Prior genital tract infection with a murine or human biovar of Chlamydia trachomatis protects mice against heterotypic challenge infection.

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar of Chlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars of C. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murine C. trachomatis induces immunity against challenge with either of two human biovars.

Reactivation of chlamydial genital tract infection in mice.

A model was developed to study chlamydial quiescence in C3H/HeN (C3H) and C57BL/6N (C57) mice following genital tract infection by Chlamydia trachomatis MoPn. Reactivation of chlamydial shedding following immunosuppression indicated that viable MoPn remained in the genital tract for up to 4 or 5 weeks after the apparent clearance of a primary infection. Either cyclophosphamide or cortisone acetate treatment could cause reactivation, but cyclophosphamide was more effective. However, the frequency of reactivation by either drug diminished with time in both mouse strains. Progesterone treatment prior to infection of C57 mice greatly reduced the frequency of reactivation by cyclophosphamide and also correlated with the development of marked fluid accumulation and distension of the uterine horns in the vast majority of those animals. This pathology was apparent by 5 to 7 weeks postinfection and was consistently seen through 110 days postinfection. Neither of these phenomena was observed in C57 mice that had not been treated with progesterone or in C3H mice under any conditions tested. The infecting dose of MoPn did not clearly influence the frequency of reactivation in either inbred strain as defined by this model.

Dissemination of Chlamydia trachomatis chronic genital tract infection in gamma interferon gene knockout mice.

Mice (C57BL/6), treated with progesterone and infected intravaginally with the mouse pneumonitis strain of Chlamydia trachomatis (MoPn), acquired genital tract disease that ascended from the endocervix to the uterine horns, oviducts, and ovaries in a temporal fashion before the occurrence of spontaneous microbiological resolution by about 28 days after infection. Surprisingly, dissemination of MoPn in small numbers to draining lymph nodes, the peritoneal cavity, spleen, liver, kidneys, and lungs occurred in normal mice during the early stages of disease (7 to 14 days) in a portion of infected animals but resolved from these tissues, by microbiological criteria, prior to resolution of genital tract involvement. In contrast, gamma interferon knockout (IFN-gamma KO) mice exhibited dissemination of infection to a greater extent and for longer periods in a variety of tissues, and a portion of infected IFN-gamma KO mice failed to microbiologically resolve their genital tract disease. By comparison, C57BL/6 SCID mice uniformly failed to resolve their genital tract disease and exhibited high levels of dissemination to all tissues tested for extended (50-day) periods of times. Interestingly, although IFN-gamma KO mice failed to completely clear organisms from their genital tracts, they exhibited an attenuated infection indistinguishable from that of heterozygous littermates when challenged 112 days after primary infection. These data support a role for IFN-gamma in containing dissemination of MoPn from the genital tract to extragenital sites and in the microbiological resolution of infection. Data also indicate that IFN-gamma is not required for modulating reinfections, which normally follow a shorter and less dramatic course.

Protective efficacy of major outer membrane protein-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection.

The protective efficacy of immunoglobulin A (IgA) and IgG monoclonal antibodies (MAbs) specific for the major outer membrane protein of Chlamydia trachomatis MoPn was evaluated in a murine genital tract infection model. MAbs were delivered into serum and vaginal secretions of naive mice by using the backpack hybridoma tumor system, and protective efficacy was assessed over the first 8 days following challenge by quantitative determination of chlamydial recovery from cervicovaginal swabs, histopathological evaluation of genital tract tissue, and immunohistochemical detection of chlamydial inclusions. IgA and IgG significantly reduced the incidence of infection following vaginal challenge with 5 50% infectious doses, but such protection was overwhelmed by 10- and 100-fold higher challenge doses. Both MAbs also consistently reduced vaginal shedding from infected animals with all three challenge doses compared with the negative control MAb, although the magnitude of this effect was marginal. Blinded pathological evaluation of genital tract tissues at 8 days postinfection showed a significant reduction in the severity of the inflammatory infiltrate in oviduct tissue of infected IgA- and IgG-treated animals. Immunohistochemical detection of chlamydial inclusions revealed a marked reduction in the chlamydial burden of the oviduct epithelium; this finding is consistent with the reduced pathological changes observed in this tissue. These studies indicate that the presence of IgA or IgG MAbs specific to major outer membrane proteins has a marginal effect in preventing chlamydial colonization and shedding from the genital tract but has a more pronounced effect on ascending chlamydial infection and accompanying upper genital tract pathology.

A conjugation procedure for Bdellovibrio bacteriovorus and its use to identify DNA sequences that enhance the plaque-forming ability of a spontaneous host-independent mutant.

Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.

Identification of a Bdellovibrio bacteriovorus genetic locus, hit, associated with the host-independent phenotype.

Bdellovibrios invade and grow within the periplasmic space of suitable gram-negative bacteria. Wild-type bdellovibrios are obligately dependent on host cells for growth, but spontaneous host-independent (H-I) mutants that grow axenically on standard rich culture media can be isolated. Such mutants generally retain the ability to grow intraperiplasmically, although the plaques that they produce on lawns of host cells are smaller and more turbid than those produced by wild-type bdellovibrios. Here, we identify the first genetic locus associated with the H-I phenotype: hit (host interaction). We show that three individual H-I mutants suffered mutations at the hit locus and that recombination of wild-type hit sequences into the genomes of the H-I mutants greatly enhanced their plaquing ability. DNA sequence analysis localized the hit mutation in each of the H-I mutants to a 135-bp region of the genome. Mutations at hit may not fully account for the H-I phenotype, however, as recombination of wild-type hit sequences into the genomes of the H-I mutants had little effect on the axenic-growth phenotype of the mutants. Possible explanations for this result and potential roles for the hit locus are discussed.