Hadi Sumanta， Tri Wibawa， Suwarno Hadisusanto， Anik Nuryati， Hari Kusnanto

1Center for Environmental Health Engineering and Disease Control， Yogyakarta， Indonesia

2Department of Microbiology， Faculty of Medicine， Universitas Gadjah Mada， Yogyakarta， Indonesia

3Department of Biology， Faculty of Biology， Universitas Gadjah Mada， Yogyakarta， Indonesia

4Department of Health Analyst， Yogyakarta Health Polytechnic， Yogyakarta， Indonesia

5Department of Public Health， Faculty of Medicine， Gadjah Mada University， Yogyakarta， Indonesia

Genetic variation of Leptospira isolated from rats catched in Yogyakarta Indonesia

Hadi Sumanta1*， Tri Wibawa2， Suwarno Hadisusanto3， Anik Nuryati4， Hari Kusnanto5

1Center for Environmental Health Engineering and Disease Control， Yogyakarta， Indonesia

2Department of Microbiology， Faculty of Medicine， Universitas Gadjah Mada， Yogyakarta， Indonesia

3Department of Biology， Faculty of Biology， Universitas Gadjah Mada， Yogyakarta， Indonesia

4Department of Health Analyst， Yogyakarta Health Polytechnic， Yogyakarta， Indonesia

5Department of Public Health， Faculty of Medicine， Gadjah Mada University， Yogyakarta， Indonesia

ARTICLE INFO

Article history：

in revised form 20 July 2015

Accepted 15 August 2015

Available online 20 September 2015

Pathogenic Leptospira

16S rRNA

Rats

Genetic variation

Objective： To detect genetic variations among pathogenic Leptospira isolated from rats using 16S rRNA gen as chronometer. Methods： This is an observational study with cross sectional design. Rats saples were taken in Yogyakarta Special Region of Indonesia. Leptospira in the rats was detected by two methods ie. real time PCR （qPCR） by using primers correspond to16S rRNA gene of Leptospira， and standard PCR by using different set of primer correspond to the 16S rRNA gene of Leptospira. The standard PCR amplicon then subjected for DNA sequencing. Analysis genetic variation was performed using MEGA 6.2. Software. Results：There were 99 DNA samples from rats included in this study. Detection of Leptospira by using qPCR revealed 25 samples positive for pathogenic Leptospira， while only 6 samples were able to be detected using standard PCR. The new primer set correspond to 16S rRNA gene was able to detect specifically pathogenic Leptospira in the rats. Sequencing analysis of 6 PCR amplicons showed that the Leptospira which infect the rats catched in Yogyakarta genetically close related with pathogenic Leptospira which were isolated from human， animal， rodents， and environment. Conclusions： It can be considered that rats are the most important vector and reservoir of Leptospira.

1. Introduction

Leptospirosis is an infectious disease caused by a direct zoonotic Leptospira［1-3］. Leptospira is transmitted through direct contact with urine and body fluids of an infected animal， or through indirect contact with water and soil environments contaminated with Leptospira［4-6］. The urine of infected animals and rodents can pollute the environment such as soil， water，and vegetation［7,8］. Leptospira may reside in the kidney of reservoir and when its passing urine， it may contaminate theenvironment［9-11］.

Rats are reservoir with chronic leptospirosis infection. The infection is transmitted from rats to another through direct contact at a young age or older［12］. There were reported that some Leptospira species has specific susceptibility to particular rodents. Leptospira icterohaemorrhagiae species commonly infect Bandicota indica and Rattus diardii， whereas Leptospira ballum infect Mus musculus. Rattus norvegicus， Rattus diardii， Rattus exulans and Suncus murinus allegedly having an important role in the incidence of leptospirosis［13］.

Leptospira has three types of ribosomal RNA ie. 5S， 16S，and 23S rRNA. Among the three， 16S rRNA gene is the most frequently used molecular markers［14-18］. The 16S rRNA is building block of the 30S subunit， which is important for translation. The 16S rRNA gene is the most commonly used andaccepted to study the genetic relationship among bacteria［19］.

The 16S rRNA gene of Leptospira can be detected using standard polymerase chain reaction （PCR）. Detection may be done by using a primer set， targeting the 16S rRNA gene［14-18］. It was also reported that Leptospira could be detected by using real time-PCR（qPCR）， which was employed primer set and probe correspond to 16S rRNA gene［20,21］.

There is limited information about genetics of Leptospira which are circulating in Indonesia. Our work gives new information concerning the genetics variation in Leptospira which was isolated from rats. It is important also to understand its relationship with previously reported Leptospira which were isolated from human，rodents， animal and environment.

2. Materials and methods

2.1. DNA samples

Rats were catched at Bantul District， Yogyakarta， Indonesia. Rats were sucrificed and kidney tissue was obtained. DNA was isolated by using PCR High Pure Template Preparation Kit（Roche?）. DNA samples were stored at -80 ℃ before subsequent analysis. This work has been aprooved by Medical and Health Research Ethics Committee of Faculty of Medicine， Universitas Gadjah Mada.

2.2. PCR amplification

DNA was amplified using Dream TaqTMGreen PCR Master Mix （2×）. The PCR reaction was done according to the manufacturer protocol. Reaction mixture was cosist of green buffer， dATP， dCTP， dGTP， dTTP， MgCl2， destilated water，and mineral oil （Promega?）. One set of primer correspond to 16S rRNA gene were employed. Primer sequences are 508F （5'-GGCGGACATGTAAGT CAGGT-3'） and 1217R （5'-GGGACCGGATTTTTGAGATT-3'）. The PCR amplification generated 709 bp PCR products. Amplification products were mounted and run on 2% agarose gel.

2.3. DNA sequence analysis

Amplicon of PCR product then subjected to be sequenced by using direct sequencing method. Multiple alignment analysis was done to analyze the genetic diversity between our results and 16S rRNA gene sequences deposited in the gene bank. Phylogenetic analysis was perfromed by using MEGA 6.2 software.

3. Results

There were 99 rats involved in this study. DNA samples obtained from kidney tissue of rats. Leptospira detection by using qPCR was showed positive in 25 samples ［21］. However， only 6 samples were confirmed as pathogenic Leptospira by using standard PCR（Figure 1）. Cohen's Kappa test between the two methods showed value of 0.32 （P＜0.01）. Detection of Leptospira using qPCR and standard PCR， which was targeted 16S rRNA gene， showed weak agreement.

DNA sequencing correspond to the 16S rRNA gene of 6 Leptospira were obtained. By using BLAST analysis， it was noticed that all 6 Leptospira isolates are closely similar with 16S rRNA of pathogenic Leptospira. This means that the primer set for standard PCR is fully spesific to the pathogenic Leptospira， though less sensitive than qPCR methods.

The phylogentic tree of 6 DNA sequence of 16S rRNA showed that the Leptospira isolated from rats catched in Bantul district，Yogyakarta has close relationship with pathogenic Leptospira reported in gene bank， which is isolated from various host and environment. Leptospira isolated from rats： CP001221， KC662445，KC662446， KC662447， KC662448， KC662449， KC662450，KC662451， KC662452， KC662453， KC662455， NR_074524，and NR_074481. Leptospira isolated from human： EF596782，F(xiàn)J154561 FJ154567， JQ765633， JQ988854， JQ988855，JQ988858， and JX040543. Leptospira isolated from Cow：JQ765632 and JQ765635. Leptospira isolated from Mus muculus HM776722， and environment （GU254504， JQ288733） （Figure 2）.

4. Discussion

Leptospira detection from rat's tissues samples is commonly done by using standard PCR and qPCR methods. Our result showed that qPCR is more sensitive compare to the standard PCR. Previous report by Desvars et al， showed simillar result in which detection by using qPCR successfully detect 29.8% of the sample， whereas by using standard PCR was able to detect 14.18% of the samples［22］.

Rats as reservoir， plays an important role for Leptospira infection to humans. Although dogs， pigs， cows， horses， cats， rabbits， bats，squirrels， raccoons can also serve as a reservoir. However， rats are the most prominent reservoir among others. Rats are the most potential vector and reservoir for Leptospira transmision to human being［21］. Furthermore， Barcellos et al reported that the distribution of leptospirosis in human were concentrated in areas where rats were highly populated， as well as areas with unfavorable trash management and poor sanitary conditions［23］.

Certain Leptospira serovar has been associated with particular animals such as Leptospira icterohaemorrhagiae and Leptospira copenhageni were associated with rats， Leptospira canicola with dogs， Leptospira pomona with pigs， Leptospira hardjo with cows， and Leptospira gryppotyphosa with voles ［8,24］. However， It is interesting that our result showed pathogenic Leptospira isolated from rats have close relationship with Leptospira isolated from other host and environment［25-30］. This means that the Leptospira circulating in the rats， animals， humans， and environtment were genetically similar. There is no evidence that particular genetics constitution has tropism for spesific host and resorvoir. In conclusion， we should be considered rats are the most important vector and reservoir of Leptospira. Further studies are needed for detail elaboration of transmission pathway from rats to other animals， environment， and human.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

This paper is part of the dissertation of HS.

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15 June 2015

Hadi Sumanta， Center for Environmental Health Engineering and Disease Control， Jl. Gedongkiwo MJ I / 984， RT. 54 RW. 11 Yogyakarta， 55142，Yogyakarta， Indonesia.

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E-mail： hadisumanta@yahoo.com