Molecular and serological characterization of Leptospira kirschneri serogroup Pomona isolated from a human case in a Brazilian rural area

Ilana Teruszkin Balassiano Juliana Magalhães Vital-Brazil Tatiane Mendes Varela Ramos Loeci Natalina Timm Martha Maria Pereira About the authors

Abstract

INTRODUCTION:

Leptospirosis is an important health concern in Brazil. Currently, information on the epidemiology of the disease in the rural areas of the country is lacking.

METHODS:

Serological and molecular techniques were used to characterize a clinical isolate of Leptospira.

RESULTS:

The strain CLEP 00060, isolated from a 59-year-old man in a rural area of Rio Grande do Sul state, Brazil, was identified as belonging to L. kirschneri serogroup Pomona serovar Mozdok.

CONCLUSIONS:

This study contributes to the local epidemiological knowledge of leptospirosis, prevention of the disease by vaccines, and improvements in its diagnosis.

Keywords:
Leptospira kirschneri; Leptospirosis; Typing

Leptospirosis is a worldwide zoonosis, being more common in tropical regions and predominantly found in impoverished populations inhabiting developing countries, where incidence peaks are observed during the raining season11. Adler B, de la Peña Moctezuma A. Leptospira and leptospirosis. Vet Microbiol. 2010; 40 (3-4): 287-96.. Although leptospirosis is currently recognized as a disease of epidemic potential with a significant impact on public health in many countries, it remains neglected22. Schneider MC, Najera P, Pereira MM, Machado G, Dos Anjos CB, Rodrigues RO, et al. Leptospirosis in Rio Grande do Sul, Brazil: an ecosystem approach in the animal-human interface. PLoS Negl Trop Dis. 2015;9(11):e0004095.. This can be explained by its common incidence in areas where socioeconomic (poverty, lack of water and sanitation, poor housing conditions) and environmental (heavy rains or floods) factors are crucial for the maintenance of leptospires and subsequent occurrence of the disease22. Schneider MC, Najera P, Pereira MM, Machado G, Dos Anjos CB, Rodrigues RO, et al. Leptospirosis in Rio Grande do Sul, Brazil: an ecosystem approach in the animal-human interface. PLoS Negl Trop Dis. 2015;9(11):e0004095.,33. Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009;7(10):736-47..

Although considered the definitive diagnosis for leptospirosis, the isolation of Leptospira is not a timely aid for the clinical care of human patients, being a method rarely achieved44. Chiani Y, Jacob P, Varni V, Landolt N, Schmeling MF, Pujato N, et al. Isolation and clinical sample typing of human leptospirosis cases in Argentina. Infect Genet Evol. 2016; 37: 245-51.. Moreover, the identification of clinical isolates at the serovar level is a laborious process and is restricted to a few reference laboratories11. Adler B, de la Peña Moctezuma A. Leptospira and leptospirosis. Vet Microbiol. 2010; 40 (3-4): 287-96..

Despite these challenges, identifying the infective serovar is very important for diagnosis and epidemiological studies, because it can provide information related to the host reservoirs involved in pathogen transmission and, therefore, contribute to the adoption of multidisciplinary control strategies22. Schneider MC, Najera P, Pereira MM, Machado G, Dos Anjos CB, Rodrigues RO, et al. Leptospirosis in Rio Grande do Sul, Brazil: an ecosystem approach in the animal-human interface. PLoS Negl Trop Dis. 2015;9(11):e0004095.

3. Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009;7(10):736-47.
-44. Chiani Y, Jacob P, Varni V, Landolt N, Schmeling MF, Pujato N, et al. Isolation and clinical sample typing of human leptospirosis cases in Argentina. Infect Genet Evol. 2016; 37: 245-51..

Concerning the epidemiology of leptospirosis, there is a lack of information on the countryside areas of Brazil, compared to that on the urban areas where serovars Copenhageni and Icterohaemorrhagiae are predominant22. Schneider MC, Najera P, Pereira MM, Machado G, Dos Anjos CB, Rodrigues RO, et al. Leptospirosis in Rio Grande do Sul, Brazil: an ecosystem approach in the animal-human interface. PLoS Negl Trop Dis. 2015;9(11):e0004095.,55. Miraglia F, Matsuo M, Morais ZM, Dellagostin OA, Seixas FK, Freitas JC, et al. Molecular characterization, serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil. Diagn Microbiol Infect Dis. 2013;77(3):195-9.. However, it should be highlighted that there are reports of other serovars found in different species of domestic and wild animals in those areas66. Cosate MR, Barouni AS, Moreira EC, Veloso IF, Gomes MT, Salas CE. Molecular characterization by LSSP-PCR and DNA sequencing of a pathogenic isolate of Leptospira interrogans from Brazil. Zoonoses Public Hlth. 2012;59(6):379-88.,77. da Cunha CE, Felix SR, Neto AC, Campello-Felix A, Kremer FS, Monte LG, et al. Infection with Leptospira kirschneri serovar Mozdok: first report from the southern hemisphere. Am J Trop Med Hyg. 2016;94 (3):519-21..

Leptospirosis is an important public health problem in the State of Rio Grande do Sul (Southern Brazil), with an average of 428 cases reported annually. A study conducted using the One Health approach showed that rural populations of the state have an approximately eight times higher risk of contracting leptospirosis than their urban counterparts do, even though the number of reported cases was high in both areas22. Schneider MC, Najera P, Pereira MM, Machado G, Dos Anjos CB, Rodrigues RO, et al. Leptospirosis in Rio Grande do Sul, Brazil: an ecosystem approach in the animal-human interface. PLoS Negl Trop Dis. 2015;9(11):e0004095..

In the present study, we demonstrate the identification of a clinical isolate obtained from a human anicteric case in a rural area of Southern Brazil.

A 59-year-old male worker from a rice-planting farm in Southern Brazil (Santa Vitória do Palmar City, Rio Grande do Sul State), presenting with acute fever, myalgia, malaise, headache, vomiting, and diarrhea, had his blood and serum collected on the fifth and ninth day of symptoms, respectively. Serological tests immunoglobulin M-enzyme-linked immunosorbent assay (IgM-ELISA) (Bio-Manguinhos/FIOCRUZ) and microscopic agglutination test (MAT)88. World Health Organization (WHO). Human leptospirosis: guidance for diagnosis, surveillance and control. Geneva: WHO; 2003. 109p. - showed positive results for Leptospira spp. The reactive serogroups and their respective titers obtained with MAT were as follows: Icterohaemorrhagiae, 400; Australis, 200; and Pomona, 100.

Blood culture was positive for the presence of Leptospira, visualized using dark field microscopy88. World Health Organization (WHO). Human leptospirosis: guidance for diagnosis, surveillance and control. Geneva: WHO; 2003. 109p.. The culture was purified, maintained by subculturing in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium88. World Health Organization (WHO). Human leptospirosis: guidance for diagnosis, surveillance and control. Geneva: WHO; 2003. 109p., and stored at the Collection of Leptospira [(CLEP) Oswaldo Cruz Institute/FIOCRUZ)] under the registration number CLEP 00060.

Microscopic agglutination test was also carried out against reference antisera (obtained from the Royal Tropical Institute, Netherlands) representative of the 15 most prevalent serogroups in Brazil: Icterohaemorrhagiae, Canicola, Grippotyphosa, Pomona, Australis, Bataviae, Ballum, Cynopteri, Javanica, Panama, Pyrogenes, Sejroe, Tarassovi, Autumnalis, and Hebdomadis. The results demonstrated the antigenic relationship between CLEP 00060 and the Pomona serogroup (data not shown).

To identify the strain at the genomospecies level, we used different methodologies previously described as alternative tools to overcome the complex relatedness among serogroups, serovars, and species of Leptospira99. Kawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H. flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp. Microbiol Immunol. 2001;45(6):491-6.

10. Fenner JS, Anjum MF, Randall LP, Pritchard GC, Wu G, Errington J, et al. Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC. Res Vet Sci. 2010;89(1):48-57.
-1111. Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Bailey MS, Holden MT, et al. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop Dis . 2013;7(1):e1954..

Multilocus sequence typing (MLST)1111. Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Bailey MS, Holden MT, et al. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop Dis . 2013;7(1):e1954. was performed and purified amplicons were sequenced using the Platform of deoxyribonucleic acid (DNA) Sequencing - RPT01A-PDTIS/FIOCRUZ (http://plataformas.cdts.fiocruz.br/). Sequences were aligned with Clustal W and analyzed at the MLST website (http://pubmlst.org/leptospira). The analysis revealed that CLEP 00060 could be clustered with 100% similarity in the ST 117 and most likely belongs to Pomona serogroup (data not shown).

Pulsed Field Gel Electrophoresis (PFGE)1212. 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(2):289-91. was performed using the restriction enzyme NotI (Sinapse Biotecnologia, Brazil). Fingerprints of the clinical isolate and different reference strains from serogroup Pomona were analyzed using GelCompar (Applied Maths) software, and the patterns produced were compared with the Dice coefficient and clustered using the unweighted pair-group method using arithmetic averages method. The dendrogram revealed a similarity index of approximately 92% between CLEP 00060 and strain 5621 (L. kirschneri serogroup Pomona serovar Mozdok) (Figure 1), indicating that these two strains belong to closely related serovars, according to the previously established criteria for PFGE interpretation1313. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995;33(9):2233-9..

FIGURE 1
Dendrogram generated by PFGE analysis showing the relationship among the clinical isolate CLEP 00060 and some reference strains from the serogroup Pomona. L.: Leptospira; PFGE: Pulsed Field Gel Electrophoresis; CLEP: Collection of Leptospira.

Collection of Leptospira 00060 and two reference strains (L. kirschneri serogroup Grippotyphosa serovar Grippotyphosa strain Moskva V and L. interrogans serogroup Pomona serovar Pomona strain Pomona) were submitted to DNA extraction, followed by flaB amplification by polymerase chain reaction (PCR). The amplicons were digested separately by the restriction enzymes HindIII and HaeIII (Sinapse Biotecnologia, Brazil) and the fingerprints were analyzed99. Kawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H. flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp. Microbiol Immunol. 2001;45(6):491-6.. DNA was also used to amplify the rrs gene, and the amplified products were sequenced1010. Fenner JS, Anjum MF, Randall LP, Pritchard GC, Wu G, Errington J, et al. Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC. Res Vet Sci. 2010;89(1):48-57.,1414. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173(2):697-703.. Sequences were compared with reference Leptospira sequences in the genome database of the National Center for Biotechnology Information (NCBI), using the basic local alignment search tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The fingerprints generated by flaB-restriction fragment length polymorphism (RFLP) clearly showed the genetic relationship between the clinical isolate and L. kirschneri (Figure 2). Phylogenetic studies based on 16S rRNA gene sequences confirmed the result (data not shown).

FIGURE 2
Polyacrylamide gel electrophoresis (5%) of the PCR products resulting from the digestion of flaB by the restriction endonucleases HindIII and HaeIII. M: 100bp DNA ladder (Sinapse Biotecnologia, Brazil); 1: L. kirschneri reference strain Moskva V; 2: L. interrogans reference strain Pomona; 3: strain CLEP 00060 (1-3 digested with HaeIII); 4: L. kirschneri reference strain Moskva V; 5: L. interrogans reference strain Pomona; 6: strain CLEP 00060 (4-6 digested with HindIII). PCR: polymerase chain reaction; DNA: deoxyribonucleic acid;L.: Leptospira; CLEP: Collection of Leptospira.

Taken together, we assume that the isolate CLEP 00060 belongs to L. kirschneri serogroup Pomona and is closely related to serovar Mozdok, although more refined molecular methods, such as secY sequencing, and a comparison with a larger number of reference strains at PFGE could improve the identification. Even though patient sera presented low titers against serogroup Pomona in MAT, the identification of the strain can still be inferred because MAT presents low accuracy (about 33%) considering the correct prediction of the infective serogroup/serovar1515. Smythe LD, Wuthiekanun V, Chierakul W, Suputtamongkol Y, Tiengrim S, Dohnt MF, Symonds ML, Slack AT, Apiwattanaporn A, Chueasuwanchai S, Day NP, Peacock SJ. The microscopic agglutination test (MAT) is an unreliable predictor of infecting Leptospira sorovar in Thailand. Am J Trop Med Hyg . 2009;81(4):695-7..

Based on the available information, serovar Mozdok is rarely isolated from humans in Brazil77. da Cunha CE, Felix SR, Neto AC, Campello-Felix A, Kremer FS, Monte LG, et al. Infection with Leptospira kirschneri serovar Mozdok: first report from the southern hemisphere. Am J Trop Med Hyg. 2016;94 (3):519-21.. This is only the second report concerning the isolation of L. kirschneri putative serovar Mozdok from a human case in a rural area of Brazil. The first report77. da Cunha CE, Felix SR, Neto AC, Campello-Felix A, Kremer FS, Monte LG, et al. Infection with Leptospira kirschneri serovar Mozdok: first report from the southern hemisphere. Am J Trop Med Hyg. 2016;94 (3):519-21. describes the characterization of two isolates, from human and canine cases, in Pelotas, also in Rio Grande do Sul and about 240km from Santa Vitória do Palmar. Besides, the isolation of strains belonging to serogroup Pomona is more frequent in animals than in humans. Our finding reinforces the need for further studies with larger numbers of strains to define the epidemiological situation of leptospirosis in the rural areas of Brazil. It should be noted that awareness about the presence of uncommon serogroups and serovars in our territory would allow, according to the One Health approach, the prevention and control of leptospirosis by employing specific measures in accordance with our socio-economic scenario, bringing a long-term benefit to the Brazilian population.

Acknowledgements

The authors acknowledge Elisabete Coli for laboratory assistance, André Felipe Mercês Santos for PFGE dendrogram production, and Dr. Eliane de Oliveira Ferreira for revising the text. We are also grateful for access to the Leptospira MLST database, which is located at Imperial College London and is funded by the Wellcome Trust, and the Platform of DNA Sequencing - RPT01A-PDTIS/FIOCRUZ for sequencing procedures.

REFERENCES

  • 1
    Adler B, de la Peña Moctezuma A. Leptospira and leptospirosis. Vet Microbiol. 2010; 40 (3-4): 287-96.
  • 2
    Schneider MC, Najera P, Pereira MM, Machado G, Dos Anjos CB, Rodrigues RO, et al. Leptospirosis in Rio Grande do Sul, Brazil: an ecosystem approach in the animal-human interface. PLoS Negl Trop Dis. 2015;9(11):e0004095.
  • 3
    Ko AI, Goarant C, Picardeau M. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009;7(10):736-47.
  • 4
    Chiani Y, Jacob P, Varni V, Landolt N, Schmeling MF, Pujato N, et al. Isolation and clinical sample typing of human leptospirosis cases in Argentina. Infect Genet Evol. 2016; 37: 245-51.
  • 5
    Miraglia F, Matsuo M, Morais ZM, Dellagostin OA, Seixas FK, Freitas JC, et al. Molecular characterization, serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil. Diagn Microbiol Infect Dis. 2013;77(3):195-9.
  • 6
    Cosate MR, Barouni AS, Moreira EC, Veloso IF, Gomes MT, Salas CE. Molecular characterization by LSSP-PCR and DNA sequencing of a pathogenic isolate of Leptospira interrogans from Brazil. Zoonoses Public Hlth. 2012;59(6):379-88.
  • 7
    da Cunha CE, Felix SR, Neto AC, Campello-Felix A, Kremer FS, Monte LG, et al. Infection with Leptospira kirschneri serovar Mozdok: first report from the southern hemisphere. Am J Trop Med Hyg. 2016;94 (3):519-21.
  • 8
    World Health Organization (WHO). Human leptospirosis: guidance for diagnosis, surveillance and control. Geneva: WHO; 2003. 109p.
  • 9
    Kawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H. flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp. Microbiol Immunol. 2001;45(6):491-6.
  • 10
    Fenner JS, Anjum MF, Randall LP, Pritchard GC, Wu G, Errington J, et al. Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC. Res Vet Sci. 2010;89(1):48-57.
  • 11
    Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Bailey MS, Holden MT, et al. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop Dis . 2013;7(1):e1954.
  • 12
    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(2):289-91.
  • 13
    Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995;33(9):2233-9.
  • 14
    Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173(2):697-703.
  • 15
    Smythe LD, Wuthiekanun V, Chierakul W, Suputtamongkol Y, Tiengrim S, Dohnt MF, Symonds ML, Slack AT, Apiwattanaporn A, Chueasuwanchai S, Day NP, Peacock SJ. The microscopic agglutination test (MAT) is an unreliable predictor of infecting Leptospira sorovar in Thailand. Am J Trop Med Hyg . 2009;81(4):695-7.

  • Financial support: Secretaria de Vigilância em Saúde, Ministério da Saúde.

Publication Dates

  • Publication in this collection
    May-Jun 2017

History

  • Received
    19 Oct 2016
  • Accepted
    24 Mar 2017
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