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Acta Tropica 126 (2013) 164– 166

Contents lists available at SciVerse ScienceDirect

Acta Tropica

journa l h o me pag e: www.elsev ier .com/ locate /ac ta t ropica

hort communication

olecular characterization of virulent Leptospira interroganserogroup Icterohaemorrhagiae isolated from Cavia aperea

eonardo G. Montea, Sérgio Jorgeb, Marina A. Xaviera, Fernanda M.A. Leala,arta G. Amarala, Fabiana K. Seixasc, Odir A. Dellagostinb, Cláudia P. Hartlebena,∗

Laboratório de Imunodiagnóstico, Núcleo de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Campusniversitário Capão do Leão, CP 354, CEP: 96010-900 Pelotas, RS, BrazilLaboratório de Vacinologia, Universidade Federal de Pelotas, Campus Universitário Capão do Leão, CP 354, CEP: 96010-900 Pelotas, RS, BrazilLaboratório de Genômica Funcional, Universidade Federal de Pelotas, Campus Universitário Capão do Leão, CP 354, CEP: 96010-900 Pelotas, RS, Brazil

r t i c l e i n f o

rticle history:eceived 17 October 2012eceived in revised form 31 January 2013

a b s t r a c t

Leptospirosis is a worldwide zoonotic infection caused by pathogenic Leptospira. Synanthropic rodentsare recognized carriers of leptospires; however, the role of wild rodents in the epidemiology of the diseaseis still incipient. In this work, we describe Leptospira strain isolated from Cavia aperea (Brazilian guinea

ccepted 11 February 2013vailable online 19 February 2013

eywords:eptospirosisrazilian guinea pig

pig). The isolated strain was characterized by partial rpoB gene sequencing, variable-number tandem-repeats and histopathological analysis. The strain was identified as Leptospira interrogans, serogroupIcterohaemorrhagiae and caused clinical signs of leptospirosis in the hamster model, attesting to itsvirulence. In conclusion, these findings could be useful for elucidating the epidemiological role of C.aperea in leptospirosis.

ild rodent

. Introduction

Leptospirosis is a zoonosis of global distribution caused by infec-ion with pathogenic spirochetes of the genus Leptospira (Adler ande la Pena, 2010). Leptospires can survive in the environment foreveral weeks and usually gain access to new hosts by passagecross mucous membrane or through skin abrasions (Levett, 2001;aine et al., 1999). In Latin America the prevalence of leptospirosis isigh because of environmental conditions and distinct leptospiraleservoir species (Ko et al., 2009; Levett, 2001). The epidemiology ofeptospirosis involves more than 260 pathogenic serovars and theeservoirs or maintenance hosts are difficult to identify, moreover,hey can be persistently infected without clinical signs and have

low serological response (Adler and de la Pena, 2010; Monahant al., 2009).

Synanthropic rodents were the first recognized carriers of lep-

ospires and the major animal species that can shed the bacteriahroughout their lifespan (WHO, 2003). They have been incrimi-ated as a primary source of infection of human beings (Adler and

∗ Corresponding author. Tel.: +55 53 32757517; fax: +55 53 32757551.E-mail addresses: leonardogmonte@hotmail.com (L.G. Monte),

ergiojorgevet@hotmail.com (S. Jorge), xaviermarinaa@gmail.com (M.A. Xavier),ehleal@yahoo.com.br (F.M.A. Leal), seixas.fk@gmail.com (F.K. Seixas),dir@terra.com.br (O.A. Dellagostin), clauhart@terra.com.br,artlebenclaudia@gmail.com (C.P. Hartleben).

001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.actatropica.2013.02.009

© 2013 Elsevier B.V. All rights reserved.

de la Pena, 2010; Clark and Olfert, 1986). Wild rodents also havebeen considered potential Leptospira reservoirs; however, their rolein the epidemiology of the disease is unclear (Turk et al., 2003).Among these animals, the Brazilian guinea pig (Cavia aperea), asmall wild herbivore rodent, may have potential as a source of infec-tion on the cycle of leptospirosis in Latin America since C. apereais found in Colombia, Ecuador, Venezuela, Guyana, Bolivia, northArgentina, Uruguay, Paraguay and Brazil (Redford and Eisenberg,1992; Dunnum et al., 2008). In this work, we reported the isolationof pathogenic Leptospira from the Brazilian guinea pig in SouthernBrazil.

2. Materials and methods

2.1. Isolation of leptospires

A free-living Brazilian guinea pig was admitted to the Fed-eral University of Pelotas for veterinary medical care in the cityof Pelotas, RS, Brazil (31◦46′′S, 52◦20′′W). After death of animal,the kidneys were aseptically removed and macerated under ster-ile conditions in liquid Ellinghausen–McCullough–Johnson–Harris(EMJH) medium without antibiotics, supplemented with Lep-tospira Enrichment EMJH (Difco, BD Diagnostics, Sparks, MD,

USA). The kidney cultures were incubated at 30 ◦C and exam-ined weekly by dark-field microscopy. The procedures usedin the present study were approved by the federal author-ities (Instituto Brasileiro do Meio Ambiente e dos Recursos

L.G. Monte et al. / Acta Tropica 126 (2013) 164– 166 165

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ig. 1. Electrophoresis in 1% agarose gel. M: molecular marker (1 kb plus DNA laddererovar Copenhageni strain Fiocruz L1-130) and (c) genomic DNA isolated from Bra

aturais Renováveis – IBAMA/authorization for scientific activitiesumber 24501-1).

.2. Genomic DNA extraction

For DNA extraction, a 7-day culture was harvested by centrifu-ation (15,000 × g for 30 min) at 4 ◦C and the pellet was frozent −20 ◦C. Genomic DNA was extracted using the illustra bacteriaenomicPrep Mini Spin Kit (GE Healthcare®) following the protocolesigned for Gram-negative bacteria. The extracted DNA was sub-itted to agarose gel electrophoresis in order to be quantified and

valuated with regard to its integrity and then stored at −20 ◦C.

.3. Polymerase chain reaction and partial rpoB gene sequencing

The partial rpoB gene was amplified and sequenced usingrimers previously described for Leptospira genomic specie iden-ification (La et al., 2006). PCR amplification was carried out withne cycle at 94 ◦C for 5 min, 35 cycles at 94 ◦C for 30 s, 51 ◦Cor 30 s, 72 ◦C for 2 min and a final extension at 72 ◦C for 7 min.mplicons were evaluated by agarose gel electrophoresis. Beforeequencing, PCR products were purified by GFX PCR DNA and Geland purification kit according to manufacturers’ instructions (GEealthcare®). The sequencing was performed in a MegaBACE 1000NA sequencer (GE Healthcare®) using Dynamic ET-terminator

echnology. Chromatograms were assembled and analyzed usingontigExpress® module of Vector NTI 10.0 suite (Invitrogen®).he assembled sequence was submitted to BLAST alignmentwww.ncbi.nlm.nih.gov/BLAST) against other rpoB sequences avail-ble in GenBank.

.4. Variable-number tandem-repeat (VNTR) analysis

For Leptospira serogroup and serovar identification of the newsolated seven discriminatory primers were used: VNTR4, VNTR7,NTR9, VNTR10, VNTR11, VNTR19 and VNTR23 (Majed et al., 2005).he PCR reaction was carried out using a cycle of 94 ◦C for 5 min, fol-owed by 35 cycles of amplification at 94 ◦C for 30 min, 55 ◦C for 30 s,2 ◦C for 1 min and a final extension of 72 ◦C for 30 s. Aliquots werevaluated by agarose gel electrophoresis and the sizes of amplifiedroducts were estimated by comparison (Majed et al., 2005). DNArom previously characterized Leptospira interrogans strain Fiocruz1-130 was chosen as positive control (Ko et al., 2009; Nascimentot al., 2004).

.5. Leptospira virulence evaluation

The isolated was grown in EMJH medium and 1 mL (108 bacterialells) was inoculated intraperitoneally in 28-day-old Golden Syr-

an hamster. The animal was monitored daily for the presence oflinical signs. A hamster injected with PBS served as the negativeontrol. For histopathological studies, lung, liver and kidney tissueamples were fixed in 10% formalin (pH 7.0), and then embedded

egative control (water template), (b) positive control (genomic DNA of L. interrogans guinea pig.

in paraffin. Sections of 5–6 �m thickness from each organ werestained with hematoxylin and eosin. Animal procedures carried outin this study were reviewed and approved by the Committee forAnimal Care and Use of the Federal University of Pelotas.

3. Results

A PCR product of approximately 600 bp for rpoB gene was ampli-fied and sequenced to perform the taxonomic classification. ABLAST analysis of the sequences of this amplicon demonstrated100% identity only to L. interrogans. In addition, VNTR profilerevealed the presence of tandem repeats for the seven discrimina-tory primers used (Fig. 1), according to the dendrogram previouslyproposed as L. interrogans serogroup Icterohaemorrhagiae (Majedet al., 2005).

The Brazilian guinea pig isolated was virulent in the hamstermodel. The animal inoculated developed acute lethal infectioncharacterized by renal and pulmonary lesions (Fig. 2). Renal tissuespresented infiltration with leucocytes cells (Fig. 2A) and tubularnecrosis (Fig. 2B); microscopic foci of alveolar hemorrhage occurredin infected animals (Fig. 2C). Macroscopic pulmonary hemorrhagewith widespread bleeding was observed. None of these featureswere observed in the healthy control animal (data not shown).

4. Discussion

Leptospirosis occurs in both rural and urban areas, in temperateand tropical climates and rodents are believed to play an importantrole in the continuous maintenance of Leptospira and its dissemi-nation in the environment (Faine et al., 1999). Among those majorcarriers, there are three widely distributed species of synanthropicrodents that act as reservoirs of host-related serovars: the brownrat (Rattus norvegicus), Icterohaemorrhagiae and Copenhageniserovars (Icterohaemorhagiae serogroup); the black rat (Rattusrattus), Icterohaemorrhagiae and Ballum serovars (Icterohaemor-rhagiae and Ballum serogroups, respectively); and the house mouse(Mus musculus), Ballum serovar (Ballum serogroup) (Pereira andAndrade, 1988; Hathaway et al., 1981; Faine et al., 1999). However,there is limited information concerning pathogenic leptospiresinfecting wild rodents living in urban/suburban areas and its rolein the epidemiology of the leptospirosis is poorly understood Faineet al. (1999).

The first report of isolation and serological characterization ofpathogenic Leptospira from Brazilian guinea pig was done by DeCastro et al. (1961). More recently, a serological survey of lep-tospiral infection in Brazilian guinea pig detected agglutinatingantibodies against Leptospira kirschineri (Butembo serovar) andL. interrogans species (Hardjo and Bratislava serovars), serovarsthat are frequently associated to leptospirosis outbreak in domes-

tic animals (Faine et al., 1999; Gressler et al., 2010). Thus,the close interaction between wild and synanthropic rodents inurban/suburban areas defines a considerable hazard of human andanimal leptospiral infection.

166 L.G. Monte et al. / Acta Tropica 126 (2013) 164– 166

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B) renal tubular necrosis and (C) widespread pulmonary hemorrhage.

For Leptospira from Brazilian guinea pig molecular typing, weerformed analysis of VNTR, which has proved to be a highly pow-rful and discriminating method for bacterial population structuretudies, characterizing isolates even from mono-morphic bacterialopulations and could differentiate serogroup and serovar from L.

nterrogans sensu stricto (Pourcel et al., 2003; Farlow et al., 2002;ajed et al., 2005). Moreover, we performed rpoB gene sequenc-

ng to confirm the Leptospira species (Renesto et al., 2000; La et al.,006). The sequencing of the rpoB gene and VNTR profile allowedhe classification of the Brazilian guinea pig isolate strain as belong-ng to L. interrogans serogroup Icterohaemorrhagiae. The serogroupdentification of pathogenic Leptospira should be useful in lepto-pirosis outbreaks and epidemiological surveys (Levett, 2001). Weecently reported the same serogroup from capybara in Southernrazil (Jorge et al., 2012). The Icterohaemorrhagiae serogroup isommonly found in synanthropic rodents responsible for numer-us outbreaks of urban leptospirosis (Adler and de la Pena, 2010).

L. interrogans from the Brazilian guinea pig was used to inoc-lated the hamster, the animal species that mimics pathologyssociated with acute lethal forms of human and experimentaleptospirosis. Lesions were observed in lung and kidney tissues. Thesolate caused clinical signs of leptospirosis and lesions in hamsternoculated, attesting to its virulence.

Summing up, we reported the isolation and characterization of L.nterrogans serogroup Icterohaemorrhagiae from Brazilian guineaig. These findings can help to understand the role of this small wildodent in leptospirosis epidemiology in Latin America and showhat the Brazilian guinea pig may harbor pathogenic Leptospira.

cknowledgments

This work was supported by Coordenac ão de Aperfeic oamentoe Pessoal de Nível Superior (CAPES) and Conselho Nacional deesenvolvimento Científico e Tecnológico (CNPq). The authors arerateful to Instituto Brasileiro do Meio Ambiente e dos Recursosaturais Renováveis (IBAMA).

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