The Chlamydia muridarum Organisms Fail to Auto-Inoculate the Mouse Genital Tract after Colonization in the Gastrointestinal Tract for 70 days

Abstract

Chlamydia muridarum is known to colonize in the gastrointestinal tract for long periods of time, which has been hypothesized to serve as a reservoir for spreading to the genital tract. To test this hypothesis, a luciferase-expressing C. muridarum was used to establish a long-lasting infection in the mouse gastrointestinal tract following either intragastric or intrarectal inoculations. In vivo imaging revealed significant bioluminescent signals mainly in the mouse abdominal area throughout the experiments. Ex vivo imaging localized the signals to the mouse gastrointestinal tract, which was confirmed by monitoring the C. muridarum organisms in the mouse organs/tissues. Despite the long-lasting colonization in the gastrointestinal tract and active shedding of infectious organisms in the rectal swabs, the organisms did not cause any significant infection or pathology in the genital tract throughout the experiments, which was reproduced in multiple strains of mice and with an increased inoculation dose to the gastrointestinal tract. The above observations have demonstrated that the long-lasting C. muridarum organisms from the gastrointestinal tract are inefficient in auto-inoculating the genital tract, suggesting that the gastrointestinal tract Chlamydia may utilize an indirect mechanism to affect its pathogenicity in the genital tract.

Fig 1. In vivo imaging of mice infected intragastrically or intrarectally with luciferase-expressing C. muridarum.

CBA/J mice were intragastrically (i.g., n = 5, panels a-i) or intrarectally (i.r., n = 5, j-r) inoculated with 5 x 10⁴ IFUs of luciferase-expressing C. muridarum. On different days after the inoculations as indicated on top of the figure, a whole body in vivo imaging was used to detect the luciferase-generated bioluminescence signals as displayed in red/green/blue colors (in the order of decreasing intensity). Images taken from one of the mice in each group were shown. It is worth noting that the bioluminescence signals were detectable as early as day 3 after inoculation at the initial inoculation sites either stomach (panel a) or anorectum (j). Starting on day 7, the signals persisted in the mouse abdominal area throughout the experiments with similar distribution patterns regardless of the initial inoculation routes.

Fig 2. Ex vivo imaging of luciferase-expressing C. muridarum and recovery of C. muridarum genomes and infectious organisms from mouse organs.

CBA/J mice were intragastrically (panels a1-5 & b1-5) or intrarectally (panels c1-5 & d1-5) inoculated with 5 x 10⁴ IFUs of luciferase-expressing C. muridarum as described in Fig 1 legend. On days 28 & 70 after infection as indicated on the left of the figure, a whole body in vivo imaging was taken (panels a1-d1). At the same time, a group of 3 to 5 mice were sacrificed at each time point for harvesting the internal organs. Organs from the same mouse were arrayed in a Petri dish as shown in panels a2-d2. Ex vivo imaging was immediately taken to acquire bioluminescence signals as shown in panels a3-d3 (column labeled with “original”). Green arrows point to signals from the rectum, yellow to the ceccum and red to the stomach. The arrow-pointed areas were magnified and displayed on the right with the same arrow indication (columns labeled with “magnified”, panels a3a-d3b). At each time point, only one representative mouse-derived in vivo whole body image and ex vivo image of the organs from the same mouse are shown. The same organs after ex vivo imaging were subjected to tissue homogenization with some organs separated into different tissues as shown along the X-axis at the bottom. On the same day of sacrificing, both vaginal and rectal swabs were taken from each mouse. All organs/tissues/swabs, including lungs, heart, spleen, liver, stomach, small intestine (SI), ceccum, colon, rectum, rectal (R)-swab (from stomach to R-swab, grouped as gastrointestinal tract, or GI), kidneys, left (L) or right (R) ovary-oviduct (ov) or uterine horn (uh), vagina-cervix (CV) and vaginal (V) swab (from L-ov, grouped as genital tract) were titrated for Chlamydia muridarum genome copies (panels a4-d4) and live organisms (as inclusion forming units, IFUs, panels a5-d5). The genome copies and IFUs were expressed in total numbers per organ/tissue/swab (log10) as mean ± standard error and displayed along the Y-axis on the right. Note that bioluminescence signals were mainly detected in the mouse gastrointestinal tract throughout the experiments regardless of the inoculation routes, which is confirmed by the detection of C. muridarum genomes and live organisms from the same organs/tissues. The C. muridarum organisms detected from the rectal and vaginal swabs reflect the chlamydial presence in the GI and genital tract respectively.

Fig 3. Recovering live C. muridarum organisms from GI versus genital tracts of CBA/J mice.

The same two groups of mice as described in Fig 1 legend and an additional group of CBA/J (n = 5) infected intravaginally (i.vag.) with the same amount of luciferase-expressing C. muridarum were monitored for live chlamydial organism shedding in their rectal (panels a, c & e) and vaginal (b, d & f) swabs on different days after infection as indicated along the X-axis at the bottom of the figure. The results were expressed as Log10 IFUs displayed along the Y-axis. Note that no significant live organisms were detected in the vaginal swabs of mice infected in the GI tract throughout the experiments.

Fig 4. Monitoring C. muridarum infection in both the GI and genital tracts following an intrarectal inoculation with 1 x 10⁷ IFUs.

CBA/J mice were intrarectally (n = 4) inoculated with 1 x 10⁷ IFUs of luciferase-expressing C. muridarum. (A) On different days after the inoculations as indicated on top of the figure, a whole body in vivo imaging was used to detect the luciferase-generated bioluminescence signals as displayed in red/green/blue colors (in the order of decreasing intensity). Images taken from one of the mice in each group were shown. (B) At the same time, these mice were monitored for live chlamydial organism shedding in their rectal (panel a) and vaginal (b) swabs on different days after infection as indicated along the X-axis at the bottom of the figure. The results were expressed as Log10 IFUs displayed along the Y-axis. Note that no significant live organisms were detected in the vaginal swabs throughout the experiment.

Fig 5. Evaluating the gross pathology in the genital tracts of CBA/j mice infected with C. muridarum.

The three parallel groups of CBA/J mice infected with luciferase-expressing C. muridarum intragastrically (i.g., panel a, n = 5), intrarectally (i.r., b, n = 5) or intravaginally (i.vag., panels c, n = 5) as described in Fig 3 legend were sacrificed on day 70 after infection for visually identifying (arrow) and scoring (white numbers) hydrosalpinx. Mice with hydrosalpinx on either side of the genital tract were defined positive for hydrosalpinx. The severity of hydrosalpinx was scored from each oviduct independently and the scores from both sides of the same mouse were added together as the score for that mouse. Both incidence rates and severity scores of hydrosalpinx from each group were listed beside the corresponding images. Note that significant hydrosalpinx was detected only in mice infected intravaginally but intragastrically or intrarectally.

Fig 6. Evaluating the microscopic pathology in the mouse genital tracts.

The same mouse genital tissues harvested on day 70 after infection as described in Fig 5 legend were examined for inflammatory infiltration in the genital tract tissues. (A) Representative images of H&E-stained sections of vagina (panels a, d & g under 10x objective lens while a1, d1 & g1 under 100x), uterine/uterine horn (b, e & h, 10x; b1, e1 & h1, 100x) and oviduct (c, f & i, 10x; c1, f1 & i1, 100x) from mice infected intragastrically (i.g., a-c), intrarectally (i.r., d-f) and intravaginally (i.vag., g-i) were shown. (B) The Inflammatory infiltration from the vagina (a), uterine/uterine horn (b) and oviduct (c) tissues were semi-quantitatively scored (shown along the Y-axis) as described in the Materials and Method section. Kruskal-Wallis Test was used to calculate the p values listed in the figure. Note that only the tissues from the intravaginally infected mice displayed significant levels of inflammatory infiltration.

Fig 7. Monitoring C. muridarum infection in the GI and genital tracts of C57BL/6J and C3H/HeJ mice following an intrarectal inoculation.

C57BL/6J (panels a1-a3, n = 4) and C3H/HeJ (b1-3, n = 3) mice were intrarectally infected with C. muridarum and both anorectal and cervico-vaginal swabs were taken on different days after infection as listed along the X-axis in the bottom of the figure. The number of live organisms recovered from the swabs was expressed as Log10 IFUs. "ND” denotes not detected. On day 70 after infection, mice were sacrificed for harvesting the genital tracts for visually identifying and scoring hydrosalpinx (a3 & b3) as described in Fig 3 legend. Note that although significant levels of live organisms were continuously detectable in the mouse GI tract throughout the experiment, no significant level of live organisms was detected in the mouse vaginal swabs and there was no significant hydrosalpinx in any of the mice.