Experimental chlamydial salpingitis in immunosuppressed guinea pigs infected in the genital tract with the agent of guinea pig inclusion conjunctivitis.

At necropsy indication of spread of infection to fallopian tubes was found in 25 of 41 (60%) female guinea pigs infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis and immunosuppressed with cyclophosphamide. Eighteen were examined histologically, and the diagnosis of acute salpingitis was confirmed in 10, based on inflammatory reaction, detection of guinea pig inclusion conjunctivitis in tissue, and formation of cysts (pyosalpinx and hydrosalpinx). Infection of fallopian tube tissue was confirmed by indirect immunofluorescence and electron microscopy. Infection of endometrial tissue and peritoneum was also recognized. Data suggested that the immunosuppression mediated by cyclophosphamide resulted in a prolonged genital tract infection and concomitant ascending infection leading to salpingitis.     

Effect of estradiol on chlamydial genital infection of female guinea pigs.

Female guinea pigs were treated daily with 1 mg of beta-estradiol-3-benzoate intramuscularly beginning 14 days before intravaginal inoculation with the chlamydial agent of guinea pig inclusion conjunctivitis and continuing during the course of the infection. Treatment with estradiol was found to markedly influence the course of genital infection with the chlamydial agent of guinea pig inclusion conjunctivitis, producing infections of greater intensity and longer duration than those in control animals. Moreover, pathogenesis was altered in that ascending infection was observed, resulting in endometritis, cystic salpingitis, and cystitis. Infection in the controls was limited to the cervix and vagina. Estradiol treatment increased the apparent number of infected cells in the cervix and vagina as detected by histopathology and immunofluorescent staining. Humoral and cell-mediated immune responses to the chlamydial agent of guinea pig inclusion conjunctivitis were comparable in estradiol-treated and untreated animals. These data indicate that hormonal manipulation may have profound effects on the course of chlamydial genital infections.     

Analysis of the humoral immune response to chlamydial genital infection in guinea pigs.

Studies using the guinea pig model of chlamydial genital infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) have shown that serum and local antibodies play a role both in the resolution of infection and in protection against reinfection. Thus, this model is suited for further exploration of immune mechanisms and for vaccine studies with chlamydial macromolecules. We have further characterized the model by assessing the antigen-specific antibody response to experimental genital infection by using immunoblotting to assay both genital secretions and serum. The GPIC agent was characterized by analysis of outer membrane proteins, which indicated that the GPIC agent possessed a major outer membrane protein (MOMP), with a molecular mass of 39 kilodaltons (kDa), and a 61-kDa protein, analogous to cysteine-rich 60-kDa proteins or doublets of Chlamydia trachomatis strains. As indicated by immunoblotting, most infected animals produced serum immunoglobulin G antibodies to MOMP, the 61-kDa proteins, an 84-kDa outer membrane protein, and lipopolysaccharide. Such serum antibodies persisted for at least 813 days after primary genital infection. Immunoglobulin A antibodies against the 61-kDa proteins, lipopolysaccharide, and MOMP, but not the 84-kDa protein, were detected in secretions. Animals challenged with GPIC 825 days after primary infection became infected again despite the presence of serum antibodies, but the period of chlamydial shedding was significantly shorter and less intense than in primary infections. Although the specific mechanism is not known, these data suggest that a long-lasting immune effect is capable of altering the course of infection late after primary infection. Correlation of the antigen-specific antibody response and other immune parameters with the duration and degree of protective immunity induced by infection or vaccination may be helpful in further understanding the nature of such protective immunity.     

Role of cell-mediated immunity in the resolution of secondary chlamydial genital infection in guinea pigs infected with the agent of guinea pig inclusion conjunctivitis.

Guinea pigs which have recovered from a genital infection with the agent of guinea pig inclusion conjunctivitis demonstrate strong immunity to reinfection for a short period of time but then become susceptible to reinfection. The secondary infection is markedly shortened in duration and decreased in intensity. Previous studies have indicated an important role for humoral immunity in resistance to and in recovery from reinfection. However, the contribution of cell-mediated immunity to immunity toward or recovery from a secondary infection is not clear. Guinea pigs were infected in the genital tract with guinea pig inclusion conjunctivitis and were challenged at either 30 or 75 days after the primary infection. Prior to challenge, one group of animals were injected with rabbit anti-guinea pig thymocyte serum (ATS) while control groups received either normal rabbit serum or no treatment. Treatment was continued daily for the course of the experiment. On day 30, ATS-treated guinea pigs had a slightly higher rate of reinfection, and generally the infection persisted longer than in controls. On day 75, all animals became reinfected upon challenge, but control animals resolved their infections in 3 to 9 days. In contrast, most ATS-treated animals remained infected throughout the course of the experiment. Although the animals became reinfected, the levels of chlamydiae were much lower than those observed during the primary infection. ATS treatment abrogated T-cell responses, but serum and secretory antibody responses remained normal. Histopathological examination revealed some decrease in mononuclear infiltration of endocervical and uterine tissues in ATS-treated animals. These data indicate that previously infected guinea pigs require both cell-mediated immunity and humoral immunity for resolution of a challenge infection.     

Inactivated Orf-virus shows disease modifying antiviral activity in a guinea pig model of genital herpesvirus infection

Background

Inactivated Orf virus (iORFV) has been used as a preventative as well as a therapeutic immunomodulator in veterinary medicine in different species. iORFV elicits strong effects on cytokine secretion in mice and human immune cells leading to an auto-regulated loop of initial up-regulation of inflammatory and Th1-related cytokines followed by Th2-related cytokines that attenuate immunopathology. The therapeutic potential of iORFV has been recognized in several models for difficult-to-treat disease areas such as chronic viral diseases, liver fibrosis or various forms of cancer.

Influence of the estrous cycle on the development of upper genital tract pathology as a result of chlamydial infection in the guinea pig model of pelvic inflammatory disease.

Guinea pigs were infected intravaginally with the chlamydial agent of guinea pig inclusion conjunctivitis at varying times during the estrous cycle. Genital tract tissues were collected 30 days after infection and processed for histopathological analysis. No difference was seen in the course of lower genital tract infection. However, a significantly greater percentage of tissues from animals infected on day 11 of the cycle were found to have chronic inflammation and fibrosis in the mesosalpinx compared to those from animals infected on day 6 or day 16. In addition, a significantly greater percentage of oviduct tissues from day 11-infected guinea pigs had marked tubal dilatation when compared to oviducts from day 6-or day 16-infected animals. The increased incidence of pathological changes was also noted in the endocervix, uterine fundus, and uterine horns but not the exocervix. These data indicate that the time of the estrous cycle and the corresponding hormonal influences may be an important influence on the development of upper genital tract disease.     

Susceptibility to reinfection after a primary chlamydial genital infection.

Female guinea pigs which had been infected genitally with the agent of guinea pig inclusion conjunctivitis were challenged at various times after infection with fresh inocula to determine the duration of immunity resulting from the primary infection. At 30 days after infection, most guinea pigs were resistant to reinfection, as indicated by the inability to isolate chlamydiae from cervical swabs. However, at 77, 155, and 294 days, all animals became reinfected, although the course of the infection was abbreviated and of lower intensity. When various immune parameters were examined, a decrease in antibodies in both serum (immunoglobulin G [( IgG]) and genital secretions (IgA, IgG) was observed after 30 days. A decrease in antibodies to the major outer membrane protein and an 84K component was noted in serum. In genital secretions, IgA antibodies to all major chlamydial components declined markedly after 30 days. Cell-mediated immunity as measured by proliferation of peripheral blood lymphocytes to guinea pig inclusion conjunctivitis antigen also was at a peak response 30 days after infection and decreased thereafter. Thus, loss of complete immunity could not be associated with a particular immune parameter. When genital secretions were examined 14 days after the challenge infection, IgA antibody levels to the lipopolysaccharide and 61K protein components had increased in intensity, whereas other antibodies were relatively low. In addition, complete immunity to a third infection was not increased in duration when animals had recovered from two previous genital infections.     

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.     

Characterization of lymphocyte response in the female genital tract during ascending Chlamydial genital infection in the guinea pig model

It is well known that pathology caused by chlamydial infection is associated closely with the host response to the organism and that both innate and adaptive host responses contribute to tissue damage. While it is likely that the organism itself initiates the acute inflammatory response by eliciting cytokine and chemokine production from the host cell, the adaptive response is the result of activation of the cell-mediated immune response. While there are several studies describing the nature of the pathologic response in primate, guinea pig, and murine models, there is less information on the kinetics of the CD4 and CD8 response following primary and challenge infections. In this study, we have quantified by flow cytometry the mononuclear cell response to genital infection with the agent of guinea pig inclusion conjunctivitis in the cervix, endometrium, and oviducts at various times following a primary intravaginal infection and after a challenge infection. Tissues from individual animals were assessed for cells expressing CD4, CD8, or Mac-1 and for B cells. Peak responses of each subset occurred 10 to 14 days after a primary infection. The number of Mac-1-expressing cells in each tissue site was found to be dependent on the size of the inoculating dose of chlamydiae. The responses of each cell type were generally stronger in the cervix than in the upper genital tract. In contrast to the murine model but consistent with the primate models, there were equal numbers of CD4 and CD8 cells present in the infiltrates. Twenty-one days after challenge infection, which was performed 50 days after the primary infection, there was a significant increase in the number of CD4, CD8, and B cells in the oviduct compared to the number of these cells at the same time after a primary infection, providing clear cellular evidence for a cell-mediated immune pathologic response.     

Pathogenesis of attenuated Junin virus in the guinea pig model

The purpose of this work was to elucidate the pathogenesis of attenuated Junin virus (JV) strains in the guinea pig model. Three groups of guinea pigs were infected by the IM route with 10(3) PFU of the XJC13 and XJO-attenuated strains or with the XJ pathogenic strain of JV, respectively. Viremia was studied at 3, 5, 7, 9, 12, and 14 days postinfection (pi) (a) in serum samples of all animals and in washed cells from XJC13-infected guinea pigs by conventional techniques and (b) in whole blood samples from XJC13 and XJO animals by coculture with Vero cells. Virus spread was studied at 14 days pi in brain, spleen, lymph nodes, and bone marrow by parallel suckling mouse inoculation or organ homogenates and coculture of cell suspensions with Vero cells. By coculture techniques of whole blood, an otherwise undetectable viremia was demonstrated for both attenuated strains throughout the observation period. In contrast, XJ viremia was easily detected by direct techniques, as has already been shown. Attenuated virus was also shown to reach brain and bone marrow when coculture methods were employed. But titers were always markedly lower than those of the pathogenic strain. The sustained viremia demonstrated in guinea pigs infected with either attenuated strain explains the mode of viral dissemination and accounts for viral rescue and antigen detection from some organs. These results suggest that attenuated strains do not differ greatly in their invasive capacity in guinea pigs, but later on viral replication is impaired. Therefore, these findings reveal potential risks and should be noted when developing human vaccines.     

Immunization against chlamydial genital infection in guinea pigs with UV-inactivated and viable chlamydiae administered by different routes.

Female guinea pigs were immunized with viable or UV light-inactivated chlamydiae (agent of guinea pig inclusion conjunctivitis), belonging to the species Chlamydia psittaci, by intravenous, subcutaneous, oral, or ocular routes. All animals were then inoculated vaginally with viable chlamydiae to determine the extent of protection against challenge infection induced by the various regimens. The course of genital infection was significantly reduced in intensity in all groups of animals except the unimmunized controls and those animals immunized orally with inactivated antigen. Guinea pigs immunized with viable antigen were more likely to develop resistance to challenge infection and, in general, had a significantly greater degree of protection than animals immunized with inactivated antigen. No one route seemed superior in producing a protective response. Animals in all groups demonstrating protection developed serum and secretion immunoglobulin G antibody responses to chlamydiae. Lymphocyte proliferative reactions to chlamydial antigen were variable among groups. Immunoblot analysis of serum and secretions indicated a wide range of antibody specificities, but most protected animals produced antibodies to the major outer membrane protein, lipopolysaccharide, and the 61-kilodalton protein. No definitive associations could be made between the increased ability of immunization with viable organisms to produce resistance to challenge infection and a particular immune parameter. These data indicate that viable chlamydiae given by various routes are able to induce a strong immune response which can provide resistance against reinfection in some cases or at least reduce the degree of infection to a greater degree than inactivated antigen. However, complete resistance to genital tract infection may be difficult to obtain and alternate immunizations strategies may have to be developed.     

Effect of antithymocyte serum on the course of chlamydial genital infection in female guinea pigs

The treatment of female guinea pigs, infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis, with rabbit anti-guinea pig thymocyte serum extended the course of the infection by 20 to 30 days. The rabbit anti-guinea pig thymocyte serum was shown to suppress delayed hypersensitivity responses to the guinea pig inclusion conjunctivitis agent and the contact allergen oxazolone. The appearance of antibody in genital secretions was delayed, but the infection persisted at low levels even when normal serum and secretory antibody titers were attained, indicating that cell-mediated immunity may play a role in the resolution of chlamydial genital infections.     

Chronic chlamydial genital infection in congenitally athymic nude mice.

Congenitally athymic nude mice and their heterozygous counterparts were inoculated intravaginally with the chlamydial agent of mouse pneumonitis, a Chlamydia trachomatis biovar. Heterozygous mice resolved their infections in 20 days, whereas nude mice developed chronic infections which lasted at least 265 days and did not resolve within the time course of the experiments. Heterozygous mice produced high levels of antibody in both serum and secretions in contrast to nude mice, which produced very low levels of antibody in serum alone.     

Inbred guinea pig model of intrauterine infection with cytomegalovirus.

Outbred guinea pigs have previously been utilized in an experimental model for the study of congenital infection with cytomegalovirus (CMV). Development of an inbred model of intrauterine CMV infection would allow analysis of the cells involved in CMV immunity, studies of transplacental CMV transfer, and investigation of the cellular immune factors that participate in intrauterine CMV infections. This study was therefore designed to assess the inbred guinea pig as a model for the study of congenital CMV infection. Intrauterine fetal and placental infection with CMV was demonstrated in inbred Strain 2 guinea pigs, and the maternal factors influencing transplacental transmission of CMV were evaluated. Infectious virus was recovered from placentas and offspring of mothers that experienced primary CMV infection during pregnancy, but not from placentas and offspring of mothers that were inoculated with CMV prior to pregnancy. However, histologic lesions consisting of focal necrosis and inflammation were seen in tissues of offspring from both groups of mothers. Inoculation of seronegative pregnant Strain 2 animals with low doses of virus (2.5 to 3.5 log10 TCID50) resulted in both placental and fetal CMV infection without significant maternal death. Infection of placentas and offspring occurred in utero regardless of the stage of pregnancy. In addition, infectious virus was detectable in fetal tissues at the time of maternal viremia but also later during the course of maternal infection, ie, 4 weeks after inoculation. These findings indicate that the inbred guinea pig model can be used to investigate the pathogenesis of intrauterine CMV infections.     

Resolution of chlamydial genital infection with antigen-specific T-lymphocyte lines.

To determine cell-mediated immune mechanisms involved in the resolution of chlamydial genital infection of mice, we utilized an established murine model in which it has been demonstrated that resolution of infection occurs independently of the antibody response. Splenic T lymphocytes were obtained from mice that had previously been immunized with viable elementary bodies of the mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. Antigen-reactive T lymphocytes were maintained and expanded in vitro by frequent restimulation with UV light-inactivated MoPn in the presence of antigen-presenting cells and recombinant interleukin-2 (rIL-2). Flow cytometry indicated that this cell line was at least 92% positive for the pan-specific T-cell marker Thy1.2. Stimulation of the cells in the presence of syngeneic antigen-presenting cells plus MoPn antigen and in the absence of exogenous IL-2 induced the cells to produce IL-2 activity in culture supernatants. Following adoptive transfer, this T-lymphocyte line was effective in inducing resolution of an ongoing MoPn genital infection in congenitally athymic nude mice which otherwise maintain chronic unresolved infections. The line was less efficient in resolving the infection after longer periods in culture. An additional T-lymphocyte line was derived from the spleens of athymic mice that had received the first line and had resolved the infection. These T cells were also capable of inducing resolution of the infection. Lastly, this cell line was treated with specific antibody and complement to delete either CD4+ or CD8+ T lymphocytes in an attempt to enrich for T-cell subpopulations prior to transfer into infected athymic mice. The anti-CD4-treated line was essentially depleted of CD4 cells, while the anti-CD8-treated line was only partially enriched for CD4 cells, with a large proportion of CD8 cells still present. Nude mice that received either of the treated T-cell lines or the parental cell line were capable of resolving the infection, although the line with increased numbers of CD4 cells was more efficient than either the parental line or the CD8 line.     

Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection.

The purpose of this investigation was to determine the relative roles of the humoral and cell-mediated immune responses in the resolution of chlamydial genital infection of mice and resistance to reinfection. To this end, female BALB/c mice were rendered B cell deficient by treatment with heterologous anti-immunoglobulin M (IgM) serum from birth. Controls were similarly treated with either normal serum or phosphate-buffered saline. Before inclusion in each experiment, anti-IgM-treated mice were screened for the absence of IgM in serum and for the presence of cell-mediated immune responses. In addition, spleen cells from anti-IgM-treated mice responded to concanavalin A and phytohemagglutinin but not to lipopolysaccharide. By these criteria, mice were designated B cell deficient. B-cell-deficient mice and controls were inoculated intravaginally with a suspension of mouse pneumonitis agent (MoPn), a Chlamydia trachomatis biovar. All B-cell-deficient mice resolved the infection. Additionally, no significant difference was seen in the course of the infection in B-cell-deficient mice when compared with controls. In contrast to control mice, B-cell-deficient mice displayed no detectable antibody responses to MoPn in serum or in genital secretions. However, both B-cell-deficient mice and controls developed delayed-type hypersensitivity and T-cell proliferative responses to MoPn. When challenged 53 days after primary infection, no significant difference was seen in the resistance of B-cell-deficient mice to reinfection when compared with that of the controls. These data indicate that cell-mediated immune mechanisms play an important role in the resolution of and resistance to chlamydial genital infection in this model.     

Protective role of serum antibody in immunity to chlamydial genital infection.

Female guinea pigs were injected intraperitoneally with pooled immunoglobulin derived from animals immunized to the chlamydial agent of guinea pig inclusion conjunctivitis. Genital infections in animals receiving pooled immunoglobulin from immune animals were markedly decreased with regard to the number of inclusions detected compared with control animals. These data indicated that serum-derived antibody was able to provide a degree of protection against a chlamydial genital tract infection.     

Pathogenesis of Lassa virus infection in guinea pigs.

A rodent model for human Lassa fever was developed which uses inbred (strain 13) and outbred (Hartley) guinea pigs. Strain 13 guinea pigs were uniformly susceptible to lethal infection by 2 or more PFU of Lassa virus strain Josiah. In contrast, no more than 30% of the Hartley guinea pigs died regardless of the virus dose. In lethally infected strain 13 guinea pigs, peak titers of virus (10(7) to 10(8) PFU) occurred in the spleen and lymph nodes at 8 to 9 days, in the salivary glands at 11 days, and in the lung at 14 to 16 days. Virus reached low titers (10(4) PFU) in the plasma and brain and intermediate titers in the liver, adrenal glands, kidney, pancreas, and heart. In moribund animals, the most consistent and severe histological lesion as an interstitial pneumonia. In contrast, the brain was only minimally involved. The immune response of lethally infected strain 13 guinea pigs, as measured by the indirect fluorescent antibody test, was detectable within 10 days of infection and was similar in timing and intensity to the fluorescent antibody test response of both lethally infected and surviving outbred animals. In contrast to the fluorescent antibody response, neutralizing antibody developed late in convalescence and was thus detected only in surviving outbred guinea pigs. The availability of a rodent model for human Lassa fever in uniformly susceptible strain 13 guinea pigs should facilitate detailed pathophysiological studies and efficacy testing of antiviral drugs, candidate vaccines, and immunotherapy regimens to develop control methods for this life-threatening disease in humans.     

Humoral immunity in the resolution of genital infection in female guinea pigs infected with the agent of guinea pig inclusion conjunctivitis.

Female guinea pigs infected in the genital tract with the chlamydial agent of guinea pig inclusion conjunctivitis were selectively immunosuppressed by varying regimens of cyclophosphamide (Cy) treatment. Temporary suppression of both humoral and cell-mediated immunity by daily treatment of Cy (25 mg/kg) for 13 days resulted in a prolonged infection, whereas daily treatment for the duration of the experiment totally prevented the development of humoral and cell-mediated responses and produced an intense and prolonged infection which did not resolve. When humoral immunity alone was suppressed by treatment with Cy (250 and 150 mg/kg) at 9-day intervals, the infection again did not resolve. Treatment with 100 mg of Cy per kg at 9-day intervals resulted in an extended infection which resolved concomitantly with the development of antibody to guinea pig inclusion conjunctivitis. These data indicate that humoral immunity is essential for the recovery of female guinea pigs from guinea pig inclusion conjunctivitis genital infection. A market weight loss was observed which could not be attributed to Cy treatment alone. Edematous and ulcerative changes of the external genitalia were also noted.