Serovar identification of clinical isolates of Leptospira is generally not performed on a routine basis, yet the identity of an infecting serovar is valuable from both epidemiologic and public health standpoints. Only a small number of reference laboratories worldwide have the capability to perform the cross agglutinin absorption test (CAAT), the reference method for serovar identification. Pulsed-field gel electrophoresis (PFGE) is an alternative method to CAAT that facilitates rapid identification of leptospires to the serovar level. We employed PFGE to evaluate 175 isolates obtained from humans and animals submitted to the Centers for Disease Control and Prevention (CDC) between 1993 and 2007. PFGE patterns for each isolate were generated using the NotI restriction enzyme and compared to a reference database consisting of more than 200 reference strains. Of the 175 clinical isolates evaluated, 136 (78%) were identified to the serovar level by the database, and an additional 27 isolates (15%) have been identified as probable new serovars. The remaining isolates yet to be identified are either not represented in the database or require further study to determine whether or not they also represent new serovars. PFGE proved to be a useful tool for serovar identification of clinical isolates of known serovars from different geographic regions and a variety of different hosts and for recognizing potential new serovars.
References
[1]
World Health Organization (1999) Leptospirosis worldwide, 1999. Weekly Epidemiological Record 74: 237–242.
[2]
Babudieri B (1958) Animal reservoirs of leptospirosis. Annals of the New York Academy of Sciences 70: 393–413. doi: 10.1111/j.1749-6632.1958.tb35398.x
Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, et al. (2003) Leptospirosis: a zoonotic disease of global importance. Lancet Infectious Diseases 3: 757–771. doi: 10.1016/S1473-3099(03)00830-2
[5]
Matthias MA, Ricaldi JN, Cespedes M, Diaz MM, Galloway RL, 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: e213. doi: 10.1371/journal.pntd.0000213
[6]
Slack AT, Kalambaheti T, Symonds ML, Dohnt MF, Galloway RL, et al. (2008) Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand. Int J Syst Evol Microbiol 58: 2305–2308. doi: 10.1099/ijs.0.64947-0
[7]
Kmety E, Dikken H (1993) Classification of the species Leptospira interrogans and history of its serovars. Groningen: University Press Groningen.
[8]
Mgode GF, Machang'u RS, Goris MG, Engelbert M, Sondij S, et al. (2006) New Leptospira serovar Sokoine of serogroup Icterohaemorrhagiae from cattle in Tanzania. Int J Syst Evol Microbiol 56: 593–597. doi: 10.1099/ijs.0.63278-0
[9]
Rossetti CA, Liem M, Samartino LE, Hartskeerl RA (2005) Buenos Aires, a new Leptospira serovar of serogroup Djasiman, isolated from an aborted dog fetus in Argentina. Vet Microbiol 107: 241–248. doi: 10.1016/j.vetmic.2005.01.015
[10]
Valverde Mde L, Ramirez JM, Oca LG, Goris MG, Ahmed N, et al. (2008) Arenal, a new Leptospira serovar of serogroup Javanica, isolated from a patient in Costa Rica. Infect Genet Evol 8: 529–533. doi: 10.1016/j.meegid.2008.02.008
[11]
Faine S, Adler B, Bolin C, Perolat P (1999) Leptospira and leptospirosis. Melbourne: MedSci.
[12]
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: 628–632.
[13]
Naigowit P, Charoenchai S, Biaklang M, Seena U, Wangroongsarb P, et al. (2007) Identification of clinical isolates of Leptospira spp by pulsed field gel-electrophoresis and microscopic agglutination test. Southeast Asian J Trop Med Public Health 38: 97–103.
[14]
Herrmann JL, Bellenger E, Perolat P, Baranton G, Saint Girons I (1992) Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification. J Clin Microbiol 30: 1696–1702.
[15]
Romero EC, Blanco RM, Galloway RL (2009) Application of pulsed-field gel electrophoresis for the discrimination of leptospiral isolates in Brazil. Lett Appl Microbiol 48: 623–627. doi: 10.1111/j.1472-765X.2009.02580.x
[16]
Brenner DJ, Kaufmann AF, Sulzer KR, Steigerwalt AG, Rogers FC, 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: 839–858. doi: 10.1099/00207713-49-2-839
[17]
Hunter SB, Vauterin P, Lambert-Fair MA, Van Duyne MS, Kubota K, et al. (2005) Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol 43: 1045–1050. doi: 10.1128/JCM.43.3.1045-1050.2005
[18]
Majed Z, Bellenger E, Postic D, Pourcel C, Baranton G, et al. (2005) Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto. J Clin Microbiol 43: 539–545. doi: 10.1128/JCM.43.2.539-545.2005
[19]
Thaipadungpanit J, Wuthiekanun V, Chierakul W, Smythe LD, Petkanchanapong W, et al. (2007) A Dominant Clone of Leptospira interrogans Associated with an Outbreak of Human Leptospirosis in Thailand. PLoS Negl Trop Dis 1: e56. doi: 10.1371/journal.pntd.0000056
[20]
Dikken H, Kmety E (1978) Serological typing methods of leptospires. In: Bergan T, Norris JR, editors. Methods in Microbiology. London: Academic Press. pp. 259–307.
[21]
Morey RE, Galloway RL, Bragg SL, Steigerwalt AG, Mayer LW, et al. (2006) Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing. J Clin Microbiol 44: 3510–3516. doi: 10.1128/JCM.00670-06
[22]
Mandel M, Igambi L, Bergendahl J, Dodson ML Jr, Scheltgen E (1970) Correlation of melting temperature and cesium chloride buoyant density of bacterial deoxyribonucleic acid. J Bacteriol 101: 333–338.
[23]
Vijayachari P, Ahmed N, Sugunan AP, Ghousunnissa S, Rao KR, et al. (2004) Use of fluorescent amplified fragment length polymorphism for molecular epidemiology of leptospirosis in India. J Clin Microbiol 42: 3575–3580. doi: 10.1128/JCM.42.8.3575-3580.2004
[24]
Turk N, Milas Z, Mojcec V, Ruzic-Sabljic E, Staresina 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: 174–179. doi: 10.1111/j.1574-6968.2009.01776.x
[25]
Romero EC, Yasuda PH (2006) Molecular characterization of Leptospira sp. strains isolated from human subjects in Sao Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool. Mem Inst Oswaldo Cruz 101: 373–378. doi: 10.1590/S0074-02762006000400005
[26]
Bourhy P, Collet L, Clement S, Huerre M, Ave P, et al. (2010) Isolation and characterization of new Leptospira genotypes from patients in Mayotte (Indian Ocean). PLoS Negl Trop Dis 4: e724. doi: 10.1371/journal.pntd.0000724
[27]
Zakeri S, Sepahian N, Afsharpad M, Esfandiari B, Ziapour P, et al. (2010) Molecular epidemiology of leptospirosis in northern Iran by nested polymerase chain reaction/restriction fragment length polymorphism and sequencing methods. Am J Trop Med Hyg 82: 899–903. doi: 10.4269/ajtmh.2010.09-0721
[28]
Ellis WA, Montgomery JM, Thiermann AB (1991) Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans. J Clin Microbiol 29: 957–961.
[29]
Slack AT, Dohnt MF, Symonds ML, Smythe LD (2005) Development of a Multiple-Locus Variable Number of Tandem Repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia. Ann Clin Microbiol Antimicrob 4: 10. doi: 10.1186/1476-0711-4-10
[30]
Ciceroni L, Ciarrocchi S, Ciervo A, Petrucca A, Pinto A, et al. (2002) Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies, pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction. Res Microbiol 153: 37–44. doi: 10.1016/S0923-2508(01)01284-0
[31]
Ahmed N, Devi SM, Valverde Mde L, Vijayachari P, Machang'u RS, et al. (2006) Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species. Ann Clin Microbiol Antimicrob 5: 28. doi: 10.1186/1476-0711-5-28
[32]
Levett PN (2007) Sequence-based typing of Leptospira: epidemiology in the genomic era. PLoS Negl Trop Dis 1: e120. doi: 10.1371/journal.pntd.0000120
[33]
Vimont S, Mnif B, Fevre C, Brisse S (2008) Comparison of PFGE and multilocus sequence typing for analysis of Klebsiella pneumoniae isolates. J Med Microbiol 57: 1308–1310. doi: 10.1099/jmm.0.2008/003798-0
[34]
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, et al. (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33: 2233–2239.