Advertisement

Current Microbiology

, Volume 62, Issue 2, pp 518–524 | Cite as

Evaluation of the Use of Selective PCR Amplification of LPS Biosynthesis Genes for Molecular Typing of Leptospira at the Serovar Level

  • Josefa Bezerra da Silva
  • Eneas Carvalho
  • Rudy A. Hartskeerl
  • Paulo L. HoEmail author
Article

Abstract

Leptospirosis is an important epidemic zoonosis worldwide. Currently, there are more than 250 Leptospira pathogenic serovars known that can potentially infect humans. Conventional classification of leptospires with the serovar as the basic taxon, based on serological recognition of lipopolysaccharide (LPS) composition does not correlate well with species determination, based on general genomic features. Here, we investigate the selective amplification of polymorphic regions from the LPS biosynthesis loci (rfb) as a potential tool for serovar typing of Leptospira interrogans species. Eight pairs of primers were designed to target six ORFs from the rfb operon with varying levels of sequence polymorphism. They were tested both separately and multiplexed. Half of these primer pairs produced serovar-specific amplicons, allowing the identification of some specific serovars and also groups of serovars. It was shown that the serovar classification of Leptospira can be accessed by selective amplification of rfb operons in some cases, which may permit a parallel between the serological and the genomic classifications of Leptospira. As a conclusion, the selective amplification of rfb generated promising and already useful results, but it appears necessary to characterize a larger variety of Leptospira genomes or rfb operons to fully develop this method.

Keywords

Multiplex Polymerase Chain Reaction Leptospirosis Naam Serological Classification Predict Amplicon Size 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank FAPESP, CNPq, and Fundação Butantan for financial support.

Supplementary material

284_2010_9738_MOESM1_ESM.docx (446 kb)
Individual PCR amplification of LPS biosynthesis loci fragments using a subset of four primer pairs (pair 37–420, 10–268, 14–201, and 37–110), across several L. interrogans serovars. The use of these primer pairs produced amplicons in serovars in which it was not predicted to occur (according to the alignments performed) and also non-specific amplicons, whose size are incompatible with the expected amplicon size (indicated by an arrow). Serovars in which unpredicted amplicons occurred are presented in bold, and serovars in which non-specific amplicons occurred are italicized; otherwise, when the predicted result was obtained for the serovar, it was underlined. An approximate size marker (bp) is shown for each figure subset. Serovar Hardjo belongs to subtype Hardjoprajitno. (DOCX 446 kb)

References

  1. 1.
    Adler B, de la Pena-Moctezuma A (2009) Leptospira and leptospirosis. Vet Microbiol 140:287–296CrossRefPubMedGoogle Scholar
  2. 2.
    Ahmed N, Devi SM, Valverde ML et al (2006) Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species. Ann Clin Microbiol Antimicrob 5:28CrossRefPubMedGoogle Scholar
  3. 3.
    Ahmed A, Engelberts MF, Boer KR et al (2009) Development and validation of a real-time PCR for detection of pathogenic leptospira species in clinical materials. PLoS ONE 4:e7093CrossRefPubMedGoogle Scholar
  4. 4.
    Bharti AR, Nally JE, Ricaldi JN et al (2003) Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3:757–771CrossRefPubMedGoogle Scholar
  5. 5.
    Brenner DJ, Kaufmann AF, Sulzer KR et al (1999) Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. Int J Syst Bacteriol 49(Pt 2):839–858CrossRefPubMedGoogle Scholar
  6. 6.
    Cai CS, Zhu YZ, Zhong Y et al (2010) Development of O-antigen gene cluster-specific PCRs for rapid typing six epidemic serogroups of Leptospira in China. BMC Microbiol 10:67CrossRefPubMedGoogle Scholar
  7. 7.
    Cerqueira GM, Picardeau M (2009) A century of Leptospira strain typing. Infect Genet Evol 9:760–768CrossRefPubMedGoogle Scholar
  8. 8.
    Corney BG, Colley J, Djordjevic SP et al (1993) Rapid identification of some Leptospira isolates from cattle by random amplified polymorphic DNA fingerprinting. J Clin Microbiol 31:2927–2932PubMedGoogle Scholar
  9. 9.
    Corney BG, Slack AT, Symonds ML et al (2008) Leptospira weilii serovar Topaz, a new member of the Tarassovi serogroup isolated from a bovine source in Queensland, Australia. Int J Syst Evol Microbiol 58:2249–2252CrossRefPubMedGoogle Scholar
  10. 10.
    de la Pena-Moctezuma A, Bulach DM, Adler B (2001) Genetic differences among the LPS biosynthetic loci of serovars of Leptospira interrogans and Leptospira borgpetersenii. FEMS Immunol Med Microbiol 31:73–81PubMedGoogle Scholar
  11. 11.
    Djadid ND, Ganji ZF, Gouya MM et al (2009) A simple and rapid nested polymerase chain reaction-restriction fragment length polymorphism technique for differentiation of pathogenic and nonpathogenic Leptospira spp. Diagn Microbiol Infect Dis 63:251–256CrossRefPubMedGoogle Scholar
  12. 12.
    Faine S, Adler B, Bolin C et al (1999) Leptospira and Leptospirosis, 2nd edn. MediSci, MelbourneGoogle Scholar
  13. 13.
    Galloway RL, Levett PN (2008) Evaluation of a modified pulsed-field gel electrophoresis approach for the identification of Leptospira serovars. Am J Trop Med Hyg 78:628–632PubMedGoogle Scholar
  14. 14.
    Gouveia EL, Metcalfe J, de Carvalho AL et al (2008) Leptospirosis-associated severe pulmonary hemorrhagic syndrome, Salvador, Brazil. Emerg Infect Dis 14:505–508CrossRefPubMedGoogle Scholar
  15. 15.
    Koizumi N, Watanabe H (2005) Leptospirosis vaccines: past, present, and future. J Postgrad Med 51:210–214PubMedGoogle Scholar
  16. 16.
    Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  17. 17.
    Leon A, Pronost S, Fortier G et al (2010) Multilocus sequence analysis for typing Leptospira interrogans and Leptospira kirschneri. J Clin Microbiol 48:581–585CrossRefPubMedGoogle Scholar
  18. 18.
    Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14:296–326CrossRefPubMedGoogle Scholar
  19. 19.
    Majed Z, Bellenger E, Postic D et al (2005) Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 43:539–545CrossRefPubMedGoogle Scholar
  20. 20.
    Matthias MA, Ricaldi JN, Cespedes M et al (2008) Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon. PLoS Negl Trop Dis 2:e213CrossRefPubMedGoogle Scholar
  21. 21.
    McBride AJ, Athanazio DA, Reis MG et al (2005) Leptospirosis. Curr Opin Infect Dis 18:376–386CrossRefPubMedGoogle Scholar
  22. 22.
    Morey RE, Galloway RL, Bragg SL et al (2006) Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing. J Clin Microbiol 44:3510–3516CrossRefPubMedGoogle Scholar
  23. 23.
    Perolat P, Chappel RJ, Adler B et al (1998) Leptospira fainei sp. nov., isolated from pigs in Australia. Int J Syst Bacteriol 48(Pt 3):851–858CrossRefPubMedGoogle Scholar
  24. 24.
    Postic D, Riquelme-Sertour N, Merien F et al (2000) Interest of partial 16S rDNA gene sequences to resolve heterogeneities between Leptospira collections: application to L. meyeri. Res Microbiol 151:333–341CrossRefPubMedGoogle Scholar
  25. 25.
    Ramadass P, Jarvis BD, Corner RJ et al (1992) Genetic characterization of pathogenic Leptospira species by DNA hybridization. Int J Syst Bacteriol 42:215–219CrossRefPubMedGoogle Scholar
  26. 26.
    Ramadass P, Meerarani S, Venkatesha MD et al (1997) Characterization of leptospiral serovars by randomly amplified polymorphic DNA fingerprinting. Int J Syst Bacteriol 47:575–576CrossRefPubMedGoogle Scholar
  27. 27.
    Salaun L, Merien F, Gurianova S et al (2006) Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol 44:3954–3962CrossRefPubMedGoogle Scholar
  28. 28.
    Sambrook J, Russell RW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  29. 29.
    Slack AT, Kalambaheti T, Symonds ML et al (2008) Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand. Int J Syst Evol Microbiol 58:2305–2308CrossRefPubMedGoogle Scholar
  30. 30.
    Slack AT, Khairani-Bejo S, Symonds ML et al (2009) Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia. Int J Syst Evol Microbiol 59:705–708CrossRefPubMedGoogle Scholar
  31. 31.
    Turk N, Milas Z, Mojcec V et al (2009) Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis. FEMS Microbiol Lett 300:174–179CrossRefPubMedGoogle Scholar
  32. 32.
    Valverde ML, Ramirez JM, Montes de Oca LG et al (2008) Arenal, a new Leptospira serovar of serogroup Javanica, isolated from a patient in Costa Rica. Infect Genet Evol 8:529–533CrossRefGoogle Scholar
  33. 33.
    Victoria B, Ahmed A, Zuerner RL et al (2008) Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira. PLoS ONE 3:e2752CrossRefPubMedGoogle Scholar
  34. 34.
    Yasuda PH, Steigerwalt AG, Sulzer KR et al (1987) Deoxyribonucleic-acid relatedness between serogroups and serovars in the family Leptospiraceae with proposals for 7 new leptospira species. Int J Syst Bacteriol 37:407–415CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Josefa Bezerra da Silva
    • 1
  • Eneas Carvalho
    • 1
  • Rudy A. Hartskeerl
    • 2
  • Paulo L. Ho
    • 1
    Email author
  1. 1.Centro de BiotecnologiaInstituto ButantanSão PauloBrazil
  2. 2.KIT Biomedical ResearchRoyal Tropical InstituteAmsterdamThe Netherlands

Personalised recommendations