Murine Model of Ocular Infection by a Human Biovar of Chlamydia trachomatis

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1 Murine Model of Ocular Infection by a Human Biovar of Chlamydia trachomatis Judith A. Whittum-Hudson, Terrence P. O'Brien, and Robert A. Prendergast 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 1 mice/group) were topically infected in the conjunctiva with C serovar of C. trachomatis. The effects were tested of single and repeated infection with 5 inclusion-forming units (IFU) in 5 [A 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 1 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 tests of vaccine candidates derived from clinically relevant human biovars. Invest Ophthalmol Vis Sci. 1995;36: \jhlamydia trachomatis infections of the eye are the leading cause of preventable infectious blindness in the world. 1 " 3 Immunologic studies of experimental ocular chlamydial infections in animals have been hampered by a lack of a good inbred animal model susceptible to the human biovars. Several animal models using C. psittati or the mouse pneumonitis strain (MoPn) of C. trachomatis have contributed important information regarding the pathogenesis of chlamydial infection.' 1 " l(> Nevertheless, it would be helpful to have The Wilmer Oplilhalmolagiml Institute, Johns Hopkins University, Baltimore, Maryland. Supported in pan l/y giants from the Edna Mr.Connell Clark Foundation (New York, Neiv York) and l/y United States Public. Health Seivice grant EY3324. Submitted for publication June 22, 1994; mused April 1, 1995; accepted April 25, hoprietary interest category: N. Ref)rint requests: Judith Whitlum-Hudson, Ocular Immunology Uiboratories, The Wilmer Institute, 457 Wiliner-Woods, 6 N. Wolfe Street, Baltimore, Ml) an inbred animal model of ocular infection induced by a human biovar of C. trachomatis for detailed immunologic studies. Subhuman primate experimental models of ocular C. trachomatis infection induced by human chlamydial biovars have provided useful information regarding the immunopathogenesis of disease; the disease induced in these primates is similar to that observed in humans. 2 ' 3 ' 17 " 32 However, drawbacks of the various monkey models include their outbred background, variability in responses to infection or immunization, and enormous cost. Inbred mice provide a desirable alternative for studies of immunopathogenesis and vaccine development against many infectious organisms, including C. trachomatis. 6 ' 44 The immune responses of inbred mice, including mucosal immunity, are well characterized, and many reagents are available for the detection of lymphocyte markers, cytokines, and immunoglobu Investigative Ophthalmology & Visual Science, September 199.5, Vol. 3fi, No. 1 Copyright Association for Research in Vision and Ophthalmology Downloaded From: on 11/12/218

2 Ocular Chlamydial Infection in Mice 1977 lins. :V7 ' ir> ll The lack of such knowledge in cynomolgus monkeys and a lack of appropriate immunologic reagents for guinea pigs have been impediments to interpretation of some approaches to protective immunization against ocular chlamydial infection. With the advent of new generations of vaccine candidates and vaccine carriers, a murine model of ocular disease induced by a human biovar of C. trachomatis would be valuable. In the experiments reported here, we demonstrate a reproducible murine model of ocular C. trachomatis infection induced by a human ocular biovar (serovar C; TW/3). MATERIALS AND METHODS Mice Young adult female BALB/c (H-2 fl ), C3H/HeN (H- 2 k ), and C57B1/6J (H-2 h ) mice were obtained from Charles River Breeders (Wilmington, MA) or Jackson Labs (Bar Harbor, ME) and maintained in our animal facilities. Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the National Institutes of Health Guidelines for the Use of Experimental Animals. All ocular procedures were performed on anesthetized mice (.66 mg/kg ketamine hydrochloride and promazine [Aveco, Fort Dodge, IA] or ether). Inoculum Preparation Stocks of C. trachomatis (serovar C; TW/3) were grown in HeLa cells using standard methods. 3 Purified elementary bodies (EB) were obtained by density gradient centrifugation in Percoll (Pharmacia, Piscataway, NJ). Stocks were titrated on McCoy cells to determine the infectious titer of inclusion-forming units (IFU)/ ml. A freshly thawed EB aliquot was diluted in cold sucrose-phosphate-glutamic acid buffer, and the inoculum was kept on ice during inoculations. Deeply anesthetized mice were challenged with 5 IFU/eye in 5 fa by topical application to the upper fornix with a micropipettor using new sterile pipette tips for each eye. Approximately half of the inoculum remained under the lid when the eyes were closed. Immediately after completion of eye inoculations, the remaining inoculum was retitrated on McCoy monolayers to confirm the challenge dosage. In some experiments, separate groups of mice (4 to 1 each) were infected with additional concentrations of EB ranging from 3 to 1 (> IFU/eye, or they received repeated infectious challenges of 1 or 5 IFU. Number of inoculations, doses, and times of inoculation are noted under specific experimental results. Microbiologic Assessment of Infection Conjunctivae of both eyes were swabbed individually to test for the presence of chlamydia by isolation culture or direct fluorescent antibody assay using standard methods. 2 ' 1 " 27 ' 5 Unless otherwise noted, both eyes were assayed by the same method. At various times after infectious ocular challenge, conjunctival surfaces were swabbed with sterile dacron urethral swabs (Pur- Fybr, Munster, IL) for culture isolation. Individual swabs were placed in.5 ml medium (Eagle's minimal essential medium containing 1% fetal bovine serum, 2 mm L-glutamine, 5.9% glucose, 1 mm HEPES, vancomycin hydrochloride [1 /xg/ml], gentamicin sulfate (1 /xg/ml), and mycostatin [1 /ig/ml]) 2 ' 1 in 5-ml plastic tubes containing two sterile 4-mm glass beads. Samples were vortexed for 2 minutes, and 1 fx\ was added to McCoy cell monolayers in two wells each of two separate flatbottom microtiter plates. McCoy cells were pretreated with DEAE-Dextran (Pharmacia, Piscataway, NJ) for 15 to 2 minutes before the addition of samples. After centrifugation (lloogfor 6 minutes at room temperature) and incubation (37 C for 6 minutes), complete medium containing 1 /ig/ml cycloheximide was added to each well. After 48 to 72 hours, one set of plates was washed, and monolayers were stained with the Culture Confirmation Reagent (Syva, San Jose, CA). Inverted plates were read under a X4 objective by epifluorescence microscopy (Carl Zeiss, Thornwood, NY). Wells were graded on a to 4+ scale for total inclusions (inclusion-forming units; IFU) in each well (1 +, 1 to 9 inclusions; 2+, 1 to 2 inclusions; 3+, >2 1 to 1 inclusions/15 field count; 4+, >2 inclusions, >1 inclusions/15 field count). The total IFU yield for each sample was determined by enumeration of total inclusions in two wells, each of which received 1 (A of sample, and was expressed as IFU/ml. The infectious yield was calculated from first-passage values and represented sampling of the organism accessible to swabs, not the total replicating organism within the conjunctivae at any one time. All samples were subjected to second passage from the remaining two wells/sample to detect more weakly positive samples or to confirm first-passage positive cultures. For the second passage, McCoy cell monolayers containing additional duplicates of samples cultured in the first passage were scraped and transferred to a new plate of McCoy indicator cells for an additional 48 to 72 hours. Those monolayers were fixed and stained as for first-passage cells, and inclusions were counted in a masked fashion. Results were expressed either as the mean total IFU isolated per eye or per mouse (sum of results for both eyes) or as the percent of total eyes or mice that were culture positive at designated time points. For direct fluorescent antibody staining of EB, conjunctival smears from individual eyes were applied to Teflon-coated slides with sterile dacron urethral swabs. Slides were immediately fixed in absolute MeOH and stored at 2 C until staining with the Direct Reagent (Syva) according to Downloaded From: on 11/12/218

3 1978 Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 1 the manufacturer's instructions. The detecting reagents for both assays were monoclonal mouse antimajor outer membrane protein (MOMP) of C. trachomatis. Slides were read under X4 final magnification by fluorescence microscopy. Smears were graded for EB using a to 4+ DFA scale, where = 1 to 5 EB; 1+ = 6 to 5 EB; 2+ = 5 to 1 EB; 3+ = >1 EB 24 ' 5 and total EB counts were obtained for smears containing at least 2 epithelial cells. Uninfected control mice kept in separate cages were examined and swabbed at the same intervals as infected mice, and they served as negative control samples included in each assay. Equivalent results were obtained in all assays for controls receiving mock inoculum (5 /Ltl sucrose -phosphate-glutamic acid buffer) or no inoculum. Clinical and Histopathologic Evaluation of Ocular Disease Mice were examined by slit lamp or binocular microscopy (XlO to X6) using blunt, nontoothed forceps to determine whether ocular inflammation was induced by infectious challenge with the human biovar of C. trachomatis. Uninfected mice also were examined to control for the trauma of lid manipulations. A scoring system similar to that used for humans and monkeys was modified for the mouse system. 1 " 3 ' 2226 Both superior and inferior conjunctivae were scored for discharge, lid thickening (chemosis), and lid redness (hyperemia) on a to 3+ scale. Because no follicles, a hallmark of trachoma, 12 were induced under the experimental conditions used, this clinical feature was not included in the scoring system. Histologic examination of conjunctivae was performed to correlate ocular pathology with exposure to infectious chlamydia and the microbiologic criteria for infection. At various times during infection, mice were killed and conjunctival surfaces from both lids of both eyes were removed, frozen-embedded, and stored at 85 C until sectioning. Frozen serial sections (8 to 1 /itm) were cut on a cryostat, placed on gelatincoated slides, and fixed for 1 seconds in cold acetone. Multiple slides, each containing four to eight sections, were stored at 2 C until immunohistochemical staining; at least one slide was stained with Diff-Quick (Baxter, McGaw Park, IL) and graded for histopathology using the scoring system shown in Table 1. The total histopathologic disease score (THDS) was obtained by grading several features of conjunctivae on a scale of to 4+, including stromal infiltration, exudate overlying the epithelium, and goblet cell loss (Table 1). Values were obtained by evaluating at least two to four serial sections from each conjunctival specimen for the histologic features. Serologic Assays Plasma or serum was obtained in each experiment from matched infected and uninfected mice by tailbleeding into.5 ml Alsever's solution or axillary exsanguination. After allowing the red cells to settle in the former, tubes were centrifuged and the supernatants saved. Processed samples were stored at 85 C. During tailbleeding, the volume of blood was recorded as the total drops obtained, and subsequent dilutions were made based on the calculated initial dilution in Alsever's solution. Enzyme-linked immunosorbent assay was performed on selected samples using our published methods. 2 "' Whole C-serovar EB diluted in phosphate-buffered saline (PBS) served as antigen (~1 6 IFU/well). Serial dilutions of serum (5 fil) were added to each well after blocking with PBS-1% bovine serum albumin, followed by sequential washes in PBS-.5% Tween and the addition of alkalinephosphatase conjugated secondary antibody to mouse immunoglobulin (Ig)G+IgM+IgA (Kirkegaard & Perry, Gaithersburg, MD). Reactions were developed at 37 C with nitrophenylphosphate (Sigma, St. Louis, MO) in diethanolamine buffer, and the OD 4 5 of each well was taken after 6 to 9 minutes. Positive and negative control sera were included in each assay. Immunoblotting was performed as previously described, 51 except that EB (.5 to 1 mg/ml) solubilized in sodium dodecyl sulfate and.5 M 2-mercaptoethanol-containing sample buffer were subjected to preparative minigel polyacrylamide gel electrophoresis (12.5%), and proteins were transferred to nitrocellulose paper (NCP) using an LKB Multiphor II Transfer System (Pharmacia). Unoccupied binding sites on the NCP were blocked with BLOTTO (5% dry milk in 15 mm NaCl-1 mm Tris,.5% Tween). Sera (1:2 in BLOTTO) from known infected and uninfected mice were incubated overnight at 4 C with NCP strips. After washes, AP-conjugated anti-mouse IgG (1:5) was applied to each strip, except for an additional positive control of monkey serum that received AP-labeled anti-human IgG. Reactions were visualized by development with nitroblue tetrazolium and 5-bromo-4- chloro-3-indolyl phosphate (Sigma). Lymphoproliferative Assay Lymphoproliferative responses by cells obtained from infected and matched uninfected control mice (n s= 2 mice/group) were used to determine whether cellmediated immunity to chlamydia developed as a function of ocular infection. Lymphocytes obtained from cervical draining lymph nodes pooled from at least two mice or from individual spleens were tested for proliferative responses to chlamydial EB and the nonspecific mitogens, concanavalin A (ConA; Sigma) and bacterial lipopolysaccharide (Sigma) using standard Downloaded From: on 11/12/218

4 Ocular Chlamydial Infection in Mice 1979 TABLE l. Histopathologic Grading of Mouse Conjunctivae Feature Grading Scale Explanation Exudate -4+ Epithelial loss -4+ Stromal infiltrate -4+ Goblet cell loss -4+ = none; 1 = minimal; 2 = moderate; 3 = severe patchy; 4 = severe, layer = none; 1 = patchy or slight; 2 = moderate; 3 = severe; 4 = no normal epithelium remains = scattered, control; 1 = focal, limited to subepithelium; 2 = many foci, some deeper; 3 = dense, or large foci, <5% clear stroma; 4 = solid infiltrate, no normal stroma = none; 1 = moderate, partial; 2 = severe, partial; 3 = a few left; 4 = none present A total histopathologic disease score based on equal numbers of conjunctival surfaces for each mouse is obtained by adding the scores for each pathologic criterion. This score can be adjusted for one or two surfaces per eye for both eyes. methods.* 1 Uninfected control mice from the same shipment were tested in the same assay. Briefly, single cell suspensions (1 6 /ml) were prepared, and 1 /xl of cells was added in triplicate to round-bottomed wells for each stimulant. Several concentrations of EB (serovar C) were tested, ranging from 1.5 X 1 7 IFU/ ml to 1.5 X 1 4 IFU/ml; freshly thawed aliquots of EB with known infectious titers were used in each assay. ConA and lipopolysaccharide were tested at two or more optimal concentrations each (2.5 to 2 //g/ml). Plates were incubated for 4 days at 37 C, and, during the last 16 to 2 hours, they received 1 /ici/well of 3 H-thymidine. Plates were harvested on filters with a PhD cell harvester (Cambridge Technology, Watertown, MA), and label incorporation was determined by scintillation counting (Opti-Fluor; Packard Instruments, Meridan, CT) in a liquid scintillation counter. Mean counts per minute (cpm) for each set of triplicate wells were determined, and results were expressed as delta cpm = mean Experimental cpm mean Control cpm (medium only), or as a Stimulation Index where SI = mean Experimental cpm + mean Control cpm. An SI of >2. was considered positive. Student's /-test was used to determine significance of differences between responses by cells from infected and uninfected mice (SigmaStat; Jandel Scientific, San Raphael, CA). RESULTS Clinical Disease and Histopathology Clinical and histologic criteria were used to determine whether ocular disease was induced by topical challenge with a human biovar of chlamydia. A single infectious ocular challenge with 5 IFU of C. trachomatis (C serovar) induced a mild mucopurulent discharge for 7 to 14 days in all mice. Discharge persisted, and lid thickening and erythema were worse in repeatedly infected eyes; control eyes were normal during this time. Grossly, detectable inflammation waned after 14 to 21 days, and all eyes returned to normal by 6 to 8 weeks after inoculation. Globes remained normal and free of inflammation in all mice. Histologic staining of conjunctival samples showed clear differences between infected and uninfected eyes consistent with infection-induced pathology. At least two tarsal conjunctival surfaces were examined from both eyes of each mouse in these experiments; upper and lower lid conjunctivae yielded similar pathologic scores. The mean THDS was derived from at least two conjunctival surfaces by masked grading of the condition of the epithelium, the extent of an inflammatory cellular infiltrate, amount of exudate overlying the epithelium, and the numbers of goblet cells, using the grading scheme shown in Table 1. A comparison of mean histopathologic scores of conjunctivae from mice obtained in two experiments at several times after single infectious challenge (5 IFU) and uninfected, but swabbed, controls are shown in Figure 1. Even at day 7 after infection, THDS were significantly higher than those observed for controls (P <.8); histologic disease remained significantly higher than in controls, whereas eyes were culture positive (P <.1 to.1) and peaked at days 28 to 35. At days 28 to 35, THDS were significantly higher than scores for earlier and later time points in infection (P <.2). By days 41 to 56, when all eyes were culture negative, THDS were no longer significandy different from control THDS. The THDS for control mice represents an average for tissues removed throughout two 6-week experiments. Microscopic evidence of infection-induced pathol- Downloaded From: on 11/12/218

5 Investigative Ophthalmology 8c Visual Science, September 1995, Vol. 36, No ters of mononuclear cells were observed in some cases (Fig. 2c). These cumulative results demonstrate that ocular disease is induced in BALB/c mice by one or more exposures to a human ocular biovar of C. tracho- LLJ 12 - S 1-j <u to (U CO CD } matis *p<.1 a o n.s. 4 - Control d. 7 "* p<.8 ** p<.1 d d d Experimental Group FIGURE l. Total histopathologic disease scores (THDS) compared for infected BALB/c (n = 6 to 17) mice and uninfected control mice at several times after a single ocular infection with C/TW3 (5 IFU/eye). The data are from two separate experiments, and results are expressed as the mean THDS SEM based on masked histologic grading under X25 final magnification as described in Table 1. Disease scores were significantly different from controls (n = 36 eyes) by Student's Rest at day 7 (P <.8; n = 6), days 14 to 21 (P <.1; «= 17), and days 28 to 35 (P <.1; n = 1) except at days 41 to 59, when all mice (n = 1 eyes) had become culture negative and inflammation was subsiding. THDS of control mice represents an average of tissues removed at various times after mock infection. IFU = inclusion-forming units. ogy was detectable within 7 days. A single infectious challenge with 5 IFU of serovar C-EB resulted in a moderate subepithelial mononuclear infiltrate, a serous exudate overlying the epithelium, and loss of normal goblet cell distribution on the epithelial surface. Though a minor degree of inflammation was observed in mock-infected controls swabbed and subjected to slit lamp examination, the extent of inflammation and tissue pathology observed histologically clearly differed from infected eyes, as shown in Figure 2. Much greater pathology was induced by repeated inoculation of 5 IFU, most notably the extent of subepithelial mononuclear infiltrate. The infiltrate extended deeper into the conjunctival substantia propria after repeated infection, and evidence of exudate at the epithelial surface increased with the dose or number of infectious challenges. Both eyes from each mouse were found to have similar pathology, regardless of whether two or four conjunctival surfaces were examined. Representative examples of the effects of single and repeated infection are shown (Figs. 2a to 2d). Follicles, the hallmark of human ocular infection by C. trachomatis, were not observed by clinical or histologic examination. However, small subepithelial clus- Microbiologic Evidence of Ocular Infection To confirm that active infection correlated with signs of ocular disease induced by chlamydial serovar C, experiments were performed to test whether microbiologic criteria of infection were fulfilled. Two independent microbiologic methods were used in several experiments: isolation of replicating organisms in tissue culture or identification of chlamydial EB by direct fluorescence antibody (DFA) assay.32'1*"5 To reduce potential trauma of multiple sample collections from the limited mouse conjunctival surface, a single swab for either culture confirmation or DFA was used to identify infected eyes on any given day. A dose-response experiment was performed with BALB/c mice to determine if 5 IFU/5 /xl were an optimal inoculating dosage. Conjunctival swabs were collected at days 7, 1 to 12, 14 to 17, 21, 28, and 35 or 42 after infection. Longitudinal culture results from an experiment comparing four inoculum concentrations (5 to 5 IFU/eye) using four to six mice per group are shown in Figure 3. All mice were infected and sampled on the same days. Data are expressed as the percent of eyes culture positive by either first or second passage. Titers of each inoculum concentration were reconfirmed by titration cultures on the day of ocular infection; the actual infectious challenge delivered is indicated. The two highest inoculum concentrations induced infection for 7 to 21 days in 1% of mice assessed, although in mice receiving 174 IFU not all eyes were culture positive after day 14 (Fig. 3). Less than 1% of eyes (and mice) were infected when the inoculum contained <1 IFU, and eyes of those mice cleared infection earlier (Fig. 3). Cumulative time course results after a single ocular infection with 5 IFU/eye dosage from more than 12 experiments demonstrated that the majority of eyes were culture positive through day 28 (not shown). The direct fluorescence antibody assay diat detects free EB in cytologic smears was used as another microbiologic criterion to confirm infection of mice by the human chlamydial biovar. We compared culture and DFA results in BALB/c mice over a typical infection course by performing culture on one eye and DFA staining on the other from each of seven mice that received 5 IFU/eye. The results of one comparison are shown in Table 2 and demonstrate that, except on day 1, DFA and culture results were completely congruent. We attribute the lower number of DFA-positive eyes at this time point to inadequate sample collection because DFA was positive at this Downloaded From: on 11/12/218

6 1981 Ocular Chlamydial Infection in Mice V%*-^ J..i v FIGURE 2. Histopathologic results of single and repeated ocular infection in BALB/c mice. Frozen sections of BALB/c conjunctiva were stained with Lee's stain (Diff-Quik). (A) Normal conjunctiva. Goblet cells (arrow) are plentiful and intact, there is no evidence of exudate at the epithelial surface, and subepithelial mononuclear cells are sparse and scattered. The total histopathologic disease score (THDS) for this tissue is < 1. (B,C) Conjunctiva? removed 7 to 1 days after a single infectious challenge. Notable loss of goblet cells and subepithelial clustering of mononuclear cells are present. A 1+ exudate is seen (B; arrow), as is a possible subclinical follicle (C; arrow). THDS for B and C are 7.5 and 6.5, respectively. (D) Repeated infection (5 times/week for 3 weeks) results in a massive inflammatory infiltrate and a homogeneous 4+ exudate (e) overlying the epithelium. Goblet cells and the outer epithelial layer are missing. THDS grade is 16. Original magnifications, XlOO V 4IFU 174 IFU 815 IFU O 375 IFU 35 Time after Ocular Infection (days) FIGURE 3. Dose dependence of murine ocular infection. Groups of four to six mice were infected once in both eyes with 375 to 4 IFU/eye of C/TVV3 elementary bodies. Swabs were taken at the times indicated for first- and secondpassage cultures. Data are expressed as the percent of culture-positive mice (one or both eyes positive) at each inoculum concentration. = 4 IFU; = 174 IFU; = 815 IFU; and J = 375 IFU per eye. Inoculum concentrations were confirmed by titration on McCoy cells at the time of infection. IFU = inclusion-forming units. time point in other experiments. By day 35 alter infection, all eyes were negative by both assays. To confirm that we were not assaying inoculum carryover in the day 7 samples, parallel groups of mice were topically challenged with equivalent concentrations of live or ultraviolet-inactivated EB. Eyes were sampled immediately (time ), at 7 hours, and daily through day 7 for culture or DFA analyses. Figure 4 shows culture and DFA results of two such experiments. For both assays, eyes rapidly cleared the organism regardless of its viability, so that the majority of eyes were DFA negative at 48 hours and culture negative by day 3 (cultures were not performed at 24 or 48 hours). At day 4, eyes challenged with infectious organism began to show increasing positivity by both assays, and 1% of eyes were positive by day 6. No eyes receiving ultravioletinactivated EB became culture positive at any time, and 1% were DFA negative by day 4 after infection (Fig. 4). These results support our conclusion that active conjunctival infection developed because extracellular chlamydia remain viable and infectious only a short time. DFA scores and the number of EB detected in smears were lower for mouse conjunctiva? than observed for larger monkey and human eyes, Downloaded From: on 11/12/218

7 1982 Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 1 and they may relate to differences in the surface area sampled, swab size, numbers of infected conjunctival cells, or a combination of these. Nonetheless, these data provide unequivocal microbiologic evidence, using the two established diagnostic criteria for chlamydial infection, that BALB/c mice are susceptible to ocular infection with a human biovar of C. trachomatis. Susceptibility of Various Inbred Mouse Strains to Ocular Chlamydial Infections Experiments were performed to compare the susceptibility of two additional inbred mouse strains to C. trachomatis infection. Groups of 5 to 1 C3H/HeN and C57B1/6J were infected with 5 IFU/eye at the same times as groups of BALB/c mice. Both BALB/c and C3H/HeN exhibited peak infection at 14 to 21 days and recovered from infection by day 28 to 35 (Table 3). C57B1/6 (B6) mice were also included in one experiment and were found to be resistant to ocular infection. B6 mice exhibited fewer inclusions (mean peak of 2 to 3 IFU/ocular sample versus 1 to 2 IFU for C3H and BALB/c) over a shorter time course after infection. One hundred percent of B6 eyes were culture-positive for only 1 week, though some eyes remained positive for 3 weeks (Table 3). These experiments demonstrate that both BALB/c or C3H/HeN strains can be used as ocular infection models, but B6 mice develop more limited ocular infections with the C serovar. Immunologic Evidence for Ocular Chlamydial Infection One immunologic criterion of primary chlamydial infection is the presence of specific serum antibody. Selected sera from groups of infected and uninfected mice were tested for anti-chlamydial antibody by enzyme-linked immunosorbent assay and immunoblotting. No animals were seropositive by either assay before exposure to chlamydia. At the dosage of 5 IFU used for a single infection, enzyme-linked immunosorbent assay was negative (not shown). By immunoblotting, responses to homologous EB were detected only in mice exposed to chlamydia. Bands consistent with reactivity to MOMP (4 kda), 6 to 75 kda, and 3 to 31 kda proteins were seen. Results of one immunoblot experiment are shown in Figure 5 for individual plasmas obtained by tailbleedings before and 26 weeks after ocular infection. The blot demonstrates that even a mild ocular exposure results in longlasting reactivity with one or more chlamydial antigens, as seen in lanes 6 to 8, whereas no immunoreactivity was seen for preinfection samples. The two other mice in this experiment exhibited responses intermediate between those seen in lanes 6 to 8 after infection. All these mice were equally culture positive after primary infectious challenge and on rechallenge immediately after the 26-week bleed (data not shown). Preinfection plasmas from all five mice (lanes 1 to 5), and from others in independent experiments, failed to exhibit any reactivity with blotted chlamydial antigens and provided evidence that mice were not exposed to chlamydia before ocular infection. Positive immunoblots also were obtained with sera from other mice killed 3 weeks after single infection (not shown). Experiments are planned to determine whether mice develop neutralizing serum or tear antibodies to chlamydia after ocular infection. As another immunologic measure of successful infection of BALB/c mice with the human chlamydial biovar, chlamydia-specific responses by regional draining cervical LN and spleen cells were tested in proliferative assays. 3 As shown in Figure 6, cervical LN cells obtained at 9 days after infection exhibited dose-dependent responses to live EB in 4-day assays that were significantly higher than responses by cells from normal BALB/c mice at the two lowest antigen concentra- TABLE 2. Time Course of Infection After Inoculation With 5 IFU: Direct Fluorescent Antibody Analysis Time After Inoculation (days) DFA Score (mean SEM) Number of EB/Smear (mean SEM) Number of DFA- Positive Eyes Number of Culture- Positive Eyes /7 4/7 7/7 6/7 1/7 /7 7/7 7/7 7/7 6/7 1/7 /7 Mice (n = 7) received 5 IFU in 5 fi\ SPG on day ; swabs were taken from conjunctival surfaces of one eye at each time point, using sterile dacron urethral swabs. Slides were fixed in MeOH and stained with the DFA reagent. DFA scores were based on the standard -3+ scale. Total elementary bodies per smear of s2 epithelial cells were counted. Isolation cultures were performed with swabs from the second eye of each mouse; culture results are expressed as number positive by first or second passage. IFU = inclusion-forming units; DFA = direct fluorescence antibody. Downloaded From: on 11/12/218

8 Ocular Chlamydial Infection in Mice i 4 - LLj C/J , 2 - C CO 1 - Days after Ocular Challenge g Days after Ocular Challenge -#- Live EB -O- UV-EB -#- Live EB -O- UV-EB FIGURE 4. Transition from inoculum carryover to in vivo infection. Mice received either live or ultraviolet-inactivated elementary bodies (EB) topically into each eye. Swab samples were collected at the times shown. Similar results were obtained in two independent experiments. The data shown were obtained from five mice at to 3 days and from 1 mice at 4 to 7 days and are expressed as mean SEM. (A) Isolation cultures. (B) Direct fluorescence antibody results. = live EB; J = ultraviolet-inactivated EB. tions (P <.5). In most experiments, antigen concentrations > 5 X 1 6 IFU/ml were inhibitory or toxic and stimulation indices declined (Fig. 6). Responses were also inoculum-dose-dependent because higher doses or multiple ocular challenges resulted in proportionately higher stimulation indices (not shown). Proliferative responses by cells from draining lymph nodes were positive through day 21 but appeared to depend on active infection because stimulation indices declined after day 28 to 3. DISCUSSION The studies reported here demonstrate that conjunctivae of BALB/c mice have been infected reproducibly with a human biovar of C. trachomatis (serovar C). A moderate dose of infectious C. trachomatis (2 X 1* 5 X 1 3 IFU/eye) induced highly reproducible histopathologic, microbiologic, and immunologic evidence of infection. The intensity of these parameters was dose-dependent because either a higher inoculum dosage (1 h IFU) or multiple challenges resulted in increased numbers of inclusions isolated in duplicate cultures (1 to 2/2 //I versus 1 to 1/2 //I), increased conjunctival inflammation, and stronger immunologic responses. The numbers of culture-positive eyes and the inclusion counts increased over 7 to 14 days, and 1% of mice remained positive though 21 to 28 days, indicating true conjunctival infection. The results obtained by DFA were similar. Because chlamydia are obligate intracellular parasites and do not replicate extracellularly, detection of inclusions in isolation cultures from 7 to 28 days after infection can only represent organisms that replicated within conjunctival cells. Previous studies showed that after topical ocular challenge of monkey eyes with 1 (> ultraviolet-inactivated organisms, the majority of EB were cleared within 24 hours, and none were detectable by DFA by 6 days after challenge. 52 Similar results were obtained for BALB/c mice when live and ultravioletinactivated EB were compared directly. Taken together, these data support the establishment of a replicating chlamydial infection in mouse conjunctivae and demonstrate the feasibility of the murine model of ocular disease induced by a human biovar of C. trachomatis. Longitudinal sample collection from infected eyes clearly showed there was an early window of time when cultures were negative (Fig. 4a). The murine model, as for other ocular models, permits repeated sampling from the same eyes to identify initiation or termination of infection. The most marked histopathology was observed during active infection, and this resolved as infection cleared. Diagnosis of most experimental chlamydial infections of mice relies on microbiologic assays without benefit of direct visualization of the infected tissue until biopsy or necropsy is performed (e.g., lung, genital tract). Our data also suggest that ocular infections in mice begin to clear earlier than they do in humans and monkeys. 323 Several possible explanations are that proportionately fewer mouse conjunctival cells are infected by a human chlamydial biovar, mice may have more efficient immune (or nonimmune) mechanisms to deal with mucosal infections, or infection may enter an inapparent state more rapidly in mice than observed in humans or monkeys " 53 Additional immunologic and molecular studies will help to distinguish among these possibilities. Compared to other reports, 34 " 35 the success in establishing ocular infections in the current studies Downloaded From: on 11/12/218

9 1984 Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 1 TABLE 3. Comparison of Susceptibility to Ocular Chlamydia trachomatis Infection in Three Inbred Mouse Strains BALB/c Days After Infection % Mice Positive 7 IFU/Eye H H/HeN % Mice Positive C57BI/6 IFU/Eye % Mice Positwe ND IFU/Eye MX Eyes were infected with 49 1FU/5 fj\ of viable C. trachomatis (C/TW-3) on day. Results represent fluorescent antibody staining for inclusions in two welts/eye using an FITC-labeled and-momp monoclonal antibody. The number of culture-positive mice were determined from first- and second-passage cultures. Infectious yields from swabs of individual eyes were obtained from inclusions counted in duplicatefirst-passagecultures and are given for each mouse strain as mean IFU SEM ml of ocular sample for three experiments with BALB/c and C3H/HeN and one experiment with C57B1/6J mice (n mice/strain). ND = not determined; IFU = inclusion-forming units; FITC = fluorescein isothiocyanate; MOMP = major outer membrane protein a. 12 o 1 line Incoiporat< CD kda production or receptors for this important family of cytokines have been shown.9'3'1'1'"''13'54fi5 Interferon-y has been shown to downregulate chlamydial development,13'51' but the role of type 1 interferons (IFNa//5) in chlamydial infection is unknown. Resistance also may relate to route of infection for particular chlamydial serovars. Immunoblotting and lymphoproliferative assays X1 probably relates in great measure to our choices of BALB/c as the inbred mouse strain and of the specific human biovar of C. trachomatis, C serovar. Clearly, mice of other genetic backgrounds are also susceptible to chlamydial infection, and it will be of interest to test additional human biovars in the mouse eye model. Mouse strain-dependent differential resistance to viruses, as well as to C. trachomatis, has been reported.5'6'4'41'4"1* The genetic basis for resistance is not completely understood, but differences in interferon I FIGURE 5. Chlamydia-specific antibody after ocular infection with 5 IFU of C serovar. Immunoblot results For plasma from five mice comparing responses to solubilized elementary bodies before and after infection. Lanes 1 to 5 contain preinfection samples. Results for 3 of 5 mice 26 weeks after ocular infection with 5 IFU/eye are shown in lanes 6 to 8, compared to a positive control mouse from the same experiment after subcutaneous immunization with purified major outer membrane protein and ocular challenge (lane 9). Molecular weights are indicated on the left of the figure. All mouse sera were tested at 1:2. IFU = inclusion-forming units. 8 6 O 4 Infected Normal * p<.5 2 *& n Antigen concentration (IFU ml"1 x 1.5) FIGURE 6. Chlamydia-specific, cell-mediated immunity 9 days after a single ocular infection. Lymphoproliferation to whole organism is dose dependent over several log concentrations. The intensity of in vitro responses is related to ocular challenge dose. Cervical lymph node cells pooled from two to three mice (15 cells/well) were cultured with varying concentrations of viable C-EB (1.5 X X 1(i IFU/well) for 4 days; 1 to 2 (xd of ^H-thymidine was added to each well for the final 16 to 2 hours of culture. Results are expressed as mean counts per minute of incorporated label obtained from scintillation counting of triplicate wells; standard deviations were <1%. IFU = inclusionforming units; EB = elementary bodies. Downloaded From: on 11/12/218

10 Ocular Chlamydial Infection in Mice 1985 demonstrated that animals converted from negative to chlamydia-specific responders after a single infectious ocular challenge. Although antibody responses were only moderate, they persisted for months. We detected positive serum IgG responses to the 4-kDa MOMP and several other chlamydial proteins. Nevertheless, seropositive mice were not significandy protected from infectious rechallenge 7 months after primary infection (not shown). Both serologic and cellular responses were inoculum dose-dependent and suggest that this model will be useful for immunologic manipulations designed to stimulate protective immunity. Evidence for chlamydia-specific immune responsiveness during murine ocular infection was consistent with studies in humans and in other animal models of chlamydial ocular infection " We have demonstrated in the cynomolgus monkey model high frequencies of chlamydia-specific T and B lymphocytes in draining cervical lymph nodes and conjunctivae after experimental ocular infection. 19 ' 2 Similar studies are planned in the mouse model. The development of an inbred mouse model of ocular infection with a human serovar provides a new means to study the immunopathogenesis of ocular chlamydial infections in larger numbers of animals. Most murine studies of chlamydia have used the mouse pneumonitis agent (MoPn) of C. trachomatis in which microbiologic and histopathologic studies of lung have precluded collection of longitudinal samples from single animals. Guinea pigs have been used for studies of ocular chlamydial infections since the identification of guinea pig inclusion conjunctivitis agent, a C. psittaci strain. 4 ' 8 ' 141 ' 15 ' 57 Unfortunately, there is a paucity of reagents available for detailed cell-mediated immunologic study in this species. In addition, unlike humans and other animal models, guinea pigs develop total immunity to reinfection for 3 to 45 days, which may indicate a unique response to chlamydia by guinea pigs that could complicate interpretations during tests of protective immunity.' 1 N1> The baseline parameters of ocular disease have been fulfilled in an inbred mouse model of chlamydial infection in our experiments. The murine ocular infection model should aid in identification of promising antichlamydial vaccine candidates derived from human chlamydial biovars more quickly based on both immunogenicity and protection from disease. Vaccine candidates that prove to be successful in mice could then be tested further for induction of protective immunity in the subhuman primate model of trachoma or other chlamydial infection models. Key Wards Chlamydia trachomatis, conjunctiva, eye, immunopathogenesis, mouse, trachoma Acknowledgments The authors thank Vivian Velez, Lori Dejong, and Timothy Conn for invaluable technical assistance in developing and characterizing this model and Donnell Berry for preparing histologic specimens. They also thank Drs. Hugh Taylor, Thomas Quinn, and Paul Montgomery for their critical readings of the manuscript. References 1. Dawson CR, Jones BR, Tarizzo ML. Guide to trachoma control. WHO Bull. 1981; Taylor HR. Trachoma. Int Ophthalmol. 199; 14: Taylor HR, Siler JA, Mkocha HA, Munoz B, West S. The natural history of endemic trachoma: A longitudinal study. AmJ Trop Med Hyg. 1992;46: Batteiger BE, Rank RG. Analysis of the humoral immune response to chlamydial genital infection in guinea pigs. Infect Immun. 1987; 55: Byrne GI, Padilla M, Lacy D, Paulnock D, Guo X. Mouse model for protective immunity to chlamydia. In: Bowie WR, Caldwell HD, Jones RP, et al, eds. Chlamydial Infections. Cambridge: Cambridge University Press; 199: Fuentes V, Orfila J. Genetic control of natural resistance to Chlamydia psittaci Loth strain in mice. In: Bowie WR, Caldwell HD, Jones RP et al, eds. Chlamydial Infections. Cambridge: Cambridge University Press; 199: Hough AJ Jr, Rank RG. Induction of arthritis in C57B1/6 mice by chlamydial antigen: Effect of prior immunization or infection. AmfPathol. 1988; 13: Murray ES, Charbonnet LT, MacDonald AB. Immunity to chlamydial infections of the eye: The role of circulatory and secretory antibodies in resistance to re-infection with guinea pig inclusion conjunctivitis./ Immunol. 1973; 11: Murray HW. Gamma interferon, cytokine-induced macrophage activation, and antimicrobial host defense: In vitro, in animal models, and in human beings. Diagn Microbiol Infect Dis. 199; 13: Ramsey KH, Rank RG. Resolution of chlamydial genital infection with antigen-specific T-lymphocyte lines. Infect Immun. 1991;59: Ramsey KH, Soderberg LS, Rank RG. Resolution of chlamydial genital infection in B-cell-deficient mice and immunity to reinfection. Infect Immun. 1988;56: Rank RG. Role of the immune response. In: Barron AL, ed. Microbiology of Chlamydia. Boca Raton, FL: CRC Press; 1988: Rank RG, Ramsey KH, Pack EA, Williams DM. Effect of gamma interferon on resolution of murine chlamydial genital infection. Infect Immun. 1992; 6: Rank RG, Sanders MM. Pathogenesis of endometritis and salpingitis in a guinea pig model of chlamydial genital infection. AmfPathol. 1992; 14: Rank RG, Soderberg LS, Sanders MM, Batteiger BE. Downloaded From: on 11/12/218

11 1986 Investigative Ophthalmology & Visual Science, September 1995, Vol. 36, No. 1 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. Infect Immun. 1989;57: Williams DM, Grubbs B, Schachter J. Primary murine Chlamyida trachomatis pneumonia in B-cell-deficient mice. Infect Immun. 1987;55: Holland SM, Hudson AP, Bobo L, Whittum-Hudson JA, Viscidi RP, Quinn TC, et al. Demonstration of chlamydial RNA and DNA during a culture-negative state. Infect Immun. 1992;6: Hudson AP, McEntee CM, Reacher M, Whittum- Hudson JA, Taylor HR. Inapparent ocular infection by Chlamydia trachomatis in experimental and human trachoma. Curr Eye Res. 1992; 11: Pal S, Taylor HR, Huneke RB, Prendergast RA, Whittum-Hudson JA. Frequency of antigen-specific B cells during experimental ocular Chlamydia trachomatis infection. Infect Immun. 1992; 6: Pal S, Pu Z, Huneke RB, Taylor HR, Whittum-Hudson JA. Chlamydia-specific lymphocytes in conjunctiva during ocular infection: Limiting dilution analysis. Reg Immunol. 199;3:l7l-l Patton DL, Cosgrove PA, Grutzmacher RD, Kuo CC, Wang SP. Experimental trachoma in subcutaneous conjunctival autografts in macaques. Invest Ophthalmol VisSci. 1987;28: Taylor HR. Ocular models of chlamydial infection. Rev Infect Dis. 1985;7: Taylor HR. Development of immunity to ocular chlamydial infection. Am f Trop Med Hyg. 199;42: Taylor HR, Agarwala N, Johnson SL. Detection of experimental Chlamydia trachomatis eye infection in conjunctival smears and in tissue culture by use of fluorescein-conjugated monoclonal antibody./clin Microbiol. 1984; 2: Taylor HR, Johnson SL, Schachter J, Caldwell HD, Prendergast RA. Pathogenesis of trachoma: The stimulus for inflammation. / Immunol. 1987; 138: Taylor HR, Prendergast RA, Dawson CR, Schachter J, Silverstein AM. An animal model for cicatrizing trachoma. Invest Ophthalmol Vis Sti. 1981;21: Taylor HR, Whittum-Hudson J, Schachter J, Caldwell HD, Prendergast RA. Oral immunization with chlamydial major outer membrane protein (MOMP). Invest Ophthalmol Vis Sti. 1988;29: Wang S, Grayston JT. Pannus with experimental trachoma and inclusion conjunctivitis agent infection of Taiwan monkeys. Am f Ophthalmol. 1967; 63: Whittum-Hudson JA, Prendergast RA, Taylor HR. Changes in conjunctival lymphocyte populations induced by oral immunization with Chlamydia trachomatis. Curr Eye Res. 1986; 5: Whittum-Hudson JA, Taylor HR, Farazdaghi M, Prendergast RA. Immunohistochemical study of the local inflammatory response to chlamydial ocular infection. Invest Ophthalmol Vis Sti. 1986;27: Whittum-Hudson JA, Taylor HR. Anti-chlamydial specificity of conjunctival lymphocytes during experimental ocular infection. Infect Immun. 1989; 57: Young E, Taylor HR. Immune mechanisms in chlamydial eye infections: Development of T suppressor cells. Invest Ophthalmol Vis Sti. 1986;27: An L. Production and characterization of anti-idiotypic antibodies, biological mimicry of a glycolipid exoantigen of Chlamydia trachomatis in vivo. Amherst: University of Massachusetts; Thesis. 34. Barsoum IS, Hardin LK, Colley DG. Immune responses of mice after conjunctival exposure to Chlamydia trachomatis serovar A. Med Microbiol Immunol (Berl). 1988; 177: Colley DG, Goodman TG, Barsoum IS. Ocular sensitization of mice by live (but not irradiated) Chlamydia trachomatis serovar A. Infect Immun. 1986; 54: Ellermann-Eriksen S, Justesen J, Mogensen SC. Genetically determined difference in the antiviral action of a//?-interferon in cells from mice resistant or susceptible to herpes simplex virus type 2. / Gen Virol. 1986;67: Ishizaki M, Allen JE, Beatty PR, Stephens RS. Immune specificity of murine T-cell lines to the major outer membrane protein of Chlamydia trachomatis. Infect Immun. 1992;6O: Levitt D, Danen R, Bard J. Both species of chlamydia and two biovars of Chlamydia trachomatis stimulate mouse B lymphocytes. / Immunol. 1986; 136: Levitt D, Corlett R. Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro. /Immunol. 1988; 14: Lopez C. Genetics of natural resistance to herpesvirus infections in mice. Nature. 1975; 258: PeposeJS, Whittum-Hudson JA. An immunogenetic analysis of resistance to herpes simplex virus retinitis in inbred strains of mice. Invest Ophthalmol Vis Sti. 1987:28: Su Y, Oakes JE, Lausch RN. Ocular virulence of a HSV type 1 strain is associated with heightened sensitivity to a/ interferon. / Virol. 199; 64: Whittum-Hudson JA, PeposeJS. Herpes simplex virus type 1 induces anterior chamber-associated immune deviation (ACAID) in mouse strains resistant to intraocular infection. Curr Eye Res. 1988; 7: Zawatsky R, Gresser I, DeMaeyer E, Kirchner H. The role of interferon in the resistance of C57B1/6 mice to various doses of HSV type 1. / Infect Dis. 1982;146: Jackson S, Mestecky J, Childers NK, Michalek SM. Liposomes containing anti-idiotypic antibodies: An oral vaccine to induce protective secretory immune responses specific for pathogens of mucosal surfaces. Infect Immun. 199;58: Kaufmann SH. Immunity to bacteria. Curr Opin Immunol. 1989;2: Kiyono H, Mosteller LM, Eldridge JH, Michalek SM, McGhee JR. IgA responses in xid mice: Oral antigen primes Peyer's patch cells for in vitro immune re- Downloaded From: on 11/12/218

12 Ocular Chlamydial Infection in Mice 1987 sponses and secretory antibody production. JImmunol. 1983; 131: Michalek SM, Childers NK, Katz J, Denys FR, Berry AK, Eldridge JH, et al. Liposomes as oral adjuvants. Curr Top Microbiol Immunol. 1989; 146: Michalek SM, McGhee JR, Kiyono H, Colwell DE, Eldridge JH, Wannemuehler MJ, et al. The IgA response: Inductive aspects, regulatory cells, and effector functions. Ann NY Acad Sci. 1983; 49: Rapoza PA, Quinn TC, Kiessling LA, Green WR, Taylor HR. Assessment of neonatal conjunctivitis with a direct immunofluorescent monoclonal antibody stain for chlamydia. JAMA. 1986;255: Taylor HR, Maclean IW, Brunham RC, Pal S, Whittum-Hudson J. Chlamydial heat shock proteins and trachoma. Infect Immun. 199;58: Taylor HR, Velez VL. Clearance of chlamydial elementary bodies from the conjunctival sac. Invest Ophthalmol Vis Sci. 1987;28: Cheema MA, Schumacher HR Jr, Hudson AP. RNAdirected molecular hybridization screening: Evidence for inapparent chlamydial infection. Am J Med Sci. 1991;32: Eriksen SE, Sommerlund M, Mogensen SC. Differential sensitivity of macrophages from HSV-resistant and -susceptible mice to respiratory burst priming by interferon a//3.jgen Virol. 1989; 7: Mogensen SC, Virelizier J. The interferon-macrophage alliance. Interferon. 1987; 8: Beatty WL, Byrne GI, Morrison RP. Morphologic and antigenic characterization of interferon-gamma-mediated persistent Chlamydia trachomatis infection in vitro. Proc Natl Acad Sci USA. 1993; 9: Murray ES, Radcliffe FT. Experimental conjunctival infection of guinea pigs with the guinea pig inclusion conjunctivitis (GP-ic) Bedsonia. Am J Ophthalmol. 1967; 63: Downloaded From: on 11/12/218