Züchtung der Spirochaete pallida (Schaudinn)

Schereschewsky, J.

doi:10.1055/s-0029-1201453

Cited by 12 publications

(6 citation statements)

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“…4 Strain typing was performed on testes samples that yielded PCR+ results. 5 Weakly positive TP-PA titer +/-1:80 6 Isolate could not be typed due to low number of spirochetes after rabbit propagation 7 Strain type observed with residual lesion swab specimen in NRS https://doi.org/10.1371/journal.pone.0227769.t002…”

Section: Propagation Of T Pallidum From Cryopreserved Penile Ulcer Smentioning

confidence: 99%

“…Research on the utility of various culture media and methods for in vitro propagation have yielded inconsistent results over the years. Sustained passage has been limited or unattainable, with low yields, contamination, and/or loss of viability, virulence and pathogenicity being reported [1][2][3][4][5][6][7]. However, a recent study suggests that in vitro propagation of T. pallidum is possible using a microaerobic, nutrient-defined rabbit cell culture system, with sustained propagation of viable treponemes for >6 months [8].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Propagation Of T Pallidum From Cryopreserved Penile Ulcer Smentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…Members of this group of organisms are morphologically indistinguishable and have nearly identical genomes, with the T. pallidum subspecies sharing >99.78% sequence identity with one another and ∼99.2% identity with T. paraluiscuniculi ( 9 , 10 ). Attempts to culture the T. pallidum -related pathogens had been unsuccessful for over 100 years despite concerted efforts ( 11 – 14 ), necessitating the maintenance of strains through serial infection of rabbits or other mammals ( 15 , 16 ). This fastidious nature appears to be associated with an extreme adaptation to life within mammalian tissue, accompanied by genome reduction and the substantial loss of metabolic and biosynthetic capabilities ( 3 , 14 , 17 , 18 ).…”

Section: Introductionmentioning

confidence: 99%

Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains

Edmondson

DeLay

Kowis

et al. 2021

mBio

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The bacterium that causes syphilis, Treponema pallidum subsp. pallidum, has now been cultured in vitro continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the T. pallidum subsp. endemicum Bosnia A strain has been achieved. During in vitro passage, T. pallidum subsp. pallidum exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle’s minimal essential medium as the basal medium was not effective in sustaining growth of T. pallidum subsp. pallidum beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture. T. pallidum subsp. pallidum growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol, d-mannitol, or l-histidine did not dramatically affect survival or growth. Additionally, T. pallidum subsp. pallidum can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the T. pallidum subsp. pallidum culture system that are required for sustained, long-term survival and multiplication. IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum. Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.

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“… pallidum ( T. pallidum ), the causative agent of syphilis, was first identified by Schaudinn and Hoffman in 1905 ( 5 , 6 ) as “very light, thin spiraled microorganisms, turning around their largest length and moving back and forth.” Rapid progress in the study of this bacterium was made within 5 years, with the verification of the presence of spirochetes in experimentally infected animals by Metchnikoff and Roux ( 7 ), the invention of dark-field microscopy for easy visualization of T. pallidum by Karl Landsteiner, development of the first serological test for syphilis by von Wassermann et al ( 8 ), and the introduction of arsphenamine as an effective, relatively nontoxic antisyphilis agent by Paul Ehrlich ( 9 , 10 ). Successful culture of T. pallidum was reported almost immediately and during the subsequent decades ( 11 , 12 ), but these reports were found to be either irreproducible or the result of contamination with nonpathogenic Treponema species that colonize human skin ( 13 ). During the 1970s, progress was made in characterizing some T. pallidum physiological properties, most notably its microaerophilic nature and improved survival in the presence of mammalian cells ( 14 ).…”

Section: Introductionmentioning

confidence: 99%

Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum

Edmondson

Hu²,

Norris

2018

mBio

179

173

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Investigation of Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, has been hindered by an inability to culture the organism continuously in vitro despite more than a century of effort. In this study, long-term logarithmic multiplication of T. pallidum was attained through subculture every 6 to 7 days and periodic feeding using a modified medium (T. pallidum culture medium 2 [TpCM-2]) with a previously described microaerobic, rabbit epithelial cell coincubation system. Currently, cultures have maintained continuous growth for over 6 months with full retention of viability as measured by motility and rabbit infectivity. This system has been applied successfully to the well-studied Nichols strain of T. pallidum, as well as to two recent syphilis isolates, UW231B and UW249B. Light microscopy and cryo-electron microscopy showed that in vitro-cultured T. pallidum retains wild-type morphology. Further refinement of this long-term subculture system is expected to facilitate study of the physiological, genetic, pathological, immunologic, and antimicrobial susceptibility properties of T. pallidum subsp. pallidum and closely related pathogenic Treponema species and subspecies.

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Züchtung der Spirochaete pallida (Schaudinn)

Cited by 12 publications

References 0 publications

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

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

Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains

Long-Term In Vitro Culture of the Syphilis Spirochete Treponema pallidum subsp. pallidum

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