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Comparative Study
. 2009 Aug;77(8):3284-93.
doi: 10.1128/IAI.00147-09. Epub 2009 May 26.

Strain and Virulence Diversity in the Mouse Pathogen Chlamydia Muridarum

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Free PMC article
Comparative Study

Strain and Virulence Diversity in the Mouse Pathogen Chlamydia Muridarum

Kyle H Ramsey et al. Infect Immun. .
Free PMC article

Abstract

The mouse chlamydial pathogen Chlamydia muridarum has been used as a model organism for the study of human Chlamydia trachomatis urogenital and respiratory tract infections. To date, two commonly used C. muridarum isolates have been used interchangeably and are essentially taken to be identical. Herein, we present data that indicate that this is not the case. The C. muridarum Weiss isolate and C. muridarum Nigg isolate varied significantly in their virulences in vivo and possessed different growth characteristics in vitro. Distinct differences were observed in intravaginal 50% infectious doses and in challenge infections, with the Weiss isolate displaying greater virulence. Respiratory infection by the intranasal route also indicated a greater virulence of the Weiss isolate. In vitro, morphometric analysis revealed that the Weiss isolate produced consistently smaller inclusions in human cervical adenocarcinoma cells (HeLa 229) and smaller plaques in monolayers of mouse fibroblasts (L929) than did the Nigg isolate. In addition, the Weiss isolate possessed significantly higher replicative yields in vitro than did the Nigg isolate. In plaque-purified isolates derived from our stocks of these two strains, total genomic sequencing identified several unique nonsynonymous single nucleotide polymorphisms and insertion/deletion mutations when our Weiss (n = 4) and Nigg (n = 5) isolates were compared with the published Nigg sequence. In addition, the two isolates shared 11 mutations compared to the published Nigg sequence. These results prove that there is genotypic and virulence diversity among C. muridarum isolates. These findings can be exploited to determine factors related to chlamydial virulence and immunity.

Figures

FIG. 1.
FIG. 1.
Primary and challenge infection courses with C. muridarum strains. (A) Course of primary and challenge infections of mice inoculated intravaginally with 104 IFU of MoPn Weiss (closed circles). The primary infection is shown in the left half of the graph. Each point represents the mean and standard deviation (error bars) for IFU isolated from cervical-vaginal swabs (n = 38; two experiments). Following the resolution of infection, mice were then challenged intravaginally at day 56 after primary infection either homotypically with MoPn Weiss (open circles) or heterotypically with MoPn Nigg (open squares) at 105 IFU. Only mice challenged with MoPn Weiss displayed any susceptibility to challenge infection (P < 0.02 by repeated-measures ANOVA). (B) Course of primary infection of mice inoculated intravaginally with MoPn Nigg (closed squares) (n = 20; one experiment). Mice were then challenged intravaginally at day 56 after primary infection with MoPn Weiss (open circles) (n = 10) or MoPn Nigg (open squares) (n = 10) at 105 IFU. A tendency toward higher rates of culture-positive mice and higher infectious burdens was observed in the mice challenged with MoPn Weiss, but this did not prove to be significant (P = 0.07 by repeated-measures ANOVA). Primary infection courses between MoPn Weiss and MoPn Nigg were not significantly different (P = 0.789 by repeated-measures ANOVA). Comparison between any primary infection and any challenge infection yielded highly significant differences (P < 0.0001).
FIG. 2.
FIG. 2.
Morbidity in mice with MoPn respiratory infection. The solid line in each panel represents the mean daily percent change in body weight of 10 mice inoculated intranasally with MoPn Nigg. The dashed line represents the mean daily percent change in body weight of 10 mice inoculated with MoPn Weiss. (A) Mice were inoculated with 103 IFU of either strain. MoPn Weiss-infected mice gained less weight over time than did the MoPn Nigg-infected mice (P < 0.02 by repeated-measures ANOVA). (B) Mice were inoculated with 104 IFU of either strain. MoPn Weiss-infected mice had a significant weight loss compared to the weight gain experienced by mice infected with MoPn Nigg (P < 0.005). One mouse died in the MoPn Weiss-infected group on day 17. (C) Mice were inoculated with 105 IFU of either strain. All but two mice died by day 8 postinfection in the MoPn Weiss-infected group, and two mice died in the MoPn Nigg-infected group on days 9 and 17 (Fig. 3). The mean weight losses of the groups over time were significantly different (P < 0.02).
FIG. 3.
FIG. 3.
Kaplan-Meier survival curve. Mice were infected with 105 IFU of either MoPn Weiss or MoPn Nigg intranasally. Nine of ten mice infected with MoPn Weiss died through day 12 postinfection, whereas 2 of 10 mice infected with MoPn Nigg died on days 9 and 17 postinfection. The survival curves are significantly different (P < 0.004 by log-rank test).
FIG. 4.
FIG. 4.
Replication rate of MoPn isolates in vitro. Each point represents the mean IFU at the specified time postinfection. Error bars are standard errors of the means of triplicate cultures (20 fields per replicate). Open circles represent MoPn Weiss; closed circles represent MoPn Nigg. (A) Results from sonicated coverslips representing cell-associated EBs. (B) Results from supernatants representing extracellular EBs. For both panels, the results represent a single experiment. A separate experiment yielding essentially identical results was also conducted (not shown). In both cases, MoPn Weiss displayed a higher yield of progeny than did MoPn Nigg (P < 0.001 by repeated-measures ANOVA).
FIG. 5.
FIG. 5.
Inclusion size variation in MoPn isolates. Inclusion size was measured as the area of each inclusion at 12, 18, and 24 h postinfection for the two strains. Each point represents the morphometric analysis of a single inclusion (n = 150 to 220 per time point). Closed circles are values for MoPn Nigg; open circles are values for MoPn Weiss. Horizontal bars are the means of each group at each time. P values provided at each time point represent those derived by comparing the two isolates using an unpaired, two-tailed t test.
FIG. 6.
FIG. 6.
Plaque size variation in MoPn isolates. Plaque sizes in cultures of MoPn isolates grown for 5 days were measured. Each point represents the morphometric analysis of a single plaque of either strain (n = 118 for Nigg and n = 69 for Weiss). Closed circles are values for MoPn Nigg; open circles are values for MoPn Weiss. The means (horizontal bars) of the MoPn Nigg plaque sizes were larger than those of MoPn Weiss. The P value shown was derived by comparing the two isolates using an unpaired, two-tailed t test.

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