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CHLORAMPHENICOL LEVELS IN TIGER SHRIMPS (Penaeus monodon) REARED IN A LABORATORY CONTROLLED SYSTEM AND ITS EFFECT ON Vbrio parahaemolyticus Lim Mui Hua ý Master of Science 2012

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CHLORAMPHENICOL LEVELS IN TIGER SHRIMPS (Penaeus monodon) REARED IN A LABORATORY CONTROLLED

SYSTEM AND ITS EFFECT ON Vbrio parahaemolyticus

Lim Mui Hua

ý

Master of Science 2012

O! +sat hhidmat itilaklu®atAkadetuik UN[VERSITI MALAYSIA SARAWAK

CHLORAMPHENICOL LEVELS IN TIGER SHRIMPS (Penaeus monodon) REARED IN A LABORATORY-CONTROLLED SYSTEM AND ITS EFFECT

ON Vibrio parahaemolyticus

LIM MUI HUA

A thesis submitted in fulfillment of the requirements for the degree of

Master of Science

r ýT

ý IL

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK

2012

ACKNOWLEDGEMENT

First of all, I wish to express my deepest gratitude to my dearest supervisor,

Prof. Dr. Kasing Apun and co-supervisor Prof. Dr. Lau Seng for their guidance, support,

advice and care throughout the whole project.

I would like to dedicate my special thanks to my family, for their continuous

care, love and support throughout my project.

My sincere thanks to the Director of Fisheries Research Institute Sarawak, Mr.

Albert Chuan Gambang, Head of Biotechnology and Fisheries Product Division, Miss

Yong Ai Hua, Head of Aquaculture Division, Mr. Mohammed bin Mohidin and Research

Officer Miss Imelda Riti Rantty for their advice, encouragement and assistance

throughout the lengthy project. Not forgetting my fellow Laboratory Assistants in the

biotechnology laboratory, Mr. Haji Marzukhi bin Omar, Miss Sofina binti Saufi and Miss

Christina John Willy Nonon for their technical assistance who had unselfishly helped me

in this project.

Finally, I would like to thank all my friends for their guidance a;;. '

throughout my project.

ii

ABSTRACT

Chloramphenicol is a potent, broad-spectrum antibiotic suitable for the treatment of a variety of

infectious diseases. It is banned in Europe and the United States because of the risk of aplastic

anaemia and carcinogenic properties. According to the European Commission Decision

2001/699/EC and 2001/1705/EC certain fishery and aquaculture products imported for human

consumption must be subjected to a test in order to ensure the absence of choramphenicol

residues. This study was conducted to determine the level of chloramphenicol in tiger shrimps

(Penaeus monodon) through its life cycle, the withdrawal period of tiger shrimps and to study the

relationship between levels of chloramphenicol in the tissue of tiger shrimps and water samples.

A commercial Enzyme-linked immunosorbent assay (ELISA) test was applied for the qualitative

screening analysis in this experiment at a level corresponding to the European Union (EU)

minimum required performance limit (MRPL) set for chloramphenicol analysis. The formulation

which consisted of two different concentrations of Chloramphenicol: Treatment 1, Ti (50

mg/kg), and Treatment 2, T2 (75 mg/kg) were tested on tiger shrimps reared in experimental

tanks. There was no significant differences (P>0.05) in water quality parameters such as pH,

temperature, salinity and Dissolved Oxygen (DO). There was significant differences (P<0.05)

between the levels of chloramphenicol in both treatments in the shrimp tissue and in the rearing

water samples. However, the analysis of the level of chloramphenicol in the tissues in both

treatment showed a similar trend of absorption. After termination of chloramphenicol in the feed.

chloramphenicol residue was not detected at the eleventh and twelfth week for both water and

tissue analysis. This study suggests that the withdrawal period of chloramphenicol in the shrimp

iii

tissue was two weeks. The antimicrobial susceptibility of V. parahaemolyticus isolated from

aquaculture farms towards chloramphenicol and eight other antibiotics were also studied. V.

parahaemolyticus was chosen as it was the most common bacteria isolated from the aquaculture

ponds. The incidence of antimicrobial resistance pattern was compared in V. parahaemolyticus

strains isolated from the hepatopancreas of tiger shrimps, water and sediment samples taken from

the inlet, centre, outlet, reservoir and discharge section of four aquaculture farms in Kuching. In

vitro resistance tests were performed by the standardized disk diffusion method on Mueller-

Hinton agar (MHA). Three types of bacteria were isolated namely Vibrios, Chromobacterium

violaceum and Hafnia alvei but vibrios were the most common bacteria. Among the Vibrios, the

species commonly isolated was V. parahaemolyticus. A total of 140 V. parahaemolyticus strains

isolated from the four different farms were examined for their antimicrobial resistance to nine

commonly used antimicrobials: ampicillin, gentamicin, neomycin, cephalothin, tetracycline,

nalidixic acid, kanamycin, chloramphenicol and streptomycin. In general, the most frequently

encountered form of resistance in all the samples were resistance to ampicillin (100%),

tetracycline (60%) and nalidixic acid (37.5%). On the other hand, all the strains from the

samples were totally susceptible to gentamicin and chloramphenicol. Low levels of resistance of

less than 30% were demonstrated in the other antimicrobial agents. The results in this study

confirm that all V. parahaemolyticus strains were susceptible to chloramphenicol. However, the

resistance towards ampicillin, tetracycline and rQlidixic acid suggest that the use of

antimicrobials in tiger shrimps should be controlled to overcome future resistance problem.

Keywords: Chloramphenicol, ELISA, Aquaculture, Penaeus monodon, Antimicrobial agents, V. parahaemolyticus

iv

Paras kloramfenikol dalam udang harimau (Penaeus monodon) yang dipelihara dalam sistem kawalan makmal dan kesan terhadap Vibrio parahaemolyticus

ABSTRAK

Kloramfenikol merupakan sejenis antibiotik digunakan secara meluas untuk merawat pelbagai

jenis penyakit berjangkit. Namun, penggunaan kloramfenikol sudah diharamkan di negara

seperti Eropah dan Amerika Syarikat kerana kloramfenikol boleh menyebabkan `aplastic

anaemia' dan mengandungi ciri-ciri karsinogenik. Mengikut European Commission Decision

2001/699/EC dan 2001/1705/EC, hasil-hasil perikanan dan akuakultur yang diimport untuk

pemakanan manusia mesh diuji supaya ia bebas daripada residu kloramfenikol. Kajian ini

dijalankan untuk menentukan paras kloramfenikol dalam udang harimau (Penaeus monodon)

menerusi kitar hidupnya, mengenalpasti `withdrawal period' bagi udang harimau dan untuk

menguji hubungan antara paras kloramfenikol dalam tisu udang harimau dan sampel air. Ujian

`Enzime-Linked immunosorbent Assay' (ELISA) telah digunakan dalam experimen ini untuk

analisa penyaringan kualitatif pemantauan mengikut paras Minimum Required Performance

Limit (MRPL) yang ditentukan oleh Kesatuan Eropah (EU) untuk analisa kloramfenikol.

Formulasi yang terdiri daripada dua kepekatan Kloramfenikol yang berbeza iaitu: rawatan 1,

RI (50 mg/kg) dan rawatan 2, R2 (75 mg/kg) telah diuji pada udang harimau dalam tangki

eksperimen. Keputusan menunjukkan tiada perbezaan yang ketara (P>0.05) antara parameter ..

kualiti air seperti pH, suhu, saliniti dan kandungan oksigen terlarut (DO). Terdapat perbezaan

yang signifikan (P<0.05) di antara paras Kloramfenikol bagi kedua-dua rawatan dalam tisu

udang dan sampel air ternakan. Namun, keputusan analisa yang dijalankan menunjukkan paras

V

penyerapan yang sama. Residu kloramfenikol tidak dapat dikesan dalam minggu ke-11 dan ke-

12 bagi kedua-dua analisa sampel air dan tisu. Kajian ini mencadangkan bahawa "withdrawal

period" untuk kloramfenikol dalam tisu udang ialah dua minggu. V. parahaemolyticus adalah

bakteria yang sangat biasa diasingkan. Oleh sebab itu, ia telah dipilih untuk kajian ini. Kajian

rintangan antibiotik V. parahaemolyticus yang diasingkan dari ladang aquakultur terhadap

kloramfenikol dan lapan antibiotik yang lain juga dijalankan. Corak rintangan antimikrob telah

dibandingkan dengan strain V. parahaemolyticus daripada hepatopankreas udang harimau,

sampel air dan sedimen dari dalam, tengah, luar, reservoir dan seksyen buangan dari empat

ladang akuakultur di Kuching. Ujian rintangan in vitro telah dUalankan melalui kaedah

"standardized disk diffusion" ke atas Mueller-Hinton agar (MHA). Tiga jenis bakteria telah

diasingkan iaitu Vibrios, Chromobacterium violaceum dan Hafnia alvei tetapi vibrios

merupakan bakteria yang sangat biasa. Antara semua Vibrios, spesies yang paling biasa ialah

V. parahaemolyticus. Sejumlah 140 V. parahaemolyticus yang diekstrak dari empat ladang yang

berlainan telah diuji untuk rintangan antimikrob kepada Sembilan antimikrob yang biasa

digunakan iaitu ampicillin, gentamicin, neomicin, cefalothin, tetrasiklin, asid nalidixic,

kanamicin, kloramfenikol dan streptomicin. Secara keseluruhannya, antimikrob yang paling

biasa dirintang oleh semua sampel ialah ampicillin (100%), tetrasiklin (60%) dan asid nalidixic

(37.5%). Selain daripada itu, semua strain dari kesemua sampel langsung tidak rintang kepada

gentamicin dan kloramfenikol. Bagi antimikrob lain, naras rintangan adalah kurang daripada

30%. Keputusan dalam kajian ini menunjukkan semua V. parahaemolyticus strain adalah tidak

rintang kepada kloramfenikol. Walau bagaimanapun, rintangan kepada ampicillin, tetrasiklin

vi

dan asid nalidixic mengesyorkan bahawa penggunaan antimikrobial dalam udang harimau

dikawal untuk menandatangani masalah rintangan pada masa yang akan datang.

Katakunci : Kloramfenikol, ELISA, Akuakultur, Penaeus monodon, Agen antimikrob, V. parahaemolyticus

vii

Pusat Khidmat Makiumat Akademik UNIVERSITI MALAYSIA SARAWAK

TABLE OF CONTENTS

ACKNOWLEDGEMENT ABSTRACT ABSTRAK TABLE OF CONTENTS CONFERENCE PROCEEDINGS PUBLISHED PAPERS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER 1 INTRODUCTION

CHAPTER 2 LITERATURE REVIEW

11

111

V

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xi

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xvi

I

2.1 Chloramphenicol 12 2.1.1 Pharmaceutical mechanism 13 2.1.2 Absorption, distribution and secretion 14

in human body 2.1.3 Toxic effect 14 2.1.4 Drug administration 15 2.1.5 Use of chloramphenicol in Aquaculture industry 16 2.1.6 Quantitative Analysis of Chloramphenicol 17 2.1.7 Reports of chloramphenicol use in aquaculture and 18

its effect

2.2 Common bacteria species found in shrimp aquaculture farms 19 2.2.1 Vibrio parahaemolyticus 20 2.2.2 Characteristics of V. parahaemolyticus 20 2.2.3 Growth and survival of V. parahaemolyticus 21

in the environment 2.2.4 Incidence of V. parahaemolyticus in shrimp farming 21 2.2.5 Symptoms and outbreaks caused by 22

V. parahaemolyticus 2.2.6 Mode of transmission 23 2.2.7 Isolation and itcntification 23

2.3 Use of antibiotics in aquaculture 24 2.3.1 The concern over antibiotics usage in aquaculture 25 2.3.2 Antibiotic susceptibility test 26 2.3.3 Antibiotic susceptibility of V. parahaemolyticus 27

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CHAPTER 3 MATERIALS AND METHODS

3.1 Experimental design for the studies on the levels of 31 chloramphenicol in the life cycle of tiger shrimps (P. monodon)

3.1.1 Samples of tiger shrimps 31 3.1.2 Design of experimental tanks 31 3.1.3 Sampling technique and frequency 33 3.1.4 Physico-chemical water parameters 34 3.1.5 Screening of chloramphenicol using ELISA 35 3.1.6 Confirmatory analysis using LC-MS/MS 37

3.2 Experimental Design for the antimicrobial susceptibilities of 39 Vibrio parahaemolyticus isolates from the aquaculture farms towards chloramphenicol 3.2.1 Sampling sites 39 3.2.2 Sample collection 39 3.2.3 Physical parameters of pond water 40 3.2.4 Bacterial isolation 40

3.2.4.1 Water samples 41 3.2.4.2 Sediment samples 41 3.2.4.3 Shrimp samples 42

3.2.5 Bacterial identification 42 3.2.5.1 Gram staining 43 3.2.5.2 Biochemical test 43 3.2.5.3 Commercial Identification Systems kit (BBL)43

3.2.6 Antimicrobial susceptibility test 45

3.3 Statistical Analysis 46

CHAPTER 4 RESULTS

4.1 Studies on the level of chloramphenicol in the life cycle of 47 tiger shrimps (P. monodon) in a laboratory-controlled environment

4.1.1 Biological parameters of tiger shrimps 47 4.1.2 Level of chloramphenicol in the tiger shrimps 50

during the treatm4pt period (week 1-8) 4.1.3 Level of chloramphenicol in the water of 52

experimental tanks from week 1 to 8 4.1.4 Level of chloramphenicol in the waste/sediment 55

of the experimental tanks 4.1.5 The withdrawal period of tiger shrimps 57

following termination of chloramphenicol feeding

IX

4.2 Isolation of V. parahaemolyticus from aquaculture ponds 59

4.2.1 Bacterial Isolation 59 4.2.2 Biochemical test 61 4.2.3 Chloramphenicol and Antimicrobial resistance of V. 63

parahaemolyticus isolates 4.2.4 Comparison of mean disk diffusion zone diameters, 65

p-value and resistance breakpoints for V. parahaemolyticus isolates from water, sediment and tiger shrimps

CHAPTER 5 DISCUSSION

CHAPTER 6 CONCLUSION

REFERENCES

APPENDIX

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CONFERENCE PROCEEDINGS

Lim, M. H. 2005.6 Posters on the Analytical Services of Antibiotic Residues (Chloramphenicol and Nitrofurans) for Jabatan Perikanan Malaysia for the lauunching of the Analytical Services of Antibiotic Residues by the laboratories of Jabatan Perikanan Malaysia by the Honourable Director-General, Dato Junaidi bin Che Ayub at Hotel Vistana, Pulau Pinang on the 16 September 2005.

Lim, M. H and Yong, A. H. (2006). Determination of chloramphenicol residues in tiger shrimps from aquaculture farms in Sarawak by electrospray triple quadrupole liquid chromatography tandem mass spectrometry (LC-MS/MS). A paper presented as poster in the 4`h National Fisheries Symposium 2006. Advancing R&D towards fisheries business opportunity, Kuching.

Lim, M. H., Apun, K. and Lau, S. (2007). Studies on the accumulation of chloramphenicol in the life cycle of tiger shrimps (Penaeus monodon). A paper presented as oral in the "International Conference on Natural Resources and Environmental Management 2007 and Environmental Safety and Health" on 27-29 November 2007 at Hotel Crowne Plaza Riverside, Kuching, Sarawak.

Lim, M. H., Apun, K. and Lau, S. (2008). Studies on the accumulation of chloramphenicol in the life cycle of tiger shrimps (Penaeus monodon). Presented in the Research Symposium on Biotechnology, 9 April 2008, UNIMAS, Kota Samarahan, Sarawak.

Lim, M. H and Yong, A. H. (2009). Study of Chloramphenicol Residues in Shrimps samples from Private Processing Plants in Sarawak. A paper presented as poster in the 5`h National Fisheries Symposium 2008. Fish for wealth creation, Kuala Terengganu.

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PUBLISHED PAPERS

Lim, M. H. 2005. The application of LC-MS/MS in the detection of antibiotics in IPPCS. FRI Newsletter. Research Updates. Vol. 10. No. 2. July 2005, ISSN: 0128-9403, p. 8.

Lim, M. H. 2005. Quality and Food Safety. FISHWAK, a publication of IPPS Vol. 1 Issue]. 2005 p. 24-26.

Lim, M. H. 2006. An assessment of Chloramphenicol levels in marine shimps in Sarawak. FRI Newsletter. Short Comm, Vol. 11. No. 1 &2. July 2006, ISSN: 0128-9403, p. 9.

Lim, M. H. 2006. Peruntukan Mengurus Dasar Baru Peralatan LC-MS/MS. FISHWAK, a publication of IPPS Vol. 1 Issuel. 2005 pp. 41-44.

Lim, M. H. 2006. Introduction to instrumentation- LC-MS/MS (Demonstration and analysis). Training programme in sample preparation for antibiotics analyses (chloramphenicol and nitrofurans) 8-12 August, 2006. Department of Fisheries Malaysia.

Lim, M. H and Yong, A. H. (2007). Determination of chloramphenicol residues in tiger shrimps from aquaculture farms in Sarawak by electrospray triple quadrupole liquid chromatography tandem mass spectrometry (LC-MS/MS). 423-433 pp. In Chee, P. E. et al., (eds). Proceedings of the 4th National Fisheries Symposium 2006. Advancing R&D towards fisheries business opportunity, Kuching. 488 pp.

Lim, M. H and Yong, A. H. (2009). Study of Chloramphenicol Residues in Shrimps samples from Private Processing Plants in Sarawak. 329-341 pp. In Abu, T. A. et al., (eds). Proceedings of the 5th National Fisheries Symposium 2008. Fish for wealth creation, Kuala Terengganu. 347 pp.

Lim, M. H and Yong, A. H. (2010). Preliminary study of chloramphenicol residues in frozen raw shrimps from processing plants in Sarawak. Malaysian Fisheries Journal, 9: 37-45

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LIST OF TABLES

Table Page

Table 3.1 The amount of chloramphenicol given to tiger shrimps for Treatment 33 1 and Treatment 2

Table 3.2 SRM transitions monitored for chloramphenicol and chloramphenicol- D5 38

Table 3.3 Zone Diameter Interpretive Standards 46

Table 4.1 Biological parameters of tiger shrimps recorded for 8 weeks in the 48 experimental tanks

Table 4.2 Initial physical parameters recorded before treatment of tiger shrimps 49

Table 4.3 Physical parameters recorded during treatment of tiger shrimps with 49 chloramphenicol after 8 weeks in experimental tanks

Table 4.4 The percentage of chloramphenicol accumulation in the tiger shrimps 50 weekly in Treatment I

Table 4.5 The percentage of chloramphenicol accumulation weekly in the tiger 51 shrimps in Treatment 2

Table 4.6 Statistical analysis by One way Anova analysis of chloramphenicol 54 concentration in tissue and water samples

Table 4.7 Summary of the result of the concentration of accumulation of 56 chloramphenicol in the tissue of tiger shrimps, water and waste in Treatment I and Treatment 2 weekly

Table 4.8 Water quality parameters recorded from the four tiger shrimp aquaculture 59

ponds

Table 4.9 Biochemical characteristics of V. parahaemolyticus 62

Table 4.10 V. parahaemolyticus isolates from tiger shrimp tissue exhibiting 64 resistance to antimicrobial agent

Table 4.11 V. parahaemolyticus isolates exhibiting resistance to antimicrobial agent 65

Table 4.12 Mean disk diffusion zone diameters, p-value and resistance breakpoints 66 for V. parahaemolyticus isolates recovered from different ponds

Xlll

LIST OF FIGURES

Figure Page

Figure 2.1 The chemical composition of Chloramphenicol 12

Figure 2.2 The structure of Chloramphenicol (Clarke's, 2004) green represents 13 chloride molecules , red represents oxygen molecule, white represents hydrogen molecule, blue represents nitrogen molecule and black represents carbon molecule

Figure 2.3 Electron microscopic image of V. parahaemolyticus 20

Figure 2.4 The structure of V. parahaemolyticus under compound microscope 20

Figure 2.5 Antibiotic susceptibility test using the different antibiotic discs 27

Figure 3.1 Shrimp feed before mixing 33

Figure 3.2 Shrimp feed after mixing 33

Figure 3.3 Layout diagram of the aquaculture pond 40

Figure 3.4 The red spot showing the hepatopancreas of tiger shrimp 42

Figure 4.1 The level of chloramphenicol in the tissue of tiger shrimp 52 (P. monodon) during the first 8 weeks treatment period in the tanks

Figure 4.2 The level of chloramphenicol in the water samples of the experimental 53 tanks in Treatment I and Treatment 2 from week I to 8

Figure 4.3 The level of chloramphenicol in the tiger shrimps waste in experimental 55 tanks of Treatment I and Treatment 2

Figure 4.4 The level of chloramphenicol in the tissue of tiger shrimp in Treatment 1 58 and Treatment 2 from week I to 12

Figure 4.5 Level of chloramphenicol in the tissues of*tiger shrimps from week 8 to 58 week 12 in Treatment I and 2

Figure 4.6 Colonies on TCBS agar plate. Blue-green colonies on TCBS agar 60 indicated colonies are Vibrio parahaemolyticus

Figure 4.7 V. parahaemolyticus Gram stain under I 0,000x magnification 61

XIV

Figure 4.8 BBL Crystal ID System 62

Figure 4.9 Recording test result using BBL Crystal Viewer 63

xv

LIST OF ABBREVIATION

%

µg/L +

µg µL A AAM APW CLSI cm DO DoF DoF ELISA ERIC-PCR EU FDA FRIS g/kg g/mol GAP GHP GMP HACCP HPLC HUKM 1 KIA km LCMS LC-MS/MS m2

mg mg/day mg/kg min mL MRL

negative percentage microgram per litre positive less than and equal microgram microlitre

acid American Academy of Microbiology alkaline peptone water Clinical and Laboratory Standards Institute centimetre Dissolved Oxygen Department of Fisheries Department of Fisheries Malaysia Enzyme-linked Immunosorbent Assay Enterobacterial Repetitive Intergenic Consensus sequence European Union Food and Drug Act Fisheries Research Institute Sarawak gram per kilogram gram per molar Good Aquaculture Practices Good Hygienic Practices Good Manufacturing Practices Hazard Analysis Critical Control Point High Performance Liquid Chromatography Hospital Universiti Kebangsaan Malaysia Intermediate Kligler Iron Agar kilometre Liquid Chromatography Mass Spectrometer liquid chromatography-electrospray negative ionization tandem metre square mass spectrometry milligram milligram per day milligram per kilogram minute millilitre

maximum residue limit

xvi

MRPL MRVP n NaCL NCCLS ng/g ng/mL NIAID nm NOAH °C PCR ppt PVC r R RAPD-PCR S SPB spp SRM B TCBS tRNA TSA TSB TSIA wssv

Minimum Required Performance Limit Methyl Red and Voges-Proskauer number Sodium Chloride National Committee for Clinical Laboratory Standards nanogram per gram nanogram per millilitre National Institute of Allergy and Infectious Diseases nanometer National Office of Animal Health degree centigrade Polymerase Chain Reaction parts per trillion Polyvinyl Chloride correlation Resistant Randomly Amplified Polymorphic DNA Susceptible salt polymyxin broth species selected reaction monitoring beta thiosulfate citrate bile salts sucrose Transfer ribonucleic acid Tryptic Soy Agar Trytone Salt Broth Triple sugar iron agar White Spot Syndrome Virus

rt

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CHAPTER 1

INTRODUCTION

Aquaculture is the fastest growing food sector globally. It is established as a high protein

resource to fulfill the food demand since the natural resources exhibits over exploitation. In

recent years, aquaculture has experienced vigorous development in the world. Aquaculture

already produces nearly half of the world's food fish and is forecast to increase. The growing

importance of tiger shrimp aquaculture as a source of food has been well documented

worldwide. They are highly priced on most markets throughout the world. Thus, aquaculture

supports many important fisheries. Global aquaculture production for the year 2000 exceeded 35

million metric tonnes worth of over 52 thousand million US dollars with crustaceans

contributing up to 21 percent of the value (Ahmad et al., 2006). Shrimp production from both

wild harvest and farm culture estimate levels at approximately 6624 million metric tonnes

totalling a value of more than USD$ 23 billion (FAO, 2009). The giant tiger shrimp is the major

species contributing to global shrimp aquaculture and is ranked first by value at over four

thousand million US dollars (Deachamag et al., 2006).

In the state of Sarawak, Malaysia, the tiger shrimp farming industry is also an

important economic sector. The tiger shrimp, Penaeus monodon, is the main species cultured in

aquaculture industry. The production value of tiger shrimpsin Sarawak in the year 2005 was

estimated at RM 171 million at 8,147 metric tonnes whereas the tiger shrimp fry production

locally was 416,814,900 pieces (Anon, 2005). Globally aquaculture has been increasing rapidly

and already accounts for nearly half of all food fish consumed (Hishamunda et al., 2008). The

I

shrimp farming industry is also an important economic sector in many Asian countries and the

export of farmed shrimps generates large amounts of foreign exchange into the country

(Graslund et al., 2003). This success has increased the need to intensify farming practices to

maximize profits (Tendencia and dela Pena, 2002).

Although tiger shrimp farming is an important economic sector, it encountered

problems of diseases and the repeated outbreaks of infectious diseases. Diseases are pathological

conditions of part, organ, or system of an organism resulting from various causes such as

infection, genetic defect, or environmental stress. There are a number of bacterial diseases that

are lethal to shrimp such as Yellowhead disease, Whitespot syndrome, Taura syndrome and

Infectious hypodermal and hematopoietic necrosis. The shrimp becomes weak and disoriented

and may have dark wounds on the cuticle. The mortality rate can exceed 70%. Vibriosis is one of

the most common and major disease problems in shellfish aquaculture. Vibriosis is a bacterial

disease caused by the Vibrio species and responsible for mortality of cultured shrimp worldwide

(Chen et al., 2000). Vibrio species are widely distributed in culture facilitates throughout the

world. Vibrio-related infections frequently occur in hatcheries. According to Saulnier et al.,

(2000), Cai et al., (2006) and Kleter et al., (2009), the major vibriosis diseases were caused by

Vibrio spp. which caused mass mortalities of cultured tiger shrimps. These have resulted in a

great loss to the tiger shrimps farming.

The deterioration of environmental conditions and the pressure to ensure continuous

production had led to reliance on the use of antibiotics. Antibiotics have been used in animal

2

health since the early 1950's. Their use can be either therapeutic (treatment of disease) or

subtherapeutic (prevention of disease or improved production) according to Eastshire

communications 2002. Different drugs are often administered to farmed shrimp to promote

growth and to treat or prevent disease as previous researchers reported (Wang et al., 2004, Uno

et al., 2006 and Nogueira-Lima, 2006). The benefits of antibiotics as additives in animal feeds

are numerous. Antibiotics are usually given incorporated or mixed in the shrimp feed by the

farmers. In Malaysia, antibiotics such as oxytetracycline, oxolinic acid, chloramphenicol and

furazolidone are incorporated in artificial feeds as treatment against luminescent vibriosis in

grow-out ponds (The Department of Fisheries, 1999). Holmstrom et al., 2003 reported 74% of

farmers used 13 different antibiotics in shrimp pond management in Thailand, the world's largest

producer of cultured shrimps. A wide variety of antibiotics and other therapeutants are used to

control diseases of fish and other aquatic animals (Tu et al., 2009).

In addition, a variety of chemicals such as fertilizers, liming materials, disinfectants,

oxidants, pesticides, herbicides, absorbents and minerals are often applied to aquaculture systems

(Ngo et al., 2009). Concern has also been expressed regarding the use of chemicals in shrimp

farms and its potential impacts on the environment and human health (Subasinghe et al., 1996;

Graslund and Bengtsson, 2001). Among the many potential environmental impacts of

aquaculture, the potential toxic effect of the chemicals usfi for the control of fish diseases must

be considered (Lalumera et al., 2004). The negative and potential toxic effect affect the health of

the tiger shrimp such as increase their stress levels and their susceptibilities to infections. These

antibiotics residues in the shrimps thus pose dangers to human health.

3

An important antibiotic commonly used in the aquaculture industry is

chloramphenicol. As early as 1949, chloramphenicol, produced from Streptomyces venezuelae

bacteria, which was discovered in soil sample from Venezuela, isolated by David Gottlieb was

introduced into clinical practice (Hanekamp and Wijnands, 2004). Chloramphenicol is a potent,

broad-spectrum antibiotic drug, effective for both gram positive and gram negative bacteria, is

associated to serious toxic effects, especially bone marrow depression and fatal aplastic anaemia

in humans, and therefore is limited only to life-threatening cases (Forti et al., 2005 and Lucia and

Fernando, 2006). It has also been shown that chloramphenicol is toxic to humans, and diseases

such as leucopenia occurred. Eating shrimp contaminated with chloramphenicol does not make

people sick right away. The amount of chloramphenicol will built up in the body over time, if a

person continues to consume contaminated shrimps. Feder et al., 1981 has identified two

potentially fatal adverse effects after consuming chloramphenicol such as aplastic anemia and

grey syndrome. A daily dose of 2 mg over 40 days can cause death (Fraunfelder et al., 1982).

Chloramphenicol is colourless and soluble in water (Clarke, 2004). Apart from its use

in human, chloramphenicol is widely used in veterinary because it is cheap and effective (Shen et

al., 2006 and Vinas et al., 2005). Due to the well-known risk of irreversible bone marrow

disorders and the absence of safe residue levels, the European Union (EU) has prohibited it for

veterinary use in 1994 and no maximum residue limit (MRL) has been established for this

antibiotic.

4

Nusat Khidmat Maklunwt Aliademik UNNERSIT[ MALAYSLI 9ARAWAK

The high chloramphenicol doses lead to significant residual levels in the shrimp

products, the import of which has been banned since 2002 by the USA, Japan and the EU

(Ponprateep et al., 2009). Despite this legal ban, chloramphenicol has recently been found in

several animal-derived foods, mainly aquaculture products (Kleter et al. 2009; Kesarcodi-

Watson et al. 2008; Lucia and Fernando, 2006). This drug is still used in Asian countries, which

are known to be the greatest producers of seafood (Storey et al., 2003). Chloramphenicol has

been found in several imported foodstuffs such as shrimp from Asia (Ding et al., 2005). The

European Union has reported finding chloramphenicol in shrimp from Malaysia (Shrimp News

International May 17,2004). As a consequence, these food imports have been rejected by

European authorities because significant amount of chloramphenicol residues had been detected.

Another finding of chloramphenicol containing shrimps was found in the United States (FDA,

2002). Due to this, the Malaysian government has made it compulsory to monitor

chloramphenicol in all shrimp produces. Thus, it is very important that no chloramphenicol or

products containing it are used in the raising and processing of shrimp, not at any stage or any

dose (Saw, 2007). In addition, the possible impact of chloramphenicol on the environment is a

risk for development of resistance.

Antibiotics are also commonly detected in the environment as contaminants

(Castiglioni et al., 2008). The major part of the drugs enäüp in the environment, either directly

due to excessive feeding and reduced appetite of the cultured organism or indirectly after having

passed through the organism under treatment. Antibiotics normally used for therapeutic

treatment may not be completely metabolized and can therefore contaminate waste, surface and

ground waters (Calamari et al., 2003; Castiglioni et al., 2005 and Sacher et al., 2007).

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Antibiotics also enter the environment as a result of leaching from faeces and uneaten

antibiotic feed. It has been estimated that a minimum of 75% of most of the antibiotics in feed

used in aquaculture systems are transported to the surrounding environment and accumulate in

the sediment (Castiglioni et al., 2005). These antibiotics used by the farmers have negative

effects on the cultured shrimps, cause a risk for food safety, occupational health, and negative

effects on adjacent ecosystems (Graslund et al., 2003; Holmstrom et al., 2003; Biyela et al.,

2004 and D'Costa et al., 2006). The usage of antibiotics has therefore brought about great

concern. The unconsumed food and shrimp faecescontaining antibiotics reach the sediment or

can be washed by currents to distant sites. Once in theenvironment, these antibiotics can be

ingested by wild fish, shrimp and other organisms.

The widespread use of antibiotics and the common practice of preventive usage is a

risk for development of resistance towards antibiotics (American Academy of Microbiology,

1999; Inglis, 2000 and Balcazar et al., 2006). Therefore, the use of antimicrobial agents in

aquaculture has been reported to result in the increase in the frequency of strains resistant to

these agents (Balcazar et al., 2006; Nakayama et al., 2006 and Tu et al., 2009). Potentially these

resistant strains can have an impact on the therapy of fish diseases, the therapy of human

diseases or the environment of the fish farms. The presence of antimicrobial agents even at low

concentration through leaching or continued usage clll still promote the development of

antimicrobial-resistant strains (Smith et al., 2003; Kummerer, 2003 and Cabello, 2006). The

aquatic and terrestrial ecosystems are the biggest known reservoirs of antibiotic-resistant

bacteria. Resistance may cause difficulties to treat bacterial infections in the cultured shrimp, and

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