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

• Type of Material: Article
• Authors: Lara E. Pereira, Centers for Disease Control and Prevention; Samantha S. Katz, Centers for Disease Control and Prevention; Yongcheng Sun, Centers for Disease Control and Prevention; Patrick Mills, Centers for Disease Control and Prevention; Willie Taylor, Centers for Disease Control and Prevention; Patricia Atkins, Charles River Labs; Charles M. Thurlow, Oak Ridge Institute for Science and Education; Kai-Hua Chi, Centers for Disease Control and Prevention; Damien Danavall, Centers for Disease Control and Prevention; Nicholas Cook, Oak Ridge Institute for Science and Education; Tamanna Ahmed, Oak Ridge Institute for Science and Education; Alyssa Debra, Oak Ridge Institute for Science and Education; Susan Philip, San Francisco Department of Public Health; Stephanie Cohen, San Francisco Department of Public Health; Kimberly Workowski, Emory University; Ellen Kersh, Centers for Disease Control and Prevention; Yetunde Fakile, Centers for Disease Control and Prevention; Cheng Y. Chen, Centers for Disease Control and Prevention; Allan Pillay, Centers for Disease Control and Prevention
• Language: English
• Date: 2020-01-13
• Publisher: PUBLIC LIBRARY SCIENCE
• Publication Version: Final Published Version
• Copyright Statement: This is an open access article, free of all copyright
• License: Creative Commons Universal : Public Domain Dedication
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0227769
• Title of Journal or Parent Work: PLOS ONE
• Volume: 15
• Issue: 1
• Start Page: e0227769
• End Page: e0227769
• Grant/Funding Information: This work was made possible through CDC Division of STD Prevention with partial support from the Advanced Molecular Detection (AMD) program at the CDC. Patricia Atkins [PA] is employed by Charles River Laboratories. Charles River Laboratories provided support in the form of salary for author Patricia Atkins [PA], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
• Keywords: COMPLETE GENOME SEQUENCE; SUBSP PALLIDUM; THERAPY; DIVERSITY; MACROLIDE RESISTANCE; MUTATIONS; Science & Technology; EXPERIMENTAL SYPHILIS; Multidisciplinary Sciences; ACTIVE SYPHILIS; EFFICACY; Science & Technology - Other Topics; CENTRAL-NERVOUS-SYSTEM
• Research Categories: Biology, Anatomy; Biology, Genetics

Abstract

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 (CDCSF003, 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.