UNIVERSITI PUTRA MALAYSIA

AETIOPATHOGENICITY OF ULCERATIVE DISEASE IN KOI CARP, CYPRINUS CARPIO LINNAEUS

SUREERAT BUTPROM

FPV 2004 18

AETIOPATHOGENICITY OF ULCERATIVE DISEASE

IN KOI CARP, CYPRINUS CARPIO LINNAEUS

SUREERAT BUTPROM

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2004

AETIOPATHOGENICITY OF ULCERATIVE DISEASE IN KOI CARP, CYPRINUS CARPIO LINNAEUS

By

SUREERAT BUTPROM

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

November 2004

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Dedicated to

My parent: Charat & Varee Butprom

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

AETIOPATHOGENICITY OF ULCERATIVE DISEASE

IN KOI CARP, CYPRINUS CARPIO LINNAEUS

By

SUREERAT BUTPROM

November 2004

Chairman: Associate Professor Dr. Hassan Hj Mohd Daud, Ph.D. Faculty: Veterinary Medicine

A chronic persistent ulcerative lesion of the skin in Koi carp (Cyprinus carprio L.)

was reported in several commercial aquarium shops in Klang Valley. A study was

conducted to chart the epizootiology and pathogenicity of the disease and its

aetiological agents which included histopathological, bacteriological, virological and

experimental infection.

Thirty-six diseased Koi carps with skin ulcer were examined. Findings showed that

the ulcerative lesion development involved the deep muscle layers showing tissue

necrosis and inflammation in chronic lesions. The fish abdomen was filled with a

clear or red-tinged ascitic fluid. The liver and kidneys were pale in colour, swollen

and friable. In the histopathological study, lesions were found in the skin, gill,

kidneys, liver, spleen and intestine. The changes were characterized by diffuse

haemorrhages, cell degeneration and necrosis in the skin, liver and kidneys. Lamellar

epithelial cells showed hyperplasia and hypertrophy at the base of gill lamellae.

Depositions of haemosiderin were seen in kidneys, liver, hepatopancreas and spleen.

In the intestine, haemorrhage in the tunica propria and atrophy of mucosal epithelium

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were seen. Electron microscopy revealed two type of virus like particles which were

associated with the histopathological changes in the kidneys. The virus-like particles

were presumably coronavirus and reovirus based on their morphology.

Morphological and biochemical characteristics of bacteria isolated from the diseased

Koi were determined by routine biochemical tests in combination with the BBL

Crystal KitTM. In the present study, Gram-negative non-lactose fermenting rods were

isolated from the skin lesions and kidneys. In total, 11 bacteria species were

identified and Aeromonas hydrophila was the dominant species isolated from the fish

in this study. The other species of the ulcer-associated bacteria were (i) Shewanella

putrefaciens, (ii) Vibrio cholerae, (iii) Pseudomonas diminuta, (iv)

Chryseobacterium meningosepticum, (v) Empedobacter brevis, (vi) Pseudomonas

aeruginosa, (vii) Pantoea agglomerans, (viii) Enterobacter sakazakii, (viiii)

Morganella morganii and (x) Aeromonas veronii.

Primary cell cultures were initiated from the brain, gonad and kidneys of goldfish

(Carassius auratus) by using trypsinization technique. The cells were cultured in

Leibovitz-15 (L-15) medium supplemented with 10 to 20% fetal calf serum (FCS)

and L-glutamine. The primary cell cultures from brain and gonad tissues were

successfully established and reached monolayer confluence within 15 to 30 days.

The attachment efficiency was serum-dependent though increasing FCS

concentration did not stimulate further growth of cells. Virological examinations of

ulcerated tissues on the primary cell cultures and established cell line, FHM, were

negative for cytopathic effect (CPE) even after three blind passages of 10 days

interval.

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Aeromonas hydrophila, S. putrefaciens and V. cholerae isolated from natural Koi

carp with skin ulcer were used in experimental infection. The fish were injected 0.1

ml bacteria suspension at 1x107 cfu/ml. Bacteria were reisolated from ulcerative

lesion and kidneys and had the same biochemical characteristics as those isolated

from naturally infected fish. Ulcers began to appear three post infection as small and

flat lesions. No apparent mortality was observed in all groups during the 30 days

of the experiment. Histopathological studies revealed that A. hydrophila

individually or in combination with other bacteria could have caused the small

superficial ulcerative lesions. Haemorrhages and inflammation were seen in

spleen, adipose tissue and kidneys. While individually or in combination, injection

of S. putrefaciens and V. cholerae displayed localized lesion which was restricted

only to the injection site.

In conclusion, ulcerative lesion in Koi carp was primarily caused by multiple

infection of several bacteria species, although the possibility of viral

involvement must not be ruled out.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains

ETIOPATOGENISITI PENYAKIT ULSER PADA

IKAN KOI, CYPRINUS CARPIO LINNAEUS

Oleh

SUREERAT BUTPROM

November 2004

Pengerusi: Profesor Madya Dr. Hassan Hj Mohd Daud, Ph.D. Fakulti: Perubatan Veterinar

Penyakit ulser yang kronik pada ikan Koi carp (Cyprinus caprio L.) telah dilaporkan

di beberapa pusat akuarium komersil dan pusat pengumpulan ikan di sekitar Lembah

Klang. Satu penyelidikan telah dijalankan untuk mengetahui agen-agen etiologin

penyakit ini dan pathogenisitinya yang mana merangkumi kajian histopatologi,

bakteriological, virologi dan ujian jangkitan

Sebanyak tiga puluh enam ekor ikan kap Koi yang berpenyakit ulser telah diperiksa.

Hasil pemerhatian telah menunjukkan bahawa pertumbuhan ulser adalah melibatkan

lapisan otot dalam yang menunjukkan nekrosis tisu dan inflamasi bagi lesi-lesi yang

kronik. Bahagian abdomen ikan telah dipenuhi oleh cecair asites yang berwarna

merah atau jernih. Bahagian hati dan ginjal pula bewarna pucat, membengkak dan

mudah hancur. Di dalam kajian histopatologi, lesi-lesi dapat dilihat di bahagian kulit,

insang, ginjal, hati, limpa dan usus. Perubahan-perubahan ini telah dicirikan

mengikut hemoraj resap, degenerasi sel dan nekrosis di dalam kulit, hati dan ginjal.

Sel epithelial lamella telah menunjukkan hyperplasia dan hipertrofi pada bahagian

lamella insang. Deposit haemosiderin telah dilihat di dalam ginjal, hati, pankreas dan

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limpa. Di dalam usus kecil hemoraj di dalam tunika propria dan atrofi mucosa

epithelium juga telah dilihat. Mikroskopi elektron pula telah menunjukkan dua

partikel seperti virus yang mana dikaitkan dengan perubahan-perubahan

histopatologikal di ginjal. Partikel seperti virus ini berkemungkinan adalah

koronavirus atau reovirus berdasarkan ciri-ciri morfologinya.

Ciri-ciri morfologi dan biokimia bagi isolat bakteria daripada ikan kap Koi yang

berpenyakit telah ditentukan dengan cara ujian kimia berserta dengan BBL Crystals

KitTM. Di dalam kajian ini, rod gram negatif fermentasi bukan laktas telah diisolat

daripada lesi di bahagian kulit dan ginjal. Secara keseluruhannya,11 spesis bakteria

telah dikenalpasti di mana Aeromonas hydrophila merupakan spesis yang paling

dominan diisolat bagi keseluruhan persampelan yang telah dibuat dalam kajian ini.

Spesis-spesis bakteria lain yang juga menyebabkan ulser adalah seperti (i)

Shewanella putrefaciens, (ii) Vibrio cholerae, (iii) Pseudomonas diminuta, (iv)

Chryseobacterium meningosepticum, (v) Empedobacter brevis, (vi) Pseudomonas

aeruginosa, (vii) Pantoea agglomerans, (viii) Enterobacter sakazakii, (viiii)

Morganella morganii dan (x) Aeromonas veronii.

Kultur-kultur sel primer telah dihasilkan daripada tisu otak, gonad dan ginjal ikan

emas (Carassius auratus) melalui teknik pentripsinin. Sel-sel ini dikultur di dalam

media L-15 yang tambah dengan 10-20% serum anak lembu (FCS) dan L-glutamine.

Kultur-kultur sel primer daripada tisu otak dan gonad telah berjaya ditumbuhkan

serta mencapai konfluen ekalapis dalam masa 15-30 hari. Keupayaan perlekatan sel

adalah bergantung kepada kandungan serum walaupun peningkatan kepekatan FCS

tidak seterusnya merangsang pertumbuhan sel yang lebih menggalakkan. Kajian

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virus ke atas tisu-tisu ulser ke di dalam kultur sel primer dan sel kekal, FHM pula

adalah negatif bagi kesan sitopatiknya (CPE) walaupun selepas tiga peringkat pasaj

buta yang berselang 10 hari bagi setiap pasaj.

Aeromonas hydrophila, S. putrefaciens dan V. cholerae yang diisolat daripada ikan

kap Koi yang mempunyai ulser telah digunakan di dalam jangkitan eksperimen. Ikan

kap telah disuntik dengan 0.1 ml bakteria pada kepekatan 1x107 cfu/ml. Bakteria ini

boleh diisolat semula daripada lesi ulser dan ginjal dan mempunyai ciri-ciri biokimia

yang sama seperti bakteria yang diisolat daripada ikan yang mempunyai infeksi

semulajadi. Tanda-tanda penyakit mula dilihat tiga hari selepas jangkitan dibuat yang

mana berupa lesi ulseratif yang kecil dan rata. Tiada sebarang kematian diperhatikan

bagi semua kumpulan selama 30 hari ujikaji dijalankan. Histopatologi telah

menunjukkan, sama ada secara individu atau kombinasi dengan bakteria lain, A.

hydrophila boleh menyebabkan lesi permukaan berulser yang kecil. Hemoraj dan

inflamasi telah dikesan pada bahagian limpa, tisu adipos dan ginjal.

Walaubagaimanapun, sama ada secara individu atau kombinasi, suntikan S.

putrefaciens dan V. cholerae telah menunjukkan lesi setempat di mana ianya hanya

terdapat di bahagian yang disuntik sahaja.

Pada kesimpulanya penyakil ulseratif pada ikan kap Koi pada asasnya disebabkan

oleh jangkitan majmuk beberapa spesis bakteria, tetapi kemungkinan penglibatan

virus tidak boleh ditolak.

ACKNOWLEDGMENTS

I would like to express my deep sense of gratitude to my main supervisor, Associate

Professor Dr. Hassan Hj Mohd Daud, for his invaluable guidance, kindness and

constructive suggestions throughout the thesis research. I sincerely appreciate the

innumerable hours he spent reading the draft and the suggestions to improve the

thesis. Gratitude is also expressed to my co-supervisors Associate Professor Dr.

Noordin Mohamed Mustapha and Dr. Zunita Zakaria, for their valuable suggestions,

opinions and kind assistance throughout this study.

My sincere thanks to the SEAMEO Regional Center for Graduate Study and

Research in Agriculture (SEAMEO SEARCA) for supporting me to pursue my

Master degree study at Universiti Putra Malaysia. My sincere gratitude is also

extended to my home university “Ubonratchathani Rajabhat University” and my

faculty “Agricultural Technology”, Thailand for granting me study leave.

Appreciations are also extended to all members of the Faculty of Veterinary

Medicine for providing the research facilities and technical assistance during my

graduate study. I could not have achieved success or finished my work without them.

I must also thank to Mr. Ho Oi Kuan, Ms Azilah Jalil and all staff in the Electron

Microscopy Unit, UPM. Their assistance facilitated my electron microscopic work

successfully.

I am indebted to all my friends, especially Ms. Chua Mei Ling for her kind help, Dr.

Lee Kok Leong, Dr. Sanjoy Banarjee, Ms. Fennie Fong, Dr. Najiah Musa, Ms.

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Noraini Abd Said, Dr. Ng Chi Foon, Ms. Benchamaporn Pimpa, Mr. Pitoon

Nopnakorn, Mr. Anut Chatiratikul, Mr. Boodee Khamseekieow, Ms. Narumon

Sumalee, Ms. Wanna Ammawath and Ms. Apinya Vanichpun for their marvelous

friendships and especially Ms. Doothsadee Luelon for her inspiration thoughout the

period of my study.

My deepest gratitude to my parents, Charat and Wannee Butprom for their

understanding, love, affections and moral support during the study. My great and

best family members, thank you all for the encouragement and concerns. I also owe

my special thanks to my best sister Dr. Phakamat Chirathipnichakul for her spiritual

support throughout the duration of my study.

I certify that an Examination Committee met on 15 September 2004 to conduct the final examination of Sureerat Butprom on her Master of Science thesis entitled “Aetiopathogenicity of Ulcerative Disease in Koi Carp, Cyprinus carpio Linnaeus” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Jasni Sabri, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Abdul Rani Bahaman, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Mohamed Shariff Mohamed Din, Ph.D. Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Chalor Limsuwan, Ph.D. Assistant Associate Professor Faculty of Fisheries Kasetsart University, Thailand (Independent Examiner)

GULAM RUSUL RAHMAT ALI, Ph.D. Professor /Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Hassan Hj Mohd Daud, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Noordin Mohamed Mustapha, Ph.D. Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Zunita Zakaria, Ph.D. Faculty of Veterinary Medicine Universiti Putra Malaysia (Member)

AINI IDERIS, Ph.D. Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been dully acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

SUREERAT BUTPROM Date:

TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATIONS xxi CHAPTER 1 INTRODUCTION 1.1 2 LITERATURE REVIEW 2.1

2.1 Koi Fish 2.1 2.2 Characteristics of Ulcerative Disease 2.2 2.3 Characteristics of Known Ulcerative Disease in Fish 2.4

2.3.1 Epizootic ulcerative syndrome (EUS) 2.4 2.3.2 Goldfish ulcer disease 2.6 2.3.3 Carp erythrodermatitis 2.7 2.3.4 Motile aeromonad septicaemia 2.8

2.4 Pathogens Associated and Related with Ulcerative 2.10 Disease in Koi 2.5 Laboratory Procedures for Isolation and Identification 2.14 of Bacterial Agents 2.6 Laboratory Procedures for Isolation and Identification 2.17 of Viral Agent

3 ISOLATION AND IDENTIFICATION OF BACTERIA 3.1 ASSOCIATED WITH ULCERATIVE DISEASE

3.1 Introduction 3.1 3.2 Materials and Methods 3.2

3.2.1 Fish sampling procedure 3.2 3.2.2 Sources of bacterial isolates 3.3 3.2.3 Colonies morphology and biochemical test 3.4 3.2.4 Maintenance of bacterial isolates 3.4 3.2.5 BBL Crystal KitTM 3.4

3.3 Results 3.5 3.4 Discussion 3.14

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4 DETECTION AND ISOLATION OF THE VIRUS ASSOCIATED WITH ULCERATIVE DISEASE USING FISH CELL CULTURE 4.1

4.1 Introduction 4.1 4.2 Materials and Methods 4.2

4.2.1 Established cell line culture 4.2 4.2.2 Preparation of establishing primary cell culture 4.2 4.2.3 Virus isolation 4.4 4.2.4 Detection of viral replication in cell cultures 4.5

4.3 Results 4.5 4.4 Discussion 4.10

5 LIGHT AND ELECTRON MICROSCOPIC EXAMINATION

OF ULCERATIVE DISEASE IN KOI CARP 5.1 5.1 Introduction 5.1 5.2 Materials and Methods 5.2

5.2.1 Gross examination 5.2 5.2.2 Histopathological examination 5.2 5.2.3 Scanning electron microscopy 5.3 5.2.4 Transmission electron microscopy examination 5.3

5.3 Results 5.4 5.3.1 Gross examination 5.4 5.3.2 Histopathological examination 5.10

5.4 Discussion 5.24

6 EXPERIMENTAL INFECTION OF ULCERATIVE DISEASE 6.1 6.1 Introduction 6.1 6.2 Materials and Methods 6.2

6.2.1 Experimental design 6.2 6.2.2 Bacterial preparation 6.3 6.2.3 Experimental fish 6.3 6.2.4 Histopathological examinations 6.4

6.3 Results 6.4 6.3.1 Bacterial isolation 6.4 6.3.2 Physiochemical properties of water 6.5 6.3.3 Clinical signs and pathological changes 6.5

6.4 Discussion 6.15

7 GENERAL DISCUSSION AND CONCLUSION 7.1 REFERENCES R.1 APPENDICES A.1 BIODATA OF THE AUTHUR B.1

LIST OF TABLES

Table Page

1 Koi samples collected from commercial farms and aquarium shops 3.3 in the Federal Territory and Selangor between February to August 2003.

2 Grouping of bacteria isolated following protocols as described by 3.10 Jang et al. (1990) and BBL Crystal KitTM 3 Biochemical reactions of isolated bacteria by using conventional 3.11

test and BBL Crystal KitTM 4 Bacteria isolated from ulcerative lesion of the skin and kidney 3.13

of 36 diseased Koi carps

5 Number of ulcerative lesions on the body surface of affected fish 5.5 6 Number of bacterial isolates from experimental infection in Koi carps 6.5

7 Enumeration of water samples collected at day 1, 15 and 30 from 6.7

the experimental tank.

LIST OF FIGURES

Figure Page

1 Phase contrast micrograph of the fibroblast-like morphology 4.7 of goldfish brain cells forming a matrix-like network after 15 days of culture. (scale bar = 20 μm)

2 Stained cell monolayer preparation of brain cells showing 4.7 fibroblast morphology after 15 days of culture. May-Grunwald/Giemsa. (scale bar = 20 μm) 3 Phase contrast micrograph of the fusiform fibroblastic cells 4.8

with filiform processes from the goldfish brain cells after 15 days of culture. (scale bar = 15 μm)

4 Stained cell monolayer preparation of gonad cells showing 4.8

epithelial morphology after 15 days of culture. May-Grunwald/Giemsa. (scale bar = 15 μm)

5 Phase contrast micrograph of goldfish gonad culture showing 4.9 characteristics of epithelial-like (arrowhead) and fibroblast-like cells (arrow) after 15 days of culture. (scale bar = 20 μm) 6 Fathead minnow cells at 7 days of third passages after overlaid 4.9

with virus supernatant. The cells show normal appearance. May-Grunwald/ Giemsa. (scale bar = 20 μm)

7 Diseased carp displayed ulcer lesion (arrowhead) in the lateral 5.6 body wall with intracutaneous hemorrhage

8 Koi carp with advanced ventral ulcerative and extensive 5.6

hemorrhage (arrowhead) 9 Diseased Koi carp displayed skin ulcer surround by 5.7

intracutaneous hemorrhage (arrowhead) in the head

10 Koi carp with skin ulcer (arrowhead) and underlying muscle 5.7 with Argulus spp. (arrow) infection

11 Distention of abdomen and accumulation of ascitic fluid in 5.8 the abdomen. Also note scale protrusion was observed in Koi carp with skin ulcer

12 The abdominal cavity contained serosanguineous ascetic fluid 5.8

(arrow) and fibrinous adhesions (arrowhead) between visceral organs

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13 Pale and soft kidneys (K) with ascetic fluid (arrowhead) 5.9 14 Cross section of muscle fibers (M) showing vacuolar degeneration 5.12

and cellular invasion (arrow). H&E (scale bar = 10 μm) 15 An ulcerative lesion showed extensive hemorrhage (arrow). 5.12

Muscle fibers (M) were fragmented and showed vacuolar degeneration (arrowhead) H&E (scale bar = 30 μm)

16 Caseation necrosis of muscle fibers was infiltrated massive 5.13

numbers of muscular lymphocytes (arrowhead) and macrophages (arrow). H&E (scale bar = 10 μm)

17 Electron micrograph of myofibrils (M) were markedly 5.13

fragmented and separated (scale bar = 1µm). 18 Electron micrograph of macrophages (arrowhead) in dermal 5.14

loose connective tissue. (scale bar = 5µm) 19 Presence of PMN leukocytes (arrowhead) and bacterial rods 5.14 aggragating at ulcerative lesion (arrow). (scale bar = 3µm) 20 Hypertrophy and hyperplasia (arrowhead) of gill (G) epithelial 5.15

cells with fusion of adjoining lamellae. H&E (scale bar = 50 µm)

21 Monogenean (MO) on the gill (G) lamellae of fish. H&E 5.15

(scale bar = 50 µm) 22 Monogenean (arrow) parasites on the gill (G) of fish. H&E 5.16

(scale bar = 50 µm) 23 Myxobolus spp. (arrow) cyst presence within the gill (G) of fish. 5.16

H&E (scale bar = 10 µm)

24 Renal tubular (RT) exhibited vacuolar degeneration changes 5.17 (arrow). Pyknotic nuclei (arrowhead) in the renal parenchyma were also noted. H&E (scale bar = 10 µm)

25 Extensive hemorrhages (arrowhead) in the renal parenchyma 5.17

as well as cloudy swelling (arrow) of the tubules. H&E (scale bar = 30 µm)

26 Section through kidney showed characteristic pale coloured 5.18

focal of haemosiderin (arrowhead). H&E (scale bar = 10 µm)

27 Fungal hyphae (arrowhead) in stained squash preparations from 5.18 kidneys. May-Grunwald/Giemsa (scale bar = 10 µm)

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28 Numerous bacterial rods (arrowhead) occur singly or in pairs 5.19 in kidney squash preparations. May-Grunwald/Giemsa (scale bar = 10 µm)

29 Transmission electron micrograph of cell in kidneys tissue 5.19

undergoing degeneration, markedly expended mitochondria with destroyed tubular cristae (arrowhead); RBC= red blood cell. (scale bar = 2µm)

30 Coronavirus-like virions showing hexagonal capsid with crowns 5.20 seen in the vacuolized cytoplasm of kidney. (scale bar = 0.1µm).

31 Viral particles packed in paracrystalline arrangement seen in 5.20

haematopoietic cell of kidney. (scale bar = 1µm) 32 Transmission electron micrograph of virus-like particle in 5.21

luminae seen in haematopoietic cell. (scale bar = 0.2 µm) 33 Hemosiderin (arrowhead) in the hepatopancreas (HP). H&E 5.21

(scale bar = 50 µm) 34 In liver, hepatocytes (arrowhead) showed granular degeneration 5.22

and haemosiderin granules. H&E (scale bar = 10 µm) 35 Vacuolar degeneration of hepatocyte cells (arrowhead) and 5.22

congested hepatic blood vessel (arrow). (scale bar = 10 µm) 36 Small intestine showed necrosis of luminal layer. H&E 5.23 (scale bar = 50 µm) 37 Inoculated Koi carp displayed scales loss and small superficial 6.9

haemorrhagic ulcer (arrowhead) at injection site at 3 dpi

38 On the 3rd dpi, experimental fish showed inflamed bruised 6.9 (arrowhead) appearance at injection site

39 The spleen showed marked hyperaemia (arrowhead). Clear 6.10

ascitic fluid (AF) and fibrinous (FB) adhesions between The viscera have also observed at 5 dpi

40 Healing response at site of injection in Koi carp at 10 dpi 6.10 41 On the 1st dpi, acute inflammatory reactions of the muscle 6.11

fibers (M) which contained massive numbers of inflammatory cells (IC) and erythrocytes (E). H&E (scale bar = 30 μm)

42 Injection site contained bacterial exudates (B) and macrophages 6.11

(MC) surrounded by erythrocytes (E) and inflammatory cells (IC) at 5 dpi. H&E (scale bar = 10 μm)

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43 Muscular section showed necrotic core of granuloma (G) at 6.12 injection site surrounded by fibrotic tissue (F) andinflammatory cells (IC) at 5 dpi. H&E (scale bar = 30 μm)

44 Inflammatory cells like PMN leukocytes (P) as well as macrophages 6.12

(MC) were seen near degeneration and necrosis of the muscle fibers (M) at 5 dpi. H&E (scale bar = 10 μm)

45 Adipose tissue (A) showed haemorrhage at 5 dpi; hepatopancreas 6.13

(PC). H&E (scale bar = 30 μm)

46 Presence of haemosiderin (H) in hepatopancreas (PC) and spleen (S) 6.13 at 10 dpi. H&E (scale bar = 50 μm)

47 Section of spleen (S) with depletion macrophages centre 6.14

(MC) at 10 dpi. H&E (scale bar = 10 μm)

48 Kidneys section showed haemosiderin (H) deposition in 6.14 haematopoietic tissue (HT) at 10 dpi. H&E (scale bar = 10 μm)

LIST OF ABBREVIATIONS

AS Atlantic salmon gonad cell line

BB brown bullhead trunk cell line

BF-2 bluegill fry

CCO channel catfish ovary

CCVD channel catfish virus disease

cfu colony forming unit

CHSE-214 Chinook salmon embryo

CPE cytopathic effect

CRV channel catfish reovirus

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetatic acid

ELISA enzyme-linked-immunosorbent assay

EPC epithelioma papulosum cyprini

EUS epizootic ulcerative syndrome

FBS fetal bovine serum

FHM fathead minnow peduncle

HCl hydrochloride acid

HEPES N-[2-hydroxyethyl] piperazine N1- [2-ethanesulfonic acid]

H&E hematoxylin and eosin

H2 S hydrogen sulphide

IHNV infectious haemopoietic necrosis virus

IM intramuscular

IPNV infectious pancreatic necrosis virus

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IU/ml international unit per milliliter

MEM minimum essential medium

MEM-4 minimum essential medium with 4% serum

MEM-10 minimum essential medium with 10% serum

mM millimolar

NaHCO3 sodium bicarbonate

NEAA non-essential amino acids

OD optical density

O/F oxidative-fermentative

PCR polymerase chain reaction

PTA phosphotungstic acid

RNA ribonucleic acid

rpm revolution per minute

RTG-2 rainbow trout gonad

SEM scanning electron microscopy

SGV sand goby virus

SHRV snakehead rhabdovirus

SVCV spring viraemia of carp virus

TCBS thiosulphate citrate bilesalt sucrose agar

TEM transmission electron microscopy

TSA tryptic soy agar

TSI triple sugar iron agar

UDRV ulcerative disease rhabdovirus

UV ultra violet

VHSV viral hemorrhagic septicaemia virus

1.1

CHAPTER 1

INTRODUCTION

Ornamental fish keeping is a world-wide hobby. In Malaysia, aquarium fish trade

started in the 1950's within the southern state. Initially, the activity was mainly

limited to the collection of fish from the wild for subsequent distribution to

Singapore, which began to export fish to Europe in the late 1940's (Tan, 2002).

Significant development of aquarium fish farms only began in the 1980's. Today,

there are more than 400 farms, 90% of these produce ornamental fishes, and about

10% produce natural feed and aquatic plants (Anon, 2003a). The potential for further

expansion of the industry in Malaysia is enormous.

In tandem with the development of ornamental fish farms, there has been a

significant increase in the export of ornamental fish from Malaysia in the last few

years. Within the five year period, from 1991 to 1995, export had significantly

increased from 101.4 million tails valued at RM14.8 million to 175.6 million tails in

1994 valued at RM25.9 million, an increase of more than 150%. Statistical data from

1995 to 2001 showed that in terms of production, this has increased from about 100

million tails in 1995 to 340 million tails in 2001 giving an increase of 240%.

Ornamental fish farming is largely an export oriented industry. It was estimated that

about 96% of ornamental fish produced in this country are exported. This made the

ornamental fish industry the fastest growing component in the fisheries sector and a

significant source of foreign currency earner (Tan, 2002; Anon, 2003a; 2003b)