Effects of cationic liposome-encapsulated doxycycline on experimental Chlamydia trachomatis genital infection in mice

L. Sangaréa, R. Morisseta, L. Gabouryb and M. Ravaoarinoroa,*

a Department of Microbiology and Infectiology, and b Department of Pathology, CHUM-Hôtel-Dieu, 3840 St Urban Street, Montreal, Quebec, Canada H2W 1T8


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a previous study, we demonstrated that cationic liposome-encapsulated doxycycline (CaL-Dox) was two-fold more effective than free doxycycline against Chlamydia trachomatis in vitro. Here, we evaluated the effects of two CaL-Dox regimens in comparison with unencapsulated doxycycline on the course of chlamydial genital infection in mice. Progesterone-treated, female CF-1 mice were challenged intravaginally with 1.2 x 105 inclusion-forming units (ifu) of C. trachomatis. Two days post-infection, the animals were divided into four treatment groups for im injection of doxycycline at 10 mg/kg body weight bd for 3 (3 Dox) or 7 days (7 Dox), or of CaL-Dox at the same dose level for 3 (3 CaL-Dox) or 7 days (7 CaL-Dox) consecutively. An infected fifth group served as a control and was given an empty CaL preparation. C. trachomatis were isolated after five blind passages from 82% of infected control mice, 61.4% of 3 Dox, 52.2% of 3 CaL-Dox, 29% of 7 Dox and 20% of 7 CaL-Dox animals. Histopathological reactions were found in various tissues of the genital tract in 79.5% of infected control mice, 80.9% of 3 Dox, 65.2% of 3 CaL-Dox, 47.1% of 7 Dox and 25.7% of 7 CaL-Dox animals. Total antichlamydial antibody titres were lower in 7 CaL-Dox mice than in all the other groups (P < 0.005). The results showed that progesterone-treated CF-1 mice are suitable for investigation of both lower and upper genital tract infection with a lymphogranuloma venereum biovar strain of C. trachomatis. Neither 7 CaL-Dox nor 3 CaL-Dox treatment was more effective than unencapsulated 7 Dox doses in the bacteriological cure of chlamydial genital infection in mice. However, 7 CaL-Dox prevented tissue damage in the genital tract significantly more than all the other regimens (P < 0.05). These results suggest that liposome-encapsulated doxycycline, particularly CaL-Dox, may have potential for the clinical treatment of chlamydial infections.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chlamydia trachomatis is a major bacterial agent in sexually transmitted diseases worldwide.1,2 In women, chlamydial cervicitis may ascend in the genital tract and result in endometritis and salpingitis, which, in turn, can lead to ectopic pregnancy or infertility.1,3,4 Newborn infants exposed to C. trachomatis in the birth canal may develop pneumonitis or conjunctivitis.1,4 In men, the pathogen is responsible for urethritis, and coinfections with Neisseria gonorrhoeae are frequent in both hetero- and homosexual men. Moreover, C. trachomatis is associated with epididymitis and prostatitis. Lymphogranuloma venereum (LGV) occurs in both men and women. Recent studies have revealed that chlamydial infections of the lower genital tract can be an important risk factor for acquired sexual transmission of HIV.5 These bacterial infections are a public health problem in both industrialized and developing countries.2

Currently, tetracyclines, macrolides including azalides and the newer fluoroquinolones are the drugs of choice in monomicrobial urogenital infections due to C. trachomatis.6,7 However, some other pathogens are often associated with C. trachomatis in genital infections. In such polymicrobial infections, the drugs used should be active against all the pathogens that could be involved. However, these drugs must be given twice or more and for at least 7 days, except for azithromycin, and they often cause adverse reactions that lead to poor compliance as well as therapy failure.6 In such cases, Chlamydia is not completely eradicated so that the infection may persist and contribute to the development of complications. Moreover, relative resistance in vitro of C. trachomatis clinical strains to certain tetracyclines and macrolides has been reported.810 Although fluoroquinolone-resistant clinical strains of C. trachomatis have not been isolated to date, a quinolone-resistant mutant has been found in vitro.11

Various strategies, including the development of new molecules and the improvement of existing drug formulations by the use of carriers such as liposomes, have been elaborated to overcome these problems. We have reported previously that liposome-encapsulated tetracycline and doxycycline are more active than their free forms against C. trachomatis in vitro.12 Several animal models, including mice, have been used to study the pathogenesis of chlamydial strains in urogenital infections and to evaluate the actions of antibiotics.13,14 Tuffrey & Taylor-Robinson15 were able to infect mouse genitals with strain SA-2f, an LGV serotype of C. trachomatis. Development of infection was dependent on the subcutaneous injection of progesterone. Ito et al.16 have shown that progesterone-pretreated CF-1 mice are variously susceptible to chlamydial strains of the trachoma biovar. No detailed description of the histopathological reactions in tissues of the upper genital tract is available for this model. To the best of our knowledge, no study has yet reported infection of these animals with a strain belonging to the LGV biovar of C. trachomatis.

In the present investigation, we have demonstrated that progesterone-treated CF-1 mice are susceptible to genital infection with C. trachomatis L2/434/Bu, and we have evaluated the effects of two cationic liposome-encapsulated Dox regimens in this model in comparison with unencapsulated doxycycline on the course of chlamydial genital infection.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organism

C. trachomatis L2/434/Bu, provided by LSPQ (Laboratoire de Santé Publique du Québec, Montreal, Québec, Canada), was grown in HeLa 229 cells (ATCC CCL 2.1).12 Elementary bodies (EBs) were purified from infected, harvested and ultrasonically disrupted cells,17 then resuspended in sucrose–phosphate–glutamic acid (SPG) buffer, pH 7.4, in 0.5 mL aliquots of 6.02 x 106 inclusion-forming units (ifu)/ mL. Before serological assay of the antigen preparations, EBs in SPG buffer were further concentrated by centrifugation in 0.01 M phosphate-buffered saline (PBS), pH 7.0, to achieve a concentration of 6.02 x 109 ifu/mL. Aliquots were stored at –70°C until their use.

Drugs

The drugs used in the investigation were doxycycline hydrochloride (Pfizer, New York, NY, USA) and its cationic liposomal formulation CaL-Dox (93.5 ± 38.9 µg/mL). The phospholipids consisted of egg lecithin, stearylamine and cholesterol in a 7:2:1 molar ratio. CaL-Dox was prepared by sonication and stored at 4°C for use in the same week.12,18

Inoculation of mice

Three hundred and forty female CF-1 mice, 31–36 days old and weighing 16–18 g, were obtained from Charles River Canada Inc. (Montreal, QC, Canada). Before the experiments, mice were verified free from Chlamydia and Mycoplasma contamination by examination of animals. Ten and 3 days before inoculation, the mice were injected sc with 2.5 mg of progesterone (Depo-Provera, Upjohn, Don Mills, Ontario, Canada) in 0.1 mL of injectable sterile water: 320 mice, under ketamine–xylazine (Bayer, Etobicoke, ON, Canada) anaesthesia, were inoculated intravaginally by pipette tips with 1.2 x 105 ifu in 20 µL of SPG buffer, and 20 uninfected controls received 20 µL of sterile SPG buffer. They were maintained in an inverted position until recovery from anaesthesia.19

Treatment

Two days post-inoculation, the animals were separated into four treatment groups of 70 mice each: in the first and second groups, each mouse received im injections bd of doxycyline at 10 µg/g body weight for 3 (3 Dox) or 7 (7 Dox ) consecutive days, respectively. In the remaining two groups, each animal was given im injections of CaL-Dox at 10 µg/g for 3 (3 CaL-Dox) or 7 (7 CaL-Dox) consecutive days, respectively. Forty infected mice received 0.1 mL of empty CaL preparation, and the 20 uninfected controls received only 0.1 mL of sterile water for 7 days.

Specimen collection

To monitor the course of infection and the effect of the treatment regimens, the first (s1) samples were collected 2 days post-inoculation, before the treatments began, 3 days (s2) after the completion of treatments and weekly thereafter (s3 to s7) until cortisone administration. Cervico-vaginal swabs were taken from one to 11 mice under Enflurane (Abbott Laboratories Ltd, Montreal, Québec, Canada) anaesthesia in each treatment and control group, and expressed in 1 mL of SPG buffer, pH 7.4, containing vancomycin (100 µg/mL), gentamicin (50 µg/mL) and amphotericin B (25 µg/mL), before freezing at –70°C until cultivation. The mice were then dissected and blood samples were collected for serological assays. The entire genital tract was removed and placed in 10% buffered formalin for histological examination.

Cortisone injection

Two days after the seventh specimen collection (s7), all the remaining mice in each treatment and control group were injected with hydrocortisone sodium succinate (Abbott Laboratories Ltd), at 125 mg/kg of body weight per day for 6 consecutive days. Two days after the last injection, all these mice were killed and samples were collected as described previously.

Assessment of drug efficacy of antichlamydial regimens

Cell culture isolation.
An aliquot of 0.25 mL of each cervico-vaginal specimen in SPG buffer was cultured in drug-free medium on 24-h-old HeLa 229 cell monolayers in 24-well plates. Four wells were used for each sample, and after five blind passages, the coverslips were subjected to May– Grünwald–Giemsa staining to detect chlamydial inclusions by microscopic examination.12 The Giemsa technique was chosen because it was convenient for the large number of samples that were to be tested, and studies have shown that it gives comparable results to immunofluorescence.20

Serology.
Serum was diluted two-fold (1:2 to 1:512) in 0.01 M PBS, pH 7.0, and the total antibody titre was determined by a variation of the microimmunofluorescence (micro-IF) technique described by Wang & Grayston,21 in which a goat anti-mouse polyvalent immunoglobulin (IgG, IgA, IgM)–fluorescein isothiocyanate conjugate (Sigma Immuno Chemicals, St Louis, MO, USA) was used. The titre was defined as the highest serum dilution producing definite fluorescence of C. trachomatis EB on slide examinations under a fluorescence microscope.

Histopathology.
The genital tract was embedded in paraffin, and sections were stained with haematoxylin and eosin. The sections examined included the cervico-vaginal portion, uterus, Fallopian tubes and ovaries. Histopathological changes occurring at various sites of the genital tract were evaluated according to inflammatory reactions:14 any genital tract section with histopathological reactions was considered abnormal.

Statistical analysis
The results were analysed by the {chi}2 test, Student's t-test or Fisher's exact test, with SAS monospace software. P values <=0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteriological tests

Progesterone pre-treatment contributed to effective genital tract infection of mice with human isolates of C. trachomatis. Five blind passages in drug-free medium were run for each cervico-vaginal swab. Cultures were negative in all uninfected control mice, positive in 82% of infected controls, 61.4% of 3 Dox, 52.2% of 3 CaL-Dox, 29% of 7 Dox and 20% of 7 CaL-Dox animals: the infection rates were significantly lower in all treatment regimens than in infected control mice (P < 0.05), except for the 3 Dox regimen. There was no difference between the overall rates of positive culture in Dox- and CaL-Dox-treated mice (P = 0.112); the difference from positive control mice was not due to the drug formulation. However, mice in the three-dose regimen (56.8%) had a significantly higher infection rate than those given seven doses (24.5%; P = 0.001). Although there were fewer infected animals in the 7 CaL-Dox group than in the 7 Dox group, the difference was not statistically significant (P > 0.05).

Two days post-inoculation (s1), chlamydial inclusions were found in cervico-vaginal swab cultures from all positive controls and from mice in all treatment regimens, except for one mouse from the 7 CaL-Dox group. In week 7 (s7), four of six mice among the positive controls and six of 11 in the 3 Dox group were culture positive, whereas in this same time period, none was found to be positive with the 3 CaL-Dox regimen. One mouse was culture positive in each of the 7 Dox and 7 CaL-Dox groups. After cortisone therapy, cervico-vaginal swab culture in s8 showed an increase in the infection rate among untreated infected controls (6/8), 3 Dox (7/10), 3 CaL-Dox (5/10) and 7 Dox (3/10) mice, while only 1/10 was positive in the 7 CaL-Dox group.

Serology

Total serological antibody (IgM, IgG and IgA) titres were measured by micro-IF, and their kinetics were analysed using the titre detected in 50% of the samples (T50), from each period of sampling (s1 to s8) (Table IGo). Nine out of 20 uninfected mice (negative controls) showed total antibody titres of 1:8, thus, any serum with a total antibody titre equal to or greater than 1:16 was considered positive. The results of serological tests in positive controls and mice from the various treatment groups are presented in Table IGo. The antichlamydial antibody titres detected in s1 were lower in 7 CaL-Dox mice than in those from all other groups. Total antibodies reached peak titres in the positive controls by s6, later than in 3 Dox (s4), 3 CaL-Dox, 7 Dox and 7 CaL-Dox (s5) groups. After cortisone therapy, there was no increase in the total antibody titre detected in the 7 CaL-Dox-treated mice, in contrast to the other groups.


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Table I. Total antichlamydial antibody titres at various periods of sampling of positive control and treated mice
 
Histopathological reactions

All sections of the genital tract were examined with an optical microscope to assess the presence of pathological changes such as infiltration with polymorphonuclear leucocytes (acute inflammation), mononuclear leucocytes (chronic inflammation), plasma cells, fibrosis, epithelial erosion (excluding mesosalpinx) and oviduct dilatation. Examples of these pathological changes are shown in Figures 1–3GoGoGo. Genital tract tissues were considered abnormal when at least one of these parameters was present. The results are given as the percentage of animals with abnormal tissue sections in their genital tract. Abnormal ovaries were not found, and uninfected mice did not have pathological changes in their genital tract. Table IIGo presents the overall results in positive controls and treated mice. Pathological changes occurred more frequently in untreated mice than in treated animals (P = 0.001). Likewise, the overall rate of abnormal genital tract tissues was higher with the three-dose antichlamydial regimens than with the seven-dose regimens (P = 0.001). Moreover, the overall rate was higher in mice treated with doxycycline than in those given the CaL-Dox regimens: this difference, due to the drug formulation, was significant (P = 0.002). The results were comparable between the 3 CaL-Dox- and 7 Dox-treated groups (P > 0.05), whereas the rates were significantly lower in 7 CaL-Dox- than in 7 Dox- and in 3 CaL-Dox-treated groups.



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Figure 1. Sections of mouse vagina stained with haematoxylin and eosin. (a) Normal mucus-containing epithelial cells of the vaginal mucosa during dioestrus in an uninfected animal in the 7 CaL-Dox group at s4; magnification x20. (b) Abscess formation in mucus-containing epithelial cells of the vagina during dioestrus. Presence of cell debris, mononuclear and polymorphonuclear leucocytes in both abscess and vaginal lumen: chronic inflammation in 3 Dox animals at s7; magnification x40.

 


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Figure 2. Sections of mouse uterus stained with haematoxylin and eosin. (a) Normal uterus in 7 CaL-Dox animals at s3; magnification x20. (b) Chronic infiltration of the uterine stroma in positive control animals at s4; magnification x40.

 


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Figure 3. Sections of mouse oviduct stained with haematoxylin and eosin. (a) Normal oviduct in 3 CaL-Dox animals at s7; magnification x20. (b) Polymorphonuclear leucocytes in a dilated lumen of the oviduct and infiltrates in a layer of columnar cells: 3 Dox animals at s7; magnification x40.

 

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Table II. Incidence of pathological changes in various sites of the genital tract of mice from the positive control and treatment regimen groups
 
Histopathological reactions were detected in cervico-vaginal sections in the s1 period in the positive controls and in all treatment regimen mice. Endometritis and salpingitis appeared in the s1 period in the positive controls, 3 Dox- and 3 CaL-Dox-treated mice, while in the 7 Dox group, endometritis was detected from s6 and salpingitis from s8. In 7 CaL-Dox mice, endometritis was noted in s7 and s8, and the sole case of salpingitis was found in s7. Inflammatory reactions reached their peak level in the uterus and Fallopian tube in s3 and s4, respectively. Inflammatory infiltrates with necrosis and abscess formation (Figure 1bGo) were found in cervico-vaginal, uterine and oviduct tissues. Filling of the lumens of the cervix, uterus and oviducts with polymorphonuclear leucocytes and fibrosis of the endometrial stroma were not frequent findings. In a few positive control animals, histopathological reactions were present in both tubes. Histopathological changes over time were similar in severity among the positive controls and 3 Dox animals, unlike those in the 7 CaL-Dox group, in which the inflammatory lesions were less extensive and pronounced. These results demonstrated that the 7 CaL-Dox regimen was the most protective against ascending chlamydial genital infection in mice.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A number of experimental systems have been employed to study the pathogenesis of chlamydial diseases and to evaluate the action of antimicrobial drugs on these pathogens. Mice inoculated by various routes have been widely used for experimental genital infection with Chlamydia and to prove that C. trachomatis is an aetiological agent in pelvic inflammatory disease.13,14 However, Ito et al.22 were able to establish genital tract infection in mice with a human oculogenital strain by intravaginal inoculation. They showed that without progesterone, the animals could only be infected during three specific stages of the oestrous cycle, when leucocytes are present. Later, they inoculated progesterone-treated, female CF-1 mice intravaginally with seven human trachoma biovar strains of C. trachomatis, and observed differences in the duration of infection and their invasiveness; although they demonstrated that virulent strains such as immunotypes E and D cause uterine infection, no histopathological data were provided.16 The LGV chlamydial serotypes are thought to be more invasive than trachoma biovar strains. To date, no genital infection with an LGV strain in CF-1 mice has been reported. In the present study, we infected mice with a strain of C. trachomatis serovar L2 and showed that it was able to induce ascending infection and histopathological changes in the upper genital tract. With serial blind passages in drug-free medium, the pathogen could be detected from cervico-vaginal swab cultures for up to 9 weeks.

To counter the decreased microbial susceptibility to available drugs, several strategies have been developed: these include the use of liposomes as carriers to achieve high concentrations of active drugs in the target infectious site and possibly to reduce their required dose levels. Sangaré et al.18 and Al-Awadhi et al.23 showed that liposome-encapsulated tetracyclines are more effective than their free forms on C. trachomatis in cellular systems in vitro. In a previous study, we also demonstrated in vitro that CaL-Dox possesses antimicrobial and pharmacokinetic properties that could allow its use in vivo.12,18 The inhibitory effect on the growth of C. trachomatis in vitro was two-fold greater with CaL-Dox than with free doxycycline. Thus, we evaluated two treatment regimens of CaL-Dox in the mouse model of genital tract infection developed in our laboratory, in comparison with the same regimen of the free drug. Usually, even in experimental animal models, doxycycline is given bd at a dose of 10 µg/g of body weight for 7 days or longer: a comparative study of both free and liposomal doxycycline given for shorter periods could provide more data on the therapeutic effectiveness of CaL-Dox. The treatment regimen of 3 CaL-Dox doses was assessed to determine whether reduced doses are as effective as the seven dose regimen of free doxycycline. A number of investigators have evaluated the effect of tetracycline antibiotics on chlamydial genital infections in murine models.13 Most of them used either non-human isolates of Chlamydia or unnatural routes to inoculate the animals.2426 They showed that early treatment, starting before inoculation or within the first few days, could lead to bacteriological cure and substantially reduce severe tubal damage and elevation of total serological antichlamydial antibody titres.

Various factors, including virulence of the chlamydial strain used, influence the induction of pathological changes in upper genital tissues and subsequent infertility.27 Jones et al.28 reported that chlamydial culture from urogenital specimens could be substantially improved by serial passage in tissue culture. In our study, we used an invasive strain to inoculate progesterone-pretreated, normal mice and we started antichlamydial therapy 2 days post-infection. Although CaL-Dox was two-fold more active in vitro than free doxycycline,12 under our experimental conditions in vivo, bacteriological cure was not higher with 3 CaL-Dox or 7 CaL-Dox than with 7 Dox. The high rates of positive cultures may have been due in part to the invasiveness of the chlamydial strain used. Despite the early commencement of all treatment regimens, antichlamydial antibody titres were detected from day 2 post-inoculation, and the T50 remained high until weeks 7 (s7) and 8 (s8). A similar profile of high antichlamydial antibody titres has been reported in women,29 and in a mouse model of salpingitis infection with the mouse pneumonitis (MoPn) strain of C. trachomatis25 while they were under antichlamydial therapy. However, lower T50 results were obtained with the 7 CaL-Dox treatment regimen. Likewise, ascending infections in the genital tract and their histopathological changes occurred less with 7 CaL-Dox than with all the other regimens: this drug formulation had more effect on the rate of inflammation than did the addition of anti-inflammatory agents to tetracycline in the mouse model of salpingitis infection with C. trachomatis MoPn.25 Empty CaL alone did not moderate the histopathological changes in infected mice, but the 3 CaL-Dox regimen had effects comparable to those of 7 Dox, and by increasing the dosage to 7 CaL-Dox, genital tract tissues were significantly more protected than with the 7 Dox regimen.

These findings suggest that liposomes could act as a reservoir for doxycycline, allowing the slow release of entrapped drug. They could facilitate antichlamydial drug diffusion through the cell wall to its site of action.30 Thus, liposome-encapsulated doxycyline may have potential for the treatment of chlamydial infections. Furthermore, experimental antichlamydial therapies with other types of liposome-encapsulated antibiotics could be realized. The effect of liposome-encapsulated doxycycline on cellular immune responses and its pharmacokinetic profile are currently under investigation in our laboratory.


    Acknowledgments
 
We thank Marie-Claude Guertin (Centre de recherche, CHUM–Hôtel-Dieu, Montreal) for her valuable assistance with the statistical analyses and Ovid Michael Da Silva for editing this manuscript. This work was supported by a grant from MRC/PMAC (Pfizer Canada Inc.). L.S. is the recipient of a ‘Programme Canadien des Bourses de la Francophonie' scholarship.


    Notes
 
* Corresponding author. Tel: +1-514-843-2675; Fax: +1-514-843-2625; E-mail: madeleine.ravaoarinoro.chum{at}ssss.gouv.qc.ca Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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11 . Dessus-Babus, S., Bébéar, C. M., Charron, A., Bébéar, C. & De Barbeyrac, B. (1998). Sequencing of gyrase and topoisomerase IV quinolone resistance-determining regions of Chlamydia trachomatis and characterization of quinolone-resistant mutants obtained in vitro. Antimicrobial Agents and Chemotherapy 42, 2474–81.[Abstract/Free Full Text]

12 . Sangaré, L., Morisset, R. & Ravaoarinoro, M. (1999). In-vitro anti-chlamydial activities of free and liposomal tetracycline and doxycycline. Journal of Medical Microbiology 48, 689–93.[Abstract]

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18 . Sangaré, L., Morisset, R., Omri, A. & Ravaoarinoro, M. (1998). Incorporation rates, stabilities, cytotoxicities and release of liposomal tetracycline and doxycycline in human serum. Journal of Antimicrobial Chemotherapy 42, 831–4.[Abstract]

19 . Stagg, A. J., Tuffrey, M., Woods, E., Wunderink, E. & Knight, S. C. (1998). Protection against ascending infection of the genital tract by Chlamydia trachomatis is associated with recruitment of major histocompatibility complex class II antigen-presenting cells into uterine tissue. Infection and Immunity 66, 3535–40.[Abstract/Free Full Text]

20 . Munday, P. E., Johnson, A. P., Thomas, B. J. & Taylor-Robinson, D. (1980). A comparison of the sensitivity of immunofluorescence and Giemsa for staining Chlamydia trachomatis inclusions in cycloheximide-treated McCoy cells. Journal of Clinical Pathology 33, 177–9.[Abstract]

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22 . Ito, J. I., Jr, Harrison, H. R., Alexander, E. R. & Billings, L. J. (1984). Establishment of genital tract infection in the CF-1 mouse by intravaginal inoculation of a human oculogenital isolate of Chlamydia trachomatis. Journal of Infectious Diseases 150, 577–82.[ISI][Medline]

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25 . Landers, D. V., Sung, M. L., Bottles, K. & Schachter, J. (1993). Does addition of anti-inflammatory agents to antimicrobial therapy reduce infertility after murine chlamydial salpingitis? Sexually Transmitted Diseases 20, 121–5.[ISI][Medline]

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Received 13 October 1999; returned 30 December 1999; revised 14 August 2000; accepted 5 December 2000





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PubMed
PubMed Citation
Articles by Sangaré, L.
Articles by Ravaoarinoro, M.