Serotyping and Enzyme Characterization of Pasteurella haemolytica and Pasteurella multocida Isolates Recovered from Pneumonic Lungs of Stressed Feeder Calves

Ninety-one isolates of Pasteurella multocida (Pm) and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34 (27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories. Received: 29 August 1996 / Accepted: 15 October 1996     

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.     

Isopycnic characterization of lipopolysaccharides from Pasteurella multocida

In a novel application of an established procedure, isopycnic density gradient centrifugation procedures were used to analyze material obtained from the Westphal phenol extraction procedure of Pasteurella multocida cells. The initial phenol phase contained most of the lipopolysaccharides (LPS) and the major component had a buoyant density of 1.38 g/ml in CsCl density gradients. Repartitioning the phenol phase with an equal volume of water produced a second aqueous phase which contained most of the LPS. This LPS appeared as a single symmetrical band with a buoyant density of 1.40 g/ml. Buoyant density patterns obtained with schlieren optics in CsCl density gradients were useful in characterizing LPSs from P. multocida.     

Lipase Activity from Strains of Pasteurella multocida

Thirteen clinical isolates of Pasteurella multocida from a variety of different animals and humans were examined for their ability to produce lipase. Lipase substrates used included Tween 20, Tween 40, Tween 80, and Tween 85. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in Roswell Park Memorial Institute-1640 defined media (RPMI-1640), but activity increased in the filtrates when the cultures were allowed to proceed to the stationary phase. All strains examined (except for serotype 2) showed lipase activity against at least one of the Tweens. Tween 40 was the best substrate to demonstrate lipase activity. Pasteurella multocida serotype 8 produced the most active lipase against Tween 40 (3,561.7 units of activity/μg of protein). This activity continued to increase after P. multocida entered a stationary growth phase. P. multocida lipase activity was optimal at pH 8.0. Lipase activity of P. multocida serotype 8 was eluted from a Sepharose 2B column at several points, indicating that several lipases may be produced in vitro by this organism. These data demonstrate that clinical isolates of P. multocida produce lipase; therefore, this enzyme should be considered a potential virulence factors for this organism. Received: 16 September 1999 / Accepted: 22 November 1999     

Molecular characterization of Pasteurella multocida isolates from rabbits

Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.     

Morphology of Pasteurella multocida bacteriophages

Twenty-one tailed phages with icosahedral heads belong to the Myoviridae, Siphoviridae, and Podoviridae families and to four morphological types. Type AU, with 10 phages, has a contractile tail and is morphologically identical with coliphage P2. Lysates contain contracted tail sheaths assembled end-to-end and abnormal structures with long tails and multiple tail sheaths. Types C-2 and 32, with one and three phages, respectively, have long, noncontractile tails. Type 22 includes seven phages, has a short tail, and resembles coliphage T7. Our results agree with previous biological data and suggest that types AU, C-2, 32, and 22 correspond to four different phage species.     

Heat-labile toxin-producing isolates of Pasteurella multocida from rabbits.

Five of one hundred forty seven isolates of Pasteurella multocida from rabbits were found to produce heat-labile toxin. Each isolate was assayed for the ability of potassium thiocyanate (KSCN) extracts to cause dermonecrosis in guinea pig skin, ability of bacteria or filtrates to cause cytotoxicity in cell cultures, and reactivity with monoclonal antibodies to heat-labile P. multocida toxin. Five capsular type D isolates produced dermonecrosis and reacted with monoclonal antibodies to toxin. Filtrates of all five of these isolates were cytotoxic for cell cultures. Potassium thiocyanate extracts of all five isolates caused pleuritis and pneumonia in rabbits after intranasal inoculation. Turbinate atrophy was seen in 5 of 19 rabbits inoculated intranasally with toxic extracts. Heat-labile toxin was not produced by 109 capsular type A isolates or 19 nontypable isolates.     

Partial purification of an osteolytic toxin from Pasteurella multocida

A protein toxin apparently composed of one polypeptide with an estimated Mr of 155,000 was purified from sonicated cells of a type D strain of Pasteurella multocida (LFB3) by preparative polyacrylamide gel electrophoresis (PAGE) and DEAE-Sephadex A50 chromatography. Its specific activity was 150-fold greater than that of the crude extract. The partially purified protein was cytotoxic for embryonic bovine lung cells, lethal for mice and caused turbinate atrophy in gnotobiotic pigs; a single intraperitoneal injection of approximately 360 ng kg-1 caused 50% turbinate atrophy. Reversal of the two-step purification procedure using DEAE-Sephacel chromatography followed by preparative PAGE increased the yield of toxin 30-fold; the specific activity of the partially purified toxin was 1970-fold greater than that of the crude extract.     

R-Plasmids in Pasteurella multocida.

Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r_k^?m_k^?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm³, while the density of pSR11 was 1.709 g/cm³.     

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.     

Constitutive expression of Pasteurella multocida toxin

Abstract The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of β-galactosidase from a cps-lacZ fusion ( lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.     

Transmission of Pasteurella multocida in rabbits

Contact and airborne transmission of P. multocida in rabbits was evaluated in an artificially controlled environment. Transmission by contact occurred more readily from rabbits with acute infections than from rabbits with chronic infections. However, airborne transmission to rabbits in adjacent cages did not occur.     

Partial characterization of plasmids from rabbit isolates of Pasteurella multocida.

Plasmids have not been reported for isolates of Pasteurella multocida from rabbits. We assayed 28 isolates of rabbit P. multocida for plasmids and sought to determine whether or not plasmid presence correlated with clinical or pathologic findings, serotype, toxin production, possession of pili, or biochemical characteristics. Fourteen isolates bore a single 1.6 Md (covalently closed circular form in 0.7% agarose gels) plasmid. An additional isolate had two plasmids which migrated as a closely-spaced doublet, centered around 1.6 Md. Eleven isolates appeared to have identical plasmids, according to Hae III and Hinf I digests. The apparent linear size of this common plasmid in 2% agarose gels was 2.1 Md, as calculated from the sums of the sizes of Hae III or Hinf I digestion fragments. Linearization of the common plasmid with Msp I produced an apparent size of 2.5 Md in 0.7% agarose gels. No correlations between presence of the common plasmid and somatic serotype, toxigenicity, presence of pili, antimicrobial resistance, selected biochemical characteristics, anatomic site from which the bacteria were cultured, or disease status of the host were found.     

Pili of Pasteurella multocida of porcine origin

Abstract Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pasteurella multocida isolated from pigs with atrophic rhinitis. Rigid pili were found on 60–80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocida and as a result frequently were difficult to see. After growth in vitro, piliated P. multocida cells produced few pili (approx. 3–5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.