Differentiation of phase I and variant strains of Bordetella pertussis on Congo red media

Parton, R.

doi:10.1099/00222615-26-4-301

Cited by 11 publications

(9 citation statements)

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“…2001), haemolysin production (Hly + ) (Glaser et al . 1988), and the differential affinity to bind Congo red (CR) dye (Parton 1988). On the other hand, it has been demonstrated that a 40 or 45 kDa protein observed in SDS‐PAGE OMP profiles from Bvg – and Bvg + mod B. bronchiseptica strains corresponds to flagellin isotypes and its presence has been proposed as a marker of the avirulent phenotype (Passerini de Rossi et al .…”

Section: Introductionmentioning

confidence: 99%

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

Friedman

Rossi

Messina

et al. 2001

Lett Appl Microbiol

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Aims: The present study shows that Congo red binding and urease activity assays are useful for selection of virulent (Bvg + ) Bordetella bronchiseptica cultures. Methods and Results: Congo red binding and urease activity of Bvg + B. bronchiseptica cultures in different liquid media were compared with the expression of virulence markers such as ®lamentous haemagglutinin and some outer membrane proteins (OMP). The correlation with the reference virulence markers allowed the establishment of cut-off values for the proposed markers to assure the virulent phenotype (³ 26 nmol ml ±1 of CR and £ 2á6 U). Using both assays, modulated cultures with avirulent phenotype (Stainer-Scholte broth, with MgSO 4 20 mmol l ±1 and brain heart infusion broth) and semi-modulated cultures with intermediate phenotypes (tryptose phosphate broth and 83% Stainer-Scholte with MgSO 4 5 mmol l ±1 cultures) could be distinguished. Conclusions: CR binding assay and urease activity are speci®c and sensitive enough to detect intermediate phenotypes that could only be detected by subtle changes in OMP pro®les. Signi®cance and Impact of the Study: The production of effective veterinary vaccines is hampered by reversible B. bronchiseptica antigenic modulation. The proposed assays are technically suitable for selection of virulent cultures to optimize vaccine production. INTRODUCTIONBordetella bronchiseptica is a primary aetiologic agent of atrophic rhinitis and pneumonia in swine, and also causes tracheobronchitis in dogs and rhinitis in rabbits and guinea pigs (Goodnow 1980). Whole-cell vaccines for B. bronchiseptica are used but their use has not always been successful (Register 2000). The production of effective veterinary vaccines is hampered by the high frequency of B. bronchiseptica phase variation and antigenic modulation.The virulent strains (Bvg + ) are characterized by the expression of virulence factors, codi®ed by vag genes (virulence-activated genes), which are positively regulated by the bvgAS locus (Cotter and Miller 1997). The avirulent variants (Bvg ± ), originated in bvgAS mutations, express vrg genes (virulence-repressed genes) that encode the production of¯agellin and urease in B. bronchiseptica (Monack et al. 1989;Akerley et al. 1992;McMillan et al. 1996). Bvg + strains grown in the presence of modulators (MgSO 4 20 mmol l ±1 ) express an avirulent phenotype (Bvg + mod) similar to that of Bvg ± strains. Intermediate phenotypes (Bvg i ) obtained under semi-modulating conditions, i.e. lower concentrations of modulating agents, have been described (Lacey 1960;Cotter and Miller 1997;McMillan et al. 1999).In order to characterize the virulent phenotype,`phase markers' are used including: haemagglutinating activity (HA) due to the expression of ®lamentous haemagglutinin (FHA) (Bemis and Plotkin 1982), polypeptides of M r 30±32, 90±95 and 200 kDa in the outer membrane protein (OMP) pro®les (Armstrong and Parker 1986;Passerini de Rossi et al. 2001), haemolysin production (Hly + ) (Glaser et al. 1988), and the differential af®nity to bin...

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Section: Introductionmentioning

confidence: 99%

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

Friedman

Rossi

Messina

et al. 2001

Lett Appl Microbiol

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“…Binding or uptake of Congo red dye can differentiate between Vir+ and Vir-strains of B. pertussis (23 Figure 2C shows 124 ng of cAMP produced by B. pertussis cells with adenylate cyclase activity. Importantly, the authenticity of the cAMP peak from reactions like those shown in Fig.…”

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confidence: 99%

“…To differentiate further the insertion mutations of these strains, we tested their effect on the production of FHA, the 69-kDa OMP, and the ability of the bacteria to bind Congo red dye, all of which are positively controlled by the vir locus (23,30,32). The insertion mutations in BC70, BC71, and BC77 eliminated the expression of FHA and the 69-kDa OMP and cellular binding of Congo red dye as well as hemolysin and adenylate cyclase activities (Table 2).…”

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Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes

1990

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“…Casamino acids (Difco) were added to a final concentration of 1% (w/v) where minimal growth conditions were not required. Congo red (Sigma) was included in the agar to a final concentration of 0.002% (w/v) for differentiating virulent and avirulent B. pertussis strains (Parton 1988). Escherichia coli strains were grown in nutrient broth or agar (Oxoid).…”

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confidence: 99%

“…The Congo red-binding (Crb+) phenotype in B. pertussis is associated with virulent strains and is lost during both antigenic modulation and phase variation (Parton 1988). The present study of transposon mutagenesis was done in an attempt to identify genes associated with the expression of Congo red-binding and also other virulence-regulatory genes which, like the bug operon, would be expected to have a pleiotropic effect on the expression of virulence-associated factors.…”

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High frequency of independent IS50 transposition during Tn5 mutagenesis of Bordetella pertussis virulence-associated genes

Ward

Duggleby

Parton

et al. 1992

Lett Appl Microbiol

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Transposon Tn5-generated mutants of Bordetella pertussis were selected on the basis of their inability to bind the dye Congo red (Crb-). No mutants which were solely Crb- were found. Ten mutants were phenotypically equivalent to previously described strains with mutations in the virulence regulatory (bvg) locus and failed to express a range of virulence-associated factors. Two of these mutants were shown to have Tn5 insertions within the bvg locus, while another two mutants showed deletions in this regulatory region. Complementation studies indicated that the other six mutants had spontaneous mutations in the bvg locus, but with Tn5 inserted elsewhere in the chromosome. Several of the mutants, besides having a single Tn5 insertion, also showed additional IS50 insertions, indicating that the IS50 element contained within Tn5 had transposed independently. Such additional insertion events, which themselves would have the potential to cause mutation, could complicate the interpretation of mutant phenotypes which could thus arise from the insertional inactivation of more than one gene.

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Differentiation of phase I and variant strains of Bordetella pertussis on Congo red media

Cited by 11 publications

References 11 publications

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

Phenotype evaluation of Bordetella bronchiseptica cultures by urease activity and Congo red affinity

Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes

High frequency of independent IS50 transposition during Tn5 mutagenesis of Bordetella pertussis virulence-associated genes

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