nurhidayu binti al-saari
TRANSCRIPT
UNIVERSITI PUTRA MALAYSIA
SELECTION OF POTENTIAL BACTERIAL PROBIOTICS FOR TIGER GROUPER (Epinephelus fuscoguttatus Forsskal) LARVICULTURE
NURHIDAYU BINTI AL-SAARI
FP 2012 31
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SELECTION OF POTENTIAL BACTERIAL PROBIOTICS FOR TIGER GROUPER (Epinephelus fuscoguttatus Forsskal) LARVICULTURE
By
NURHIDAYU BINTI AL-SAARI
Thesis Submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science
June 2012
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Specially dedicated to my lovely parents to whom I indebted my life.
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science
SELECTION OF POTENTIAL BACTERIAL PROBIOTICS FOR TIGER GROUPER (Epinephelus fuscoguttatus Forsskal) LARVICULTURE
By
Nurhidayu Binti Al-saari
June 2012 Chairman: Ina Salwany Md Yasin, PhD Faculty: Agriculture
Rapid development in today’s aquaculture industries have expanded to the level
of commercial scale and has indirectly led to environmental degradation and
catastrophic losses due to bacterial diseases. Thus, the application of probiotics
was proposed as an environmental-friendly preventive measure. This study was
carried out specifically to search for probiotic candidates for use in the
larviculture of Epinephelus fuscoguttatus. Hypothetically, selected intestinal
microfloras of E. fuscoguttatus are able to confer protection on larvae by
exhibiting antagonistic activity against pathogenic bacteria.
A set of criteria for bacterial probiotics was used in this study, focusing on
phenotypic and genotypic characterizations of the candidate strains, in vitro
capacity to inhibit the growth of targeted competitive strains, the haemolytic
activity and antibiotic susceptibility of selected candidates and virulence
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assessment to the host. Initially, the intestines of healthy individual grouper
which were randomly collected from three geographically distant farms in
Malaysia were processed and bacterial isolation was performed. A preliminary
identification and grouping method successfully identified 31 species, while 22
isolates as unidentified from a total of 123 isolated aerobes and facultative
anaerobes.
The first screening process was undertaken to assess the antagonistic activity
exhibited by the isolates against four common fish pathogens. Of the 123
isolates, 40 (32.5%) displayed strong antagonistic activity, mostly Gram-positive
bacteria (n=32; 80%). The haemolytic nature of the short listed probionts was
then assessed where only nine (22.5%) showed no breakdown of red blood cells
and they were grouped into five genera based on 16S rRNA gene sequence
analysis. This genotypic strategy was used to eliminate another four isolates
considering their potential pathogenic nature to human and sequences similarity
from which they were considered identical. Screening process was ended with
the antibiotic susceptibility assay of five selected isolates which resulted in the
elimination of V. harveyi i.e. JAQ01 and JAQ02 due to their opportunistic nature
and the availability of incomprehensible evidence that these strains may
potentially carry resistance genes for β-lactamase in a transferrable genetic
material.
Considering the needs for an advanced study to measure the antagonism
capacity of three selected candidates, bacteriocin-like inhibitory substances
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(BLIS) and co-culture assays were performed. The former assay nominated
Bacillus cereus JAQ04 as the most promising candidates with initial addition at
108 and/or 109 cfu/mL with 48 and/or 72 h pre-incubation time. Alternatively, for
co-culture method, the growth of pathogenic V. alginolyticus can also be
inhibited at 105 cfu/mL of all probionts tested. Both methods revealed that the
highest inhibition of pathogens has always associated with the introduction of
significantly higher densities of probionts.
Lastly, virulence assessment was made by determining the relationship
observed between the probiont concentrations with larval survival. Conclusively,
in relation to the previous antagonistic assay, 105 cfu/mL of B. cereus (JAQ04)
and Micrococcus luteus (JAQ07) were recommended as the safest and
adequate initial amount for use in field trial. Both bacteria needed at least 48 h to
proliferate before being able to counteract the pathogens. Thorough studies on
these bacteria including in-depth assessment on their effect in vivo are
recommended.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PEMILIHAN BAKTERIA BERPOTENSI PROBIOTIK BAGI KULTUR LARVA KERAPU HARIMAU (Epinephelus fuscoguttatus Forsskal)
Oleh
Nurhidayu Binti Al-saari
Jun 2012
Pengerusi: Ina Salwany Md Yasin, PhD Fakulti: Pertanian
Pembangunan pesat sektor akuakultur pada hari ini telah berkembang kepada
skala komersial dan secara tidak langsung telah mengakibatkan pencemaran
alam sekitar dan kerugian besar yang disebabkan oleh penyakit bakteria. Oleh
itu, penggunaan probiotik telah dicadangkan sebagai langkah pencegahan yang
mesra alam. Kajian ini telah dijalankan khusus untuk mencari calon-calon
probiotik untuk digunakan dalam ternakan larva Epinephelus fuscoguttatus.
Diandaikan, microflora terpilih dari usus E. fuscoguttatus dapat memberi
perlindungan kepada larva dengan cara menentang bakteria pathogen.
Satu set kriteria pemilihan bakteria probiotik telah digunakan dalam kajian ini
memberi tumpuan kepada pencirian fenotip dan genotip terikan calon, kapasiti
in vitro untuk menghalang pertumbuhan strain sasaran, aktiviti hemolitik dan
kerentanan antibiotik calon-calon yang dipilih dan penilaian kebisaan tuan
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rumah. Pada mulanya, usus kerapu dari individu sihat yang dikumpulkan secara
rawak dari tiga ladang berasingan di Malaysia telah diproses dan diikuti dengan
pemencilan bakteria. Kaedah pengenalan dan pengkelasan awal berjaya
mengenal pasti 31 spesies, manakala 22 isolat dikelaskan sebagai tidak dikenali
daripada jumlah keseluruhan 123 aerobes dan anaerobes fakultatif terpencil.
Proses saringan pertama telah dijalankan untuk menilai aktiviti antagonistik
yang dipamerkan oleh isolat-isolat yang dikaji terhadap empat patogen yang
biasa bagi ikan. Daripada 123 isolat, 40 (32.5%) memaparkan aktiviti
antagonistik yang kuat, yang mana kebanyakannya adalah bakteria Gram-positif
(n = 32; 80%). Sifat hemolitik probionts yang telah disenarai pendek
kemudiannya dinilai di mana hanya sembilan (22.5%) tidak menunjukkan
pecahan sel darah merah dan mereka dikumpulkan kepada lima genus
berdasarkan analisis jujukan gen 16S rRNA. Strategi genotip ini telah digunakan
untuk menghapus kira empat lagi isolat disebabkan mereka berpotensi menjadi
patogen terhadap manusia dan mempunyai persamaan jujukan gen di mana
mereka dianggap serupa. Proses saringan diakhiri oleh penilaian kerentanan
antibiotik oleh lima isolat terpilih yang menyebabkan penghapusan V. harveyi
JAQ01 dan JAQ02 kerana sifat oportunis mereka dan kewujudan bukti sedia
ada yang masih belum jelas yang menerangkan bahawa terikan ini mungkin
membawa gen rintangan bagi β-lactamase dalam bahan genetik boleh pindah.
Melihat kepada keperluan kajian yang lebih dalam untuk mengukur kapasiti
antagonisma tiga calon yang dipilih, bahan-bahan rencatan menyerupai
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bacteriocin (BLIS) dan ujian kultur bersama telah dilakukan. Asai pertama telah
menamakan Bacillus cereus JAQ04 sebagai calon yang paling berpotensi
digunakan pada kepekatan 108 dan/atau 109 cfu/mL dengan 48 dan/atau 72 jam
masa pengeraman. Sebagai alternatif, bagi kaedah kultur bersama,
pertumbuhan patogen V. alginolyticus juga boleh direncatkan dengan
penambahan 105 cfu/mL probionts yang diuji. Kedua-dua kaedah mendedahkan
bahawa perencatan tertinggi patogen sentiasa dikaitkan dengan penambahan
probionts pada ketumpatan yang ketara lebih tinggi.
Akhir sekali, penilaian kebisaan telah dilakukan dengan cara menentukan
hubungan diantara kepekatan probiont dengan kemandirian larva.
Kesimpulannya, dengan merujuk kepada penilaian antagonistik terdahulu, 105
cfu/mL B. cereus (JAQ04) dan Micrococcus luteus (JAQ07) adalah jumlah yang
disyorkan paling selamat dan mencukupi bagi kegunaan ladang. Kedua-dua
bakteria memerlukan sekurang-kurangnya 48 jam untuk berkembang sebelum
dapat mengatasi patogen. Kajian yang menyeluruh ke atas bakteria ini termasuk
penilaian terperinci mengenai kesan mereka secara in vivo adalah disyorkan.
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ACKNOWLEDGEMENTS
First and foremost, greatest praises and gratitude were dedicated to Allah, the
Almighty, Beneficial and the most Merciful, who has enabled me to finish this
thesis.
I would like to express my gratitude to everybody who helped me and gave me
the courage and possibility to complete this project. This work would not have
been possible without the support and encouragement of my supervisors,
colleagues and family and it is a great honor that I now have the opportunity to
express my gratitude to them.
The first person I would like to thank is my direct supervisor Dr. Ina Salwany Md.
Yasin whose help, stimulating suggestions and encouragement which eventually
helped me a lot in all the time of doing this project and writing this thesis. She is
the one who kept an eye on the progress of my work and always available when
I need her advices. Thanks to you for your untiring support, motivation and
encouragement in both morally and financially.
Many thanks go to my co-supervisors, Assoc. Professor Dr. Hassan Mohd Daud
and Professor Dr. Sharr Azni Harmin who has also been abundantly helpful, and
have assisted me in numerous ways. I owe them lots of appreciation in return to
their comments, ideas, critical suggestion and constant encouragement.
I furthermore want to acknowledge my fellow labmates and dearest friends,
Shikin, Kak Hidayah, Kak Zalina, Wanie and Edi for their unconditional support
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and warm friendship. Million thanks to Professor Dr. Mohd Zamri Saad and staff
from Faculty of Veterinary Medicine, En. Fadhil Syukri, Pn. Zaiton, En. Azmi, En.
Jasni, Pn. Shafika, Cik Azlina and En. Abdullah for their technical assistance,
critical reviews and guidance. I am deeply indebted to all of you and my prayer
and love will always be with you.
Finally, my special thanks were dedicated to my family (Abah, Mak, Isyam, Awi
and Ashraf), who I owed the most for their constant encouragement, love and
patience. I am grateful to be blessed by having them beside me. My parents’
courage and conviction will always inspire me. It is to them that I dedicate my
work.
I also wished to thank all farms involved in specimen collection and financial
support from Agro-Biotechnology Institute (ABI), Ministry of Science, Technology
and Innovation (MOSTI) Malaysia
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I certify that a Thesis Examination Committee has met on 12 June 2012 to conduct the final examination of Nurhidayu binti Al-saari on her thesis entitled " Selection of Potential Bacterial Probiotics for Tiger Grouper, (Epinephelus fuscoguttatus Forsskal) Larviculture " in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science. Members of the Thesis Examination Committee were as follows: Mohd Salleh Kamarudin, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Kamaruzaman Sijam, PhD
Associate Professor Name of Faculty Universiti Putra Malaysia (Internal Examiner) Che Roos Saad, PhD
Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Najiah Musa, PhD
Associate Professor Faculty of Fisheries and Aqua-Industry Universiti Malaysia Terengganu Malaysia (External Examiner)
SEOW HENG FONG, PhD
Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Ina Salwany Md Yasin, PhD Senior Lecturer Faculty of Agriculture Universiti Putra Malaysia (Chairman) Hassan Mohd Daud, PhD
Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Sharr Azni Harmin, PhD
Professor Faculty of Science and Biotechnology Universiti Selangor (Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date :
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.
NURHIDAYU BINTI AL-SAARI
Date: 12 June 2012
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TABLE OF CONTENTS
Page
DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xxi CHAPTER 1 INTRODUCTION 1
2 LITERATURE REVIEW 2.1 Aquaculture for Food Security 7
2.2 Background of Epinephelus fuscoguttatus (Forsskål,1775)
8
2.3 Larval Rearing and Bacterial Diseases of Grouper 10 2.4 Common Practices to Overcome Bacterial Diseases in
Fish Culture and Probiotics as an Alternative 13
2.5 Probiotics in Marine Larviculture 2.5.1 Probiotics in General 16
2.5.2 Current Status of Probiotics in Larviculture and Relevent Researches
17
2.6 Selection Protocols of Bacterial Probiotics for MarineFish Larvae
2.6.1 Acquisition of Bacterial Strains from Healthy Animals
19
2.6.2 In vitro Screening 21 2.6.3 Organism Identification 24 2.6.4 Safety Consideration of Potential Candidates 25
3 ISOLATION AND CHARACTERIZATION OF INTESTINAL MICROFLORA OF FARMED TIGER GROUPER (Epinephelus fuscoguttatus)
3.1 Introduction 29 3.2 Materials and Methods
3.2.1 3.2.1 Fish Samples 30 3.2.2 Bacterial Isolation 32 3.2.3 Bacterial Identification and Grouping 33
3.3 Results and Discussion 3.3.1 Bacterial Isolation 34
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3.3.2 Bacterial Identification and Grouping 37 3.4 Conclusion 43 4 IN VITRO SCREENING OF BACTERIAL PROBIONTS AND
16S rRNA SEQUENCE-BASED IDENTIFICATION
4.1 Introduction 44 4.2 Materials and Methods
4.2.1 Preparation of Bacterial Strains 45 4.2.2 Preliminary Screening based on the Antagonism
to Pathogens 46
4.2.3 Haemolysis Assay 47 4.2.4 DNA Extraction and 16S rRNA Gene Sequence
Analysis 47
4.2.5 Antibiotics Susceptibility Assay 48 4.2.6 Further Assesment on the Antagonistic Activity of
Probionts Against Pathogenic Bacteria 49
4.2.6.1 Preparation of selected probionts and target strains
49
4.2.6.2 Modified cross-streak method for BLIS assay
50
4.2.6.3 Co-culture of Vibrio alginolyticus with probionts
51
4.3 Results and Discussion 4.3.1 Screening of Antagonistic Activity and Haemolytic
Property 52
4.3.2 16S rRNA Gene Sequence Analysis 57 4.3.3 Antibiotics Susceptibility Assay 60 4.3.4 Bacteriocin-Like Inhibitory Substances (BLIS)
Assays 61
4.3.5 Co-culture Method 70 4.3.6 Relation between BLIS Assay and Co-culture
Method 74
4.4 Conclusion 74
5 TOLERANCE LEVEL ASSESSMENT OF PROBIOTIC CANDIDATES
5.1 Introduction 78 5.2 Materials and Methods 5.2.1 Experimental Animals 80 5.2.2 Preparation of Bacterial Suspension 80 5.2.3 Treatments and Experimental Design 80 5.2.4 Statistical Analysis 81 5.3 Results and Discussion 5.3.1 Effect of Bacterial Probionts on the Hatching
Rate of E. fuscoguttatus Eggs 82
5.3.2 Tolerance Level Assessment of Bacterial Probionts Against E. fuscoguttatus Larvae
84
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5.4 Conclusion 93 6 GENERAL DISCUSSION AND CONCLUSION 94 REFERENCES 102 APPENDICES 116 BIODATA OF STUDENT 122 LIST OF PUBLICATIONS 123