THE EFFICACY OF HERBAL PLANTS EXTRACT AGAINST INFECTION OF AEROMONAS HYDROPHILA, STAPHYLOCOCCUS XYLOSUS AND STREPTOCOCCUS AGALACTIAE IN SNAKEHEAD (CHANNA STRIATA) FISH
AZIRAH BINTI AKBAR ALI
UNIVERSITI SAINS MALAYSIA 2014 THE EFFICACY OF HERBAL PLANTS EXTRACT AGAINST INFECTION OF AEROMONAS HYDROPHILA, STAPHYLOCOCCUS XYLOSUS AND STREPTOCOCCUS AGALACTIAE IN SNAKEHEAD (CHANNA STRIATA) FISH
by
AZIRAH BINTI AKBAR ALI
Thesis submitted in fulfilment of the requirements for the degree of Master of Sciences
February 2014 KEBERKESANAN EKSTRAK TUMBUH-TUMBUHAN HERBA TERHADAP PENYAKIT AEROMONAS HYDROPHILA, STAPHYLOCOCCUS XYLOSUS DAN STREPTOCOCCUS AGALACTIAE PADA IKAN HARUAN (CHANNA STRIATA)
oleh
AZIRAH BINTI AKBAR ALI
Tesis yang diserahkan untuk memenuhi keperluan bagi Ijazah Sarjana Sains
Februari 2014 ACKNOWLEDGEMENT
In the name of Allah S.W.T, the Most Gracious, Most Merciful thus this thesis has been possible.
With great honour, my deepest appreciation to my supervisor, Dr. Zary Shariman
Yahaya, for his excellent guidance, unlimited support and motivation, patience, willingness of time in sharing ideas and knowledge and also his Do’a. Thank you very much Dr.
A special thanks goes to Mr. Sinan Al-Hakeem for his assistance, suggestion, and patience. Not forgotten thanks to Ms. Ainul Farhana, in helping me a lot during at laboratory time, collecting the plants samples, being patience and supportive.
I would like to convey my heartfelt thanks to all my friends, Kak Amelia and Nahid for their support and help during in fish trials experiment, lab 418 members (Kak
Shamim, Kak Khairiah, Firdaus, Rajiv, Keat Lay, Boon Aun, Julian, Gaikling, Sally and others) and also Nik aisyah, and Mimi for their word inspiration and encourage.
Finally, I express my sincere gratitude to my beloved parents, sister and brothers for their prayer, patience, trust, untiring support in every way, valuable motivation, constant love, inspiration and made my dream come true.
Last but not least, I would like to thank Universiti Sains Malaysia (USM), Penang for providing the necessary facilities especially Staffs in Biology School. Not forgotten thanks to government which provide me the financial support during my study and also project grant which able the project to carry on.
Azirah Akbar Ali, February 2014
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TABLE OF CONTENTS
PAGES ACKNOWLEDGEMENT…………………………………………………. ii
TABLE OF CONTENTS………………………………………………...... iii
LIST OF TABLES…………………………………………………………. xi
LIST OF FIGURES……………………………………………………….. xiv
LIST OF PLATES…………………………………………………………. xv
LIST OF ABBREVIATIONS……………………………………………… xvii
ABSTRAK…………………………………………………………………. xix
ABTRACT……………………………………………….…………………. xxi
CHAPTER ONE: INTRODUCTION
1.1 Problem Statement……..……………………………………………...... 2
1.2 Research objectives…………………………………………………… 3
CHAPTER TWO: LITERATURE REVIEW
2.1 Aquaculture…………………………………………………………….. 4
2.1.1 Aquaculture in Malaysia…………………………………………. 5
2.2 Snakehead fish………………………………………………………….. 7
2.2.1 Taxonomy and Synonymy of Snakehead fish…………………… 7
2.2.2 Snakehead fish (Channa straita)………………………………... 8
2.2.2. (a) Taxonomy and distribution………………………………… 9
2.2.2. (b) Biology of C. striata…………………………………………… 9
2.2.2. (c) Morphology of C. striata……………………………………… 10
2.2.2. (d) Importance medicinal properties of C. striata……………. 11
A. Amino acids……………………………………………… 12
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B. Fatty acids………………………………………………... 12
2.2.2. (e) Traditional health treatment………………………………. 13
2.2.2. (f) Pharmacological properties of Channa striata…………….. 13
A. Post-partum and energy booster meal……………………. 14
B. Wound healing…………………………………………… 14
C. Anti-pain…………………………………………………. 14
D. Anti-inflammatory and anti-pyretic……………………… 15
E. Anti-oxidants…………………………………………….. 15
F. Anti-fungal and anti-bacterial……………………………. 15
G. Cardiological effect………………………………………. 16
H. Haematological treatments………………………………. 16
I. Neurology and neurophysiology…………………………. 16
J. Skin disease………………………………………………. 16
K. Platelet-aggregation……………………………………… 17
L. Antinociceptive properties……………………………….. 17
M. Other uses………………………………………………… 17
2.3 Fish diseases……………………………………………………………. 18
2.3.1 Types of fish diseases……………………………………………… 19
2.3.1(a) Microbial diseases……………………………………………. 19
2.3.1. (a).1Viral….………………………………………………. 19
2.3.1. (a).2 Bacteria………………………………………………... 20
2.3.1. (a).3 Fungi………………………………………………….. 22
2.3.1. (a).4 Protozoa…………………………………………………. 23
2.4 Bath treatments for sick fish……………………………………………. 24
2.4.1 Types of bath treatment…………………………………………… 24
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2.4.1(a) Dip Bath………………………………………………….... 24
2.4.1(b) Short Bath…………………………………………………. 24
2.4.1(c) Prolonged Bath……………………………………………… 24
2.4.2 Types of Chemotherapeutants used in Malaysia and Singapore….. 25
2.5 Phytotherapeutics in Aquaculture……………………………………… 27
2.5.1 Role of phytotherapeutic in aquaculture.………………………… 29
2.5.1. (a) Growth promoter………………………………………… 29
2.5.1. (b) Immunostimulants………………………………………... 29
2.5.1. (c) Antibacterial, Antiviral and Antifungal agents…………… 30
2.5.1. (d) Anti-stress agent………………………………………….. 31
2.5.1. (e) Appetite stimulators………………………………………. 31
2.6 Plants used in present study……………………………………………. 32
2.6.1 Senna spectabilis………………………………………………………... 32
2.6.2 Jacaranda filicifolia……………………………………………………. 33
2.6.3 Samanae saman……………………………………………………...... 36
2.6.4 Tamarindus indica…………………………………………………….... 37
2.6.5 Carica papaya……………………………………………………...... 39
2.6.6 Andira inermis……………………………………………………...... 40
2.6.7 Morinda elliptica……………………………………………………..... 41
2.6.8 Coleus aromaticus benth………………………………………… 42
2.6.9 Citrus hystrix……………………………………………………...... 44
2.6.10 Milletia pinnata……………………………………………………..... 45
2.6.11 Cymbopogon nardus…………………………………………………. 47
2.6.12 Cymbopogon citratus………………………………………………… 48
2.6.13 Polyalthia longifolia………………………………………………… 49
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CHAPTER THREE: MATERIALS AND METHODS
3.1 Flow chart……………………………………………………………… 51
3.2 Plant materials……………………………………………………...... 52
3.3 Preparation of water plants extract…………………………………….. 54
3.4 Antimicrobial activity screening of the crude plants extracts………….. 54
3.4.1 Microorganisms…………………………………………………… 54
3.4.2 Antimicrobial activity test (Well Diffusion Method)……………... 55
3.4.2. (a) 1. Nutrient Agar preparation………………………………. 55
3.4.2. (a) 2 Mueller-Hinton Agar preparation……………………….. 55
3.4.2. (b) Water plants extract preparation…………………………... 55
3.4.2. (c) Bacteria suspension preparation…………………………... 56
3.4.2. (d) Well Diffusion Method…………………………………… 56
3.5 Minimum Inhibitory Concentration (MIC)…………………………….. 56
3.5.1 Justification of using three plants extract…………………………. 56
3.5.2 Mueller-Hinton Broth Preparation………………………………… 57
3.5.3 Water plants extract preparation…………………………………... 57
3.5.4 Bacteria suspension preparation…………………………………... 57
3.5.5 Microtitre Plate Assay…………………………………………….. 57
3.6 Brine Shrimp toxicity test……………………………………………… 58
3.6.1 Artificial seawater preparation…………………………………… 58
3.6.2 Artermia salina hatchings and collection………………………… 58
3.6.3 Water plants extract preparation………………………………….. 59
3.6.4 Toxicity test Assay……………………………………………….. 59
3.6.5 Toxicity test determination……………………………………….. 59
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3.7 Scanning Electron Microscope (SEM)………………………………… 60
3.7.1 Justification of using S. saman water extract……………………… 60
3.7.2 Bacteria preparation……………………………………………….. 60
3.7.3 Water extracts preparation………………………………………… 60
3.7.4 Sample preparation for SEM……………………………………… 61
3.8 Phytochemical test of water plants extract of S. saman………………….. 62
3.8.1 Alkaloids test…………………………………………………….... 62
3.8.2 Saponins test……………………………………………………..... 62
3.8.3 Flavonoids test…………………………………………………….. 62
3.8.4 Tannins and Polyphenolic test……………………………………. 63
3.8.5 Triterpenes or Steroids test………………………………………... 63
3.9 Gas chromatography and Mass-spectrum (GC-MS) of S. saman……….. 63
3.9.1 Samanae saman extract preparation………………………………. 63
3.9.2 Gas chromatography and Mass-spectrum for S. saman…………….. 63
3.10 Pathogenicity test…………………………………………………….. 64
3.10.1 Justification of using gram positive bacteria…………………… 64
3.10.2 Bacteria suspension preparation for injection………………….. 64
3.10.3 Snakehead fish………………………………………………….. 65
3.10.3. (a) Collection and storage experimental animal……………… 65
3.10.3. (b) Challenging fish pathogens……………………………….. 65
3.11 Therapeutic investigation of S. saman extracts in-vivo……………….. 66
3.11.1 Preparation of pathogenic bacteria and injection………………… 66
3.11.2 Collection and storage of experimental animals…………………. 67
3.11.3 Treatment protocol………………………………………………. 67
3.11.4 Preparation of S. saman water extract…………………………… 67
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3.11.5 Bath treatment……………………………………………………. 67
3.11.6 Exposure design experimental…………………………………… 68
3.12 Molecular identification and confirmation of S. xylosus……………… 68
3.12.1 Bacteria preparation……………………………………………… 68
3.12.2 DNA extraction from bacteria…………………………………… 68
3.12.3 Polymerase Chain Reaction (PCR)………………………………. 69
3.12.4 Agarose gel electrophoresis……………………………………… 71
3.12.4. (a) Agarose gel preparation…………………………………. 71
3.12.4. (b) Agarose gel running…………………………………….. 71
3.12.4(c) Agarose gel visualizing…………………………………….. 71
3.12.5 Purification of DNA……………………………………………... 71
3.12.6 Sequencing and Analysis………………………………………… 72
3.13 Confirmation of S. xylosus using API STAPH test kit……………… 72
3.13.1 Bacteria suspension preparation ……………………………… 72
3.13.2 API STAPH method…………………………………………….. 73
3.13.3 API STAPH analysis……………………………………………. 73
CHAPTER FOUR: RESULTS
4.1 Antimicrobial activity test (Well Diffusion Method)…………………... 75
4.2 Minimum Inhibitory Concentration (MIC)…………………………….. 78
4.3 Brine Shrimp Toxicity Test…………………………………………….. 80
4.4 Scanning Electron Microscope (SEM)…………………………………. 83
4.5 Phytochemical test of Samanae saman…………………………………….. 85
4.6 Gas chromatography and Mass spectrum (GC-MS) of S. saman……….. 85
4.7 Pathogenicity test………………………………………………………. 87
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4.8 Therapeutic treatment…………………………………………………... 89
4.8.1 Day One…………………………………………………………… 89
4.8.2 Day Two……………………………………………………...... 89
4.8.3 Day Three……………………………………………………...... 90
4.8.4 Day Four……………………………………………………...... 91
4.8.5 Day Five……………………………………………………...... 91
4.8.6 Day Six……………………………………………………...... 92
4.8.7 Day Seven……………………………………………………...... 92
4.8.8 Day Eight……………………………………………………...... 93
4.8.9 Day Nine……………………………………………………...... 93
4.8.10 Day Ten……………………………………………………...... 94
4.8.11 Day Eleven…………………………………………………….... 94
4.8.12 Day Twelve……………………………………………………... 95
4.8.13 Day Thirteen…………………………………………………….. 95
4.8.14 Day Fourteen……………………………………………………. 96
4.8.15 Day Fifteen……………………………………………………... 96
4.9 Molecular identification and confirmation of S. xylosus…………………. 99
4.10 Confirmation of S. xylosus using API STAPH test kit……………… 100
CHAPTER FIVE: DISCUSSIONS
5.1 Antimicrobial activity test (Well Diffusion Method)…………………... 103
5.2 Minimum Inhibitory Concentration (MIC)…………………………….. 105
5.3 Brine Shrimps Toxicity test…………………………………………….. 106
5.4 Scanning Electron Microscope (SEM)…………………………………. 109
5.5 Gas chromatography and Mass spectrum(GC-MS) of Samanae saman 110
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5.6 Phytochemical test of Samanae saman…………………………………….. 111
5.7 Pathogenicity test……………………………………………………….. 114
5.8 Therapeutic treatments…………………………………………………. 116
5.9 Molecular identification and API STAPH of S .xylosus………………….. 119
CHAPTER SIX: CONCLUSION
6.1 Conclusions……………………………………………………...... 120
6.2 Recommendations for further research…………………………………. 121
REFERENCES……………………………………………………...... 122
APPENDIX……………………………………………………...... 148
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LIST OF TABLES
PAGES
Table 2.1 Fish capture production in Asia and the Pacific region 5
Table 2.2 Species recognized for the family Channidae 8
Table 2.3 Important amino acids and fatty acids in different sources 11 of Channa striata extract Table 2.4 Bacterial pathogens present in Fresh water and Marine 21 water Table 2.5 Occurrence of fungi on fishes 22
Table 2.6 Protozoa detected in infected blood and organs of 23 Oreochromis niloticus and Clarias gariepinus Table 2.7 Chemotherapeutants used in aquaculture sector in Malaysia 26 and Singapore Table 2.8 List of plants herbal against fish and shell fish pathogen 28 diseases administrated through oral, injection or bath treatments Table 2.8 The activity on the crude extracts of Jacaranda species on 35 various bacteria pathogens Table 3.1 List of plants samples used in the study 53
Table 3.2 List of microorganisms used in the study 54
Table 3.3 Instruments temperature 64
Table 3.4 Grading for fish lesion 66
Table 3.5 Primers used in the study 70
Table 3.6 Components used in PCR 70
Table 3.7 PCR profiles used 70
Table 3.8 Reading table for API STAPH 74
Table 4.1 Antimicrobial screening of 13 species of plants extracts 76 against three pathogenic bacteria Table 4.2 MIC values of three plants extracts against three 78
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pathogenic bacteria Table 4.3 Artemia nauplii tested with three plants extracts 81
Table 4.4 Phytochemical results of Samanae saman 85
Table 4.5 Lesion and cumulative mortality induced by S. xylosus and 87 S. agalactiae on Snakehead (Channa striata) Table 4.6 Lesion and cumulative mortality induced by S. xylosus in 89 Day one post injection Table 4.7 Lesion and cumulative mortality induced by S. xylosus in 90 Day two post injection Table 4.8 Lesion and cumulative mortality induced by S. xylosus in 90 Day three post injection Table 4.9 Lesion and cumulative mortality induced by S. xylosus in 91 Day four post injection Table 4.10 Lesion and cumulative mortality induced by S. xylosus in 91 Day five post injection Table 4.11 Lesion and cumulative mortality induced by S. xylosus in 92 Day six post injection Table 4.12 Lesion and cumulative mortality induced by S. xylosus in 92 Day seven post injection Table 4.13 Lesion and cumulative mortality induced by S. xylosus in 93 Day eight post injection Table 4.14 Lesion and cumulative mortality induced by S. xylosus in 93 Day nine post injection Table 4.15 Lesion and cumulative mortality induced by S. xylosus in 94 Day ten post injection Table 4.16 Lesion and cumulative mortality induced by S. xylosus in 94 Day eleven post injection Table 4.17 Lesion and cumulative mortality induced by S. xylosus in 95 Day twelve post injection Table 4.18 Lesion and cumulative mortality induced by S. xylosus in 95 Day thirteen post injection Table 4.19 Lesion and cumulative mortality induced by S. xylosus in 96 Day fourteen post injection
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Table 4.20 Lesion and cumulative mortality induced by S. xylosus in 97 Day fifteen post injection Table 4.21 Comparison of three groups of treatment using S. saman to 97 control mortality and reduce per cent of severity on Snakehead (C. striata) Table 4.22 Identification of strain using BLAST analysis 99
Table 4.23 Confirmation of S. xylosus using API STAPH test kit 100
Table 4.24 Identification of Staphylococcus species using apiweb 101
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LIST OF FIGURES
PAGES
Figure 2.1 Interaction between host, pathogens, environment and the 19 outbreak of diseases Figure 3.1 Overall flow chart 51
Figure 4.1 Mortality percentage of Artemia nauplii against variety 82 concentration of Jacaranda filicifolia within 6 Hours Figure 4.2 Mortality percentage of Artemia nauplii against variety 82 concentration of Samanae saman within 24 Hours Figure 4.3 Presence of bioactive compound in Samanae saman extract 86
Figure 4.4 Fingerprint of the spectrum 86
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LIST OF PLATES
PAGES
Plate 2.1 Snakehead fish, Channa striata 11
Plate 2.2 Senna spectabilis, local name Kasia Kuning 33
Plate 2.3 Jacaranda filicifolia, local name Jambul Merak 34
Plate 2.4 Samanae saman, local name Pukul Lima 37
Plate 2.5 Tamarindus indica, local name Asam Jawa 38
Plate 2.6 Carica papaya, local name Betik 40
Plate 2.7 Andira inermis local name Brown Heart 41
Plate 2.8 Morinda elliptica, local name Mengkudu kecil 42
Plate 2.9 Coleus aromaticus benth, local name Lemuju 43
Plate 2.10 Citrus hystrix, local name Limau Purut 45
Plate 2.11 Milletia pinnata, local name Mempari 46
Plate 2.12 Cymbopogon nardus, local name Serai Wangi 47
Plate 2.13 Cymbopogon citratus, local name Serai 49
Plate 2.14 Polyalthia longifolia, local name Asoka 50
Plate 4.1 Antimicrobial activity of certain plants extracts against 77 Staphylococcus xylosus bacteria Plate 4.2 MIC values of T. indica, J. filicifolia, and S. saman against 79 A. hydrophila Plate 4.3 MIC values of T. indica, J. filicifolia and S. saman against 79 S. agalactiae Plate 4.4 MIC values of T. indica, J. filicifolia and S. saman against 80 S. xylosus Plate 4.5 SEM of S. agalactiae treated with S. saman extract 83
Plate 4.6 SEM of S. xylosus treated with S. saman extract 84
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Plate 4.7 Extensive blanching lesion with furuncle-like ulcerated 88 core on Day three post-injection of S. xylosus pathogens Plate 4.8 Therapeutic treatments on Day three post injection of S. 98 xylosus pathogens Plate 4.9 Agarose gel 1% stained with Ethidium bromide (EtBr) 99
Plate 4.10 API STAPH strip results 101
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LIST OF ABBREVIATIONS
ppm Part per million
ppt Part per thousand
rpm Rotation per minutes
BLAST Basic Local Alignment Search Tool
BSE Bovine Spongiform Encephalopathy
CPK Creatine phosphokinase
CTF II CArdiotoxic factor II
CFU Colony forming unit
CNS Central Nerve System
DDS Department Delivery System (DDS)
DNA Deoxyribonucleic acid
DOF Department of fisheries
DHA Decosahexaenoic acid
EtBr Ethdium Bromide
EPA Eicosapentaenoic acid
FRIM Forest Research Institute Malaysia
GC-MS Gass Chromatography and Mass Spectrum
HMDS Hexamethyldisilazane
IHNV Infectious Haematopoietic Necrosis Virus
LC50 Lethal Concentration that kill half of sample population
MIC Minimum Inhibitory Concentration
MG Mycotic Granulomatosis
MOA Ministry of Agriculture and Agro-based industry
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PCR Polymerase chain reaction
RBC Red Blood Cell
SFSE Shol fish skin extract
SEM Scanning electron microscope
SVCV Spring Viremia Carp Virus
TEM Transmission electronmicroscope
TAE Tris-Acetate-EDTA
VHSV Viral Haemorrhagi Septicaemia Virus
WBC White Blood Cell
ZIA Aquaculture Zone Industry
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KEBERKESANAN EKSTRAK TUMBUH-TUMBUHAN HERBA TERHADAP PENYAKIT Aeromonas hydrophila, Staphylococcus xylosus DAN Streptococcus agalactiae PADA IKAN HARUAN (Channa striata)
ABSTRAK
Ujian saringan telah dilakukan untuk 13 spesis tumbuhan tergolong dalam 9 jenis famili seperti Senna spectabilis, Jacaranda filicifolia, Samanae saman, Tamarindus indica, Carica papaya, Andira inermis, Morinda elliptica, Coleus aromaticus benth,
Citrus hystrix, Milletia pinnata, Cymbopogon nardus, Cymbopogon citratus dan
Polyalthia longifolia untuk mencari aktiviti antibakteria terhadap tiga patogen ikan seperti; Aeromonas hydrophila, Staphylococcus xylosus dan Streptococcus agalactiae. Kepekatan perencatan minimum (MIC) menunjukkan, S. xylosus direncat oleh S. saman dengan kepekatan terendah iaitu 0.625 mg/ml berbanding dengan T. indica dan J. filicifolia; 2.5 mg/ml. Tamarindus indica dan J. filicifolia menunjukkan aktiviti terhadap semua patogen yang diuji. Ujian ketoksikan dengan udang air masin telah dijalankan ke atas tiga ekstrak tumbuhan dengan menggunakan nauplii Artemia salina sebagai sampel ujian dan keputusan menunjukkan , T. indica dan J. filicifolia adalah sangat toksik dan S. saman menunjukkan ketosikan yang terendah (> 1.0 mg/ml) ; dengan nilai LC50, 2.22 mg/ml. Mikroskop imbasan elektron (SEM) digunakan ke atas dua sel patogen; S. xylosus dan S. agalactiae yang di rawat dengan ekstrak daun S. saman dan menunjukkan kerosakan struktur pada permukaan sel. Di samping itu, analisis fitokimia dilakukan untuk melihat komponen kimia yang hadir dalam ekstrak daun S. saman; menunjukkan kewujudan Alkaloid, Flavonoid, Tanin dan Sebatian
Polifenolik. Kromatografi Gas dan Spektrum Jisim (GC-MS) dijalankan ke atas
xix sampel ekstrak S. saman dan menunjukkan kehadiran aktif kompaun hidrazin, 1-etil-
1-(1-methylpropyl). Potensi S. xylosus dan S. agalactiae untuk menyebabkan jangkitan penyakit pada C. striata disiasat melalui ujian patogen secara in-vivo.
Suntikan intra-muskular dilakukan ke atas sirip ikan dengan kepekatan 109, 107 dan
105/CFU larutan anggaran bakteria. Ikan disuntik dengan kepekatan 109 S. xylosus menunjukkan bisul seperti luka dengan erosi dan juga bisul seperti luka dengan pembentukan ulser, pada hari ke-3 selepas suntikan. Manakala, ikan yang disuntik dengan kepekatan 109 S. agalactiae menunjukkan sedikit bengkak pada hari pertama dan sembuh dengan lebih cepat. Keberkesanan ekstrak daun S. saman terhadap penyakit yang disebabkan oleh patogen S. xylosus diuji secara in-vivo dalam ikan haruan. Ikan dibahagikan kepada tiga kumpulan, Kumpulan 1 (dua kali rawatan),
Kumpulan 2 (empat kali rawatan) dan Kumpulan 3 (kawalan tanpa rawatan).
Daripada keputusan yang diperolehi, Kumpulan 2 (empat kali rawatan) menunjukkan bilangan kematian dan peratusan tahap keterukan iaitu 1 dan 5%, berbanding kawalan, 4 dan 25%.
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THE EFFICACY OF HERBAL PLANTS EXTRACT AGAINST INFECTION OF Aeromonas hydrophila, Staphylococcus xylosus AND Streptococcus agalactiae IN SNAKEHEAD (Channa striata) FISH
ABSTRACT
Preliminary screening was done for 13 plants species belonging to member of nine families such as Senna spectabilis, Jacaranda filicifolia, Samanae saman,
Tamarindus indica, Carica papaya, Andira inermis, Morinda elliptica, Coleus aromaticus benth, Citrus hystrix, Milletia pinnata, Cymbopogon nardus,
Cymbopogon citratus and Polyalthia longifolia were screened to find an antibacterial activity against three common fish pathogens namely Aeromonas hydrophila,
Staphylococcus xylosus and Streptococcus agalactiae. Minimum Inhibitory
Concentration (MIC) showed, S. xylosus was inhibited by S. saman with lowest concentration at 0.625 mg/ml compared to T. indica and J. filicifolia; 2.5 mg/ml respectively. Tamarindus indica and J. filicifolia showed activity against all pathogens tested. Toxicity test of brine shrimp was conducted on three selected plants extract by using Artemia salina nauplii as a test sample and from the results,
T. indica and J. filicifolia were highly toxic; and S. saman shows lower toxicity (>1.0 mg/ml); with LC50 value of 2.22 mg/ml. Scanning electron microscope (SEM) were used to observe on two pathogens; S. xylosus and S. agalactiae treated with S. saman leaf extracts and showed structural damage in the treated cells. In addition, phytochemical analysis was conducted to investigate the chemical constitution in S. saman leaf extract; and found presence of alkaloid, flavonoid, tannins and polyphenolic compounds. Gas chromatography and mass spectrum (GC-MS) on S. saman extract samples showed presence of active compounds Hydrazine, 1-ethyl-1-
(1-methylpropyl). The potential of the S. xylosus and S. agalactiae to induce disease
xxi infection in C. striata were investigated through in-vivo pathogenicity test. The intramuscular injection is done into dorsal fin of the fishes with concentration of 109,
107 and 105/CFU bacteria suspension. Fish injected with 109 of S. xylosus suspension showed furuncle like lesion with erosion and also furuncle like lesion with ulcerated core, in day 3 post injections. Fish injected with 109 of S. agalactiae suspension showed slight swelling in day 1 and healed faster. The efficacy of S. saman leaf extract against disease caused by S. xylosus pathogens was tested in-vivo in the to Snakehead fish. The experiment of fish were divided into three groups;
Group 1 (two times treatment), Group 2 (four times treatment) and Group 3 (control without treatment). From the results obtained, Group 2 (four times treatment) showed lower number in mortality and percentage of severity which is 1 and 5 % compared to the control of 4 and 25 %.
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CHAPTER 1
INTRODUCTION
Aquaculture provides big opportunities for the production of wide variety of aquatic foods including fish and shell fish. It provides important sources of protein and also source of food. Consumption of food fish is increasing had risen from 40 million tonnes in 1970 to 86 million tonnes in 1998 and reached 110 million tonnes in 2010 (Sihag and Sharma, 2012). A review on world fishery production showed that, the capture aquaculture production has maintained a level at 90 million tonnes for more than a decade, with fisheries showing an increasement from 34.6 million tonnes in 2001 to 55.7 million tonnes in 2009 (Pridgeon and Klesius,2012).
Aquatic diseases are the biggest constraint in aquaculture farmed production.
Major pathogens been reported in aquaculture sector include: bacteria, fungi, viruses and parasites. In addition, as reported by Shariff and Subasinghe (1993) identified a number of parasitic, bacterial, viral and fungal pathogens common in the Malaysian aquaculture industry. Losses through diseases are considered significant, although hard data are lacking. Bacterial infection is commonly reported in aquaculture production especially in eggs, intestines, skin surface and gills (Cahill, 1990). This is because bacteria can survive in aquatic environment independently of their hosts and become major impediment to aquaculture. In Malaysia, the estimated losses in finfish cultured in floating cages in Peninsular Malaysia due to pathogenic bacteria was reported to have amounted to RM 20 million (Najiah et al., 2011). Recent disease outbreaks reported in Kedah are thought to have caused the industry up to
$10 million (Shariff 1995). The pathogenic bacteria such as Aeromonas hydrophila,
1
Staphylococcus xylosus and Streptococcus agalactiae are among the common fish pathogens which have infected fish farms and caused mortality (Schaperclaus et al.,
1992).
The presence of antibiotic residues in fish farms is increasing due to their extensive used such as in animal feeds, bath treatment, in order to treat animal diseases and promote growth (Pericas et al., 2010). Generally, chemical residues in aquaculture farms can accumulate in fish and could cause chronic health effects to consumers. Among the health problems caused by the chemical residues are cancer, nerve problems and immunological problems. In year 2001, antibiotic residue especially chloramphenicol and nitrofurans again became important issues due to their detection in shrimp farms in China, Vietnam and Thailand. Similarly, in
Malaysia, chemical hazards including pesticides and antibiotic residues have been reported occur in freshwater aquaculture fish (Abu bakar et al., 2010).
1.1 Problem Statement
Uses of antibiotics and chemotherapeutic agents for treatment and prophylaxis in aquaculture production have been criticized due to it negative side effect (Cristea et al., 2012). An urgent research on plant-derived based for treatment is needed; which have been reported by various workers; as an antistress, antimicrobial, immunostimulant agents, growth promoter and appetite enhancement (Citarasu et al.,
2001; Sivaram et al., 2004; Magdelin,2005). In addition, there is a lack of study done on water-based herbal plants extracts. Although, not many volatile active compounds have been found in these water based extracts, but, it is eco-friendly, safer to use, and also cheaper sources for farmers, which can prepare their own water
2 extracts with minimal training. Furthermore, this also can help to reduce management cost by not purchasing any chemotherapeutic chemicals.
1.2 Research objectives
The aim of the study is:
i. To determine the effects of the plants extracts such as Samanae saman,
Jacaranda filicifolia, Tamarindus indica and others on the activity of three
common pathogenic bacteria namely; Aeromonas hydrophila, Staphylococcus
xylosus and Streptococcus agalactiae.
ii. To gain a better understanding about herbal plants extracts and to find the
efficacy in treating bacterial fish disease in-vitro and also in-vivo.
iii. To explore the possibility of using herbal plants extracts as an alternative
treatment for Snakehead fish farmers in Malaysia.
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CHAPTER 2
LITERATURE REVIEW
2.1 Aquaculture
Asia and the pacific region is the world’s largest producer of fish. In 2008, the amount from capture fisheries reached 48.3 million tonnes, which was an increase of 2.9 per cent compared to 2006 (Lymer et al., 2010). Fish production growth in Southeast Asia has also been very high in the past four decades with marine capture production increasing almost linearly throughout this period (Table
2.1) (Lymer et al., 2010). The total capture production in 2006 was 15.4 million tonnes, of which marine capture was 88% while in 2008 was 16.1 million tonnes, which consisted mainly of pelagic marine fish. The increase in marine production in
2004 could be attributed to the increases in production from Myanmar, Indonesia,
Vietnam and the Philippines, but slight drop in production occurred in Thailand and
Malaysia, 2 and 3% respectively (Lymer et al., 2008). In 2008, aquaculture production in this region recorded an increase, of which ninety-four different species were reported (Lymer et al., 2010).
4
Table 2.1: Fish capture production in Asia and the Pacific region (by sub region) (x1000 tones) China Southeast South Other Oceania Asia Asia Asia Marine fishes 2103 4868 991 495 60 Marine molluscs 646 80 12 2 4 Marine crustaceans 0 9 81 17 2 Freshwater fishes 1615 1588 1361 13 8 Freshwater molluscs 268 63 0 2 2 Freshwater crustaceans 0 4 80 0 0 Total 4633 6613 2524 529 77 Total capture production 15141 16133 6591 6467 1096 Total contribution (%) 31 41 38 8 7 Source: Lymer et al. (2010)
2.1.1 Aquaculture in Malaysia
Aquaculture is one of the important branches in agriculture. In 1994, the total aquaculture production in Malaysia amounted to 114,114 metric ton of food fish valued at USD 145.8 million and USD 227.8 million aquarium fish valued at USD
17.5 million. Malaysian Department of Fisheries (DOF) has predicted that the aquaculture sector will generate almost USD 400 million (RM 1.39 billion) of revenue per year for the nation. Cage culture is one of the aquaculture systems in
Malaysia that have a lot of potentials and can be further developed in the future.
Cage culture has been producing more than 22,000 tons of fish per year and the value is about USD 106 millon (RM 371 million) per year (Ahmad Faiz et al., 2010).
In 2010, the total fishery sector showed an increase of 3.77 % in production compared to the year before. According to Department of Statistics Malaysia, the value of gross output generated by the fisheries sub sector was RM 689.6 million in
2008. Out of this, the states of Sabah, Perak and Pahang accounted for more than half of the total values of gross output (54.6%). Department of Fisheries stated that, deep sea fisheries showed an increase in value in a total of RM 1.271 million
5 compared to RM 1.164 million in 2009. On the other hand, revenue from coastal fisheries was RM 5.305 million compared to RM 4.907 million in 2009 (Inside
Malaysia, 2012)
The Ministry of Agriculture and Agro-based industry (MOA) through the
Department of Fisheries (DOF) has developed an aquaculture industry zone (ZIA) program throughout Malaysia (Ahmad Faiz et al., 2010). The aims of the program are:
(a) To create permanent areas for Aquaculture Industry Zone
(b) To increase the production of fish in line with the goal of balance of trade
(BoT) plan.
(c) To increase the net income of aquaculturist to at least USD 850 per month
(d) To ensure the production of fish and fish products which have high quality
and safe for consumption
(e) To increase private sector participation through the provision of ZIA areas,
infrastructure and Department Delivery System (DDS) and
(f) To create a chain of efficient aquaculture fish production areas.
However, fish disease is considered a serious issue in aquaculture fish farming.
Diseases in Malaysia aquaculture have been reported since the 1980s (Anderson,
1988). The common pathogens found in the Malaysia aquaculture industry are parasitic, bacterial, viral and fungal pathogens (Shariff and Subasinghe, 1993).
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2.2 Snakehead fish
2.2.1 Taxonomy and Synonymy of Snakehead fish
Snakeheads (family Channidae) have two genera, Channa which is native to
Asia, Malaysia and Indonesia; while Parachanna is endemic to a tropical Africa
(Courtenay and Williams, 2004). Currently, Channidae are recognized for 26 species of Channa and 3 of Parachanna (Table 2.2). According to Nelson (2006), the snakeheads are classified into:
Class : Actinopterygii
Subclass : Neopterygii
Order : Perciformers
Suborder : Channoidae
Family : Channidae
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Table 2.2: Species recognized for the family Channidae
No Species Name References 1* Channa amphibeus Chel snakehead McClelland,1845 2 Channa argus Northern snakehead Cantor,1842 3 Channa asiatica Chinese snakehead Linnaeus,1758 4 Channa aurantimaculata Orangespotted snakehead Musikasinthorn,2000 5 Channa bankanensis Bangka snakehead Bleeker,1852 6 Channa baramensis Baram snakehead Steindachner,1901 7 Channa barca Barca snakehead Hamilton,1822 8 Channa bleheri Rainbow snakehead Vierke,1991 9 Channa burmanica Burmese snakehead Chaudhuri,1919 10 Channa cyanospilos Bluespotted snakehead Bleeker,1853 11 Channa gachua Dwarf snakehead Hamilton,1822 12 Channa harcourtbutleri Inle snakehead Annandale,1918 13 Channa lucius Splendid snakehead Cuvier,1831 14 Channa maculate Blotched snakehead Lacepede,1802 15* Channa maurulius Bullseye snakehead Hamilton,1822 16 Channa marulioides Emperor snakehead Bleeker,1851 17 Channa melanoptera Blackfinned snakehead Bleeker,1855 18 Channa melasoma Black snakehead Bleeker,1851 19* Channa micropeltus Giant snakehead3 Cuvier,1831 20 Channa nox Night snakehead1 Zhang et al., 2002 21 Channa orientalis Ceylon snakehead Schneider,1801 22 Channa panaw Panaw snakehead Musikasinthorn,1998 23 Channa pleurophthalma Ocellated snakehead Bleeker,1851 24* Channa punctata Spotted snakehead Bloch,1793 25 Channa stewartii Golden snakehead Playfair,1867 26* Channa striata Chevron snakehead Bloch,1797 27 Parachanna africana Niger snakehead Steindachner,1879 28 Parachanna insignis Congo snakehead Sauvage,1884 29 Parachanna obscura African snakehead Gunther,1861 Source: Courtenay and Williams (2004) Note (*): Purpose as Food fish (Haniffa et al., 2013) Highlighted Channa spp indicated occurred in Malaysia
2.2.2 Snakehead fish (Channa striata)
Channa striata is well-known as Haruan in Malaysia and is a native freshwater fish of tropical Africa and Asia (Ng and Lim, 1990). It belongs to the family Channidae and is also known as Murrels or Serpent-Headed fish. Some species of snakeheads are highly valued as food fishes, particularly in India, South eastern Asia, and China and to a lesser extent in Africa. Because of its popularity as
8 a food in southern China and adjacent south-eastern Asia, the chevron snakehead (C. striata) has been reported as widely introduced into islands from the western Indian
Ocean eastward to Hawaii. The northern snakehead (C. argus) has been a market leader and cultured in China and Korea. Other snakeheads utilized as food fishes include the Chinese snakehead (C. asiatica), Blotched snakehead (C. maculata), and
Spotted snakehead (C. punctata).
2.2.2. (a) Taxonomy and distribution
Channa striata is a tropical, freshwater, carnivorous, air-breathing fish species, indigenous to Malaysia and widely distributed within the country. A study by Kajima et al., (1994) and Kumar (1995) on the genetic variability of Snakehead based on fish mitochondrial DNA revealed that this species has been present in
Malaysia for more than 600,000 years providing evidence that the fishes are truly
Malaysia indigenous species.
2.2.2. (b) Biology of Channa striata
Channa striata is not a good swimmer but with a fast flip action, is quite an efficient predator. It is also known as air-breathing fish that can survive in the environments with low dissolved oxygen and high ammonia contents (Marimuthu and Haniffa, 2007) and stay alive without water as long as its gills remain moist.
However, C. striata have mostly been found in waters up to 12 meters deep and 4 to
80 meters wide. In addition, C. striata is usually found in rivers with salinity about
10 ppt and temperature around 20.7 to 26.4 °C with pH range of 4.3 to 7.9.
Although, C. striata is known as a hardy fish and able to tolerate to some extend any deterioration of the water quality, it is very sensitive to contamination of its habitat and has developed a unique physiological adaptation namely the ability to move
9 from pond to pond by crossing on land in order to find suitable and clean water (Mat
Jais, 1991). It is carnivorous and feed on frogs, fish, insects, tadpole and earthworms (Muntaziana et al., 2013).
In addition, C. striata is not a good swimmer and prefers stagnant, slow running and shallow water not more than two metre deep with dead log and aquatic plants so that it can easily hide and hunt for food (Mat Jais, 2007). The fish has a unique habit of settling itself into bottom mud of ponds during drought season and going deep and deeper into mud during the dry period and only comes out when the situation is better (Rahman et al., 2012).
2.2.2. (c) Morphology of Channa striata
Channa striata possesses an angular head without patches of scales and with large mouth as shown in Plate 2.1. The pectoral fin length is about half the length of the head. The dorsal fin contains 37-46 fin rays; the anal fin has 23-29 fin rays; the pectoral fins bear about 15-17 rays and the pelvic fins with 6 rays, with rounded caudal fin. The colours of snakehead fish can be highly variable or complex but most often the dorsum appears to be dark brown to black (Courtenay and Williams,
2004).
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Plate 2.1: Snakehead fish, Channa striata
2.2.2. (d) Importance Medicinal properties of Channa striata
The medicinal effects of C. striata are attributed to two major components, the amino acids and the fatty acids as described in Table 2.3.
Table 2.3: Important amino acids and fatty acids in different sources of C. striata extract
Fillet Roe Mucus Amino acids Glycine No published data No published data Glutamine acid Arginine Aspartic acid Fatty acids Eicosapentaenoic Eicosapentaenoic Oleic acid acid (EPA) acid (EPA) Linoleic acid Docosahexaenoic Docosahexaenoic acid (DHA) acid (DHA) Palmitic acid Arachidonic acid Oleic acid Hexadeconoic acid Stearic acid Oleic Arachidonic acid Linoleic Source: Mohd Shafri and Abdul Manan (2012)
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A. Amino acids
Previously, Mat Jais et al., (1994) have conduct the study on amino acid profile in the fillet extract of C. striata which found the extract to be rich in glycine, a non-essential amino acid. In addition, amino acid such as glutamic acids, arginine, aspartic acid and glycine have been found in C. striata. These amino acids are important in influencing the sense of pain and in healing wounds.
B. Fatty acids
The first study on lipid profiles on C. striata was reported by Endinkeau and
Kiew (1993) which showed a high level of fat (11- 17%) wet weight and a high ratio unsaturated: saturated (1.2-2.3) and low omega-3 in general. The ability of C. striata to produce unsaturated fatty acids such as Eicosapentaenoic acid (EPA) and
Decosahexaenoic acid (DHA) (Jaya Ram et al., 2011) in high amounts (Abdul
Rahman et al., 1995) showed the efficiency of the fish fatty acids as a wound healing agent. In addition, some of the most abundant fatty acids in C. striata are C16
(Palmitic acid), C22:6 (DHA), C18:1 (Oleic acid) and C18:0 (Stearic acid) (Zakaria et al., 2007; Dahlan-Daud et al., 2010).
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2.2.2. (e) Traditional health treatment
Channa striata is commonly consumed as a food fish as, freshwater fish consumption in Malaysia provides an important source of protein constituting up to
70% of total protein requirements (Osman et al., 2001) and is also recognised as a source of omega-3 fatty acids (Ng, 2006). In addition, C. striata is also highly valued for its medicinal properties. Among many types of fishes in Malaysia, only the Malaysian Channidae (including C. micropeltes, C. striata and C. gachua), the mudskipper, Periophtalmus spp., and the freshwater eel, Monopterus albus
(Abdullah et al., 2010) are known to be used in traditional Malay medicine. Other
Southeast Asian countries such as Thailand, Vietnamese and Cambodians as well as the Chinese also use C. striata in the treatment of diseases (Wee, 1982). The popularity of C. striata as a therapeutic agent is related to folk belief in its efficacy in treating wounds, relieving pain and boosting energy in the sick and elderly. Mat Jais
(1997) reported that C. striata is consumed to fasten healing especially for mothers who underwent caesarean operations and become supplementary among illnesses like diabetic gangrene and cancer.
2.2.2. (f) Pharmacological properties of Channa striata
The snakehead meat has good taste, high nutrient and also has high pharmaceutical medicinal values (Khanna, 1978). In addition, many of its health benefits have been studied and described in detail
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A. Post-partum and energy booster meal
There has been a dearth of studies looking at current attitude of post-partum mothers towards C. striata as a medicinal food fish. Furthermore, among the
Malays, C. striata is cooked in the form of curried, spiced, fried or roasted fish, playing the role of functional foods which provide health benefit beyond basic nutrition. Other forms of product such as broth or a tonic of C. striata extracts are quite popular. The energy-restoring properties of C. striata are also recognised in the Malay society where it is consume for recovery process from minor to major illnesses as well as a diet supplement for elderly people.
B. Wound healing
The uses of C. striata as a wound healing agent is thought to be influenced by high level of specific amino acids (e.g. glycine) and fatty acids (arachidonic acid), believed to be involved in the promotion of wound healing by the initiation of a series of reactions involving remodelling of collagen, re-epithelialisation of wound and induction of wound contraction. Channa striata extracts also has the ability to cause proliferation of mesenchymal cells and maintain sufficient cell viability for use as a biochemical agent and promoter of healing (Abdul Wahid et al., 2009) which is not limited to dermal wounds but possibly involving other types of organs as well.
C. Anti-pain
In addition, the anti-nociceptive property of C. striata is thought to be due to its glycine and arachidonic acid constituents which are known to be involved in the anti-nociceptive pathway (Kapoor et al., 2006). The extracts of C. striata have better antinociceptive properties compared to extracts from other Channidae (Mohd
14
Hasan, 2005) and work in a concentration dependent manner (Zakaria, 2005) in a wide range of temperatures and pH (Dambisya et al., 1999).
D. Anti-inflammatory and anti-pyretic
The anti-inflammatory effect of C. striata extracts towards acute and chronic inflammation appears better than other Channidae (Somchit et al., 2004; Mohd
Hasan, 2005). It is also used in treating diseases such as osteoarthritis (Michelle et al., 2004). In addition, C. striata may have a role in the treatment of joint diseases with a clearer inflammatory component such as rheumatoid arthritis. The anti- inflammatory property may also be the reason behind the observable antipyretic activity of the aqueous extract (Zakaria et al., 2008).
E. Anti-oxidants
Among freshwater fishes, C. striata appears to have a medium level of anti- oxidant activities (Lokman, 2006) possibly contributed by some of the major amino acids and fatty acids which it contains.
F. Anti-fungal and anti-bacterial
The skin and intestinal mucus extract of C. striata showed antibacterial activity against Aeromonas hydrophila and Pseudomonas aeruginosa (Dhanaraj et al., 2009). As reported by Mat Jais et al. (2008), antifungal activities of C. striata ethanolic extract showed activity on Neurospora crassa, Aleurisma keratinophilum and Cordyceps militaris.
15
G. Cardiological effect
Fish oil supplementation is widely regarded as an effective preventative measure against cardiovascular problems. Calo et al. (2005) concluded that, fish oil supplementation could be useful in preventing post-operative atrial. The skin extract of C. striata known as Shol fish skin extract (SFSE) which, has been found to contain potent active compound, cardiotoxic factor II (CTF-II) (Karmakar et al.,
2002), with hypotensive effect and cardiotoxic property that influence the increase in cardiac marker enzyme creatine phosphokinase (CPK) and creatine phosphokinase-
MB (CPK-MB) values (Karmakar et al., 2004).
H. Haematological treatments
The cardiotoxic factor II (CTF-II factor) found in SFSE also has blood- modulating properties. This factor could induce a decrease in haemoglobin, total
Red Blood Cells (RBC), White Blood Cell (WBC), and platelet count (Karmakar et al., 2004).
I. Neurology and neurophysiology
The skin extract of C. striata could initiate apnoea and irreversible blockade of nerve-muscle preparation (Karmakar et al., 2002) and also influence the serotonergic receptor system which possible role as anti-depressant (Saleem et al.,
2011).
J. Skin disease
Channa striata is very useful for treating skin disease problems such as acne, pimples, allergy, psoriasis, sclerosis, infection and other related skin problems.
Basically, it is due to the content of fatty acid, Docosahexaenoic acid (DHA) in C.
16 striata which was recognized as a nutraceutical with clinical value in skin problem treatment (Mat Jais et al. 1998; Mori et al., 1999).
K. Platelet-aggregation
Aggregation of platelet is one of the steps in blood clotting and wound healing. Channa striata extract produced positive results in diabetic patients whom undergoing drug treatments. This is beneficial to those suffering diabetes mellitus and it will also contribute as an alternative treatment for dengue haemorrhagic patients (Mat Jais 2007).
L. Antinociceptive properties
Hydromethanolic fraction of Haruan fillets extracts produces a dose- dependent anti-nociceptive property, which also plays a role in the healing process
(Mat Jais et al., 1998).
M. Other Uses
Due to high demand for a special diet which increases awareness on the benefits of food from beef products and public concern over bovine spongiform encephalopathy (BSE), the demand for fish meat has increased dramatically, which in turn has led to higher amounts of unwanted fish parts being disposed such as fish skin. Thus, in order to avoid wasteful disposal of fish skin, researchers looked at alternative ways to utilize this valuable food source (Babji et al., 2011). For instance, the production of gelatin has been shown to be comparable to commercial gelatins made from cold-water fishes and bovine skin (See et al., 2010).
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2.3 Fish diseases
Aquatic diseases are one of the major problem in aquaculture production.
Adding to that, water is a very suitable medium for disease transmission in aquatic animals (Sihag and Sharma, 2012). According to Kinne (1980), a disease may be caused by genetic disorders, physical injury, nutritional imbalance and abiotic factors such as pollution and pathogens. The presence of the disease outbreak are varied due to the complex interaction between the host and the disease-causing situation.
The interrelationships between the host organisms, the pathogens and environmental factors are shown in Figure 2.3. The natural balances between these three factors are correlated with unpolluted environment with the normal fluctuations in ambient conditions. However, a reduction in the quality of environment (E), may lead to the frequency and severity of disease (D). In addition, an increase in the host population density may cause an increase in the risk of disease outbreaks and the increase in virulence of the pathogens (P).
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Figure 2.1: Interaction between host, pathogen, environment and the outbreak of diseases Source: Bohl (1989)
2.3.1 Types of fish diseases
2.3.1. (a) Microbial Diseases
The causative agents of diseases are divided into four categories; Viral, Bacteria,
Fungi and Protozoa
2.3.1. (a). 1 Viral
Viral diseases in fishes are very serious due to the fact that the diagnosis is difficult and in a significant number of cases they are acute or sub-acute (SANO,
1995). Viral Haemorrhagic Septicaemia Virus (VHSV) and Infectious
Haematopoietic Necrosis Virus (IHNV) are caused by viruses of the genus
Novirhabdovirus, family Rhabdoviridae (Brudesth et al., 2002). According to
William et al., (1999), this virus genus can infect Salmonid fish of all ages and cause
80% to 100% mortality. Disease outbreaks are found mainly in fingerlings less than
6 months of age. Infectious pancreatic necrosis virus (IPNV) belongs to family
19
Birnaviridae, and genus Aquabirnavirus (Roberts, 2001). The IPNV and IHNV are both important disease agents and can lead to disease outbreaks with huge losses in fish farming worldwide among wild and cultured salmonid fish (La Patra et al.,
2001). Spring Viremia of carp virus (SVCV) belongs to genus Vesiculovirus, family
Rhabdoviridae which causes severe disease in wild and cultured in the common carp
(Cyprinus carpio) with very high mortality (Oreshkova et al., 1999).
2.3.1. (a). 2 Bacteria
Bacterial pathogen diseases are responsible for heavy mortalities in both cultured and wild shell fish species all over the world (Jakhar et al., 2010a). In addition, certain bacteria have been reported as pathogenic to fish and shell fish.
Three gram positive cocci (Micrococcus, Streptococcus and Staphylococcus) and six gram negative rods (Aeromonas, Proteus, Citrobacter, Pseudomonas,
Flavobacterium and Chromobacterium) genera of bacteria have been categories as potential pathogens to Aristichthys nobilis and Ctenopharyngdon idella fish fingerlings (Welker et al., 2005; Verma et al., 2006). Dahiya and Sihag (2009) also stated that, three bacteria (viz Vibrio anguillarum, Vibrio alginolyticus and
Aeromonas hydrophila) have been reported to cause pathogenicity in the Indian magur (Clarias batrachus L.). In addition, bacterial necrosis in freshwater prawn,
Macrobrachium rosenbergii was caused by Aeromonas hydrophila, Pseudomonas flourescens and Enterobacter aerogenes (Jakhar et al., 2010b) while, Micrococcus luteus, Micrococcus varians, Cellabiosococcus scuri, Streptococcus group Q1 and
Staphylococcus aureus were found to cause tail rot disease (Jakhar et al., 2010c).
Table 2.4 below is a list of all the known pathogenic bacteria species found in fresh water and marine water fish farms around the world.
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Table 2.4: Occurrence of bacterial pathogens in Fresh water and marine water Taxonomy Presence in fresh Presence in Marine water water Acinetobacter calcoaceticus + + Aeromonas hydrophila + Agrobacterium spp. + Alcaligenes denitrificans + Alcaligenes Faecalis + Alcaligenes Piechaudii + Alteromonas haloplanktis + Arthrobacter spp. + Asticaccaulis spp. + Bacillus cereus + + Bordetella bronchiseptica + Caulobacter spp. + Coryneforms + + Cytophaga spp. + + Cytophaga fermentans + + Cytophaga hutchinsonii + Cytophaga salmonicolor + Enterobacter aerogenes + + Erwinia herbicola + + Erwinia stewartii + Escherichia coli + + Flavobacterium spp. + + Flexibacter spp. + + Hafnia alvei + Hyphomicrobium vulgare + Hyphomonas polymorpha + Janthinobacterium lividum + Klebsiella spp. + Listeria spp. + Lucibacterium harveyi + Micrococcus spp. + + Micrococcus roseus + Moraxella spp. + Photobacterium angustum + Prosthecomicrobium spp. + Pseudomonas fluorescens + + Serratia spp. + + Serratia liquefaciens + Serratia marinorubra + Staphylococcus spp. + + Vibrio alginolyticus + Vibrio parahaemolyticus + Yersinia spp. + Source: Austin and Austin (1987)
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2.3.1. (a). 3 Fungi
Fungi, which cause fungal disease, are present in both marine and freshwater fishes. Fungi are commonly known as ‘Fish-molds’ which attack eggs, fry, fingerlings and adult fishes and usually in the initial stage of infection, the fungal infections starts when the host has been injured. Fungal diseases are easily recognized by relatively superficial, colony of fluffy growth on the skin and gill of fishes. Various workers have reported the mycotic infection in fishes which occurred in India (Srivastava and Srivastava, 1978). Myazaki and Egusa (1973) observed an invasive component in histological sections of ulcerative disease affected as mycotic granulomatosis. Achyla, Saprolegnia and Aphanomyces were commonly identified from the lesion surface of affected fish (Roberts et al., 1993).
Below in Table 2.5 is the list of common fungi species that can cause infection in fish:
Table 2.5: Occurrence of fungi on fishes
Fungus Scientific name of Fishes Achlya sp. Labeo calbasu Channa gachua Notopterus chitala Puntius conchonius Labeo bata Puntius ticto Labeo calbasu Dictyuchys sterile Anabas testudineus Channa punctatus Puntius conchonius Saprolegnia sp. Colisa fasciatus Channa punctatus Cirrhinus mrigala Labeo calbasu Channa punctatus Channa striata Anabas testudineus Source: Srivastava and Srivastava (1978)
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2.3.1. (a). 4 Protozoa
Fishes are hosts for many protozoan parasites. In fish farm, parasitic protozoa may lead to epidemics and mortalities resulting in economic losses (Khalil and
Polling, 1997). A study by Neave (1906) reported the presence of Trypanosomes in the blood of Synodontis shall, Bagarus bajad and Mugil species. Studies done by Al
Wasila (1976) reported the presence of parasites in Sudan which infect Oreochromis niloticus fish. Trypanosoma alhusaini and Myxobolus sp. are the protozoa that live in freshwater fishes such as Polypterues sengalus, Clarias lazera, Synodontis shall and Serrtatus (Idris, 1986). Below in Table 2.6 shows a list of parasitic protozoa species which can infect the blood and internal organs of both Oreochromis niloticus and Clarias gariepinus.
Table 2.6: Protozoa detected in infected blood and organs of Oreochromis niloticus and Clarias gariepinus.
Fish species Infected blood/organs Detected protozoan Oreochromis niloticus Liver, kidneys and ovaries Myxobolus sp. Liver Cryptobia sp. Clarias gariepienus Blood and livers Trypanosoma sp. Livers, kidneys and ovaries Myxobolus sp. Blood Haemogregarine sp. Source: Adam et al. (2009)
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2.4 Bath treatments for sick fish
2.4.1 Types of bath treatment
The aim of bath treatment is to eliminate the external infections which occur on skin, gills and fins of the fish that may be caused by bacterial, parasite or fungal diseases (Francis-Floyd, 1996). There are three basic of the bath treatments, which are listed below:
2.4.1. (a) Dip bath
The fishes are dipped into concentrated chemical or solution for a short time period which is often less than one minute. The prolonged exposure of fishes in the chemicals at a high concentration would make stressful condition and cause fatality to the fishes.
2.4.1. (b) Short bath
The fishes are subjected into moderate concentration of chemical or solution for time period between 30 minutes to several hours. The duration of exposure to the chemical is determined by the types of chemical used, the concentration used and the facility in which the fish are housed. This is an excellent method for administering many medications to fish which are placed in aquaria, tanks or raceways.
2.4.1. (c) Prolonged bath
Usually, this bath treatment is applied to pond fishes. The small concentration of chemicals used and left in the water without changing or flow through where the chemicals will eventually disappear or break down.
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