteen lungs without pneumonia. The lesions ranged from fatal acute exudative necrotising pneumonia to predominantly proliferative pneumonia. Half of the caprine herpesvirus-inoculated goats developed a clinical catarrhal rhinitis five days post-inoculation and the only virus-specific histopathological lesion was a mild tracheitis. Canine herpesvirus was recovered from the nasal swabs of all caprine herpesvirus- inoculated goats developed a clinical catarrhal rhinitis five days post-inoculation and the only virus-specific histopathological lesion was a mild tracheitis. Canine herpesvirus was recovered from the nasal swabs of all canine herpesvirus-inoculated goats and from the lungs of three goats inoculated with caprine herpesvirus alone. The experimental inoculations demonstrated that P. haemolytica alone can produce pneumonia in goats. In addition, the study showed that caprine herpesvirus readily proliferates in the upper respiratory tract and lungs of goats but the role of caprine herpesvirus in the aetiology of pneumonia remains uncertain.

Journal Article↗
PubMed1993

Comparison between the minimal inhibitory concentration of tilmicosin and oxytetracycline for bovine pneumonic Pasteurella haemolytica isolates.

The minimal inhibitory concentration (MIC) of tilmicosin and oxytetracycline was determined for Pasteurella haemolytica isolated from Dutch cattle in 1991. The agar dilution method was used. Of the Pasteurella haemolytica strains examined, 96% (24) had a MIC for tilmicosin of 2 micrograms/ml or lower; in one strain the MIC was 8 micrograms/ml. The MIC for oxytetracycline was equal to or higher than 64 micrograms/ml in 19 strains (76%), whereas for 5 strains the MIC was 1 microgram/ml and for 1 strain 2 micrograms/ml.

Animals↗
PubMed1999

Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction-modification system.

In a previous study, a recombinant plasmid that contains a CACAG pentanucleotide repeat was isolated from a Pasteurella haemolytica A1 library. Southern hybridization analysis using a (CACAG)5probe indicated the presence of two loci that contain the pentanucleotide repeats on the genome of P.haemolytica A1. Additional hybridization analyses against genomic DNA from related microorganisms indicated that the repeats are only present in P.haemolytica and Pasteurella trehalosi T3. The various serotypes of P.haemolytica werefound to have either one or two of the CACAG repeat-containing loci. Examination of the locus designated Rpt2 by PCR and sequence analysis indicated that the number of CACAG repeats could change upon serial subculture which most likely occurs as a result of DNA slipped-strand mispairing. A plasmid carrying the Rpt2 locus was isolated and characterized. Sequenceanalysis indicated that the CACAG repeats are contained within the 5'-end of a gene that showed homology to mod genes of type III restriction-modification systems. A second open reading frame downstream was identified which showed homology to res genes of type III restriction-modification systems. Both the modification and restriction proteins could be expressed and polypeptides of the expected sizes were detected by SDS-PAGE. Restriction activity could also be detected in crude cytoplasmic extracts of Escherichia coli strains carrying the mod and res genes on recombinant plasmids.

Amino Acid Sequence↗
PubMed1998

Hemoptysis as the sole presentation of Pasteurella multocida infection.

Pasteurella multocida has been implicated as the cause of a variety of respiratory conditions (eg, bronchitis, pneumonia, lung abscess, empyema), but hemoptysis has been noted only in conjunction with other lung conditions. We report a case in which hemoptysis was the sole manifestation of Pasteurella infection. The patient was a middle-aged man with severe obstructive lung disease and exposure to cats. Diagnosis was made by bronchoscopy and high-dose penicillin was required for resolution.

Animals↗
PubMed1996

Phylogenetic relationships and diversity within the Pasteurella haemolytica complex based on 16S rRNA sequence comparison and outer membrane protein and lipopolysaccharide analysis.

The outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 30 untypeable isolates of Pasteurella haemolytica were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with the profiles of typeable isolates. The phylogenetic relationships of 28 isolates representing each of the serotypes of P. haemolytica and Pasteurella trehalosi, as well as untypeable isolates of P. haemolytica, were determined by comparing 16S rRNA sequences. The analysis of the OMP and LPS profiles of the untypeable isolates revealed five groups, which were designated untypeable groups 1 (UG1) through UG5. The UG1 and UG2 isolates had OMP and LPS profiles identical to the profiles of certain serotype A1 and A2 isolates, respectively. Furthermore, UG1 isolates originating from cattle and sheep could be clearly differentiated on the basis of their OMP profiles. The OMP and LPS profiles of UG3 isolates were similar appearance to the profiles of serotype A11 isolates, suggesting that these two groups are closely related. The OMP profiles of UG4 and UG5 isolates were unique and different from the OMP profiles of the UG1 through UG3 isolates. A comparison of 16S rRNA sequences revealed that typeable isolates of P. haemolytica could be divided into the following three groups: (i) serotype A1, A5 through A9, A12 through A14, and A16 isolates, (ii) serotype A2 isolates, and (iii) serotype A11 isolates. the isolates belonging to the first group all had identical sequences, whereas the sequences of isolates belonging to the second and third groups differed from the sequences of the isolates belonging to the first group at two and four base positions, respectively. The sequence data for the untypeable isolates confirmed the conclusions derived from the OMP and LPS analysis. Isolates belonging to UG1 and UG2 were identical to serotype A1 and A2 isolates, respectively; isolates belonging to UG3 were related to serotype A11 isolates, although there was some sequence heterogeneity within this group; and isolates belonging to UG4 and UG5 were more distantly related to P. haemolytica than were isolates belonging to UG1 through UG3 and were clearly members of two different species. As expected, isolates of P. trehalosi were event more distantly related to P. haemolytica than were the untypeable isolates, but there was significantly more sequence variation among the four serotypes of this species than there was among the serotypes of P. haemolytica. The correlation of the OMP and LPS data with the 16S rRNA sequence data suggested that OMP and LPS analyses might be useful for preliminary screening and comparing large numbers of isolates in taxonomic and epidemiological studies of the Pasteurellaceae.

Animals↗
PubMed1999

Taxonomic relationships of the [Pasteurella] haemolytica complex as evaluated by DNA-DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov., Mannheimia granulomatis comb. nov., Mannheimia glucosida sp. nov., Mannheimia ruminalis sp. nov. and Mannheimia varigena sp. nov.

The present paper presents the conclusions of a polyphasic investigation of the taxonomy of the trehalose-negative [Pasteurella] haemolytica complex. Clusters previously identified by ribotyping and multilocus enzyme electrophoresis (MEE) have been evaluated by 16S rRNA sequencing and DNA-DNA hybridizations. Results obtained by the different techniques were highly related and indicated that the [P.] haemolytica complex contains distinct genetic and phenotypic groups. At least seven species were outlined, five of which were named. We refrained in formal naming of more groups until additional strains are characterized. Five 16S rRNA clusters were identified corresponding to distinct lineages previously outlined by MEE. Within 16S rRNA cluster I two distinct genotypic groups have been outlined in addition to [P.] haemolytica sensu stricto (biogroup 1). Each of the clusters II, III, IV and V represent at least one new species. The investigations underline that [P.] haemolytica sensu stricto only contains strains that do not ferment L-arabinose even though they are referred to as 'biotype A' of [P.] haemolytica. The five 16S rRNA clusters identified had a common root relative to the other species within the family Pasteurellaceae, and the overall sequence similarity among these five clusters was higher than what is observed within the existing genera of the family. The allocation of the trehalose-negative [P.] haemolytica complex to a new genus seems to be indicated. Based on the polyphasic investigation performed a new genus Mannheimia is proposed for the trehalose-negative [P.] haemolytica complex. At the present stage two previously named species are transferred to this new genus and three new species are described. [P.] haemolytica is reclassified as Mannheimia haemolytica comb. nov., whereas Pasteurella granulomatis, Bisgaard taxon 20 and [P.] haemolytica biovar 3J are reclassified and combined in the species Mannheimia granulomatis comb. nov. Mannheimia glucosida sp. nov. corresponds to [P.] haemolytica biogroups 3A-3H and the beta-glucosidase and meso-inositol-positive strains of [P.] haemolytica biogroup 9. All typable strains within M. glucosida belong to serotype 11. Mannheimia ruminalis sp. nov. consists of strains previously classified as Bisgaard taxon 18 and [P.] haemolytica biogroup 8D. Finally, Mannheimia varigena sp. nov. includes [P.] haemolytica biogroup 6 as well as Bisgaard taxon 15 and Bisgaard taxon 36. The type strains are NCTC 9380T (M. haemolytica), ATCC 49244T (M. granulomatis), CCUG 38457T = P925T (M. glucosida), CCUG 38470T = HPA92T (M. ruminalis) and CCUG 38462T = 177T (M. varigena).

Base Composition↗
PubMed1991

Genomic distribution of a serotype 1-specific antigen-coding DNA fragment of Pasteurella haemolytica.

A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.

Animals↗
PubMed1970

Intracellular Pasteurella pseudotuberculosis: Multiplication in Cultured Spleen and Kidney Cells.

Pasteurella pseudotuberculosis multiplied within rabbit cells in primary culture. Spleen cells from rabbits were either dispersed mechanically, exposed to the organism immediately after cell preparation, and grown as a pellet or the cells were dispersed enzymatically and grown as a monolayer for 4 to 6 days before the bacteria were introduced. Intracellular multiplication proceeded at a logarithmic rate for 1 to 2 days, with a generation time of about 70 min in pelleted cells and 4 to 5 hr in monolayered cells. Under the conditions employed, the wild-type virulent strain and an avirulent mutant multiplied at similar rates. Atmosphere, constituents of the medium, and multiplicity of infection influenced intracellular proliferation. The organism also proliferated in kidney cells. Microscopic observation of stained cells indicated limited growth of the pathogen in spleen cells at 37 C. In kidney cells, the pasteurellae localized in compartments; frequently, a single compartment with masses of proliferating organisms almost filled the cytoplasm.

Journal Article↗
PubMed1992

Ovine pulmonary surfactant induces killing of Pasteurella haemolytica, Escherichia coli, and Klebsiella pneumoniae by normal serum.

Pulmonary surfactant has been shown to play an increasingly important role in bacterial clearance at the alveolar surface in the lung. This study describes a bactericidal mechanism in which ovine pulmonary surfactant induces killing of Pasteurella haemolytica by normal serum. To demonstrate killing, six bacterial species were incubated first with pulmonary surfactant for 60 min at 37 degrees C and then with serum for an additional 60 min at 37 degrees C. P. haemolytica type A1 strains 82-25 and L101, a P. haemolytica type 2 strain, Escherichia coli, and Klebsiella pneumoniae were susceptible and Pasteurella multocida, Serratia marcescens, and Pseudomonas aeruginosa were not susceptible to killing by ovine pulmonary surfactant and normal serum. No bacteria incubated with bovine pulmonary surfactant were killed by normal serum. Although the species origin of pulmonary surfactant was selective, the species origin of serum was not. P. haemolytica incubated with ovine pulmonary surfactant was killed by fetal calf serum, gnotobiotic calf serum, pooled normal sheep serum, pooled normal rabbit serum, and pooled guinea pig serum. Ultrastructurally, killed P. haemolytica suspensions contained dead cells and cells distorted with vacuoles between the cytoplasmic membrane and the cytoplasm. The mechanism of killing did not correlate with concentrations of complement or lysozyme or titers of residual antibody in either the pulmonary surfactant or the serum, and killing was reduced by preincubation of surfactant with P. haemolytica lipopolysaccharide. Preliminary characterization of both surfactant and serum implicate a low-molecular-weight proteinaceous component in the surfactant and serum albumin in the serum. This mechanism may help clear certain gram-negative bacteria from the lungs of sheep as a part of the pulmonary innate defense system.

Animals↗
PubMed1997

Generation of targeted nonpolar gene insertions and operon fusions in Pasteurella haemolytica and creation of a strain that produces and secretes inactive leukotoxin.

An efficient method for targeted gene inactivation and generation of chromosomal gene fusions in Pasteurella haemolytica has been devised and used to create an lktC::cat operon fusion by allelic exchange at the leukotoxin gene cluster (lktCABD). A copy of the lktC gene was insertionally inactivated by using a nonpolar, promoterless cat cassette and then delivered into P. haemolytica on a shuttle vector. Plasmid incompatibility was used to detect clones where double recombination events had occurred at the chromosomal locus. The insertion in lktC did not affect expression of the downstream genes, and the mutant strain secreted an antigenic proleukotoxin that was neither leukotoxic nor hemolytic. Expression of the lktC gene in trans restored the wild-type phenotype, confirming that LktC is required for activation of the proleukotoxin to the mature leukotoxin. Construction of the lktC::cat operon fusion allowed us to quantitate leukotoxin promoter activity in P. haemolytica and to demonstrate that transcription was maximal during early logarithmic growth phase but was reduced following entry into late logarithmic phase. This allelic exchange system should be useful for future genetic studies in P. haemolytica and could potentially be applied to other members of Haemophilus-Actinobacillus-Pasteurella family, where genetic manipulation is limited.

Bacterial Toxins↗
PubMed1972

Gene transfer in Pasteurella pestis harboring the F'Cm plasmid of Escherichia coli.

A strain of Pasteurella pestis, harboring the F'Cm plasmid from Escherichia coli, was able to donate its chromosome to auxotrophic recipient strains of P. pestis. The frequency of gene transfer in P. pestis was approximately 10(-6) per donor cell, 100 times less efficient than gene transfer in Pasteurella pseudotuberculosis, but efficient enough to determine entry times for the markers histidine, threonine, and tryptophan and to show linkage to the markers arginine and pigmentation. An attempt to extend the conjugation system to different serotypes of P. pseudotuberculosis and to Yersinia enterocolitica did not succeed.

Arginine↗
PubMed2001

Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) haemolytica.

The molecular evolution of the leukotoxin structural gene (lktA) of Mannheimia (Pasteurella) haemolytica was investigated by nucleotide sequence comparison of lktA in 31 bovine and ovine strains representing the various evolutionary lineages and serotypes of the species. Eight major allelic variants (1.4 to 15.7% nucleotide divergence) were identified; these have mosaic structures of varying degrees of complexity reflecting a history of horizontal gene transfer and extensive intragenic recombination. The presence of identical alleles in strains of different genetic backgrounds suggests that assortative (entire gene) recombination has also contributed to strain diversification in M. haemolytica. Five allelic variants occur only in ovine strains and consist of recombinant segments derived from as many as four different sources. Four of these alleles consist of DNA (52.8 to 96.7%) derived from the lktA gene of the two related species Mannheimia glucosida and Pasteurella trehalosi, and four contain recombinant segments derived from an allele that is associated exclusively with bovine or bovine-like serotype A2 strains. The two major lineages of ovine serotype A2 strains possess lktA alleles that have very different evolutionary histories and encode divergent leukotoxins (5.3% amino acid divergence), but both contain segments derived from the bovine allele. Homologous segments of donor and recipient alleles are identical or nearly identical, indicating that the recombination events are relatively recent and probably postdate the domestication of cattle and sheep. Our findings suggest that host switching of bovine strains from cattle to sheep, together with inter- and intraspecies recombinational exchanges, has played an important role in generating leukotoxin diversity in ovine strains. In contrast, there is limited allelic diversity of lktA in bovine strains, suggesting that transmission of strains from sheep to cattle has been less important in leukotoxin evolution.

Alleles↗
PubMed1965

Isolation of Antigens of Pasteurella pestis I. Lipopolysaccharide-Protein Complex and R and S Antigens.

Larrabee, Allan R. (Fort Detrick, Frederick, Md.), John D. Marshall, and Dan Crozier. Isolation of antigens of Pasteurella pestis. I. Lipopolysaccharide-protein complex and R and S antigens. J. Bacteriol. 90:116-119. 1965.-Pasteurella pestis contains at least 18 different antigens, 2 of which will protect experimental animals from challenge infection. A specific polysaccharide isolated and described as a hapten was isolated as a complete antigen. Two additional antigens were isolated from P. pestis. The preparation of antisera directed against these three antigens and the content of protein, lipid, and carbohydrate of each preparation were studied. None of the preparations will protect mice from challenge infection with virulent P. pestis. A basis for naming the new antigens which does not conflict with previously published designations is presented.

Journal Article↗
PubMed1966

Pasteurella pestis: growth temperature, virulence, and the graded response.

Rosenwald, Albert J. (Fort Detrick, Frederick, Md.), and Ralph E. Lincoln. Pasteurella pestis: growth temperature, virulence, and the graded response. J. Bacteriol. 91:1693-1695. 1966.-A comparison of the virulence of Pasteurella pestis was made by the graded and quantal response methods. Both tests reflected the difference in virulence of cultures grown at three temperatures. Cultures grown at lower temperatures gave the most variable response in virulence tests, and cultures grown at higher temperatures were more virulent. Results from the graded response test were obtained more quickly and more economically than those from the quantal response test.

Animals↗
PubMed1994

Fatal pneumonia following inoculation of healthy bighorn sheep with Pasteurella haemolytica from healthy domestic sheep.

In a series of three experiments, isolates of Pasteurella haemolytica biotype A, serotype 2, ribotype reference WSU-1, from healthy domestic sheep, were inoculated intratracheally into eight bighorn sheep (Ovis canadensis canadensis) and seven domestic sheep with doses of bacteria ranging from 5.3 x 10(8) to 8.6 x 10(11) colony forming units. Seven of eight inoculated bighorn sheep died from acute pneumonia within 48 hr of inoculation, whereas all seven domestic sheep inoculated with comparable or greater doses of bacteria remained healthy. One contact control bighorn sheep also died 6 days after its penmates received P. haemolytica. Three other noncontact control bighorn sheep remained healthy during the experiments. Pasteurella haemolytica biotype A, serotype 2, ribotype reference WSU-1 in the inocula was recovered from one or more tissues from all bighorns that died; whereas, it was not detected in any bighorn sheep before inoculation. Three different ribotypes of P. haemolytica A2 were recovered from bighorn sheep; however, only the ribotype reference WSU-1 in the domestic sheep-origin inoculum was recovered from all dead bighorn sheep, and was not recovered from bighorn sheep that survived the experiments. Thus, a relatively nonpathogenic and common isolate of P. haemolytica from healthy domestic sheep was lethal in bighorn sheep under experimental conditions.

Animals↗
PubMed1999

Pasteurella multocida Splenic Abscess Causing Fever of Unknown Origin: Report of One Case.

Pasteurella multocida has been associated with several different types of human infections but there is only a single report in the literature of splenic abscess caused by this bacteria. We report here a case of splenic abscess in an 81 year old male, with type II diabetes and a synthetic arterial graft in the leg, but considered to be an immunocompetent host. The patient presented to the hospital for FUO investigation. No portal of entry of the organism was identified. The patient responded well to laparoscopy drainage of the abscess and 20 days of antibiotic therapy. Attention must be paid to primary splenic abscess as a cause of FUO, and that Pasteurella multocida could be the etiologic agent in such a case.

Journal Article↗
PubMed2002

p53 and c-myc activation by Pasteurella haemolytica leukotoxin is correlated with bovine mononuclear cells apoptosis.

To analyse the role of Pasteurella haemolytica Leukotoxin (LKT) in the mechanism of apoptotic cell death of bovine lymphocytes, we evaluated DNA fragmentation and p53 and c-myc expression. P. haemolytica strain ATCC 14003 was cultivated for LKT production. DNA fragmentation was analysed by electrophoresis on Agarose gel. DNA strand breaks in individual apoptotic cells were also detected by an in situ Terminal deoxy nucleotidyl Transferase (TdT). The Polymerase Chain Reaction (PCR) procedure was used for verified p53 and c-myc activation by P. haemolytica LKT. LKT was able to induce DNA fragmentation in a dose and time-dependent fashion. The greatest apoptotic effect was obtained using LKT at a concentration of 0.25 U. The results show that p53 and c-myc activation by LKT is correlated with apoptosis of bovine lymphocytes and monocytes. Our data suggest that LKT may have an important role in the bacterial virulence of Pasteurella haemolytica.

Animals↗