FIG 3.

The durable transmucosal protection induced by intragastric immunization is not dependent on long-term gastrointestinal infection. Groups of C57BL/6J mice immunized intragastrically with 2 × 10⁵ IFU of CM-mCherry (n = 5 for panel a and n = 7 for panel b) or not immunized (n = 6) (c) were treated on day 28 with doxycycline (20 μg/kg of body weight intragastrically once daily) for 2 weeks (days 28 to 42) (b and c) or were not treated with doxycycline (a). The doxycycline-treated mice were then rested for 2 weeks (days 43 to 56). On day 56 after immunization in the GI tract, all mice were intravaginally challenged with 2 × 10⁵ IFU of clone G13.32.1. (A) Mice were monitored for the shedding of chlamydiae by evaluation of both vaginal (a to c) and rectal (a1 to c1) swab specimens over the course of infection (the days after challenge infection are designated 3′ to 56′ in parentheses). Results are expressed as the log₁₀ number of IFU per swab specimen. Mice in the immunization plus doxycycline treatment group displayed no IFU in the rectal swab specimens prior to the intravaginal challenge (days 31 to 56) (b) but maintained transmucosal protection against chlamydial infection in the genital tract (*, P < 0.05, Wilcoxon rank-sum test, for panel b1 versus panel c1), equivalent to the findings for immunized mice not treated with doxycycline (*, P < 0.05, Wilcoxon rank-sum test, for panel a1 versus panel c1). These two groups maintained similar levels of protection (*, P > 0.05, Wilcoxon rank-sum test, for panel b1 versus panel a1). (b and c) The genital tract G13.32.1 organisms spread to the GI tracts. (a and a1) Black bars, G13.32.1 alone; dark red bars; both CM-mCherry and G13.32.1. (B) On day 114 after intragastric immunization, all mice were sacrificed to evaluate the upper genital tract pathology macroscopically. Representative images of the entire genital tracts from the groups receiving immunization without doxycycline treatment (a2), immunization plus doxycycline treatment (b2), or doxycycline treatment without immunization (c2) are shown. White arrows, oviducts positive for hydrosalpinges. Magnified images of oviduct/ovary regions are shown on the right of the overall genital tract images, with the white numbers indicating the hydrosalpinx scores. Both the incidence of hydrosalpinx and the hydrosalpinx severity score (mean ± standard deviation) are listed above the corresponding images. Regardless of doxycycline treatment, immunized mice were significantly protected from the development of hydrosalpinx (*, P < 0.05, Wilcoxon rank-sum test, for the immunization alone group in panel a2 versus panel c2 and for the immunization plus doxycycline treatment group in panel b2 versus panel c2).