Background: More than 260 leptospiral serovars by serology have been recorded. New genotypic methods have classified Leptospira into 20 species. Serovar identification is essential for epidemiological study of this disease.
Objective: We identified fingerprints from reference serovar strains of Leptospira spp. and representative serovars from field rat isolates by pulsed-field gel electrophoresis (PFGE).
Methods: Extract genomic DNA from 28 reference serovars of Leptospira spp. and 13 leptospiral isolates from field rats, selected as representative serovars Pyrogenes, Bataviae, Autumnalis, and Australis, using PFGE following NotI restriction digest.
Results: PFGE with NotI restriction enzyme successfully differentiated 28 reference serovars into 27 fingerprint patterns, with the exception of serovars Copenhageni and Icterohaemorrhagiae. The discriminatory power of these reference strains was 0.99. Isolates that yielded patterns identical to their corresponding serovars were serovars Pyrogenes strain Salinem and Bataviae. Patterns for isolates of serovars Autumnalis and Australis were different from reference serovar Autumnalis strain Akiyami A and serovar Australis strain Ballico used in this study.
Conclusion: PFGE can be useful for identifying serovars of leptospiral isolates from reservoirs and for identifying new serovars of Leptospira for epidemiological study.
2. Bharti AR Nally JE Ricaldi JN Matthias MA Diaz MM Lovett MA et al. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis. 2003; 3:757-71.
3. Terpstra WJ. Typing leptospira from the perspective of a reference laboratory. Acta Leiden. 1992; 60:79-87.
4. Faine S Adler B Bolin C Perolat P. Systematics (Taxonomy Nomenclature and Classification). In: Leptospira and leptospirosis. 2 ed. Melbourne: MedSci; 1999. p. 57-66.
5. Leptospira. Available from: http://www.en.wikipedia.org/wiki/Leptospira
6. Haapala DK Rogul M Evans LB Alexander AD. Deoxyribonucleic acid base composition and homology studies of Leptospira. J Bacteriol. 1969; 98:421-8.
7. Zuerner RL Bolin CA. Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays. J Clin Microbiol. 1997; 35:2612-7.
8. Ellis WA Montgomery JM Thiermann AB. Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans. J Clin Microbiol. 1991; 29: 957-61.
9. Ciceroni L Ciarrocchi S Ciervo A Petrucca A Pinto A Calderaro A et al. Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction. Res Microbiol. 2002; 153: 37-44.
10. Woodward MJ Redstone JS. Differentiation of leptospira serovars by the polymerase chain reaction and restriction fragment length polymorphism.Vet Rec. 1993; 132:325-6.
11. Natarajaseenivasan K Prabhu N Selvanayaki K Raja SS Ratnam S. Human leptospirosis in Erode South India: serology isolation and characterization of the isolates by randomly amplified polymorphic DNA (RAPD) fingerprinting. Jpn J Infect Dis. 2004; 7: 193-7.
12. Perolat P Grimont F Regnault B Grimont PA Fournie E Thevenet H et al. rRNA gene restriction patterns of Leptospira: a molecular typing system. Res Microbiol. 1990; 141:159-71.
13. Kositanont U Chotinantakul K Phulsuksombati D Tribuddharat C.Assessment of Southern blot ribotyping for differentiation of Leptospira strains isolated from field rats. J. Microbiol Methods. 2007; 69:288-97.
14. Herrmann JL Bellenger E Perolat P Baranton G Saint Girons I. Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification. J Clin Microbiol. 1992; 30: 1696-702.
15. Ellighahausen HC McCullough WG. Nutrition of Leptospira Pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am J Vet Res. 1965; 26:45-51.
16. Galloway RL Levett PN. Evaluation of a modified pulsed-field gel electrophoresis approach for the identification of Leptospira serovars. Am J Trop Med Hyg. 2008; 78:628-32.
17. Nascimento AL Verjovski-Almeida S Van Sluys MA Monteiro-Vitorello CB Camargo LE Digiampietri LA et al. Genome features of Leptospira interrogans serovar Copenhageni. Braz J Med Biol Res. 2004; 37: 459-77.
18. Tamai T Sada E Kobayashi Y. Restriction endonuclease DNA analysis of Leptospira interrogans serovars Icterohaemorrhagiae and Copenhageni. Microbiol Immunol. 1988; 32:887-94.
19. Herrmann JL Baril C Bellenger E Perolat P Baranton G Saint Girons I. Genome conservation in isolates of Leptospira interrogans. J Bacteriol. 1991; 173:7582-8.
20. Ralph D McClelland M Welsh J Baranton G Perolat P. Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes. J Bacteriol 1993; 175:973-81.
21. Majed Z Bellenger E Postic D Pourcel C Baranton G Picardeau M. Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol. 2005; 43:539-45.
22. Perolat P Merien F Ellis WA Baranton G. Characterization of Leptospira isolates from serovar hardjo by ribotyping arbitrarily primed PCR and mapped restriction site polymorphisms. J Clin Microbiol. 1994; 32:1949-57.