OCCURRENCE OF Shigella, Yersinia AND Salmonella SPECIES FROM SELECTED WILDLIFE AND HABITATS

IN SARAWAK

Chen Yik Ming

Master of Science 2012

Pusat Khidmat Maklumat Akademik [JTIIVERSIT[ MALAYSIA SARAWAK

OCCURRENCE OF Shigella, Yersinia AND Salmonella SPECIES FROM SELECTED WILDLIFE AND HABITATS IN SARAWAK

CHEN YIK MING

A thesis submitted in fulfillment of the requirement for the Degree of

Master of Science (Microbiology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2012

ACKNOWLEDGEMENT

I would like to express my deepest appreciation and thankfulness to my supervisor, Professor

Dr. Kasing Apun, co-supervisor and Eco-zoonosis project leader, Professor Dr. Mohd.

Tajuddin Abdullah for their advice, guidance and supervision throughout this project.

I would like to express a special gratitude to Mr. Wahap Marni, Mr. Besar Ketol, Mr.

Azis Ajim, Mr. Dahlan Rambli, Mr. Nasron Ahmad, Mr. Isa Sait, Miss Rahah bt Mohd Yakup

and all the staff, lab assistants and students from the Department of Zoology and Institute of

Biodiversity and Environmental Conservation (IBEC) in the Faculty of Resource Science and

Technology, Universiti Malaysia Sarawak (UNIMAS) who had assisted in the sampling trips.

Sampling trip in Nanga Merit were approved by Sarawak Planning Unit with permit reference

number (13) UPN/S/G1/I/10.1 Vol. 26. This project was funded by UNIMAS grant

E I4006/F07/06/ZRC/03/2007(03).

A special appreciation is attributed to Dr. Lesley Maurice Bilung and Dr. Samuel

Lihan for their advice and guidance in conducting the research. Sincere thanks to Miss

Hashimatul Fatma Hashim for her advice and assistance throughout the field work. I would

like to thank the Microbiology Laboratory assistant, Mr. Azis Ajim and the Immunology

Laboratory assistant, Miss Limjatai for their technical assistance. My sincere appreciation to

all my labmates, friends and fellow partners in this project, especially Mr. Adorn Benjamin,

Miss Kho Kai Ling, Miss Anita Tahir, Miss Lee Jong Jen, Miss Harttini Neeni Hatta and Miss

Sarina Niyup for their companionship, advice and knowledge.

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I would like to thank all the lecturers and staffs in Faculty of Resource Science and

Technology for their guidance and assistance. Lastly, I would like to express my gratitude to

my family for their support and patience towards me during the progress of the project.

Thank you.

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ABSTRACT

Shigella spp., Salmonella spp. and Yersinia spp. are gastrointestinal pathogens which

can infect birds, bats, rodents and other small mammals. These animals may serve as reservoir

for many diseases and play an important role in transmission of zoonoses. The destruction or

disturbances of the wildlife habitats have been identified as a factor that lead to the emergence

of these zoonoses outbreaks as habitats can influence the composition of bacterial species.

Therefore, this study was conducted to determine the occurrence and association of Shigella

spp., Salmonella spp. and Yersinia spp. in selected wildlife from two natural and three

disturbed habitats., Three sampling trips to Nanga Merit, Pueh and Mount Singai were held for

the samples collection in the study. These areas represented disturbed (Nanga Merit's village,

Pueh and foot of Mount Singai) and natural habitats (Nanga Merit's forest and Mount

Singai's forest). Anal or cloacal swabs, feacal and small intestinal of birds, bats and rodents

were collected aseptically. In addition, soil and water samples were collected along the

trapping routes to represent environmental samples. A total of 714 samples were collected

from the animal and environment from the three sampling. XLD and CIN agar were used for

the isolation of Shigella, Salmonella and Yersinia. Shigella, Salmonella and Yersinia were

absent in all the animal and environmental samples collected from Pueh and Mount Singai.

The isolated bacteria were then identified through a series of standard biochemical tests and

multiplex PCR. Through multiplex PCR, virulence genes of Shigella (ipaH and ial) were

detected in three bats and one bird sample from Nanga Merit Village which represent a

disturbed habitat. One bird, two bats, a soil and a water sample from the forest in Nanga Merit

which represent natural habitat were positive for the ipaH and ial gene. One bat isolate from

both sites at Nanga Merit were positive for the flic gene which is a virulence gene in

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Salmonella Typhimurium. No Yersinia was found in any of the samples from all the sampling

trips indicating none of the wild animals harboured Yersinia. Birds and bats from village

showed higher percentage for the presence of virulence genes of Shigella spp. and Salmonella

spp. compared to the forest in Nanga Merit. The Shigella isolates were sequenced and

identified as Shigella boydii strain 3555-77. Two Salmonella strains which were positively

detected for flic gene in bat samples were sequenced as Salmonella enteric subsp enteric

serovar Typhimurium. The low occurrence of Shigella and Salmonella in birds and bats

samples showed the low transmission risk of these pathogenic bacteria from human, animals

and environment. The absence of Shigella, Yersinia and Salmonella species in soil and water

indicate the low contamination of the bacteria through soil and water in Nanga Merit. Even

though the number of isolates detected per species examined is low, the potential of infection

risk of Shigella, Yersinia and Salmonella in the wildlife and environment had to be monitored

to prevent infection in future.

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ABSTRAK

Shigella spp., Salmonella spp. dan Yersinia spp. merupakan patogen sistem

penghadaman yang selalu menjangkit burung, kelawar, tikus dan haiwan liar yang lain.

Haiwan-haiwan ini merupakan reservoir dan memainkan peranan penting dalam penyebaran

wabak zoonotik. Pencerobohan atau pengangguan habitat haiwan liar merupaken salah satu

faktor yang menbawa kepada kemunculan zoonotik kerana habitat mempengaruhi komposisi

bagi bakteria spesies. Oleh itu, kajian ini dilakukan untuk memperoleh kadar pemulihan dan

hubungan antara Shigella spp., Salmonella spp. dan Yersinia spp. dalam haiwan tertentu dari

dua habitat semulajadi dan tiga habitat terganggu. Tiga kajian ke Nanga Merit, Pueh and

Gunung Singai telah diadakan demi pengumpulan sampel bagi kajian ini. Lokasi-lokasi ini

mewakili habitat terganggu (Kampung Nanga Merit, Pueh and tapak gunung Gunung Singai)

dan habitat semulajadi (Hutan Nanga Merit and Hutan Gunung Singai). Anal atau kloakal

swabs, najis dan usus kecil daripada burung, kelawar dan tikus telah dikumpul dengan

kaedah aseptik. Selain itu, sampel tanah dan air juga dikumpul di sepanjang laluan

pemerangkapan haiwan. Sebanyak 714 sampel dikumpul daripada haiwan dan alam

semulajadi dari ketiga-tiga tempat. XLD dan CIN agar digunakan dalam pengasingan

Shigella. Salmonella dan Yersinia. Shigella. Salmonella dan Yersinia tidak hadir dalam

semua sampel yang dikumpul daripada haiwan dan alam semulajadi dari Pueh and Gunung

Singai. Bakteria - bakteria yang telah diasingkan akan dikenalpasti melalui ujian-ujian

biokimia dan kaedah tindak balas berantai polimeras multipleks (multipleks PCR). Melalui

multipleks, gen virulen bagi Shigella (i aH dan ial telah dikesan dalam tiga sampel kelawar

dan satu sampel burung dari kampung Nanga Merit yang mewakili habitat terganggu. Satu

sampel burung, dua sampel kelawar, satu sampel tanah dan satu sampel air daripada hutan

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Nanga Merit yang mewakili habitat semulajadi positif dengan kehadiran gen ipaH dan ial.

Satu sampel kelawar darf kampung dan satu sampel kelawar darf hutan di Nanga Merit

adalah positif dengan kehadiran is gen iaitu virulen gen bagi Salmonella Typhimurium.

Tiada Yersinia dikesan dalam semua sampel yang dikumpul darf ketiga-tiga tempat yang

dikaji. Ini menunjukkan tiada hidupan liar yang mengandungi bakteria Yersinia. Keputusan

menunjukkan burung dan kelawar darf kampung mempunyai peratusan yang lebih tinggi bagi

Shigella dan Salmonella gen virulen berbanding sampel yang diperolehi darf hutan

semulajadi di Nanga Merit. Keputusan selepas penjujukan bagi semua sampel yang

menpunyai gen virulen menunjukkan dua isolat dikenal pasti sebagai Shigella boydii strain

3555- 77. Dua Salmonella yang mempunyai his gen darf sampel kelawar telah dikenalpasti

sebagai Salmonella enteric subsp enteric serovar Typhimurium. Kehadiran Ship-ells dan

Salmonella yang rendah dalam burung dan kelawar menunjukkan kadar risiko yang rendah

untuk patogenik bakteria ini menjangkiti manusia, dan haiwan di Nanga Merit.

Ketidakhadiran Shigella" Yersinia dan Salmonella spesies dalam sampel tanah dan air telah

menunjukkan kadar pencemaran yang rendah oleh bakteria-bakteria ini melalui tanah dan

air di Nanga Merit. Walaupun bilangan bakteria yang dikenalpasti per spesies adalah rendah,

namun penyebaran Shi ells Yersinia dan Salmonella oleh haiwan dan alam semulajadi

harusjuga dikawal bagi mengelakkanjangkitan darf berlaku pada masa hadapan.

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Pusat kri; umat Maktumat Akademik UNIVERSTfI MALAYSIA SARAWAK

TABLE OF CONTENTS

ACKNOWLEDGEMENTS

ABSTRACT

ABSTRAK

TABLE OF CONTENTS

LIST OF CONFERENCE PROCEEDINGS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER 1 GENERAL INTRODUCTION

CHAPTER 2 LITERATURE REVIEW

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Vii

xi

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1

9

2.1 The relation of zoonoses, wildlife and human. 9

2.2 Shigella outbreaks, infections and symptoms in human and 12

wildlife.

2.3 Yersinia outbreaks, infections and symptoms in human and 17

wildlife.

2.4 Salmonella infections, outbreaks and symptoms in human and 21

wildlife.

2.5 Roles of birds, bats and rodents in transmission of Shigella, 24

Yersinia and Salmonella.

2.6 Isolation and identification of Shigella, Yersinia and Salmonella. 29

2.7 The detection of virulence gene of Shigella, Salmonella and 33

Yersinia by Multiplex Polymerase Chain Reaction (PCR).

vii

2.8 16S ribosomal RNA (rRNA) gene sequencing for identification 37

of bacteria.

CHAPTER 3 MATERIALS AND METHODS 39

3.1 Study Area. 39

3.2 Specimens Collection. 43

3.3 Collection of swab samples from birds, bats and rodents. 44

3.4 Collection of soils and waters samples. 44

3.5 Bacterial Isolation and Identification. 45

3.5.1 Swabs, intestines and feces samples. 45

3.5.2 Soil and water samples. 46

3.5.3 Isolation and identification of Shigella, Yersinia and 47

Salmonella.

3.6 Bacterial Genomic DNA Isolation. 49

3.6.1 Direct Lysis Method. 49

3.6.2 DNA Extraction Kit. 50

3.7 Polymerase Chain Reaction (PCR). 51

3.7.1 Multiplex PCR. 51

3.7.2 Gel visualization and documentation. 55

3.8 Molecular identification via Polymerase Chain Reaction (PCR) 56

amplification of 16S ribosomal rRNA (rRNA) gene.

3.8.1 Gel visualization and documentation. 57

3.8.2 Purification of PCR products before DNA sequencing. 58

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3.9 DNA sequencing of the isolates and data analysis. 59

CHAPTER 4 RESULTS 60

4.1 Total samples collected from three sampling trips. 60

4.2 Bacterial Isolation and identification. 64

4.2.1 Isolation of Shigella, Salmonella, Yersinia and other 64

bacteria on XLD and CIN agar.

4.2.2 Identification and confirmation of bacteria through gram - 66

staining and standard biochemical tests.

4.3 Occurrence of Shigella, Salmonella, Yersinia in different animal 67

hosts, soil and water samples collected from Nanga Merit, Pueh

and Mount Singai.

4.4 Amplification of virulence genes for Shigella, Yersinia and 69

Salmonella isolates by multiplex PCR.

4.5 Identification of bacterial strain through 16S rRNA gene 76

sequencing.

CHAPTER 5 DISCUSSION 79

5.1 The occurrence of Shigella, Salmonella and Yersinia in bats, 79

birds, rodents, soil and water in Nanga Merit, Pueh and Mount

Singai, Sarawak.

5.2 Detection of virulence genes of Shigella, Salmonella and 86

Yersinia in all the samples through multiplex PCR and the

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identification of the bacteria through 16S rRNA gene

sequencing.

5.3 A comparison of the bacterial identification results between 90

standard biochemical test and molecular methods.

CHAPTER 6 GENERAL CONCLUSION

REFERENCES

APPENDIX A

APPENDIX B

APPENDIX C

APPENDIX D

APPENDIX E

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116

118

120

122

123

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LIST OF CONFERENCE PROCEEDINGS

Chen Yik Ming, Kasing Apun, Lesley Maurice Bilung, Hashimatul Fatma Hashim, Mohd.

Tajuddin Abdullah, Kho Kai Ling and Adom Benjamin (2009). Isolation of Shigella spp. and

Yersinia spp. from wild animals, soil and water from different habitats in Sarawak.

Proceedings of the International Congress of Malaysian Society for Microbiology (ICMSM)

1S`-4`h December 2009, Parkroyal Hotel Penang, pg 273. ISBN 978-983-41487-4-4.

Chen Yik Ming, Kasing Apun, Lesley Maurice Bilung, Mohd. Tajuddin Abdullah (2010).

Prevalence of Shigella, Salmonella and Yersinia spp in Disturbed (Human Settlement) and

Natural (Forest) Habitats in Nanga Merit, Kapit, Sarawak. Proceedings of the 3rd UNIMAS

Colloquium, 24" -25th May 2010, Universiti Malaysia Sarawak (UNIMAS), pg 114. ISBN

978-967-5418-08-2.

Chen Yik Ming, Kasing Apun, Mohd. Tajuddin Abdullah, Lesley Maurice Bilung,

Hashimatul Fatma Hashim, Kho Kai Ling and Adom Benjamen (2010). Detection of

virulence genes in Shigella, Salmonella and Yersinia spp. from wild animals in disturbed and

natural habitats in Sarawak. Proceedings of the Seminar of Universiti Malaysia Sarawak

(UNIMAS) and The Field Museum of National History (FMNH), 17`h December 2010,

UNIMAS.

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LIST OF TABLES

Table Page

Table 3.1 Volume and concentration of each PCR mixture for the detection of 52 virulence genes in Shigella isolates.

Table 3.2 Volume and concentration of each PCR mixture for the detection of 53 virulence genes in Salmonella isolates.

Table 3.3 Volume and concentration of each PCR mixture in the detection of 53 virulence genes in Yersinia isolates.

Table 3.4 PCR cycles profile used for detection of virulence genes with 35 cycles. 53

Table 3.5 Primer sequence for virulence genes detection of Shigella, Salmonella and 54 Yersinia species.

Table 3.6 Primer sequence of 16S rRNA gene. 56

Table 3.7 Volume and concentration of each reagents in PCR used in 16S rRNA 57 gene.

Table 3.8 The PCR cycles profile used for 16S rRNA sequencing with 30 cycles. 57

Table 4.1 Total number of birds, bats and rodents captured from both Nanga Merit 60 Village and Nanga Merit's forest at Nanga Merit, Kapit, Sarawak.

Table 4.2 Total number of soil and water samples collected from both Nanga Merit 61 Village and Nanga Merit's forest, Nanga Merit, Kapit, Sarawak.

Table 4.3 Total number of birds, bats and rodents captured during the fieldtrips to 62 Pueh, Sarawak.

Table 4.4 Total number of soil and water samples collected during the fieldtrip to 62 Pueh, Sarawak.

Table 4.5 Total number of birds, bats and rodents captured during the fieldtrips to 63 Mount Singai, Bau, Sarawak.

Table 4.6 The total of soil and water samples collected during the fieldtrip to 63 Mount Singai, Bau, Sarawak.

Table 4.7 The occurrence of Shigella, Yersinia and Salmonella in birds, bats and 68 rodents in Nanga Merit Village and Nanga Merit forest in Nanga Merit, Kapit, Sarawak in percentage (%)based on biochemical tests results.

xii

Table 4.8 The occurrence of Shigella, Yersinia and Salmonella in soil and water 68 samples in Nanga Merit Village and forest in Nanga Merit, Kapit, Sarawak in percentage (%) based on biochemical tests results.

Table 4.9 The occurrence of Shigella virulence genes (ial, ipaH and setiB) among all 72 the birds, bats and rodents samples in Nanga Merit, Kapit.

Table 4.10 The occurrence of Shigella virulence genes (ial, ipaH and set1B) among 72 isolates from soil and water samples in Nanga Merit, Kapit.

Table 4.11 Results of multiplex PCR for positive virulence genes of Shigella and 75 Salmonella from the samples obtained in Nanga Merit Village (Site 1) and forest (Site 2) in Nanga Merit, Kapit, Sarawak.

Table 4.12 The occurrence of Shigella and Salmonella isolates in total birds, water and 78 bats, respectively in Nanga Merit.

Table 5.1 Comparison of the results of standard biochemical tests and 16S rRNA 92 gene sequencing for the identification of Shigella, Salmonella and Yersinia in different samples from Nanga Merit.

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LIST OF FIGURES

Figure Papae

Figure 2.1 Life cycle of Shigella bacteria. (Source: http: // 13 microbewiki. kenyon. edu/index. php/Shigella).

Figure 2.2 The mode of transmission of Yersinia enterocolitica from different 19 sources such as contaminated food and water, infected animals and humans (Sources: http: //www. hindawi. com/joumals/jpath/2011/429069/figl/).

Figure 2.3 Potential routes of transmission of zoonotic pathogens from 25 livestocks animals, wild animals to human and environment (Source: Rogers and Haines, 2005).

Figure 3.1 Map showing sampling sites in Nanga Merit (Source: Jabatan 40 Pemetaan Malaysia cawangan Sarawak).

Figure 3.2 Map showing sampling sites of Nanga Merit, Pueh and Mount 42 Singai, Sarawak. (Source: http: //www. maplandia. com/malaysia/sarawak/kampung- pueh/register/)

Figure 4.1 The morphology of Shigella, Salmonella and Yersinia on XLD and 65 CIN agar.

Figure 4.2 Amplicons obtained by multiplex PCR for eight Shigella isolates 70 isolated from Nanga Merit with ipaH, ial and set1B genes with expected size of approximately 423 bp, 320 bp and 147 bp respectively fragmented by 2% agarose gel electrophoresis.

Figure 4.3 Amplicons obtained by multiplex PCR for Salmonella isolates from 73 Nanga Merit with fliC gene in with expected size of 559 bp fragmented by 2% agarose gel electrophoresis.

Figure 4.4 Amplicons obtained by multiplex PCR for Yersinia isolates from 74 Nanga Merit for detection of virF and ail gene in with expected size of 519 bp and 170 bp respectively fragmented by 2% agarose gel electrophoresis.

Figure 4.5 Agarose gel electrophoresis of the PCR products for detection of 16S 76 rRNA gene in Shigella and Salmonella isolates with expected size of 1500 bp.

xiv

LIST OF ABBREVIATIONS

ATCC American Type Culture Collection

kb Kilo base pair

H2S Hydrogen sulphide

VP Voges Proskauer

MAC MacConkey agar

XLD Lysine Decarboxylase agar

DCA Deoxycholate citrate agar

HE Hekteon Enteric agar

KIA Kligler iron agar

SS Salmonella-Shigella agar

CIN Cefsulodin-Irgasan-novobiocin agar

TSI Triple sugar iron agar

SEL Selenite broth

NA Nutrient agar

dNTPs Deoxynucleotide triphosphates

DNA Deoxyribonucleic acid

ShETI Shigella enterotoxin 1

bp Base pair

U Unit

ddH2O Distilled water

Taq Thermus aquaticus DNA polymerase

NaCl Sodium chloride

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NCBI National Center of Biotechnology Information

BLASTN Basic Local Alignment Search Tool

rpm Revolution per minute

ml Mililitre

g Gram

µ1 Microlitre

s Second

min/s Minute or second

% Percentage

µm Micrometer

mm milimeter

°C Degree celcius

xvi

CHAPTER 1

GENERAL INTRODUCTION

Zoonoses are infectious diseases that are naturally transmitted between vertebrate animals and

humans (Yu, 1993). The infectious agents can be protozoa, fungi, bacteria, chlamydia or

viruses. These agents have potential to cause diseases in both humans and wildlife. There are

several pathways whereby pathogens can invade humans such as through air, drinking water,

food, or hand-to-mouth contact. According to Taylor (2001), over 1400 zoonotic diseases

have been identified worldwide, with approximately 61% significant for human health.

Wildlife serves as a reservoir for many diseases common to domestic animals and humans. It

has played an important role in transmissible zoonotic diseases to human. Transmission can

occur directly and indirectly from the reservoir to the susceptible animals, humans and

environment (Kruse et al., 2004; Kemper et al., 2006). Close contact between humans and

animals, their body fluids and feces will also cause transmission of zoonotic disease.

Despite the occurrence of bacteria in wildlife, various bacteria also exist on or in the

surface of both soil and water. The bacterial species that exist in soil and water is usually

complex and populated. According to Torsvik et al. (1998), the bacterial communities in

pristine soil and sediments may contain a large diversity of more than 10,000 different

bacterial types. Water is a common vehicle for the transmission of infectious microorganisms

especially extraintestinal infection (Boyd, 1995) and cause a serious threat to surrounding

residents, wildlife and environment. Waterborne transmission is a highly effective means for

1

spreading infectious agents to a large portion of the population into the environment (Moe,

1997).

Waterborne pathogens can be transmitted into human and animals through intact or

abraded skin, inhalation or simply application of water to eyes, ears, nose, oral cavity and

mucosa of the genitourinary tracts (Boyd, 1995). Large quantities of enteric microorganisms

can be introduced into environment and transmit to human through fecal and urine

contamination from the infected persons or animals that is discharged into sewers or

unprotected waterways and soils (Moe, 1997).

Enteric bacterial species of Shigella, Yersinia and Salmonella are some of the

important pathogenic enteric bacteria which can be found in soils and water (Stevika et al.,

2004). These pathogens can be commonly found in animals such as primates, birds, insects,

rodents, mice and tigers (King, 1998; Lal et. al, 2003). Rodents are among the major vectors

or carriers in transmitting Shigella and Yersinia in animals (King, 1998). Special types of

Salmonella enterica subsp. Enteric serovar Typhimurium (S. Typhimurium) was found to

occurr endemically in hedgehogs and wild passerine birds which causing sporadic cases and

small outbreaks in humans in Norway (Kruse et al., 2004). These zoonotic agents can be

transmitted from wildlife to humans indirectly via contaminated food and water.

As shigellosis is a primarily disease of humans, there are major research on Shigella

has been human oriented and published in medical literature but this information has not been

readily available to veterinary. Shigella has been known to infect humans in developing

countries and primates (Renquist and Whitney, 1987). However, sparse information of

2

Shigella being found in other animal species such as bats and birds are available. These

animals may serve as important carriers for Shigella in human-animal-human transmission or

environmental-human transmission and vice versa.

Shigella has a world-wide geographic distribution and can commonly be found in

tropical or subtropical developing countries. Shigella is commonly found in water

contaminated with human feces and fecal-oral route is the primary mode of transmission

(Bhunia, 2007). Shigella can also be found in animals such as primates, birds, insects, rodents,

mice, tigers (King, 1998). Among the Shigella spp., Shigella flexneri is one of the species

which is capable of producing severe gastrointestinal disease and death in wildlife and

humans. In Malaysia, Shigella spp. is endemic and was reported to be third most common

bacterial agent responsible for childhood diarrhoea (Thong et al., 2005).

The genus Yersinia consists of three principal pathogenic species which are Yersinia

enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis (Butler, 1998). Yersinia

enterocolitica is considered a serious intestinal pathogen for many animal species as well as

for humans. Yersinia enterocolitica is an emerging waterborne bacterial pathogen. It has been

isolated all over the world from animals, water, environment, raw foods materials and human

beings during last two decades (Lal et al., 2003). The pathogen is widespread in the

environment including soil and water and it is frequently found in the intestinal tract of

mammals particularly pigs, cattle, rodents and fish. The first case report of Yersinia

enterocolitica infection in Malaysia was in 1984 from an Indian woman which confirmed the

presence of the infection in Malaysia (Jegathesan et al., 1984).

3

Infections by Salmonella caused salmonellosis in human. Zoonotic disease caused by

Salmonella in which birds and rodents have been suggested as the reservoir (Meerburg and

Kijstra, 2007). Salmonella enteritidis and Salmonella Typhimurium are zoonotic in origin

where rodents, cattle, reptiles and birds served as the primary reservoir. Besides that,

Salmonella spp. was also detected in the wild bird in several countries, for example, Norway

(Kapperud and Rosef, 1983), Japan (Kobayashi et al., 2007) and Trinidad (Adesiyun et al.,

1998).

Application of molecular techniques is now frequently used in characterizing

virulence genes in bacteria. One of the techniques is multiplex PCR which has been widely

used in the detection of virulence genes in bacteria. Multiplex PCR is easily performed,

reproducible, sensitive and is able to give a presumptive identification of pathogenic strains of

Shigella, Salmonella and Yersinia species and allow simultaneous detection of two or more

virulence genes in bacteria (Wang et al., 1997). Multiplex PCR also provides a new tool with

the potential to be employed for epidemiological and diagnostic purposes. In this research,

multiplex PCR was carried out to detect specific virulence genes of Shigella, Salmonella and

Yersinia spp.

Traditional microbiology methods for the isolation and identification of Shigella,

Salmonella and Yersinia species including enrichment of the cultures, plating on the selective

agar plates and the identification of the colonies through biochemical tests are labour

extensive and time consuming which may lead to false positive results due to atypical

phenotypic properties of some strains (Head, 1982). Thus, 16S ribosomal RNA (rRNA) gene

sequencing was used to identify and validate the species of the bacterial strains. 16S rRNA

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Pusat IVllamat Makiumat Akademik ONIVERSTIT MALAYSIA SARAWAK

gene sequencing is a widely accepted tool for the molecular identification of bacteria

(Bosshard et al., 2006), and, is also used to detect species that cannot be cultured to provide a

better identification for gram-negative bacteria where 89.2% of strains may be identified

down to the species level (Mignard and Flandrois, 2006).

Habitats have been reported to influence the composition of the bacterial species in

animals (Shade et al., 2008). The disturbance to habitats has been reported to have an effect

on diversity of microbes in wildlife (Meier et al., 2008; Berga et al., 2012). In this study, two

habitats were chosen as study sites which were disturbed habitat and natural habitat. The

diversity and distribution of both bacteria in disturbed forest will differ from natural habitats

as it is possible that these bacteria are adapted to the disturbed environment for survival as a

consequence of living habits against host and habitats.

The animal (bird, bat and rodent), soil and water samples for this study were collected

from three different areas in Sarawak which are Nanga Merit, Pueh and Mount Singai. Nanga

Merit is a remote village which is located 70 kilometers from Kapit town in Sarawak. Kapit is

the seventh of the eleventh administrative division in Sarawak, east Malaysia on the island of

Borneo. Nanga Merit is a sub-district of Kapit division. Due to the remoteness of this area,

Nanga Merit was chosen as study site. In Nanga Merit, disturbed habitats consisted of human

settlement area comprising a longhouse and some secondary forest nearby were selected as

study site. Another site studied was a forest area, representing a natural habitat as it is located

two hour boat ride from Nanga Merit Village. The forest is free from the interference of

human activities such as hunting and farming.

5

Pueh is the second sampling site selected in this study. Pueh is chosen to represent a

disturbed habitat as it is a settlement area. Pueh Village is located approximately 120

kilometers from Kuching City near the coastal town of Sematan in Lundu District, Sarawak.

Villagers are still living in the only longhouse standing in the village. The third sampling area

is Mount Singai. Mount Singai is located in Bau District, between Kampung Tanjung,

Kampung Atas and Kampung Segong in Singai area, Kuching, Sarawak. Two forest areas at

Mount Singai which represent natural and disturbed habitats were chosen as study area. The

potential of wildlife as a source of zoonotic infections is significant (Kruse, 2004). Bats, birds

and rodents are appropriate for the surveillance of bacteria as they are the most numerous

tropical mammals and have widely diverse food habits. They have various types of

relationships and associations to human in the nature. Thus, they are good indicators of the

presence of an agent in a tropical rural community. Furthermore, bats, birds and rodents also

play an important role as natural reservoir and vectors for zoonotic disease as they are

abundant in different habitats and significant in human environment (Arata et al., 1968;

Asideyun et al., 2009).

Birds, rodents and bats in the disturbed forest may be infected with Shigella,

Salmonella and Yersinia easily through their feeding habits and, by direct or indirect contact

with human. These zoonotic agents may also be transmitted from wildlife to humans through

direct contact with the animals or indirectly. Transmission of the bacteria could occur when

the animals ingest contaminated food and water and then transmit to other animals,

environment or human. These animals may have the potential to contaminate water and soil

through feces, nasal and any body fluids. Pathogens originating in animal feces can be

transported in flow of water from soil surfaces, wastewater discharges and other sources to

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