EPIDEMIOLOGY of EMERGING DISEASES and DISORDERS in CAGE CULTURED Pangasius Spp

EPIDEMIOLOGY OF EMERGING DISEASES AND DISORDERS IN CAGE CULTURED Pangasius spp. IN PAHANG, MALAYSIA

NUR SYAKEERA BINTI MAHMUD

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology)

Kulliyyah of Science International Islamic University Malaysia

JULY 2019

ABSTRACT

Pangasius hypophthalmus or famously known by local Malaysians as Patin Hitam is one of the most important sources of food in Malaysia. It is widely cultured in the Peninsular Malaysia especially in Pahang due to the fact that it is a popular consumed freshwater fish. Global economic interest in the fish has increased based on increasing demand in USA and Europe countries. However, high mortality rate of the fish due to bacterial and viral infections gave a negative impact towards the interest which needs to be solved. Therefore, bacteria in P. hypophthalmus in Pahang were being focused including risk factors associated to the prevalence of bacteria and virus in P. hypophthalmus. This research was conducted for two cycles (February – September 2016 and January – August 2017) in several different farms in Temerloh and Pekan, Pahang. Bacteria and virus samples were taken from three organs of P. hypophthalmus; kidney, liver and spleen. Physical parameters for water quality were measured using a multi-parameter probe sensor (YSI, USA) and chemical parameters were analyzed with DR900 colorimeter (Hach, USA). Bacteria samples were identified using biochemical test kits, API 20NE and 20E, followed by confirmation of the bacteria using Polymerase Chain Reaction (PCR). Virus samples were identified using conventional PCR. There were several bacteria isolated throughout the culture period. The highest prevalence of bacteria found was Aeromonas hydrophila (63%) followed by Photobacterium damselae (23%), Plesiomonas shigelloides (7%), Pseudomonas luteola (4%) and Pseudomonas fluorescens (3%). There was no virus isolated from these two sites. The susceptible size of Pangasius spp. that was infested by A. hydrophila was 300 – 400 g. Furthermore, risk factors such as ammonia, nitrite, iron, sulfide, temperature, pH, total suspended solid (TSS), dissolved oxygen (DO) and rainfall rate affected the presence of bacteria were different at all sites. However, only ammonia was present throughout the sampling period at all sites. Sulfide, nitrite, iron and ammonia were the most significant parameters that have strong associations with the presence of A. hydrophila. Significantly, these results could contribute to better treatment of fish disease and development of standard operating procedure of future fish culture for early disease prevention.

خالصة البحث

إن نبات )Pangasius hypophthalmus( املهشور ند اتجممع ب) Patin Hitam( هي واح ة من املصاد الغذائية املوعة يف ماليزيا. وهي تريب بهشكل واس يف ماليزيا وخاصة يف والية باهدج، وهي معروفة وهلا أمهية االقمصادية العاملية ولذلك يزداد الطلب نليوا من الراليات املمح ة األمرييكية وأو وبا. وم ذلك فإن الراقيات العالية ق يسبب االلموابات البكمريية والفريوسية هلاو تأثري سليب نلى الصحة األمر الذي يمطلب البحث .واحلل وبالمايل فإن البكمرييا املرجردة يف Pangasius hypophthalmus يف باهدج جتري د اسموا مبا يف ذلك نرامل اخلطر املرتبطة باالنمهشا البكمريي والفريوسي يف Pangasius hypophtalmus. متت هذه المجربة يف دو تني )فرباير- سبمعرب 6102 ويداير- أغسطس 6102( يف اقفاص مساك خمملفة يف Temerloh و Pekan يف والية باهداج. مت أخذ نيدات ية البكمري والفريوسات من ثالثة انظاء Pangasius hypophthalmus الكلر والكب والطحاي الطحال.مت قياس املعلعات الفيزيائية جلردة املياة باسمخ ام اجوزة اسمهشعا مبسا املعلعة املمع دة ) ,YSI AMERICA( واملعلعات الكيعيائية حتليلوا باسمخ ام ميقاس األلران ,DR900 (HACH (AMERICA. مت حتي العيدات البكمريية باسمخ ام مجعرنات اخميا الكيعياء احليرية API 20NE و 20E وبالمايل المحقق من البكمرييا باسمخ ام تفانل البلعرة املمسلسل Polymerase Chain Reaction (PCR) ومت حت ي نيدات الفريوسات باسمخ ام PCR المقلي ية. هداك بدمرييا معزولة حالل ة فت حت ي العيدات أنلى البكمرييا املرجردة هي (Aeromonas hydrophila (63% تليوا Photobacterium (Pseudomonas luteola (4%) ,Plesiomonas shigelloides (7%) ,damselae (23% و (Pseudomonas fluorescens (3%.مل يمم العثر نلى فريوس من مراق حت ي العيدات. مقاس .Pangasius spp الذي يصاب بسورلة من طريق A. hydrophila هر 400-300 جرام باإلضافة إىل ذلك نرامل اخلطر مثل األمرنيا والديتيات واحل ي والكربيمي ود اجة احلرا ة ود اجة احلعرضة واملراد الصلبة العالقة (TSS) واألكسيجني املذاب (DO) ومعل املطر يؤثر إىل وصرل البكمرييا ختملف يف مجي أماكن اليت حت د العيدات. وم ذلك ترج األمرنيا فقط طرال فتة اخذ وحت ي العيدات يف مجي املراق . تعمرب الكربيمي ات والدتيت واحل ي واألمرنيا من أهم العرامل اليت ترتبط ابمباطا قريا برجرد A. hydrophila بهشكل كبري. ميكن أن تسوم هذهالدمائج يف حتسيس نالج األمراض األمساك وتطرير إجراءات المهشغيل القياسية لهشربية األمساك يف املسمقبل للرقاية من األمراض يف وقت مبكر.

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biotechnology)

………………………………..… Nur Nazifah binti Mansor Supervisor

………………………………..… Ahmed Jalal Khan Chowdhury Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biotechnology)

………………………………..… Mohd Fuad bin Miskon Internal Examiner

………………………………..… Fahrul Zaman bin Huyop External Examiner

This thesis was submitted to the Department of Biotechnology and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology)

………………………………..… Mardiana binti Mohamad Ashaari Head, Department of Biotechnology

This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology)

………………………..………… Shahbudin bin Saad Dean, Kulliyyah of Science

iii

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Nur Syakeera binti Mahmud

Signature ...... Date ......

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

EPIDEMIOLOGY OF EMERGING DISEASES AND DISORDERS IN CAGE CULTURED Pangasius spp. IN PAHANG, MALAYSIA

I declare that the copyright holder of this thesis are jointly owned by the student and IIUM.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Nur Syakeera binti Mahmud

……..…………………….. …………………….. Signature Date

ACKNOWLEDGEMENTS

In the name of Allah, the Most Gracious, the Most Merciful. I would like to express my deepest gratitude to the Almighty Allah. Without the blessing of Him I would not have been able to complete this project. I am also would like to thank to my supervisor Dr. Nur Nazifah Mansor and my co-supervisor Prof. Dr. Ahmed Jalal Khan Chowdhury for their sincere guiding and outstanding supervising. Their wide knowledge, experience and untiring encouragement give me strength along the journey of this research. Special credits to all lab assistants especially to Br. Masrul and Br. Yahya for their kind and sincere assistance and being very supportive throughout this project. I am indebted beyond the words to my entire colleague and friends from Biotechnology department especially Nurul Fatihah, Nur Faiqah, Safwan, Hairi, Nik Nadiah, Suphia Amiera, Nur Amalin Nadia for moral support, professional help and indirect involvement during the hard time have greatly contributed to the completion of this project. I am also would like to give a big appreciation to the National Fish Health Research Division staffs especially to Dr Siti Zahrah, Madam Rimatulhana, Mr. Shahidan, Mr. Fahmi, Ms. Hilma for their guidance and help throughout this study. I wish to express my personal appreciation to my parents, Mahmud bin Makka and Rohani binti Lapo, to my little sister Nur Farha Adila, other siblings, to my husband Mohammad Faiz Hizzuan bin Hanapi and to my daughter Wardah Qaisara for their support, advice and love who always there in my hours of need. Last but not least, thank you very much for those who are directly and indirectly lends a hand in making if this project success. May Allah bless all of you.

TABLE OF CONTENTS

Abstract ...... i Abstract in Arabic ...... ii Approval page ...... iii Declaration ...... iv Copyright ...... v Acknowledgements ...... vi List of Tables ...... ix List of Figures ...... xi List of Abbreviations ...... xiii List of Symbols ...... xv

CHAPTER ONE: INTRODUCTION ...... 1 1.1 Background of the Study ...... 1 1.2 Statement of the Problem...... 2 1.3 Research Objectives...... 4 1.4 Research Questions ...... 4 1.5 Research Hypotheses ...... 4 1.6 Significance of the Study ...... 5 1.7 Limitations of the Study ...... 5

CHAPTER TWO: LITERATURE REVIEW ...... 6 2.1 Pangasius hypophthalmus (Patin Hitam) ...... 6 2.2 Pahang River ...... 8 2.3 Freshwater Fish Diseases ...... 11 2.4 Emerging diseases in Pangasius hypophthalmus ...... 15 2.5 Fish Farming Management ...... 17

CHAPTER THREE: RESEARCH METHODOLOGY ...... 20 3.1 Media Preparation ...... 22 3.2 Sample collection...... 22 3.3 Gram-staining ...... 23 3.4 Biochemical Test ...... 24 3.4.1 Oxidase Test ...... 24 3.4.2 Catalase Test ...... 24 3.5 Bacterial Identification using API System ...... 24 3.5.1 Inoculation...... 25 3.5.2 Profile Calculation and Interpretation ...... 25 3.6 Virus Identification ...... 27 3.6.1 Sample Process ...... 27 3.6.2 DNA Extraction ...... 27 3.6.3 Polymerase Chain Reaction (PCR) ...... 27 3.6.4 Gel Electrophoresis ...... 28 3.6.4.1 Agarose Preparation ...... 28 3.6.4.2 Sample Preparation ...... 29 3.6.4.3 Gel Visualization ...... 29

vii

3.7 Water Quality Observation ...... 29 3.7.1 Physical Water Quality ...... 29 3.7.2 Chemical Water Quality ...... 29 3.8 Statistical Analysis...... 30

CHAPTER FOUR: RESULTS AND DISCUSSION ...... 31 4.1 Clinical Observation ...... 31 4.2 The Prevalence of Bacteria in Pangasius hypophthalmus ...... 36 4.2.1 Bacterial Isolation Pattern ...... 40 4.3 Physicochemical Water Quality ...... 41 4.3.1 Water Quality Parameters between Months ...... 51 4.3.2 Water Quality Parameters between Different Sites ...... 52 4.4 Statistical Analysis...... 53 4.4.1 Principal Component Analysis of Water Quality...... 53 4.4.2 Canonical Correspondence Analysis of Water Quality Parameters ...... 58 4.4.3 Relationship between the Presence of Bacteria and Water Quality ...... 64 4.5 Weight of Pangasius hypophthalmus and Prevalence of Aeromonas hydrophila ...... 72 4.6 Discussion ...... 73

CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS ...... 76 5.1 Conclusion ...... 76 5.2 Recommendations...... 76

REFERENCES ...... 78

APPENDIX I: LOCATION OF PAHANG RIVER BASIN IN MALAYSIA (SRTM, 2008) ...... 87 APPENDIX II: SAMPLING SITES ...... 88 APPENDIX III: INSTRUMENTATION ...... 89 APPENDIX IV: RESULTS OF TEST FROM BACTERIAL IDENTIFICATION USING API 20NE ...... 90 APPENDIX V: VIRUS RESULTS ...... 91 APPENDIX VI: A SAMPLE OF COLONY OBSERVED ...... 92

viii

LIST OF TABLES

‎Table‎2.1 Taxonomy Classfication of P. hypophthalmus 8

Table 2.2 Standard Water Quality Parameters in Malaysia (NWQSM, 1985) 19

‎Table‎3.1 Selection of API Kit 25

‎Table‎3.2 PCR Protocol of CCV 28

‎Table‎3.3 Thermocycling Conditions of PCR 28

‎Table‎4.1 Mean Rate ± SD of Prevalence of Bacteria from P. hypophthalmus in Different Sites 40

‎Table‎4.2 Mean Rate ± SD of Physicochemical Water Quality in Kg. Pangsenam, Temerloh 42

‎Table‎4.3 Mean Rate ± SD of Physicochemical Water Quality in Kg. Pangsenam, Temerloh 43

‎Table‎4.4 Mean Rate ± SD of Physicochemical Water Quality in Kuala Krau, Temerloh 44

‎Table‎4.5 Mean Rate ± SD of Physicochemical Water Quality in Kg. Bintang, Temerloh 45

‎Table‎4.6 Mean Rate ± SD of Physicochemical Water Quality in Kg. Terlang, Pekan 46

‎Table‎4.7 Mean Rate ± SD of Physicochemical Water Quality in Kg. Acheh, Pekan 47

‎Table‎4.8 Mean Rate ± SD of Physicochemical Water Quality in Tg. Pulai, Pekan 48

‎Table‎4.9 Mean Rate ± SD of Physicochemical Water Quality in Tg. Pulai, Pekan 49

‎Table‎4.10 Mean Rate ± SD of Physicochemical Water Quality in Different Sites 50

‎Table‎4.11 Principal Component Loadings from Principal Component Analysis of Water Quality Parameters from Temerloh Site 53

‎Table‎4.12 Principal Component Loadings from Principal Component Analysis of Water Quality Parameters from Temerloh Site 54

‎Table‎4.13 Principal Component Loadings from Principal Component Analysis of Water Quality Parameters from Pekan Site 55

‎Table‎4.14 Principal Component Loadings from Principal Component Analysis of Water Quality Parameters from Pekan Site 56

‎Table‎4.15 Cannonical Correspondence Analysis Summary Statistics for Water Quality Parameters at Temerloh Site 59

‎Table‎4.16 Cannonical Correspondence Analysis Summary Statistics for Water Quality Parameters at Temerloh Site 60

‎Table‎4.17 Cannonical Correspondence Analysis Summary Statistics for Water Quality Parameters at Pekan Site 61

‎Table‎4.18 Cannonical Correspondence Analysis Summary Statistics for Water Quality Parameters at Pekan Site 62

‎

LIST OF FIGURES

‎Figure‎1.1 The Production of Pangasius in Tonnes Per Metric and the Wholesale Value of Pangasius Production 3

‎Figure‎2.1 Pangasius hypophthalmus 7

Figure 2.2 Sampling Sites along Pahang River 9

Figure 2.3 Structure of A. hydrophila 12

‎Figure‎3.1 Sampling sites along Pahang River 21

‎Figure‎3.2 Flowchart of Methodology 22

‎Figure‎3.3 Well of the Strip 26

‎Figure‎3.4 Total Well of the Strip 26

‎Figure‎4.1 Haemorrhages on All Fins 31

‎Figure‎4.2 Haemorrhages on Mouth and Discoloration 32

‎Figure‎4.3 Pale Liver 32

‎Figure‎4.4 White Nodule on Spleen 33

‎Figure‎4.5 Black Nodule on Spleen 33

‎Figure‎4.6 Ascites on Spleen 34

‎Figure‎4.7 White Nodule on Kidney 34

‎Figure‎4.8 Congested Kidney 35

‎Figure‎4.9 Bacterial Isolation Pattern from P. hypophthalmus in Temerloh 38

Figure 4.10 Bacterial Isolation Pattern from P. hypophthalmus in Pekan 39

Figure 4.11 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Kuala Krau, Temerloh 64

‎Figure 4.12 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Kg. Pangsenam, Temerloh in 2016 65

Figure 4.13 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Kg. Bintang, Temerloh 66

Figure 4.14 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Kg. Pangsenam, Temerloh in 2017 67

Figure 4.15 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Kg. Terlang, Pekan 68

Figure 4.16 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Tg. Pulai, Pekan in 2016 69

Figure 4.17 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Kg. Acheh, Pekan 70

Figure 4.18 Cannonical Correspondence Analysis Ordination Diagram Showing the Effect of Water Quality Parameters on the Presence of Bacteria at Tg. Pulai, Pekan in 2017 71

Figure 4.19 Weight of P. Hypophthalmus and the Prevalence of A. Hydrophila 72

xii

LIST OF ABBREVIATIONS

API Analytical Profile Index

BNP Bacillary Necrosis of Pangasius

CCV Channel Catfish Virus cm Centimeter dGH Water Hardness dNTP Deoxynucleotide Triphosphate

DNA Deoxyribonucleic Acid e.g. For Example ft Feet g Gram kb Kilo Base kg Kilogram km Kilometer

L Liter

MAS Motile Aeromonas Septicemia mm Millimeter m Meter

µm Micrometer m3 Cubic Meter mg/l Milligram Per Liter

µl Microliter mM Micromole

MgCl2 Magnesium Chloride mL Milliliter

xiii

mA Milliampere mm/day Millimeter Per Day ppt Part Per Thousand pmol Picomole psi Pressure rpm Revolutions Per Minute

RNA Ribonucleic Acid

RM Ringgit Malaysia

xiv

LIST OF SYMBOLS

°C Degree Celsius

% Percentage

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Fish as a food is consumed by many animals including humans. According to

Mohanty (2015), fish has been an important part of the diet for humans in most countries since the dawn of time since three quarters of Earth are covered by water. It is man’s most vital source of high quality protein, providing almost 16% of the animal protein consumed by the world’s population. Fish is advantageous as food because it is easy to digest, and it contains high nutritional value. However, fish are susceptible to a wide variety of bacterial pathogens, most of which are capable of causing disease

(Akila & Kumaran, 2018).

Fish become contaminated due to their adverse environment. For example, the environment condition of sewage, contaminated water, harvesting area, contamination by workers, utensils, equipment, and unhygienic handling can result in the presence of a great number of bacteria in the fish (Wyatt et al., 1979). Many investigations concerning the bacterial flora of fish have been increasingly carried out. Some of these investigations prove that the flesh and internal organs of healthy fresh fish are microbiological sterile, although some investigations have recorded the presence of bacteria in fish muscles (Cahill, 1990; Sugita et al., 1996). Bacterial disease is responsible of heavy mortality in both wild and cultured fish. Most of the causative bacteria are naturally occurring saprophytes. They are essentially opportunistic pathogenic bacteria which invade the tissue of a fish host rendered and survive for a period of time in the environment (Sugita et al., 1996).

Due to strong market demand, fish is being cultured in farms, also known as aquaculture. One type of fish widely cultured is Pangasius spp. It is commonly called as river or silver stripped catfish, Siamese shark, sutchi catfish, or swai catfish. This fish species live in freshwater and its endemic to the Mekong basin. It is a riverine catfish belonging to the members of the family Pangasiidae. Pangasius species exhibits fast growth when cultured in a good environment. Pangasius is an air- breathing fish that can tolerate low Dissolved Oxygen (DO) of any water body. It can be easily cultured in fish ponds, concrete tanks, fish cages, or fish pens. Common cultured species are Pangasius hypophthalmus, known as Sutchi river catfish, and

Pangasius bocourti, known as Bocourts catfish. The locations of this type of species cultured in Malaysia are in Perak River and Pahang River. Fishes that achieved 0.8 –

1.2 kg can be sold to the buyer as RM17 per kg (Bernama, 2019).

1.2 STATEMENT OF THE PROBLEM

In aquaculture, disease problem is a significant factor that affects the aquaculture system that plays a role to meet fish protein demand. Emerging diseases affect both marine and freshwater aquaculture. In Malaysia, catfish are the largest freshwater aquaculture species being cultured, including Pangasius spp., Clarias spp. and Mystus numerus (FAO, 2009). Pangasius spp. is one of the largest and most important inland fisheries in the world, and Vietnam produced more than 1.1 million tonnes in 2008.

Other producers are Thailand, Cambodia, Laos People's Democratic Republic,

Myanmar, Malaysia, Bangladesh, and China (FAO, 2009). The production of

Pangasius spp. in Malaysia showed increase from 2,990.05 tonnes in 2010 to 6,400.89 tonnes in 2017 (Figure 1.1). Other than that, the wholesale value market of Pangasius increased from RM32,872.87 in 2010 to RM64,653.75 in 2017.

7,000.00 100,000.00 90,000.00 6,000.00

80,000.00

5,000.00 70,000.00

4,000.00 60,000.00 value value (RM)

50,000.00

production (tonnes) 3,000.00 40,000.00

2,000.00 30,000.00 Pangasius 20,000.00 Pangasius 1,000.00 10,000.00 0.00 0.00 2010 2011 2012 2013 2014 2015 2016 2017 Year Pangasius production (tonnes) Wholesale value of Pangasius production (RM)

Figure 1.1 The Production of Pangasius in Tonnes Per Metric and the Wholesale Value of Pangasius Production (2010-2017) (AFS, 2018)

However, it has been reported that the production of Pangasius spp. was affected with disease problems causing almost 30% mortality, especially in Sungai

Pahang due to multiple infections of bacteria and virus. This disease problem had affected almost 150 farmers culturing Pangasius in Temerloh and Pekan, where the estimated losses are approximately RM2.4 million/year. The reported bacterial disease was Motile Aeromonas Septicemia (MAS) caused by Aeromonas hydrophila while the virus is a Channel Catfish Virus (CCV) caused by herpesvirus. MAS is caused by any of the three species of the genus Aeromonas which are Aeromonas hydrophila, A. caviae and A. sobria. These species are commonly referred to as motile aeromonads

(Hanson et al., 2014). The clinical signs to determine the disease are the presence of hemorrhages on the head, mouth and base of fins; a red, swollen vent and pink to yellow ascitic fluid. Sometimes gas in the gut can be observed too. This infection often occurs on fish when the climate changes from dry to rainy season (Nguyen,

2008). While the clinical signs of infected fish with CCV include distention of the

abdomen, exophthalmia, pale or hemorrhagic gills, and hemorrhagic areas at the bases of fins and throughout the skin-particularly on the ventral surface. Although the liver and kidneys may be pale, a general hyperemia occurs throughout the visceral cavity.

The spleen is generally enlarged and dark red. The stomach and intestine are devoid of food but filled with a mucoid secretion.

1.3 RESEARCH OBJECTIVES

In this “Epidemiology of Emerging Diseases and Disorders in Cage Cultured

Pangasius spp. in Pahang, Malaysia” study, two objectives were set. The two objectives were:

1. To determine the prevalence of bacterial and virus infections of Pangasius

hypophthalmus in Pahang, Malaysia.

2. To investigate the risk factors associated with the infections in Pangasius

hypophthalmus including environment factors.

1.4 RESEARCH QUESTIONS

1. What are the common pathogens that cause disease in Pangasius

hypophthalmus culture?

2. What are the risk factors that enhance the infectious disease in Pangasius

hypophthalmus culture?

1.5 RESEARCH HYPOTHESES

Prevalence of bacterial and virus infection in Pangasius spp. cultured in Pahang River are due to combination factors such as seasons, low water quality, introduction of outside pathogens and poor farm management.

1.6 SIGNIFICANCE OF THE STUDY

This study will contribute to farmers in their farm management system as to educate on the important to know the suitable time to culture Pangasius, determine the disease status of Pangasius in Pahang River which are the famous state culturing Pangasius and give information about the risk factors that may contribute to the infectious disease in Pangasius hypophthalmus.

1.7 LIMITATIONS OF THE STUDY

The study parameters were limited to only nine water quality parameters which were ammonia, nitrite, iron, sulfide, temperature, pH, Dissolved Oxygen (DO), Total

Suspended Solid (TSS) and rainfall rate. The sampling sites also limited to three sampling sites per district which started from the most upstream site in Temerloh to the most downstream site in Pekan.

CHAPTER TWO

LITERATURE REVIEW

2.1 Pangasius hypophthalmus (PATIN HITAM)

Patin is the popularly-consumed freshwater fish in Peninsular Malaysia with global economic interest, increasing demand in the USA and Europe (Haslawati et al., 2014).

Pangasius hypophthalmus, binomial name of P. hypophthalmus species. The local name of Pangasius is Patin meanwhile its common names are sutchi catfish, Siamese shark, iridescent shark and striped catfish which come from the family of Pangasiidae.

This freshwater fish originally comes from an area extending from the Mekong River basin in Vietnam to the Chao Phraya River in Thailand (Rohul Amin et al., 2005), which indicates the tropical climate of the areas. In Malaysia, the most popular place culturing P. hypophthalmus are Temerloh, Pekan and Bera in Pahang, located in

Peninsular Malaysia. According to Nur Farhana (2012), this species prefer water with a 6.5 – 7.5 pH, a water hardness of 2.0 – 29.0 dGH and a temperature range of 22 –

26°C. Pangasius spp. is an air breathing fish thus, they don’t need to have a much higher DO in the water column.

The common names given are based on the physical characteristics of

Pangasius which are striped, shiny and iridescent color. The fins are dark grey or black that looks like a shark. The maximum total length of the fish is 130 cm that is equivalent to 4 ft and the weight can be up to a maximum of 44 kg depending on its size (Roberts & Vidthayanon, 1991). The body is elongated and laterally compressed where upper surface of head is unpolished and has an obtusely rounded snout. Dorsal spine is serrated anteriorly meanwhile pectoral spine is serrated internally which

makes the pectoral spine comparatively stronger than dorsal spine. Its upper jaw is longer than lower jaw and the mouth is moderately gap.

Figure 2.1 Pangasius hypophthalmus

Pangasius is scientifically classified in the Actinopterygii class, Siluriformes order, Pangasiidae family and in Pangasius genus. There are several recognized species in this genus. According to Nelson (1994), there are 21 species of family

Pangasiidae with genus Pangasius. They are Pangasius bocourti, Pangasius conchophilus, Pangasius djambal, Pangasius elongatus, Pangasius humeralis,

Pangasius kinabatanganensis, Pangasius krempfi, Pangasius kunyit, Pangasius lamaudii, Pangasius lithostoma, Pangasius macronema, Pangasius mahakamensis,

Pangasius mekongensis, Pangasius myanmar, Pangasius nasutus, Pangasius nieuwenhuisii, Pangasius pangasius, Pangasius polyuranodon, Pangasius rheophilus,

Pangasius sabahensis and Pangasius sanitwongsei. Two common species which is

widely cultured in Malaysia are P. hypophthalmus and P. nasutus or locally known as

Patin Hitam and Patin Buah.

Table 2.1 Taxonomy Classification of P. hypophthalmus

Taxonomy Classification

Kingdom Animalia

Phylum Chordata

Class Actinopterygii

Order Siluriformes

Family Pangasiidae

Genus Pangasius

Species Pangasius hypophthalmus

Habitats of P. hypophthalmus are freshwater and brackish water such as river, floodplains, estuaries and canals. They usually can be found in lower portions of large rivers and estuaries. (Talwar & Jhingran, 1991). This fish is not only a carnivorous species but also voracious that consumes decaying animal and vegetative matter

(Talwar & Jhingran, 1991).

2.2 PAHANG RIVER

Located in the eastern part of Peninsular Malaysia, Pahang River spans between the latitude of N 2° 48’ 45’’ and N 3° 40’ 24’’ and between longitude of E 101° 16’ 31’’ and E 103° 29’ 34’’ (Figure 2.1). It is 435 km long and stretches from Ulu Tembeling