Maximum likelihood genome-wide SNP tree and murine lung colonization of the classical bordetellae.

<p>(A) Genomes of bordetellae <i>B</i>. <i>pertussis</i> strains Tohama I, <i>B</i>. <i>parapertussis</i> strains Bpp5, 12822, and <i>B</i>. <i>bronchiseptica</i> strains 253, 1289, MO149 were mapped against reference genome RB50. The ML tree (100 bootstrap replicates, 50% bootstrap cut-off) was estimated with RAxML. (B) Bacterial numbers are represented as percent change in the average CFU from day 0 to 7 post-inoculation (100% recovery cut-off) of three to four mice per time point.</p

Sheep serum does not kill Bpp5.

<p><i>B</i>. <i>bronchiseptica</i> RB50 (diamond), RB50Δ<i>wbm</i> (square), <i>B</i>. <i>parapertussis</i>_<i>hu</i> 12822 (empty triangle) or <i>B</i>. <i>parapertussis</i>_<i>ov</i> Bpp5 (filled triangle) or HI (circle) were incubated with PBS (solid lines) or CVF treated (dashed lines) sheep serum for 1 hour at the indicated concentrations. The average percent survival of three independent experiments is shown +/- standard error. * indicates a <i>p</i> value of ≤0.05 between <i>B</i>. <i>parapertussis</i>_<i>ov</i> strains and <i>B</i>. <i>bronchiseptica</i> strain RB50.</p

Sequence similarity of <i>Bordetella</i> Virulence Factors.

<p>Percent sequence similarity of virulence factor genes of <i>B</i>. <i>pertussis</i> strains (Tohama I, 18323, CS), <i>B</i>. <i>bronchiseptica</i> strains (253, 1289, MO149, R77, D445) <i>B</i>. <i>parapertussis</i>_<i>hu</i> strain 12822 and <i>B</i>. <i>parapertussis</i>_<i>ov</i> strain Bpp5 compared to <i>B</i>. <i>bronchiseptica</i> strain RB50.</p

Host Specificity of Ovine <i>Bordetella parapertussis</i> and the Role of Complement

<div><p>The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, <i>Bordetella parapertussis</i>, is composed of two distinct clades that have specialized to different hosts: one to humans (<i>Bpp_hu</i>), and the other to sheep (<i>Bpp_ov</i>). While <i>Bpp_hu</i> and the other classical bordetellae can efficiently colonize mice, <i>Bpp_ov</i> strains are severely defective in their ability to colonize the murine respiratory tract. <i>Bpp_ov</i> genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on <i>B</i>. <i>bronchiseptica</i> and <i>Bpp_hu</i> strains. <i>Bpp_ov</i> lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing <i>in vitro</i>. Based on these results, we hypothesized that <i>Bpp_ov</i> failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the <i>Bpp_ov</i> defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, <i>Bpp_ov</i> strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of <i>Bpp_ov</i> to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.</p></div

Complement contributes to control of <i>B</i>. <i>parapertussis</i>_<i>ov</i> strains.

<p>C57BL/6 mice (solid lines) or C3 deficient mice (dashed lines) were inoculated with <i>B</i>. <i>bronchiseptica</i> strain RB50 (A) or <i>B</i>. <i>parapertussis</i>_<i>ov</i> strain Bpp5 (B). Bacterial colonization was enumerated and Log₁₀ average of three to four mice per group +/- standard deviation was determined at the indicated time points. Dashed line indicates limit of detection. * indicates <i>p</i> value <0.05 between wild-type and C3 deficient groups.</p

Complement efficiently deposits onto the cell surface and kills Bpp5.

<p>(A) <i>B</i>. <i>bronchiseptica</i> RB50 (diamond), RB50Δ<i>wbm</i> (square) or <i>B</i>. <i>parapertussis</i>_<i>ov</i> Bpp5 (triangle) were incubated with complement active (solid lines) or complement inactive serum (dashed lines) at the indicated concentrations for 1 hour. The average percent survival of three independent experiments is shown +/- standard error. (B) Flow cytometry analysis of C3b deposition onto RB50, RB50Δ<i>wbm</i> or Bpp5 incubated with complement sufficient (solid line) or deficient (dashed line) serum. Samples were unstained or stained with FITC-anti-mouse C3 antibodies and analyzed and a representative image was shown. The average percentage of FITC-positive cells of three replicates is indicated +/- standard error. * indicates <i>p</i> value <0.05 in comparison to RB50.</p

Extra-intestinal dissemination of SEE1.

<p>Bacterial burden of the liver (A) and spleen (B) from three experiments was taken and normalized with corresponding controls, and analyzed by Tukey’s post-test of one-way ANOVA, and significance of <i>p</i><0.05 is indicated by *. There were five mice per group in the first two experiments and four mice per group in the last experiment. Mice that received the same treatment in each of the three experiments were considered one group for final data analysis.</p

Overall histopathology scores of ceca.

<p>The primary site of colonization for SE in mice is the cecum; so the histopathology of the ceca of three mice from each control group and four mice from each infection group was determined. Statistical significance was determined by one-way ANOVA Tukey’s pairwise comparison test, and significance was set at <i>p</i><0.05; indicated by *.</p

Gross pathological changes in mice.

<p>Overall changes in the mice internal organs from the third experiment were photographed and analyzed. Mice infected with SEE1 grown in egg yolk (B) appear to have greater outward pathology than the mice infected with SEE1 from LB broth (A) or from mouse feces (C). Major changes in gross pathology and organs of interest are indicated by the arrows and Roman numerals. I, The liver displaying marked paling, and prominent blood vessels. II, The small intestine showing signs of paling compared to the small intestine in A and C. III, The cecum that is emptied and shriveled. IV, The colon section of the large intestine that is extremely pale and empty. V, Fluid build-up in the visceral tissues. There were four mice in each experimental group and three mice in each control group.</p

Cytokine analysis of ceca of mice infected with SEE1 from three sources.

<p>Pro-inflammatory and anti-inflammatory cytokine profiles had been determined by ELISA for ceca of mice infected with SEE1 from egg yolk (four mice), LB broth (four mice), and passed through mice (four mice) as well as corresponding controls for each source used (three mice each). Results were normalized with corresponding controls and analyzed through one-way ANOVA and subsequent Tukey’s Pairwise Comparison Test. Significance of <i>p</i><0.05 is indicated by *.</p