Brit. 7. vener. Dis. (1973) 49, 239 Br J Vener Dis: first published as 10.1136/sti.49.3.239 on 1 June 1973. Downloaded from

Syphilitic immunofluorescence in experimental relapsing fever

D. J. M. WRIGHT* Department of Venereal Diseases, Guy's Hospital, London, S.E. 1 AND C. D. GINGER Division of Parasitology, National Institute for Medical Research, Mill Hill, London, NW7 1AA

Immunological cross-reactivity has been shown The USPHS Manual of Syphilis Serology (1969) was between related genera of the family Treponemataceae. followed when carrying out the fluorescent treponemal Extracts of Borrelia cross-react with antibodies in antibody test on T. pallidum (Nichols strain) with the syphilitic serum (Saurino and De Lamater, 1952) following exceptions: it was not found necessary to inactivate the serum at 56°C. and the test was conducted and produce positive skin tests in the later stages of at room temperature. When staining with serum and syphilis (Ranque, Quilici, and Assadourian, 1967). conjugate, the buffer did not contain 2 per cent. Tween 80. The fluorescent treponemal antibody absorption The method for B. duttoni was that described by Coffey test (FTA-ABS) is at present considered to be and Eveland (1967) and for various leptospirae, L. specific, as its name implies, for treponemal disease. icterohaemorrhagiae, L. canicola, and L. pomona, that of However, we have found that Borrelia infection in Coffin and Maestrone (1962). Both techniques involved mice also induces an antibody that reacts in the preliminary fixation in acetone and then through graded FTA-ABS test, which would suggest that Treponema alcohols with final fixation in methanol. Sera were taken and Borrelia share a common antigen. in 5 ,ul. amounts, either undiluted or as reciprocal dilu- tions of 5, 25, 125, and 625 in phosphate buffered saline. http://sti.bmj.com/ Specific goat anti-mouse globulin conjugated with Material and methods fluorescein isothiocyanate (Hyland) was used at a dilution ORIGIN OF TEST SERA of 1/80. Results were recorded as negative (-), equivocal B. duttoni (Wellcome mouse strain), the causative (±), weak positive (wk.+), and positive (+). There was organism of tick-borne replasing fever, was used. Inocula no differentiation between + + + and + + + + as used were obtained by diluting infected mouse blood with in the routine FTA-ABS test. The microscope used was citrated saline. Male Parkes strain mice (18-20 g.) were a 'Zetopan' (Reichert) with an HB 200 high pressure infected by intraperitoneal injection and blood smears mercury vapour lamp. All tests were carried out in on October 1, 2021 by guest. Protected copyright. were taken from each mouse immediately before death. duplicate, and read independently by two observers, The ratio of Borrelia to erythrocytes was determined by concordance being required for assessment of results as microscopical examination after Leishman staining. positive. Three to four mice were killed and bled at each time of ABSORPTION TESTS sampling and the pooled sera were stored at -20°C. The effects of with the Two infections were absorption 'sorbent' (Burroughs without preservative. experimental Wellcome) used in the FTA absorption test and Reiter produced, after the administration of 4 x 108 organisms protein (Burroughs Wellcome) were tested on sera from into 25 mice and 1-3 x 108 into 35 mice. Six pooled mice experimentally infected with B. duttoni and T. samples of sera were collected in the first experiment pallidum. Indirect immunofluorescence was then carried and nine in the second experiment. Further studies were out on Borrelia and T. pallidum substrates. The absorbing carried out on four sera reactive in the unabsorbed FTA potency of the sorbent was assessed using human non- test, with titres greater than 320, from mice with asympto- specific positive control sera. These were tested at matic syphilis. Control sera were obtained from forty dilutions of 1 in 5, 1 in 25, and 1 in 125 in saline and in normal Parkes strain mice and pooled into twelve samples. sorbent against T. pallidum and Reiter antigens. IMMUNOFLUORESCENT TESTS TESTS FOR CARDIOLIPIN ANTIBODIES Suspensions of spirochaetes were dried in advance on The Wassermann antibody was assayed by the VDRL clear areas of slides, treated with a Teflon (Hiflon PTFE test (USPHS Manual, 1969) and the cardiolipin (fluores- Gallenkamp) water repellent (Goldman, 1968). cent) antibody (Wright, Doniach, Lessof, Turk, Grimble, Received for publication August 21, 1972 and Catterall, 1970) using frozen rat, mouse, and human *.In receipt of an M.R.C. grant kidney antigen. 240 British 7ournal of Venereal Diseases Br J Vener Dis: first published as 10.1136/sti.49.3.239 on 1 June 1973. Downloaded from

Results TABLE Development offluorescent antibodies during The sera from mice infected with B. duttoni gave Borrelia parasitaemia, using different diluents positive immunofluorescence with T. pallidum anti- Borrelia Treponemal antibodies gen (see Table). The appearance of the fluorescent Days after Experiment Borrelia Reiter treponemal antibody (FTA) followed the observed no. infection Saline Saline Sorbent protein parasitaemia, with the maximum antibody levels on I 0 Day 4, when spirochaetes began to disappear from 1 the blood (see Figure). A subsequent fall in antibody 2 - + 5 wk 5 - levels occurred and sera were 3 + 5 + 5 + 5 - FTA-negative by 6 + 5 + 25 + 25 - Day 28, indicating the transient nature ofthe response 14-18 + 5 + 5 + 25 - (Figure and Table). Titres were established against 2 0 the homologous B. duttoni antigen. The rise and fall 1 of the titre was as for T. pallidum substrates but the 2 3 + 5 + 5 height of the titres was lower and the duration of 4 + 25 + 25 ± 125 positive reactions was shorter. 5 + 25 wk 25 ± 25 14 +5 + 25 + 25 21 + 5 + 5 2*5 Ex p 1 4 0 X 108 B. duttoni 28 Exp 2 3 X 108 B. duttoni o ho. v Antibody absent A u 2.0- -,lt Antibody present B. duttoni antigens gave a positive immunofluores- co * * Borre/li found in s mears cent reaction with the four sera from asymptomatic from Exp I on doy 10 syphilitic mice. This reactivity was likewise abolished 8 by the undiluted Reiter protein antigen. L. 1.5 The VDRL and cardiolipin fluorescence tests were negative and no antinuclear factors were seen 0. at any stage. Developmentof FTAAbs Exp.2 http://sti.bmj.com/ 0 4'- 4 /A/A/I Discussion -J O1.0 - The FTA-ABS test is a standard confirmatory test 0- Relapse1 Exp 2 for treponemal infection, but there is no doubt that the common group antigen which occurs in a variety of spirochaetes (Deacon and Hunter, 1962) can *. I I I I I I .I "V produce antibody in experimental animals which is 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 21 28 detected by the FTA test (Tringali and Cox, 1970). on October 1, 2021 by guest. Protected copyright. Days after injection Therefore, antibody to commensal spirochaetes could account for some anomalous false positive FIGURE FTA-ABS test after parasitaemia due to results in human sera. inoculation with B. duttoni (Wellcome mouse strain) In our experiments, Borrelia induced a positive FTA-ABS reaction wbich appeared at the height of Forty control sera from uninfected mice, with the B. duttoni parasitaemia and then rapidly declined. phosphate buffered saline as diluent, gave one Borrelia agglutinating (Balteanu, Russ, andVoiculescu definite positive and one doubtful positive result at 1948) and adhering antibodies (Schuhardt, 1942) a dilution of 1/5. Sorbent abolished both these behave in an identical way, quite unlike Borrelia positives. No pooled specimens of the control sera immobilizing antibodies whose persistence and were positive using phosphate-buffered saline diluent. strain specificity is analogous to the treponeme The treponemal antibody titre in the mice infected immobilisins (Coffey and Eveland, 1967). Sorbent with Borrelia was not reduced using sorbent, and Reiter protein antigen represent different despite the ability of sorbent to abolish treponemal extracts of T. reiteri. It has been recently shown that fluorescence in non-specific control sera. The sorbent is a less efficient absorbent (Wilkinson and highest titres of these sera were 1/25 (two sera) Wiseman, 1971). This finding is confirmed here by and 1/125 (one serum). Undiluted Reiter protein the failure of sorbent diluent to remove the antibody antigen abolished the fluorescence in both the control detected in the FTA test. This treponemal fluores- and test sera (see Table). cence was abolished by using the more effective Syphilitic immunofluorescence in experimental relapsing fever 241 Br J Vener Dis: first published as 10.1136/sti.49.3.239 on 1 June 1973. Downloaded from

Reiter protein. Effective absorption implies that References there is a group antigen shared between T. pallidum BALTEANU, I., Russ, M., and VOICULESCU, M. (1948) and T. reiter and B. duttoni. Conversely, the cross- Arch. roum. Path. exp. Microbiol., 15, 310 reacting fluorescence of mouse syphilitic sera with BRYCESON, A. D. M., PARRY, E. H. O., PERINE, P. L., Borrelia antigen, which resisted sorbent but was WARRELL, D. A., VUKOTICH, D., and LEITHEAD, C. S. abolished by Reiter protein could well be an expres- (1970) Quart. J3. Med., 39, 129 the COFFEY, E. M., and EVELAND, W. C. (1967) J. infect. Dis., sion of the common group antibody. However, 117, 23, 29 failure of Borrelia-infected mice to produce higher COFFIN, D. L., and MAESTRONE, G. (1962) Amer. J3. vet. titres against B. duttoni was possibly due to de- Res., 23, 159 naturation of the B. duttoni antigen. This cross- DEACON, W. E., and HUNTER, E. F. (1962) Proc. Soc. exp. reactivity does not extend to the Leptospirae since Biol. (N.Y.), 110, 352 these organisms were negative in this test. GOLDMAN, M. (1968) 'Fluorescent Antibody Methods', The FTA-ABS test detects an antibody unrelated p. 148. Academic Press, New York and London to cardiolipin antibodies because both the VDRL PLAUT and STEINER (1944)Quoted by Stokes, Beerman, and and the syphilis tissue fluorescence tests were Ingraham antibodies RANQUE, J., QUILICI, M., and ASSADOURIAN, Y. (1967) negative. Mice rarely develop cardiolipin Med. trop., 27, 519 in treponemal disease (Wright and Doniach, 1971), SAURINO, V. R., and DE LAMATER, E. D. (1952) Amer. this hence the negative finding on occasion. J. Syph., 36, 353 Patients with syphilis, when subjected to fever SCHUHARDT, V. T. (1942) In 'A Symposium on Re- therapy using relapsing fever as the pyrogen (Plaut lapsing Fever in the Americas', ed. F. R. Moulton. and Steiner, 1944), showed no cross-protective Amer. Acad. Adv. Sci., Publication 18, p. 58 immunity. The Borrelia agglutination tests (Saurino STOKES, J. H., BEERMAN, H., and INGRAHAM, N. R. (1944) and De Lamater, 1952) and complement-fixing tests 'Modern Clinical Syphilology', 3rd ed. Saunders, kWolstenholm and Gear, 1948) were also negative in Philadelphia of TRINGALI, G. R., and Cox, P. M. (1970) Brit. J. vener. syphilis. Nevertheless, high blood counts Borrelia Dis., 46, 313 are recorded with relapsing fever in man (Bryceson, USPHS (1969) 'Manual of Tests for Syphilis'. Parry, Perine, Warrell, Vukotich, and Leithead, WILKINSON, A. E., and WISEMAN, C. C. (1971) Proc. roy. 1970), and it is possible that the FTA-ABS test with Soc. Med., 64, 422 sorbent may be found positive in such patients. WOLSTENHOLME, B., and GEAR, J. H. S. (1948) Trans. http://sti.bmj.com/ roy. Soc. trop. Med. Hyg., 41, 513 WRIGHT, D. J. M., and DONIACH, D. (1971) Proc. roy. Summary Soc. Med., 64, 419 The development of a transient positive FTA test -, , LESSOF, M. H., TURK, J. L., GRIMBLE, A. S., was found in an experimental B. duttoni infection in and CATTERALL, R. D. (1970) Lancet, 1, 740 mice. This antibody could be removed by undiluted Reiter protein antigen but not by sorbent. The Immuno-fluorescence syphilitique dans la fiavre on October 1, 2021 by guest. Protected copyright. possibility that a positive FTA-ABS test could arise recurrente exp6rimentale in human relapsing fever is discussed. SOMMAIRE We should like to thank Miss A. Parr (Interdepartmental On a observe l'apparition d'une positivite transitoire du Laboratory, Guy's Hospital) and Miss S. Denton (Mill test FTA-ABS dans l'infection experimentale de la Hill) for their technical help, Dr. A. Grimble and Prof. souris par B. duttoni. L'anticorps peut etre dlimine par M. H. Lessof (Guy's Hospital) for their valuable advice l'antigene prot6lque de Reiter non dilue mais non par le and encouragement and Dr. R. Broughton (Department sorbant. La possibilite que l'epreuve FTA-ABS soit of Aerobic Bacteriology, Burroughs Wellcome) for the positive chez l'homme dans la fievre recurrente est strains of Leptospirae. discutee.