)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with...

8
Research Article Molecular Characterization of Leptospira spp. in Environmental Samples from North-Eastern Malaysia Revealed a Pathogenic Strain, Leptospira alstonii Muhammad Azharuddin Azali, 1 Chan Yean Yean, 2 Azian Harun, 2 Nurul Najian Aminuddin Baki, 2 and Nabilah Ismail 2 1 School of Agriculture Science and Biotechnology, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Tembila Campus, 22200 Besut, Terengganu, Malaysia 2 Department of Medical Microbiology and Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia Correspondence should be addressed to Nabilah Ismail; [email protected] Received 17 December 2015; Revised 16 March 2016; Accepted 17 March 2016 Academic Editor: Marcel Tanner Copyright © 2016 Muhammad Azharuddin Azali et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e presence of pathogenic Leptospira spp. in the environment poses threats to human health. e aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% ( = 33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii ( = 1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, = 1/33, and L. licerasiae, = 7/33) and nonpathogenic leptospire, L. meyeri ( = 22/33) in 24.2% and 66.7%, respectively. is study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors. 1. Introduction Leptospirosis is one of the most important zoonoses infecting both developing and developed countries in the world [1]. Traditionally, leptospirosis was classified as an occupational disease [2]. Later, leptospirosis was also associated with recreational activities through exposure to the contaminated soil or water [3–5]. An increasing number of people involved in outdoor activities have increased the chances of infection. Pets and rodents are the sources of infection in the housing areas. High density of rats in the markets poses potential threats to visitors and town service workers [6]. e expan- sion of housing areas also increases the opportunistic contact between humans and the infected wildlife. Environments are frequently associated with nonpathogenic leptospires. How- ever, a novel pathogenic species, L. kmetyi, has been isolated from environmental samples in Malaysia [7]. Malaysia is a tropical country with high seasonal rainfall, warm temperatures, and wet and humid climate. ese condi- tions lengthen the survival of leptospires in the environment. It is common that floods occur following heavy rainfall dur- ing monsoon season in the East Coast. e presence of lep- tospires in the environment during flooding may potentially cause an outbreak of leptospirosis. Kelantan which is located in the East Coast of Malaysia is among the affected states. e number of cases increased during flooding from on average 20 cases to 31 cases [8]. Many inhabitants in this state are also involved in agricultural activities which pose the highest risk as 50 cases were reported from the workers in this sector [8]. It was also reported that, in 2011, one death was recorded out of 276 cases [9]. Previously, one death was reported from a waterfall in Kelantan which is Jeram Pasu [10]. In order to identify the potential threats of leptospirosis in the envi- ronments, the present study aims to detect and characterize Hindawi Publishing Corporation Journal of Tropical Medicine Volume 2016, Article ID 2060241, 7 pages http://dx.doi.org/10.1155/2016/2060241

Transcript of )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with...

Page 1: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

Research ArticleMolecular Characterization of Leptospira spp inEnvironmental Samples from North-Eastern MalaysiaRevealed a Pathogenic Strain Leptospira alstonii

Muhammad Azharuddin Azali1 Chan Yean Yean2 Azian Harun2

Nurul Najian Aminuddin Baki2 and Nabilah Ismail2

1School of Agriculture Science and Biotechnology Faculty of Bioresources and Food Industry Universiti Sultan Zainal AbidinTembila Campus 22200 Besut Terengganu Malaysia2Department of Medical Microbiology and Parasitology School of Medical Sciences Health Campus Universiti Sains Malaysia16150 Kubang Kerian Kelantan Malaysia

Correspondence should be addressed to Nabilah Ismail drnabilahusmmy

Received 17 December 2015 Revised 16 March 2016 Accepted 17 March 2016

Academic Editor Marcel Tanner

Copyright copy 2016 Muhammad Azharuddin Azali et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Thepresence of pathogenicLeptospira spp in the environment poses threats to humanhealthThe aimof this studywas to detect andcharacterize Leptospira spp from environmental samples A total of 144 samples comprised of 72 soil and 72 water samples werecollected from markets and recreational areas in a north-eastern state in Malaysia Samples were cultured on Ellinghausen andMcCullough modified by Johnson and Harris media Leptospires were positive in 229 (119899 = 33) of the isolates Based on partialsequences of 16S rRNA a pathogenic leptospire Leptospira alstonii (119899 = 133) was identified in 3 of the isolates followed byintermediate leptospire (L wolffii 119899 = 133 and L licerasiae 119899 = 733) and nonpathogenic leptospire L meyeri (119899 = 2233)in 242 and 667 respectively This study demonstrates the presence of a clinically significant pathogenic L alstonii in theenvironments which could pose health risks to the occupants and visitors

1 Introduction

Leptospirosis is one of themost important zoonoses infectingboth developing and developed countries in the world [1]Traditionally leptospirosis was classified as an occupationaldisease [2] Later leptospirosis was also associated withrecreational activities through exposure to the contaminatedsoil or water [3ndash5] An increasing number of people involvedin outdoor activities have increased the chances of infectionPets and rodents are the sources of infection in the housingareas High density of rats in the markets poses potentialthreats to visitors and town service workers [6] The expan-sion of housing areas also increases the opportunistic contactbetween humans and the infected wildlife Environments arefrequently associated with nonpathogenic leptospires How-ever a novel pathogenic species L kmetyi has been isolatedfrom environmental samples in Malaysia [7]

Malaysia is a tropical country with high seasonal rainfallwarm temperatures andwet andhumid climateThese condi-tions lengthen the survival of leptospires in the environmentIt is common that floods occur following heavy rainfall dur-ing monsoon season in the East Coast The presence of lep-tospires in the environment during flooding may potentiallycause an outbreak of leptospirosis Kelantan which is locatedin the East Coast ofMalaysia is among the affected statesThenumber of cases increased during flooding from on average20 cases to 31 cases [8] Many inhabitants in this state are alsoinvolved in agricultural activities which pose the highest riskas 50 cases were reported from the workers in this sector [8]It was also reported that in 2011 one death was recordedout of 276 cases [9] Previously one death was reported froma waterfall in Kelantan which is Jeram Pasu [10] In orderto identify the potential threats of leptospirosis in the envi-ronments the present study aims to detect and characterize

Hindawi Publishing CorporationJournal of Tropical MedicineVolume 2016 Article ID 2060241 7 pageshttpdxdoiorg10115520162060241

2 Journal of Tropical Medicine

Thailand

Kelantan

Peninsular Malaysia

Pahang Terengganu

South China Sea

(6132∘N 102238∘E)(6087∘N 102237∘E)

(5742∘N 102374∘E)(5794596∘N 102336681∘E)

South marketNorth market North waterfall

South waterfall

Figure 1 Location of sampling sites in north-eastern state of Malaysia

Leptospira spp isolated from the selected environments inMalaysia

2 Material and Methods

21 Sampling Sites Samplings were conducted from Decem-ber 2012 to November 2013 at the selected recreational areas(waterfalls with spots of running and still water) and marketsin the north-eastern state of Malaysia Kelantan (Figure 1)Those sampling sites which cover urban and rural areasof three districts were selected based on previous reportsof leptospirosis cases rat infestations and improper wastemanagement

22 Isolation of Leptospires

221 Sample Collection Water samples in recreational areaswere collected from shaded suspected areas for the presenceof animals and in-between rock areas Water samples werecollected about 1 foot below the water surface Water sampleswere filtered through 02 120583mNalgene Filter Unit and 40mLof the samples were transferred into centrifuge tubes andthen centrifuged at 4000timesg 27∘C for 20mins Two mL ofthe samples were inoculated into 5mL of liquid EMJHmediawith the addition of 5-fluorouracil (100 120583gmL) For soil sam-ples sampling locations were selected from wet and shadedareas from garbage sites and in the places where spoiled foodwas spotted In sterile 50mL falcon tubes the soil sampleswere mixed with sterile water and shaken vigorously The

suspension was allowed to settle for 5ndash10 minutes beforebeing filtered using 02120583mNalgene Filter Unit

222 Culture of Leptospires Several drops of the filtrateswere inoculated into EMJH media supplemented withantimicrobial agents (5-fluorouracil 100 120583gmL) and incu-bated at 30∘C in a shaker incubator at 25 rpm to accelerate thegrowth of leptospires The presence of leptospires was exam-ined under dark-field microscopy using 20x and 40x mag-nification daily for 28 days Leptospires can be distinguishedby other spirochetes based on their characteristic thin helicalstructures with prominent hooked ends and motility

223 Culture Purification In an event of contamination1000 120583L of contaminated cultures were transferred into freshliquid EMJH media supplemented with sulfamethoxazoleand trimethoprim (408 120583gmL) amphotericin B (5 120583gmL)and 5-fluorouracil (100 120583gmL) according to Chakrabortyet al with some modifications [14] The concentration oftrimethoprim was changed from 20120583gmL to 8 120583gmL andfosfomycin was not added to the media The cultures wereexamined under dark-field microscopy using 20x and 40xmagnification daily for 28 days If the contaminants were stillpresent the cultures were diluted in sterile distilled waterusing a serial dilution technique starting from dilutions 10minus310minus6 10minus9 and 10minus12 and then transferred into solid EMJHmediaThe diluted culture was incubated for 3 weeks or untilthe leptospires colonies were observed on plates Single

Journal of Tropical Medicine 3

Table 1 List of primers used

Primer Targeted genes Annealing temperature (∘C) Product size Sequences SourcesG1 secY 285 bp 51015840-CTGAATCGCTGTATAAAAGT 31015840

[11]G2 55 51015840-GGAAAACAAATGGTCGGAAG 31015840

B64-I flaB 563 bp 51015840-CTGAATTCTCATCTCAACTC 31015840

B64-II 51015840-GCAAAATCAGATGGACG AT 31015840

Pathogenic gene LipL32 58 120 bp 51015840-AGG TCT TTACAGAATTTCTTTCA 31015840 This study51015840-TTACTTAGTCGCGTCAGAA 31015840

Sapro 1 rrs 55 240 bp 51015840-AGAAATTTGTGCTAATACCGAATGT 31015840 [12]Sapro 2 51015840-GGCGTCGCTGCTTCAGGCTTTCG 31015840

Bak2-F 16S rRNA 52 796 bp 51015840-AGTTTGATCMTGGCTCAG 31015840 [13]Bak2-R 51015840-GGACTACHAGGGTATCTAAT 31015840

isolated colonies were transferred from the plates into liquidmedia using sterile Pasteur pipettes The cultures were exam-ined under dark-field microscopy daily for 28 days

23 Serological Characterization The microscopic aggluti-nation test (MAT) was performed using a set of hyper-immune rabbit antisera purchased from the QueenslandHealth Clinical and Statewide Services Australia includingAutumnalis Tarassovi Pyrogenes Javanica GrippotyphosaCopenhageni Canicola Ballum Hardjoparjitno CelledoniPatoc Djasiman Icterohaemorrhagiae Pomona Australisand Hebdomadis using the available method [15] Culturesof 4 to 7 days of age were used as antigens The turbidity ofthe cultures was adjusted to 05 McFarland The plates weregently shaken to mix contents covered to exclude debris andprevent evaporation and incubated at 30∘C for 2 hours Allof the isolates were screened for agglutination at titre ge1 100under dark-field microscope Agglutination of at least 50 ofthe leptospires was considered as positive

24 Molecular Characterization of the Isolates DNA extrac-tion was performed on isolates by using Qiagen DNeasyBlood amp Tissue Kit (Qiagen USA) according to the man-ufacturerrsquos protocols for Gram negative bacteria and storedat 20∘C until use A 20120583L-PCR reaction mixture containing1x PCR buffer 25mMMgCl

2 016mMdNTPrsquos premixed

004 120583Mof each primer and 075 units of Taq Polymerase wasused in all PCR amplifications Amplification was performedin MJ Research thermocycler with an initial denaturationat 94∘C for 5 minutes followed by 30 cycles of 94∘C for30 seconds annealing for 30 seconds at Ta of the primers(Table 1) and extension at 72∘C for 30 seconds The cycleswere then followed by the final extension at 72∘C for 5minutes Primers G1G2 and B64-IB64-II were used todetect the Leptospira spp Primer targeting virulence genewas used for the detection of lipL32 gene Bak2 primer pairtargeting 16SrRNA gene was used for species identificationThe primer sets Sapro 1 and Sapro 2 were used to detect thenonpathogenic leptospires Amplified products were charac-terized by electrophoresis of 5120583L of each reaction on 15agarose gel for 50min at 90V The PCR product amplifiedby Bak2 primer pair was purified using QIAquick PCRPurification Kit (Qiagen USA) and subsequently sequenced

using Sanger sequencing kits and instruments supplied byApplied Biosystems US performed by First Base Labora-tories (Selangor Malaysia) The DNA sequences were editedin BioEdit [16] and compared against the GenBank databaseusing BLAST The 16S rRNA gene partial sequences of allisolates were aligned with the 16S rRNA sequences of the typestrains obtained from GenBank by using Multiple SequenceComparison by Log-Expectation (MUSCLE) in MEGA 6software [17]The annotated sequences were submitted to theGenBank

3 Results

31 Isolation of Leptospires A total of 144 samples comprisedof 72 water (markets 119899 = 36 recreational areas 119899 = 36)and 72 soil (markets 119899 = 36 recreational areas 119899 = 36)samples were collectedOut of those samples 33were positivefor leptospires based on their characteristic morphology andmotility Among positive samples 7 (5) were water samplesand 26 (18) were soil samples A total of 29 positive samples(water 119899 = 7 soil 119899 = 22) were collected from markets andonly four positive samples (water 119899 = 0 soil 119899 = 4) werecollected from recreational areas

32 Serological Characterization All of the leptospiral iso-lates in this study were not agglutinated with any of thereference hyperimmune sera used in MAT (Table 2) Hencethe isolates did not belong to any of the tested serogroups

33 Molecular Characterization Out of 33 isolates 18 isolateswere detected as Leptospira spp using G1G2 and B64-IB64-II primers (Table 2) None of the isolates were found to con-tain the virulence geneMolecular identification by 16S rRNAverified that from 33 positive samples in this study 31 isolateswere identified as Leptospira spp and two isolates wereidentified as leptospiral closest relatives Leptonema illini Apathogenic leptospire L alstonii (3) was isolated from asoil sample in a market Eight isolates (242) were identifiedas intermediate pathogenic species comprised of seven Lwolffii and one L licerasiae A total of 22 isolates (667) wereidentified as nonpathogenic leptospires L meyeri

The phylogenetic tree constructed using the Neighbour-Joining method was illustrated in Figure 2 Leptospira spp

4 Journal of Tropical Medicine

Table 2 Results for molecular and serological tests

Species (119899 = 33) G1G2 B64-IB64-II SAPRO1SAPRO2 Virulence gene (LipL32) MAT(119899) (119899)

L alstonii (119899 = 1) 1 1 mdash mdashL wolffii (119899 = 7) mdash 7 mdash mdashL licerasiae (119899 = 1) mdash 1 mdash mdashL meyeri (119899 = 22) 17 22 mdash mdashLeptonema illini (119899 = 2) mdash 2 mdash mdashTotal (33) 18 33 0 0

Leptospira interrogans AY631984Leptospira kirschneri AY631895Leptospira noguchii AY631886

Leptospira alexanderi AY631880Leptospira borgpetersenii AY887899

Leptospira weilii AY631877Leptospira santarosai AY631883

WS1 Leptospira alstonii KT002380Leptospira kmetyi AB279549Leptospira alstonii AY631881

Leptospira inadai AY631896Leptospira broomii AY796065Leptospira fainei AY631885

Leptospira licerasiae EF612284

Leptospira wolffii KT002374Leptospira wolffii KT002375

Leptospira wolffii KT002378Leptospira wolffii KT002379Leptospira wolffii KT002384Leptospira wolffii KT002385

Leptospira wolffii EF025496Leptospira wolffii KT002394

Leptospira vanthielli AY631897Leptospira wolbachii AY631879Leptospira biflexa AY631876

Leptospira terpstrae AY631888Leptospira meyeri KT002389

Leptospira meyeri KT002372Leptospira meyeri KT002373Leptospira meyeri KT002376Leptospira meyeri KT002377

Leptospira meyeri KT002381Leptospira meyeri KT002382Leptospira meyeri KT002383

Leptospira meyeri KT002390Leptospira meyeri KT002391Leptospira meyeri KT002392Leptospira meyeri KT002393Leptospira meyeri KT002395Leptospira meyeri KT002396Leptospira meyeri KT002397Leptospira meyeri KT002398Leptospira meyeri KT002399Leptospira meyeri KT002400Leptospira meyeri KT002401Leptospira meyeri KT002402Leptospira meyeri KT002403Leptospira meyeri KT002404

Leptonema illini AY714984Leptonema illini KT002386Leptonema illini KT002387

WS9 Leptospira licerasiae KT002388LS12LS18

SS5SS6WS5WS6

WS15

WS10LS1LS7SS3SS4WS2WS3WS4WS11

WS14WS16WS17WS18WW2WW3WW4WW5WW6WW7WW8

WS7WS8

WS12WS13

8032

2134

91 6738

819999

9792

6388

60901369

10029

49

62

80

10076

16

55 63

Pathogenic

Intermediate

Nonpathogenic

002

Figure 2 A phylogenetic tree generated by the Neighbour-Joining method using MEGA 6 based on 16S rRNA gene sequence of Leptospiraisolates and the selected strains

isolated in this study were clearly separated into three cladesStrain WS1 was discovered from soil samples included in thepathogenic clade It was closely related to type strains of Lalstonii and L kmetyi LS12 LS18 SS5 SS6 WS5 WS6 WS9and WS15 were grouped into intermediate pathogenic clade

LS1 LS7 SS4 WS1 WS2 WS3 WS4 WS10 WS11 WS12WS13WS14WS16WS17WS18WW2WW3WW4WW5WW6 WW7 and WW8 were grouped into nonpathogenicclade These isolates were closely related to type strains of Lmeyeri and L yanagawa

Journal of Tropical Medicine 5

4 Discussion

Even though this is not the first study reporting the detectionand characterization of leptospiral isolates from environ-mental samples its isolation from recreational and marketareas would give an impact to the community Ganoza et alreported that the prevalence of leptospires in water samplesfrom markets was 679 [18] In the present study positivewater samples from markets were found in 194 onlyLeptospires may be unable to survive in the drainage waterin this market The water was found to contain detergent andappeared oily most probably due to its close proximity tothe nearby food stalls It has been reported that 30 ppm ofdetergent (Ceepryn Fixanol and Sapamine) were lethal toleptospires in water in 5 minutes [19] The prevalence of lep-tospires in water samples from recreational areas in Malaysiawas 1167 [20] In the current study a lower prevalenceof 556 was noted which could be due to the effect ofinappropriate choice of sampling points The chosen pointsmay have not been exposed to the leptospiral contamination

All isolates recovered in this study showed negativereactions to 16 reference hyperimmune sera tested in MATSimilarly negative results of MAT for environmental isolateswere also reported from another study in which eight isolatesfrom the selected urban sites in Malaysia were negative fora total of 25 hyperimmune sera tested [20] The availabilityof the reference hyperimmune sera was very limited becausenone of local reference laboratories supplies the hyperim-mune sera Since the sera used in this study were procuredoverseas the available panel of sera may not cover thecirculating local serovar A total of 37 serovars of Leptospirafrom 13 serogroups have been identified in Malaysia [21]This highlights the importance of having locally producedhyperimmune sera available for local use

In our study G1G2 and B64-IB64-II primers detected18 out of 33 isolates as Leptospira spp However 16S rRNAgene sequencing identified 31 out of 33 isolates as Leptospiraspp G1G2 and B64-IB64-II primers were not able to detectall strains of Leptospira spp The occurrence of new strainsof leptospires in humans animals and environment hasincreased the need for new primers that are more specificthan G1G2 and B64-IB64-II Leptospiral putative virulencegene LipL32 rises as a new target for the detection ofpathogenic leptospires though its role in virulence mecha-nisms remains unknown [22] This is further complicatedby the absence of this gene in pathogenic and intermediateleptospires isolated in this studyThis gene was either absenceor undetectable by PCR because in some cases the LipL32gene was not detectable in L licerasiae by PCR but its productwas detectable by both Southern blot hybridization andWestern immunoblot [23] In the previous study by Murgiaet al primer sets Sapro 1 and Sapro 2 were used to detectthe saprophytic leptospires [11] However these primers werefound to be unspecific [11] as pathogenic leptospire Lalstonii was amplified by this primer in this study

In this study a pathogenic species L alstonii was isolatedfrom a soil sample in market area Our finding was in linewith previous studies that also isolated L alstonii from theenvironment [24] Exposure to the soil in market areas which

harboured pathogenic strains may explain the high seropos-itivity of leptospiral antibodies among garbage collectorsand town cleaners in Kelantan [6] L alstonii was possiblyharboured by frogs as it was initially isolated from frogs[25] The isolation of leptospires from frogs specifically fromkidney was reported as early as 1964 [26] Several pathogenicserovars or serogroups such as bim Australis and Ballumhave been isolated from frogs [27 28] However inoculationof frogs with pathogenic serovars to establish experimentalleptospirosis led to unsatisfactory results as leptospires werenot recovered in their organs [29]

The intermediate species L wolffii and L licerasiae wereisolated from markets and recreational areas They are ableto cause diseases in humans although less frequent [12] Thepresence of pathogenic and intermediate species warrantsadherence to precautions and preventive measures amongthe visitors to those areas It was reported that Malaysianswho were involved in recreational activities especially waterrelated are 24 times more likely to acquire leptospiro-sis compared to those who were not involved in similaractivities [30] Leptospirosis was detected in wildlife inMalaysia including wild mammals-monkeys bats squirrelsandmongoose [31]Those animals could be the reservoirs forleptospires in recreational areas located in remote areas

A previous study reported high numbers of L meyeri(881) from environmental samples [24] Our study alsodemonstrated the predominance of L meyeri in Malaysiaenvironment Though this species was nonpathogenic thereis controversy surrounding L meyeri pathogenicity sta-tus L meyeri serovar ranarum ICF which was previouslyconsidered as nonpathogenic was shown to be related topathogenic strain suggesting that this species consisted ofboth pathogenic and nonpathogenic strains [32] This strainwas also amplified by sets of primers specific for pathogenicLeptospira and not the sets of primers specific for saprophyticstrain [11] This result is also supported by phylogeneticanalysis in another study [13] This disagreement demands anovel typingmethod of Leptospira spp which covers not onlypathogenic species but also intermediate and nonpathogenicstrains to resolve the uncertainties in speciesrsquo designation inthe genus of Leptospira The accurate species designation andclassification are crucial because the presence of clinicallyimperative leptospires in the environment would not beoverlooked

Phylogenetic analysis of the studied isolates using 16SrRNA gene sequences concurred with the findings of pre-vious report by Morey et al in 2006 that the pathogenicintermediate and nonpathogenic species each formed oneclade [33] Intermediate groups were closely related to thepathogenic rather than nonpathogenic group Isolates inthe nonpathogenic group were closely related to both Lmeyeri and L yanagawa It was reported that species withinthe nonpathogenic groups were separated by no more than10 bp [33] Using the 16S rRNA gene L meyeri recoveredin this study were not separated In order to demonstrateintraspecies genetic variations the use of other gene loci isnecessary either singly or in combination It was also sug-gested that a cut-off point of 1000 base pairs (bp) was appliedfor leptospiral species identification [34] The phylogenetic

6 Journal of Tropical Medicine

analysis of environmental isolates using 16S rRNA was alsofound to be better than using PFGE profiles

5 Conclusion

Thedata presented in this study demonstrates the presence ofa clinically significant pathogenic L alstonii and the predom-inance of Lmeyeri in the environmentThe pathogenic strainwas found to not contain one of the highly conserve putativeleptospiral virulence genes Early prevention is required toreduce the risks of infection amongst the population Hencecontinuous control and surveillance are essential in loweringthe burden of the disease

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported in part by the KPI Fund UniversitiSains Malaysia Short Term Grant (304PPSP61311069) andRU Grant (1001PPSP812064)

References

[1] G Pappas P Papadimitriou V Siozopoulou L Christou andN Akritidis ldquoThe globalization of leptospirosis worldwideincidence trendsrdquo International Journal of Infectious Diseasesvol 12 no 4 pp 351ndash357 2008

[2] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[3] C Radl M Muller S Revilla-Fernandez et al ldquoOutbreak ofleptospirosis among triathlon participants in Langau Austria2010rdquo Wiener Klinische Wochenschrift vol 123 no 23-24 pp751ndash755 2011

[4] S Brockmann I Piechotowski O Bock-Hensley et al ldquoOut-break of leptospirosis among triathlon participants in Germany2006rdquo BMC Infectious Diseases vol 10 article 91 2010

[5] J Morgan S L Bornstein A M Karpati et al ldquoOutbreak ofleptospirosis among triathlon participants and community res-idents in Springfield Illinois 1998rdquo Clinical Infectious Diseasesvol 34 no 12 pp 1593ndash1599 2002

[6] M N Shafei M R Sulong N A Yaacob et al ldquoSeroprevalenceof leptospirosis among town service workers in northeast-ern state of Malaysiardquo International Journal of CollaborativeResearch on Internal Medicine amp Public Health vol 4 no 4 pp395ndash403 2012

[7] A T Slack S Khairani-Bejo M L Symonds et al ldquoLeptospirakmetyi sp nov isolated from an environmental source inMalaysiardquo International Journal of Systematic and EvolutionaryMicrobiology vol 59 no 4 pp 705ndash708 2009

[8] A Hanim ldquoPenyakit kencing tikus meningkatrdquo 2014 httpww1utusancommyutusanTimur20140106wt 02Penyakit-kencing-tikus-meningkat

[9] Bernama ldquoSix died from leptospirosis in Kelantan in first sixmonths of 2013rdquo 2015 httpwwwmysinchewcomnode87855

[10] Kencing tikus Air Terjun Jeram Pasu lengang 2013 httpww1utusancommyutusanTimur20131003wt 03Kencing-tikus-Air-Terjun-Jeram-Pasu-lengang

[11] R Murgia N Riquelme G Baranton andM Cinco ldquoOligonu-cleotides specific for pathogenic and saprophytic leptospiraoccurring in waterrdquo FEMS Microbiology Letters vol 148 no 1pp 27ndash34 1997

[12] J N Ricaldi D E Fouts J D Selengut et al ldquoWhole genomeanalysis of Leptospira licerasiae provides insight into leptospiralevolution and pathogenicityrdquo PLoS Neglected Tropical Diseasesvol 6 no 10 Article ID e1853 2012

[13] J V Hookey J Bryden and L Gatehouse ldquoThe use of16S rDNA sequence analysis to investigate the phylogeny ofLeptospiraceae and related spirochaetesrdquo Journal of GeneralMicrobiology vol 139 no 11 pp 2585ndash2590 1993

[14] A Chakraborty SMiyahara S Y AMVillanuevaM Saito NG Gloriani and S-I Yoshida ldquoA novel combination of selectiveagents for isolation of Leptospira speciesrdquo Microbiology andImmunology vol 55 no 7 pp 494ndash501 2011

[15] J R Cole Jr C R Sulzer and A R Pursell ldquoImprovedmicrotechnique for the leptospiral microscopic agglutinationtestrdquo Journal of AppliedMicrobiology vol 25 no 6 pp 976ndash9801973

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[18] C A Ganoza M A Matthias D Collins-Richards et alldquoDetermining risk for severe leptospirosis bymolecular analysisof environmental surface waters for pathogenic LeptospirardquoPLoS Medicine vol 3 no 8 pp 1329ndash1340 2006

[19] S L Chang M Buckingham and M P Taylor ldquoStudieson Leptospira Icterohaemorrhagiae IV Survival in water andsewage destruction in water by Halogen compounds syntheticdetergents and heatrdquo Journal of Infectious Diseases vol 82 no3 pp 256ndash266 1948

[20] D Benacer P YWho S NM Zain F Amran and K LThongldquoPathogenic and saprophytic Leptospira species in water andsoils from selected urban sites in peninsularMalaysiardquoMicrobesand Environments vol 28 no 1 pp 135ndash140 2013

[21] D Benacer S N Mohd Zain F Amran R L Gallowayand K L Thong ldquoIsolation and molecular characterizationof Leptospira interrogans and Leptospira borgpetersenii Isolatesfrom the urban rat populations of Kuala LumpurMalaysiardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 88 no4 pp 704ndash709 2013

[22] AHabarta P A EAbreuNOlivera et al ldquoIncreased immuno-genicity to LipL32 of Leptospira interrogans when expressedas a fusion protein with the cholera toxin B subunitrdquo CurrentMicrobiology vol 62 no 2 pp 526ndash531 2011

[23] M A Matthias J N Ricaldi M Cespedes et al ldquoHumanleptospirosis caused by a new antigenically unique Leptospiraassociated with a Rattus species reservoir in the PeruvianAmazonrdquo PLoS Neglected Tropical Diseases vol 2 no 4 articlee213 2008

[24] M Saito S Y AMVillanueva A Chakraborty et al ldquoCompar-ative analysis of Leptospira strains isolated from environmentalsoil and water in the Philippines and Japanrdquo Applied andEnvironmental Microbiology vol 79 no 2 pp 601ndash609 2013

[25] L Smythe B Adler R A Hartskeerl R L Galloway CY Turenne and P N Levett ldquoClassification of Leptospira

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 2: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

2 Journal of Tropical Medicine

Thailand

Kelantan

Peninsular Malaysia

Pahang Terengganu

South China Sea

(6132∘N 102238∘E)(6087∘N 102237∘E)

(5742∘N 102374∘E)(5794596∘N 102336681∘E)

South marketNorth market North waterfall

South waterfall

Figure 1 Location of sampling sites in north-eastern state of Malaysia

Leptospira spp isolated from the selected environments inMalaysia

2 Material and Methods

21 Sampling Sites Samplings were conducted from Decem-ber 2012 to November 2013 at the selected recreational areas(waterfalls with spots of running and still water) and marketsin the north-eastern state of Malaysia Kelantan (Figure 1)Those sampling sites which cover urban and rural areasof three districts were selected based on previous reportsof leptospirosis cases rat infestations and improper wastemanagement

22 Isolation of Leptospires

221 Sample Collection Water samples in recreational areaswere collected from shaded suspected areas for the presenceof animals and in-between rock areas Water samples werecollected about 1 foot below the water surface Water sampleswere filtered through 02 120583mNalgene Filter Unit and 40mLof the samples were transferred into centrifuge tubes andthen centrifuged at 4000timesg 27∘C for 20mins Two mL ofthe samples were inoculated into 5mL of liquid EMJHmediawith the addition of 5-fluorouracil (100 120583gmL) For soil sam-ples sampling locations were selected from wet and shadedareas from garbage sites and in the places where spoiled foodwas spotted In sterile 50mL falcon tubes the soil sampleswere mixed with sterile water and shaken vigorously The

suspension was allowed to settle for 5ndash10 minutes beforebeing filtered using 02120583mNalgene Filter Unit

222 Culture of Leptospires Several drops of the filtrateswere inoculated into EMJH media supplemented withantimicrobial agents (5-fluorouracil 100 120583gmL) and incu-bated at 30∘C in a shaker incubator at 25 rpm to accelerate thegrowth of leptospires The presence of leptospires was exam-ined under dark-field microscopy using 20x and 40x mag-nification daily for 28 days Leptospires can be distinguishedby other spirochetes based on their characteristic thin helicalstructures with prominent hooked ends and motility

223 Culture Purification In an event of contamination1000 120583L of contaminated cultures were transferred into freshliquid EMJH media supplemented with sulfamethoxazoleand trimethoprim (408 120583gmL) amphotericin B (5 120583gmL)and 5-fluorouracil (100 120583gmL) according to Chakrabortyet al with some modifications [14] The concentration oftrimethoprim was changed from 20120583gmL to 8 120583gmL andfosfomycin was not added to the media The cultures wereexamined under dark-field microscopy using 20x and 40xmagnification daily for 28 days If the contaminants were stillpresent the cultures were diluted in sterile distilled waterusing a serial dilution technique starting from dilutions 10minus310minus6 10minus9 and 10minus12 and then transferred into solid EMJHmediaThe diluted culture was incubated for 3 weeks or untilthe leptospires colonies were observed on plates Single

Journal of Tropical Medicine 3

Table 1 List of primers used

Primer Targeted genes Annealing temperature (∘C) Product size Sequences SourcesG1 secY 285 bp 51015840-CTGAATCGCTGTATAAAAGT 31015840

[11]G2 55 51015840-GGAAAACAAATGGTCGGAAG 31015840

B64-I flaB 563 bp 51015840-CTGAATTCTCATCTCAACTC 31015840

B64-II 51015840-GCAAAATCAGATGGACG AT 31015840

Pathogenic gene LipL32 58 120 bp 51015840-AGG TCT TTACAGAATTTCTTTCA 31015840 This study51015840-TTACTTAGTCGCGTCAGAA 31015840

Sapro 1 rrs 55 240 bp 51015840-AGAAATTTGTGCTAATACCGAATGT 31015840 [12]Sapro 2 51015840-GGCGTCGCTGCTTCAGGCTTTCG 31015840

Bak2-F 16S rRNA 52 796 bp 51015840-AGTTTGATCMTGGCTCAG 31015840 [13]Bak2-R 51015840-GGACTACHAGGGTATCTAAT 31015840

isolated colonies were transferred from the plates into liquidmedia using sterile Pasteur pipettes The cultures were exam-ined under dark-field microscopy daily for 28 days

23 Serological Characterization The microscopic aggluti-nation test (MAT) was performed using a set of hyper-immune rabbit antisera purchased from the QueenslandHealth Clinical and Statewide Services Australia includingAutumnalis Tarassovi Pyrogenes Javanica GrippotyphosaCopenhageni Canicola Ballum Hardjoparjitno CelledoniPatoc Djasiman Icterohaemorrhagiae Pomona Australisand Hebdomadis using the available method [15] Culturesof 4 to 7 days of age were used as antigens The turbidity ofthe cultures was adjusted to 05 McFarland The plates weregently shaken to mix contents covered to exclude debris andprevent evaporation and incubated at 30∘C for 2 hours Allof the isolates were screened for agglutination at titre ge1 100under dark-field microscope Agglutination of at least 50 ofthe leptospires was considered as positive

24 Molecular Characterization of the Isolates DNA extrac-tion was performed on isolates by using Qiagen DNeasyBlood amp Tissue Kit (Qiagen USA) according to the man-ufacturerrsquos protocols for Gram negative bacteria and storedat 20∘C until use A 20120583L-PCR reaction mixture containing1x PCR buffer 25mMMgCl

2 016mMdNTPrsquos premixed

004 120583Mof each primer and 075 units of Taq Polymerase wasused in all PCR amplifications Amplification was performedin MJ Research thermocycler with an initial denaturationat 94∘C for 5 minutes followed by 30 cycles of 94∘C for30 seconds annealing for 30 seconds at Ta of the primers(Table 1) and extension at 72∘C for 30 seconds The cycleswere then followed by the final extension at 72∘C for 5minutes Primers G1G2 and B64-IB64-II were used todetect the Leptospira spp Primer targeting virulence genewas used for the detection of lipL32 gene Bak2 primer pairtargeting 16SrRNA gene was used for species identificationThe primer sets Sapro 1 and Sapro 2 were used to detect thenonpathogenic leptospires Amplified products were charac-terized by electrophoresis of 5120583L of each reaction on 15agarose gel for 50min at 90V The PCR product amplifiedby Bak2 primer pair was purified using QIAquick PCRPurification Kit (Qiagen USA) and subsequently sequenced

using Sanger sequencing kits and instruments supplied byApplied Biosystems US performed by First Base Labora-tories (Selangor Malaysia) The DNA sequences were editedin BioEdit [16] and compared against the GenBank databaseusing BLAST The 16S rRNA gene partial sequences of allisolates were aligned with the 16S rRNA sequences of the typestrains obtained from GenBank by using Multiple SequenceComparison by Log-Expectation (MUSCLE) in MEGA 6software [17]The annotated sequences were submitted to theGenBank

3 Results

31 Isolation of Leptospires A total of 144 samples comprisedof 72 water (markets 119899 = 36 recreational areas 119899 = 36)and 72 soil (markets 119899 = 36 recreational areas 119899 = 36)samples were collectedOut of those samples 33were positivefor leptospires based on their characteristic morphology andmotility Among positive samples 7 (5) were water samplesand 26 (18) were soil samples A total of 29 positive samples(water 119899 = 7 soil 119899 = 22) were collected from markets andonly four positive samples (water 119899 = 0 soil 119899 = 4) werecollected from recreational areas

32 Serological Characterization All of the leptospiral iso-lates in this study were not agglutinated with any of thereference hyperimmune sera used in MAT (Table 2) Hencethe isolates did not belong to any of the tested serogroups

33 Molecular Characterization Out of 33 isolates 18 isolateswere detected as Leptospira spp using G1G2 and B64-IB64-II primers (Table 2) None of the isolates were found to con-tain the virulence geneMolecular identification by 16S rRNAverified that from 33 positive samples in this study 31 isolateswere identified as Leptospira spp and two isolates wereidentified as leptospiral closest relatives Leptonema illini Apathogenic leptospire L alstonii (3) was isolated from asoil sample in a market Eight isolates (242) were identifiedas intermediate pathogenic species comprised of seven Lwolffii and one L licerasiae A total of 22 isolates (667) wereidentified as nonpathogenic leptospires L meyeri

The phylogenetic tree constructed using the Neighbour-Joining method was illustrated in Figure 2 Leptospira spp

4 Journal of Tropical Medicine

Table 2 Results for molecular and serological tests

Species (119899 = 33) G1G2 B64-IB64-II SAPRO1SAPRO2 Virulence gene (LipL32) MAT(119899) (119899)

L alstonii (119899 = 1) 1 1 mdash mdashL wolffii (119899 = 7) mdash 7 mdash mdashL licerasiae (119899 = 1) mdash 1 mdash mdashL meyeri (119899 = 22) 17 22 mdash mdashLeptonema illini (119899 = 2) mdash 2 mdash mdashTotal (33) 18 33 0 0

Leptospira interrogans AY631984Leptospira kirschneri AY631895Leptospira noguchii AY631886

Leptospira alexanderi AY631880Leptospira borgpetersenii AY887899

Leptospira weilii AY631877Leptospira santarosai AY631883

WS1 Leptospira alstonii KT002380Leptospira kmetyi AB279549Leptospira alstonii AY631881

Leptospira inadai AY631896Leptospira broomii AY796065Leptospira fainei AY631885

Leptospira licerasiae EF612284

Leptospira wolffii KT002374Leptospira wolffii KT002375

Leptospira wolffii KT002378Leptospira wolffii KT002379Leptospira wolffii KT002384Leptospira wolffii KT002385

Leptospira wolffii EF025496Leptospira wolffii KT002394

Leptospira vanthielli AY631897Leptospira wolbachii AY631879Leptospira biflexa AY631876

Leptospira terpstrae AY631888Leptospira meyeri KT002389

Leptospira meyeri KT002372Leptospira meyeri KT002373Leptospira meyeri KT002376Leptospira meyeri KT002377

Leptospira meyeri KT002381Leptospira meyeri KT002382Leptospira meyeri KT002383

Leptospira meyeri KT002390Leptospira meyeri KT002391Leptospira meyeri KT002392Leptospira meyeri KT002393Leptospira meyeri KT002395Leptospira meyeri KT002396Leptospira meyeri KT002397Leptospira meyeri KT002398Leptospira meyeri KT002399Leptospira meyeri KT002400Leptospira meyeri KT002401Leptospira meyeri KT002402Leptospira meyeri KT002403Leptospira meyeri KT002404

Leptonema illini AY714984Leptonema illini KT002386Leptonema illini KT002387

WS9 Leptospira licerasiae KT002388LS12LS18

SS5SS6WS5WS6

WS15

WS10LS1LS7SS3SS4WS2WS3WS4WS11

WS14WS16WS17WS18WW2WW3WW4WW5WW6WW7WW8

WS7WS8

WS12WS13

8032

2134

91 6738

819999

9792

6388

60901369

10029

49

62

80

10076

16

55 63

Pathogenic

Intermediate

Nonpathogenic

002

Figure 2 A phylogenetic tree generated by the Neighbour-Joining method using MEGA 6 based on 16S rRNA gene sequence of Leptospiraisolates and the selected strains

isolated in this study were clearly separated into three cladesStrain WS1 was discovered from soil samples included in thepathogenic clade It was closely related to type strains of Lalstonii and L kmetyi LS12 LS18 SS5 SS6 WS5 WS6 WS9and WS15 were grouped into intermediate pathogenic clade

LS1 LS7 SS4 WS1 WS2 WS3 WS4 WS10 WS11 WS12WS13WS14WS16WS17WS18WW2WW3WW4WW5WW6 WW7 and WW8 were grouped into nonpathogenicclade These isolates were closely related to type strains of Lmeyeri and L yanagawa

Journal of Tropical Medicine 5

4 Discussion

Even though this is not the first study reporting the detectionand characterization of leptospiral isolates from environ-mental samples its isolation from recreational and marketareas would give an impact to the community Ganoza et alreported that the prevalence of leptospires in water samplesfrom markets was 679 [18] In the present study positivewater samples from markets were found in 194 onlyLeptospires may be unable to survive in the drainage waterin this market The water was found to contain detergent andappeared oily most probably due to its close proximity tothe nearby food stalls It has been reported that 30 ppm ofdetergent (Ceepryn Fixanol and Sapamine) were lethal toleptospires in water in 5 minutes [19] The prevalence of lep-tospires in water samples from recreational areas in Malaysiawas 1167 [20] In the current study a lower prevalenceof 556 was noted which could be due to the effect ofinappropriate choice of sampling points The chosen pointsmay have not been exposed to the leptospiral contamination

All isolates recovered in this study showed negativereactions to 16 reference hyperimmune sera tested in MATSimilarly negative results of MAT for environmental isolateswere also reported from another study in which eight isolatesfrom the selected urban sites in Malaysia were negative fora total of 25 hyperimmune sera tested [20] The availabilityof the reference hyperimmune sera was very limited becausenone of local reference laboratories supplies the hyperim-mune sera Since the sera used in this study were procuredoverseas the available panel of sera may not cover thecirculating local serovar A total of 37 serovars of Leptospirafrom 13 serogroups have been identified in Malaysia [21]This highlights the importance of having locally producedhyperimmune sera available for local use

In our study G1G2 and B64-IB64-II primers detected18 out of 33 isolates as Leptospira spp However 16S rRNAgene sequencing identified 31 out of 33 isolates as Leptospiraspp G1G2 and B64-IB64-II primers were not able to detectall strains of Leptospira spp The occurrence of new strainsof leptospires in humans animals and environment hasincreased the need for new primers that are more specificthan G1G2 and B64-IB64-II Leptospiral putative virulencegene LipL32 rises as a new target for the detection ofpathogenic leptospires though its role in virulence mecha-nisms remains unknown [22] This is further complicatedby the absence of this gene in pathogenic and intermediateleptospires isolated in this studyThis gene was either absenceor undetectable by PCR because in some cases the LipL32gene was not detectable in L licerasiae by PCR but its productwas detectable by both Southern blot hybridization andWestern immunoblot [23] In the previous study by Murgiaet al primer sets Sapro 1 and Sapro 2 were used to detectthe saprophytic leptospires [11] However these primers werefound to be unspecific [11] as pathogenic leptospire Lalstonii was amplified by this primer in this study

In this study a pathogenic species L alstonii was isolatedfrom a soil sample in market area Our finding was in linewith previous studies that also isolated L alstonii from theenvironment [24] Exposure to the soil in market areas which

harboured pathogenic strains may explain the high seropos-itivity of leptospiral antibodies among garbage collectorsand town cleaners in Kelantan [6] L alstonii was possiblyharboured by frogs as it was initially isolated from frogs[25] The isolation of leptospires from frogs specifically fromkidney was reported as early as 1964 [26] Several pathogenicserovars or serogroups such as bim Australis and Ballumhave been isolated from frogs [27 28] However inoculationof frogs with pathogenic serovars to establish experimentalleptospirosis led to unsatisfactory results as leptospires werenot recovered in their organs [29]

The intermediate species L wolffii and L licerasiae wereisolated from markets and recreational areas They are ableto cause diseases in humans although less frequent [12] Thepresence of pathogenic and intermediate species warrantsadherence to precautions and preventive measures amongthe visitors to those areas It was reported that Malaysianswho were involved in recreational activities especially waterrelated are 24 times more likely to acquire leptospiro-sis compared to those who were not involved in similaractivities [30] Leptospirosis was detected in wildlife inMalaysia including wild mammals-monkeys bats squirrelsandmongoose [31]Those animals could be the reservoirs forleptospires in recreational areas located in remote areas

A previous study reported high numbers of L meyeri(881) from environmental samples [24] Our study alsodemonstrated the predominance of L meyeri in Malaysiaenvironment Though this species was nonpathogenic thereis controversy surrounding L meyeri pathogenicity sta-tus L meyeri serovar ranarum ICF which was previouslyconsidered as nonpathogenic was shown to be related topathogenic strain suggesting that this species consisted ofboth pathogenic and nonpathogenic strains [32] This strainwas also amplified by sets of primers specific for pathogenicLeptospira and not the sets of primers specific for saprophyticstrain [11] This result is also supported by phylogeneticanalysis in another study [13] This disagreement demands anovel typingmethod of Leptospira spp which covers not onlypathogenic species but also intermediate and nonpathogenicstrains to resolve the uncertainties in speciesrsquo designation inthe genus of Leptospira The accurate species designation andclassification are crucial because the presence of clinicallyimperative leptospires in the environment would not beoverlooked

Phylogenetic analysis of the studied isolates using 16SrRNA gene sequences concurred with the findings of pre-vious report by Morey et al in 2006 that the pathogenicintermediate and nonpathogenic species each formed oneclade [33] Intermediate groups were closely related to thepathogenic rather than nonpathogenic group Isolates inthe nonpathogenic group were closely related to both Lmeyeri and L yanagawa It was reported that species withinthe nonpathogenic groups were separated by no more than10 bp [33] Using the 16S rRNA gene L meyeri recoveredin this study were not separated In order to demonstrateintraspecies genetic variations the use of other gene loci isnecessary either singly or in combination It was also sug-gested that a cut-off point of 1000 base pairs (bp) was appliedfor leptospiral species identification [34] The phylogenetic

6 Journal of Tropical Medicine

analysis of environmental isolates using 16S rRNA was alsofound to be better than using PFGE profiles

5 Conclusion

Thedata presented in this study demonstrates the presence ofa clinically significant pathogenic L alstonii and the predom-inance of Lmeyeri in the environmentThe pathogenic strainwas found to not contain one of the highly conserve putativeleptospiral virulence genes Early prevention is required toreduce the risks of infection amongst the population Hencecontinuous control and surveillance are essential in loweringthe burden of the disease

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported in part by the KPI Fund UniversitiSains Malaysia Short Term Grant (304PPSP61311069) andRU Grant (1001PPSP812064)

References

[1] G Pappas P Papadimitriou V Siozopoulou L Christou andN Akritidis ldquoThe globalization of leptospirosis worldwideincidence trendsrdquo International Journal of Infectious Diseasesvol 12 no 4 pp 351ndash357 2008

[2] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[3] C Radl M Muller S Revilla-Fernandez et al ldquoOutbreak ofleptospirosis among triathlon participants in Langau Austria2010rdquo Wiener Klinische Wochenschrift vol 123 no 23-24 pp751ndash755 2011

[4] S Brockmann I Piechotowski O Bock-Hensley et al ldquoOut-break of leptospirosis among triathlon participants in Germany2006rdquo BMC Infectious Diseases vol 10 article 91 2010

[5] J Morgan S L Bornstein A M Karpati et al ldquoOutbreak ofleptospirosis among triathlon participants and community res-idents in Springfield Illinois 1998rdquo Clinical Infectious Diseasesvol 34 no 12 pp 1593ndash1599 2002

[6] M N Shafei M R Sulong N A Yaacob et al ldquoSeroprevalenceof leptospirosis among town service workers in northeast-ern state of Malaysiardquo International Journal of CollaborativeResearch on Internal Medicine amp Public Health vol 4 no 4 pp395ndash403 2012

[7] A T Slack S Khairani-Bejo M L Symonds et al ldquoLeptospirakmetyi sp nov isolated from an environmental source inMalaysiardquo International Journal of Systematic and EvolutionaryMicrobiology vol 59 no 4 pp 705ndash708 2009

[8] A Hanim ldquoPenyakit kencing tikus meningkatrdquo 2014 httpww1utusancommyutusanTimur20140106wt 02Penyakit-kencing-tikus-meningkat

[9] Bernama ldquoSix died from leptospirosis in Kelantan in first sixmonths of 2013rdquo 2015 httpwwwmysinchewcomnode87855

[10] Kencing tikus Air Terjun Jeram Pasu lengang 2013 httpww1utusancommyutusanTimur20131003wt 03Kencing-tikus-Air-Terjun-Jeram-Pasu-lengang

[11] R Murgia N Riquelme G Baranton andM Cinco ldquoOligonu-cleotides specific for pathogenic and saprophytic leptospiraoccurring in waterrdquo FEMS Microbiology Letters vol 148 no 1pp 27ndash34 1997

[12] J N Ricaldi D E Fouts J D Selengut et al ldquoWhole genomeanalysis of Leptospira licerasiae provides insight into leptospiralevolution and pathogenicityrdquo PLoS Neglected Tropical Diseasesvol 6 no 10 Article ID e1853 2012

[13] J V Hookey J Bryden and L Gatehouse ldquoThe use of16S rDNA sequence analysis to investigate the phylogeny ofLeptospiraceae and related spirochaetesrdquo Journal of GeneralMicrobiology vol 139 no 11 pp 2585ndash2590 1993

[14] A Chakraborty SMiyahara S Y AMVillanuevaM Saito NG Gloriani and S-I Yoshida ldquoA novel combination of selectiveagents for isolation of Leptospira speciesrdquo Microbiology andImmunology vol 55 no 7 pp 494ndash501 2011

[15] J R Cole Jr C R Sulzer and A R Pursell ldquoImprovedmicrotechnique for the leptospiral microscopic agglutinationtestrdquo Journal of AppliedMicrobiology vol 25 no 6 pp 976ndash9801973

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[18] C A Ganoza M A Matthias D Collins-Richards et alldquoDetermining risk for severe leptospirosis bymolecular analysisof environmental surface waters for pathogenic LeptospirardquoPLoS Medicine vol 3 no 8 pp 1329ndash1340 2006

[19] S L Chang M Buckingham and M P Taylor ldquoStudieson Leptospira Icterohaemorrhagiae IV Survival in water andsewage destruction in water by Halogen compounds syntheticdetergents and heatrdquo Journal of Infectious Diseases vol 82 no3 pp 256ndash266 1948

[20] D Benacer P YWho S NM Zain F Amran and K LThongldquoPathogenic and saprophytic Leptospira species in water andsoils from selected urban sites in peninsularMalaysiardquoMicrobesand Environments vol 28 no 1 pp 135ndash140 2013

[21] D Benacer S N Mohd Zain F Amran R L Gallowayand K L Thong ldquoIsolation and molecular characterizationof Leptospira interrogans and Leptospira borgpetersenii Isolatesfrom the urban rat populations of Kuala LumpurMalaysiardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 88 no4 pp 704ndash709 2013

[22] AHabarta P A EAbreuNOlivera et al ldquoIncreased immuno-genicity to LipL32 of Leptospira interrogans when expressedas a fusion protein with the cholera toxin B subunitrdquo CurrentMicrobiology vol 62 no 2 pp 526ndash531 2011

[23] M A Matthias J N Ricaldi M Cespedes et al ldquoHumanleptospirosis caused by a new antigenically unique Leptospiraassociated with a Rattus species reservoir in the PeruvianAmazonrdquo PLoS Neglected Tropical Diseases vol 2 no 4 articlee213 2008

[24] M Saito S Y AMVillanueva A Chakraborty et al ldquoCompar-ative analysis of Leptospira strains isolated from environmentalsoil and water in the Philippines and Japanrdquo Applied andEnvironmental Microbiology vol 79 no 2 pp 601ndash609 2013

[25] L Smythe B Adler R A Hartskeerl R L Galloway CY Turenne and P N Levett ldquoClassification of Leptospira

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 3: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

Journal of Tropical Medicine 3

Table 1 List of primers used

Primer Targeted genes Annealing temperature (∘C) Product size Sequences SourcesG1 secY 285 bp 51015840-CTGAATCGCTGTATAAAAGT 31015840

[11]G2 55 51015840-GGAAAACAAATGGTCGGAAG 31015840

B64-I flaB 563 bp 51015840-CTGAATTCTCATCTCAACTC 31015840

B64-II 51015840-GCAAAATCAGATGGACG AT 31015840

Pathogenic gene LipL32 58 120 bp 51015840-AGG TCT TTACAGAATTTCTTTCA 31015840 This study51015840-TTACTTAGTCGCGTCAGAA 31015840

Sapro 1 rrs 55 240 bp 51015840-AGAAATTTGTGCTAATACCGAATGT 31015840 [12]Sapro 2 51015840-GGCGTCGCTGCTTCAGGCTTTCG 31015840

Bak2-F 16S rRNA 52 796 bp 51015840-AGTTTGATCMTGGCTCAG 31015840 [13]Bak2-R 51015840-GGACTACHAGGGTATCTAAT 31015840

isolated colonies were transferred from the plates into liquidmedia using sterile Pasteur pipettes The cultures were exam-ined under dark-field microscopy daily for 28 days

23 Serological Characterization The microscopic aggluti-nation test (MAT) was performed using a set of hyper-immune rabbit antisera purchased from the QueenslandHealth Clinical and Statewide Services Australia includingAutumnalis Tarassovi Pyrogenes Javanica GrippotyphosaCopenhageni Canicola Ballum Hardjoparjitno CelledoniPatoc Djasiman Icterohaemorrhagiae Pomona Australisand Hebdomadis using the available method [15] Culturesof 4 to 7 days of age were used as antigens The turbidity ofthe cultures was adjusted to 05 McFarland The plates weregently shaken to mix contents covered to exclude debris andprevent evaporation and incubated at 30∘C for 2 hours Allof the isolates were screened for agglutination at titre ge1 100under dark-field microscope Agglutination of at least 50 ofthe leptospires was considered as positive

24 Molecular Characterization of the Isolates DNA extrac-tion was performed on isolates by using Qiagen DNeasyBlood amp Tissue Kit (Qiagen USA) according to the man-ufacturerrsquos protocols for Gram negative bacteria and storedat 20∘C until use A 20120583L-PCR reaction mixture containing1x PCR buffer 25mMMgCl

2 016mMdNTPrsquos premixed

004 120583Mof each primer and 075 units of Taq Polymerase wasused in all PCR amplifications Amplification was performedin MJ Research thermocycler with an initial denaturationat 94∘C for 5 minutes followed by 30 cycles of 94∘C for30 seconds annealing for 30 seconds at Ta of the primers(Table 1) and extension at 72∘C for 30 seconds The cycleswere then followed by the final extension at 72∘C for 5minutes Primers G1G2 and B64-IB64-II were used todetect the Leptospira spp Primer targeting virulence genewas used for the detection of lipL32 gene Bak2 primer pairtargeting 16SrRNA gene was used for species identificationThe primer sets Sapro 1 and Sapro 2 were used to detect thenonpathogenic leptospires Amplified products were charac-terized by electrophoresis of 5120583L of each reaction on 15agarose gel for 50min at 90V The PCR product amplifiedby Bak2 primer pair was purified using QIAquick PCRPurification Kit (Qiagen USA) and subsequently sequenced

using Sanger sequencing kits and instruments supplied byApplied Biosystems US performed by First Base Labora-tories (Selangor Malaysia) The DNA sequences were editedin BioEdit [16] and compared against the GenBank databaseusing BLAST The 16S rRNA gene partial sequences of allisolates were aligned with the 16S rRNA sequences of the typestrains obtained from GenBank by using Multiple SequenceComparison by Log-Expectation (MUSCLE) in MEGA 6software [17]The annotated sequences were submitted to theGenBank

3 Results

31 Isolation of Leptospires A total of 144 samples comprisedof 72 water (markets 119899 = 36 recreational areas 119899 = 36)and 72 soil (markets 119899 = 36 recreational areas 119899 = 36)samples were collectedOut of those samples 33were positivefor leptospires based on their characteristic morphology andmotility Among positive samples 7 (5) were water samplesand 26 (18) were soil samples A total of 29 positive samples(water 119899 = 7 soil 119899 = 22) were collected from markets andonly four positive samples (water 119899 = 0 soil 119899 = 4) werecollected from recreational areas

32 Serological Characterization All of the leptospiral iso-lates in this study were not agglutinated with any of thereference hyperimmune sera used in MAT (Table 2) Hencethe isolates did not belong to any of the tested serogroups

33 Molecular Characterization Out of 33 isolates 18 isolateswere detected as Leptospira spp using G1G2 and B64-IB64-II primers (Table 2) None of the isolates were found to con-tain the virulence geneMolecular identification by 16S rRNAverified that from 33 positive samples in this study 31 isolateswere identified as Leptospira spp and two isolates wereidentified as leptospiral closest relatives Leptonema illini Apathogenic leptospire L alstonii (3) was isolated from asoil sample in a market Eight isolates (242) were identifiedas intermediate pathogenic species comprised of seven Lwolffii and one L licerasiae A total of 22 isolates (667) wereidentified as nonpathogenic leptospires L meyeri

The phylogenetic tree constructed using the Neighbour-Joining method was illustrated in Figure 2 Leptospira spp

4 Journal of Tropical Medicine

Table 2 Results for molecular and serological tests

Species (119899 = 33) G1G2 B64-IB64-II SAPRO1SAPRO2 Virulence gene (LipL32) MAT(119899) (119899)

L alstonii (119899 = 1) 1 1 mdash mdashL wolffii (119899 = 7) mdash 7 mdash mdashL licerasiae (119899 = 1) mdash 1 mdash mdashL meyeri (119899 = 22) 17 22 mdash mdashLeptonema illini (119899 = 2) mdash 2 mdash mdashTotal (33) 18 33 0 0

Leptospira interrogans AY631984Leptospira kirschneri AY631895Leptospira noguchii AY631886

Leptospira alexanderi AY631880Leptospira borgpetersenii AY887899

Leptospira weilii AY631877Leptospira santarosai AY631883

WS1 Leptospira alstonii KT002380Leptospira kmetyi AB279549Leptospira alstonii AY631881

Leptospira inadai AY631896Leptospira broomii AY796065Leptospira fainei AY631885

Leptospira licerasiae EF612284

Leptospira wolffii KT002374Leptospira wolffii KT002375

Leptospira wolffii KT002378Leptospira wolffii KT002379Leptospira wolffii KT002384Leptospira wolffii KT002385

Leptospira wolffii EF025496Leptospira wolffii KT002394

Leptospira vanthielli AY631897Leptospira wolbachii AY631879Leptospira biflexa AY631876

Leptospira terpstrae AY631888Leptospira meyeri KT002389

Leptospira meyeri KT002372Leptospira meyeri KT002373Leptospira meyeri KT002376Leptospira meyeri KT002377

Leptospira meyeri KT002381Leptospira meyeri KT002382Leptospira meyeri KT002383

Leptospira meyeri KT002390Leptospira meyeri KT002391Leptospira meyeri KT002392Leptospira meyeri KT002393Leptospira meyeri KT002395Leptospira meyeri KT002396Leptospira meyeri KT002397Leptospira meyeri KT002398Leptospira meyeri KT002399Leptospira meyeri KT002400Leptospira meyeri KT002401Leptospira meyeri KT002402Leptospira meyeri KT002403Leptospira meyeri KT002404

Leptonema illini AY714984Leptonema illini KT002386Leptonema illini KT002387

WS9 Leptospira licerasiae KT002388LS12LS18

SS5SS6WS5WS6

WS15

WS10LS1LS7SS3SS4WS2WS3WS4WS11

WS14WS16WS17WS18WW2WW3WW4WW5WW6WW7WW8

WS7WS8

WS12WS13

8032

2134

91 6738

819999

9792

6388

60901369

10029

49

62

80

10076

16

55 63

Pathogenic

Intermediate

Nonpathogenic

002

Figure 2 A phylogenetic tree generated by the Neighbour-Joining method using MEGA 6 based on 16S rRNA gene sequence of Leptospiraisolates and the selected strains

isolated in this study were clearly separated into three cladesStrain WS1 was discovered from soil samples included in thepathogenic clade It was closely related to type strains of Lalstonii and L kmetyi LS12 LS18 SS5 SS6 WS5 WS6 WS9and WS15 were grouped into intermediate pathogenic clade

LS1 LS7 SS4 WS1 WS2 WS3 WS4 WS10 WS11 WS12WS13WS14WS16WS17WS18WW2WW3WW4WW5WW6 WW7 and WW8 were grouped into nonpathogenicclade These isolates were closely related to type strains of Lmeyeri and L yanagawa

Journal of Tropical Medicine 5

4 Discussion

Even though this is not the first study reporting the detectionand characterization of leptospiral isolates from environ-mental samples its isolation from recreational and marketareas would give an impact to the community Ganoza et alreported that the prevalence of leptospires in water samplesfrom markets was 679 [18] In the present study positivewater samples from markets were found in 194 onlyLeptospires may be unable to survive in the drainage waterin this market The water was found to contain detergent andappeared oily most probably due to its close proximity tothe nearby food stalls It has been reported that 30 ppm ofdetergent (Ceepryn Fixanol and Sapamine) were lethal toleptospires in water in 5 minutes [19] The prevalence of lep-tospires in water samples from recreational areas in Malaysiawas 1167 [20] In the current study a lower prevalenceof 556 was noted which could be due to the effect ofinappropriate choice of sampling points The chosen pointsmay have not been exposed to the leptospiral contamination

All isolates recovered in this study showed negativereactions to 16 reference hyperimmune sera tested in MATSimilarly negative results of MAT for environmental isolateswere also reported from another study in which eight isolatesfrom the selected urban sites in Malaysia were negative fora total of 25 hyperimmune sera tested [20] The availabilityof the reference hyperimmune sera was very limited becausenone of local reference laboratories supplies the hyperim-mune sera Since the sera used in this study were procuredoverseas the available panel of sera may not cover thecirculating local serovar A total of 37 serovars of Leptospirafrom 13 serogroups have been identified in Malaysia [21]This highlights the importance of having locally producedhyperimmune sera available for local use

In our study G1G2 and B64-IB64-II primers detected18 out of 33 isolates as Leptospira spp However 16S rRNAgene sequencing identified 31 out of 33 isolates as Leptospiraspp G1G2 and B64-IB64-II primers were not able to detectall strains of Leptospira spp The occurrence of new strainsof leptospires in humans animals and environment hasincreased the need for new primers that are more specificthan G1G2 and B64-IB64-II Leptospiral putative virulencegene LipL32 rises as a new target for the detection ofpathogenic leptospires though its role in virulence mecha-nisms remains unknown [22] This is further complicatedby the absence of this gene in pathogenic and intermediateleptospires isolated in this studyThis gene was either absenceor undetectable by PCR because in some cases the LipL32gene was not detectable in L licerasiae by PCR but its productwas detectable by both Southern blot hybridization andWestern immunoblot [23] In the previous study by Murgiaet al primer sets Sapro 1 and Sapro 2 were used to detectthe saprophytic leptospires [11] However these primers werefound to be unspecific [11] as pathogenic leptospire Lalstonii was amplified by this primer in this study

In this study a pathogenic species L alstonii was isolatedfrom a soil sample in market area Our finding was in linewith previous studies that also isolated L alstonii from theenvironment [24] Exposure to the soil in market areas which

harboured pathogenic strains may explain the high seropos-itivity of leptospiral antibodies among garbage collectorsand town cleaners in Kelantan [6] L alstonii was possiblyharboured by frogs as it was initially isolated from frogs[25] The isolation of leptospires from frogs specifically fromkidney was reported as early as 1964 [26] Several pathogenicserovars or serogroups such as bim Australis and Ballumhave been isolated from frogs [27 28] However inoculationof frogs with pathogenic serovars to establish experimentalleptospirosis led to unsatisfactory results as leptospires werenot recovered in their organs [29]

The intermediate species L wolffii and L licerasiae wereisolated from markets and recreational areas They are ableto cause diseases in humans although less frequent [12] Thepresence of pathogenic and intermediate species warrantsadherence to precautions and preventive measures amongthe visitors to those areas It was reported that Malaysianswho were involved in recreational activities especially waterrelated are 24 times more likely to acquire leptospiro-sis compared to those who were not involved in similaractivities [30] Leptospirosis was detected in wildlife inMalaysia including wild mammals-monkeys bats squirrelsandmongoose [31]Those animals could be the reservoirs forleptospires in recreational areas located in remote areas

A previous study reported high numbers of L meyeri(881) from environmental samples [24] Our study alsodemonstrated the predominance of L meyeri in Malaysiaenvironment Though this species was nonpathogenic thereis controversy surrounding L meyeri pathogenicity sta-tus L meyeri serovar ranarum ICF which was previouslyconsidered as nonpathogenic was shown to be related topathogenic strain suggesting that this species consisted ofboth pathogenic and nonpathogenic strains [32] This strainwas also amplified by sets of primers specific for pathogenicLeptospira and not the sets of primers specific for saprophyticstrain [11] This result is also supported by phylogeneticanalysis in another study [13] This disagreement demands anovel typingmethod of Leptospira spp which covers not onlypathogenic species but also intermediate and nonpathogenicstrains to resolve the uncertainties in speciesrsquo designation inthe genus of Leptospira The accurate species designation andclassification are crucial because the presence of clinicallyimperative leptospires in the environment would not beoverlooked

Phylogenetic analysis of the studied isolates using 16SrRNA gene sequences concurred with the findings of pre-vious report by Morey et al in 2006 that the pathogenicintermediate and nonpathogenic species each formed oneclade [33] Intermediate groups were closely related to thepathogenic rather than nonpathogenic group Isolates inthe nonpathogenic group were closely related to both Lmeyeri and L yanagawa It was reported that species withinthe nonpathogenic groups were separated by no more than10 bp [33] Using the 16S rRNA gene L meyeri recoveredin this study were not separated In order to demonstrateintraspecies genetic variations the use of other gene loci isnecessary either singly or in combination It was also sug-gested that a cut-off point of 1000 base pairs (bp) was appliedfor leptospiral species identification [34] The phylogenetic

6 Journal of Tropical Medicine

analysis of environmental isolates using 16S rRNA was alsofound to be better than using PFGE profiles

5 Conclusion

Thedata presented in this study demonstrates the presence ofa clinically significant pathogenic L alstonii and the predom-inance of Lmeyeri in the environmentThe pathogenic strainwas found to not contain one of the highly conserve putativeleptospiral virulence genes Early prevention is required toreduce the risks of infection amongst the population Hencecontinuous control and surveillance are essential in loweringthe burden of the disease

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported in part by the KPI Fund UniversitiSains Malaysia Short Term Grant (304PPSP61311069) andRU Grant (1001PPSP812064)

References

[1] G Pappas P Papadimitriou V Siozopoulou L Christou andN Akritidis ldquoThe globalization of leptospirosis worldwideincidence trendsrdquo International Journal of Infectious Diseasesvol 12 no 4 pp 351ndash357 2008

[2] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[3] C Radl M Muller S Revilla-Fernandez et al ldquoOutbreak ofleptospirosis among triathlon participants in Langau Austria2010rdquo Wiener Klinische Wochenschrift vol 123 no 23-24 pp751ndash755 2011

[4] S Brockmann I Piechotowski O Bock-Hensley et al ldquoOut-break of leptospirosis among triathlon participants in Germany2006rdquo BMC Infectious Diseases vol 10 article 91 2010

[5] J Morgan S L Bornstein A M Karpati et al ldquoOutbreak ofleptospirosis among triathlon participants and community res-idents in Springfield Illinois 1998rdquo Clinical Infectious Diseasesvol 34 no 12 pp 1593ndash1599 2002

[6] M N Shafei M R Sulong N A Yaacob et al ldquoSeroprevalenceof leptospirosis among town service workers in northeast-ern state of Malaysiardquo International Journal of CollaborativeResearch on Internal Medicine amp Public Health vol 4 no 4 pp395ndash403 2012

[7] A T Slack S Khairani-Bejo M L Symonds et al ldquoLeptospirakmetyi sp nov isolated from an environmental source inMalaysiardquo International Journal of Systematic and EvolutionaryMicrobiology vol 59 no 4 pp 705ndash708 2009

[8] A Hanim ldquoPenyakit kencing tikus meningkatrdquo 2014 httpww1utusancommyutusanTimur20140106wt 02Penyakit-kencing-tikus-meningkat

[9] Bernama ldquoSix died from leptospirosis in Kelantan in first sixmonths of 2013rdquo 2015 httpwwwmysinchewcomnode87855

[10] Kencing tikus Air Terjun Jeram Pasu lengang 2013 httpww1utusancommyutusanTimur20131003wt 03Kencing-tikus-Air-Terjun-Jeram-Pasu-lengang

[11] R Murgia N Riquelme G Baranton andM Cinco ldquoOligonu-cleotides specific for pathogenic and saprophytic leptospiraoccurring in waterrdquo FEMS Microbiology Letters vol 148 no 1pp 27ndash34 1997

[12] J N Ricaldi D E Fouts J D Selengut et al ldquoWhole genomeanalysis of Leptospira licerasiae provides insight into leptospiralevolution and pathogenicityrdquo PLoS Neglected Tropical Diseasesvol 6 no 10 Article ID e1853 2012

[13] J V Hookey J Bryden and L Gatehouse ldquoThe use of16S rDNA sequence analysis to investigate the phylogeny ofLeptospiraceae and related spirochaetesrdquo Journal of GeneralMicrobiology vol 139 no 11 pp 2585ndash2590 1993

[14] A Chakraborty SMiyahara S Y AMVillanuevaM Saito NG Gloriani and S-I Yoshida ldquoA novel combination of selectiveagents for isolation of Leptospira speciesrdquo Microbiology andImmunology vol 55 no 7 pp 494ndash501 2011

[15] J R Cole Jr C R Sulzer and A R Pursell ldquoImprovedmicrotechnique for the leptospiral microscopic agglutinationtestrdquo Journal of AppliedMicrobiology vol 25 no 6 pp 976ndash9801973

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[18] C A Ganoza M A Matthias D Collins-Richards et alldquoDetermining risk for severe leptospirosis bymolecular analysisof environmental surface waters for pathogenic LeptospirardquoPLoS Medicine vol 3 no 8 pp 1329ndash1340 2006

[19] S L Chang M Buckingham and M P Taylor ldquoStudieson Leptospira Icterohaemorrhagiae IV Survival in water andsewage destruction in water by Halogen compounds syntheticdetergents and heatrdquo Journal of Infectious Diseases vol 82 no3 pp 256ndash266 1948

[20] D Benacer P YWho S NM Zain F Amran and K LThongldquoPathogenic and saprophytic Leptospira species in water andsoils from selected urban sites in peninsularMalaysiardquoMicrobesand Environments vol 28 no 1 pp 135ndash140 2013

[21] D Benacer S N Mohd Zain F Amran R L Gallowayand K L Thong ldquoIsolation and molecular characterizationof Leptospira interrogans and Leptospira borgpetersenii Isolatesfrom the urban rat populations of Kuala LumpurMalaysiardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 88 no4 pp 704ndash709 2013

[22] AHabarta P A EAbreuNOlivera et al ldquoIncreased immuno-genicity to LipL32 of Leptospira interrogans when expressedas a fusion protein with the cholera toxin B subunitrdquo CurrentMicrobiology vol 62 no 2 pp 526ndash531 2011

[23] M A Matthias J N Ricaldi M Cespedes et al ldquoHumanleptospirosis caused by a new antigenically unique Leptospiraassociated with a Rattus species reservoir in the PeruvianAmazonrdquo PLoS Neglected Tropical Diseases vol 2 no 4 articlee213 2008

[24] M Saito S Y AMVillanueva A Chakraborty et al ldquoCompar-ative analysis of Leptospira strains isolated from environmentalsoil and water in the Philippines and Japanrdquo Applied andEnvironmental Microbiology vol 79 no 2 pp 601ndash609 2013

[25] L Smythe B Adler R A Hartskeerl R L Galloway CY Turenne and P N Levett ldquoClassification of Leptospira

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 4: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

4 Journal of Tropical Medicine

Table 2 Results for molecular and serological tests

Species (119899 = 33) G1G2 B64-IB64-II SAPRO1SAPRO2 Virulence gene (LipL32) MAT(119899) (119899)

L alstonii (119899 = 1) 1 1 mdash mdashL wolffii (119899 = 7) mdash 7 mdash mdashL licerasiae (119899 = 1) mdash 1 mdash mdashL meyeri (119899 = 22) 17 22 mdash mdashLeptonema illini (119899 = 2) mdash 2 mdash mdashTotal (33) 18 33 0 0

Leptospira interrogans AY631984Leptospira kirschneri AY631895Leptospira noguchii AY631886

Leptospira alexanderi AY631880Leptospira borgpetersenii AY887899

Leptospira weilii AY631877Leptospira santarosai AY631883

WS1 Leptospira alstonii KT002380Leptospira kmetyi AB279549Leptospira alstonii AY631881

Leptospira inadai AY631896Leptospira broomii AY796065Leptospira fainei AY631885

Leptospira licerasiae EF612284

Leptospira wolffii KT002374Leptospira wolffii KT002375

Leptospira wolffii KT002378Leptospira wolffii KT002379Leptospira wolffii KT002384Leptospira wolffii KT002385

Leptospira wolffii EF025496Leptospira wolffii KT002394

Leptospira vanthielli AY631897Leptospira wolbachii AY631879Leptospira biflexa AY631876

Leptospira terpstrae AY631888Leptospira meyeri KT002389

Leptospira meyeri KT002372Leptospira meyeri KT002373Leptospira meyeri KT002376Leptospira meyeri KT002377

Leptospira meyeri KT002381Leptospira meyeri KT002382Leptospira meyeri KT002383

Leptospira meyeri KT002390Leptospira meyeri KT002391Leptospira meyeri KT002392Leptospira meyeri KT002393Leptospira meyeri KT002395Leptospira meyeri KT002396Leptospira meyeri KT002397Leptospira meyeri KT002398Leptospira meyeri KT002399Leptospira meyeri KT002400Leptospira meyeri KT002401Leptospira meyeri KT002402Leptospira meyeri KT002403Leptospira meyeri KT002404

Leptonema illini AY714984Leptonema illini KT002386Leptonema illini KT002387

WS9 Leptospira licerasiae KT002388LS12LS18

SS5SS6WS5WS6

WS15

WS10LS1LS7SS3SS4WS2WS3WS4WS11

WS14WS16WS17WS18WW2WW3WW4WW5WW6WW7WW8

WS7WS8

WS12WS13

8032

2134

91 6738

819999

9792

6388

60901369

10029

49

62

80

10076

16

55 63

Pathogenic

Intermediate

Nonpathogenic

002

Figure 2 A phylogenetic tree generated by the Neighbour-Joining method using MEGA 6 based on 16S rRNA gene sequence of Leptospiraisolates and the selected strains

isolated in this study were clearly separated into three cladesStrain WS1 was discovered from soil samples included in thepathogenic clade It was closely related to type strains of Lalstonii and L kmetyi LS12 LS18 SS5 SS6 WS5 WS6 WS9and WS15 were grouped into intermediate pathogenic clade

LS1 LS7 SS4 WS1 WS2 WS3 WS4 WS10 WS11 WS12WS13WS14WS16WS17WS18WW2WW3WW4WW5WW6 WW7 and WW8 were grouped into nonpathogenicclade These isolates were closely related to type strains of Lmeyeri and L yanagawa

Journal of Tropical Medicine 5

4 Discussion

Even though this is not the first study reporting the detectionand characterization of leptospiral isolates from environ-mental samples its isolation from recreational and marketareas would give an impact to the community Ganoza et alreported that the prevalence of leptospires in water samplesfrom markets was 679 [18] In the present study positivewater samples from markets were found in 194 onlyLeptospires may be unable to survive in the drainage waterin this market The water was found to contain detergent andappeared oily most probably due to its close proximity tothe nearby food stalls It has been reported that 30 ppm ofdetergent (Ceepryn Fixanol and Sapamine) were lethal toleptospires in water in 5 minutes [19] The prevalence of lep-tospires in water samples from recreational areas in Malaysiawas 1167 [20] In the current study a lower prevalenceof 556 was noted which could be due to the effect ofinappropriate choice of sampling points The chosen pointsmay have not been exposed to the leptospiral contamination

All isolates recovered in this study showed negativereactions to 16 reference hyperimmune sera tested in MATSimilarly negative results of MAT for environmental isolateswere also reported from another study in which eight isolatesfrom the selected urban sites in Malaysia were negative fora total of 25 hyperimmune sera tested [20] The availabilityof the reference hyperimmune sera was very limited becausenone of local reference laboratories supplies the hyperim-mune sera Since the sera used in this study were procuredoverseas the available panel of sera may not cover thecirculating local serovar A total of 37 serovars of Leptospirafrom 13 serogroups have been identified in Malaysia [21]This highlights the importance of having locally producedhyperimmune sera available for local use

In our study G1G2 and B64-IB64-II primers detected18 out of 33 isolates as Leptospira spp However 16S rRNAgene sequencing identified 31 out of 33 isolates as Leptospiraspp G1G2 and B64-IB64-II primers were not able to detectall strains of Leptospira spp The occurrence of new strainsof leptospires in humans animals and environment hasincreased the need for new primers that are more specificthan G1G2 and B64-IB64-II Leptospiral putative virulencegene LipL32 rises as a new target for the detection ofpathogenic leptospires though its role in virulence mecha-nisms remains unknown [22] This is further complicatedby the absence of this gene in pathogenic and intermediateleptospires isolated in this studyThis gene was either absenceor undetectable by PCR because in some cases the LipL32gene was not detectable in L licerasiae by PCR but its productwas detectable by both Southern blot hybridization andWestern immunoblot [23] In the previous study by Murgiaet al primer sets Sapro 1 and Sapro 2 were used to detectthe saprophytic leptospires [11] However these primers werefound to be unspecific [11] as pathogenic leptospire Lalstonii was amplified by this primer in this study

In this study a pathogenic species L alstonii was isolatedfrom a soil sample in market area Our finding was in linewith previous studies that also isolated L alstonii from theenvironment [24] Exposure to the soil in market areas which

harboured pathogenic strains may explain the high seropos-itivity of leptospiral antibodies among garbage collectorsand town cleaners in Kelantan [6] L alstonii was possiblyharboured by frogs as it was initially isolated from frogs[25] The isolation of leptospires from frogs specifically fromkidney was reported as early as 1964 [26] Several pathogenicserovars or serogroups such as bim Australis and Ballumhave been isolated from frogs [27 28] However inoculationof frogs with pathogenic serovars to establish experimentalleptospirosis led to unsatisfactory results as leptospires werenot recovered in their organs [29]

The intermediate species L wolffii and L licerasiae wereisolated from markets and recreational areas They are ableto cause diseases in humans although less frequent [12] Thepresence of pathogenic and intermediate species warrantsadherence to precautions and preventive measures amongthe visitors to those areas It was reported that Malaysianswho were involved in recreational activities especially waterrelated are 24 times more likely to acquire leptospiro-sis compared to those who were not involved in similaractivities [30] Leptospirosis was detected in wildlife inMalaysia including wild mammals-monkeys bats squirrelsandmongoose [31]Those animals could be the reservoirs forleptospires in recreational areas located in remote areas

A previous study reported high numbers of L meyeri(881) from environmental samples [24] Our study alsodemonstrated the predominance of L meyeri in Malaysiaenvironment Though this species was nonpathogenic thereis controversy surrounding L meyeri pathogenicity sta-tus L meyeri serovar ranarum ICF which was previouslyconsidered as nonpathogenic was shown to be related topathogenic strain suggesting that this species consisted ofboth pathogenic and nonpathogenic strains [32] This strainwas also amplified by sets of primers specific for pathogenicLeptospira and not the sets of primers specific for saprophyticstrain [11] This result is also supported by phylogeneticanalysis in another study [13] This disagreement demands anovel typingmethod of Leptospira spp which covers not onlypathogenic species but also intermediate and nonpathogenicstrains to resolve the uncertainties in speciesrsquo designation inthe genus of Leptospira The accurate species designation andclassification are crucial because the presence of clinicallyimperative leptospires in the environment would not beoverlooked

Phylogenetic analysis of the studied isolates using 16SrRNA gene sequences concurred with the findings of pre-vious report by Morey et al in 2006 that the pathogenicintermediate and nonpathogenic species each formed oneclade [33] Intermediate groups were closely related to thepathogenic rather than nonpathogenic group Isolates inthe nonpathogenic group were closely related to both Lmeyeri and L yanagawa It was reported that species withinthe nonpathogenic groups were separated by no more than10 bp [33] Using the 16S rRNA gene L meyeri recoveredin this study were not separated In order to demonstrateintraspecies genetic variations the use of other gene loci isnecessary either singly or in combination It was also sug-gested that a cut-off point of 1000 base pairs (bp) was appliedfor leptospiral species identification [34] The phylogenetic

6 Journal of Tropical Medicine

analysis of environmental isolates using 16S rRNA was alsofound to be better than using PFGE profiles

5 Conclusion

Thedata presented in this study demonstrates the presence ofa clinically significant pathogenic L alstonii and the predom-inance of Lmeyeri in the environmentThe pathogenic strainwas found to not contain one of the highly conserve putativeleptospiral virulence genes Early prevention is required toreduce the risks of infection amongst the population Hencecontinuous control and surveillance are essential in loweringthe burden of the disease

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported in part by the KPI Fund UniversitiSains Malaysia Short Term Grant (304PPSP61311069) andRU Grant (1001PPSP812064)

References

[1] G Pappas P Papadimitriou V Siozopoulou L Christou andN Akritidis ldquoThe globalization of leptospirosis worldwideincidence trendsrdquo International Journal of Infectious Diseasesvol 12 no 4 pp 351ndash357 2008

[2] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[3] C Radl M Muller S Revilla-Fernandez et al ldquoOutbreak ofleptospirosis among triathlon participants in Langau Austria2010rdquo Wiener Klinische Wochenschrift vol 123 no 23-24 pp751ndash755 2011

[4] S Brockmann I Piechotowski O Bock-Hensley et al ldquoOut-break of leptospirosis among triathlon participants in Germany2006rdquo BMC Infectious Diseases vol 10 article 91 2010

[5] J Morgan S L Bornstein A M Karpati et al ldquoOutbreak ofleptospirosis among triathlon participants and community res-idents in Springfield Illinois 1998rdquo Clinical Infectious Diseasesvol 34 no 12 pp 1593ndash1599 2002

[6] M N Shafei M R Sulong N A Yaacob et al ldquoSeroprevalenceof leptospirosis among town service workers in northeast-ern state of Malaysiardquo International Journal of CollaborativeResearch on Internal Medicine amp Public Health vol 4 no 4 pp395ndash403 2012

[7] A T Slack S Khairani-Bejo M L Symonds et al ldquoLeptospirakmetyi sp nov isolated from an environmental source inMalaysiardquo International Journal of Systematic and EvolutionaryMicrobiology vol 59 no 4 pp 705ndash708 2009

[8] A Hanim ldquoPenyakit kencing tikus meningkatrdquo 2014 httpww1utusancommyutusanTimur20140106wt 02Penyakit-kencing-tikus-meningkat

[9] Bernama ldquoSix died from leptospirosis in Kelantan in first sixmonths of 2013rdquo 2015 httpwwwmysinchewcomnode87855

[10] Kencing tikus Air Terjun Jeram Pasu lengang 2013 httpww1utusancommyutusanTimur20131003wt 03Kencing-tikus-Air-Terjun-Jeram-Pasu-lengang

[11] R Murgia N Riquelme G Baranton andM Cinco ldquoOligonu-cleotides specific for pathogenic and saprophytic leptospiraoccurring in waterrdquo FEMS Microbiology Letters vol 148 no 1pp 27ndash34 1997

[12] J N Ricaldi D E Fouts J D Selengut et al ldquoWhole genomeanalysis of Leptospira licerasiae provides insight into leptospiralevolution and pathogenicityrdquo PLoS Neglected Tropical Diseasesvol 6 no 10 Article ID e1853 2012

[13] J V Hookey J Bryden and L Gatehouse ldquoThe use of16S rDNA sequence analysis to investigate the phylogeny ofLeptospiraceae and related spirochaetesrdquo Journal of GeneralMicrobiology vol 139 no 11 pp 2585ndash2590 1993

[14] A Chakraborty SMiyahara S Y AMVillanuevaM Saito NG Gloriani and S-I Yoshida ldquoA novel combination of selectiveagents for isolation of Leptospira speciesrdquo Microbiology andImmunology vol 55 no 7 pp 494ndash501 2011

[15] J R Cole Jr C R Sulzer and A R Pursell ldquoImprovedmicrotechnique for the leptospiral microscopic agglutinationtestrdquo Journal of AppliedMicrobiology vol 25 no 6 pp 976ndash9801973

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[18] C A Ganoza M A Matthias D Collins-Richards et alldquoDetermining risk for severe leptospirosis bymolecular analysisof environmental surface waters for pathogenic LeptospirardquoPLoS Medicine vol 3 no 8 pp 1329ndash1340 2006

[19] S L Chang M Buckingham and M P Taylor ldquoStudieson Leptospira Icterohaemorrhagiae IV Survival in water andsewage destruction in water by Halogen compounds syntheticdetergents and heatrdquo Journal of Infectious Diseases vol 82 no3 pp 256ndash266 1948

[20] D Benacer P YWho S NM Zain F Amran and K LThongldquoPathogenic and saprophytic Leptospira species in water andsoils from selected urban sites in peninsularMalaysiardquoMicrobesand Environments vol 28 no 1 pp 135ndash140 2013

[21] D Benacer S N Mohd Zain F Amran R L Gallowayand K L Thong ldquoIsolation and molecular characterizationof Leptospira interrogans and Leptospira borgpetersenii Isolatesfrom the urban rat populations of Kuala LumpurMalaysiardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 88 no4 pp 704ndash709 2013

[22] AHabarta P A EAbreuNOlivera et al ldquoIncreased immuno-genicity to LipL32 of Leptospira interrogans when expressedas a fusion protein with the cholera toxin B subunitrdquo CurrentMicrobiology vol 62 no 2 pp 526ndash531 2011

[23] M A Matthias J N Ricaldi M Cespedes et al ldquoHumanleptospirosis caused by a new antigenically unique Leptospiraassociated with a Rattus species reservoir in the PeruvianAmazonrdquo PLoS Neglected Tropical Diseases vol 2 no 4 articlee213 2008

[24] M Saito S Y AMVillanueva A Chakraborty et al ldquoCompar-ative analysis of Leptospira strains isolated from environmentalsoil and water in the Philippines and Japanrdquo Applied andEnvironmental Microbiology vol 79 no 2 pp 601ndash609 2013

[25] L Smythe B Adler R A Hartskeerl R L Galloway CY Turenne and P N Levett ldquoClassification of Leptospira

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 5: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

Journal of Tropical Medicine 5

4 Discussion

Even though this is not the first study reporting the detectionand characterization of leptospiral isolates from environ-mental samples its isolation from recreational and marketareas would give an impact to the community Ganoza et alreported that the prevalence of leptospires in water samplesfrom markets was 679 [18] In the present study positivewater samples from markets were found in 194 onlyLeptospires may be unable to survive in the drainage waterin this market The water was found to contain detergent andappeared oily most probably due to its close proximity tothe nearby food stalls It has been reported that 30 ppm ofdetergent (Ceepryn Fixanol and Sapamine) were lethal toleptospires in water in 5 minutes [19] The prevalence of lep-tospires in water samples from recreational areas in Malaysiawas 1167 [20] In the current study a lower prevalenceof 556 was noted which could be due to the effect ofinappropriate choice of sampling points The chosen pointsmay have not been exposed to the leptospiral contamination

All isolates recovered in this study showed negativereactions to 16 reference hyperimmune sera tested in MATSimilarly negative results of MAT for environmental isolateswere also reported from another study in which eight isolatesfrom the selected urban sites in Malaysia were negative fora total of 25 hyperimmune sera tested [20] The availabilityof the reference hyperimmune sera was very limited becausenone of local reference laboratories supplies the hyperim-mune sera Since the sera used in this study were procuredoverseas the available panel of sera may not cover thecirculating local serovar A total of 37 serovars of Leptospirafrom 13 serogroups have been identified in Malaysia [21]This highlights the importance of having locally producedhyperimmune sera available for local use

In our study G1G2 and B64-IB64-II primers detected18 out of 33 isolates as Leptospira spp However 16S rRNAgene sequencing identified 31 out of 33 isolates as Leptospiraspp G1G2 and B64-IB64-II primers were not able to detectall strains of Leptospira spp The occurrence of new strainsof leptospires in humans animals and environment hasincreased the need for new primers that are more specificthan G1G2 and B64-IB64-II Leptospiral putative virulencegene LipL32 rises as a new target for the detection ofpathogenic leptospires though its role in virulence mecha-nisms remains unknown [22] This is further complicatedby the absence of this gene in pathogenic and intermediateleptospires isolated in this studyThis gene was either absenceor undetectable by PCR because in some cases the LipL32gene was not detectable in L licerasiae by PCR but its productwas detectable by both Southern blot hybridization andWestern immunoblot [23] In the previous study by Murgiaet al primer sets Sapro 1 and Sapro 2 were used to detectthe saprophytic leptospires [11] However these primers werefound to be unspecific [11] as pathogenic leptospire Lalstonii was amplified by this primer in this study

In this study a pathogenic species L alstonii was isolatedfrom a soil sample in market area Our finding was in linewith previous studies that also isolated L alstonii from theenvironment [24] Exposure to the soil in market areas which

harboured pathogenic strains may explain the high seropos-itivity of leptospiral antibodies among garbage collectorsand town cleaners in Kelantan [6] L alstonii was possiblyharboured by frogs as it was initially isolated from frogs[25] The isolation of leptospires from frogs specifically fromkidney was reported as early as 1964 [26] Several pathogenicserovars or serogroups such as bim Australis and Ballumhave been isolated from frogs [27 28] However inoculationof frogs with pathogenic serovars to establish experimentalleptospirosis led to unsatisfactory results as leptospires werenot recovered in their organs [29]

The intermediate species L wolffii and L licerasiae wereisolated from markets and recreational areas They are ableto cause diseases in humans although less frequent [12] Thepresence of pathogenic and intermediate species warrantsadherence to precautions and preventive measures amongthe visitors to those areas It was reported that Malaysianswho were involved in recreational activities especially waterrelated are 24 times more likely to acquire leptospiro-sis compared to those who were not involved in similaractivities [30] Leptospirosis was detected in wildlife inMalaysia including wild mammals-monkeys bats squirrelsandmongoose [31]Those animals could be the reservoirs forleptospires in recreational areas located in remote areas

A previous study reported high numbers of L meyeri(881) from environmental samples [24] Our study alsodemonstrated the predominance of L meyeri in Malaysiaenvironment Though this species was nonpathogenic thereis controversy surrounding L meyeri pathogenicity sta-tus L meyeri serovar ranarum ICF which was previouslyconsidered as nonpathogenic was shown to be related topathogenic strain suggesting that this species consisted ofboth pathogenic and nonpathogenic strains [32] This strainwas also amplified by sets of primers specific for pathogenicLeptospira and not the sets of primers specific for saprophyticstrain [11] This result is also supported by phylogeneticanalysis in another study [13] This disagreement demands anovel typingmethod of Leptospira spp which covers not onlypathogenic species but also intermediate and nonpathogenicstrains to resolve the uncertainties in speciesrsquo designation inthe genus of Leptospira The accurate species designation andclassification are crucial because the presence of clinicallyimperative leptospires in the environment would not beoverlooked

Phylogenetic analysis of the studied isolates using 16SrRNA gene sequences concurred with the findings of pre-vious report by Morey et al in 2006 that the pathogenicintermediate and nonpathogenic species each formed oneclade [33] Intermediate groups were closely related to thepathogenic rather than nonpathogenic group Isolates inthe nonpathogenic group were closely related to both Lmeyeri and L yanagawa It was reported that species withinthe nonpathogenic groups were separated by no more than10 bp [33] Using the 16S rRNA gene L meyeri recoveredin this study were not separated In order to demonstrateintraspecies genetic variations the use of other gene loci isnecessary either singly or in combination It was also sug-gested that a cut-off point of 1000 base pairs (bp) was appliedfor leptospiral species identification [34] The phylogenetic

6 Journal of Tropical Medicine

analysis of environmental isolates using 16S rRNA was alsofound to be better than using PFGE profiles

5 Conclusion

Thedata presented in this study demonstrates the presence ofa clinically significant pathogenic L alstonii and the predom-inance of Lmeyeri in the environmentThe pathogenic strainwas found to not contain one of the highly conserve putativeleptospiral virulence genes Early prevention is required toreduce the risks of infection amongst the population Hencecontinuous control and surveillance are essential in loweringthe burden of the disease

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported in part by the KPI Fund UniversitiSains Malaysia Short Term Grant (304PPSP61311069) andRU Grant (1001PPSP812064)

References

[1] G Pappas P Papadimitriou V Siozopoulou L Christou andN Akritidis ldquoThe globalization of leptospirosis worldwideincidence trendsrdquo International Journal of Infectious Diseasesvol 12 no 4 pp 351ndash357 2008

[2] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[3] C Radl M Muller S Revilla-Fernandez et al ldquoOutbreak ofleptospirosis among triathlon participants in Langau Austria2010rdquo Wiener Klinische Wochenschrift vol 123 no 23-24 pp751ndash755 2011

[4] S Brockmann I Piechotowski O Bock-Hensley et al ldquoOut-break of leptospirosis among triathlon participants in Germany2006rdquo BMC Infectious Diseases vol 10 article 91 2010

[5] J Morgan S L Bornstein A M Karpati et al ldquoOutbreak ofleptospirosis among triathlon participants and community res-idents in Springfield Illinois 1998rdquo Clinical Infectious Diseasesvol 34 no 12 pp 1593ndash1599 2002

[6] M N Shafei M R Sulong N A Yaacob et al ldquoSeroprevalenceof leptospirosis among town service workers in northeast-ern state of Malaysiardquo International Journal of CollaborativeResearch on Internal Medicine amp Public Health vol 4 no 4 pp395ndash403 2012

[7] A T Slack S Khairani-Bejo M L Symonds et al ldquoLeptospirakmetyi sp nov isolated from an environmental source inMalaysiardquo International Journal of Systematic and EvolutionaryMicrobiology vol 59 no 4 pp 705ndash708 2009

[8] A Hanim ldquoPenyakit kencing tikus meningkatrdquo 2014 httpww1utusancommyutusanTimur20140106wt 02Penyakit-kencing-tikus-meningkat

[9] Bernama ldquoSix died from leptospirosis in Kelantan in first sixmonths of 2013rdquo 2015 httpwwwmysinchewcomnode87855

[10] Kencing tikus Air Terjun Jeram Pasu lengang 2013 httpww1utusancommyutusanTimur20131003wt 03Kencing-tikus-Air-Terjun-Jeram-Pasu-lengang

[11] R Murgia N Riquelme G Baranton andM Cinco ldquoOligonu-cleotides specific for pathogenic and saprophytic leptospiraoccurring in waterrdquo FEMS Microbiology Letters vol 148 no 1pp 27ndash34 1997

[12] J N Ricaldi D E Fouts J D Selengut et al ldquoWhole genomeanalysis of Leptospira licerasiae provides insight into leptospiralevolution and pathogenicityrdquo PLoS Neglected Tropical Diseasesvol 6 no 10 Article ID e1853 2012

[13] J V Hookey J Bryden and L Gatehouse ldquoThe use of16S rDNA sequence analysis to investigate the phylogeny ofLeptospiraceae and related spirochaetesrdquo Journal of GeneralMicrobiology vol 139 no 11 pp 2585ndash2590 1993

[14] A Chakraborty SMiyahara S Y AMVillanuevaM Saito NG Gloriani and S-I Yoshida ldquoA novel combination of selectiveagents for isolation of Leptospira speciesrdquo Microbiology andImmunology vol 55 no 7 pp 494ndash501 2011

[15] J R Cole Jr C R Sulzer and A R Pursell ldquoImprovedmicrotechnique for the leptospiral microscopic agglutinationtestrdquo Journal of AppliedMicrobiology vol 25 no 6 pp 976ndash9801973

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[18] C A Ganoza M A Matthias D Collins-Richards et alldquoDetermining risk for severe leptospirosis bymolecular analysisof environmental surface waters for pathogenic LeptospirardquoPLoS Medicine vol 3 no 8 pp 1329ndash1340 2006

[19] S L Chang M Buckingham and M P Taylor ldquoStudieson Leptospira Icterohaemorrhagiae IV Survival in water andsewage destruction in water by Halogen compounds syntheticdetergents and heatrdquo Journal of Infectious Diseases vol 82 no3 pp 256ndash266 1948

[20] D Benacer P YWho S NM Zain F Amran and K LThongldquoPathogenic and saprophytic Leptospira species in water andsoils from selected urban sites in peninsularMalaysiardquoMicrobesand Environments vol 28 no 1 pp 135ndash140 2013

[21] D Benacer S N Mohd Zain F Amran R L Gallowayand K L Thong ldquoIsolation and molecular characterizationof Leptospira interrogans and Leptospira borgpetersenii Isolatesfrom the urban rat populations of Kuala LumpurMalaysiardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 88 no4 pp 704ndash709 2013

[22] AHabarta P A EAbreuNOlivera et al ldquoIncreased immuno-genicity to LipL32 of Leptospira interrogans when expressedas a fusion protein with the cholera toxin B subunitrdquo CurrentMicrobiology vol 62 no 2 pp 526ndash531 2011

[23] M A Matthias J N Ricaldi M Cespedes et al ldquoHumanleptospirosis caused by a new antigenically unique Leptospiraassociated with a Rattus species reservoir in the PeruvianAmazonrdquo PLoS Neglected Tropical Diseases vol 2 no 4 articlee213 2008

[24] M Saito S Y AMVillanueva A Chakraborty et al ldquoCompar-ative analysis of Leptospira strains isolated from environmentalsoil and water in the Philippines and Japanrdquo Applied andEnvironmental Microbiology vol 79 no 2 pp 601ndash609 2013

[25] L Smythe B Adler R A Hartskeerl R L Galloway CY Turenne and P N Levett ldquoClassification of Leptospira

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 6: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

6 Journal of Tropical Medicine

analysis of environmental isolates using 16S rRNA was alsofound to be better than using PFGE profiles

5 Conclusion

Thedata presented in this study demonstrates the presence ofa clinically significant pathogenic L alstonii and the predom-inance of Lmeyeri in the environmentThe pathogenic strainwas found to not contain one of the highly conserve putativeleptospiral virulence genes Early prevention is required toreduce the risks of infection amongst the population Hencecontinuous control and surveillance are essential in loweringthe burden of the disease

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported in part by the KPI Fund UniversitiSains Malaysia Short Term Grant (304PPSP61311069) andRU Grant (1001PPSP812064)

References

[1] G Pappas P Papadimitriou V Siozopoulou L Christou andN Akritidis ldquoThe globalization of leptospirosis worldwideincidence trendsrdquo International Journal of Infectious Diseasesvol 12 no 4 pp 351ndash357 2008

[2] P N Levett ldquoLeptospirosisrdquo Clinical Microbiology Reviews vol14 no 2 pp 296ndash326 2001

[3] C Radl M Muller S Revilla-Fernandez et al ldquoOutbreak ofleptospirosis among triathlon participants in Langau Austria2010rdquo Wiener Klinische Wochenschrift vol 123 no 23-24 pp751ndash755 2011

[4] S Brockmann I Piechotowski O Bock-Hensley et al ldquoOut-break of leptospirosis among triathlon participants in Germany2006rdquo BMC Infectious Diseases vol 10 article 91 2010

[5] J Morgan S L Bornstein A M Karpati et al ldquoOutbreak ofleptospirosis among triathlon participants and community res-idents in Springfield Illinois 1998rdquo Clinical Infectious Diseasesvol 34 no 12 pp 1593ndash1599 2002

[6] M N Shafei M R Sulong N A Yaacob et al ldquoSeroprevalenceof leptospirosis among town service workers in northeast-ern state of Malaysiardquo International Journal of CollaborativeResearch on Internal Medicine amp Public Health vol 4 no 4 pp395ndash403 2012

[7] A T Slack S Khairani-Bejo M L Symonds et al ldquoLeptospirakmetyi sp nov isolated from an environmental source inMalaysiardquo International Journal of Systematic and EvolutionaryMicrobiology vol 59 no 4 pp 705ndash708 2009

[8] A Hanim ldquoPenyakit kencing tikus meningkatrdquo 2014 httpww1utusancommyutusanTimur20140106wt 02Penyakit-kencing-tikus-meningkat

[9] Bernama ldquoSix died from leptospirosis in Kelantan in first sixmonths of 2013rdquo 2015 httpwwwmysinchewcomnode87855

[10] Kencing tikus Air Terjun Jeram Pasu lengang 2013 httpww1utusancommyutusanTimur20131003wt 03Kencing-tikus-Air-Terjun-Jeram-Pasu-lengang

[11] R Murgia N Riquelme G Baranton andM Cinco ldquoOligonu-cleotides specific for pathogenic and saprophytic leptospiraoccurring in waterrdquo FEMS Microbiology Letters vol 148 no 1pp 27ndash34 1997

[12] J N Ricaldi D E Fouts J D Selengut et al ldquoWhole genomeanalysis of Leptospira licerasiae provides insight into leptospiralevolution and pathogenicityrdquo PLoS Neglected Tropical Diseasesvol 6 no 10 Article ID e1853 2012

[13] J V Hookey J Bryden and L Gatehouse ldquoThe use of16S rDNA sequence analysis to investigate the phylogeny ofLeptospiraceae and related spirochaetesrdquo Journal of GeneralMicrobiology vol 139 no 11 pp 2585ndash2590 1993

[14] A Chakraborty SMiyahara S Y AMVillanuevaM Saito NG Gloriani and S-I Yoshida ldquoA novel combination of selectiveagents for isolation of Leptospira speciesrdquo Microbiology andImmunology vol 55 no 7 pp 494ndash501 2011

[15] J R Cole Jr C R Sulzer and A R Pursell ldquoImprovedmicrotechnique for the leptospiral microscopic agglutinationtestrdquo Journal of AppliedMicrobiology vol 25 no 6 pp 976ndash9801973

[16] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series no 41 pp 95ndash98 1999

[17] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[18] C A Ganoza M A Matthias D Collins-Richards et alldquoDetermining risk for severe leptospirosis bymolecular analysisof environmental surface waters for pathogenic LeptospirardquoPLoS Medicine vol 3 no 8 pp 1329ndash1340 2006

[19] S L Chang M Buckingham and M P Taylor ldquoStudieson Leptospira Icterohaemorrhagiae IV Survival in water andsewage destruction in water by Halogen compounds syntheticdetergents and heatrdquo Journal of Infectious Diseases vol 82 no3 pp 256ndash266 1948

[20] D Benacer P YWho S NM Zain F Amran and K LThongldquoPathogenic and saprophytic Leptospira species in water andsoils from selected urban sites in peninsularMalaysiardquoMicrobesand Environments vol 28 no 1 pp 135ndash140 2013

[21] D Benacer S N Mohd Zain F Amran R L Gallowayand K L Thong ldquoIsolation and molecular characterizationof Leptospira interrogans and Leptospira borgpetersenii Isolatesfrom the urban rat populations of Kuala LumpurMalaysiardquoTheAmerican Journal of Tropical Medicine and Hygiene vol 88 no4 pp 704ndash709 2013

[22] AHabarta P A EAbreuNOlivera et al ldquoIncreased immuno-genicity to LipL32 of Leptospira interrogans when expressedas a fusion protein with the cholera toxin B subunitrdquo CurrentMicrobiology vol 62 no 2 pp 526ndash531 2011

[23] M A Matthias J N Ricaldi M Cespedes et al ldquoHumanleptospirosis caused by a new antigenically unique Leptospiraassociated with a Rattus species reservoir in the PeruvianAmazonrdquo PLoS Neglected Tropical Diseases vol 2 no 4 articlee213 2008

[24] M Saito S Y AMVillanueva A Chakraborty et al ldquoCompar-ative analysis of Leptospira strains isolated from environmentalsoil and water in the Philippines and Japanrdquo Applied andEnvironmental Microbiology vol 79 no 2 pp 601ndash609 2013

[25] L Smythe B Adler R A Hartskeerl R L Galloway CY Turenne and P N Levett ldquoClassification of Leptospira

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 7: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

Journal of Tropical Medicine 7

genomospecies 1 3 4 and 5 as Leptospira alstonii sp novLeptospira vanthielii sp nov Leptospira terpstrae sp nov andLeptospira yanagawae sp nov respectivelyrdquo International Jour-nal of Systematic and Evolutionary Microbiology vol 63 no 5pp 1859ndash1862 2013

[26] S L Diesch W F McCulloch J L Braun and H C Elling-hausen Jr ldquoLeptospires isolated from frog kidneysrdquoNature vol209 no 5026 pp 939ndash940 1966

[27] C O R Everard D G Carrington H Korver R Burke J DEverard and C Gravekamp ldquoLeptospires in the whistling frog(Eleutherodactylus johnstonei) on Barbadosrdquo Journal of TropicalMedicine and Hygiene vol 93 no 2 pp 140ndash145 1990

[28] C Gravekamp H Korver J Montgomery et al ldquoLeptospiresisolated from toads and frogs on the Island of BarbadosrdquoZentralblatt fur Bakteriologie vol 275 no 3 pp 403ndash411 1991

[29] S L Diesch W F McCulloch and J L Braun ldquoExperimentalleptospirosis in frogsrdquo Nature vol 214 no 5093 pp 1139ndash11401967

[30] AAN Rafizah BDAziah YNAzwany et al ldquoRisk factors ofleptospirosis among febrile hospital admissions in northeasternMalaysiardquo Preventive Medicine vol 57 pp S11ndashS13 2013

[31] S Thayaparan I Robertson F Amraan L Sursquout and MT Abdullah ldquoSerological prevalence of leptospiral infectionin wildlife in Sarawak Malaysiardquo Borneo Journal of ResourceScience and Technology vol 2 no 2 pp 71ndash74 2013

[32] D Postic N Riquelme-Sertour F Merien P Perolat and GBaranton ldquoInterest of partial 16S rDNA gene sequences toresolve heterogeneities between Leptospira collections applica-tion to L meyerirdquo Research in Microbiology vol 151 no 5 pp333ndash341 2000

[33] R E Morey R L Galloway S L Bragg A G SteigerwaltL W Mayer and P N Levett ldquoSpecies-specific identificationof Leptospiraceae by 16S rRNA gene sequencingrdquo Journal ofClinical Microbiology vol 44 no 10 pp 3510ndash3516 2006

[34] G M Cerqueira A J A McBride A Queiroz et al ldquoMonitor-ing Leptospira strain collections the need for quality controlrdquoAmerican Journal of Tropical Medicine and Hygiene vol 82 no1 pp 83ndash87 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Page 8: )JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jtm/2016/2060241.pdf · et al., with some modi cations [ ]. e concentration of trimethoprim was changed from g/mL to

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom