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Infection and Immunity, December 2000, p. 7209-7211, Vol. 68, No. 12
0019-9567/00/$04.00+0

LETTERS TO THE EDITOR

Murine Models of Chlamydia trachomatis Genital Tract Infection: Use of Mouse Pneumonitis Strain versus Human Strains

	LETTER

Morrison and Morrison (11) analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice. They provided an important foundation from which we can study the effects of experimentally induced perturbations in the systemic immune response on the development of local genital tract immunity. The observed cytokine responses were obtained using the Chlamydia trachomatis strain mouse pneumonitis (MoPn).

The MoPn agent, the mouse biovar of C. trachomatis, is much more virulent in mice than human strains (14), causing acute pathology throughout the genital tract and characteristically resulting in systemic infection (12). In addition, the developmental cycle of MoPn is more rapid, its duration being approximately half that of human strains, and the strain is more prolific. However, in humans the urogenital C. trachomatis serovars cause no systemic infections and upper genital tract progression, followed by pathology, usually resulting from multiple infections, is only seen in a small percentage of women (3, 13).

We have shown an important role for gamma interferon (IFN-) in the early clearance of chlamydia from the genital tract (4). When we infected IFN-^/ mice with MoPn we observed no significant differences in the clearance of chlamydiae between infected and control mice. However, when we infected animals with the human genital isolate (5, 6, 9) serovar D, we saw striking differences in infection kinetics between IFN-^/ and control animals beginning as early as day 4 and continuing throughout the observation period (70 days). Thus, the MoPn strain and the human C. trachomatis strains differ in response to at least one important cytokine and most likely will be found to differ in response to other cytokines.

Recently, a novel high-resolution technique has been introduced for whole-genome analysis: amplified fragment-length polymorphism (AFLP). This technique has proven its usefulness as a tool in bacterial taxonomy and epidemiology. It was shown that human isolates were clearly different from the MoPn strain of C. trachomatis (10). Both the AFLP and DNA-DNA hybridization demonstrate that the MoPn strain should be considered a separate Chlamydia species. Indeed, Everett et al. (2) proposed a reclassification of the order Chlamydiales and its current taxa based on phylogenetic analyses of the 16S and 23S rRNA genes with corroborating genetic and phenotypic information. This interesting proposal was the first topic on the program of the 4th European Chlamydia Congress in August this year (Helsinki, Finland). One of the proposed new species in this reclassification is Chlamydia muridarum sp. nov. Thus, it is the considered opinion of bacterial taxonomists that the MoPn strain differs considerably from human C. trachomatis strains.

Finally, although both in animal models and in humans specific major histocompatibility complex class I alleles and HLA types were found to be independently associated with pelvic inflammatory disease and tubal infertility (1, 7, 8), repeated pelvic inflammatory disease periods and repeated C. trachomatis infections increase the risk for tubal infertility significantly (3, 13). This is most likely based on enhanced autoimmune reactivities, including heat shock protein 60 autoimmunity. This demonstrates that proteins expressed by the microorganism are the determinants for the immunologic response of the host, emphasizing the importance of the choice of the chlamydial strain to be used as a model for genital tract infection.

Thus, it seems to be worthwhile and potentially clinically more relevant to investigate the cytokine profiles in the murine genital tract during the course of infection using human C. trachomatis strains and to compare those cytokine responses to the results obtained with the MoPn agent. Through this comparison, a basis for the selection of the most appropriate strain for use in murine models of human genital tract infection can be made.

	FOOTNOTES

^* Phone: (626) 359-8111

Fax: (626) 301-8954

E-mail: jlyons{at}coh.org

	REFERENCES

1.	Cohen, C. R., S. S. Sinei, E. A. Bukusi, J. J. Bwayo, K. K. Holmes, and R. C. Brunham. 2000. Human leukocyte antigen class II DQ alleles associated with Chlamydia trachomatis tubal infertility. Obstet. Gynecol. 95:72-77[Abstract/Free Full Text].
2.	Everett, K. D., R. M. Bush, and A. A. Andersen. 1999. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of the organisms. Int. J. Syst. Bacteriol. 49:415-440[Abstract/Free Full Text].
3.	Hillis, S. D., L. M. Owens, P. A. Marchbanks, L. E. Amsterdam, and W. R. MacKenzie. 1997. Recurrent chlamydial infections increase the risk of hospitalisation for ectopic pregnancy and pelvic inflammatory disease. Am. J. Obstet. Gynecol. 1:103-106.
4.	Ito, J. I., and J. M. Lyons. 1999. Role of gamma interferon in controlling murine chlamydial genital tract infection. Infect. Immun. 67:5518-5521[Abstract/Free Full Text].
5.	Ito, J. I., J. M. Lyons, and L. P. Airo-Brown. 1990. Variation in virulence among oculogenital serovars of Chlamydia trachomatis in experimental genital tract infection. Infect. Immun. 58:2021-2023[Abstract/Free Full Text].
6.	Ito, J. I., H. R. Harrison, E. R. Alexander, and L. J. Billings. 1984. Establishment of genital tract infection in the CF-1 mouse by intravaginal inoculation of a human oculogenital isolate of Chlamydia trachomatis. J. Infect. Dis. 150:577-582[Medline].
7.	Kimani, J., I. W. Maclean, J. J. Bwayo, K. MacDonald, J. Oyugi, G. M. Maitha, R. W. Peeling, M. Cheang, N. J. Nagelkerke, F. A. Plummer, and R. C. Brunham. 1996. Risk factors for Chlamydia trachomatis pelvic inflammatory disease among sex workers in Nairobi, Kenya. J. Infect. Dis. 173:1437-1444[Medline].
8.	Lichtenwalner, A. B., D. L. Patton, Y. T. Cosgrove Sweeney, L. K. Gaur, and W. E. Stamm. 1997. Evidence of genetic susceptibility to Chlamydia trachomatis-induced pelvic inflammatory disease in the pig-tailed macaque. Infect. Immun. 65:2250-2253[Abstract].
9.	Lyons, J. M., and J. I. Ito. 1995. Reducing the risk of Chlamydia trachomatis genital tract infection by evaluating the prophylactic potential of vaginally applied chemicals. Clin. Infect. Dis. 21(Suppl. 2):S174-S177.
10.	Meijer, A., S. A. Morré, A. J. C. van den Brule, P. H. M. Savelkoul, and J. M. Ossewaarde. 1999. Genomic relatedness of Chlamydia isolates determined by amplified fragment length polymorphism analysis. J. Bacteriol. 181:4469-4475[Abstract/Free Full Text].
11.	Morrison, S. G., and R. P. Morrison. 2000. In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection. Infect. Immun. 68:2870-2879[Abstract/Free Full Text].
12.	Nigg, C. 1942. Unidentified virus which produces pneumoniae and systemic infection in mice. Science 95:49[Free Full Text].
13.	Weström, L., R. Joesoef, G. Reynolds, A. Hadgu, and S. E. Tompson. 1992. Pelvic inflammatory disease and fertility. A cohort study of 1844 women with laparoscopically verified disease and 657 control women with normal laparoscopic results. Sex. Transm. Dis. 19:185-192[Medline].
14.	Williams, D. M., J. Schachter, D. J. Drutz, and C. V. Sumaya. 1981. Pneumonia due to Chlamydia trachomatis in the immunocompromised (nude) mouse. J. Infect. Dis. 143:238-241[Medline].

					Servaas A. Morré Section of Molecular Pathology Department of Pathology University Hospital Vrije Universiteit Amsterdam, The Netherlands
					Joseph M. Lyons* James I. Ito Jr. Department of Infectious Diseases City of Hope National Medical Center 1500 E. Duarte Rd. Duarte, California 91010

	AUTHOR'S REPLY

Morré, Lyons, and Ito, Jr., raise several issues that relate to the legitimacy of the Chlamydia trachomatis mouse pneumonitis (MoPn) murine model of genital tract infection (7). The MoPn model is far more comparable to human chlamydial genital tract infection than alluded to by Morré et al. Vaginal inoculation of MoPn results in an infection that naturally ascends from the lower genital tract (vagina and cervix) to infect upper genital tract tissues (uterine horns and oviducts) (1, 6). The inflammatory response and postinfection sequelae that develop following MoPn infection, such as tubal occlusion, hydrosalpinx, and infertility, are characteristic of chlamydial genital tract infection in women (16). Conversely, vaginal inoculation of mice with human biovars of C. trachomatis produces a mild infection of the lower genital tract, which is characterized by the shedding of fewer chlamydiae for a shorter duration (9). Human biovars cause postinfection sequelae in mice only when high doses are inoculated directly into the uterine horns or ovarian bursa (14, 15). Systemic MoPn infection per se does not develop following vaginal inoculation. Occasionally, low numbers of MoPn are isolated from systemic sites (2; R. P. Morrison, unpublished data), but pathological changes are only observed in genital tract tissues. Perhaps vaginal infection of mice with human biovars also results in low levels of systemic chlamydiae, but such information is not presently available.

Comparing the incidence of upper genital tract pathology in the MoPn model to that of natural infection of women is potentially problematic. A plethora of confounding variables, such as host genetic influences, antibiotic treatment early during the course of infection, etc., profoundly influence the progression of chlamydial disease in experimental models (3, 12, 13) and perhaps similarly affect disease progression in humans. Nevertheless, the pathology of the upper genital tract of MoPn infected mice is comparable to that of women with post-chlamydial infection sequelae (6, 7, 16). The assertion that tubal infertility or other postinfection sequelae result from "enhanced autoimmune reactivities, including heat shock protein 60 autoimmunity," is scientifically unsubstantiated. There exists no direct evidence that autoimmune responses contribute to the pathogenesis of chlamydial genital tract disease.

Amplified fragment-length polymorphism provides only a glimpse of the relative genetic relatedness among chlamydial isolates (5). On the other hand, the genomic sequence analyses of MoPn and serovar D conclusively reveal striking similarities in gene content and the order of genes within the chromosomes (10, 11). MoPn and human biovars clearly display differences in host range, but that does not a priori dismiss the MoPn infection model as an inappropriate correlate of human infection. In fact, the authors of the above-mentioned genomic study state that "the extraordinary conservation offers encouragement for investigators using the MoPn biovar to model disease caused by human biovars of C. trachomatis."

Differences in the response of MoPn and human biovars of C. trachomatis to gamma interferon is an important consideration, and perhaps a possible shortcoming of the MoPn model of genital tract infection. The role of gamma interferon in both MoPn and human biovar models of infection has been extensively evaluated by several research groups not cited by Morré et al. (2, 4, 8, 9). Particularly applicable is the study by Perry et al. (9), who quite eloquently address in their discussion how differences in sensitivity to gamma interferon may influence the pathogenesis of murine and human chlamydial infection.

Although not flawless, MoPn genital tract infection of mice is a reasonable model for the study of Chlamydia-host interactions. It is important to recognize, however, that regardless of the model, the pathogenesis of human chlamydial infections will only be understood through a more thorough investigation of chlamydial infection in its natural human host.

	REFERENCES

1.	Barron, A. L., H. J. White, R. G. Rank, B. L. Soloff, and E. B. Moses. 1981. A new animal model for the study of Chlamydia trachomatis genital infections: infection of mice with the agent of mouse pneumonitis. J. Infect. Dis. 143:63-66[Medline].
2.	Cotter, T. W., K. H. Ramsey, G. S. Miranpuri, C. E. Poulsen, and G. I. Byrne. 1997. Dissemination of Chlamydia trachomatis chronic genital tract infection in gamma interferon gene knockout mice. Infect. Immun. 65:2145-2152[Abstract].
3.	de la Maza, L. M., S. Pal, A. Khamesipour, and E. M. Peterson. 1994. Intravaginal inoculation of mice with the Chlamydia trachomatis mouse pneumonitis biovar results in infertility. Infect. Immun. 62:2094-2097[Abstract/Free Full Text].
4.	Johansson, M., K. Schon, M. Ward, and N. Lycke. 1997. Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor-deficient mice despite a strong local immunoglobulin A response. Infect. Immun. 65:1032-1044[Abstract].
5.	Meijer, A., S. A. Morré, A. J. C. van den Brule, P. H. M. Savelkoul, and J. M. Ossewaarde. 1999. Genomic relatedness of Chlamydia isolates determined by amplified fragment length polymorphism analysis. J. Bacteriol. 181:4469-4475.
6.	Morrison, R. P., K. Feilzer, and D. B. Tumas. 1995. Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection. Infect. Immun. 63:4661-4668[Abstract].
7.	Morrison, S. G., and R. P. Morrison. 2000. In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection. Infect. Immun. 68:2870-2879.
8.	Perry, L. L., K. Feilzer, and H. D. Caldwell. 1997. Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN--dependent and -independent pathways. J. Immunol. 158:3344-3352[Abstract].
9.	Perry, L. L., H. Su, K. Feilzer, R. Messer, S. Hughes, W. Whitmire, and H. D. Caldwell. 1999. Differential sensitivity of distinct Chlamydia trachomatis isolates to IFN--mediated inhibition. J. Immunol. 162:3541-3548[Abstract/Free Full Text].
10.	Read, T. D., R. C. Brunham, C. Shen, S. R. Gill, J. F. Heidelberg, O. White, E. K. Hickey, J. Peterson, T. Utterback, K. Berry, S. Bass, K. Linher, J. Weidman, H. Khouri, B. Craven, C. Bowman, R. Dodson, M. Gwinn, W. Nelson, R. DeBoy, J. Kolonay, G. McClarty, S. L. Salzberg, J. Eisen, and C. M. Fraser. 2000. Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res. 28:1397-1406[Abstract/Free Full Text].
11.	Stephens, R. S., S. Kalman, C. Lammel, J. Fan, R. Marathe, L. Aravind, W. Mitchell, L. Olinger, R. Tatusov, Q. Zhao, E. V. Koonin, and R. W. Davis. 1998. Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282:754-759[Abstract/Free Full Text].
12.	Su, H., R. P. Morrison, R. Messer, W. Whitmire, S. Hughes, and H. D. Caldwell. 1999. The effect of doxycycline treatment on the development of protective immunity in a murine model of chlamydial genital infection. J. Infect. Dis. 180:1252-1258[CrossRef][Medline].
13.	Swenson, C. E., M. L. Sung, and J. Schachter. 1986. The effect of tetracycline treatment on chlamydial salpingitis and subsequent fertility in the mouse. Sex. Transm. Dis. 13:40-44[Medline].
14.	Tuffrey, M., P. Falder, J. Gale, R. Quinn, and D. Taylor-Robinson. 1986. Infertility in mice infected genitally with a human strain of Chlamydia trachomatis. J. Reprod. Fertil. 78:251-260[CrossRef][Medline].
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