Figure 1.
Vaginal infection by G. vaginalis results in minimal histological inflammation.
G. vaginalis JCP8151B-SmR4 titers were determined by enumerating colony forming units (CFU) in vaginal washes (A) and tissue homogenates (B) at 24 and 72 hpi. For samples containing no colonies, the limit of detection for each was determined and is displayed instead of a value of 0. Results are meta data from 2 independent experiments, each with 10 mice/infection group. (C) Histological inflammation was assessed by hematoxylin-eosin (H&E) staining of formalin-fixed, paraffin-embedded vaginal tissue sections.
Figure 2.
G. vaginalis titers in vaginal washes reflect titers in vaginal tissue and correlate directly with titers of G. vaginalis in uterine horns.
Vaginal wash and tissue titers determined as in Fig. 1 were plotted and analyzed using GraphPad® Prism 5.0. ***P<0.001; *P<0.05. Results are meta data from 2 independent experiments, each with 10 mice/infection group.
Figure 3.
Vaginal sialidase activity correlates with G. vaginalis vaginal infection and is associated with ascending uterine horn infection.
(A) Sialidase enzymatic activity was determined in vaginal washes from mock and G. vaginalis infected mice at 24 and 72 hpi and statistical significance determined by the Mann-Whitney test. The dotted line represents the cutoff value used to distinguish sialidase (+) from sialidase (−) vaginal washes for performing Fisher’s exact test, shown in the box above. Results are meta data from 4 independent experiments (24 hpi-mock n = 44, G. vaginalis n = 70; 72 hp-mock n = 29, G. vaginalis n = 40). The sialidase activity value from each G. vaginalis-infected mouse was plotted against G. vaginalis CFU in the corresponding vaginal wash (B) or tissue homogenate (C) and Spearman correlations were determined. ***P<0.0001; **P<0.005. (D) Fisher’s exact test to determine whether ascending uterine horn infection by G. vaginalis was contingent upon the presence of vaginal sialidase activity. Results shown represent combined data from both 24 and 72 hpi, though each time point was significant when analyzed individually.
Figure 4.
G. vaginalis adheres to murine vaginal epithelial cells in vitro and in vivo.
(A) Phase contrast light microscopy of vaginal wash wet mounts (panels a and b) and H&E staining of vaginal tissue sections (panels c and d) from mock (a and c) and G. vaginalis (b and d) infected mice. Arrows in panel b and dark purple puncta in panel d illustrate potentially adherent bacteria in G. vaginalis-infected samples. (B) Fluorescent confocal microscopy images (with corresponding bright-field images) of vaginal epithelial cells either mock (top) or RBITC labeled G. vaginalis (bottom) infected in vitro for 3 h at 37°C. (C) Fluorescent confocal microscopy images of vaginal washes from mock or RBITC labeled G. vaginalis infected mice, collected at 4 hpi.
Figure 5.
G. vaginalis induces a robust histological epithelial exfoliation and proliferation response.
(A) Histological exfoliation scoring. H&E stained, formalin-fixed, paraffin-embedded vaginal tissue sections from the 24 hpi time point were assessed for evidence of epithelial exfoliation (eosin-rich layers of epithelial cells superficial to the transitional epithelium) and assigned a numerical value from 0–3, with 0 = none and 3 = very robust. The Kruskal-Wallis test was used to for statistical evaluation of differences between groups (P = 0.0161). For pairwise comparisons, post-hoc testing was performed using the Mann-Whitney U-test, with significance indicated in the figure. *P<0.05. Upon conservative correction for multiple comparisons with Bonferroni-Holm, the non-parametric tests remain significant. (B) Representative images of epithelial scoring, with white, capped lines representing the measured thickness of the transitional epithelial layer shown in (C). (C) Using the same samples from (A), average thickness of the transitional epithelium was determined from five measurements per sample using StreamStart® software. Results in each graph are meta-data from 4 independent experiments (mock n = 36, G. vaginalis n = 25, heat-killed (HK) G. vaginalis n = 10). The data passed the D’Agostino & Perason omnibus normality test. Therefore, a one-way ANOVA was used detected significant differences between groups (P = 0.0004), followed by post-hoc pairwise comparisons using the unpaired t-test. **P<0.01; ***P<0.001. Again, differences remained significant with the conservative Bonferroni correction. (D) Transitional epithelium thickness values from (C) were plotted against exfoliation scores from (A) and Spearman correlation was determined. ***P<0.001.
Figure 6.
G. vaginalis-induced exfoliation is evident in vaginal washes and correlates with vaginal titers and sialidase activity.
(A) Representative images of phase contrast light microscopy fields of vaginal wash wet mounts from mock, G. vaginalis and HK G. vaginalis infection groups, as indicated, used in (B) to enumerate epithelial cells. (B) The average numbers of epithelial cells per field of view were calculated from 5 images per sample. Results are meta-data from 4 independent experiments (mock n = 51, G. vaginalis n = 74, HK G. vaginalis n = 19). The Kruskal-Wallis test was used to for statistical evaluation of differences between groups (P = 0.0065). The Mann-Whitney U-test was used for post-hoc pairwise comparisons, with significance indicated in the figure. **P<0.01; *P<0.05. Differences remained significant with the conservative Bonferroni-Holm correction for multiple comparisons. (C) and (D) Epithelial counts were plotted against their corresponding vaginal wash CFU (C) or sialidase activities (D) and Pearson correlations were determined.
Figure 7.
Human clinical samples display evidence of epithelial exfoliation associated with bacterial vaginosis.
Heat-fixed, Gram stained slides were prepared from fresh human vaginal swabs and classified as BV (+) or no BV (−) by Nugent scoring (scores 7–10 and 0–3 respectively). The average number of epithelial cells per field of view was calculated from 3 images per sample. BV (−) n = 28, BV (+) n = 27. The Mann-Whitney U-test was used for statistical evaluation of differences between groups. ***P<0.0001.