Vaccination with the recombinant major outer membrane protein elicits long-term protection in mice against vaginal shedding and infertility following a Chlamydia muridarum genital challenge.

Implementation of a vaccine is likely the best approach to curtail Chlamydia trachomatis infections. The aim of this study was to determine the ability of a vaccine formulated with the recombinant major outer membrane protein (MOMP) and Th1 and Th2 adjuvants, delivered by combinations of systemic and mucosal routes, to elicit long-term protection in mice against a genital challenge with Chlamydia muridarum. As a negative control, mice were vaccinated with the recombinant Neisseria gonorrhoeae porinB, and the positive control group was immunized with C. muridarum live elementary bodies (EB). The four vaccines formulated with MOMP, as determined by the titers of IgG and neutralizing antibodies in serum, proliferative responses of T-cells stimulated with EB and levels of IFN-γ in the supernatants, elicited robust humoral and cellular immune responses over a 6-month period. Groups of mice were challenged genitally at 60, 120, or 180 days postimmunization. Based on the number of mice with positive vaginal cultures, number of positive cultures, length of time of shedding, and number of inclusion forming units recovered, MOMP vaccinated groups were significantly protected. To assess fertility, when the vaginal cultures became negative, female mice were caged with male mice and the outcome of the pregnancy evaluated. As determined by the number of pregnant mice and the number of embryos, two of the vaccine formulations protected mice up to 180 days postimmunization. To our knowledge this is the first subunit of Chlamydia vaccine that has elicited in mice significant long-term protection against a genital challenge.

Differences in infectivity and induction of infertility: a comparative study of Chlamydia trachomatis strains in the murine model.

Chlamydia trachomatis, although commonly asymptomatic in women, can result in chronic sequelae, such as pelvic inflammatory disease, ectopic pregnancy and infertility. However, a clear relationship has not been determined between specific serovars and the ability to lead to upper genital tract infection or infertility. Thus, in order to investigate differences in pathogenicity, C3H/HeN mice were infected in the ovarian bursa with the C. trachomatis strains D (UW-3/Cx), F (N.I.1), F (IC-Cal-3) and E (Bour). Differences both in the amount of vaginal shedding as well as subsequent fertility rates were observed between D (UW-3/Cx) and F (N.I.1) compared to F (IC-Cal-3) and E (Bour). Approximately 50% of the mice infected with the D (UW-3/Cx) and F (N.I.1) strains had vaginal shedding for up to 3-4 weeks after infection and fertility rates of less than 25%. Furthermore, mice inoculated with D (UW-3/Cx) and F (N.I.1) showed infertility even in the absence of medroxy progesterone acetate (MPA) treatment. In contrast, both MPA and non-MPA treated mice infected with F (IC-Cal-3) or E (Bour) did not show vaginal shedding and had fertility rates between 45 and 88%. Mutations in the CT135 open reading frame have been associated with virulence. However, no nucleotide differences were found among the four isolates for CT135. This murine model of infection with C. trachomatis may help with the understanding of disease pathology in humans and ultimately vaccine development.

Induction of protection against vaginal shedding and infertility by a recombinant Chlamydia vaccine.

A vaccine formulated with the Chlamydia muridarum recombinant major outer membrane protein, plus the adjuvants CpG and Montanide, was tested for its ability to protect BALB/c mice against a vaginal challenge. Mice were immunized by mucosal [intravaginal (i.vag.) plus colonic (col.), or intranasal (i.n.) plus sublingual (s.l.)], or systemic [intramuscular (i.m.) plus subcutaneous (s.c.)] routes, and a combination of mucosal priming and systemic boosting routes. A negative control group was vaccinated with the Neisseria gonorrhoeae porin B (Ng-rPorB) and a positive control group was inoculated in the nares with live Chlamydia. The strongest Chlamydia-specific humoral and cell-mediated immune responses were observed in the groups immunized by a combination of mucosal and systemic routes. Following the vaginal challenge, groups immunized using mucosal priming followed by systemic immunization had a significant decrease in the number of mice with positive vaginal cultures. For example, of the mice immunized i.n./s.l.+i.m./s.c., 24% had positive cultures during the six weeks of the experiment versus 69% for the negative control group immunized with Ng-rPorB (P<0.05). Similarly, the groups of mice primed by the mucosal routes and boosted by the systemic routes had significantly less IFU in the vaginal cultures when compared to the Ng-rPorB animals (P<0.05). These combination groups were also protected against infertility. The two groups had fertility rates of 100% (i.n./s.l.+i.m./s.c.) and 81% (i.vag./col.+i.m./s.c.) equivalent to the positive-control group immunized with live Chlamydia (100% fertility; P>0.05). These results show the importance of the schedule and routes of vaccination and represent the first study to show protection against infertility by a Chlamydia recombinant subunit vaccine.

Molecular detection of Rickettsia bellii, Rickettsia montanensis, and Rickettsia rickettsii in a Dermacentor variabilis tick from nature.

Rickettsial diseases, such as Rocky Mountain spotted fever, pose a public health threat because of humans' interrelationship with common arthropod species, such as ticks, mites, fleas, and lice. Individuals may come in contact with these vectors of disease on a fairly regular basis either directly or indirectly through pets or wildlife species, at home or in recreational areas. Therefore, it is of vital importance to know and understand the geographical distribution and prevalence of disease and rickettsial-infected arthropods. We analyzed Dermacentor variabilis ticks from nature found positive for Rickettsia sp. to determine the specific species present. Rickettsiae were detected through a 17-kDa surface antigen seminested PCR. Seminested PCR represents a sensitive and specific molecular technique in which to identify the presence of bacteria within arthropod hosts. Through sequence analysis of this gene, three Rickettsia species, Rickettsia bellii, Rickettsia montanensis, and Rickettsia rickettsii, were detected in a single tick specimen. Further molecular analyses of the 17-kDa surface antigen and the citrate synthase gene were also performed to support this finding. This is the first report of the detection of multiple Rickettsia species from a single D. variabilis tick in nature.

A rickettsial mixed infection in a Dermacentor variabilis tick from Ohio.

We present the first report of superinfection in a Dermacentor variabilis tick from nature. The single tick, collected in Ohio, was found infected with Rickettsia belli, R. nontanensis, and R. rickettsii.

Identification of Balamuthia mandrillaris by PCR assay using the mitochondrial 16S rRNA gene as a target.

Balamuthia mandrillaris is an opportunistic pathogen that causes granulomatous amebic meningoencephalitis in animals, including humans. Based on sequence analysis of mitochondrial small-subunit-rRNA genes, we developed primers that amplify a Balamuthia-specific PCR product. These primers will be useful for retrospective analyses of fixed tissues and possible identification of Balamuthia in vivo.

Genotyping of Balamuthia mandrillaris based on nuclear 18S and mitochondrial 16S rRNA genes.

Balamuthia mandrillaris is an opportunistically pathogenic ameba that causes fatal granulomatous amebic encephalitis (GAE) in vertebrates. Previous phylogenetic analyses that included the sequence of a single nuclear small subunit ribosomal RNA gene (18S or ssu rDNA) from this ameba suggested that Balamuthia is closely related to Acanthamoeba, another opportunistically pathogenic amebic genus, which includes multiple ssu rDNA genotypes. We tested whether this also is true for Balamuthia. The nuclear ssu rDNA from 4 isolates and the mitochondrial ssu rDNA from 7 isolates of B. mandrillaris have been sequenced. No variation in the nuclear rDNA sequences and low levels of variation in the mitochondrial rDNA were found. Both gene sequences were consistent with a single genotype for B. mandrillaris. The mitochondrial sequences of B. mandrillaris are unique and should be useful for development of genus-specific diagnostic probes for use with clinical, environmental, and archived specimens.