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ISOLATION OF VIBRIO PARAHAEMOLYTICUS FROM RAW VEGETABLES IN LOCAL MARKETS AT KUCHING AND SAMARAHAN Ling Mee Huong 16562 This project is submitted in partial fulfillment of requirements for degree of Bachelor of Science with Honours in Resource Biotechnology Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK 2009

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Page 1: ISOLATION OF VIBRIO PARAHAEMOLYTICUS … of Vibrio...ISOLATION OF VIBRIO PARAHAEMOLYTICUS FROM RAW VEGETABLES IN LOCAL MARKETS AT KUCHING AND SAMARAHAN Ling Mee Huong 16562 This project

ISOLATION OF VIBRIO PARAHAEMOLYTICUS FROM RAW VEGETABLES

IN LOCAL MARKETS AT KUCHING AND SAMARAHAN

Ling Mee Huong

16562

This project is submitted in partial fulfillment of requirements for degree of

Bachelor of Science with Honours in Resource Biotechnology

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2009

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I

ACKNOWLEDGEMENTS

I would like to express my sincere appreciations and deepest gratitude to my supervisor,

Dr Lesley Maurice Bilung for her continuous guidance and support throughout the final year

project. Special thanks dedicated to all the lecturers for their efforts on laboratory knowledge

and skills. Besides, I would like to thank master students in Microbiology laboratory, Miss

Kho Kai Ling, Miss Chan Yik Ming and Miss Jennifer Goh, as well as master students from

other laboratories for their advice and assistance throughout the laboratory work. Not

forgetting my parents and siblings who gave me great support physically and mentally

throughout the university years. Last but not least thanks to my laboratory mates and course

mates for their supports and knowledge.

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TABLE OF CONTENTS

CONTENTS PAGE NUMBER

ACKNOWLEDGEMENTS I

TABLE OF CONTENTS II

LIST OF ABBREVIATIONS IV

LIST OF TABLES VI

LIST OF FIGURES VIII

ABSTRACT IN ENGLISH AND BAHASA MALAYSIA XI

CHAPTER 1 INTRODUCTION

1.1 Introduction 1

1.2 Objectives 4

CHAPTER 2 LITERATURE REVIEW

2.1 Characteristics of V.parahaemolyticus 5

2.2 Antigenic Characteristics of V.parahaemolyticus 7

2.3 Epidemiology 9

2.4 Prevalence of Vibrio parahaemolyticus in Raw Vegetables 10

2.5 Route of Transmission 12

2.5 Diseases caused and Symptoms 13

2.6 Method of Detection 14

2.7 Treatments and Preventions 15

CHAPTER 3 MATERIALS AND METHODS

3.1 Sample Collection 18

3.2 Enrichment and Detection of V.parahaemolyticus 18

3.3 Isolation and Detection Method 19

3.4 Biochemical /Confirmation tests 19

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CHAPTER 4 RESULTS AND DISCUSSIONS

4.1 Isolation and Detection of Vibrio parahaemolyticus 22

from raw vegetables on Thiosulfate-Citrate-Bile-

Sucrose (TCBS)

4.2 Isolation and Detection of V.parahaemolyticus 28

from Raw Vegetables on CHROMagar Vibrio

4.3 Biochemical Tests for Positive Colonies Isolated

from TCBS and CHROMagar Vibrio

4.3.1 Salt Tolerance Test 33

4.3.2 Triple Sugar Ion Test 34

4.3.3 Gram Staining 35

4.3.4 Citrate Utilization Test 36

4.3.5 Motility Test 38

4.3.6 Oxidase Test 39

4.4 Effects of Different Factors on the Occurrences of

V.parahaemolyticus in Raw Vegetables in Kuching

and Samarahan

4.4.1 Effects of Types of Vegetables on the 42

Occcurrences of V.parahaemolyticus

in Raw Vegetables

4.4.2 Effects of Time or Season on the Occurrences 53

of V.parahaemolyticus in Raw Vegetables

4.4.3 Effects of Different Stalls in the Wet Markets 61

on the Occurrences of V.parahaemolyticus in Raw

Vegetables

CHAPTER 5 CONCLUSION 75

REFERENCES 78

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LIST OF ABBREVIATIONS

AFP American Free Press

AGS Arginine Glucose Slant

APW Alkaline Peptone Water

BAM Bacteriological Analytical Manual

°C Degree Celcius

CDC Centers for Disease Control

CFU Colony Forming Unit

CIFOR Council to Improve Foodborne Outbreak Response

Cm Centimetre

CV CHROMagar Vibrio

DNA Deoxyribose Nucleic Acid

FDA Food and Drug Administration

g Gram

H2S Hydrogen sulfide

HCl Hydrogen chloride

ml Milliliter

mm Milimeter

NaCl Sodium Chloride

PCR Polymerase Chain Reaction

RAPD Random Amplified Polymorphic DNA

SIM Sulfide, Indole, Motility

TCBS Thiosulfate-Citrate-Bile Salt

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TDH Thermostable Direct Hemolysin

TLH Thermolabile Hemolysin

TRH TDH-related Hemolysin

TSI Triple Sugar Ion

T1No Tryptone Broth with no NaCl

T1N1 Tryptone Broth with 1% NaCl

T1N3 Tryptone Broth with 3% NaCl

T1N6 Tryptone Broth with 6% NaCl

T1N8 Tryptone Broth with 8% NaCl

URE Urease

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LIST OF TABLES

Tables Pages

Table 3.1: Types of sample tested for the prevalence of Vibrio 18

parahaemolyticus.

Table 4.1: Number of TCBS-positive samples of raw vegetables obtained 25

from three stalls in Stutong wet markets.

Table 4.2: Number of TCBS-positive samples of raw vegetables obtained 26

from three stalls in 7th

Mile wet markets.

Table 4.3: Number of TCBS-positive samples of raw vegetables obtained 27

from three stalls in Samarindah wet markets.

Table 4.4: Number of CHROMagar Vibrio-positive samples of raw 30

vegetables obtained from three stalls in Stutong wet markets.

Table 4.5: Number of CHROMagar Vibrio-positive samples of raw 31

vegetables obtained from three stalls in 7th

Mile wet markets.

Table 4.6: Number of CHROMagar Vibrio -positive samples of raw 32

vegetables obtained from three stalls in Samarindah wet markets.

Table 4.7: Biochemical tests for Vibrio parahaemolyticus isolated from raw 41

vegetables from the three stalls in Stutong, 7th

mile and

Samarindah wet markets.

Table 4.8: Occurrences of V.parahaemolyticus in raw vegetables based on 43

TCBS to see the effects of types of vegetables.

Table 4.9: Occurrences of V.parahaemolyticus in raw vegetables based on 44

CHROMagar Vibrio to see the effects of types of vegetables.

Table 4.10: Occurrences of Vibrio parahaemolyticus in raw vegetables 54

according to TCBS to see the effect of plating hours on sampling

trips.

Table 4.11: Occurrences of Vibrio parahaemolyticus in raw vegetables 55

according to CHROMagar Vibrio to see the effect of plating

hours on sampling trips.

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Tables Pages

Table 4.12: Occurrences of Vibrio parahaemolyticus isolated from raw 62

vegetables on TCBS to see the effect of different stalls in

different wet markets.

Table 4.13: Occurrences of Vibrio parahaemolyticus isolated from raw 63

vegetables on CHROMagar Vibrio to see the effects of different

stalls in different wet markets.

Table 4.14: Percentage of occurrences of Vibrio parahaemolyticus in raw 64

vegetable isolated from Kuching and Samarahan by using TCBS

based on effect of different wet markets.

Table 4.15: Percentage of occurrences of Vibrio parahaemolyticus in raw 64

vegetables isolated from Kuching and Samarahan by using

CHROMagar Vibrio based on the effect of different wet markets.

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LIST OF FIGURES

Figures Pages

Figure 4.1: Vibrio parahaemolyticus colonies (seen as green colonies) 23

isolated from raw vegetables samples in Kuching and Samarahan

by using TCBS.

Figure 4.2: Purple colonies are identified as positive result for Vibrio 29

parahaemolyticus on CHROMagar Vibrio.

Figure 4.3: Positive result (growth at 3%, 6% and 8% (w/v) of salted broth) 33

for Vibrio parahaemolyticus.

Figure 4.4a (left): Positive result for V.parahaemolyticus on TSI agar. 35

Figure 4.4b (right): Negative result for V.parahaemolyticus on TSI agar. 35

Figure 4.5: V.parahaemolyticus is seen as gram negative, rod shaped cells. 36

Figure 4.6a (left): Negative result for citrate utilization test of 37

V.parahaemolyticus.

Figure 4.6b (right): Positive result for citrate utilization test of 37

V.parahaemolyticus.

Figure 4.7: Circular outgrowth V.parahaemolyticus on SIM agar. 39

Figure 4.8: V.parahaemolyticus is oxidase positive. 40

Figure 4.9: Graph on the occurrences of Vibrio parahaemolyticus in raw 45

vegetables isolated from Kuching and Samarahan by using TCBS

based on effects of different types of vegetables.

Figure 4.10: Occurrences of Vibrio parahaemolyticus in raw vegetables 46

isolated from Kuching and Samarahan by using CHROMagar

Vibrio based on effects of different types of raw vegetables.

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Figures Pages

Figure 4.11: Percentage of occurrences of Vibrio parahaemolyticus in 47

raw vegetables isolated from Kuching and Samarahan by using

TCBS based on effects of different types of vegetables.

Figure 4.12: Percentage of occurrences of Vibrio parahaemolyticus in 48

raw vegetables isolated from Kuching and Samarahan by using

CHROMagar Vibrio based on effects of different types of raw

vegetables.

Figure 4.13: Total percentage of occurrences of Vibrio parahaemolyticus in 50

raw vegetables isolated from Kuching and Samarahan by using

TCBS based on effects of types of vegetables.

Figure 4.14: Total percentage of occurrences of Vibrio parahaemolyticus in 51

raw vegetables isolated from Kuching and Samarahan by using

CHROMagar Vibrio based on effects of different types of raw

vegetables.

Figure 4.15: Occurrences of Vibrio parahaemolyticus in raw vegetables 56

isolated from Kuching and Samarahan by using TCBS based

on effects of sampling trips and plating hours.

Figure 4.16: Occurrences of Vibrio parahaemolyticus in raw vegetables 57

isolated from Kuching and Samarahan by using CHROMagar

Vibrio based on effects of sampling trips and plating hours.

Figure 4.17: Percentage of occurrences of Vibrio parahaemolyticus in 65

raw vegetables isolated from Kuching and Samarahan by using

TCBS based on effect of different stalls and wet markets.

Figure 4.18: Percentage of occurrences of Vibrio parahaemolyticus in 66

raw vegetables isolated from Kuching and Samarahan by using

CHROMagar Vibrio based on effect of different stalls and wet

markets.

Figure 4.19: Percentage of occurrences of Vibrio parahaemolyticus in 68

raw vegetables isolated from Kuching and Samarahan by using

TCBS based on effect of different wet markets.

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Figures Pages

Figure 4.20: Percentage of occurrences of Vibrio parahaemolyticus in raw 69

vegetables isolated from Kuching and Samarahan by using

CHROMagar Virbio based on effect of different wet markets.

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Isolation of Vibrio parahaemolyticus from Raw Vegetables in Local Markets

at Kuching and Samarahan

Ling Mee Huong

Resource Biotechnology Programme

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Currently, Vibrio parahaemolyticus are considered as one of the main cause for bacterial gastroenteristis worldwide. Various

researches had been conducted and stated that the outbreaks involving raw fruits and vegetables occurred besides those of

animal and seafood originated products. This study aims to detect and identify the presence of Vibrio parahaemolyticus in raw

vegetables found in local wet markets, Kuching, Sarawak. In this study, samplings were conducted from three different stalls

found from three different wet markets (Stutong, 7th Mile and Samarindah) respectively. Three types of vegetables were

bought and sampled, namely cucumber, tomato and water spinach. Enrichment was done in alkaline peptone water (APW).

Thiosulfate-citrate-bile salt (TCBS) agar and CHROMagar Vibrio was applied in the methodology in selection of Vibrio

parahaemolyticus.The bacteria appeared as green colonies on TCBS agar and purple on CHROMagar Vibrio. The

presumptive colonies were then be streaked on nutrient agar and later stored as stock culture. Biochemical tests were

performed in order to identify the isolated colonies. Vibrio parahaemolyticus was found in raw vegetables samples obtained

from local wet markets but in little quantity (20 occurrences from 270 total samples or 10.7% isolated from TCBS and 9

occurrences from 270 total samples or 3.3% isolated from CHROMagar Vibrio due to the poor hygiene level in the markets as

well as improper handling of vegetables and cross-contamination with other food sold in the market.

Keywords: Vibrio parahaemolyticus; gastroenteritis; raw vegetables; TCBS agar; CHROMagar Vibrio

ABSTRAK

Vibrio parahaemolyticus merupakan salah satu faktor dalam gastroenteritis bakteria di seluruh dunia kini. Pelbagai aktiviti

kajian telah dijalankan dan menyatakan bahawa pencetusan yang melibatkan pemakanan buah-buahan dan sayur-sayuran

mentah berlaku selain daripada produk haiwan dan makanan laut. Kajian ini bertujuan untuk mengesan dan mengenal pasti

kehadiran Vibrio parahaemolyticus dalam sayur-sayuran mentah yang dijual di pasar tempatan, Kuching, Sarawak. Dalam

kajian ini, pensampelan dijalankan untuk tiga gerai yang berlainan yang masing-masingnya berada dalam tiga pasar

tempatan (Stutong, Batu 7 dan Samarindah). Tiga jenis sayur-sayuran yang dibeli dan disampel, iaitu timun, tomato dan

kangkung. Pengkayaan koloni bakteria dijalankan dalam air pepton beralkali (APW). Agar Thiosulfat-citrat-garam hempedu

(TCBS) dan CHROMagar Vibrio digunakan dalam methodologi untuk pemilihan Vibrio parahaemolyticus. Bakteria tersebut

berwarna hijau atas agar TCBS dan ungu atas CHROMagar Vibrio. Koloni andaian kemudiaannya dikulturkan atas agar

nutrien dan sejurusnya disimpan sebagai kultur simpanan. Ujian biokimia dijalankan untuk mengenal pasti koloni yang

dipilih. Vibrio parahaemolyticus dijumpai dalam sampel sayur-sayuran yang dibeli dari pasar tempatan tetapi dalam kuantiti

yang sedikit (20 kes daripada 270 jumlah sampel ataupun 10.7% pilihan daripada TCBS dan 9 kes daripada 270 jumlah

sampel ataupun 3.3% pilihan daripada CHROMagar Vibrio kerana keadaan yang kurang bersih dalam pasar, pengendalian

sayur-sayuran yang tidak betul dan kontaminasi campuran dengan bahan makanan lain dalam pasar tempatan.

Kata kunci: Vibrio parahaemolyticus; gastroenteritis; sayur-sayuran segar; agar TCBS; CHROMagar Vibrio

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CHAPTER 1

INTRODUCTION

1.1 Introduction

According to Centers for Disease Control and Prevention (CDC), foodborne disease happens

when one or two similar illness occurs after the uptake of a common food. Foodborne illness

can happen when the food is contaminated with pathogenic bacteria due to improper preparing

and storage of food or poor hygiene practice of the food processors that can be harmful upon

consumption (Chang and Chen, 2003). Contaminated or hazardous food has moisture,

temperature, pH and nutrients that promote bacterial growth and multiplication. Handling of

raw food and serving of meals throughout the day, preparation for different diets and delay in

serving can bring problem to food serving venues (Benett et al., 2007). Direct infection or

secondary transmission through contact can lead to the transmission of disease.

V.parahaemolyticus had been recognized as one of the cause for bacterial

gastroenteritis. V.parahaemolyticus is a gram negative cells that require salt to survive (Wong,

2003). This vibrio can lead to foodborne illness such as gastroenteritis and traveler’s diarrhea

in human if raw or undercooked food is consumed or contact with aquatic environment (Khan

et al., 2002). Japan was the first country to be infected by vibrio through food uptake in the

early 1950s (Daniels et al., 2000). Matsumoto et al. (2000) and Wong (2003) mentioned that

illness caused by vibrio had affected North America, India, Southest Asia and Japan.

Pandemic strains of V.parahaemolyticus have been isolated in most diarrhea cases reported in

many Asian countries, for example India, Japan, Bangladesh, Taiwan, Thailand, Vietnam, and

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also in United States (Nandi et al., 1999; Matsumoto et al., 2000; Nishibuchi et al., 2000;

Wong et al., 2000; Bhuiyan et al., 2002). Serovar O3:K6 are mostly found in epidemic isolates,

but the other serovars can also be a part of pandemic strains (Laohapretthisan et al., 2003).

Foodborne illness caused by V.parahaemolyticus is usually sporadic type. The illness

mainly occurs through consumption of raw or improperly cooked seafood, especially during

the warmer seasons (Sakazaki et al., 2006). Clinical symptoms for V.parahaemolyticus

infections are diarrhea, abdominal cramps, vomiting, headache, nausea, fever and chills with

incubation time of 4 to 96 hours (Vuddhakul et al., 2006). Most of the illness happens during

warm season (Daniels et al., 2000). During winter, V.parahaemolyticus will survive in

sediments and hardly be detected (Sakazaki et al., 2006). In unfavorable condition,

V.parahaemolyticus can turn into viable but non-culturable state (Bates et al., 2000).

Bean (1990) and Chang and Chen (2003) stated that fruits and vegetables can act as

vehicles to transmit food borne diseases apart from other food products in Taiwan and

Western countries. According to Linton et al. (2006), many foodborne outbreaks related to

fresh vegetables, fruits and fruit juices had been reported in the past ten years. An example of

the recent outbreak related to vibrio is in Vietnam where there were 130 people infected due to

the contaminated food and water. This had drawn the public attention on the poor hygiene

condition in wet markers and restaurants (AFP, 2008). Another latest outbreak involving

Vibrio parahaemolyticus was reported in Singapore in the April 2009. The outbreaks at ‘Rojak

Geylang Serai’ Stall were believed to be cross contamination of rojak and raw seafood

ingredients that can be contaminated by Vibrio parahaemolyticus. In this outbreak, 154 cases

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were reported where 2 deaths were reported and 84 hospitalizations (Channel NewsAsia,

2009).

This worries local people since vegetables are part of their daily diet. Fresh or

minimally processed food, such as fruits and vegetables, for example, melon, carrots, alfalfa

sprouts and apple ciders have increasing demands among the public. Fresh products have

gained their popularity among consumers as they are convenient and ready to be eaten. These

products are mostly found in wet markets, supermarkets and convenience stores. However, the

degree of hygiene and handling conditions are doubted. The increase of consumption of these

fresh products can result in the elevation of food borne disease outbreaks (Labbe and Shetty,

1998). Berrang et al. (1989) stated that some of the pathogens can grow in refrigerated food

with little or no change in sensory and taste. Therefore, isolation and detection of Vibrio

parahaemolyticus will aid in preventing the spreading of disease outbreaks. The investigation

of foodborne outbreaks can help in determining short term control of the transmission as well

as long term prevention strategies in the near future (CDC, 2005).

In this study, Vibrio parahaemolyticus was isolated and identified by conventional

method. The vibrio was plated on thiosulfate-citrate-bile salts (TCBS) and CHROMagar

Vibrio. The suspected colonies were confirmed with biochemical tests in the laboratories such

as oxidase test, arginine glucose slant, arginine dihydrolase test, salt tolerance test, motility

test, and urease test (FDA, 2004). V.parahaemolyticus form green colonies on TCBS agar

since it cannot ferment sucrose (Sakazaki et al., 2006) and they appear as purple colonies on

CHROMagar Vibrio (Hara-Kudo et al., 2001). CHROMagar Vibrio allows better detection of

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V.parahaemolyticus since it is able to differentiate the species by color and it is more efficient

than classical TCBS agar.

1.2 Objectives

The objectives of this study are:

1. To isolate and detect the presence of Vibrio parahaemolyticus in raw vegetables found

in local market (Kuching and Samarahan) by using selective agar medium namely

TCBS and CHROMagar Vibrio.

2. To identify Vibrio parahaemolyticus isolates from raw vegetables through a series of

conventional methods (biochemical tests).

3. To compare the probability of Vibrio parahaemolyticus occurrence between :

1. Different types of raw vegetables

2. Different time or season

3. Different stalls in the wet markets

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CHAPTER 2

LITERATURE REVIEW

2.1 Characteristics of Vibrio parahaemolyticus

Vibrio parahaemolyticus is one of the 12 Vibrios that are known to cause foodborne disease in

human (Texas Department of Health, 1999). Most of the Vibrio members are gram negative,

with straight rod shape or with single curve (Doyle, 1989). Out of the members, V.cholerae,

V.parahaemolyticus and V.vulnificus are famous as foodborne pathogens (Hartantyo et al.,

2006). V.prarahaemolyticus is found mostly in fresh water during summer. Due to this, the

occurrence happens every year in seasons, mostly during warmer days of the year (Sakazaki et

al., 2006). They mainly live in coastal waters at temperate regions (Baross and Liston, 1973).

According to Cavallo and Stabili (2002), Vibrio parahaemolyticus is usually involved

in foodborne related disease that are linked to uptake of raw or undercooked seafood, poor

post harvest storage conditions or improper handling of food during preparation.

V.parahaemolyticus is an important bacterial pathogens for causing gastroenteritis (Mead et

al., 1999) and also foodborne diseases in some Asian countries (Joseph et al., 1982). However,

the outbreaks reported in Europe are rare (Scientific Committee on Veterinary Measures

Relating to Public Health, 2001). The strains of V.parahaemolyticus can be recognized by

using O:K serotyping scheme (Center for Food Safety and Applied Nutrition, 2005). It is a

useful way to identify the species present and to investigate on its epidemiology.

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V.parahaemolyticus is moderately halophilic, motile, fermentative bacteria

(Kandhasamy et al., 2008). They are non-spore forming rods with 0.5 to 0.8 μM in width and

1.4 to 2.6 μM in length (Drake, 2008). V.parahaemolyticus are motile as most of them have

single polar flagellum when they are grown in medium while peritrichorus flagellum can be

found in young culture on the surface of solid medium (Sakazaki et al., 2006). Vibrio

members are oxidase and catalase positive, but they have the ability to ferment glucose

without production of gas (U.S. Food and Drug Administration, 2004). They are able to

tolerate with salinity of 1 to 8% sodium chloride but they cannot survive without salt

(Sakazaki et al., 2006). They survive the best when the media has around 2-3% NaCl (Doyle,

1989).

V.parahaemolyticus can undergo respiratory and fermentative metabolism. This vibrio

is chemo-organotroph which can grow in medium with D-glucose and NH4Cl. It has the

ability to ferment D-glucose but produce no gas (Doyle, 1989). It retains the ability to

increase its population when the temperature lies between 20°C and 42°C (Miwa et al., 2005).

V.parahaemolyticus survives well at temperature range of 10 to 44°C, but not at 4°C

(Sakazaki et al., 2006). Miwatani and Takeda (1976) and Oliver and Kaper (2001) stated that

V.parahaemolyticus can grow very fast as it has the generation time of 9 minutes in the

medium and 12 minutes in the seafood.

Environmental stresses such as starvation, cold temperature and suboptimal pH can

induce viable but non-culturable state (Gauthier, 2000). The bacteria are still alive as their

metabolisms are still on going but they do not form colonies on nutrient media (Gauthier, 2000;

Oliver, 2000). According to Chai and Jiang (1996) and Wand and Wong (2004),

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V.parahaemolyticus can also enter viable but non-culturable state. They can be revived within

3 days after the temperature are optimal for their growth.

2.2 Antigenic characteristics of Vibrio parahaemolyticus

V.parahaemolyticus assembles three antigens, namely thermostable somatic O antigen,

thermolabile capsular K antigen and flagellar H antigen. All V.parahaemolyticus have the

common H antigen (Drake, 2008). K antigen is a type of polysaccharide with many sugar

components such as pentoses, hexoses or hexosamines. K antigen is free from the bacterial

cell surface when they are exposed to 100°C for 1 to 2 hours (Doyle, 1989). By this, somatic

O antigen is released. O antigen is a type of lipopolysaccharide with glucose, galactose,

glucosamine, heptose, phosphorus, nitrogen compounds and fatty acid ester (Torii et al., 1969).

Therefore, serotyping of V.parahaemolyticus can be conducted by using antibodies specific to

O and K antigens (Drake, 2008). Until today, there are 12 O antigen and over 70 types of K

antigens discovered while many other strains still remain ungrouped (FDA BAM, 2001).

It is believed that some virulence factors take part in the pathogenicity of

V.parahaemolyticus. Some of the examples are hemolysin, adherence factors, enzymes,

products of TDH, TRH and URE genes (Drake, 2008). In the past, the pathogenicity of

V.parahaemolyticus is linked to Kanagawa phenomenon, which is beta-hemolysis on

Wagatsuma agar. Later, it was discovered that Kanagawa reaction is influenced by production

of TDH protein. It was called TDH proteins because inactivation by heat and its hemolytic

activity.

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According to Centre of Disease Control (2005), strains that are pathogenic are those

that produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH).

Although TDH and TRH genes are related to pathogenicity in V.parahaemolyticus, the strains

with these genes are rarely found in aquatic environment (DePaola et al., 2003; Kaufman et al.,

2003). Strains of V.parahaemolyticus from patients with gastroenteritis were hemolytic but

mostly non-hemolytic strains were found on a modified agar for those bacteria isolated from

sea organisms (Kato et al., 1965). Kanagawa reaction can occur when there is hemolytic

reaction due to the presence of thermostable extracellular substance called thermostable direct

hemolysin (TDH). TDH gene is believed to be involved in pathogenicity of the bacteria.

Sakazaki et al. (1986) and Kaper and Nishibuchi (1995) reported most of the strains isolated

from patients infected were Kanagawa positive but only a little is positive for the strains found

in environment. According to Honda et al. (1992), TDH is a pore-forming toxin that

contributes to hemolysis, cytotoxicity, enterotoxicity, and cardiotoxicity (Jong and Young,

2001). TDH is active against erythrocytes of dogs, mice, rats, and human, weak against

erythrocytes of rabbit and sheep and inactive against horse erythrocytes.

Kanagawa negative outbreaks can also happen when TDH-related hemolysin (TRH)

produced by those Kanagawa negative strains (Honda et al., 1988). Shirai et al. (1990) and

Kishishita et al. (1992) discovered that TRH-positive vibrio have the ability to cause

gastroenteritis too. Apart from this, some of the characteristics that participate in the

pathogenicity of V.parahaemolyticus are lipase, gelatinase and hemolysin (enzymatic),

adhensiveness, cytotoxicity and enterotoxicity (biological) and enteropathogenic activities of

the strains (Baffone et al, 2001).

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2.3 Epidemiology

After the first cases concerning V.parahaemolyticus infection reported by Fujino et al. (1953),

there are other reports identified in Japan (Aiiso and Matsuno, 1961; Sakai et al., 1970;

Takikawa, 1958). V.parahaemolyticus contributes to 70% of the foodborne illeness cases

reported in Japan during the early 1960s (Sakai et al., 1970; Sakazaki, 1979). Other countries

affected are such as North America, Central America, South America, Africa, Asia, Europe,

Australia and New Zealand (Doyle, 1989; Texas Department of Health, 1999; Daniels et al.,

2000; Centre for Disease Control, 2005; Chang and Wong, 2005; Drake, 2008). Pandemic

strains of V.parahaemolyticus have been isolated in most diarrhea cases reported in many

Southeast Asian countries, for example India, Japan, Bangladesh, Taiwan, Thailand, Vietnam,

and also in United States (Nandi et al., 1999; Matsumoto et al., 2000; Nishibuchi et al., 2000;

Wong et al., 2000; Bhuiyan et al., 2002). Serovar O3:K6 are mostly found in epidemic isolates,

but the other serovars can also be a part of pandemic strains (Laohapretthisan et al., 2003).

The number of victims can be a single person to a population (Barker, 1974; Okabe,

1974). Barker (1974) mentioned that secondary spread can happen among the family members.

Seasonal trend of the outbreaks can be observed, especially between June and October (Sakai

et al., 1965). Asymptomatic person can also be affected by this vibrio during summer (Sakai et

al., 1965).

V.parahaemolyticus can survive well in aquatic environment due to its ability to

endure halophilic condition. Many reports mentioned that V.parahaemolyticus can be isolated

from seafood such as fish, shellfish and crustacean as well as sea water (Drake, 2008;

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Kandhasamy et al., 2008). V.parahaemolyticus can attach to chitin (a part of exoskeleton in

plankton) through production of chitinase and it can degrade this plankton to recycle the

organic contents (Kaneko and Colwell, 1978; Sakazaki et al., 2006). V.parahaemolyticus can

also be found in digestive tracts of shellfish (Kandhasamy et al., 2008).

2.4 Prevalence of Vibrio parahaemolyticus in Raw Vegetables

Fruits and vegetables constitute an important part in our daily diets. According to Canada

Paediatric Society (2008), there are more people eating fresh fruits and vegetables as these

food products are recommended for healthier diet. These food products can be imported since

the demands for fresh produce increases as a result of increased consumption of fruits and

vegetables. If the food products are imported from countries that do not practice proper and

hygienic methods of production, there is a chance for the occurrences of microbial

contaminations.

Zhang (2005) stated that as there are more people consuming vegetables, foodborne

illness related to fresh products consumption increases as well. Some of the factors

contributing to this scenario are the change in eating habit, the centralization of produce

distribution, rising popularity of ready fruits and vegetables and the rise in produce imports.

CIFOR (2008) stated that some of the reasons for changes in diet are industrial consolidation

and globalization, health concerns, dietary suggestions and culinary trends. The consumption

of food will contribute to foodborne outbreaks if the food is not cooked or handled properly.

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According to Chang and Chen (2003), food borne illness in Taiwan occurs most

frequently at homes and followed by schools, restaurants and working places respectively.

This is because the food products can be obtained easily. Todd (1992) stated that in Canada,

restaurants are the most frequent location for outbreaks, followed by food stands and homes.

From above, it is clearly seen that there is difference among occurrence locations for Asia and

developed Western countries due to different eating habit.

CDC (2005) reported that foodborne disease linked to uptake of fresh fruits and

vegetables make up 12% of the reported cases in United States. Bean (1990) and Chang and

Chen (2003) stated that fruits and vegetables can act as vehicles to transmit food borne

diseases apart from other food products in Taiwan and Western countries. Wong et al. (1999)

and Chang and Chen (2003) revealed that Vibrio parahaemoyticus can be detected from

seafood, meat and meat products, cereal products, egg products, fruits and vegetables, boxed

meals and others.

Fresh or minimally processed food, such as fruits and vegetables, for example, melon,

carrots, alfalfa sprouts and apple ciders have increasing demands among the public. Thus, this

can lead to elevation of food borne disease outbreaks (Labbe and Shetty, 1998). Fresh

products have gained their popularity among consumers as they are convenient and ready to be

eaten. These products are mostly found in supermarket and convenience stores. Temperature is

an important issue in preventing food borne disease outbreaks. Berrang et al. (1989) stated that

some pathogens can grow in refrigerated food with little or no change in sensory and taste.

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Sushi had been famous among the public for its taste and healthfulness. The

ingredients in sushi are vinegar rice, seafood, vegetables, and raw fish. Some of sushi is

prepared with cooked seafood while some are served raw (such as sashimi). Food-borne

illness involves sushi are Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella

species and Listeria monocytogenes (Safe Food News, 2006). Cross contamination can happen

during sushi preparation which uses raw and cooked fish.

2.5 Route of Transmission

The minimum dose for infection by V.parahaemolyticus is around 105 to 10

7 Kanagawa

positive cells after the uptake of contaminated food products. Virulent cell population can be

expanded through genetic transformation of Kanagawa negative cells or host selection and

colonization of Kanagawa positive cells in vivo (Doyle, 1989).

Seafood is always linked to foodborne illness caused by V.parahaemolyticus

(Sakazaki et al., 2006). In Japan, raw fish meat and shellfish are the main source for the

infection as the residents like to eat raw fish and products in their custom. Raw vegetables can

also be a vector to the illness through contamination of kitchen utensils. In European countries,

V.parahaemolyticus seldom occurs because eating raw fish is not famous among the residents

(Sakazaki et al., 2006).

Cases involving foodborne outbreak caused by V.parahaemolyticus are reported

mostly in East European, United States, United Kingdom and also Africa (Sakazaki et al.,

2006). Seafood consumed is cooked for a short period before they are eaten. Crab and shrimp