Interaction of Pasteurella multocida with free-living amoebae

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

Processing of Free-Living Amoebae for Transmission Electron Microscopy

Ultrastructural features have proved useful in identifying and classifying free-living amoebae (Page 1985). A simple technic which facilitates embedding amoebae for transmission electron microscoov is described here.     

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.     

Immunization of Mice with Components of Pasteurella multocida

Log-phase cells of Pasteurella multocida strain P-1059 were used to prepare isolated culture filtrate, cell wall, and cytoplasmic components. Culture filtrate was further separated by column chromatography. A portion of cytoplasm and culture filtrate was conjugated to ferritin by means of metaxylylene diisocyanate. Cell walls induced more protection in mice than the conjugated or unconjugated cytoplasm or culture filtrate. The cell walls caused edema and erythema when given intradermally in rabbits, whereas cytoplasm and culture filtrate produced dermal necrosis. The first of four chromatographically separated fractions of culture filtrate was possibly more immunogenic in mice than cell walls. This fraction was less reactive intradermally in rabbits than cell walls but more reactive than the other fractions.     

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.     

Vertical Distribution of Potentially Pathogenic Free-Living Amoebae in Freshwater Lakes1

The vertical distribution of thermotolerant (37°C and 45°C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic paniculate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by participates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P < 0.005) numbers of FLA were isolated from two paniculate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe²⁺ state was predominant. In this midsummer period, 13 Naegieria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.     

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.     

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.     

Seasonal Distribution of Thermotolerant Free-Living Amoebae. II. Lake Issaqueena1

A quantitative study of the seasonal distribution of thermotolerant (37°C and 45°C), small free-living amoebae (FLA) was conducted in Lake Issaqueena, a warm, monomictic lake with steep, sloping banks and a maximum basin depth of 10 m in the Piedmont region of South Carolina. Naegleria and Vahlkampfia were the most frequently encountered FLA in littoral sediment and surface water samples whereas Acanthamoeba was most commonly isolated from profundal sediment, especially during late summer. In the water column, FLA populations were highest in a persistent detrital layer; however, few amoebae were isolated from a massive (～1.5 m thick) layer of Oscillatoria. The only N. fowleri isolated in this study was from the detrital layer. Discussion of the influence of differences in watershed and basin morphology on variations in the size and generic composition of FLA populations for the aquatic ecosystems of Lake Issaqueena and Willard's Pond is included.     

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.     

Acute meningoencephalomyelitis in a rabbit infected with Pasteurella multocida

Pasteurella multocida was isolated in pure culture from the optic chiasm of a rabbit that was euthanatized subsequent to acute development of neurological signs. Histopathologically, there was meningoencephalomyelitis, bilateral otitis interna, retrobulbar cellulitis, optic neuritis and iritis. The ocular involvement, severity of the spinal lesions and the lack of otitis media was unusual.     

Secretion of Proteases from Pasteurella multocida Isolates

The capability of Pasteurella multocida to secrete proteases to the culture medium and their characterization were studied in five animal isolates (bovine, chicken, sheep, and two from pig). All the isolates produced proteases in a wide range of molecular mass. It is suggested that they are neutral metalloproteases, since they were optimally active between pH 6 and 7, inhibited by chelating agents but not by other protease inhibitors, and reactivated by calcium. Proteases from isolates were able to degrade IgG. Several proteins from supernatants of cultures precipitated with 70% (NH₄)₂SO₄ of all the P. multocida isolates were recognized by a polyclonal antiserum raised against a purified protease from Actinobacillus pleuropneumoniae. Protease production might play an important role during tissue colonization and in P. multocida diseases. Received: 26 May 1998 / Accepted: 15 August 1998     

Characterization of Pasteurella multocida associated with pneumonia in bighorn sheep

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

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     

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.