Infection of newborn piglets with Bordetella pertussis: a new model for pertussis.

Bordetella pertussis is the causative agent of pertussis or whooping cough. This bacterium is a human pathogen that under experimental conditions also infects selected rodents and primates. Here, we show for the first time that newborn piglets can be infected with B. pertussis when it is delivered intrapulmonarily. Infected piglets displayed fever and respiratory symptoms, such as nasal discharge, nonparoxysmal coughing, and breathing difficulties. Eventually, all infected animals developed severe bronchopneumonia, which in some cases was combined with a fibrinous pleuritits. Immunohistochemical staining revealed the presence of large numbers of B. pertussis cells within airways, adhering to the epithelial lining or phagocytosed by macrophages and neutrophils. Viable bacteria were reisolated from bronchoalveolar lavages and lung lesions for more than 10 days postinfection. The systemic presence of pertussis toxin was shown by hypoglycemia, lymphocytosis, and induction of a clustered pattern of CHO cells by serum and bronchoalveolar lavage samples. Thus, a large-animal model for pertussis was developed, which should complement existing rodent models for identifying the immune responses relevant to the design of new vaccines. In particular, this model should help researchers analyze the roles of both maternal and mucosal immunity in disease protection against pertussis and should ultimately assist in the design of new vaccines for early life protection.

Two physically and serologically distinct lipopolysaccharide profiles in strains of Bordetella pertussis and their phenotype variants.

The lipopolysaccharide (LPS) from nine strains representing 18 phenotype variants of Bordetella pertussis could be grouped into one of two distinct profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. One group, representing the wild-type LPS profile of B. pertussis, consisted of two silver-staining bands: a dominant brown-amber a band and a faster-migrating, minor, black-staining b band. The second group, representing a variant LPS profile, consisted of a single black-staining band of similar mobility to the b band in the wild-type profile. By electrophoretic transfer (Western) blot analysis, mouse antiserum raised against whole cells of Tohama I (prototype wild-type LPS strain) recognized only the a band from all strains/phenotypes possessing the wild-type LPS profile. In contrast, mouse antiserum raised against whole cells of 134 (prototype variant LPS strain) recognized all b bands, regardless of strain/phenotype, and could be shown to cross-react weakly with the a band from Tohama I. These results and results from cohemagglutination and immunodiffusion analyses support the classification of B. pertussis into one of two physiologically and serologically distinct LPS phenotypes: Lps AB for the wild-type profile and Lps B for the variant profile. The relationship of LPS type and phenotypic, or "phase," variation is discussed.

Isolation and characterization of isogenic pairs of domed hemolytic and flat nonhemolytic colony types of Bordetella pertussis.

Four different serotype strains of Bordetella pertussis, 3779BL(2)S(4), Tohama I, 353/Z, and 2753, were plated on Bordet-Gengou agar, where they grew as domed, hemolytic (D(+)H(+)) wild-type colonies. Cloned D(+)H(+) colony types of all four strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar, and these subsequently grew as flat, nonhemolytic (D(-)H(-)) colonies when transferred back onto Bordet-Gengou agar. The frequency of D(-)H(-) organisms within a population of cloned D(+)H(+) was determined to be between 5 x 10(-5) and 5 x 10(-6). The D(-)H(-) colony types maintained their flat, nonhemolytic characteristics for over 80 single-colony passages on Bordet-Gengou agar. The isogenic pairs of D(+)H(+) and D(-)H(-) colony types from the four strains were compared for hemagglutination titer, lymphocytosis-promoting activity, adenylate cyclase activity, and presence of agglutinogens by agglutination. In all cases the D(-)H(-) colony types showed reduced activities or amounts of antigen compared with their D(+)H(+) parents. Freely diffusible antigens were markedly different between the two phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the two colony types by Triton X-100 were also markedly different as judged by radial immunodiffusion with antifimbrial hemagglutinin, antilymphocytosis-promoting factor, and anti-353/Z adsorbed with autoclaved 353/Z. In addition, autoradiographs of (125)I-surface-labeled whole cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed unique banding patterns for each colony type. Since all organisms, regardless of colony type, were grown on Bordet-Gengou agar, the differences reported could not be due to medium composition. Differences between phenotypes were also independent of passage number on Bordet-Gengou agar. By analogy to previous studies, the D(-)H(-) organisms appear to fulfill the criteria for phase III or phase IV in the system of Leslie and Gardner (P. H. Leslie and A. D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1954).

Phenotypic variation and modulation in Bordetella bronchiseptica.

Most of the isolates of Bordetella bronchiseptica obtained by this laboratory possessed a characteristic colonial morphology when grown on Bordet- Gengou agar (BGA) at 37 degrees C. The colonies appeared domed (Dom+) with a smooth colonial surface (Scs+) and a clear zone of hemolysis ( Hly +). From these Dom+ Scs+ Hly + BGA colony types arose flat (Dom-), smooth colonial surface (Scs+) and nonhemolytic ( Hly -) variants at frequencies of 10(-2) to 10(-3). Isogenic pairs of Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA phenotype variants (BGA-PVs) were picked from 11 strains of B. bronchiseptica, and their whole cell lysates were compared with each other by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Characteristic SDS-PAGE profiles were observed for each of the Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs with regard to (i) surface-exposed proteins, based on autoradiographs of 125I- Iodogen -labeled organisms, (ii) polypeptide differences, based on gels stained with Coomassie brilliant blue R-250, and (iii) lipopolysaccharide differences based on gels stained with silver after oxidation with periodic acid. SDS-PAGE profiles were then used to monitor the phenotypes expressed by Dom+ Scs+ Hly + and Dom- Scs+ Hly - BGA-PVs transferred and grown on brucella agar, Trypticase soy agar, and nutrient agar. When grown on non-BGA media, the Dom+ Scs+ Hly + BGA-PVs from six of eight strains showed SDS-PAGE profiles identical to those of Dom- Scs+ Hly - BGA-PVs. This phenotypic change was reversible even after 15 subcultures on the non-BGA media, since Dom+ Scs+ Hly + organisms passed back onto BGA expressed both Dom+ Scs+ Hly + colonial morphology and Dom+ Scs+ Hly + SDS-PAGE profiles. The influence of cultural conditions on maintenance of virulence is discussed.

Isolation and characterization of Bordetella pertussis phenotype variants capable of growing on nutrient agar: comparison with phases III and IV.

Unsupplemented nutrient agar (NA) was used to select spontaneous phenotype variants (PVs) of Bordetella pertussis Tohama I and 3779 which, by their growth on NA, could possibly be considered equivalent to phase IV in the system of Leslie and Gardner (P.H. Leslie and A.D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1953). NA growers (Gna+) were selected from the flat, nonhemolytic, non-NA grower (Dom- Hly- Gna-) PV of both strains at a rate of between 10(-7) to 10(-8) per cell per generation. When cultured on Bordet-Gengou agar (BGA), more than one colony type was observed in strain 3779; these all retained the Gna+ characteristic during 10 to 30 passages on BGA. Analysis of 125I-surface-labeled whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no major changes between the Dom- Hly- Gna+ PV and their Dom- Hly- Gna- PV parents in polypeptide profile (by Coomassie stain), in surface exposure of proteins (by autoradiography), or in lipopolysaccharide profile (by silver stain). Increased resistance to oleic acid, tetracycline, erythromycin, rifampin, and penicillin G, however, was characteristic for the Dom- Hly- Gna+ PV. Five phase IV strains and a phase III B. pertussis strain had similar antibiotic and oleic acid sensitivity profiles as the Dom- Hly- Gna+ isolates and plated with similar efficiency on NA, despite heterogeneity in BGA colonial morphology and lipopolysaccharide profile.

Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose.

We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigations into the nature of the interaction and activity of PT in host tissues.

Identification of two bvg-repressed surface proteins of Bordetella pertussis.

Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen.

Protection against group B Neisseria meningitidis disease: effect of serogroup B polysaccharide and polymyxin B on immunogenicity of serotype protein preparations.

The inability to prepare an effective polysaccharide vaccine against group B Neisseria meningitidis was the impetus for these studies. Outer membrane protein vaccines used in our initial studies failed to induce bactericidal antibodies in humans. The particulate nature of these vaccines may have led to their clearance before effective immune stimulation. Less denaturing procedures, therefore, were developed for preparation of serotype 2 protein-containing vaccines. These procedures included isolation of naturally released outer membrane vesicles and selective removal of lipopolysaccharide from the vesicles by the nonionic detergent Brij-96. The resultant protein vaccines were evaluated with and without noncovalently complexed group B meningococcal polysaccharide or polymyxin B sulfate or both. The new vaccines were at least 10-fold more immunogenic in mice and guinea pigs than the previous vaccines when assayed for bactericidal and enzyme-linked immunosorbent assay antibodies. The protein vaccines alone protected guinea pigs from intrachamber infection, and a single 0.1-microgram injection prevented meningococcal bacteremia in mice. Addition of group B polysaccharide to the protein significantly improved the immunogenicity of the protein, and this combined vaccine showed a greater protective effect. Polymyxin B generally reduced the immunogenicity of the vaccines in both mice and guinea pigs.

Protection against group B Neisseria meningitidis disease: preparation of soluble protein and protein-polysaccharide immunogens.

Although effective polysaccharide vaccines have been developed for meningococcal groups A, C, Y, and W135, the purified group B polysaccharide has proven to be nonimmunogenic. Earlier studies indicated that serotype 2 outer membrane protein vaccines induced bactericidal antibodies in animals and protected them from meningococcal challenge. However, a similar vaccine induced only low levels of antiprotein antibodies in both adults and children (C.E. Frasch et al., in J.B. Robbins et al., ed., Seminars in Infectious Disease vol. 4, p. 263-267, 1982). Methods were therefore developed to produce more immunogenic serotype 2 protein vaccines. We found that, by growing the organism for 65 to 72 h at 32 degrees C, three to four times more outer membrane protein was released into the culture medium than could be extracted from overnight-grown cells. The outer membranes were therefore purified directly from the broth by ultrafiltration followed by ammonium sulfate precipitation. Most of the lipopolysaccharide was selectively removed from the membranes by treatment with the nonionic detergent Brij-96. The Brij-96 was then removed and the resulting vaccine was filter sterilized. Some vaccines were prepared by combining equal parts of detergent-treated membrane protein and high-molecular-weight group B polysaccharide producing highly soluble vaccines. These new vaccines were compared by using an enzyme-linked immunosorbent inhibition assay to an insoluble vaccine (E-06) found to be poorly immunogenic in humans. A human serum with serotype 2 specificity was used in the inhibition assay, and 5 microgram of E-06 was required for 50% inhibition, whereas less than 1 microgram of the soluble vaccines was required. Addition of group B polysaccharide slightly increased the inhibitory capacity of the protein component.

Phorbol myristate acetate inhibits HeLa 229 invasion by Bordetella pertussis and other invasive bacterial pathogens.

The microfilament inhibitors cytochalasins B and D have been traditionally used to indirectly evaluate the requirement for actin in the uptake of invasive bacterial pathogens by nonprofessional phagocytes. Through their effects on microfilaments, both cytochalasins also impart profound alterations in cellular morphology and surface topology, which likely interfere with adherence. Alterations affecting adherence would complicate interpretation of the effect of cytochalasins on entry alone. As an alternative to cytochalasins, the effect of the tumor promoter phorbol myristate acetate (PMA) was examined for its effects on uptake of several invasive bacterial pathogens by HeLa 229 cells. In this communication, PMA was shown to induce a similar change in HeLa cell actin distribution, but, in contrast to cytochalasins B and D, PMA had no significant effect on gross cell morphology. The modified actin distribution was shown to reduce internalization of Bordetella pertussis, Yersinia pseudotuberculosis, Shigella flexneri, and Salmonella hadar in a dose-dependent manner at concentrations ranging from 1 to 1,000 ng/ml. The magnitude of reduction at a PMA concentration of 1,000 ng/ml was greater than the reduction elicited by cytochalasin B at 2.5 micrograms/ml but was less than that elicited by cytochalasin D at 2.5 micrograms/ml. Mezerein, a functional analog of PMA, caused a similar dose-dependent reduction in uptake of B. pertussis, whereas an inactive analog of PMA, alpha-4-phorbol-12,13-didecanoate was without effect on invasion. Binding studies further reveal that pretreatment of HeLa cells with PMA or mezerein did not significantly impair the ability of B. pertussis to adhere, in contrast to cytochalasins B and D, which caused a marked reduction in adherence.

Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin.

Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.

Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium.

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 degrees C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 degrees C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 degrees C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 degrees C-grown cells of SA134 (rfa13020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 degrees C; following a switch to 30 degrees C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of proteolytic enzymes, storage and reduction on the structure and biological activity of pertussigen, a toxin from Bordetella pertussis.

Pertussigen (Ptx), referred to by many different names, including pertussis toxin, was separated into five polypeptide subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous Tris-glycine buffer system. Under non-reducing conditions, the apparent molecular weights of the polypeptides (mean 10(-3)) were: S1 (26.3), S2 (24.4), S3 (22.7), S4 (12.2), and S5 (11.3). Under reducing conditions, the apparent molecular weights (mean 10(-3)) were: S1 (28.2), S2 (24.8), S3 (24.3), S4 (12.2) and S5 (13.9). The identity of the individual polypeptide subunits was further confirmed by their unique two-dimensional peptide maps. The polypeptides which showed an apparent increase in molecular weight under reducing conditions were those previously found to contain at least two cysteine residues. Reducing conditions also altered the reactivity of S3 and S2 to polyclonal rabbit antibody in electrophoretic transfer (Western) blot analysis. When Ptx was stored in solution at 4 degrees C, S1 and S5 underwent a gradual decrease in apparent molecular weight, as judged by SDS-PAGE. This decrease occurred in three different buffer systems, and was similar to a decrease in apparent molecular weight of S1 and S5 after treatment with the proteolytic enzymes subtilisin or proteinase K. Neither the changes due to storage nor proteolysis affected the activity of Ptx in regard to hemagglutination, lymphocytosis promotion or histamine sensitization. These changes did, however appear to modify the reactivity of S5 in the Western blot. Both the "endogenous" and enzyme-induced changes in S1 and S5 could be stopped by phenylmethanesulfonyl fluoride. These data suggest that S1 and S5 have exposed determinants in the intact Ptx molecule which are readily cleaved by proteases, but have little bearing on the biological activity of the intact molecule. Resistance to inactivation by proteolytic cleavage may help explain the long duration of Ptx activity within in vivo biological systems.

Immunological characterization of the lipooligosaccharide B band of Bordetella pertussis.

Two structurally and immunologically different components of Bordetella pertussis endotoxin can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining: a major A band and a faster-migrating minor B band. Certain mutant strains of B. pertussis express only the B band, while the wild-type strains produce both lipooligosaccharides (LOS). Two monoclonal antibodies (MAbs) directed against the minor LOS B band were generated, allowing the study of this surface molecule on different strains of Bordetella. These two MAbs, designated BL-8 and BL-9, reacted strongly with phenol-water-purified LOS obtained from a B. pertussis LOS B mutant strain. Sodium periodate treatment of the purified LOS prevented binding of the MAbs, indicating the carbohydrate nature of the epitope(s). Western immunoblotting experiments revealed that the epitope(s) recognized by these MAbs is conserved on all B. pertussis and Bordetella bronchiseptica Vir- (avirulent) variant strains tested but is not present on Bordetella parapertussis and B. bronchiseptica Vir+ (virulent) wild-type strains. Further studies showed that although present in the lipopolysaccharide B band expressed by Vir- strains, the epitope(s) recognized by the MAbs is not accessible on the surface of intact B. bronchiseptica cells. For B. pertussis, the density and accessibility of this epitope(s) are dependent on the virulence-associated or LOS phenotype expressed by the strain. Our data demonstrate that the expression and accessibility of the epitope(s) are significantly greater on the LOS B variant strains and LOS AB Vir- strains compared with fresh B. pertussis clinical isolates. For these latter strains, which are Vir+, this epitope(s) was barely detectable on the surface of intact bacteria, despite Western blot analyses that revealed specific reactions between the MAbs and the LOS B band. The two LOS B-specific MAbs had no bacteriolytic activity against a LOS AB wild-type strain, while the control MAb BL-2, which is specific for the B. pertussis LOS A band, significantly reduced the number of living bacteria in the same assay. Moderate lytic activity against a mutant strain expressing only the LOS B band was observed for MAb BL-8 but not for MAb BL-9 or BL-2. These data demonstrate that the type, amount, and surface exposure of the LOS are related to the phenotype expressed by a specific B. pertussis strain. In addition, the LOS B MAbs also reveal the antigenic conservation of carbohydrate epitopes among B. pertussis and B. bronchiseptica strains.

The structure of the nonreducing terminal groups in the O-specific polysaccharides from two strains of Bordetella bronchiseptica.

The structures of the polysaccharide chains of the LPS from Bordetella bronchiseptica strains 110H and Bp512 were analysed by NMR spectroscopy and mass spectrometry. The polysaccharides consist of alpha-(1-4)-linked 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid repeating units. Polysaccharides from both strains have 2,3, 4-triamino-2,3,4-trideoxy-alpha-galacturonamide derivatives at their nonreducing ends, a monosaccharide identified for the first time in nature. The polymers from the two strains differ in the nature of the acylation of the amino groups of this monosaccharide. In the strain 110H, the residue is formylated at positions 3 and 4, and has N-formyl-L-alanyl or L-alanyl substituents at N-2. In the strain Bp512, the amino group at position 2 is acetylated, at position 3 it is formylated, and the amino group at position 4 bears a 2-methoxypropionyl substituent. The distribution of the acyl groups was determined from long range 1H-13C correlation (HMBC) NMR spectra. Measurement of the spectra under different pH conditions showed that carboxyl groups of the inner uronic acid residues of the polymeric chain are free, and that carboxyl groups of the terminal residues are amidated. These conclusions were confirmed by the results of mass spectrometric analysis.

Bordetella parapertussis invasion of HeLa 229 cells and human respiratory epithelial cells in primary culture.

Bordetella parapertussis, a respiratory tract pathogen commonly regarded as noninvasive, was found to invade HeLa 229 cell monolayers. Following treatment of the monolayers with gentamicin, numbers of viable B. parapertussis recovered were comparable to those of invasive Salmonella and Shigella isolates. Invasion occurs through a cytochalasin-sensitive process which appears to be distinct from receptor-mediated endocytosis. Hyperimmune antisera raised against filaments hemagglutinin, a major adhesion of B. pertussis, did not inhibit invasion by B. parapertussis, suggesting that alternate adhesin(s) are required for invasion. In addition, B. parapertussis was found to invade human respiratory epithelial cells in primary culture, as demonstrated in ultrathin sections viewed by transmission electron microscopy. Although viable intracellular B. parapertussis persist within HeLa cells, they do not multiply there and the monolayers remain intact, suggesting a possible mechanism of carriage for these organisms.

Analysis of Mycobacterium avium complex isolates from blood samples of AIDS patients by pulsed-field gel electrophoresis.

A molecular analysis of the first Mycobacterium avium complex (MAC) blood isolates from 177 patients from 10 Canadian cities revealed that each cluster of indistinguishable strains consisted of isolates from epidemiologically unrelated patients in the same city or region. This study supports the premise that acquisition of MAC from a common environmental source occasionally occurs.

Genetic diversity and relationships in populations of Bordetella spp.

Genetic diversity in 60 strains of three nominal Bordetella species recovered from humans and other mammalian hosts was assessed by analyzing electrophoretically demonstrable allelic variation at structural genes encoding 15 enzymes. Eleven of the loci were polymorphic, and 14 distinctive electrophoretic types, representing multilocus genotypes, were identified. The population structure of Bordetella spp. is clonal, and genetic diversity is relatively limited compared with most other pathogenic bacteria and is insufficient to justify recognition of three species. All isolates of Bordetella parapertussis were of one electrophoretic type, which was closely similar to 9 of the 10 electrophoretic types represented by isolates of Bordetella bronchiseptica. Bordetella pertussis 18-323, which is used in mouse potency tests of vaccines, is more similar genetically to isolates of B. bronchiseptica and B. parapertussis than to other isolates currently assigned to the species B. pertussis. Apart from strain 18-323, the isolates of B. pertussis represented only two closely related clones, and all isolates of B. pertussis from North America (except strain 18-323) were genotypically identical. Strain Dejong, which has been classified as B. bronchiseptica, was strongly differentiated from all of the other Bordetella isolates examined.

Use of pulsed-field gel electrophoresis for epidemiological study of Bordetella pertussis in a whooping cough outbreak.

We used pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with XbaI to determine the distribution of different Bordetella pertussis strains from clinical isolates obtained during a large whooping cough outbreak that occurred in Alberta, Canada, from December 1989 to May 1991. Our initial study analyzed 28 clinical isolates, 14 from the city of Edmonton and 1 from each of 14 northern Alberta towns. These clinical isolates were randomly chosen over the course of the 18-month outbreak. The DNA profiles were more heterogeneous than anticipated and caused concern that PFGE was too sensitive a technique to characterize strains. Further analysis showed that this was not the case, as clusters of similar PFGE patterns were observed in strains isolated from the same outlying town. Identical PFGE patterns were also seen in clinical strains obtained from different members of the same family. Two PFGE pattern types, a and b, predominated in the outbreak, accounting overall for 44 of 70 B. pertussis strains tested. Results from isolates from outlying towns, however, indicated involvement of local strains rather than a single, highly infectious strain in the whooping cough outbreak in Alberta.