Trachoma-Inclusion Conjunctivitis-Lymphogranuloma

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1 INFZCTON AND IMMUNITr, Mar. 973, p Copyright 973 American Society for Microbiology Vol. 7, No. 3 Printed in U.S.A. A Simplified Method for Immunological Typing of Trachoma-Inclusion Conjunctivitis-Lymphogranuloma Venereum Organisms SAN-PIN WANG, CHO-CHOU KUO, AND J. THOMAS GRAYSTON Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 995 Received for publication November 97 Responding to the pressure of large numbers of trachoma-inclusion conjunctivitis (TRIC)-lymphogranuloma venereum (LGV) isolates from field studies requiring serotyping, we have developed a simplified, less-precise method that utilizes cell culture-grown organisms to produce mouse antisera which is tested agaist prototype TRIC-LGV antigens in the micro-immunofluorescence test. Cell cultures with as few as 5 to 5% of cells showing inclusions produced adequate antibody in mice days after single injection. Knowledge of the reaction of prototype antisera with the antigens has allowed typing of most isolates tested from the pattern of cross-reaction of their antiserum. Trachoma-inclusion conjunctivitis (TRIC) organisms are being frequently isolated from the genital tract (and occasionally from the eyes) of persons with venereal infection. New cell culture techniques have greatly simplified isolation of these microorganisms (3, 5). At the same time recent development of a micro-immunofluorescence (micro-if) test for antibody study of the Chlamydia subgroup A has allowed practical immunotyping of TRIC and lymphogranuloma venereum (LGV) organisms (6). We have described thirteen separate types of TRIC-LGV organisms (9-). Typing isolates provides an important marker that adds considerable information to the studies of human infection with these organisms. Unfortunately, untyped isolates are rapidly accumulating in studies throughout the world because isolation is much easier than typing. Typing has depended on cross testing ("two-way") of antigen and antiserum from the unknown strain against prototype antigen and antisera. Only chicken egg yolk sac-grown TRIC organism is of sufficient concentration to use as antigen in the micro-if test. The egg-grown antigen has also been used to produce mouse antiserum for the test. The transfer of a cell culture TRIC isolate to egg yolk sac growth is difficult, time consuming, and costly and does away with the advantage of original isolation in cell culture. Because of the pressure for largescale typing of isolates from field studies and with increasing kniowledge of TRIC-LGV types and their cross-reactions, we have investigated a simplified method of typing, using cell culturegrown organisms to produce mouse antiserum for a "one-way" test. MATERIALS AND METHODS TRIC-LGV strains. Prototype strains used in this study have been reported (, ): type A/G-7/OT, B/TW-5/OT, Ba/AP-/OT, C/TW- 3/OT, D/UW-3/Cx, E/UW-5/Cx, F/UW-6/Cx, G/39/OC, H/UW/Cx, I/UW-/Ur, LGV- I//Bu, LGV-II/3/Bu, and LGV-III//Bu. Types G, H, and I are newly defined immunological types (). Other UW strains were recently isolated in Seattle (5). Strains UW-9/Cx, UW-3/Cx, UW-36/Cx, UW-37/Cx, UW/Cx, UW3/Cx, UW-/Cx, UW-6/Cx, UW-5/Cx, UW-53/Cx, and UW-57/Cx were isolated with diethylaminoethyl (DEAE)-dextran-treated HeLa 9 cells, and UW-7/Cx, UW-9/Cx, UW-5/Cx, UW-5/Cx, and UW-55/Cx were isolated with DEAE-dextrantreated irradiated McCoy cells. HeLa cell culture. Our procedure for primary isolation using DEAE-dextran-treated HeLa 9 cells and centrifugation (5) was followed for passage of the strains. For each passage four vials containing a cover slip with a HeLa 9 cell monolayer were inoculated. The vial used was a disposable flat-bottomed, -dram glass vial shell (outside diameter 5 mm and height 5 mm) which was plugged with a Io. rubber stopper. After 3 days incubation at 35 C, one of the four vials was used for infectivity assay. The cover slip was fixed in methyl alcohol and stained with Giemsa. An inclusion count for 3 fields at X magnification was made () with a Bausch and Lomb micrometer (7 by 7 mm) in the ocular, and the average count 356 Downloaded from on October, by guest

2 VOL. 7, 973 IMMUNOTYPING OF TRIC-LGV ORGANISMS 357 per micrometer field was obtained. An inclusion count was also made on 3 whole fields without use of the micrometer to provide comparative data for use when the micrometer is not available. If the desired inclusion count is not present, the three remaining vials may be used for another cell culture passage. Day- mouse antiserum prepared against HeLa cell grown organisms. When an appropriate infectivity was reached, the cell monolayers of the remaining three vials were harvested in. to.5 ml of. M phosphate-buffered saline, ph 7. (PBS). The infected cell suspension was equally divided and injected intravenously to each of two 5-week-old mice. The mice were exsanguinated by axillar bleeding days later and blood pooled (day- serum). About.5 ml of serum was usually obtained from the two mice. Micro-immunofluorescence (micro-if) test. The test method has been described (6, 9-). Test antigens (-5% yolk sacs) were prepared from selected infected yolk sacs with abundant elementary bodies. Nine groups of 3 antigen dots which represent 3 different types arranged in a sequence of type C, A, B, Ba, E, D, LGV-I, LGV-II, LGV-III, G, F, H, and I were placed on a microscope slide. The slides were dried in air for at least 3 min and fixed in acetone for to 5 min at room temperature. They were stored at C lq R C A B Ba E D LGV-I LGV-N and used within weeks. Duplicate slides were used to test each serum. Antiserum against infected yolk sac antigen was prepared as described before (). Ten to mice were given two.5-ml injections of % crude infected yolk sac suspension, week apart. Ten days after the first injection, they were exsanguinated and blood pooled (day- serum). The antiserum was absorbed with normal yolk sac before testing. Twofold serial dilutions of antiserum (usivig. ml) were made in PBS and tested against 3 different type antigens. The highest dilution of serum which showed definite fluorescence to each of the antigen dots was considered the end point of the serum to that antigen. Reaction at : or lower dilution of serum was considered negative. RESULTS Serological patterns in mouse antisera of different TRIC-LGV type strains. In our laboratory 3 immunological types of TRIC- LGV strains (including Ba) are now recognized (, ). In recent years we have carried out a number of micro-if tests with antiserum of each type (prepared by immunization with infected yolk sac) tested against the 3 antigens. Although each serum produced the highest titer with its homologous antigen, the pattern of cross-re PROTOTYPE ANTIGENS C A B Ba E D LGV-ILGV-ILVl- G F H I Downloaded from on October, by guest G F H II FIG.. Micro-IF antibody pattern.s of TRIC-LGV type-specific mouse antisera. Difference of one line expresses a twofold difference from the homologous titer which i shown byfive lines.

3 35 WANG, KUO, AND GRAYSTON INFECT. IMMUNITY activity titers shown with heterologous antigens infected cells of the remaining three vials were provided a distinct pattern of reaction for each harvested in. ml of PBS. Two mice per group serum, thus for each type strain. In Fig., received two intravenous injections of.5 ml of heterologous reactivity with the 3 antisera is each suspension week apart. Each mouse was presented in graphic form. For example, type C bled from the tail using heparinized capillaries strain antiserum cross-reacted to A antigen at and days after the first injection, and plasma fourfold less dilution than to the homologous C from each pair of mice at each bleeding were antigen, but not to other antigens. A much pooled. The plasma samples were simultaneously broader cross-reactivity was observed with type tested against 3 antigens. The results are shown Ba antiserum in comparison with type B, even in Table. The inclusion counts decreased with though both types reacted identically to each dilution of the TW-5 inoculum. A micrometer other. Broad or narrow cross-reactivities of the field at X magnification contained an average other types are shown in the figure. of HeLa cells. All the plasma samples tested The results suggest that immunological typing showed the TRIC type B pattern of reactions. of a strain can be achieved by determining the Even the antigen derived from the vials of the cross-reaction pattern of its antiserum in a oneway test against the 3 antigens. Table gives production of low-titer antibody. The antibody lowest inclusion count of 3 per field stimulated examples of such tests made for seven genital produced by the suspensions with inclusion TRIC isolates. Each of these strains had been counts of 5 and were similar. The one-dose grown in the egg yolk sac, and day- mouse day- antibody was more specific and of slightly antisera were used. By comparing the crossreaction pattern in the micro-if test, each strain Immunological typing of TRIC-LGV lower homologous titer. could be classified. Separation of type D and E strains using day- mouse antisera. The previous experiment suggested that one-way immu- in the one-way test may not be possible. Mouse antibody response to antigens nological typing of strains was possible using grown in HeLa cells. Preparation of effective day- mouse antisera produced with organisms mouse antiserum with antigens grown in cell grown in three vials of HeLa cells if infectivity of culture is the key to simplification of typing. The the cells had reached approximately 5 inclusions amount of antigenic mass which can be produced per micrometer field. Based on this method, 5 in three vials of HeLa 9 cells as measured by strains of different TRIC-LGV types were production of antibody in two mice was investigated. Tenfold dilutions (-,, and -) of tested. The strains already grown in egg yolk sac were passed in HeLa cells. The strains originally an infectious material, TW-5 E9 H (9 egg passages and HeLa passages), were made in isolated by HeLa cells were passed directly from sucrose phosphate glutamate medium (7). A frozen stock in HeLa cells. The results are shown.-ml amount of each dilution was inoculated in Table 3. Each of the antigen preparations into each of four vials containing a monolayer of derived from vials with inclusion counts ranging HeLa cells. Three days later, the cover slip of from 5 to 3 per micrometer field (or 9 to 5 per one vial was used for inclusion count and the whole field) produced antibodies in two mice TABLE. One-way immunological typing of genital TRIC isolates using day- mouse antisera against the yolk sac-grown isolates and 3 prototype antigens in the micro-if test Test antigens (egg yolk sac grown) Mouse antisera C Antigenic m N~~~~~~~~~ k Io type Downloaded from on October, by guest UW-5/Cx, McEla Ob 56 E UW-53/Cx, H5E LGV-III UW-5/Cx, McE 5 F UW-55/Cx, McE 5 O F UW-7/Cx, McE G UW-3/Cx, H5E 56 H UW-9/Cx, McE3 56 I a McE: McCoy cell passages and egg passage. H is for HeLa cell. b Antibody titer, reciprocal of original dilution; = less than.

4 VOL. 7, 973 IMMUNOTYPING OF TRIC-LGV ORGANISMS 359 TABLE. Micro-IF antibody in mouse plasma following immunization with the TW-5 (type B) strain grown in HeLa cell culture TW-5 inocula Inclusion countsa Immunization doses Days after first immunization Micro-IF antibody titer Prototype antigens C A B Ba E D Li L L3 G F H I - () - 5 (33) 3-' 3 (5) Ob a Inclusion counts per micrometer field (per whole field). b = Titer less than. TABLE 3. One-way typing with day- mouse antisera derived from onew-dose intravenous immunization with HeLa-SS9-grown TRIC-LGV isolates Micro-IF antibody titer Mouse antisera Inclusion Prototype antigens Antigenic C A B Ba E D Li L Ls G F H I TW-3 E Hb 5 (7) Oc OC G-7 E53 H 6 (9) O A G-7 E53 H () A UW-9/Cx H 3 (5) B AP- E H 5 (9) 56 Ba UW-/Cx H (37) E UW-/Cx H (3) O D /Bu E6 H 6 () LGV-I /Bu E6 H 3 (9) LGV-I 3/Bu E H (3) LGV-Il 3/Bu E H () LGV-Il UW-3/Cx H5 7 () LGV-III UW-6/Cx H9 5 (6) LGV-III UW-57/Cx H 5 (3) G UW-5/Cx H () F UW-37/Cx H (). F UW-3Cx H () H UW-3/Cx H6 () 56 I a Inclusion counts per micrometer field (per whole field). b E H = egg passages and HeLa passages. c = Titer less than. Downloaded from on October, by guest which were of sufficient titer to show a reaction typing of the organisms. An inclusion count of pattern for type determination. All these strains to 33 per micrometer field was obtained after have also been grown in eggs and their type con- seven to eight passages. The day- mouse antifirmed in the regular two-way cross-reaction sera produced 'allowed typing which confirmed method (). previous two-way results (three type E-D, one F, The frozen original specimens of five cervical and one H). The entire procedure including swabs from which an isolation had been obtained reisolation and typing was completed within 5 were used for reisolation and one-way micro-if weeks.

5 36 WANG, KUO, AND GRAYSTON INFECT. IMMUNITY DISCUSSION The simplified method described in this paper depends on two findings: (i) the antibody pattern in mouse sera is sufficiently distinct for the 3 known TRIC-LGV types to enable type designation from testing unknown antisera only, one-way test, and (ii) sufficient antigenic mass can be produced in cell culture to effectively immunize mice. The distinctness of the antibody patterns has been assisted by the fact that the micro-if method allows multiple antigens to be tested simultaneously against the same serum. In addition, day- antiserum (immunoglobulin M antibody, reference 6) provides a more specific pattern as well as being easier and quicker to produce. Since today most TRIC-LGV organisms are being isolated in cell culture, the key to quick typing is to produce adequate antigenic mass with few additional passages in cell culture. Cell culture growth of TRIC isolates is promoted by sonic treatment of inoculiim before cell infection, treatment of the cells with DEAE-dextran, and centrifugation of the inoculum onto the cell monolayers. We have previously estimated the minimum antigenic mass for production of specific antibody in mice to be 5 to organisms (6, ). We have obtained a yield of 9 TRIC organisms from a -oz bottle of HeLa cell culture containing approximately 7 fully infected cells (). This provides an estimate of organisms per inclusion and suggests that, in the culture vials (5 cells) used in this study, between and inclusions per cells would provide adequate antigenic mass. This proved to be correct since a micrometer field has an average of HeLa cells, and as few as five inclusions per field were associated with adequate antigenic mass for mouse immunization. Three technical aspects of the procedure were found to be important. Each of the test antigens should be frequently checked for specificity and adequate reactivity using standardized homologous antisera. For preparation of mouse antiserum, it is important that all antigen inoculum is delivered intravenously. Antiserum titers of : or lower may be considered nonspecific or equivocal, since occasional reactions are seen with low dilutions of normal mouse sera. The primary limitation of the one-way method is differentiation between closely related types such as E and D, LGV-I and LGV-II, and perhaps G and F. The regular two-way cross tests may be required, although a precise identification may not be required for most epidemiological purposes. If there are types in addition to the 3 so far described (as seems likely), they will require two-way tests for study. The simplified method for immunotyping was also found successful with TRIC organisms grown in the irradiated McCoy cells (3) in the studies carried out in Taiwan (J. L. Gale, personal communication). ACKNOWLEDGMENT This study was supported by Public Health Service research grant 5-RO-EY-9 from the National Eye Institute. LITERATURE CITED. Alexander, E. R., S. P. Wang, and J. T. Grayston Further classification of TRIC strains from ocular trachoma and other sources by the mouse toxicity prevention test. Amer. J. Ophthalmol. 63: Furness, G., D. M. Graham, and P. Reeve. 96. The titration of trachoma and inclusion blennorrhoea viruses in cell cultures. J. Gen. Microbiol. 3: Gordon, F. B., A. L. Harper, A. L. Quan, J. D. Treharne, R. St. C. Dwyer, and J. A. Garland Detection of Chlamydia (Bedsonia) in certain infections of man. I. Laboratory procedures: comparison of yolk sac and cell culture for detection and isolation. J. Infect. Dis. : Kuo, C. C., G. E. Kenny, and S. P. Wang. 97. Trachoma and psittacosis antigens in agar gel double immunodiffusion, p In R. L. Nichols (ed.), Trachoma (Proceedings of the Symposium held in Boston, Massachusetts, August 97). Excerpta Medica, Amsterdam. 5. Kuo, C. C., S. P. Wang, B. B. Wentworth, and J. T. Grayston. 97. Primary isolation of TRIC organisms in HeLa 9 cells treated with DEAEdextran. J. Infect. Dis. 5: Wang, S. P. 97. A micro immunofluorescence method. Study of antibody response of TRIC organisms in mice, p In R. L. Nichols (ed.), Trachoma (Proceedings of a symposium held in Boston, Massachusetts, August 97). Excerpta Medica, Amsterdam. 7. Wang, S. P., and J. T. Grayston. 9. Egg infectivity assay of trachoma virus. Proc. Soc. Exp. Biol. Med. 5: Wang, S. P., and J. T. Grayston A potency test for trachoma vaccine utilizing the mouse toxicity prevention test. Amer. J. Ophthalmol. 63: Wang, S. P., and J. T. Grayston. 97. Immunological relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. Amer. J. Ophthalmol. 7: Wang, S. P., and J. T. Grayston. 97. Immunological classification of TRIC and related strains with micro immunofluorescence, p. 35- In R. L. Nichols, (ed.), Trachoma (Proceedings of a symposium held in Boston, Massachusetts, August 97). Excerpta Medica, Amsterdam.. Wang, S. P., J. T. Grayston, and J. L. Gale Three new immunologic types of trachoma-inclusion conjunctivitis organisms. J. Immunol.. Downloaded from on October, by guest