Successful isolation of Treponema pallidum strains from patients’ cryopreserved ulcer exudate using the rabbit model


Autoři: Lara E. Pereira aff001;  Samantha S. Katz aff001;  Yongcheng Sun aff001;  Patrick Mills aff002;  Willie Taylor aff002;  Patricia Atkins aff003;  Charles M. Thurlow aff004;  Kai-Hua Chi aff001;  Damien Danavall aff001;  Nicholas Cook aff004;  Tamanna Ahmed aff004;  Alyssa Debra aff004;  Susan Philip aff005;  Stephanie Cohen aff005;  Kimberly A. Workowski aff001;  Ellen Kersh aff001;  Yetunde Fakile aff001;  Cheng Y. Chen aff001;  Allan Pillay aff001
Působiště autorů: Division of STD Prevention, Centers for Disease Control and Prevention, Atlanta, GA, United States of America aff001;  Division of Scientific Resources, Centers for Disease Control and Prevention, Atlanta, GA, United States of America aff002;  Charles River Laboratories, Wilmington, MA, United States of America aff003;  Oak Ridge Institute for Science and Education, Oak Ridge, TN, United States of America aff004;  San Francisco Department of Public Health, San Francisco, CA, United States of America aff005;  Emory University Department of Medicine, Atlanta, GA, United States of America aff006
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227769

Souhrn

Clinical isolates of Treponema pallidum subspecies pallidum (T. pallidum) would facilitate study of prevalent strains. We describe the first successful rabbit propagation of T. pallidum from cryopreserved ulcer specimens. Fresh ulcer exudates were collected and cryopreserved with consent from syphilis-diagnosed patients (N = 8). Each of eight age-matched adult male rabbits were later inoculated with a thawed specimen, with two rabbits receiving 1.3 ml intratesticularly (IT), and six receiving 0.6 ml intravenously (IV) and IT. Monitoring of serology, blood PCR and orchitis showed that T. pallidum grew in 2/8 rabbits that were inoculated IV and IT with either a penile primary lesion specimen (CDC-SF003) or a perianal secondary lesion specimen (CDC-SF007). Rabbit CDC-SF003 was seroreactive by T. pallidum Particle Agglutination (TP-PA) and Rapid Plasma Reagin (RPR) testing, PCR+, and showed orchitis by week 6. Euthanasia was performed in week 7, with treponemal growth in the testes confirmed and quantified by qPCR and darkfield microscopy (DF). Serial passage of the extract in a second age-matched rabbit also yielded treponemes. Similarly, rabbit CDC-SF007 showed negligible orchitis, but was seroreactive and PCR+ by week 4 and euthanized in week 6 to yield T. pallidum, which was further propagated by second passage. Using the 4-component molecular typing system for syphilis, 3 propagated strains (CDC-SF003, CDC-SF007, CDC-SF008) were typed as 14d9f, 14d9g, and 14d10c, respectively. All 3 isolates including strain CDC-SF011, which was not successfully propagated, had the A2058G mutation associated with azithromycin resistance. Our results show that immediate cryopreservation of syphilitic ulcer exudate can maintain T. pallidum viability for rabbit propagation.

Klíčová slova:

Blood – Euthanasia – Lesions – Rabbits – Syphilis – Testes – Ulcers – Treponema pallidum


Zdroje

1. Cox DL. Culture of Treponema pallidum. Methods Enzymol. 1994;236:390–405. Epub 1994/01/01. doi: 10.1016/0076-6879(94)36029-4 7968624.

2. Fieldsteel AH, Cox DL, Moeckli RA. Cultivation of virulent Treponema pallidum in tissue culture. Infect Immun. 1981;32(2):908–15. Epub 1981/05/01. 7019081; PubMed Central PMCID: PMC351528.

3. Kast CC, Kolmer JA. Concerning the cultivation of Spirochaeta pallida. Am J Syph. 1929;13:419.

4. Noguchi H. Certain alterations in biological properties of spirochaetes through artificial cultivation. Ann Inst Pasteur. 1916;30:1–4.

5. Norris SJ, Cox DL, Weinstock GM. Biology of Treponema pallidum: correlation of functional activities with genome sequence data. J Mol Microbiol Biotechnol. 2001;3(1):37–62. Epub 2001/02/24. 11200228.

6. Norris SJ, Edmondson DG. Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system. Infect Immun. 1987;53:534–9.

7. Schereschewsky J. Züchtung der Spirochaete pallida (Schaudinn) Dtsch Med Wochenschr. 1909; 35:835.

8. Edmondson DG, Hu B, Norris SJ. Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum. MBio. 2018;9(3). Epub 2018/06/28. doi: 10.1128/mBio.01153-18 29946052; PubMed Central PMCID: PMC6020297.

9. Lukehart SA, Marra CM. Isolation and laboratory maintenance of Treponema pallidum. Curr Protoc Microbiol. 2007;Chapter 12:Unit 12A 1. Epub 2008/09/05. doi: 10.1002/9780471729259.mc12a01s7 18770607.

10. Henao-Martinez AF, Johnson SC. Diagnostic tests for syphilis: New tests and new algorithms. Neurol Clin Pract. 2014;4(2):114–22. Epub 2014/04/01. doi: 10.1212/01.CPJ.0000435752.17621.48 27606153; PubMed Central PMCID: PMC4999316.

11. Ratnam S. The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol. 2005;16(1):45–51. Epub 2007/12/27. doi: 10.1155/2005/597580 18159528; PubMed Central PMCID: PMC2095002.

12. Rice M, Fitzgerald TJ. Detection and functional characterization of early appearing antibodies in rabbits with experimental syphilis. Can J Microbiol. 1984;31:62–7.

13. Centers for Disease Control and Prevention. 2016 Sexually transmitted diseases surveillance: Syphilis US Department of Health and Human Services, CDC. 2016;https://www.cdc.gov/std/stats16/syphilis.htm.

14. CDC Call to Action. Let's work together to stem the tide of rising syphilis in the United States. https://wwwcdcgov/std/syphilis/syphiliscalltoactionapril2017pdf. 2017.

15. Centers for Disease Control and Prevention. Clinical advisory: Ocular syphilis in the United States. https://wwwcdcgov/std/syphilis/clinicaladvisoryos2015htm. 2016.

16. Baker-Zander SA, Lukehart SA. Efficacy of cefmetazole in the treatment of active syphilis in the rabbit model. Antimicrob Agents Chemother. 1989;33(9):1465–9. Epub 1989/09/01. doi: 10.1128/aac.33.9.1465 2684008; PubMed Central PMCID: PMC172684.

17. Lukehart SA, Baker-Zander SA. Roxithromycin (RU 965): effective therapy for experimental syphilis infection in rabbits. Antimicrob Agents Chemother. 1987;31(2):187–90. Epub 1987/02/01. doi: 10.1128/aac.31.2.187 3551828; PubMed Central PMCID: PMC174689.

18. Lukehart SA, Baker-Zander SA, Holmes KK. Efficacy of aztreonam in treatment of experimental syphilis in rabbits. Antimicrob Agents Chemother. 1984;25(3):390–1. Epub 1984/03/01. doi: 10.1128/aac.25.3.390 6372685; PubMed Central PMCID: PMC185528.

19. Lukehart SA, Fohn MJ, Baker-Zander SA. Efficacy of azithromycin for therapy of active syphilis in the rabbit model. J Antimicrob Chemother. 1990;25 Suppl A:91–9. Epub 1990/01/01. doi: 10.1093/jac/25.suppl_a.91 2154443.

20. Marra C, Baker-Zander SA, Hook EW, 3rd, Lukehart SA. An experimental model of early central nervous system syphilis. J Infect Dis. 1991;163(4):825–9. Epub 1991/04/01. doi: 10.1093/infdis/163.4.825 2010635.

21. Molini BJ, Tantalo LC, Sahi SK, Rodriguez VI, Brandt SL, Fernandez MC, et al. Macrolide Resistance in Treponema pallidum Correlates With 23S rDNA Mutations in Recently Isolated Clinical Strains. Sex Transm Dis. 2016;43(9):579–83. Epub 2016/08/12. doi: 10.1097/OLQ.0000000000000486 27513385; PubMed Central PMCID: PMC4982755.

22. Smith JL, Singer JA, Reynolds DH, Moore MB Jr., Yobs AR, Clark JW Jr. Experimental Ocular Syphilis and Neurosyphilis. Br J Vener Dis. 1965;41:15–23. Epub 1965/03/01. doi: 10.1136/sti.41.1.15 14275954; PubMed Central PMCID: PMC1047693.

23. Tantalo LC, Lukehart SA, Marra CM. Treponema pallidum strain-specific differences in neuroinvasion and clinical phenotype in a rabbit model. J Infect Dis. 2005;191(1):75–80. Epub 2004/12/14. doi: 10.1086/426510 15593006.

24. Cameron CE. Syphilis Vaccine Development: Requirements, Challenges, and Opportunities. Sex Transm Dis. 2018;45(9S Suppl 1):S17–S9. Epub 2018/03/13. doi: 10.1097/OLQ.0000000000000831 29528992; PubMed Central PMCID: PMC6089657.

25. Kumar S, Caimano MJ, Anand A, Dey A, Hawley KL, LeDoyt ME, et al. Sequence Variation of Rare Outer Membrane Protein beta-Barrel Domains in Clinical Strains Provides Insights into the Evolution of Treponema pallidum subsp. pallidum, the Syphilis Spirochete. MBio. 2018;9(3). Epub 2018/06/14. doi: 10.1128/mBio.01006-18 29895642; PubMed Central PMCID: PMC6016234.

26. Lithgow KV, Hof R, Wetherell C, Phillips D, Houston S, Cameron CE. A defined syphilis vaccine candidate inhibits dissemination of Treponema pallidum subspecies pallidum. Nat Commun. 2017;8:14273. Epub 2017/02/02. doi: 10.1038/ncomms14273 28145405; PubMed Central PMCID: PMC5296639.

27. LaFond RE, Centurion-Lara A, Godornes C, Rompalo AM, Van Voorhis WC, Lukehart SA. Sequence diversity of Treponema pallidum subsp. pallidum tprK in human syphilis lesions and rabbit-propagated isolates. J Bacteriol. 2003;185(21):6262–8. Epub 2003/10/18. doi: 10.1128/JB.185.21.6262-6268.2003 14563860; PubMed Central PMCID: PMC219401.

28. LaFond RE, Centurion-Lara A, Godornes C, Van Voorhis WC, Lukehart SA. TprK sequence diversity accumulates during infection of rabbits with Treponema pallidum subsp. pallidum Nichols strain. Infect Immun. 2006;74(3):1896–906. Epub 2006/02/24. doi: 10.1128/IAI.74.3.1896-1906.2006 16495565; PubMed Central PMCID: PMC1418662.

29. Morgan CA, Lukehart SA, Van Voorhis WC. Protection against syphilis correlates with specificity of antibodies to the variable regions of Treponema pallidum repeat protein K. Infect Immun. 2003;71(10):5605–12. Epub 2003/09/23. doi: 10.1128/IAI.71.10.5605-5612.2003 14500480; PubMed Central PMCID: PMC201104.

30. Reid TB, Molini BJ, Fernandez MC, Lukehart SA. Antigenic variation of TprK facilitates development of secondary syphilis. Infect Immun. 2014;82(12):4959–67. Epub 2014/09/17. doi: 10.1128/IAI.02236-14 25225245; PubMed Central PMCID: PMC4249288.

31. Beale MA, MArks M, Sahi SK, Tantalo LC, Nori AV, French P, et al. Genomic epidemiology of syphilis reveals independent emergence of macrolide resistance across multiple circulating lineages. BioRxiv. 2018;https://doi.org/10.1101/413161

32. Grimes M, Sahi SK, Godornes BC, Tantalo LC, Roberts N, Bostick D, et al. Two mutations associated with macrolide resistance in Treponema pallidum: increasing prevalence and correlation with molecular strain type in Seattle, Washington. Sex Transm Dis. 2012;39(12):954–8. doi: 10.1097/OLQ.0b013e31826ae7a8 23191949; PubMed Central PMCID: PMC3668457.

33. Rolfs RT, Joesoef MR, Hendershot EF, Rompalo AM, Augenbraun MH, Chiu M, et al. A randomized trial of enhanced therapy for early syphilis in patients with and without human immunodeficiency virus infection. The Syphilis and HIV Study Group. N Engl J Med. 1997;337(5):307–14. Epub 1997/07/31. doi: 10.1056/NEJM199707313370504 9235493.

34. Sena A, Pillay A, Cox DL, Radolf JD. Treponema and Brachyspira, human host-associated spirochetes. In: J.H. J, M.A. P, K.C. C, G. F, M.L. L, S.S. R, et al., editors. Manual of clinical microbiology. 1. 11th ed ed. Washington, D.C.: ASM Press; 2015.

35. Pillay A, Chi CH, Kwakye C, Danavall D, Taleo F, Katz S, et al. New diagnostics for syphilis and yaws and detection of haemophilus ducreyi in cutaneous lesions in children BMJ Sexual Trans Infect. 2015;19:S04.2.

36. Pillay A, Liu H, Chen CY, Holloway B, Sturm AW, Steiner B, et al. Molecular subtyping of Treponema pallidum subspecies pallidum. Sex Transm Dis. 1998;25(8):408–14. Epub 1998/10/17. doi: 10.1097/00007435-199809000-00004 9773432.

37. Smajs D, Norris SJ, Weinstock GM. Genetic diversity in Treponema pallidum: implications for pathogenesis, evolution and molecular diagnostics of syphilis and yaws. Infect Genet Evol. 2012;12(2):191–202. Epub 2011/12/27. doi: 10.1016/j.meegid.2011.12.001 22198325; PubMed Central PMCID: PMC3786143.

38. Cejkova D, Zobanikova M, Chen L, Pospisilova P, Strouhal M, Qin X, et al. Whole genome sequences of three Treponema pallidum ssp. pertenue strains: yaws and syphilis treponemes differ in less than 0.2% of the genome sequence. PLoS Negl Trop Dis. 2012;6(1):e1471. Epub 2012/02/01. doi: 10.1371/journal.pntd.0001471 22292095; PubMed Central PMCID: PMC3265458.

39. Fraser CM, Norris SJ, Weinstock GM, White O, Sutton GG, Dodson R, et al. Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science. 1998;281(5375):375–88. Epub 1998/07/17. doi: 10.1126/science.281.5375.375 9665876.

40. Giacani L, Jeffrey BM, Molini BJ, Le HT, Lukehart SA, Centurion-Lara A, et al. Complete genome sequence and annotation of the Treponema pallidum subsp. pallidum Chicago strain. J Bacteriol. 2010;192(10):2645–6. Epub 2010/03/30. doi: 10.1128/JB.00159-10 20348263; PubMed Central PMCID: PMC2863575.

41. Matejkova P, Strouhal M, Smajs D, Norris SJ, Palzkill T, Petrosino JF, et al. Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays. BMC Microbiol. 2008;8:76. Epub 2008/05/17. doi: 10.1186/1471-2180-8-76 18482458; PubMed Central PMCID: PMC2408589.

42. Petrosova H, Zobanikova M, Cejkova D, Mikalova L, Pospisilova P, Strouhal M, et al. Whole genome sequence of Treponema pallidum ssp. pallidum, strain Mexico A, suggests recombination between yaws and syphilis strains. PLoS Negl Trop Dis. 2012;6(9):e1832. Epub 2012/10/03. doi: 10.1371/journal.pntd.0001832 23029591; PubMed Central PMCID: PMC3447947.

43. Pinto M, Borges V, Antelo M, Pinheiro M, Nunes A, Azevedo J, et al. Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation. Nat Microbiol. 2016;2:16190. Epub 2016/10/18. doi: 10.1038/nmicrobiol.2016.190 27748767.

44. Strouhal M, Smajs D, Matejkova P, Sodergren E, Amin AG, Howell JK, et al. Genome differences between Treponema pallidum subsp. pallidum strain Nichols and T. paraluiscuniculi strain Cuniculi A. Infect Immun. 2007;75(12):5859–66. Epub 2007/09/26. doi: 10.1128/IAI.00709-07 17893135; PubMed Central PMCID: PMC2168363.

45. Tong ML, Zhao Q, Liu LL, Zhu XZ, Gao K, Zhang HL, et al. Whole genome sequence of the Treponema pallidum subsp. pallidum strain Amoy: An Asian isolate highly similar to SS14. PLoS One. 2017;12(8):e0182768. Epub 2017/08/09. doi: 10.1371/journal.pone.0182768 28787460; PubMed Central PMCID: PMC5546693.

46. Zobanikova M, Mikolka P, Cejkova D, Pospisilova P, Chen L, Strouhal M, et al. Complete genome sequence of Treponema pallidum strain DAL-1. Stand Genomic Sci. 2012;7(1):12–21. Epub 2013/03/02. doi: 10.4056/sigs.2615838 23449808; PubMed Central PMCID: PMC3570794.

47. Arora N, Schuenemann VJ, Jager G, Peltzer A, Seitz A, Herbig A, et al. Origin of modern syphilis and emergence of a pandemic Treponema pallidum cluster. Nat Microbiol. 2016;2:16245. Epub 2016/12/06. doi: 10.1038/nmicrobiol.2016.245 27918528.

48. Grillova L, Giacani L, Mikalova L, Strouhal M, Strnadel R, Marra C, et al. Sequencing of Treponema pallidum subsp. pallidum from isolate UZ1974 using Anti-Treponemal Antibodies Enrichment: First complete whole genome sequence obtained directly from human clinical material. PLoS One. 2018;13(8):e0202619. Epub 2018/08/22. doi: 10.1371/journal.pone.0202619 30130365; PubMed Central PMCID: PMC6103504.

49. Sun J, Meng Z, Wu K, Liu B, Zhang S, Liu Y, et al. Tracing the origin of Treponema pallidum in China using next-generation sequencing. Oncotarget. 2016;7(28):42904–18. Epub 2016/06/28. doi: 10.18632/oncotarget.10154 27344187; PubMed Central PMCID: PMC5189996.

50. Lukehart SA, Hook EW 3rd, Baker-Zander SA, Collier AC, Critchlow CW, Handsfield HH. Invasion of the central nervous system by Treponema pallidum: implications for diagnosis and treatment. Ann Intern Med. 1988;109(11):855–62. doi: 10.7326/0003-4819-109-11-855 3056164.

51. National Research Council. Guide for the care and use of laboratory animals. National Academies Press (US), Washington DC. 2011;8th Edition. doi: 10.1258/la.2010.010031

52. American Veterinary Medical Association. AVMA guidelines for the euthanasia of animals: 2013 edition. https://wwwavmaorg/KB/Policies/Documents/euthanasiapdf. 2013.

53. Pillay A, Liu H, Ebrahim S, Chen CY, Lai W, Fehler G, et al. Molecular typing of Treponema pallidum in South Africa: cross-sectional studies. J Clin Microbiol. 2002;40(1):256–8. Epub 2002/01/05. doi: 10.1128/JCM.40.1.256-258.2002 11773125; PubMed Central PMCID: PMC120137.

54. Katz KA, Pillay A, Ahrens K, Kohn RP, Hermanstyne K, Bernstein KT, et al. Molecular epidemiology of syphilis—San Francisco, 2004–2007. Sex Transm Dis. 2010;37(10):660–3. Epub 2010/07/06. doi: 10.1097/OLQ.0b013e3181e1a77a 20601928.

55. Chen CY, Chi KH, Pillay A, Nachamkin E, Su JR, Ballard RC. Detection of the A2058G and A2059G 23S rRNA gene point mutations associated with azithromycin resistance in Treponema pallidum by use of a TaqMan real-time multiplex PCR assay. J Clin Microbiol. 2013;51(3):908–13. Epub 2013/01/04. doi: 10.1128/JCM.02770-12 23284026; PubMed Central PMCID: PMC3592075.

56. Pillay A, Lee MK, Slezak T, Katz SS, Sun Y, Chi KH, et al. Increased Discrimination of Treponema pallidum Strains by Subtyping With a 4-Component System Incorporating a Mononucleotide Tandem Repeat in rpsA. Sex Transm Dis. 2019;46(4):e42–e5. Epub 2018/10/27. doi: 10.1097/OLQ.0000000000000935 30365462.

57. Collart P, Franceschini P, Durel P. Experimental rabbit syphilis. Br J Vener Dis. 1971;47(6):389–400. Epub 1971/12/01. doi: 10.1136/sti.47.6.389 5160160; PubMed Central PMCID: PMC1048248.

58. Korting HC, Haag R, Walter D, Riethmuller U, Meurer M. Efficacy of ceftizoxime in the treatment of incubating syphilis in rabbits. Chemotherapy. 1993;39(5):331–5. Epub 1993/09/01. doi: 10.1159/000239145 8370324.

59. Lukehart SA, Shaffer JM, Baker-Zander SA. A subpopulation of Treponema pallidum is resistant to phagocytosis: possible mechanism of persistence. J Infect Dis. 1992;166(6):1449–53. Epub 1992/12/01. doi: 10.1093/infdis/166.6.1449 1431264.

60. Morgan CA, Molini BJ, Lukehart SA, Van Voorhis WC. Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection. J Immunol. 2002;169(2):952–7. Epub 2002/07/05. doi: 10.4049/jimmunol.169.2.952 12097401.

61. Salazar JC, Rathi A, Michael NL, Radolf JD, Jagodzinski LL. Assessment of the kinetics of Treponema pallidum dissemination into blood and tissues in experimental syphilis by real-time quantitative PCR. Infect Immun. 2007;75(6):2954–8. Epub 2007/04/18. doi: 10.1128/IAI.00090-07 17438037; PubMed Central PMCID: PMC1932886.

62. Sell S, Baker-Zander S, Powell HC. Experimental syphilitic orchitis in rabbits: ultrastructural appearance of Treponema pallidum during phagocytosis and dissolution by macrophages in vivo. Lab Invest. 1982;46(4):355–64. Epub 1982/04/01. 7040799.

63. Liu H, Rodes B, Chen CY, Steiner B. New tests for syphilis: rational design of a PCR method for detection of Treponema pallidum in clinical specimens using unique regions of the DNA polymerase I gene. J Clin Microbiol. 2001;39(5):1941–6. Epub 2001/04/28. doi: 10.1128/JCM.39.5.1941-1946.2001 11326018; PubMed Central PMCID: PMC88053.

64. Lin LR, Zhu XZ, Liu D, Liu LL, Tong ML, Yang TC. Are non-treponemal tests suitable for monitoring syphilis treatment Efficacy? Evidence from rabbit infection models. Clin Microbiol Infect. 2019. Epub 2019/06/19. doi: 10.1016/j.cmi.2019.06.004 31212076.

65. Tong ML, Zhang HL, Zhu XZ, Fan JY, Gao K, Lin LR, et al. Re-evaluating the sensitivity of the rabbit infectivity test for Treponema pallidum in modern era. Clin Chim Acta. 2017;464:136–41. Epub 2016/11/24. doi: 10.1016/j.cca.2016.11.031 27876463.

66. Miller JN. The appearance and persistence of VDRL, RPCF, and TPI antibody during the course and treatment of experimental syphilis in the rabbit. J Invest Dermatol. 1963;42:367–71.

67. Mothershed SM, Yobs AR, Clark JW Jr., Comparison of the VDRL slide, TPI, and FTA-ABS tests in experimental syphilis in rabbits. Br J Vener Dis. 1967;43(4):267–71. Epub 1967/12/01. doi: 10.1136/sti.43.4.267 4865687; PubMed Central PMCID: PMC1047899.

68. Gao K, Shen X, Lin Y, Zhu XZ, Lin LR, Tong ML, et al. Origin of Nontreponemal Antibodies During Treponema pallidum Infection: Evidence From a Rabbit Model. J Infect Dis. 2018;218(5):835–43. Epub 2018/04/28. doi: 10.1093/infdis/jiy241 29701849.

69. Turner TB, Hollander DH. Biology of the treponematoses based on studies carried out at the International Treponematosis Laboratory Center of the Johns Hopkins University under the auspices of the World Health Organization. Monogr Ser World Health Organ. 1957;35:3–266.


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