Application of pulsed-field gel electrophoresis for molecular identification of pathogenic Leptospira species in Iran: a rapid and reliable method
Abstract
Background and Objectives: Leptospirosis is a zoonotic disease caused by pathogenic Leptospira serovars. The genus Leptospira cannot differentiated by conventional techniques. However, identity determination of pathogenic serovar is precious of public health problems and epidemiological studies. Pulsed-field gel electrophoresis facilitates rapid identification of Leptospires to the serovar levels.
Materials and Methods: In this study, we employed PFGE to evaluate 28 Leptospira isolates, with animal, human and environmental origin, obtained from Razi Vaccine and Serum Research Institute of Karaj, Iran. PFGE patterns of 28 Leptospira serovars were generated using the Not I restriction enzyme in comparison with the lambda ladder.
Results: Out of 28 serovars evaluated, we identified 22 different pulsed types, designated P1- P22. Out of 22 pulse groups, 3 were found to be a common type, but others were a single Type. Groups consisting of the common type were P3, P9, P14, and P16. The results showed that the discriminatory index of PFGE by Not I enzyme was 0.99, demonstrating heterogeneous differentiation among serovar members.
Conclusion: The PFGE methodology used in this study showed excellent interlaboratory report usability, rapid, reliable, enabling standardization and data sharing between laboratories.
2. Samrot AV, Sean TC, Bhavya KS, Sahithya CS, Chan-Drasekaran S, Palanisamy R, et al. Leptospiral infection, pathogenesis and its diagnosis-A review. Pathogens 2021; 10: 145.
3. 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: 757-771.
4. Guernier V, Allan KJ, Goarant C. Advances and challenges in barcoding pathogenic and environmental Leptospira. Parasitology 2018; 145: 595-607.
5. Lopez-Canovas L, Martinez Benitez MB, Herrera Isidron JA, Flores Soto E. Pulsed field gel electrophoresis: past, present, and future. Anal Biochem 2019; 573: 17-29.
6. Arent Z, Pardyak L, Dubniewicz K, Płachno BJ, Kotula-Balak M. Leptospira taxonomy: Then and now. Med Weter 2022; 78: 489-496.
7. Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, et al. Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics. PLoS Negl Trop Dis 2019; 13(5): e0007270.
8. Levett PN, Branch SL, Whittington CU, Edwards CN, Paxton H. Two methods for rapid serological diagnosis of acute leptospirosis. Clin Diagn Lab Immunol 2001; 8: 349-351.
9. Cordonin C, Turpin M, Bringart M, Bascands J-L, Flores O, Dellagi K, et al. Pathogenic Leptospira and their animal reservoirs: testing host specificity through experimental infection. Sci Rep 2020; 10: 7239.
10. Li SJ, Zhang CC, Li XW, Tian KC, Tang GP, Wang DM, et al. Molecular typing of Leptospira interrogans strains isolated from Rattus tanezumi in Guizhou Province, Southwest of China. Biomed Environ Sci 2012; 25: 542-548.
11. Ribeiro RL, Machry L, Brazil JM, Ramos TM, Avelar KE, Pereira MM. Technical improvement to prevent DNA degradation of Leptospira spp. in pulsed field gel electrophoresis. Lett Appl Microbiol 2009; 49: 289-291.
12. 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: 628-632.
13. Naigowit P, Charoenchai S, Biaklang M, Seena U, Wangroongsarb P, Sawanpanyalert P, et al. Identification of clinical isolates of Leptospira spp. by pulsed field gel-electrophoresis and microscopic agglutination test. Southeast Asian J Trop Med Public Health 2007; 38: 97-103.
14. Birren B, Lai E. Pulsed field gel electrophoresis: a practical guide: Academic Press; 2012.
15. Esfandiari B, Pourshafie MR, Gouya MM, Khaki P, Mostafavi E, Darvish J, et al. An epidemiological comparative study on diagnosis of rodent leptospirosis in Mazandaran Province, northern Iran. Epidemiol Health 2015; 37: e2015012.
16. Dellagostin OA, Grassmann AA, Hartwig DD, Felix SR, da Silva EF, McBride AJ. Recombinant vaccines against leptospirosis. Hum Vaccin 2011; 7: 1215-1224.
17. Galloway RL, Levett PN. Application and validation of PFGE for serovar identification of Leptospira clinical isolates. PLoS Negl Trop Dis 2010; 4(9): e824.
18. Sehgal SC, Biswas D, Vijayachari P, Sugunan AP, Roy S. Molecular tools in leptospirosis diagnosis and characterization of isolates. Southeast Asian J Trop Med Public Health 2003; 34 Suppl 2: 163-169.
19. Neoh HM, Tan XE, Sapri HF, Tan TL. Pulsed-field gel electrophoresis (PFGE): A review of the “gold standard” for bacteria typing and current alternatives. Infect Genet Evol 2019; 74: 103935.
20. Machry L, Ribeiro RL, Vital-Brazil JM, Balassiano IT, Oliveira IC, Avelar KE, et al. Characterization of Leptospira sp reference strains using the pulsed field gel electrophoresis technique. Rev Soc Bras Med Trop 2010; 43: 166-169.
21. Herrmann JL, Bellenger E, Perolat P, Baranton G, Saint Girons I. Pulsed-field gel electrophoresis of Not I digests of leptospiral DNA: a new rapid method of serovar identification. J Clin Microbiol 1992; 30: 1696-1702.
22. Romero EC, Blanco RM, Galloway RL. Application of pulsed‐field gel electrophoresis for the discrimination of leptospiral isolates in Brazil. Lett Appl Microbiol 2009; 48: 623-627.
23. Moreno LZ, Miraglia F, Lilenbaum W, Neto JS, Freitas JC, Morais ZM, et al. Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing—a continuous attempt at species and serovar differentiation. Emerg Microbes Infect 2016; 5(3): e17.
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Issue | Vol 16 No 3 (2024) | |
Section | Original Article(s) | |
DOI | https://doi.org/10.18502/ijm.v16i3.15763 | |
Keywords | ||
Leptospirosis; Pulsed-field gel electrophoresis; Leptospira serovars; Molecular typing method |
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