Detection of Pathogenic, IBtermediate and Saprophytic Leptospira Isolates from Rodents in Selected

National Park and Urban Sites ofSarawak

Nuraqeelah Binti Mohammad Shamhari

Bachelor of Science with HonoursRC (Resource Biotechnology)154.95

2015N974 2015

PU~'3t Khiomaf Maklum1lt Ak:4d('~"' UNIVERSITI (\ 'SlA SAR,\\\,.\·

Detection of Pathogenic, Intermediate and Saprophytic Leptospira Isolates from Rodents in Selected National Park and Urban Sites of Sarawak

Nuraqeelah Binti Mohammad Shamhari

A progress submitted in partial fulfillment ofdegree of Bachelor of Science with Honours

(Resource Biotechnology)

Supervisor: Dr Lesley Maurice Bilung Co-supervisor: Dr Chong Yee Ling

or

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology University Malaysia Sarawak

2015

Acknowledgement

I would like to express my appreciation and gratitude to my supervisor, Dr. Lesley Maurice

Bilung for being my supervisor, advisor and educator throughout this study. Thank you for

all the guidance and advices. Besides, I would like to thank my co-supervisor, Dr. Chong

Yee Ling and also zoology students for their guidance and assistance throughout my final

year project.

Also, a special thanks to all the postgraduates in the Microbiology Laboratory of Faculty of

Resource Science and Technology UNlMAS especially Mrs. Pui Chai Fung, Mr. Azis, and

my team that are carry out studies on Leptospira Choe Sin Pei and Nur Izzul Haziq for

helping me throughout the field trip at Gunung Gading National Park and urban sites

sampling especially in Kuching. Futhermore, special thanks to related families and those

who have donated rat samples captured from their places for my research. Without their

sincere assistance and also the time spent, I would not be able to accomplish this study.

Lastly, I also would like to honor my families, teachers, and friends for all the supports,

morally and financially .

.'

Declaration

I hereby declare that this final year project thesis entitled Detection of Pathogenic,

Intermediate and Saprophytic Leptospira Isolates from Rodents in Selected National

Park and Urban Sites of Sarawak is my own work and effort and that it has not been

submitted anywhere for any award. Where other sources of information have been used,

they have been acknowledged.

~'

Nuraqeelah binti Mohammad Shamhari

Resource Biotechnology Programme Molecular Biology Department Faculty of Resource Science and Technology University Malaysia Sarawak

JI

PUS,at.l<hidmat MakJumat Akud mil· UNJ\'ERSm MAlAYSIA SARAWAJ<

Table of Contents

Page

ACKNOWLEDGEMENT I

DECLARATION II

TABLE OF CONTENTS III

LIST OF ABBREVIATION IV

LIST OF TABLES V

LIST OF FIGURES VI

ABSTRACT

1.0 Introduction 2

2.0 Literature Review

2.1 Leptospirosis 4 2.2 Leptospira species 7 2.3 Sources of Leptospira 9 2.4 Molecular analysis using PCR 10

3.0 Materials and Method

3.1 Materials 12 3.2 Methods

3.2.1 Study Sites 12 3.2.2 Samples collection 14 3.2.3 Isolation of Leptospira 15

.. 3.2.4 Detection of Leptospira using PCR 16

184.0 Results

235.0 Discussion

6.0 Conclusion II 31

32References

Appendix A

Appendix B

III

List of Abbreviations

PCR

DFM

EtBr

EMJH

BLAST

TBE

DNA

MAT .

CAAT

EtOH

PFGE

GGNP

Polymerase chain reaction

Dark field microscope

Ethidium bromide

Ellinghausen-McCullough-Johnson-Harris medium

Basic Local Alignment Search Tool

Tris-borate-EDTA

Deoxyribonucleotide acid

Microscopic agglutination test

Cross agglutinin agglutination test

Ethanol

Pulse-field Gel Electrophoresis

Gunung Gading National Park

IY

List of Tables

Page

Table 1 Global incidence rates of leptospirosis .I- 5

Table 2 Leptospirosis cases in Malaysia from 2004 to 6

2009

Table 3 Leptospirosis cases in Sarawak from 2004 until 7

February 2015

Table 4 Evidence of Leptospira research carried out by 11

using PCR in previous research

Table 5 Sampling sites of captured rats 13

Table 6 Three sets of primer used to target specific 17

gene ofLeptospira in PCR

Table 7 Number of rats captured from selected 19

sampling sites •

Table 8 Positive samples of pathogenic Leptospira 19

Table 9 Positive samples of saprophytic Leptospira 20

Table 10 Summary of results 24

Table 11 Species of rats carry Leptospira 25

..

List of Figures

Page

Figure 1 Global leptospirosis incidence map 5

Samarahan

National Park

Leptospira using lipL32-270FllipL32-692R primers

Leptospira using SaRro] /Sapro 2 primers

Niaga Satok

Leptospira detection from captured rats

Figure 2 Map location of Gunung Gading National 13

Figure 3 Map location of selected sampling sites in Kuching 14

Figure 4 Map location of Kampung Sebayor in Kota 14

Figure 5 Set cage traps using banana baits in Gunung Gading 15

Figure 6 Samples cultured in modified EMJH media 16

Figure 7 Representative gel of PCR for pathogenic 21

Figure 8 Representative gel of PCR for saprophytic 22

Figure 9 House rat (Rattus rattus) captured from Medan 24

Figure 10 Captured rats from Gunung Gading National Park 26

Figure 11 Resident area (Kampung Tupong) with positive 27

~.

,VI

Detedion of Pathogenic, Intermediate and Saprophytic Leptospira Isolates from Rodents in Selected National Park and Urban Sites of Sarawak

Nuraqeelah Binti Mohammad Shamhari

Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology University Malaysia Sarawak

ABSTRACT

Leptospirosis is a global zoonotic disease caused by pathogenic Leptospira that greatly affect both human and animals. The infection has become an urban public health concerns due the increase number of leptospirosis cases reported. Rats are known to be the major reservoir of Leptospira. Due to limited information regarding leptospirosis in Sarawak, this study aims to isolate and detect Leptospira circulating among urban rat populations in Sarawak. In this study, rats were captured using cage trap in selected urban sites namely Kuching, Kota Samarahan, and Lundu. Kidney and liver tissue samples were removed and cultured in modified Ellinghausen McCullough Johnson Harris (EMJH) media. Cultures were analysed using polymerase chain reaction (PCR) for the detection of pathogenic, saprophytic, and intermediate Leptospira. This study found that there are 44% (33 out of 75) captured rats from 6 different species were detected with Leptospira. 21.21% (5 out of 33) captured rats were carrying pathogenic Leptospira whereas 84.85% (28 out of 33) captured rats were carrying saprophytic Leptospira. This study has shown that rats have high potential to be carriers of Leptospira. This study also indicates that PCR is a well recommended molecular tool with specificity and sensitivity for Leptospira detection in chronic carriers. The finding has proven the occurrence of pathogenic Leptospira in urban sites of Sarawak thus it helps to provide knowledge regarding the infections level of pathogenic Leptospira circulating among rats in the urban sites of Sarawak. Thus, improvement on basic sanitation facilities is required in the urban sites of Sarawak to control the prevalence of leptospirosis.

Keywords: Leptospira, rats, urban sites, polymerase chain reaction

ABSTRAK

Leptospirosis adalah penyakit zoonotik global yang disebabkan oleh patogen Leptospira yang sangat memberi kesan kepada manusia dan haiwan. Jangkitan ini telah menjadi satu masalah kesihatan dalam bandar disebabkan oleh laporan peningkatan jumlah kes leptospirosis. Tikus dikenali sebagai pembawa utama Leptospira. Oleh kerana makiumat yang terhad mengenai leptospirosis di Sarawak, kajian ini bertujuan untuk mengasingkan dan mengesan Leptospira yang berada dalam kalangan populasi tikus baridar di Sarawak. Dalam kajian ini, tikus telah ditangkap di sekitar bandar terpilih iaitu Kuching, Kota Samarahan dan Lundu. Sampeltisu daripada buah pinggang dan hati diambil dan dibiakkan dalam Ellinghausen McCullough Johnson Harris (EMJH) media yang diubahsuai. Kultur sam pel telah dianalisis dengan menggunakan tindak balas rantai polimerase (PCR) bagi pengesanan patogen, saprophytic, dan pertengahan Leptospira. Kajian ini mendapati bahawa terdapat 44% (33 daripada 75) tikus ditangkap d telah dikesan dengan Leptospira. 21.21% (5 daripada 33) tikus yang ditangkap telah membawa patogen Leptospira manakala 84.85% (28 daripada 33) tikus yang ditangkap membawa saprophytic Leptospira. Kajian ini 4elah menunjukkan bahawa tikus mempunyai potensi tinggi sebagai pembawa Leptospira. Kajian ini juga menunjukkan bahawa PCR adalah alat yang baik molecular yang amat berguna untuk mengesan Leptospira dalam pembawa kronik. Hasil kajian telah membuktikan bahawa wujudnya patogen Leptospira dalam kawasan bandar di Sarawak. Penemuan ini membantu dalam memberi pengetahuan mengenai tahap jangkitan dari patogen Leptospira yang dibawa oleh tikus bandar Sarawak. Oleh itu, penambahbaikan seperti kebersihan sesuatu kawasan amat diperlukan di bandar Sarawak untuk mengawal kes leptospirosis.

Kata kunci: Leptospira, tikus , tapak bandar, rantaian polimerave reaksi

1.0 Introduction

Leptospira is often associated with global zoonotic disease known as leptospirosis.

It causes a global health impact on human and animals (Lehmann et al., 2014).

Leptospirosis is also considered as an endemic disease especially in tropical countries that

experienced seasonal rain and floods (Dutta & Christopher, 2005; WHO, 2012). This

includes Malaysia in which leptospirosis outbreaks has increased rapidly in an alarming

rate due to poor sanitation and climate factors lead to high risk of leptospirosis infection.

Several countries in Malaysia have recorded high number of leptospirosis cases especially

Perak, Kelantan, Pahang, and Sarawak (Thayaparan et al., 2013).

Most mammals especially livestock animals and rodents have high potential to get

infected with Leptospira. However, rats are known to be the major reservoirs of Leptospira

and considered as maintenance host (Mohamed-Hasan et aI., 2010). Previously,

Thayaparan et al. (2013) stated that most infections occur among occupational workers that

involve with water sources. Presently, infections have now moved into the urban sites due

to close proximity of rats' infestation that might allow human to be in contact with

Leptospira infection either directly from rats or indirectly from contaminated environment

(Tassinari et al., 2008).

In the earlier research, leptospirosis diagnosis usually was carried out via traditional "

culturing method such as microscopic agglutinatibn test (MAT). Although MAT was

known to be the gold standard assay of Leptospira detection but MAT has failed to reveal

leptospirosis infections due to the diversity of Leptospira antigen in different areas (Mgode II

et al., 2005) In addition, due to time consuming and high costs of using MAT, rapid

diagnosis of leptospirosis have now been established by using molecular technique

especially polymerase chain reaction (PCR) in order to study and detect Leptospira which

are more beneficial in terms of specificity and sensitivity (Cameron et al., 2008). 2'

Since high number of leptospirosis cases · in Sarawak was reported from Sarawak

Health Department (2015), this has led to public health concerns among people especially

people from the urban sites of Sarawak. Due to limited infonnation regarding Leptospira in

urban sites, a research was carried out by isolating and detecting Leptospira from potential

carriers (rats) that were captured in selected urban sites of Sarawak. In this research,

collected kidney and liver tissue samples were cultured using modified Ellinghausen

McCullough Johnson Harris (EMJH). PCR was then perfonned to detect the presence of

pathogenic, saprophytic, and intennediate Leptospira from captured urban rats.

The main objectives of this research were:

1. To isolate and detect pathogenic, saprophytic, and intennediate Leptospira from

rats in selected national park and urban sites in Sarawak by using PCR method.

2. To study the occurrence of pathogenic Leptospira in the urban sites of Sarawak.

3

2.0 Literature Review

2.1 Leptospirosis

Leptospirosis is known to be an important global zoonotic disease. Wojcik-Fatla et

al (2013) stated that leptospirosis infections represent re-emerging health problem due to

the increment of incidence rates in human and animals globally, including Malaysia. This

disease is known to be caused by pathogenic Leptospira namely Leptospira interrogans

and Leptospira borgpetersenii (Balamurugan et a!., 2013). The infection occurs when

animals or human get in contact either directly from the infected host or indirectly from the

exposure of contaminated environment. Pathogenic Leptospira moves through bloodstream

and attach to kidney to multiplies and release toxin to cause disease. The infection

manifests various clinical symptoms that similar to other infectious disease such as malaria,

dengue fever, and influenza (Lim et al., 2011). Doungchawee et al. (2007) also stated that

the outcomes of infections are varied, .ranging from acute febrile illness to aseptic

meningitis followed by jaundice, renal failure, pulmonary hemorrhage, and refractory

shock. Thus, due to varied clinical symptoms manifestation, it is difficult to diagnose

leptospirosis.

Leptospirosis occurs in many countries worldwide as shown in Figure 1. Similarly,

Table 1 also shows the evidence regarding the global incidence rate of leptospirosis.

Furthermore, studies carried out by Dutta and Christopher (2005) showed that tropical

countries have the highest potential risk of leptospirosis due to favorable climate conditions

for Leptospira growth. Balamurugan et al. (2013) have conducted a research in India and 01

found out that there is high number of leptospirosis cases were reported thus making it

difficult to be controlled due to poor sanitation and climate factors. Vitale et al. (2005) also

stated that 300 leptospirosis cases occurred annually in Brazil and the number of cases is

increasing over time.

PltSiI . h rn:d lakJumat k:u:Jrmi' . UN VE IT. MALAY IA ,AJt\\-\, ­

Figure 1. Global leptospirosis incidence map (Pappas et ai., 2008).

Table 1. Global incidence rates of leptospirosis (Pappas et at., 2008)

Countries with the highest incidence

Rank Countries Annual incidence Other Annual incidence per million countries per million population population

Seychelles 432.1 Belarus 3.4

2 Trinidad and Tobago 120.4 Bulgaria 3.7

3 Barbados 100.3 Chile l.6

4 Jamaica 78 Colombia l.6

5 Costa Rica 67.2 Czech Republic l.8

6 Sri Lanka 54 France 3.9

7 Thailand 48.9 Germany 0.7

8 EI Salvador 35.8 Greece 3

9 New Zealand 26 Honduras 3.1

10 Uruguay 25 Hungary 3.1

11 Cuba 24.7 Ireland 3.2

12 Nicaragua 23.3

13 Croatia 17.3

14 Russia 17.2

15 Ukraine 15.3

16 Dominican Republic 13.8

17 Brazil 12.8

18 Ecuador 1l.6

5

19 Argentina 9.5

20 Romania 9.4

21 Australia 8.9

22 Portugal 6.8

23 Denmark 6

24 Latvia 5.6

25 Slovenia 5.4

26 Philippines 4.8

27 Slovakia 4.4

28 Taiwan 4.1

Similarly, the incidence rates of leptospirosis in Malaysia is 2 to 5 of 100 000

population (Lim et al., 2011). Seasonal rainfall that is commonly occurring in Malaysia as

apparently contributes to the increment of leptospirosis cases. Sarawak also recorded high

number of leptospirosis over time. Table 2 shows the gradual increase of leptospirosis

cases from year 2004 until 2015 in Sarawak as reported by Sarawak Health Department

(2015).

Table 2. Leptospirosis cases in Malaysia from 2004 to 2009 (Thayaparan et a/., 2013)

Year Number of cases

2004 263

2005 378 . 2006 527

2007 1418

2008 1263

2009 1418

6

Table 3. Leptospirosis cases in Sarawak from 2004 to March 2015 (Sarawak Health Department, 2015)

Year Number of cases

2004 32

2005 71

2006 37

2007 70

2008 58

2009 70

2010 49

2011 186

2012 271

2013 434

2014 616

2015 (Until week 9) 163

2.2 Leptospira Species

Leptospira is spirochete grouped under the order of Spirochetales, family of

Leptospiracaea and genus of Leptospira. Leptospira comes from Greek word 'Leptos'

which means coiled, fine and spiral that describe the morphological structures of

Leptospira (Dutta & Christopher, 2005). This coiled filamentous spirochete is about 6-20

mrn in length and 0.1 mm in width with hooked end (Dutta & Christopher, 2005; Mayer­

Scholl et al., 2014). These bacteria are considered a~obligate aerobes as they survive under

favourable condition at optimum temperature of 28°C to 30 °c (Thayaparan et al., 2013).

The history of Leptospira was first described by Adolf Weil in 1885 which leptospirosis is

known to be causing clinical symptoms include jaundice, splenomegaly, and nephritis

7'

(Vinetz, 2012). Then, in 1915, scientist in Japan named Inada was able to isolate

Leptospira from patients with Weil's syndrome disease (Dutta & Christopher, 2005). In

Malaysia, Lim et al. (2011) stated that leptospirosis was first discovered in patient by

Fletcher in 1925.

Leptospira are grouped into three known as saprophytic, intermediate and

pathogenic (Ahmed et aI., 2012). Thayaparan et al. (2013) and Lim et al. (2011) described

saprophytic Leptospira are commonly found in wet environments such as streams and

waterfalls. Saprophytic Leptospira does not harm other organisms and cause disease as

they only consume organic matters in water for survival, thus they do not require host (Lim

el ai., 2011).

Apart from that, intermediate Leptospira is another group of Leptospira species that

are closely related to both pathogenic and saprophytic Leptospira respectively. Ko et al.

(2009) stated that this species is considered as opportunistic bacterium that still has unclear

pathogenicity. Voronina et al. (2014) has carried out phylogenetic studies Leptospira strain

Bairam-Ali and found out that this strain have both characteristics of pathogenic and

saprophytic Leptospira. This unexpected finding of intermediate Leptospira has brought

essential information especially on the ability of bacteria to adapt to different

environmental conditions (Voronina et al., 2014). Thus, more studies need to be done on

intennediate Leptospira in order to investigate the. transmission of pathogenicity.

On the other hand, pathogenic Leptospira is the most well-known bacteria that

cause leptospirosis infection. Lim et al. (2011) described that pathogenic Leptospira .. require host to survive as the host has ample nutrient sources that is fundamental for

survival and reproduction. The host that carries pathogenic Leptospira is also known as

natural maintenance host (Lim et al., 2011). Most studies carried out by scientists

discovered that small mammals are the potential host of pathogenic Leptospira (Ahmed et I 8

al., 2012). Pathogenic Leptospira is commonly found in the kidney and the liver of infected

host. These organs are the most essential organs in all organisms where by liver generally

perfOim functions that are related to metabolism, immunity, and storage of nutrients in the

body whereas kidney excretes toxic waste out of the body and reabsorb back essential

nutrients that are needed for body metabolism (Inner body, 2015). The pathogen is then

released to the environment via urine that leads to environment contamination and infect

susceptible organism including human (Dutta & Christopher, 2005; Lim et al., 2011;

Ahmad et al., 2012).

2.3 Sources of Leptospira

There are two sources that lead to leptospirosis infection which are direct sources

and indirect sources respectively. WHO (2012) has stated that rodents were the first

mammals recognized to carry Leptospira. Thayaparan et al. (2013) also stated that in 1917,

rats play an important role as the main source for Leptospira that contribute to human

infections. Apart from that, house rats (Rattus rattus) are the most common rats that are

found mainly in the urban sites especially in developing countries which Fletcher has

reported that these rats species were main reservoir of Leptospira (Thayaparan et al.,

2013). The increase of rats' infestation is one of the main factors that causes urban public

health problem affecting travelers and residents in cities (Sakinah et al., 2015). In addition,

indirect sources especially water and soils have high. potential risk to be contaminated with

pathogenic Leptospira. The contamination is caused by the excretion of urine from

chronically infected host which eventually shed the pathogenic Leptospira to the

environment (Dutta & Christopher, 2005; Mort1mer, 2005; Benacer et al., 2013;

Thayaparan et al., 2013).

9 1

2.4 Molecular Analysis using peR

Many molecular studies have been carried out to study the presence of Leptospira

including microscopic agglutination test (MAT), polymerase chain reaction (PCR), dark

field microscopy (DFM), cross agglutinin absorption test (CAA T), and pulse field gel

electrophoresis (PFGE). However, recent studies show that PCR is the most common

methods to investigate Leptospira.

PCR is known to be a molecular method that amplifies DNA to generate millions

copies of DNA of particular DNA sequence. Researches have been carried out on many

types of samples from different mammals to detect Leptospira species by performing PCR

as shown in Table 3. Mgode et al. (2005) has done a research on Leptospira detection in

rats and insectivores from Tanzania. Leon et al. (2006) has performed PCR for pathogenic

Leptospira taken from tissue samples of premature foal. Besides, Benacer et al. (2013)

have also carried out previous research by using PCR in isolating Leptospira from the

urban rat popUlations in Kuala Lumpur, Malaysia. Scarcelli et al. (2003) also have carried

out in previous studies on Leptospira detection from primates using PCR method in Brazil.

Apparently, PCR has widely used in diagnosing pathogenic strains of Leptospira

from suspected sources which include rodents, water, and soils (Tulsiani et aI., 2011).

Studies have shown that PCR method has high sensitivity and specificity in detecting and

identifying Leptospira that is classified as fastidious organism (Tulsiani et ai., 2011;

Benacer et al., 2013). PCR is also very useful in diagnosing leptospirosis especially in

human acute syndrome cases whereas other techniques such as MAT and bacterial culture 01

might give false-negative results and time consuming (Vitale et al., 2005). Hence, PCR is a

molecular approach that is best applied for Leptospira detection especially in new

geographical region which the prevalence of leptospirosis is still unknown (Mgode et aI.,

2005). 10'

Table 4. Leptospira detection carried out by using peR in previous research

Researchers Year Molecular Type of Samples methods

Scarcelli et al. 2003 peR Primates

Khahani-Bejo et al. 2004 peR Rats

MAT Mgode et al. 2005 Rats and insectivorous

peR

Leon et al. 2006 peR Horse

Ahmed eta!' 2012 Real-time peR Dogs

MAT

Benacer et al. peR Rats

2013 PFGE

11

3.0 Materials and Methods

3.1 Materials

The list ofmaterials used in this study are listed in Appendix 1.

3.2 Methods

3.2.1 Study Sites

The selected sampling sites were chosen for sampling which were the urban areas

in Sarawak specifically in Kuching and Kota Samarahan as summarised in Table 5.

Selected national park namely Gunung Gading National Park (GGNP) in Lundu (Figure 2)

was chosen which is one of the recreational sites where by visitors were exposed most

with water activities as it accommodates jungle trekking trails, waterfalls and jungle pools

that might have presence of Leptospira. Other than that, Kuching and Kota Samarahan are

considered developing regions in Sarawak. Th~ selected locations were residential areas in

Kuching as shown in Figure 3. Besides, Kampung Paya Mebi and Kampung Sabayor

(Figure 4) were also selected as sampling sites as these places have poor sanitation and

drainage system which most likely attract pest animals to breed and be potential carrier

Leptospira.

12 \

Table 5. Sampling sites of captured rats

No. Sampling Sites Date of Sampling

1. Gunung Gading National Park 21.10.2014 - 25.10.2014

2. Kampung Tupong 20.10.2014-16.12.2014

3. Kampung Sebayor 25.10.2014 - 26.10.2014

4. Kampung Paya Mebi 26.10.2014-·29.10.2014

5. Kampung Gita 03.11.2014 - 02.02.2015

6. Taman Sukma 30.11.2014

7. Camp Mike, Matang 02.12.2014 - 13.12.2014

8. Medan Niaga Satok 10.12.2014 - 22.01.2015

9. Jalan Perupok 13.12.2014 - 27.01.2015

10. Jalan Padang Pasir 16.12.2014 - 18.12.2014

11. Hui Sing 14.01.2015 - 24.01.2015

Figure 2. Map location ofGunung Gading National Park (Google Maps, 2015).

13

Medan Niaga Satok Kampung Gita Figure 3. Map location of selected sampling sites in Kuching (Google Maps, 2015).

Figure 4. Map location ofKampung Sebayor in Kota Samarahan (Google Maps, 2015).

3.2.2 Samples Collection

Rats were captured using cage traps. Banana, coco9ut and salted fish were used as baits

(Figure 5). The captured rats' data were listed in Table 7. In Gunung Gading National Park,

100 cages were placed in different areas along the trail within 5 days of sampling from 21 sl

October 2014 until 251h October 2014. Besides, five cage traps were placed in each selected

14·

urban areas including residential areas and public market. In residential areas, cage traps

were placed mostly in kitchen, storage room, and also surrounding the house such as

drains. In Kuching public market named Medan Niaga Satok, cage traps were placed near

drains and garbage disposal areas.

Figure 5. Set cage traps using banana baits in Gunung Gading National Park.

3.2.3 Isolation of Leptospira

Captured rats were kept inside a plastic bag and euthanized by using chloroform. All the

measurements including ear, hind foot, total body length and weight were performed and

recorded. Then, the rats were identified for their species based on the published facts on

Borneo mammals (Payne, Francis, & Phillips, 1985). After that, selected organs

specifically kidneys and livers were removed frol!l the rat by using sterile scissors and

forceps. These organs were cut into smaller pieces before adding into modified EMJH

medium (Difco BD, USA) (Figure 6) in which 0.1 g 5'fluorouracil were added to reduce

01 contamination. Three replicates were used for each tissue samples. Inoculated media were

incubated aerobically at room temperature and kept in dark for three months. ON A

extractions of samples were carried out each month for consecutive three months by using

Wizard Genomic DN~ Purification Kit (Promega, USA).

lS

Figure 6. Samples cultured in modified EMJH media.

3.2.4 Detection of Leptospira using peR method

The presence ofLeptospira was detected using PCR method where by three sets of primers

were used to target three specific genes of Leptospira (Table 6). The first primer set was

rrsFlrrsR that is used to target 16S rRNA gene. This gene identifies Leptospira spp. The

second primer set was /ipL32-270FllipL32-692R that will target on /ipL32 gene indicating

the presence of pathogenic Leptospira (Krishna et al., 2013). Primer set Saprol/Sapro2

was used to target rrs gene for saprophytic Leptospira detection and confirmation. The

cycling condition was started with initial denaturation at 95°C for 2 minutes, 35 cycles at

95°C for 1 minute, 55°C for 30 seconds, and 72 0<,= for 1 minute. Additional extension was

done at 72 °C for 5 minutes. Then, PCR was performed for Leptospira DNA to be

amplified. 2% TBE agarose gel electrophoresis was then performed on the PCR products.

The PCR products was stained with ethidium bromide (EtBr) and visualised under UV

transluminator to observe DNA band indicating positive Leptospira.

"

16