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DETECTION OF SALMONELLA ENTERITIDIS AND SALMONELLA TYPHIMURIUM FROM WATER AND ANIMAL SAMPLES IN KUBAH NATIONAL PARK, KUCHING, SARAWAK Dellroy Donny QR 101 Bachelor of Science with Honours S15 (Resource Biotechnology) D358 2012 lOll

SALMONELLA ENTERITIDIS SALMONELLA TYPHIMURIUM … of Salmonella Enteritidis and... · detection of salmonella enteritidis and salmonella typhimurium from water and animal samples

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Page 1: SALMONELLA ENTERITIDIS SALMONELLA TYPHIMURIUM … of Salmonella Enteritidis and... · detection of salmonella enteritidis and salmonella typhimurium from water and animal samples

DETECTION OF SALMONELLA ENTERITIDIS AND SALMONELLA TYPHIMURIUM FROM WATER AND ANIMAL

SAMPLES IN KUBAH NATIONAL PARK, KUCHING, SARAWAK

Dellroy Donny

QR 101

Bachelor of Science with Honours S15 (Resource Biotechnology) D358

2012lOll

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,.. \. \ I i·, ..

Pusat Khidmat MakJumat Akademik VNlVERSm MALAYSIA SAftAWAK

Detection of Salmonella enteritidis and Salmonella typhimurium From Water and Animal Samples in Kubah National Park, Kuching, Sarawak

P.KHIDMAT MAKLUMAT AKADEMIK

111111111 111111111 1000:235571

DELLROY DONNY 23374

The project is submitted in partial fulfilment of the requirement for the degree of Bachelor

of Science with Honours

(Resource Biotechnology)

Department of Molecular Biology

Faculty ofResource Science and Technology

Universiti Malaysia Sarawak

2012

...

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l .' To .,

ACKNOWLEDMENTS

First, I would like to thank to Department of Molecular Biology, Universiti

Malaysia Sarawak for giving me the opportunity to fulfill my Final Year Project (FYP)

with the facilities provided. I really appreciate for all materials, equipment, instruments,

and other facilities provided which is very necessary for the completion of my project.

I would like to express my deepest gratitude and thanks to my supervisor, Dr.

Lesley Maurice Bilung and my co-supervisors, Professor Dr. Kasing Apun and Dr. Samuel

Lihan, for their advice, encouragement and guidance throughout my final year project. Not

to forget the postgraduate students in the Microbiology laboratory, Velneti Linang and

Christy Chan Sien Wei for the help and useful advices regarding the lab work.

Finally, I would also like to thank all my colleagues for their ideas, advices and

collaborations. I appreciate the valuable experience, knowledge and laboratory skills that I

gained during this project.

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I 0 .'

DECLARATION

I declare that this thesis entitled 'Detection of Salmonella enteritidis and

Salmonella typhimurium from water and animal samples in Kubah National Park, Kuching,

Sarawak' submitted to the Faculty of Resource Science and Technology, University

Malaysia Sarawak (UNIMAS) is a presentation of my original research work and that is

has not been submitted anywhere except as cited in the references. This work was done

under the guidance of Dr. Lesley Maurice Bilung and this report work is submitted in the

partial fulfillment of the requirement for the degree of Bachelor of Science with Honours

in Resource Biotechnology.

Dellroy Donny

Faculty of Resource Science and Technology

Department of Molecular Biology

University Malaysia Sarawak

II

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, ., .' " '" Pusat Khidmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

TABLE OF CONTENTS Page

Acknowledgement I

Declaration II

Table of Contents III

List of Abbreviations v

List of Tables VI

List of Figures VII

Abstract VIII

Introduction I

1.1 Background

1.2 Research problems 3

1.3 Objectives 3

2.0 Literature review 4

2.1 Nomenclature / Taxonomy ofSalmonella 4

2.2 Salmonella infection outbreaks 5

2.3 Multiplex peR 6

2.4 Multiplex peR for detection ofSalmonella typhimurium 6

2.5 Multiplex peR for detection ofSalmonella enteritidis 7

3.0 Materials and methods 9

3.t Sample collection 9

3.2 Isolation and identification of presumptive Salmonella species 12

3.2.1 Enrichment ofbacterial samples 12

3.2.2 Isolation of Salmonella species on different type of selective agar 12

III

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.' .'

3.2.3 Preparation of stock and working culture 13

3.2.4 Gram staining 13

3.4 Molecular analysis 14

3.4.1 DNA extraction 14

3.4.2 Multiplex peR 14

4.0 Results and discussion 16

4.1 Isolation and identification of presumptive Salmonella species 16

4.1.1 Isolation of Salmonella species on different type of selective agar 16

4.1.2 Gram staining 24

4.2 Molecular analysis 25

5.0 Conclusion and recommendation 30

References 31

Appendices 35

IV

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LIST OF ABBREVIATIONS

PCR Polymerase Chain Reaction

JUC flagellar antigen gene

sefA fimbrial protein gene

g gram

Ilm micrometer

III microliter

IlM micromolar

mM milimolar

ml milliliter

U unit

°C degree Celsius

DNA deoxyribonucleic acid

% percent

LB Luria-Bertani

XLD Xylose-Lysine-Deoxycholate

MgCh magnesium chloride

dH20 distilled water

UV ultra violet

v

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• • t I

LIST OF TABLES

Table Description Page

Table 2.1 Salmonella Nomenclature. 4

Table 3.1 List of samples positive for presumptive Salmonella spp. 11

Table 3.2 List ofwater and sediment samples positive for Salmonella spp. 12

Table 3.3 The specific peR amplification conditions of Salmonella spp. 15

Table 3.4 Oligonucleotide sequences of primers used for specific peR reaction. 15

Table 4.1 Observation of bacteria colonies grown on different selective agar. 17

Table 4.2 Appearance of organisms grown on different type of selective agar. 23

Table 4.3 Isolates which were chosen for multiplex peR. 25

VI

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I •1 I

Figure

Figure 3.1

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

,

LIST OF FIGURES

Description Page

Maps showing the sampling sites. 10

Colonies of presumptive Salmonella spp. on XLD agar. 21

Colonies of presumptive Salmonella spp. on Salmonella 21

Chromogenic Agar Base.

Colonies of presumptive Salmonella spp. on Hektoen Enteric 21

Agar.

Gram-stain image of presumptive Salmonella spp. viewed under 24

light microscope

Amplicon obtained by multiplex PCR for detectingfliC and sefA 26

genes analyzed with 1.5% agarose gel electrophoresis.

Amplicon obtained by multiplex PCR for detectingfliC and sefA 26

genes analyzed with 1.5% agarose gel electrophoresis.

Amplicon obtained by multiplex PCR for detectingfliC and sefA 27

genes analyzed with 1.5% agarose gel electrophoresis.

VII

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, .'

Detection of Salmonella enteritidis and Salmonella typhimurium from water and animal samples in Kubah National Park, Kuching, Sarawak

Dellroy Donny

Resource Biotechnology Programme Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Salmonella can cause zoonotic diseases which are transmitted between human and animals. Infection of Salmonella is called salmonellosis and most infected people may develop diarrhea, abdominal cramp and nau ea. The conventional method for detecting Salmonella serovars needed 4 to 7 days for a complete diagnosis. This study is carried out to detect the presence of S. enteritidis and S. typhimurium by using rapid method, the multiplex PCR technique. The samples were collected from anal, cloacal, feces and intestine of different type of animals including bat, squirrel, mongoose and bird in Matang Wildlife Centre and Kubah National Park, Kuching, by using swab method. Moreover, samples from water and sediment from Sungai Rayu, Matang are also taken. The samples were first inoculated into an enrichment media, Luria-Bertani broth and then cultured onto three types of selective agar; Xylose-lysine-Deoxycholate (XLD), Salmonella Chromogenic Agar Base and Hektoen Enteric (HE) agar, for isolation and identification of Salmonella spp. There are 83.3% of the total isolates which were identified as presumptive Salmonella spp. Next, the DNA extraction was carried out using cell boil method. Two primer pairs which target the sefA andjliC gene of S. enteritidis and S. typhimurium respectively, were used in the multiplex PCR. The results showed that all the isolates did not possess the targeted genes, which suggest the presence of other Salmonella serovars.

Key words: S. typhimurium, S. enteritidis, multiplex PCR, Matang Wildlife Centre, Kubah National Park

ABSTRAK

Salmonella boleh menyebabkan penyakit zooonotik yang tersebar di antara manusia dengan haiwan. Jangkitan daripada Salmonella dikenali sebagai salmonellosis dan menyebabkan cirit-birit, kekejangan abdomen dan rasa loya. Kaedah konvensional untuk mengenal pasti serotip Salmonella memerlukan 4 hingga 7 hari untuk mendapatkan diagnosis yang lengkap. Kajian ini dijalankan untuk mengesan kehadiran S. enteritidis dan S. lJphimurium dengan menggunakan kaedah multiplex PCR. Sampel-sampel diambil daripada dubur, kloaka, najis dan usus beberapa jenis haiwan yang berbeza iaitu kelawar, tupai, cerpelai dan burung di Matang Wildlife Centre and Kubah National Park, Kuching dengan menggunakan kaedah swab (mengesat). Selain itu, sampel daripada air dan mendapan dari Sungai Rayu, Matang juga diambil. Sampel-sampel tersebut diinokulasi dalam kaldu LB dan dikultur ke atas 3 jenis agar berpilih; iaitu XLD, Salmonella Chromogenic Agar Base dan HE agar untuk pengasingan dan identijikasi spesis Salmonella. Terdapat 83.3% isolasi yang dikenal pasti mempunyai kemungkinan sebagai spesis Salmonella. Pengekstrakan DNA dibuat dengan menggunakan kaedah pendidihan sel. Dua set primer yang mensasarkan gen sefA dan jliC, masing- masing daripada S. enteritidis dan S. typhimurium digunakan dalam multiplex PCR. Hasil eksperiment menunjukkan tiada isolasi yang memiliki gen yang disasarkan, menandakan kemungkinan kehadiran serotip Salmonella yang lain.

Kalil kunci: S. tyuhimurium, S. enteritidis, multiplex PCR, Matang Wildlife Centre, Kubah National Park

VIII

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1.0 INTRODUCTION

1.1 Background

Bacteria of the genus Salmonella are gram-negative, rod-shaped, and non-spore-forming.

Salmonella species are also capable to grow either with the presence or absence of oxygen.

All members of the genus are motile with the diameters about 0.7 to 1.5 J,lm, 2 11m to 5 11m

in length and can grow between 7°C to 45°C. According to Hendriksen (2003), the

metabolic characteristics of Salmonella are hydrogen sulfide producing, urea negative,

oxidase negative, indole and Voges Proskauer (VP) negative, catalase positive, Simmon

citrate and methyl red positive, and also glucose fermentation with the production of acid

and usually gas (except for Salmonella typhi).

Salmonella can cause a widespread range of diseases called salmonellosis. Salmonella

enteritidis and Salmonella typhimurium have been reported as the major contribution to

human salmonellosis (Fries et al., 2003). Infection of Salmonella are zoonotic that may

occurs to human, birds, reptiles and can be found in cold and warm blooded animals, as

well as in the environment (Mirmomeni et al., 2009). Salmonellosis transferred via

infected food and water as well as through the fecal-oral route. Chen et al. (2010) stated

that the symptoms of salmonellosis are diarrhea, vomiting, mild fever, nausea, stomach

pain and headache. The sources of infection are usually from undercooked meat, poultry,

infected eggs, and unpasteurized milk products. Insects or birds may also cause Salmonella

contamination to foods. Studies of Salmonella from environmental samples indicate that

water is an important source of contamination, particularly irrigation water containing

manure, wildlife feces or sewage effluents (Castro-Rosas et al., 2011).

1

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" ,fl'

According to Pui et al. (2011), salmonellosis continues to be the main public health

problem globally and gives negative influence on the economic as the cost of surveillance

investigation, treatment and prevention of illness increases. In the United States, it is

estimated that 1.2 million non-typhoidal Salmonella infections occur annually, resulting in

19 336 hospitalizations and 378 deaths, with the medical expenses and loss of productivity

cost between $0.5 and $2.3 billion dollars (Franklin et al., 2011). Research done by

Modarressi and Thong (2010) showed that in Malaysia, the most common non-typhoidal

Salmonella reported for the period of 2003-2005 by National Public Health Laboratory are

S. enteritidis (28.1 %), S. weltevreden (25.7%), s. corvallis (10.3%) and S. typhimurium

(6.7%). Moreover, in 2006, the incidence rate of typhoid and paratyphoid in Malaysia is

0.77 per 100,000 populations (Hamdan et al., 2008).

Established conventional methods used to detect Salmonella such as the laborious

bacteriological and serological identification needed at least 4 to 7 days for positive results

(Akiba et al., 2011; Pathmanathan et al., 2004). Therefore, alternative rapid methods are

required for diagnostic treatment. Currently, rapid method that utilize technologies such as

immunomagnetic separation, EIA- and ELISA-based assay incorporating fluorescent or

colorimetric detection and molecular techniques such as PCR-based assays and DNA

hybridization has been developed. PCR test is now widely applied as it can be used to

distinguish between bacterial strains.

Salehi et al. (2011) stated that in order to colonize a host cell, bacterial pathogen uses

adhesive appendages called fimbriae which bind glycoprotein or glycolipid receptors on

epithelial cells. Bacterial adherence is generally believed to be a prerequisite for infection

and have been associated with Salmonella pathogenicity. According to Mir et al. (2010),

the virulence of Salmonella depends on varies factors,encoded by genes which lead to

their invasiveness, colonization, intracellular survival and damage towards the host tissues.

2

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I I.' " .

1.2 Research Problems

This study was intended to investigate the prevalence of S. enteritidis and S. typhimurium

in animals and water bodies at three sampling sites; Kubah National Park, Matang Wildlife

Centre and Sungai Rayu, Matang. The selected areas are places for recreational activities,

thus this research also concerned with the spreading of zoonotic disease with the risk of

transmitting the organism and disease to human (workers and visitors at the recreational

park). On the other hand, since wild birds and bats have the ability to fly freely to any

places, they can easily transmit the pathogenic bacteria to human.

1.3 Objectives

This study was undertaken with the following objectives:

1. To determine the occurrence of S. enteritidis and S. typhimurium using 3 types of

selective agar which are XLD, HE agar and Salmonella Chromogenic Agar Base,

from samples collected in Kubah National Park, Matang Wildlife Centre and

Sungai Rayu, Matang.

2. To screen and examine the pathogenic S. enteritidis and S. typhimurium from the

animal and water samples by using multiplex PCR technique.

3

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I "'

2.0 LITERATURE REVIEW

2.1 Nomenclature / Taxonomy of Salmonella

The genus of Salmonella is a member of Enterobacteriaceae and consists of only two

species, Salmonella enterica and Salmonella bongori (Kim et ai., 2006). S. enterica

subspecies include enterica, salamae, arizonae, diarizonae, houtenae and indica. This

genus also further divided into approximately 2,500 serovars which distinguished by their

somatic (0) and flagellar (H) antigens.

According to Centres for Disease Control and Prevention (CDC) (2008),

Salmonella serotype is based on the immu_ne reactions with two surface structures; 0 and

H antigens. 0 antigen is a carbohydrate antigen and is the outermost component of

lipopolysaccharide. Meanwhile, H antigen is a protein antigen called flagellin which is

present in the flagella. Table 2.1 shows the nomenclature for Salmonella spp. while

appendix 6 shows their scientific classification.

Table 2.1 Salmonella Nomenclature (adopted from Su & Chiu, 2007)

Taxonomic Position (writing format) and nomenclature No. of Genus Species Subspecies Serotypes serotypes

(capitalized, (italic) (italic) (or serovars) in each italic) (capitalized, not species or

italic)* subspecies Salmonella enterica

bongori

enterica (or subspecies I)

salamae (or subspecies II) arizonae (or subspecies IlIa) diarizonae (or subspecies IIIb) houtenae (or subspecies IV) indica (or subspecies VI) subspecies V

Enteritidis, Paratyphi, Typhimurium 9,46:z:z39 43 :z29:­6,7:I,v:l,5,7 21 :m,t:­59:z36:­13,22:z39

1504

502 95

333 72 13 22

*: Some selected serotypes (serovars) are listed as examples.

Subspecies I is present in both warm and cold blooded animals while other

subspecies are generally associated with cold-blooded animals.

4

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Pusat Khidmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

Most serotypes belong to the species S. enterica ssp. enterica. Some of the

serotypes belonging to this subspecies are described and named by the location of an

outbreak (e.g. S. enterica subspecies enterica serotype Montevideo, referred to as S.

Montevideo), while others are identified by their antigenic formula (Fries et al., 2003).

i.e subspecies 0 antigens: Phase 1 H antigen(s): Phase 2 H antigen(s)

e.g I 4,5,12:i:2

The official name for an isolate with the antigenic structure 4,5, 12:i:2 is Salmonella

enterica subspecies enterica serotype Typhimurium. However, this isolate is normally

called Salmonella Typhimurium (CDC, 2008).

2.2 Salmonella infection outbreaks

According to the Centres for Disease Control and Prevention (CDC) (2011), a total of 241

individuals were infected with the outbreaks strain of S. typhimurium since 1 st April 2009

until 18th July 2011 in United States. The infections are associated with African dwarf

flogs and water from their habitat. Besides that, the outbreak strain of S. enteritidis has also

been reported which infected 42 persons around 20th August 2011 until 27th September

2011. The infections are linked to Turkish pine nuts (CDC, 2011). CDC also reported that

a total of 19 persons range in age from 1 year to 79 years old, were infected with the

outbreak of a multi-drug resistant strain of S. typhimurium from 7 states in United States,

which began on or after October 8, 2011 until December 2011. The illness was related to

the consumption of ground beef.

5

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Research done by Anita et al. (2009) reported that the typhoid outbreak in Sungai Congkak

Recreational Park, Selangor was resulted from contaminated river water due to the poor

sanitation. The river water was contaminated with sewage disposal from two public toilets

which effluent grew Salmonella spp. The outbreaks of typhoid fever in Malaysia are found

to be sporadic which confined to the areas with inadequate safe water supply and

sanitation, food handling and personal hygiene practices (Anita et al., 2009).

2.3 Multiplex PCR

Polymerase chain reaction (PCR) is a powerful tool in in vitro amplification of specific

DNA sequence which will generate minions of target sequence copies (Amini et al., 20 10).

Multiplex PCR is a technique which able to amplify multiple targets within a single PCR

mixture and produce outcomes of varies sizes. This technique contributes to the need for

rapid species identification and detection thus will save time and cost of sample processing

(Salem et al., 2010). In order for the PCR to work properly, the reaction conditions

including the type and concentration of enzyme, reaction buffer content, time and

temperature of the annealing/extension process must be optimized (Liang et al., 2011).

2.4 Multiplex PCR for detection of Salmonella typhimurium

Research has been done by Lim et al. (2003) on selective detection of S. typhimurium by

using multiplex PCR, which targeted on abequose synthase (rjbJ) gene, flagellar H: i (fIiC)

antigen gene and flagellar H: 1,2 (fIjB) antigen gene. In this study, DNAs from S.

typhimunum, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens were

used for evaluation of the targeted genes. Multiplex PCR proved to be capable of

6

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specifically identifying S. typhimurium and discriminate it from other Salmonella serovars

and also non-Salmonella enteric pathogens. The results of this experiment showed that

none of the other Salmonella serovars and non-Salmonella enteric pathogens gave positive

results for all of the three amplified products which derived from the rib}, fiiC, and fijB

genes, at the same time.

In addition, Kim et al. (2006) has carried out a research for identifying the common

clinical serotype of Salmonella enterica subspecies enterica by using multiplex PCR-based

method. Some of the serotypes gave unique amplification patterns when compared to each

other.

2.S Multiplex peR for detection of Salmonella enteritidis

Salehi et al. (2011) stated that bacterial pathogen uses adhesive appendages called fimbriae

which bind glycoprotein or glycolipid receptors on epithelial cells in order to colonize a

host cell. They are one of the significant characteristic which involves in the survival and

persistence in the host. Thus a research has been done to detect the sefl4, sefl 7 and sef21

fimbrial virulence genes of S. enteritidis by using multiplex PCR. The sefl4 fimbriae were

shown to be the T-cell immunogen which allows the adherence to mouse epithelial cells.

Moreover, sefl7 fimbriae are a thin aggregative fimbriae encoded by the agf operon.

Meanwhile, sef21 are nearly similar to the type 1 fimbrin of S. enteritidis and consists of

21 kDa fimbrin monomers. The samples of diarrheic feces were collected from 30 hens

and 30 others from livestock in different regions in Iran. The results showed that 45

samples which were positive for S. enteritidis presented identical bands which indicated

sefl4, sef17 and sef21 (Salehi et al., 2011).

7

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Moreover, Yasmin et ai. (2007) performed a study on the simultaneous multiplex peR for

detection of E. coli, L. monocytogenes and S. enteritidis in shrimp samples which targeted

on their specific DNA sequence. Furthermore, multiplex peR carried out on the

commercially imported shrimp samples showed that none of them contain any of the three

pathogens. This outcome suggests that the multiplex peR is a reliable and beneficial for

rapid detection of bacterial pathogens thus will save time and increase ability to assure

food safety (Yasmin et ai., 2007).

8

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3.0 MATERIALS AND METHODS

3.1 Sample coUection

Bat, squirrel, treeshrew, mongoose, bird, water and sediment samples were collected from

Kubah National Park, Matang Wildlife Centre and Sungai Rayu, Matang on 3 June 2011

by a team of UNIMAS postgraduate students. Kubah National Park is located about 20 km

west of Kuching and covers an area of 2, 230 hectares, and comprises of heavily forested

slopes and sandstone ridges. The park is consisting mostly of palms vegetation and is home

to a variety of wildlife. Meanwhile, Matang Wildlife Centre is situated at the western

comer of the Kubah National Park and covers around 180 hectares of lowland forest. This

centre is Sarawak's main centre of rehabilitation for endangered wildlife. Sungai Rayu is

located nearby the Kubah National Park. The land is not cultivated and most of the natural

vegetation is still intact. The landscape is mostly covered with rain-fed croplands. The map

showing the location of each sampling sites is shown in Figure 3.1.

9

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Figure 3.1 Maps showing the sampling sites at Kubah National Park (A), Matang Wildlife Centre (8) and

Sungai Rayu (C) (Source: Goog\e Earth, 2012).

A total of 67 samples which consist of 52 anal swabs, 9 cloacal swabs, 5 fresh feces

and I small intestine samples were obtained from different species of bat, squirrel,

mongoose, and bird collected from Matang Wildlife Centre and Kubah National (Table

3.l). The samples were enriched into 2 ml Luria-Bertani (LB) broth at 37°C for 18-24

hours. The samples were subcultured on Xylose-Lysine-Deoxycholate (XLD) agar for the

isolation of Salmonella spp. Sixteen samples of anal swabs, three samples of cloacal and

two samples of feces were positive for the presence of presumptive Salmonella spp.

Collections of water and sediment samples were conducted at Sungai Rayu,

Matang. During the first sampling, four samples of water _and sediment were coUected and

another three samples of water and sediment were collected from the second sampling. A

10

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11.

12.

13.

14.

15.

16.

series of dilution was conducted for the sediment samples. All the samples were

subcultured on XLD agar for the isolation of Salmonella spp. Three samples of sediment

were positive for the presence of Salmonella spp. while there was none from water

samples.

Table 3.1 List of samples positive for presumptive Salmonella spp.

Sample FieldAnimal Common Name Scientific name

ty~e number Short-nosed fruit

1. Bat TK172776 Cynopterus brachyotis Anal bat

Short-nosed fruit 2. Bat TK172777 Cynopterus brachyotis

bat

3. Bat TK172779 Papillose wolly bat Kerivoula papil/osa

4. Treeshrew TK172784 Large treeshrew Tupaia tana

5. Bat TK172786 Fawn roundleafbat Hipposideros cervinus

Spotted-winged6. Bat TK172794 Balionycteris maculata

fruit bat

7. Bat TK172797 Dusky fruit bat Penthetor lucasii

Short-nosed fruit 8. Bat TK172799 Cynopyterus brachyotis bat

Spotted-wingedBat TK172801 Balionycteris maculata

fruit bat Short-nosed fruit

Bat TK172802 Cynopyterus brachyotis bat

Spotted-wingedBat TK172809 Balionycteris maculata

fruit bat

Bat TK172815 Dusky fruit bat Penthetor lucasii

Spotted-wingedBat TK172824 Balionycteris maculata

fruit bat Short-nosed fruit

Bat Rl Cynopterus brachyotis . bat

Bat R2 Fawn roundleafbat Hipposideros cervinus

Short-tailedMongoose Ml Herpestes brachyurus

Mongoose

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Table 3.1 List of samples positive for presumptive Salmonella spp.

Yellow rumped 17. Bird MTOOI Prionochilus xanthopygius

flowerpecker Red-tailed tailor

18. Cloacal Bird KN4 Orthotomus sericeus bird

Yellow bellied 19. Bird KNP7 A lophoixus phaeocephalus

bulbul TK172776 Short-nosed fruit

20. Bat Cynopterus brachyotis (F) bat

Feces TKl72786

21. Bat Fawn roundleafbat Hipposideros cervinus (Fl

Table 3.2 List of water and sediment samples positive for Salmonella spp.

Sampling no. Sample code Presumptive Salmonella spp. CSl-S 1

1st sampling UCSI-SI

2nd sampling UCS2-W

3.2 Isolation and identification of presumptive Salmonella species

3.2.1 Enrichment of bacterial samples

Enrichment of all bacterial samples was done using Luria Bertani broth (Laboratorios

Conda, Spain). Five hundred microliter of the sample was inoculated into Bijoux bottle

containing 3 ml of LB broth and incubated (SHEL LAB, USA) at 37°C, overnight.

3.2.2 Isolation of Salmonella species on different type of selective agar

A loop full of sample was streaked onto XLD agar, Hektoen Enteric Agar, and Salmonella

for overnight. The bacterial growth on the plates were observed and recorded.

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3.2.3 Preparation of stock and working culture

Colonies of presumptive Salmonella spp. from XLD agar were picked and cultured onto

nutrient agar (NA) (Bendosen Laboratory Chemical, Norway) slant. The cultures were

prepared in duplicate for stock and working cultures.

3.2.4 Gram staining

Gram staining was conducted according to the protocol designed Rollins and Joseph

(2000). A loopful of distilled water was placed on the slide. Then, a loop of colony sample

was transferred to the water drop and emulsified. The film was allowed to dry then passed

briefly through the Bunsen flame. Next, the slide was covered with a few drops of crystal

violet staining solution for one minute and washed briefly with tap water. The slide was

then treated with a few drops of Gram's Iodine and allowed to act for one minute.

Subsequently, the slide was washed by using tap water and then decolorized in absolute

ethyl alcohol. Finally, the smear was treated with a few drops of safranin solution and

allowed to counterstain for 30 seconds, then washed off with tap water and dried blotting

paper. The characteristics of the bacteria then were observed under light microscope.

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3.3 Molecular analysis

3.3.1 DNA extraction

The bacterial DNA extraction was conducted according to the boiling extraction method as

described by Bilung et al. (2005) with minor modification. Salmonella isolates was

cultured in 3 ml LB broth for overnight at 37°C with agitation at 145 rpm (New Brunswick

Scientific Innova 4000). One milliliter of the sample transferred to a 1.5 ml

microcentrifuge tube. The cell suspension was centrifuged (Hettich Zentrifugen, Germany)

at 10 000 rpm for 5 min. The supernatant was discarded carefully. The pellet was then

resuspended in 300 III of distilled water by vortexing (Labnet International, USA). The

microcentrifuge tube was incubated for 20 min at 100°C and immediately chilled on ice for

20 minutes. Next, the tube was centrifuged for 5 min at 13 400 rpm. The supernatant was

carefully transferred to a new micro centrifuge tube and used as the template DNA in the

peR.

3.3.1 Multiplex peR

Multiplex PCR for detection of Salmonella enteritidis and Salmonella typhimurium was

perfonned according to Jamshidi et al. (2009), with some modifications. The PCR was

carried out in a reaction volume of 25 III containing 2.5 III lOx reaction buffer (Invitrogen,

Brazil), 0.5 III Taq Polymerase (2.5 U) (Fermentas, International Inc., Canada), 0.5 IlM of

each primer (10 mM), 1.25 III dNTPs (10 mM) (RBC Bioscience Corp., Taiwan), 1.5 III

MgCh (50 mM) (Invitrogen, Brazil), and 2 III genomic DNA. The samples were subjected

to 3S cycles of amplification using Eppendoft Mastercyc1e® Personal. Table 3.3 and Table

4 shows the specific PCR amplification conditions of Salmonella spp. and the

~ucleotide sequences ofprimers used, respectively.

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