Absence of mouse-lethal toxins in Australian isolates of Bordetella avium.

A total of 18 Australian isolates of Bordetella avium and seven reference B. avium strains from Europe and the USA were tested for the presence of a mouse-lethal toxin. Five of the seven reference strains of B. avium, but none of the 18 Australian isolates, produced the toxin.

[The phenotypic properties of freshly isolated strains of Bordetella]. / Izuchenie fenotipicheskikh svoistv svezheizolirovannykh shtammov bordetell.

As the result of our investigations, newly isolated B. pertussis and B. bronchiseptica strains were studied. The results of these investigations showed that B. pertussis strains isolated under the conditions of immunoprophylaxis were characterized by sufficient stability of the main phenotypical properties which determined their pathogenicity: B. pertussis toxin, fimbrial agglutinogens and filamentous hemagglutinin. At the same time B. bronchiseptica strains isolated from animals proved to be phenotypically variable both in vivo and in the process of in vitro passage.

Serum sensitivity and lipopolysaccharide characteristics in Bordetella bronchiseptica, B. pertussis and B. parapertussis.

The viability of four strains of Bordetella bronchiseptica, two strains of B. pertussis and one strain of B. parapertussis exposed to hyperimmune and pre-colostrum porcine serum was examined. Viable cell numbers (cfu/ml) of the B. pertussis strains and a rough strain of B. bronchiseptica (CSU-P-1) decreased by 99% and 99.99%, respectively, after exposure for 1 h to porcine hyperimmune serum. In contrast, smooth B. bronchiseptica strains and the B. parapertussis strain showed no significant decrease in viable cell numbers after the same treatment. B. bronchiseptica strain CSU-P-1 also showed a 99% decrease in viable cell numbers after exposure to pre-colostrum porcine serum for 1 h whereas the other strains tested showed no decrease in viable numbers under the same conditions. Heating the hyperimmune and pre-colostrum serum at 56 degrees C for 30 min resulted in the loss of bactericidal activity suggesting the involvement of complement in both systems. Analysis of silver-stained SDS-PAGE profiles of lipopolysaccharide (LPS) extracted from the bacterial cells indicated that the smooth strains of B. bronchiseptica and the B. parapertussis strain possessed high mol. wt O-side chain-like material, whereas the B. pertussis strains and B. bronchiseptica strain CSU-P-1 did not. Gel filtration of acid-hydrolysed LPS samples indicated two distinct carbohydrate peaks for the strains with high mol. wt O-side chain-like material, whereas the other strains each yielded one distinct peak. Western-blot analysis indicated a positive reaction for anti-B. bronchiseptica antibodies to the high mol. wt O-side chain-like material of all serum-resistant strains used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of heat-labile toxin from Bordetella parapertussis by fatty acids.

The ability of heat-labile toxin (HLT) from Bordetella parapertussis to induce skin lesions in guinea pigs was found to be inhibited by lipids isolated from skin layers of adult mice, which are refractory to the lesion-inducing activity of HLT. These lipids were identified as linoleic and oleic acids. Other long-chain unsaturated fatty acids were also found to inhibit HLT; however, fatty alcohols, neutral lipids, phospholipids, cholesterol, prostaglandin, and leukotriene had no measurable effects on HLT action. The data presented in this report indicate that the ability of HLT to induce skin lesions in animals may depend, at least in part, on the free fatty acid content of the skin layer.

Biochemical and immunological comparison of lipopolysaccharides from Bordetella species.

Lipopolysaccharides (LPS) isolated from Bordetella pertussis, B. parapertussis and B. bronchiseptica were analysed for their chemical composition, molecular heterogeneity and immunological properties. All the LPS preparations contained heptose, 3-deoxy-D-manno-2-octulosonic acid, glucosamine, uronic acid, phosphate and fatty acids. The fatty acids C14:0, C16:0 and beta OHC14:0 were common to all the LPS preparations. LPS from B. pertussis strains additionally contained isoC16:0, those from B. parapertussis contained isoC14:0 and isoC16:0, and those from B. bronchiseptica contained C16:1. By SDS-PAGE, LPS from B. pertussis had two bands of low molecular mass, and the LPS from B. parapertussis and B. bronchiseptica showed low molecular mass bands together with a ladder arrangement of high molecular mass bands. Immunodiffusion, quantitative agglutination and ELISA demonstrated that the LPS from B. pertussis strains reacted with antisera prepared against whole cells of B. pertussis and B. bronchiseptica; LPS from B. parapertussis reacted with antisera to B. parapertussis and B. bronchiseptica, and LPS from B. bronchiseptica reacted with anti-whole cell serum raised against any of the three species. From these results, it is concluded that LPS from B. bronchiseptica has structures in common with LPS from B. pertussis and B. parapertussis, while the LPS from B. pertussis and B. parapertussis are serologically entirely different from each other.

Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin.

Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.

Application of gas-liquid chromatography to the routine identification of nonfermenting gram-negative bacteria in clinical specimens.

A total of 430 strains of glucose-nonfermenting gram-negative bacteria representing 35 species were analyzed for their cellular fatty acid composition by gas-liquid chromatography (GLC). On the basis of qualitative differences in their cellular fatty acid composition, these bacteria could be divided into 19 distinct chromatographic groups. Eight Pseudomonas species, Achromobacter xylosoxidans, group Vd, and Agrobacterium radiobacter were identified from their fatty acid compositions alone. The other glucose-nonfermenting gram-negative bacterial species studied here, classified within nine distinct GLC groups, were easily recognized by using the GLC fatty acid analysis supplemented with a limited number of conventional biochemical tests. The results support the hypothesis that bacterial fatty acid composition is rather specific and that qualitative GLC fatty acid analysis can be adapted in the clinical laboratory either to provide additional criteria for differentiation of closely related groups or to serve as a rapid and highly reproducible method for their routine identification.

Novel phase-shift marker in cell surface proteins of Bordetella bronchiseptica.

Cell surface proteins of phase I cultures of Bordetella bronchiseptica strains from various species of animals were compared with those of isogenic phase III cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein band with a molecular mass of 74 kilodaltons was found only in various phase I cultures. This protein disappeared in parallel with antigenic modulation and had strong antigenicity. We found it to be an appropriate phase-shift marker for two reasons: it was readily extracted and recognized, and it was specifically identified by its distinct antigenicity.

Purification and characterization of Bordetella bronchiseptica dermonecrotic toxin.

Dermonecrotic toxin produced by Bordetella bronchiseptica was purified by chromatography on DEAE Toyopearl 650M and on Bio-Gel HTP, gel filtration on Sephadex G-200, and subsequent chromatography on Bio-Gel HTP and on SP Toyopearl 650M. The purified toxin was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance liquid chromatography. There was a 90-fold increase in the dermonecrotic titer per mg protein in guinea pigs and the recovery of activity was 17.6% of that of the original cell extract. The purified toxin is a single-chain protein with a molecular weight of 145,000 and an isolelectric point of 6.3-6.7. Its minimal necrotizing dose is approximately 0.4 ng. It was completely inactivated by heating for 20 min at 56 degrees C. It contained no endotoxin, carbohydrates, nucleic acids, or hemagglutinins.

Differentiation of Bordetella avium and related species by cellular fatty acid analysis.

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.

A note on the isoprenoid quinone content of Bordetella avium and related species.

The isoprenoid quinone content of isolates of Bordetella avium (four strains), Alcaligenes faecalis (one strain), Bordetella bronchiseptica (one strain) and a Bordetella avium-like organism (four strains) was determined by reverse-phase high-performance liquid chromatography. All the isolates contained ubiquinones with eight isoprene units as the major component. No menaquinones were detected.

Properties of dermonecrotic toxin prepared from sonic extracts Bordetella bronchiseptica.

A toxin with dermonecrotic activity (DNT) was purified from sonic extracts of Bordetella bronchiseptica L3 of pig origin at phase I by chromatographic and electrophoretic methods. The purification procedure was one developed for obtaining the Pasteurella multocida DNT from sonic extracts with some modifications. Dermonecrotizing activity of B. bronchiseptica-purified DNT was increased by 600-fold compared with that of the crude extract, and the average yield was about 3%. The toxin was homogeneous, as determined by Ouchterlony double immunodiffusion, crossed immunoelectrophoresis, and disk isoelectric focusing in polyacrylamide gels. The toxin gave a single band on polyacrylamide disk gel electrophoresis (PAGE) and sodium dodecyl sulfate-SDS PAGE. The molecular weight of the toxin was ca. 190,000 +/- 5,000, as determined by SDS-PAGE. The isoelectric point of the toxin was ca. 6.5 to 6.6. The minimal necrotizing dose of the toxin for guinea pigs was about 2 ng of protein per 0.1 ml, the 50% lethal dose per mouse was about 0.3 micrograms, and the minimal cytotoxic dose for embryonic bovine lung cells was about 2 ng/ml. The toxin was heat labile and sensitive to inactivation by trypsin, Formalin, and glutaraldehyde. The mildly trypsinized B. bronchiseptica DNT preparation dissociated into two polypeptide chains, with molecular weights of ca. 75,000 +/- 4,000 (fragment 1) and ca. 118,000 +/- 5,000 (fragment 2), after treatment with dithiothreitol-SDS or urea. Upon removal of dithiothreitol and urea from the dissociated DNT preparation, the fragments reassociated, and the DNT that was formed was indistinguishable from the native toxin.

[Isolation of a restriction endonuclease with HindIII-specificity from Bordetella bronchiseptica]. / Vydelenie restriktsionnoi éndonukleazy s HindIII-spetsifichnost'iu iz Bordetella pronchiseptica.

Site-specific restriction endonuclease BbrI has been found in bacteriophage resistant strain B. bronchioseptica 4994. The technique was elaborated for purification of BbrI to the stage free of nuclease and phosphatase contamination. The yield of purified enzyme is 6000-20 000 units per 10 g of biomass. BbrI recognises and cleaves the same DNA sequence as HindIII with the formation of four-nucleotide cohesive ends. The simplicity of cultivation, security for human, presence of the single restriction endonuclease and the high level of its production make B. bronchioseptica 4994 a promising producer of BbrI restriction endonuclease, isoshizomeric to HindIII, for use in experimental practice in industry.

Purification and subunit heterogeneity of pili of Bordetella bronchiseptica.

Pili were isolated and purified from Bordetella bronchiseptica. Electron microscopic observations revealed that pili are ubiquitous in this species. The occurrence of pili and flagella appeared to correlate with growth phase and colonial morphology. Pili were about 3 to 4 nm in diameter and morphologically similar to pili isolated from other gram-negative bacteria. Internal core structure was not evident. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified pili showed that up to three different pilus subunit variants could be observed on a single strain, depending on the colonial phase and culture condition. Enzyme immunoassay and immunoblot, however, showed that these subunit variants are serologically related. Mice vaccinated with purified pili were protected against a virulent intraperitoneal challenge of B. bronchiseptica. B. bronchiseptica pili were also found to be similar to Bordetella pertussis pili in morphology and in the molecular size and antigenic structure of pilus subunits. The intact pili of B. bronchiseptica and B. pertussis, however, appeared to have weak serological cross-reactivity.

Contribution to the taxonomy of the turkey coryza agent: cellular fatty acid analysis of the bacterium.

Gas-liquid chromatography was used to analyze bacterial cellular fatty acids to elucidate the relatedness of the turkey coryza (TC) bacterium to Alcaligenes spp., Bordetella spp., and other gram-negative bacteria. The results indicated that the TC bacterium is not closely related to Alcaligenes faecalis or any of the reference strains of Alcaligenes and Bordetella studied. Most urease-positive bacterial isolates obtained from the upper respiratory tract of turkeys were identified as Bordetella bronchiseptica. It is suggested that Bordetella avium is a suitable designation for the TC bacterium formally called Bordetella-"like" and A. faecalis type I. It is also suggested that the nonpathogenic bacterium previously identified as type II A. faecalis be designated B. avium-like until further taxonomic studies are available. Furthermore, it is proposed that the term turkey coryza be used to refer to the disease induced by this bacterium.

Intracellular locations of dermonecrotic toxins in Pasteurella multocida and in Bordetella bronchiseptica.

Location of dermonecrotic toxin (DNT) in the cells of Pasteurella multocida or Bordetella bronchiseptica was investigated. After cell lysis by various procedures, various fractions prepared from bacterial cells grown in liquid culture media were assayed for dermonecrotic activity by skin testing of guinea pigs. During the death phase of the growth tested for the 2 bacterial species, little cell-free DNT was detected in the culture supernatants. Throughout the log and stationary phases of the growth, DNT activity was cell associated, but was not seen in the culture supernatants, which indicated that DNT was not secreted by actively growing P multocida or B bronchiseptica cells. Little DNT was released by subjecting whole cells to osmotic shock, a common procedure that releases proteins from the periplasmic space of many gram-negative bacteria. After sonication and centrifugation of whole cells, a substantial amount of DNT was released; results were similar when spheroplasts were used instead of whole cells. Treatment of whole cells with trypsin did not decrease the DNT activity, but trypsin treatment of sonicated cells resulted in a significant decrease in the DNT activity (P less than 0.01). The results indicated an intracellular location of the DNT of P multocida or B bronchiseptica. The DNT of P multocida or of B bronchiseptica is probably located in the cytoplasmic space.

Bordetella heat-labile toxin: further purification, characterization and mode of action.

The heat-labile toxin (HTL) was purified from sonic extracts of Bordetella bronchiseptica and Bordetella pertussis cells by a series of hydrophobic interactions, density gradient centrifugation, gel filtration, isoelectric precipitation, and isoelectric focusing. A 114-fold purification was regularly obtained with a yield of 31%. A dose of 0.75 ng was dermonecrotizing in guinea pigs. HLT is a simple protein (pI 6.9) with a molecular weight by gel filtration of 102,000 which consists of two polypeptides of 30,000 and 24,000 molecular weight. Amino acid analysis showed 15 common amino acids and the absence of methionine. The dermonecrotizing activity was inactivated at 56 degrees C, and at above pH 10 or below pH 5. Effects of various ions, detergents, enzymes and other chemical agents on HLT were determined. When instilled on the surgically exposed peripheral blood vessels of guinea pigs or suckling mice, HLT induced vasoconstriction within 15 min resulting in the decrease of blood flow, followed by manifestations of ischemia, diapedesis and petechial hemorrhage during the following 5 h. HLT activity on arterioles was unaffected by adrenergic or cholinergic blockades. Biochemically, HLT significantly inhibited in vitro the activity of Na+ - K+ ATPase prepared from rat kidney. A possible mechanism by which HLT induces dermohemorrhagic necrosis and splenic atrophy, is discussed.

[Taxonomic significance of the fatty acid composition of bacteria of the genera Bordetella and Haemophilus]. / Taksonomicheskoe znachenie zhirnokislotnogo sostava bakterii rodov Bordetella i Haemophilus.

Bacteria of the species B. parapertussis and B. bronchiseptica have proved to be identical in their fatty-acid composition with a high level (35.7-39%) of methylene-hexadecanoic acid, found to be absent in B. pertussis in experimental conditions. At the same time the total content of methylene-hexadecanoic acid and its biosynthetic precursor, hexadecenoic acid, in the first two Bordetella species is similar to the content of hexadecenoic acid in B. pertussis, which, along with the presence of common characteristics in the sign under consideration (the low level of C18:1), indicates the close relationship of these three Bordetella species. Bacteria of the species H. influenzae, H. parainfluenzae, H. aegyptius, H. aphrophilus have similar fatty-acid composition with the prevalence of hexadecanoic and hexadecenoic acids and the low level of fatty acids with 18 carbon atoms. The data on fatty-acid composition may suggest the presence of philogenetic links between the genera Bordetella and Haemophilus.

Ornithine-containing lipid of Bordetella pertussis, a new type of hemagglutinin.

The ornithine-containing lipids of six strains (phases I-IV) of Bordetella pertussis were prepared from the total extractable cellular lipids by thin-layer chromatography and treatment with phospholipase A. They were compared with those prepared from two strains each of Bordetella parapertussis and Bordetella bronchiseptica. The structures of the ornithine-containing lipid of B. pertussis and the other two species were resolved by acid and alkaline hydrolysis, gas-liquid chromatography, infrared absorption spectroscopy, amino acid analysis and combined gas-liquid chromatography/mass spectrometry. The main structure of the aminolipid of the three species of Bordetella was 3-hydroxyhexadecanoic acid, amide-linked to ornithine and esterified to the second hexadecanoic acid. The aminolipid of B. pertussis Sakurayashiki (phase III) exhibited high hemagglutinating activity for human and rabbit erythrocytes, having a minimum hemagglutinating concentration of 1 microgram/ml against 8-16 micrograms/ml for the other strains of Bordetella. All of these aminolipids showed some degree of microheterogeneity. Because the 3-hydroxyhexadecanoic acid content was especially high in strain Sakurayashiki, it was presumed that the intensity of hemagglutinating activity of the aminolipid was affected by the chain length of the central 3-hydroxy fatty acid, that is the aminolipid containing 3-hydroxyhexadecanoic acid had high hemagglutinating activity. The hemagglutination was inhibited by phosphatidylcholine at concentrations of more than 20 micrograms/ml. Other inhibitory substances were cysteine, sphingomyelin, acidic amino acids, histidine, unsaturated fatty acid and basic amino acids. Furthermore, the divalent cations Ca2+ and Mg2+ inhibited this hemagglutination at a concentration of 1 mM. The O-deacylated ornithine-containing lipid that had lost hexadecanoic acid did not have any hemagglutinating activity but did have hemolytic activity. Observation by electron microscopy indicated that erythrocytes were combined by the liposomes of the ornithine-containing lipids. On the basis of these results, the proposed mechanism of hemagglutination by the aminolipids is that the liposomes of the aminolipids combine erythrocytes by hydrophobic interaction between the fatty acid moieties of the aminolipid and the lipids of the surface of erythrocytes, and by ionic interaction between the ornithine of the aminolipid and the protein of the surface of the erythrocytes. In addition, the hemagglutinating activity of phosphatidylserine was found to be due to its similar structure to that of the ornithine-containing lipid and the mechanism was also presumed to be similar. The mechanism of hemagglutination by these aminolipids was distinct from that of lectins.