Chlamydiae and polymorphonuclear leukocytes: unlikely allies in the spread of chlamydial infection.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

Antichlamydial and antisperm antibodies in patients with chlamydial infections.

PROBLEM: Establishing the correlation between antichlamydial antibodies (AchAbs) and antisperm antibodies (ASA) in patients with chlamydial infections. METHOD OF STUDY: ASA were studied in sera from patients (142 with genital, 57 with ocular chlamydial infections) and control group (n = 100) by gelatin and tray agglutination test (TAT), sperm immobilization test (SIT) and ELISA. AchAbs were revealed by ELISA. RESULTS: A significantly higher (P < 0.05) ASA incidence was noted in patients with genital infections as compared with controls and patients with ophthalmologic infection (P < 0.0001), but not between patients with ophthalmologic infection and controls (P > 0.05). A significant correlation was established between AchAbs and ASA for TAT (r = 0.8214, P = 0.0341), SIT (r = 0.797, P = 0.032) and ELISA (r = 0.8519, P = 0.0313) in patients with genital infections only. CONCLUSIONS: The genital Chlamydia infection may play a role in the induction of ASA. This is probably a result of the inflammatory process, but not of cross-reactivity between sperm and Chlamydia trachomatis antigens.

Prevalence of Chlamydia trachomatis pooled serotypes BDE and FGK in children with chronic follicular conjunctivitis.

PURPOSE: To evaluate type-specific antichlamydial antibody IgG in children with chronic follicular conjunctivitis. METHODS: A total of 90 serum samples from juvenile patients with chronic follicular conjunctivitis were collected in the Southeastern Anatolian region of Turkey where trachoma is still endemic. These samples were investigated regarding Chlamydia trachomatis pooled serotype-specific IgG by using the micro-immunofluorescence test. A titer of 1/32 or higher was considered positive. RESULTS: Specific IgG seropositivity to Chlamydia trachomatis titer was found in 33 (36.1%) of the 90 subjects. A higher titer was observed frequently in the serotypes pooled in BDE (21 subjects), CHIJ (10 subjects), and FGK (2 subjects), respectively. CONCLUSIONS: The presence of antichlamydial antibodies in blood should always be interpreted in accordance with the history of the patient, the clinical picture, and the course of the disease. In the diagnosis of Chlamydia trachomatis infections in a patient with chronic follicular conjunctivitis, not only genus-specific antibodies but also the presence of the subspeciespecific antibodies should be investigated.

Detection of antichlamydial antibodies in tears: a diagnostic aid?

OBJECTIVES: The antibody response in sera and tears of 167 patients with suspected chlamydial conjunctivitis was compared with the antibody response in sera and tears of 45 patients with symptoms of urogenital chlamydial infection to discover whether and which type of antichlamydial antibody detected in tears may be of diagnostic help in chlamydial conjunctivitis. METHODS: Diagnosis was based on chlamydial antigen detection from the conjunctiva and urogenital tract, done by a direct immunofluorescence assay, McCoy cell culture, and polymerase chain reaction. Additionally, antichlamydial immunoglobulin A (IgA) and immunoglobulin G (IgG) were determined in sera and tears of all patients by an immunoperoxidase assay. RESULTS: Two hundred twelve patients were examined--167 with conjunctivitis, 45 with symptoms of urogenital chlamydial infection. Cell culture, direct immunofluorescence assay, and polymerase chain reaction brought identical results. Conjunctival specimens taken from 33 (20%) of the patients with conjunctivitis were Chlamydia antigen positive; specimens taken from 134 (80%) were negative. Antichlamydial antibodies were found in tears of 29 (88%) of the patients with conjunctivitis whose specimens were Chlamydia antigen positive. Fifty-four (40%) of the patients with conjunctivitis whose specimens were Chlamydia antigen negative had antichlamydial antibodies in their tears. Twenty-five patients with urethritis (56%) were Chlamydia antigen positive in urethral swabs; 20 (44%) were negative. Antichlamydial antibodies were found in the tears of eight (32%) of the Chlamydia antigen-positive and two (10%) of the Chlamydia antigen-negative patients with urethritis. In contrast to patients with conjunctivitis, findings for patients with urethritis always were negative for antichlamydial IgG in the tears. CONCLUSION: Antichlamydial antibodies in tears were seen significantly more often in patients with conjunctivitis than in those with urethritis (P < or = 0.05). Antichlamydial IgG was found only in tears of patients with conjunctivitis. Therefore, the authors conclude that the detection of antichlamydial IgG in the tears might be helpful for diagnosis in patients with suspected chlamydial conjunctivitis who have antigen-negative conjunctival swabs.

A 28 kDa major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp. and Chlamydia trachomatis-cloning and characterization of the C. psittaci mip-like gene.

Chlamydia psittaci strain guinea-pig inclusion conjunctivitis (GPIC) produces a self-limiting ocular infection of guinea-pigs, and this condition is a representative animal model of ocular chlamydial disease. Convalescent guinea-pigs, which are resistant to reinfection, produce antibodies to several elementary-body proteins, including an uncharacterized antigen of 28 kDa. Convalescent guinea-pig sera were used to identify, from a lambda expression library, two overlapping GPIC genomic clones that produced the 28 kDa antigenic protein. Nucleotide sequence analysis revealed that the gene coding for the 28 kDa protein was similar to the mip (macrophage infectivity potentiator) genes from Legionella pneumophila and Chlamydia trachomatis. The GPIC gene and its product were accordingly designated mip and Mip, respectively. Analysis of the regions flanking mip identified three tightly linked open reading frames coding for predicted products with sequence similarity to asparagine tRNA ligase (AspS), rRNA methylase (SpoU), and thioredoxin (TrxA). The arrangement of these genes in GPIC was aspS-mip-spoU-trxA. Sequence analysis of PCR products produced using genomic DNA from an ovine abortion strain of C. psittaci and from C. trachomatis strain LGV-434 demonstrated that the arrangement of mip, spoU and trxA is common among these chlamydiae.

Immunity to reinfection and immunization of male guinea pigs against urethral infection with the agent of guinea pig inclusion conjunctivitis.

BACKGROUND AND OBJECTIVES: There is little information available on immunity of males to chlamydial infection after recovering from a primary urethral infection or after immunization with chlamydial antigen. The guinea pig model of genital infection using the chlamydial agent of guinea pig inclusion conjunctivitis was utilized to evaluate the protective immune response in these circumstances. GOAL: To determine whether immunity to reinfection develops after a primary urethral infection and whether immunity develops as a result of immunization with inactivated chlamydiae. STUDY DESIGN: Groups of five male guinea pigs each in two separate experiments were infected in the urethra with chlamydiae and challenged with a fresh inoculum at either 30, 75, or 150 days after infection. The course of the challenge infection was then determined. Similarly, guinea pigs were immunized subcutaneously with ultraviolet-inactivated chlamydial elementary bodies and the course of urethral infection was determined when inoculated 2 weeks after immunization. RESULTS: Male guinea pigs were highly resistant to reinfection after a primary urethral infection. Animals that were immunized with inactivated chlamydiae generally became infected upon challenge, but the intensity of the infection was markedly reduced. CONCLUSIONS: Male guinea pigs possess protective mechanisms that make them more resistant to repeat chlamydial genital infection for a longer period of time than is seen in female guinea pigs. In addition, immunization of males with inactivated chlamydial antigen by a parenteral route is able to elicit a protective response to urethral infection with chlamydiae.

Murine model of ocular infection by a human biovar of Chlamydia trachomatis.

PURPOSE: A human biovar of Chlamydia trachomatis was used to develop a murine model of ocular chlamydial infection. The inbred mouse model will allow detailed immunologic studies during ocular infection, and use of a human biovar for infection may aid in identification of appropriate vaccine strategies against chlamydial infections. METHODS: BALB/c, C3H/HeN, and C57B1/6J mice (n = 5 to 10 mice/group) were topically infected in the conjunctiva with C serovar of C. trachomatis. The effects were tested of single and repeated infection with 5000 inclusion-forming units (IFU) in 5 microliters and different inoculum doses. Conjunctival surfaces of both eyes were swabbed for microbiologic signs (isolation culture or direct fluorescent antibody staining) of infection over 4 to 6 weeks. Conjunctivae were removed for histopathologic study, and lymphocytes from draining cervical lymph nodes and spleens were tested for chlamydia-specific proliferative responses. Serum was obtained from all mice and tested for anti-chlamydial antibodies. RESULTS: BALB/c and C3H/HeN mice developed dose-dependent microbiologic, histopathologic, and immunologic evidence of ocular infection. Eyes of mice were culture-positive from day 7 through at least day 21, with the peak of infection at days 10 to 14 after infection. Histopathologically, the development of conjunctival subepithelial mononuclear infiltration, exudate, and loss of goblet cells occurred within 1 week. Dose-dependent lymphoproliferative responses to whole chlamydial elementary bodies were observed; anti-chlamydial antibody was detected by immunoblotting only in infected mice. CONCLUSIONS: Several strains of inbred mice are susceptible to human chlamydial biovars and may provide a useful alternative disease model in which to study the immunopathogenesis of ocular chlamydial infection and test of vaccine candidates derived from clinically relevant human biovars.

Systemic immunization with Hsp60 alters the development of chlamydial ocular disease.

PURPOSE: To determine whether immunization with recombinant Hsp60 would exacerbate ocular pathology on challenge with viable chlamydial elementary bodies. METHODS: Guinea pigs were immunized either subcutaneously with recombinant Hsp60 or both subcutaneously with recombinant Hsp60 and ocularly with attenuated Salmonella typhimurium expressing the guinea pig inclusion conjunctivitis (GPIC) Hsp60 antigen. All animals were challenged in the conjunctiva with the agent of GPIC, and the degree of gross ocular pathology was determined. Immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody titers to Hsp60 were measured in ocular secretions as a measure of the degree of immunization. RESULTS: In primary and challenge GPIC infection, the degree of gross ocular pathology was lower in the immunized group. The presence of high IgA and IgG antibody titers to Hsp60 in tears suggested that the response may have been modified by the presence of blocking antibodies that either may have removed the antigen quickly or prevented interaction with sensitized T cells. In contrast to subcutaneous immunization, the combined immunization regimen, consisting of subcutaneous recombinant Hsp60 followed by ocular inoculation of the attenuated Salmonella, resulted in no difference in gross pathology after reinfection of guinea pigs with GPIC. CONCLUSIONS: These data indicated that the immunization with Hsp60 did not produce exacerbated disease on challenge with viable organisms; however, the data suggested that the route of administration, form of antigen, or both may be critical in the disease process.

Interpretation of Chlamydia trachomatis antibody response in chlamydial oculogenital infection.

OBJECTIVE: To study: (a) the chlamydial antibody response (to the D-K serovars) using the micro-immunofluorescence (micro-IF) test in the following groups: (I) chlamydial genital infection only, (II) chlamydial ocular infection only, (III) combined chlamydial ocular and genital infection (oculo-genital infection), (IV) chlamydial ocular infection with chlamydia-negative non-gonococcal urethritis, (V) adenovirus conjunctivitis (control group 1), (VI) male partners of group I-IV with no chlamydial oculogenital infection or non-gonococcal urethritis (control group 2) (b) the cross reactivity of antibodies in patients' sera between the three chlamydial species and within the serovars of C trachomatis in those with culture-positive chlamydial oculo-genital infection. SETTING: oculogenital (diagnostic) clinic at Moorfields Eye Hospital, London, UK. SUBJECTS: 209 consecutive patients attending the clinic with Chlamydia trachomatis oculogenital infection and 86 patients with adenovirus conjunctivitis (control group 1) and 55 male partners with no evidence of chlamydial oculogenital infection or non-gonococcal urethritis (control group 2). RESULTS: Of all the patients with proven chlamydial oculogenital infection, 10.5% (22/209) and 94% (197/209) had IgM and IgG antibodies respectively. The geometric mean IgG antibody titres (GMT) were 1:98, 1:123, 1:245 and 1:101 in groups I to IV respectively. The IgG GMT values seen in control groups 1 and 2 were 1:45 and 1:36 respectively. Only 2/86(2%) patients in group V (control group 1) had IgG chlamydial antibodies of 1:32 and 1:64, whilst only 1/55(1.8%) and 4/55(7.3%) of patients in group VI(control group 2) had chlamydial IgG antibody titres of > or = 1:256 and > or = 1:128 respectively. A four-fold rise or fall in IgG antibody titre occurred in 56%(107/192) of patient groups I-IV over 2-6 weeks. Low titre cross-reactive antibody responses against different chlamydial species and serovars were commonly seen; 71%(148/209) of all patients showed cross-reactivity with Chlamydia pneumoniae or psittaci species or both, whilst 92% (193/209) of patients showed some level of cross reactivity to other pooled serovars of C trachomatis (A-C and L 1-3). CONCLUSIONS: Serological diagnosis of chlamydial infection as evidenced by a positive IgM antibody response, high IgG titre (> or = 1:256) or > or = 4-fold rise or fall in IgG antibody titre was seen in 78%(163/209) of patients with culture-positive chlamydial oculogenital infection. Chlamydial IgG antibody titres of > or = 1:256 had a sensitivity of 42.6%, specificity of 98.2%, positive predictive value of 98.8% and a negative predictive value of 31% for chlamydial infection at any site, when considering groups I-IV and control group 2. In this study of 216 patients with conjunctivitis, a positive IgG antibody response (titre > or = 1:16) had a sensitivity of 98.5%, specificity of 97.7%, positive predictive value of 98.5% and a negative predictive value of 97.7%, for chlamydial conjunctivitis. Patients with dual chlamydial infection of conjunctiva and genital tract had a higher IgG GMT titre than those with ocular or genital infection alone: infection at a second site may produce an anamnestic response. Although the micro-IF test is a useful adjunct for the diagnosis of chlamydial infection, cross-reactivity between different chlamydial species and serovars is common. Chlamydial seroepidemiological studies should be interpreted with caution, as studies may attribute a serological response to a particular species or serovar in a setting where two or more are prevalent.

Partial protection against genital reinfection by immunization of guinea-pigs with isolated outer-membrane proteins of the chlamydial agent of guinea-pig inclusion conjunctivitis.

Because partial protection against reinfection is induced by experimental infection in the guinea-pig model of genital chlamydial infection, we sought to induce immunity by immunization. Female guinea-pigs were immunized subcutaneously with the major outer-membrane protein (MOMP) and the 61 kDa cysteine-rich outer-membrane protein (61 kDa) of the agent of guinea-pig inclusion conjunctivitis (GPIC) eluted from SDS-polyacrylamide gels (SDS-MOMP, SDS-61 kDa). Post-immunization sera and secretions contained antibodies to the SDS-purified proteins at high titre as measured by immunoblotting, whereas enzyme immunoassays (EIA) using whole elementary bodies as antigen showed significantly lower titres (P < 0.001). Likewise, blastogenic responses of peripheral mononuclear cells to GPIC elementary bodies were weak. Animals immunized with SDS-MOMP and SDS-61 kDa were fully susceptible to intravaginal challenge, as were control animals immunized with buffer without protein. Another group of animals were immunized with material prepared by extraction of chlamydial outer-membrane complexes with octyl beta-D-glucopyranoside (OGP) and dithiothreitol, which consisted largely of MOMP (OGP-MOMP). In contrast to the SDS-MOMP group, sera and secretions in the OGP-MOMP group showed high titres in EIA, and high titre antibodies to MOMP by immunoblot; however, most animals also had antibodies to 61 kDa, 72 kDa and ca. 84 kDa outer-membrane proteins. OGP-MOMP animals were partially protected against genital challenge as evidenced by low inclusion scores compared to control animals, although duration of infection measured by culture isolation was similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines.

Lymphocyte proliferative responses to chlamydial antigens in human chlamydial eye infections.

In order to study the relationship between cell-mediated immune responses to Chlamydia trachomatis and the pathogenesis of human chlamydial eye disease, we have measured the peripheral blood lymphocyte proliferative responses to whole chlamydial elementary bodies in 40 subjects with oculogenital chlamydial infection of varying severity, 13 subjects with genital chlamydial infections and 12 healthy seronegative controls. The mean stimulation index was significantly higher in those with oculogenital infections than in controls. There was a strong correlation between the response to C. trachomatis serotypes B and L1. We studied the relationship between proliferative responses and four clinical parameters: follicular conjunctivitis, papillary hypertrophy, corneal pannus and epithelial punctate keratitis, but were unable to show a significant association with any of these. Nor was there any association between proliferative response and serum antibody titre to C. trachomatis (pooled serotypes D-K), duration of disease or quantitative isolation of chlamydia from the conjunctiva. The depletion of CD8+ cells had no consistent effect on proliferative responses to serotype L1 in 13 subjects.

Immune response in chlamydial conjunctivitis among neonates and adults with special reference to tear IgA.

Tear and serum samples from 128 neonates and 122 adults with conjunctivitis were examined for antibodies to Chlamydia trachomatis with a micro-immunofluorescence (MIF) technique and the results compared to antigen detection by culture, enzyme immunoassay (EIA) (Chlamydiazyme, Abbott) and direct immunofluorescence (IF) (MicroTrak, Syva and Chlamyset, Orion) tests. From the 52 culture-positive adults, chlamydial IgA (titre greater than or equal to 1:8) antibodies were detected in 81% of the tear and in 62% of the serum samples, while 88% had such serum IgG antibodies (titre greater than or equal to 1:32). The persistence of chlamydial IgA in tears and sera was related to the duration of symptoms of conjunctivitis and the antibody titres declined after institution of antibiotic treatment. In the adults, the sensitivity of the MIF tear IgA antibody test (81%) was higher than that of the EIA (71%) and the IF (MicroTrak 71% and Chlamyset 62%) tests. The specificity for the MIF test was 79%, while it was 100% for the EIA and the two IF tests. Of the 67 chlamydia-infected neonates, 36% had chlamydial tear IgA antibodies, while such antibodies were only found in 15% of the sera. No neonates with chlamydia-negative conjunctivitis had chlamydial IgA antibodies. The MIF test may be used as a diagnostic method complementary to culture, EIA and IF tests in the diagnosis of chlamydial conjunctivitis in adults, but is not applicable in neonates.

Evaluation of diagnostic procedures in chlamydial eye infection.

Sixty-six patients suffering from suspected chlamydial conjunctivitis or keratoconjunctivitis underwent direct examination and culture procedures on specimens from conjunctival swabs and corneal scraping for detection of Chlamydia trachomatis. Patient blood samples were also screened for the presence of antichlamydia IgG and IgA antibodies. Eye positivity was found in 12 and 15% of patients by using culture isolation and direct examination, respectively. In 3 out of the 8 patients with culture-proven chlamydial eye infection, all of them female, C. trachomatis was also isolated from the genital tract.

Seroprevalence of Chlamydia trachomatis types in children with clinical trachoma in New Delhi, India.

Micro-immunofluorescence test with type specific antigens of ocular Chlamydial infection types A-D was used for serotyping the causative C. trachomatis serotypes in 32 inclusion positive school children suffering from trachoma. Single serotype associated infection was seen in six of the patients. The rest of them had antibodies against more than one serotype indicating simultaneous or previous infection by more than one serotype. By geometric mean titre determination, type C appeared to be the most prevalent serotype. However the highest antibody titres in individual cases were most frequently observed for serotype A. The use of geometric mean titre versus highest titre against specific serotype observed in individual cases for population survey is discussed. Isolation of organism for absolute determination of causative serotype from each patient is emphasised.

[Study of serological diagnosis in ocular chlamydial infection with IPAZYME].

We employed a indirect immunoperoxidase assay (IPAZYME in the evaluation of IgG and IgA antibody for Chlamydia trachomatis in serum samples from 218 patients such as cicatricial trachoma 55 cases, culture-positive adult inclusion conjunctivitis 48 cases and culture-negative conjunctivitis 47 cases, aged people, 68 cases as controls respectively. Frequency of positive IgG antibody showed a significant difference between adult inclusion conjunctivitis or cicatricial trachoma and controls. IgA antibody was positive in 25/48 (52%) in adult inclusion conjunctivitis and in 7/55 (12%) in cicatricial trachoma cases. Serum IgA antibody against Chlamydia trachomatis is of value to be an index of active ocular chlamydial inflammation. The correlation between severity of conjunctival cicatrix or corneal punnus and titers of IgG antibody was also significant.

Laboratory survey of Chlamydia trachomatis ocular infections.

The authors used immunofluorescence and immunoperoxidase tests to study a group of 101 patients with acute or chronic conjunctivitis, etiologically unrelated to conventional bacterial pathogens, and a control group of 30 healthy adults. Positive titers of IgG in serum and of IgA in lacrimal secretions against Chlamydia, detected by IPA, correlated with the identification of microorganisms by direct immunofluorescence. The use of both tests allows a precise evaluation of the stage of the infection and of its evolutive pattern.

Antichlamydial specificity of conjunctival lymphocytes during experimental ocular infection.

Antigen-specific responses to chlamydiae have been demonstrated with lymphocytes isolated from the conjunctiva after primary ocular infection and after topical challenge of chlamydia-immune cynomolgus monkeys with noninfectious, Triton X-100-extracted antigen. Proliferative to viable elementary bodies homologous to the original infecting serovar were demonstrated. In addition, in vitro production of antichlamydial antibody by conjunctival B cells was demonstrated by enzyme-linked immunosorbent assay of culture supernatants collected after 7 to 21 days of culture. These findings demonstrate that antigen-specific lymphocytes appear in the conjunctiva as a result of ocular chlamydial infection and that a noninfectious chlamydial antigen stimulates their reappearance or expansion at the site of original infection.

Guineapig inclusion conjunctivitis (GPIC) in a commercial colony.

Serological findings in a commercial colony of Hartley guineapigs revealed that about 70% had antibodies to Chlamydia psittaci as detected by the microimmunofluorescence method. Conjunctivitis was evident in 14% of 86 guineapigs examined. Chlamydial antigen was detected in conjunctival scrapings by a direct immunofluorescence test using Chlamydia-specific monoclonal antibody; however, C. psittaci was not demonstrated by other methods.

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.