microbiological quality of marketed penaeus monodon shrimps in ...

MICROBIOLOGICAL QUALITY OF MARKETEDPENAEUS MONODON SHRIMPS IN NORTHWESTERN PROVINCE, SRI LANKAKAMALIKA HARSHINI UBEYRATNE JANRABELGEA THESIS SUBMITTED TO CHIANG MAI UNIVERSITY ANDFREIE UNIVERSITÄT BERLIN IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFMASTER OF VETERINARY PUBLIC HEALTHCHIANG MAI UNIVERSITY AND FREIE UNIVERSITÄT BERLINSEPTEMBER 2007

ivI am grateful to Associate Professor Dr. Peter Paulsen from the Institute of MeatHygiene, Meat Technology and Food Science, University of Veterinary MedicineVienna who offered me his guidance during the laboratory practicals.I would like to express my sincere gratitude to Ms. Iseult Rea for hercontributions to the English language edition in my thesis.I would personally like to thank all the scientists, professors and all the lecturersassociated with the Master of Veterinary Public Health Degree Programme at theFreie Universität Berlin, Germany, the University of Veterinary Medicine Vienna,Austria and Chiang Mai University, Thailand who have contributed immensely byproviding useful information, advice and skills. Besides, I would like to thank Ms.Juliane Fischer, Ms. Louise Le Bel and staff of the Veterinary Public Health Centerfor Asia Pasific who supported me throughout this study.In addition, I am greatly indebted to many of my former teachers: Dr. (Ms.)Niranjala De Silva, Professor P. Abeynayake, Dr. (Ms.) Thula Wijewardana, Dr.K.F.S.T. Silva, and Professor H.W. Cyril for serving as referees and motivating me toparticipate in this International Programme.Let me also say ‘thank you’ to the following people: Dr. Udeni Edirisinghe at theFaculty of Agriculture, University of Peradeniya, Sri Lanka, for his guidance andinsightful comments, Dr. S.K.R. Amarasekara, Dr. J. Dharmawardana, Dr. A.D.N.Chandrasiri, Dr. M. Somaratne and laboratory staff of the Veterinary ResearchInstitute, Department of Animal Production and Health, Sri Lanka, for providing mewith the facilities to carry out the laboratory analysis with a great support. Thank youalso to Professor P. Abeynayake and staff, Department of Pharmacology and PublicHealth, Faculty of Veterinary Medicine and Animal Science, University ofPeradeniya, Sri Lanka, for their continuous support in sample processing throughoutthe research period.I would like to express my deep appreciation for Dr. K.J. Cooray, ConsultantMicrobiologist, Medical Research Institute, Colombo, Sri Lanka, for providing me

vavailable data of Salmonella surveillance in Sri Lanka. I am also thankful to Ms.Lashanthi, Statistical Unit, Ministry of Fisheries and Aquatic Resources, Sri Lanka,for furnishing me with available statistical data on fisheries. I would like to thank Dr.Iddya Karunasagar, Director of Research, Karnataka Veterinary, Animal and FisheriesScience University, India for providing me valuable journal articles.I am very grateful to my friends who participated from different countries ofSouth East Asia with aesthetic beauty; we developed a long friendship throughoutthese two years. I hope we will continue our friendship.Trin Utsahachant “Wat” from Thailand deserves a special mention because shemade things easy for me by helping me on most occasions as do Mr. and Ms.Nawaratne for their encouragement during my research study.Last, but not least, I wish to thank my family: my parents, for giving me life inthe first place, for educating me in both the arts and sciences. To my eldest sister whobecame a second mother to me, for her unconditional support and encouragement. Tomy brother who is like a father to me, for him listening to my complaints and foroffering guidance and help in difficult moments. To my immediate sister, for sharingher experiences, blessings and for inspiring me. To my brother-in-laws and sister-inlawfor instigating me to build on my intrinsic capabilities and to their small childrenfor giving me pleasure throughout my life.Thanks to Lord Buddha for giving me the perseverance and the ability toaccomplish the task.Kamalika Harshini Ubeyratne Janrabelge

viThesis TitleMicrobiological Quality of Marketed Penaeus monodonShrimps in North Western Province, Sri LankaAuthorMs. Kamalika Harshini Ubeyratne JanrabelgeDegreeMaster of Veterinary Public HealthThesis Advisory Committee Prof. Dr. Goetz HildebrandtChairperson (FU-Berlin)Asst. Prof. Dr. Rutch Khattiya Chairperson (CMU)ABSTRACTThe microbiological quality of marketed Penaeus monodon shrimps wasanalysed in terms of prevalence of Salmonella (including serotypes and antimicrobialresistance), Aerobic Plate Count (APC), Enterobacteriaceae, Escherichia coli andTotal Halophilic Plate Count. Samples were collected from the retail sale points inNorth Western Province in Sri Lanka. A total of 180 samples both from captured andcultured shrimps were involved.The overall prevalence of Salmonella in shrimps was 12.78 %. The prevalence ofSalmonella in captured shrimps and cultured shrimps was 14.44 % and 11.11 %respectively and differences in prevalences were not statistically significant (p=0.66).However, the prevalences of Salmonella among 14 different retail sale points differedand the difference was statistically significant (p

viiAll 23 strains isolated from shrimps were tested for antimicrobial resistanceusing 14 different antimicrobials including nalidixic acid, amoxycillin, ampicillin,gentamycin, erythromycin, chloramphenicol, ciprofloxacin, kanamycin, trimethoprim,sulphamethoxazole/trimethoprim, sulphonamides, tetracycline, streptomycin anddoxycycline hydrochloride. 100 % of all the 23 isolates of Salmonella were observedsusceptible for nalidixic acid, ampicillin, gentamycin, sulphamethoxazole/trimethoprim, chloramphenicol, ciprofloxacin, trimethoprim and tetracycline. Onestrain was susceptible to all 14 antimicrobials tested. Twenty-two strains weredetected for resistance and all these strains were resistant to two antimicrobials(erythromycin and sulphonamides) of which one was resistant to three antimicrobials(erythromycin, sulphonamides and amoxycillin). Out of 22 resistant serotypes S.Newport showed the highest resistance (50.00 %) followed by S. Weltevreden, E15,B4, F-67 each (9.09 %). The lowest was observed for S. Mbandaka, E10 and C7 each(4.55 %). The percentage antimicrobial resistant isolates from culture shrimps washigher than from capture shrimps without being statistically significant (p>0.05).Raw shrimps which have not undergone any further processing showed a lowernumber of samples meeting the standards. 48.1 % of samples tested met the standardsof ≤10 7 CFU/g for Aerobic Plate Count (APC). 32.4 % of samples tested met thestandards of ≤10 5 CFU/g for Enterobacteriaceae count. 50 % of samples met ≤10 2CFU/g for Escherichia coli count. There were no existed standards for TotalHalophilic Plate Count (THPC).A questionnaire survey revealed that there is a connection betweenmicrobiological contamination and the routine commercial operation at different retailsale locations.

viiiชื่อเรื่องวิทยานิพนธ คุณภาพทางจุลชีววิทยาของกุงกุลาดําที่จําหนายในจังหวัดตะวันตก เฉียงเหนือ ศรีลังกาผูเขียน นางสาว คามาลิกา ฮาชินิ อุเบรัด จันราเบลปริญญาสัตวแพทยสาธารณสุขศาสตรมหาบัณฑิตคณะกรรมการที่ปรึกษาวิทยานิพนธ ศ.ดร.กอซ ฮิลเดอบรานด ประธานกรรมการ (FU-Berlin)ผศ.ดร. รัชต ขัตติยะ ประธานกรรมการ (CMU)บทคัดยอการวิเคราะหคุณภาพทางจุลชีววิทยากุงกุลาดํา (Penaeus monodon) จากตลาดเพื่อหาความชุกแซลโมเนลลา(ซีโรไทป และการดื้อยาตานจุลชีพ) จุลินทรียทั้งหมด (Aerobic PlateCount) เอนเทอโรแบค-ทีริเอซี เอสเคอริเคีย โคไล และจุลินทรียฮาโลฟลลิคทั้งหมด(Totalhalophilic plate count) ทําการเก็บตัวอยางจากจุดขายปลีก ในจังหวัดตะวันตกเฉียงเหนือ ศรีลังกาโดยเปนตัวอยางกุงจํานวน 180 ตัวอยาง จากการเลี้ยงและการจับความชุกในภาพรวมของแซลโมเนลลาในกุงเทากับ 12.78 % โดยความชุกของแซลโมเนลลาในกุงจับและกุงเลี้ยงเทากับ 14.44 % และ 11.11% ตามลําดับโดยไมแตกตางอยางมีนัยสําคัญทางสถิติ (p=0.66) อยางไรก็ตามความชุกของแซลโมเนลลาในตลาดขายปลีก 14 แหงแตกตางกันอยางมีนัยสําคัญทางสถิติ (p

ixการทดสอบการดื้อยาตานจุลชีพของเชื้อทั้งหมด 23 สายพันธุที่แยกจากกุงตอยาตานจุลชีพ14 ชนิด ไดแกนาลิดิซิค แอซิด แอม็อคซีซิลลิน แอมพิซิลลิน เจนตามัยซิน อิริโทรมัยซินคลอแรมเฟนิคอล ไซโปรฟล็อคซาซิน คานามัยซิน ไตรเมทโธพริม ซัลฟาเมทธ็อคซาโซล/ไตรเมทโธพริม ซัลโฟนาไมด เตตราไซคลิน สเตรปโตมัยซิน และ ด็อกซีไซคลิน ไฮโดรคลอไรดทําการทดสอบเพื่อหาความไวตอยาตานจุลชีพของเชื้อทั้งหมด 23 สายพันธุตอ นาลิดิซิค แอซิดแอมพิซิลลิน เจนตามัยซิน ซัลฟาเมท-ธ็อคซาโซล/ไตรเมทโธพริม คลอแรมเฟนิคอลไซโปรฟล็อคซาซิน ไตรเมทโธพริม และเตตราไซคลิน พบวาเชื้อ 1 สายพันธุไวตอยาตานจุลชีพทั้ง 14 ชนิด เชื้อ 22 สายพันธุดื้อตอยาตานจุลชีพ 2 ชนิด (อิริโทรมัยซิน และซัลโฟนาไมด) โดยเชื้อ 1 สายพันธุ ดื้อตอยาตานจุลชีพ 3 ชนิด (อิริโทรมัยซินซัลโฟนาไมดและแอม็อคซีซิลลิน)จากตัวอยางเชื้อทั้ง 22 ซีโรไทปที่ดื้อยาพบวาแซลโมเนลลานิวพอรต ดื้อยามากที่สุด (50.00%) ตามดวย แซลโมเนลลาเวลเทฟเรเดน อี15 บี4โพลี่ II ชนิดละ 9.09% ซีโรไทปที่ดื้อยานอยที่สุดคือ แซลโมเนลลาแบนดากา อี10 และซี7ชนิดละ 4.55 % อัตรารอยละของการดื้อยาตนจุลชีพของเชื้อที่แยกไดจากกุงเลี้ยงสูงกวาจากกุงจับอยางไมมีนัยสําคัญทางสถิติ (p>0.05)กุงดิบซึ่งไมไดเขาขบวนการแปรรูปมีจํานวนนอยตัวอยางที่ไดมาตรฐาน 48.1 %ของตัวอยางมีจุลินทรียทั้งหมดนอยกวา 10 7 CFU/g 32.4 % ของตัวอยางมีเอนเทอโร-แบคทีริเอซีนอยกวา 10 5 CFU/g 50 % ของตัวอยางมี เอสเคอริเคีย โคไล นอยกวา 10 2 ตัวไมมีมาตรฐานสําหรับจุลินทรียฮาโลฟลลิคทั้งหมดการสํารวจแบบสอบถามแสดงถึงการเชื่อมโยงของการปนเปอนทางจุลชีววิทยาและการดําเนินการตามวิธีการคาขายประจําของสถานที่ขายปลีกแตละแหง

xTABLE OF CONTENTSPageACKNOWLEDGEMENTSiiiABSTRACT IN ENGLISHviABSTRACT IN THAIviiiLIST OF TABLESxvLIST OF FIGURESxviiABBREVIATIONS AND SYMBOLSxviii1. INTRODUCTION AND OBJECTIVES 11.1 Introduction 11.2 Objectives 72. LITERATURE REVIEW 92.1 Introduction 92.2 Non-indigenous bacteria 102.2.1 Salmonella 102.2.1.1 Historical aspects 102.2.1.2 Taxonomy and nomenclature 112.2.1.3 Serotyping 122.2.1.4 Biochemical characteristics 142.2.1.5 Growth parameters 162.2.1.6 Incidences of Salmonella in shrimps 162.2.1.7 Food-borne infections 172.2.2 Escherichia coli 182.2.2.1 Historical aspects 182.2.2.2 Biochemical characteristics 182.2.2.3 Growth parameters 192.2.2.4 Incidences of E. coli in shrimps 19

xi2.2.2.5 Food-borne infections 202.3 Indigenous bacteria 212.3.1 Halophilic bacteria 212.3.1.1 Historical aspects 212.3.1.2 Vibrio 212.3.1.3 Biochemical characteristics 222.3.1.4 Growth parameters 242.3.1.5 Incidences of Vibrio in shrimps 242.3.1.6 Food-borne infections 252.4 Antimicrobial use in aquaculture 272.4.1 Historical aspects 272.4.2 Antimicrobial resistance 282.4.2.1 Emergence of antimicrobial resistant 28bacterial strains2.4.2.2 Mechanisms of antimicrobial resistance 29in bacteria2.4.2.3 Incidences of antimicrobial resistance in 31aquaculture2.4.3 Antimicrobial resistance in Salmonella 312.5 Public health perspectives 332.5.1 Seafood safety and microbiological standards 332.5.2 Impact of food-borne hazards on health and the 35economy in developed countries2.5.2.1 Biological hazards 352.5.2.2 Antibiotic resistance 372.5.3 Impact of food-borne hazards on health and the 38economy in developing countries2.5.3.1 Biological hazards 382.5.3.2 Antibiotic resistance 402.5.4 Prevention and control of seafood- borne diseases 42

xii3. MATERIALS AND METHODS 443.1 Study design 443.2 Study method 443.2.1 Study area 443.2.2 Population 443.2.3 Sample 453.2.4 Sample size 453.2.5 Sampling plan 463.2.6 Questionnaire 493.3 Laboratory procedures 493.3.1 Aerobic Plate Count 493.3.2 Enumeration of Enterobacteriaceae 513.3.3 Rapid method to enumerate Escherichia coli 533.3.4 Total Halophilic Plate Count 563.3.5 Conventional culture method to detect Salmonella 583.3.5.1 Selection of colonies for confirmation 593.3.5.2 Biochemical confirmation 593.3.5.3 Serotyping 643.3.6 Antimicrobial sensitivity test for Salmonella 663.4 Questionnaire survey 683.5 Data management and statistical analysis 704. RESULTS 714.1 Source of the sample 714.2 Prevalence of Salmonella in marketed shrimps 714.2.1 Overall prevalence of Salmonella in shrimps 714.2.2 Prevalence of Salmonella in shrimps sold at 14 73different retail sale locations4.3 Salmonella serogroups and serotypes in Penaeus monodon 74shrimps4.4 Antimicrobial resistance of Salmonella in marketed shrimps 784.4.1 Antimicrobial resistance of Salmonella 79

xivREFERENCES 111APPENDICES 131APPENDIX A 131APPENDIX B 134DECLARATION 136CURRICULUM VITAE 137

xvLIST OF TABLESTablePage1. Examples of Salmonella nomenclature currently used in the11literature2. Biochemical and serological reactions of Salmonella 153. Growth parameters of Salmonella 164. Growth parameters of Escherichia coli 195. Biochemical characteristics of human pathogenic Vibrionaceae 23commonly encountered in seafood6. Growth parameters of the most important Vibrio spp. 247. Estimated occurrence of bacterial infections and intoxications in 39selected regions8. Typical growth of Salmonella colonies on selective solid media 599. Biochemical reactions in TSI agar 6110. Interpretation of biochemical tests results of Salmonella 6311. List of observed factors 6912. Number of samples of shrimps for Salmonella analysis 7113. Prevalence of Salmonella in marketed shrimps 7214. Prevalence of Salmonella in shrimps sold at the different retail sale 74locations15. Number and percentage of Salmonella serogroups isolated from the 76shrimps by different locations16. Serovars of Salmonella isolated from shrimps 7717. Percentage of antimicrobial resistance Salmonella 7918. Number and percentage of Salmonella strains resistant, intermediate 80and susceptible to antimicrobials tested

xviTablePage19. Antimicrobial resistance pattern by type of shrimp 8220. Antimicrobial resistance pattern by serotype 8221. Percentage of resistant serotypes 8322. Bacteriological profile of shrimps tested in terms of log CFU/g 8423. Bacteriological quality of marketed shrimps 8524. Summary results of potential factors and the levels for Salmonella 87contamination in shrimps (univariate analysis)25. Logistic regression of the risk factor associated with Salmonella 88contamination26. Summary results of potential factors and the levels for APC (log 89CFU/g) in shrimps (univariate analysis)27. Summary results of potential factors and the levels for91Enterobacteriaceae (log CFU/g) in shrimps (univariate analysis)28. Summary results of potential factors and the levels for Escherichia 93coli (log CFU/g) in shrimps (univariate analysis)29. Summary results of potential factors and the levels for Total 95Halophilic Plate Count (log CFU/g) in shrimps (univariate analysis)

xviiiABBREVIATIONS AND SYMBOLSµm micrometer(s)°C Degree Celcius°F Degree Fahrenheita wβ-lactam3M-ECAPCBPLSCDCCFCCFU/gCICIFTCLSICTDGHMDNADPDHSEAECEHECEIAEIECEPECETECEUFAOFEHDWater ActivityBeta lactamE. coli Petrifilm TestAerobic Plate CountBrilliant-green Phenol-red Lactose-SucroseCenter for Disease ControlCeylon Fisheries CorporationColony Forming Units per gramConfidence IntervalCentral Institute of Fisheries TechnologyClinical and Laboratory Standards InstituteCholera ToxinDeutsche Gesellschaft für Hygiene und MikrobiologieDeoxyribo Nucleic AcidDeputy Director of Health ServicesEntero Adherent E. coliEntero Haemorrhagic E. coliExport Inspection AgencyEntero Invasive E. coliEntero Pathogenic E. coliEntero Toxigenic E. coliEuropean UnionFood and Agriculture OrganizationFood and Environmental Hygiene Department

xixFSOsFWgGDPGHPGMPhHACCPHCHUSICMSFISOJETACARkgKP+LIALPSMDRminMKTTnmlmmMRDMRINACANACMCFNCCLSNWPOIEORFood Safety ObjectivesFresh Watergram(s)Gross Domestic ProductionGood Hygiene PracticeGood Manufacturing Practicehour(s)Hazard Analysis Critical Control PointHaemorrhagic ColitisHaemolytic Uraemic SyndromeInternational Commission on MicrobiologicalSpecification for FoodsInternational Organization for StandardizationJoint Expert Advisory Committee on Antibiotic ResistanceKilogram(s)Kanagawa phenomenon positiveLysine Iron AgarLipopolysaccharidesMulti Drug Resistantminute(s)Muller-Kauffmann Tetra Thionate novobiocinmillilitre(s)millimetre(s)Maximum Recovery DiluentMedical Research InstituteNetwork of Aquaculture Centres in Asia-PacificNational Advisory Committee on Microbiological Criteriafor FoodsNational Committee of Clinical Laboratory StandardsNorth Western ProvinceWorld Organization for Animal HealthOdds Ratio

xxpPABAPCARHRVSSLFARASPSSPSSSTECSUMFARSWtTDHTRHTSIUCDUSFDAVRBGAWHOWTOXLDProbability valuePara-Amino Benzoic AcidPlate Count AgarRelative HumidityRappaport-Vassiliadis medium with SoyaSri Lanka Fisheries and Aquatic Resources ActSanitary and PhytosanitaryStatistical Package for Social SciencesShiga Toxin producing E. coliStatistical Unit of Ministry of Fisheries and Aquatic ResourcesSea WaterMetric tonnesThermostable Direct HaemolysinTDH-related hemolysinTriple Sugar IronUniversity of California DavisUnited States Food and Drug AdministrationViolet Red Bile Glucose AgarWorld Health OrganizationWorld Trade OrganizationXylose-Lysine-Deoxycholate

1. INTRODUCTION AND OBJECTIVES1.1 IntroductionIn Asia, Penaeus monodon shrimps have been cultivated in traditional systemsfor centuries, with low productivity aimed for the domestic markets. Shrimpaquaculture which is oriented for export market is a fairly recent industry that took offin the mid 1970’s. With improved technologies and the introduction of formulatedfeeds, the industry boomed in the following decade. In 1975, the shrimp aquacultureindustry supplied to 2.5 % of total shrimp production, progressively increased toaround 30 % in the 1990’s. Today shrimp farming comprise only 3-4 % of globalaquaculture production in terms of weight, but almost 15 % in terms of value.Shrimps are one of the most important commodities of the global fishery trade(Bhaskar et al., 1995). Participation for the global shrimp trade by developingcountries are greater and about 75 % of the shrimp catches worldwide, whether fromculture or capture, originate from the developing countries (Bhasker et al., 1998).Around 80 percent of cultured shrimps come from Asia, with Thailand, China,Indonesia and India as the top producers. In the Western hemisphere, Ecuador is themajor shrimp producing country. The giant tiger or black tiger shrimp–Penaeusmonodon–accounts for more than half of the total shrimp aquaculture output(Rönnbäck, 2001).However, in Sri Lanka where there has not been a tradition of aquaculture,shrimp farming was slower to take hold. Interest in fish culture first appeared duringthe 1950’s. Shrimp culture was initiated during the last decade and before this timeshrimp production in Sri Lanka was based solely on capture fisheries. Shrimp culturestarted in the early 1980’s and the first commercial production entered the market in1984 in very small quantities. During 1992, the total production of cultured shrimpswas around 1,200 tonnes (FAO/NACA, 1995). At present, shrimps account for a

2significant portion of export revenue, in the agricultural and fisheries sector onlysecond to tea in 1998 (Cattermoul and Devendra, 2002). The percentage contributionof shrimps to the total value of all aquatic products exported varied between 48.5 %and 70.3 % between 1985 and 1992. The Statistical Unit of the Ministry of Fisheriesand Aquatic Resources reported annual fish production, which includes marine fishcatch, inland and aquaculture, at 286,370 Metric tonnes (t) in 2004 (SUMFAR,2004a). That same year prawn production, both aquaculture and wild capture, lay at2,390 and 10,730 t respectively. Export quantities of fish and fishery productsamounted to 13,681 t, these included prawns, lobsters, crabs, Beche de Mer,ornamental fish, chank and shells, shark fins, molluscs, fish maws, fish and others.The contribution of prawns was 30 % in quantity and 26 % in value (SUMFAR,2004b). The fishery sector is an important part of Sri Lanka’s economy and itcontributed around 1.8 % to the Gross Domestic Production (GDP) in 2004(SUMFAR, 2004c). Of the total fish landings of the country, about 95 % is handledby the private sector. About 70 % of landings of fresh fish are transported to urbanmarkets. A small percentage (less than 3 %) is handled by the Ceylon FisheriesCorporation (CFC), a Government entity (FAO, 2006). Fish is the most importantsource of animal protein consumed in the country, with about 60 % of the populationdepending solely on fish for their protein requirement, and accounting for nearly 65 %of the total animal protein consumed (Wijegoonawardena and Siriwardena, 1996a).Per capita consumption of fish (Kg/year/person) was 9.39 in 2003. The proportion ofprawns in the average household fish consumption/month was 3 % that the same yearaccording to the Ministry of Fisheries and Aquatic Resources (SUMFAR, 2003).As a highly prized seafood delicacy, shrimps and prawns are cash crop grownmainly for the affluent export (and urban) markets. From farms in Southeast Asia,East Asia, South Asia and South America, shrimps are exported to the major marketsin Japan, the USA and Europe; while domestic consumption accounts for only 5-20%, foreign markets absorb 80-95 % of total farmed production. Often, productsrejected for the export market due to small size, bacterial load, or chemical residuelevels are shunted to local markets (Primavera, 1994).

3There has been an increase in awareness about the nutritional value and healthbenefits of fish consumption in the last two decades. On the other hand, seafood isalso known to have been responsible for a significant percentage of food-bornediseases (Karunasagar et al., 2005; Wallace et al., 1999).In seafood related outbreaks a wide variety of viruses, bacteria, and parasiteshave been implicated, which are reported worldwide. Consumption of raw orundercooked seafood is the factor most commonly associated with infection (Butt etal., 2004). There is ample epidemiological evidence, particularly from Japan, thatconsumption of raw fish is indeed the cause of many outbreaks of food-bornediseases. Biotoxins and histamines make up a large proportion of these outbreaks(Huss, 1997). Though viruses are the most common cause of seafood relatedinfections, most of the hospitalisation and deaths are due to bacterial agents (Butt etal., 2004). As a consequence, food safety and quality aspects in trade becameimportant, since fresh food is more prone to certain microbiological contamination(Sawhney, 2005). Incidences of Salmonella in different shrimp products have beenreported by a number of investigators in India (Kumar et al., 2003). The sources ofcontamination have been described by different studies in numerous ways. Mølbak etal., 2006 have described that it is well established that the about 2500 non-typhoidSalmonella serotypes are widely distributed in nature, including in the gastrointestinaltracts of mammals, reptiles, birds and insects. Therefore, Salmonella reaches aquaticenvironments through faecal contamination. Increasing evidence that certainSalmonella types may also be part of the indigenous micro-flora in tropicalaquaculture has been reported (Huss et al., 2000). Furthermore, contamination ofshrimp products with Salmonella may be caused by poor hygiene standards duringprocessing rather than originating from shrimp ponds according to (Dalsgaard et al.,1995). Hence any presence of Salmonella in raw frozen shrimps may constitute apotential health hazard due to the food habit of consuming raw or slightly cookedshrimps (Dalsgaard et al., 1995).Moreover, bacteria of the genus Salmonella are widespread and an importantcause of food-borne infections in humans. They are the most frequent etiologic

4bacterial agents of food-borne disease outbreaks. Thus, Salmonella has an immensevalue as a specific pathogen internationally. The global Salmonella survey,conducted in the World Health Organization (WHO) Member States by WHO and theCenter for Disease Control (CDC), reported that the three serotypes, Enteritidis,Typhimurium and Typhi accounted for 76.1 % of all Salmonella isolates reported in1995. Enteritidis was the most frequently isolated serotype in 35 countries, followedby Typhi (12 countries) and Typhimurium (8 countries). The global pandemic ofSalmonella Enteritidis continued to spread as data revealed a rise globally from 25.6% in 1990 to 36.3 % in 1995. This increase was observed in most regions: American(11.3 % in 1990 and 42.6 % in 1995), Eastern Mediterranean (2.8 %/16.0 %),European (47.2 %/58.6 %), Western Pacific (8.9 %/32.3 %), African (7.1 %/9.6 %),but not in the Southeast Asian region (11.9 %/2.6 %).The trends in the proportion of isolates that were Typhimurium were morevariable. This decreased in the American (20.8 %/8.0 %) and European region (20.2%/17.6 %), and increased in the Southeast Asia (9.5 %/19.6 %), EasternMediterranean (4.5 %/4.9 %), Western Pacific (17.4 %/ 26.0 %), and African region(12.9 %/15.9 %) between 1990 and 1995.Over the same time period, the global proportion of isolates that were Typhidecreased from 15.3 % in 1990 to 12.1 % in 1995, and sharp decreases were observedin the American (13.4 %/ 1.9 %), Eastern Mediterranean (36.1 %/18.9 %), andWestern Pacific regions (11.8 %/2.5 %), compared with the African (34.5 %/34.2 %),European (0.8 %/ 1.3 %), and the Southeast Asian regions (37.8 %/39.1 %) (Herikstadet al., 2002).Serotypes Typhi (S. Typhi), S. Paratyphi and S. Sendai are highly adapted tohumans. S. Typhi and S. Paratyphi have humans as their main reservoir, and entericfever (typhoid and paratyphoid fever) as their most important clinical manifestation.Enteric fever continues to be an important cause of morbidity and mortality indeveloping countries. In Sri Lanka, enteric fever is endemic. Many of the DeputyDirector of Health Services (DPDHS) Divisions show a low prevalence. Although

6human and animal pathogens may threaten the viability of export markets. SomeEuropean countries found residues of chloramphenicol in tiger shrimps imported fromChina, Indonesia, and Vietnam (Holmström et al., 2003).In Sri Lanka, the prevailing diseases among the crustaceans are named Yellowhead disease, Infectious hypodermal and haematopoietic necrosis, White-spot disease,Baculoviral midgut gland necrosis, Gill associated virus disease, Spawner mortalitysyndrome (Midcrop mortality syndrome) (SLFARA, 1996). During 1988-1990 and in1996 Sri Lanka experienced two major disease outbreaks due to viral infection(Jayasinghe and Macintosh, 1993). Moreover, it was reported that some localisedoutbreaks of disease in farms occurred, mainly related to bacterial infections(Wijegoonawardena and Siriwardena, 1996a). In 2001 Senarath and Visvanathanreported that disease outbreaks were due to the rapid expansion of the shrimp industryin North Western Province. There is little information on the amounts of differentchemotherapeutants used in the shrimp farming industry.It was stipulated by the Fisheries and Aquatic Resources Act, No.2 of 1996 thatuse or administration of substances such as chloramphenicol, nitrofurans (includingfurazolidone) and malachite green is prohibited; fish on a regime of antibiotics mustnot be harvested for placement on the market for human consumption until fifty daysafter completion of the regime and the supervision of the use of antibiotics in fish feed(SLFARA, 1996).The centre of shrimp farming has developed in the North Western Provincecoastal area of Sri Lanka because the coast of Sri Lanka is endowed with a number oflagoons which are ideal for the establishment of shrimp farms (Cattermoul andDevendra, 2002). Therefore, it is one of the most important fishing grounds in SriLanka. The coastline of the North Western Province is approximately 120 Km long.The major brackish water bodies in this province are Puttalam lagoon, Mundal lagoonand Chilaw lagoon (FAO/NACA, 1995). North Western Province consists of twodistricts, Kurunegala and Puttalam of which the major fishing towns are Chilaw andPuttalam. In 2005, inland and aquaculture fish production in the districts Kurunegala

7and Puttalam amounted to 2140 and 7210 t respectively (SUMFAR, 2005). There aredifferent commercial groups of fish catch available at the local markets in NorthWestern Province and various hygienic practices followed in the chain of commercialoperation. There is no monitoring system adopted for the quality assurance of localconsumption.1.1.1 Significance of this studyThe microbiological quality of marketed shrimps is an important study areabecause of the/in order:1. Lack of information on micro-flora of shrimps sold at retail sale locations.2. To assure the safety and quality of shrimps as demanded by the increasingconsumer awareness.3. To provide the information to Veterinary Public Health Authorities forestablishing safety guidelines for shrimp products.In this present study, the microbiological quality of Penaeus monodon shrimpspurchased from retail sail points at the time of sale was investigated.1.2 Objectives1.2.1 Main objectives1 . To determine the prevalence, the serotypes and the antimicrobial resistanceof Salmonella in marketed shrimps (Penaeus monodon) in local markets of NorthWestern Province in Sri Lanka.2 . To find out the associated risk factors for the Salmonella contamination.

81.2.2 Secondary objectivesTo determine the microbiological contamination and hygienic status by1.2.2.1 Escherichia coli count in Penaeus monodon shrimps1.2.2.2 Microbial load in retail shrimps in terms of Aerobic Plate Count1.2.2.3 Enterobacteriaceae count in Penaeus monodon shrimps1.2.2.4 Total Halophilic Plate Count in Penaeus monodon shrimps at differentlocations and times of the day.

2. LITERATURE REVIEW2.1 IntroductionThe epidemiology of food-borne diseases is changing. New pathogens haveemerged, and some have spread worldwide. The threats of new food-borne diseasesare occurring for a number of reasons, through the globalization of the food supply,the inadvertent introduction of pathogens into new geographic areas, throughtravellers, refugees, and immigrants exposed to unfamiliar food-borne hazards whileabroad, through changes in micro-organisms, through changes in the humanpopulation (the number of highly susceptible persons is expanding worldwide becauseof ageing, malnutrition, HIV infections and other underlying medical conditions), andchanges in lifestyle. It is well documented that raw or under-processed seafoodprovides important epidemiological pathways for food-borne disease transmission(WHO, 2002).Consumption of seafood has increased over the last decade. This trend isexpected to continue both for prepared and for fresh or frozen varieties (Ahmed,1991). The likelihood of contamination of raw material and/or foods by hazardousagents is equally applicable to seafood when compared to any other food. Changes inthe level or frequency of the hazard over time depend on processing, preservationparameters and storage conditions (Huss, 2003).Seafood may be contaminated by numerous means from the surrounding water.Some food-borne agents are permanent residents of the marine environment (Klinger,2001). The water temperature is naturally having a selective effect. Thus morepsychrotrophic organisms (C. botulinum and Listeria) are common in Arctic andcolder climates, while the more mesophilic types (V. cholerae, V. parahaemolyticus)represent part of the natural flora on fish from coastal and estuarine environments of

10temperate and warm tropical zones (Huss, 1994). Another way contaminants gainaccess to products is through contamination of the marine environments by urbaneffluents. Therefore, bacteria present in seafood mainly comprise two groups,indigenous and non-indigenous (Klinger, 2001; Huss, 1994).2.2 Non-indigenous bacteria2.2.1 Salmonella2.2.1.1 Historical aspectsThe genus Salmonella was created in 1900 by Ligniēres and named in honour ofD.E. Salmon, the American veterinary pathologist who first described Salmonellacholerae-suis (Adams and Moss, 2000). Salmonella, belongs to the family ofEnterobacteriaceae. They are Gram-negative, non-spore-forming rods ( typically0.5µm by 1-3 µm) which are facultatively anaerobic, catalase-positive, oxidasenegativeand are generally motile with peritrichous flagella, except non-flagellatedvariants, such as Salmonella enterica serovar Pullorum and Salmonella entericaserovar Gallinarum (Doyle et al., 2001; Adams and Moss, 2000). The genusSalmonella consists of over 2500 serovars, as determined by its somatic (O) andflagellar (H) antigens (Cai et al., 2005). The serotypes are closely related, many ofwhich are potentially pathogenic for humans and/or animals (Yan et al., 2003). Theantigenic formulae of Salmonella serotypes are defined and maintained by the WHOCollaborating Centre for Reference and Research on Salmonella at the PasteurInstitute, Paris, France, and new serotypes are listed in annual updates of theKauffman-White scheme (Brenner et al., 2000). An antigenic scheme for theclassification of salmonellae was first proposed by White in 1926 and subsequentlyexpanded by Kauffmann in 1941 into the Kauffmann-White scheme (Doyle et al.,2001).

112.2.1.2 Taxonomy and nomenclatureThe taxonomy and nomenclature of Salmonella is rather complex and scientistsuse different systems to refer to and communicate about this genus. The Center forDisease Control and Prevention has adopted as its official nomenclature the followingscheme (Brenner et al., 2000). The genus Salmonella contains two species, each ofwhich contain multiple serotypes. The two species are S. enterica and S. bongori. Thespecies S. enterica is divided into six subspecies, which are referred to by a Romannumeral and a name (I, S. enterica subsp. enterica; II, S. enterica subsp. salamae;IIIa, S. enterica subsp. arizonae; IIIb, S. enterica subsp. diarizonae; IV, S. entericasubsp. houtenae; and VI, S. enterica subsp. indica) (Popoff, 2001). Subspecies V isnow considered to be a separate species and moved into S. bongori. Subspeciesclassification is primarily based upon chromosomal DNA hybridization andmultilocus enzyme electrophoresis (Brenner et al., 2000; Yan et al., 2003). S.bongori has 21 serotypes which are usually isolated from cold-blooded animals or theenvironment but rarely from mammalian sources including humans (Yan et al., 2003).The most recent proposal to introduce some taxonomic rectitude is to use thenon-italicized serovar name after the species name so that nomenclature becomes S.enterica subsp. enterica ser. Typhimurium (Adams and Moss, 2000).Table 1: Examples of Salmonella nomenclature currently used in the literature(Brenner et al., 2000)Complete nameS. enterica subsp. enterica ser.TyphiS. enterica subsp. enterica ser.TyphimuriumS. enterica subsp. salamae ser.GreensideCDCdesignationSalmonellaser. TyphiS. ser.TyphimuriumS. ser.GreensideOther designationsSalmonella typhiSalmonella typhimuriumS. II 50:z:e,n,x, S.greenside

12S. enterica subsp. arizonaeser.18:z 4 ,z 23S. IIIa 18:z 4 ,z 23S. enterica subsp. diarizonae ser.60:k:zS. enterica subsp. houtenae ser.MarinaS. enterica subsp. indica ser.Srinnagar“Arizona hinshawii” ser.7a,7b:1,2,5S. IIIb 60:k:z “A. hinshawii” ser.24:29:31S. ser. Marina S. IV 48:g,z 51:-, S. marinaS. ser.SrinagarS. bongori ser. Brookfield S. ser.BrookfieldS. VI 11:b:en,x, S.srinagarS. V 66:z 41:-, S. brookfield2.2.1.3 Serotyping1. Conventional serotypingThe biochemical identification of food-borne and clinical Salmonella isolates isgenerally coupled to serological confirmation (Doyle et al., 2001). The slideagglutination test is commonly used in serotyping for Salmonella based on theKauffman-White scheme which involves more than 250 antisera (Cai et al., 2005): acomplex and labor-intensive technique involving the agglutination of bacterial surfaceantigens with Salmonella-specific antibodies. These antigens include somatic (O)lipopolysaccharides (LPS) on the external surface of the bacterial outer membrane,flagellar (H) antigens associated with the peritrichous flagella, and the capsular (Vi)antigen, which is a superficial antigen overlying the O antigen and occurs only inSalmonella serovars Typhi, Paratyphi C, and Dublin (Gianella, 1982; Adams andMoss, 2000; Doyle et al., 2001). Many Salmonella show diphasic production offlagellar antigens and each strain can spontaneously and reversibly vary betweenthese two phases with different sets of H antigens, in phase 1 or the specific phase, thedifferent antigens are designated by small letters, and in phase 2 or the group phase,the antigens are numbered. The organisms tend to change from one phase to the other(Dauga et al., 1998). Baudart et al., 2000, reported that the Polyvalent Salmonella Oand H antisera were used to obtain a presumptive diagnosis, and the definitiveantigenic formula was determined by using monovalent antisera. Because antigen O

13or H is not always expressed, sometimes no agglutination was observed for someisolates (approximately 1 % of isolates from the environment).1.1 Antigenic structure of Salmonella1.1.1 O (somatic) antigensThese somatic antigens represent the side chains of repeating sugar unitsprojecting from the outer lipopolysaccharide layer of the bacterial cell wall (Gianella,1982). More than 60 different O-antigens have been identified and given Arabicnumerals. The capsular antigen has the ability to mask O antigens, thus making thelatter unavailable for agglutination with the corresponding O antibodies. The capsularantigens are heat labile and following their destruction by boiling, the heat stable Oantigens are available for antibody agglutination (Todar, 2006).1.1.2 H (flagellar) antigensThe H antigens located in the flagella of salmonellae are typically proteinaceousin composition and relatively heat labile. These H antigens exist as two forms; aspecific, or phase one form and a group, or phase two form. A given strain ofSalmonella may contain one (monophasic) or both (biphasic) forms (Defigueiredo andSplittstoesser, 1980).1.1.3 Vi (capsular) antigensAlmost all strains of S. Typhi form the Vi-antigen as a covering layer outsidetheir cell wall. The Vi-antigen is found mostly in freshly isolated strains which mustbe destroyed before this organism will react with group D somatic antisera(Defigueiredo and Splittstoesser, 1980).2. Phage typing

14Phage typing has proven to be epidemiologically valuable in straindifferentiation within a particular Salmonella serotype. Phage typing utilizes theselective ability of bacteriophages to infect strains of Salmonella and thus enablingthe user to differentiate unique isolates. This has been used to describe pandemicclones of Salmonella, such as S. enterica serovar Typhimurium definitive type 104(DT 104) that causes severe gastrointestinal illness and is typically resistant tomultiple antimicrobials. Although phage typing is the classical epidemiological testfor S. enterica serovar Typhi, it is technically difficult and can be performed only byreference laboratories (Navarro et al., 1996).3. Molecular typingMolecular typing techniques are favoured for strain discrimination in non-phagetypableserotypes of Salmonella (Chisholm et al., 1999). These typing methodsutilize restriction endonuclease digestion, nucleic acid amplification, or nucleotidesequencing techniques (Yan et al., 2003).2.2.1.4 Biochemical characteristicsSalmonellae in general produce H 2 S, produce acid from glucose, maltose,mannitol and sorbitol, utilize citrate, but do not ferment salicin, sucrose and lactose.They are catalase positive, oxidase negative, urease negative, indole negative andreduce nitrate to nitrite (Defigueiredo and Splittstoesser, 1980).There are some exceptions regarding the typical biochemical reactions ofSalmonella. Indole positive Salmonella serotypes have been reported by Wuthe et al.,1989; Pan et al., 1989. The Department of Bacteriology, National Institute ofInfectious Bacteriology and the Yamaguchi Prefectural Research Institute of PublicHealth have reported the presence of lysine decarboxylase-negative strains ofSalmonella enterica serovar Enteritidis (Masatomo et al., 2006). A strain wasidentified by Farmer et al., 1975 as Salmonella cubana was typical in all biochemical,serological, and bacteriophage reactions, except that it produced urease strongly.

15Table 2: Biochemical and serological reactions of Salmonella ( Andrews andHammack, 2006).Test or SubstrateResultSalmonellaPositiveNegativereaction (a)1. Glucose (TSI) yellow butt red butt +2. Lysine decarboxylasepurple butt yellow butt +(LIA)3. H 2 Sblackening no blackening +(TSI and LIA)4. Urease purple-red color no color change -5. Lysine decarboxylasebrothpurple color yellow color +6. Phenol red dulcitolyellow color and/or no gas; no color changebrothgas7. KCN broth growth no growth -8. Malonate broth blue color no color change - (c)9. Indole test violet color at surface yellow color at surface -10. Polyvalent flagellaragglutination no agglutination +test11. Polyvalent somatictestagglutination no agglutination +12. Phenol red lactosebroth13. Phenol red sucrosebrothyellow color and/or no gas; no color changegasyellow color and/or no gas; no color change -gas+ (b)- (c)

1614. Voges-Proskauer test pink-to-red color no color change -15. Methyl red test diffuse red color diffuse yellow color +16. Simmons citrate growth; blue color no growth; no color vchangea+, 90 % or more positive in 1 or 2 days; -, 90 % or more negative in 1 or 2 days; v,variableb, Majority of S. arizonae cultures are negativec, Majority of S. arizonae cultures are positive2.2.1.5 Growth parametersTable 3: Growth parameters of Salmonella (Bremer et al., 2003)MinimumwaterMinpHMaxpHMaxpercentageMinTemperatureMaxTemperatureOxygenrequirementactivity a wof NaCl (°C) 1 (°C) 10.92-0.93 4.0 9.5 8 6-7 45-47 Facultativeanaerobe 21 . M ost salmonellae in foods.2 . G rows with or without oxygen.3. Min=Minimum4. Max=Maximum2.2.1.6 Incidences of Salmonella in shrimpsIncidences of Salmonella in seafoods from India have been reported by a numberof investigators. Hatha and Lakshmanaperumalsamy (1997) reported 14.25 % of thefish samples and 17.39 % of crustacean samples were contaminated with Salmonella.The percentage prevalence reported varied among the different products: frozenpeeled and deveined shrimps accounted for 7.46 %, peeled and deveined shrimps for12 %, headless shell-on shrimps for 10 %, peeled and undeveined shrimps for 14 %

17(Kumar et al., 2003). Fonseka (1994) has reported the presence of Salmonella in farmshrimps in Sri Lanka. Results of the quality of shrimps sold in the markets ofHyderabad, India showed 11 % of the shrimp samples purchased were contaminatedwith Salmonella (Jonnalagadda and Bhat, 2004).The United States Food and Drug Administration (USFDA) noted an overallincidence of 7.2 % in imported and 1.3 % in domestic seafood containing Salmonelladuring a 9-year study (1990–1998) of 11,312 imported and 768 domestic seafoodsamples (Heinitz et al., 2000). In 1989, more than 8300 tonnes of frozen raw prawnswere detained by the USFDA due to contamination with Salmonella, contrary to theJapanese who have accepted Salmonella in raw frozen shrimps (Reilly and Twiddy,1992).2.2.1.7 Food-borne infectionsSalmonellosis is one of the most common and widely distributed food-bornediseases. It constitutes a major public health burden and represents a significant costin many countries (WHO, 2005). Early reports on the pathogenicity of Salmonellaindicated that ingestion of high numbers (10 5 to 10 7 ) of food-borne salmonellae was aprerequisite for human illness but more recent evidence suggests that a singleSalmonella cell may constitute a human infectious dose (D’Aoust, 2000). The clinicalcourse of human salmonellosis is usually characterized by abdominal pain, diarrhoea,nausea and sometimes vomiting (WHO, 2005). It is generally agreed that the foodchain is the major source of Salmonella infection for humans (Bezanson et al., 1981).Many factors including inadequate supplies of clean water, inadequate sanitarymeasures, lack of food hygiene and food safety measures have been responsible forincreased incidences of food-borne salmonellosis (Miko et al., 2005).Apart from specific genuine salmonellosis (typhoid and paratyphoid fever), theSalmonella infections of humans mostly account for food poisoning, caused by nontyphoidalSalmonella such as S. Typhimurium, S. Enteritidis, S. Cholerasuis, S. Hader,S. Virchow, S. Dublin etc. (Sakai and Chalermchaikit, 1996).

18Typhoid and the paratyphoid fevers which are the most severe of all diseasescaused by salmonellae include S. Typhi, S. Paratyphi A, S. Paratyphi B and S.Paratyphi C. Typhoid fever has the longest incubation period, produces the highestbody temperature, and the highest mortality rate. S. Typhi may be isolated from theblood and sometimes from the stool and urine of the victims prior to enteric fever.The paratyphoid syndrome is milder. Typhoidal Salmonella is carried only byhumans and is usually transmitted through direct contact with the faecal matter of aninfected person (Jay, 2000).Symptoms of salmonellosis can range from self-limiting diarrhoea to entericfever. Gastroenteritis generally appears 6 to 24 hours after ingestion of thecontaminated food. Symptoms resolve in 24 to 48 hours, but can last as long as oneweek. Abdominal pain, diarrhoea, vomiting, headaches and dehydration are typicalsymptoms and severity ranges from mild pain and little diarrhoea to extreme pain andbloody, severe diarrhoea. Secondary disease syndrome can be chronic and includeendocarditis, meningitis, pneumonia and arthritis (Patterson and Isaacson, 2003).2.2.2 Escherichia coli2.2.2.1 Historical aspectsEscherichia coli, originally known as Bacterium coli commune, was identified in1885 by the German paediatrician, Theodor Escherich (Peter et al., 2002). Strains ofEscherichia coli are a common part of normal microbial flora of animals, includinghumans. Most strains are harmless, but some cause diarrhoea. Strains carryingparticularly virulent properties have emerged as a serious hazard, with theconsumption of even low numbers of these organisms bearing the risk for life–threatening illness (ICMSF, 2002).2.2.2.2 Biochemical characteristics

19Escherichia coli is in the family Enterobacteriaceae. The organism is a Gramnegative,non-spore-forming, straight rod (1.1 µm x 2.0-6.0 µm) arranged in pairs orsingly; it is motile by means of peritrichous flagella or may be non-motile and mayhave capsules or microcapsules. It is a facultatively anaerobic micro-organism withan optimum growth temperature of 37 o C. Escherichia coli is oxidase-negative,catalase- positive, fermentative (glucose, lactose, D-mannitol, D-sorbitol, arabinose,maltose), reduces nitrate and is β-galactosidase-positive. Approximately 95% of thestrains are indole and methyl red positive (Fratamico and Smith, 2006). All strains ofE. coli are negative in the Voges-Proskauer test. Most strains do not hydrolyse ureaor produce H 2 S in triple sugar iron (TSI) medium and are unable to use citrate as asole carbon source (Simmon’s citrate negative) (Wilshaw, 2000).2.2.2.3 Growth parametersTable 4: Growth parameters of Escherichia coli (Bell and Kyriakides, 1998)MinimumwaterMinpHMaxpHMaxpercentageMinTemperatureMaxTemperatureOxygenrequirementactivity a wof NaCl (°C)(°C)0.95 4.0 9.5 8.5 7 46 Facultativeanaerobe1. Min=Minimum2. Max=Maximum2.2.2.4 Incidences of E.coli in shrimpsDespite being part of the normal intestinal flora, E. coli is found in aquaticecosystems. The strains isolated from aquatic environments of 13 districts inBangladesh revealed O161 to be the predominant serotype (19 %) followed by O55and O44 (12 %) and 11 % untypable. Serotype based-pathotyping of the E. colistrains revealed 47 %, 30 % and 6 % to belong to be Entero Pathogenic E. coli(EPEC), the Entero Toxigenic E. coli (ETEC) and the Entero Haemorrhagic E. coli

20(EHEC) pathotypes repectively (Alam et al., 2006). Kumar et al., 2005, reported thatestuaries and coastal water bodies, which are the major sources of seafood in India,are often contaminated by human activities and are associated with the widespreadoccurrence of E. oli in seafood. Food-borne outbreaks associated with Shiga toxinproducing E. coli (STEC) have been well documented worldwide. The occurrence ofthe STEC has been reported in a number of food products such as beef, pork, lamb,poultry and fish, and Kumar et al., reported in 2001 that STEC is prevalent inseafoods in India. Dalwara et al., 1987, reported that shrimps destined for import andexport industry tested positive for E. coli; these were 14 for fresh water (FW) shellon;5 for FW peeled deveined; 3 for Sea Water (SW) shell-on; 3 for SW peeleddeveined and 3 for seawater cooked shrimps in Singapore respectively.2.2.2.5 Food-borne infectionsEscherichia coli is often used as an indicator for faecal contamination; however,this perspective has changed with the identification of the ability of E. coli to causehuman illness. Some strains of E. coli are capable of causing food-borne disease,ranging from mild enteritis to serious illness and death. E. coli strains can cause avariety of diseases, including diarrhoea, dysentery, hemolytic uremic syndrome, andbladder and kidney infections. Different strains are usually associated with differentdiseases; this versatility of E. coli strains is due to the fact that different strains haveacquired different sets of virulence genes (Teophilo et al., 2002). EnterohemorrhagicE. coli (EHEC) or shiga toxin-producing E. coli (STEC), which cause hemorrhagiccolitis and hemolytic uremic syndrome, enterotoxigenic E. coli (ETEC), which inducetraveller’s diarrhoea, enteropathogenic E. coli (EPEC), which cause a persistentdiarrhoea in children living in developing countries, enteroadherent E. coli (EAEC),which provoke diarrhoea in children, enteroinvasive E. coli (EIEC) that arebiochemically and genetically related to Shigella species and can induce diarrhoea,diffusely adherent E. coli, which cause diarrhoea and are distinguished by acharacteristic type of adherence to mammalian cells (Smith and Fratamico, 2005).

222.3.1.2 VibrioThe genus Vibrio is a member of the family Vibrionaceae and consists of at least34 recognized species which include harmless aquatic strains as well as strainscapable of causing epidemics of cholera (Sechi, 2000). Vibrio is found in aquatichabitats with a wide range of salinities. It is very common in marine and estuarineenvironments and on the surfaces and in the intestinal contents of marine animals.There is evidence of some species in fresh water. Several species are pathogenic formarine vertebrates and invertebrates (Holt et al., 1994). Three species, V. cholerae,V. parahaemolyticus, and V. vulnificus, are well-documented human pathogens. V.mimicus is a recognized pathogen with similar characteristics to V. cholerae, exceptan ability to ferment sucrose. Other species within the genus, such as V. alginolyticus,V. fluvialis, V. furnissii, V. metschnikovii, and V. hollisae are occasional humanpathogens. Due to the ubiquitous nature of Vibrio spp. in marine aquaticenvironments, the presence of Vibrio spp. in marine and brackish water aquaculture isto be expected and water quality control measures have not made a substantialcontribution in this context (Dalsgaard, 1998). Vibrio species account for a significantproportion of human infections from the consumption of raw or undercooked shellfish(Kaysner and DePaola, 2004).2.3.1.3 Biochemical characteristicsMembers of the genus Vibrio are defined as Gram-negative rods that are straightor have a single, rigid curve. They are motile; most have a single polar flagellum,when grown in a liquid medium. Flagella are enclosed in a sheath continuous withthe outer membrane of the cell wall. They are facultatively anaerobic andchemoorganotrophic, having both a respiratory and a fermentative type ofmetabolism. The optimal temperature varies considerably; all grow at 20 o C, mostgrow at 30 o C. D-Glucose and other carbohydrates are catabolised with the productionof acid, but not gas (except for V. furnissii, V. gazogenes, and some strains of V.

23(Listonella) damsela). Most are oxidase positive (except V. gazogenes and V.metschnikovii) and produce catalase (Dalsgaard, 1998; Holt et al., 1994) .Table 5: Biochemical characteristics of human pathogenic Vibrionaceae commonlyencountered in seafood (Kaysner and DePaola, 2004).Test 1 2 3 4 5 6 7 8 9 10 11TCBS agar Y Y Y Y NG Y G G G Y GmCPC agar NG P NG NG NG NG NG NG Y NG NGCC agar NG P NG NG NG NG NG NG Y NG NGAGS KA Ka KK KK Ka KK KA KA KA KK ndOxidase + + + + + - + + + + +Arginine - - + + - + - - - + +dihydrolaseOrnithine + + - - - - + + + - +decarboxylaseLysine + + - - - + + + + V +decarboxylase0 % NaCl + + + +Growthin (w/v):3 % NaCl + + + + + + + + + + +6 % NaCl + - + + + + - + + + -8 % NaCl + - V + - V - + - - -10 % NaCl + - - - - - - - - - -Growth at 42°C + + V - nd V + + + V +Acidfrom:Sucrose + + + + - + - - - V -D-Cellobiose - - + - - - - V + + -Lactose - - - - - - - - + V -Arabinose - - + + + - - + - V -D-Mannose + + + + + + + + + V -D-Mannitol + + + + - + + + V + -ONPG - + + + - + + - + + -Voges-Proskauer+ V - - - + - - - + -

24Sensitivityto:10 µg O/129 R S R R nd S S R S R S150 µg O/129 S S S S nd S S S S R SGelatinase + + + + - + + + + + -Urease - - - - - - - V - - -(1= V. alginolyticus; 2= V. cholerae; 3= V. fluvialis; 4= V. Furnissii; 5= V. Hollisae; 6=V. Metschnikovii; 7= V. mimicus; 8= V. parahaemolyticus; 9= V. vulnificus; 10=Aeromonas hydrophila; 11= Plesiomonas shigelloides; TCBS = thiosulfate-citrate-bilesalts-sucrose; mCPC = modified cellobiose-polymyxin B-colistin; AGS = arginine-glucoseslant; Y = yellow; NG = no or poor growth; S = susceptible; nd = not done; G = green; V= variable among strains; R = resistant; P = purple; V = variable; KK = Slant alkaline/Buttalkaline; KA = Slant alkaline /Butt acidic; Ka = Slant alkaline/Butt slightly acidic)2.3.1.4 Growth parametersTable 6: Growth parameters of the most important Vibrio spp. (Berlin et al., 1999)http://www.ecolab.com/PublicHealth/Vibrio.aspTemperature(°C)pH Water activity NaCl %V. cholerae 10 – 43 5 – 9.6 0.97 – 0.998 0.1 – 4V. parahaemolyticus 5 – 43 4.8 – 11 0.94 – 0.996 0.5 – 10V. vulnificus 8 – 43 5 – 10 0.96 – 0.997 0.5 – 52.3.1.5 Incidences of Vibrio in shrimpsStudies describing the presence of vibrions in seafood in temperate regions andin Asia are quite common, and a few instances of vibrion-related disease, apart fromVibrio cholerae 01, have been reported in tropical countries (Nascimento et al., 2001).Wong et al., 1999, reported the presence of Vibrio parahaemolyticus in seafoodimported from Hong Kong, Indonesia, Thailand and Vietnam. The contaminationrates in shrimps, crabs, snails, lobsters, sand crab, fish and crawfish were 75.8 %, 73.3%, 44.3 %, 44.1 %, 32.5 %, 29.3 % and 21.1 %, respectively but none of the isolatespossessed the hemolysin genes (tdh, trh).

25Oysters, clams and mussels (filter feeders) are reservoirs of these microorganismsand the ingestion of raw or undercooked shellfish could cause an infection(Parvathi et al., 2004). Lhafi and Kühne, 2007, revealed in their study on bluemussels from the German Wadden sea, that among Vibrio isolates, Vibrioalginolyticus was the species most frequently detected (51.2 %), followed by Vibrioparahaemolyticus (39.5 %). Vibrio vulnificus was detected in 3.5 % of the samples.V. parahaemolyticus and V. vulnificus were not found in samples collected at lowwater temperatures.Hosseini et al., 2004, reported that the presence of V. parahaemolyticus, V.damsela, V. alginolyticus and V. fluvialis in shrimps in Iran and mentioned thatVibrios such as V. damsela, V. alginolyticus and V. fluvialis are indigenous to themarine environment and shrimps. Warm water shrimps imported into Denmark in1995 were contaminated with V. vulnificus in 3 of 46 frozen, raw shrimp samples butnot in any of the cooked products. The low prevalence in frozen raw shrimps wasassociated with poor resistance to the cold (Dalsgaard, 1998). Vibrioparahaemolyticus and Vibrio cholerae were examined only in seafood samples,mainly imported fish and shellfish. It was reported that both organisms were foundexclusively in imported, raw, frozen prawns and shrimps in the Republic of Cyprus(Eleftheriadou et al., 2002).2.3.1.6 Food-borne infections2.3.1.6.1 V. choleraeV. cholerae was first isolated in a pure culture in 1883 by Robert Koch. Theorganism produces the disease cholera, usually a disease prompted by poor sanitation.Humans are the only natural hosts for this organism. These organisms are sensitive toacid pH but tolerate alkaline pH (9.0-9.6) very well. A large dose is required toproduce the disease. Indeed, 10 11 vibrios given orally failed to produce illness but ifbicarbonate (e.g. Alka-Selzer®) precedes the inoculation, then only 10 4 were required

26(Fix, 2007). However, Vibrio cholerae serogroups O1 and O139 cause cholerathrough the production of an enterotoxin cholera toxin (CT) characterised by severewatery diarrhoea (Faruque et al., 1998). Vibrio cholerae non-O1 infects only humansand other primates and has genetic differences from the "O1" strain. Non-O1 strainscause a less severe disease. Pathogenic and non-pathogenic strains of the organismare found in marine and estuarine environments (Berlin et al., 1999).2.3.1.6.2 V. parahaemolyticusV. parahaemolyticus was first isolated in Japan in 1950 as a causative bacterialagent of food poisoning named ‘Shirasu (a half dried fish product) food poisoning’.This organism has been recognized as a major cause of gastroenteritis throughout theworld (Dalsgaard, 1998). Environmental strains of V. parahaemolyticus are typicallynot human pathogens. However, these strains cause disease in shrimps, oysters,mussels and other marine invertebrates. Most clinical strains of V. parahaemolyticusproduce a major virulence factor, the thermostable direct hemolysin (TDH), and aredesignated as Kanagawa phenomenon positive (KP+). Another virulence factor, theTDH-related hemolysin (TRH) is generally associated with the Kanagawaphenomenon negative (KP-) strains or with urease positive strains of V.parahaemolyticus (Nascimento et al., 2001). When ingested, V. parahaemolyticuscauses watery diarrhoea often with abdominal cramping, nausea, vomiting, fever andchills. Usually these symptoms occur within 24 hours of ingestion. Illness is usuallyself-limited and lasts three days. Severe disease is rare and occurs more commonly inpersons with weakened immune systems. V. parahaemolyticus can also cause aninfection of the skin when an open wound is exposed to warm sea water (CDC,2005b).2.3.1.6.3 V. vulnificus

27V. vulnificus is a rare cause of disease, it can cause disease in those who eat rawseafood, particularly oysters, or who have an open wound that is exposed to sea water.Persons who are immunocompromised, especially those with chronic liver disease,are at risk of V. vulnificus. Among healthy people, ingestion of V. vulnificus cancause vomiting, diarrhoea, and abdominal pain. In immunocompromised persons,particularly those with chronic liver disease, V. vulnificus can infect the bloodstream,causing a severe and life-threatening illness characterized by fever and chills,decreased blood pressure (septic shock), and blistering skin lesions. V. vulnificusbloodstream infections are fatal in about 50 % of cases. V. vulnificus can also causean infection of the skin when open wounds are exposed to warm sea water; theseinfections may lead to skin breakdown and ulceration (CDC, 2005a).2.4 Antimicrobial use in aquaculture2.4.1 Historical aspectsAntibacterial chemotherapy has been applied in aquaculture for more than 50years, with early attempts to use sulphonamides in the treatment of furunculosis introut and the tetracyclines against a range of Gram-negative pathogens. However,they didn't come into general use until the 1970’s when the sulphonamides were used,potentiated with trimethoprim. Since then, their use has grown both in numbers andquantity, as the problem of bacterial disease has increased (Inglis, 1996). The risk ofdisease in shrimp farming often increases with the culture intensity and high stockingdensities. High pond densities facilitate the spread of pathogens between ponds. Ashortage of clean water supply and insufficient waste removal lead to an overload ofmetabolites, shrimps became stressed by bad water quality, and are ultimately moreprone to becoming affected by disease (Kautsky et al., 2000). Otta et al., 1999,reported that aquatic animals such as fish and shrimp are in direct contact with themicro flora in the environment and most diseases are aroused by opportunisticpathogens which are already present in the environment and attack theimmunocompromised host.

28The potential of most veterinary antibacterials for use in aquaculture has beenconsidered, and now countries vary widely in the drugs they use in their aquaculturesystems (Inglis, 1996). A wide range of chemicals and drugs are being used, both forprophylactic treatment and to prevent or control parasitic, fungal and bacterialdiseases in shrimp hatcheries in Sri Lanka (Wijegoonawardena and Siriwardena,1996b).Antibiotic usage in aquaculture is predominantly applied with three methods ofadministration, namely: oral therapy (in feed), immersion therapy (bath, dip, flow orflush) or injection (Inglis, 1996). Other than fighting capabilities of antibioticsagainst bacterial diseases, fish farmers and livestock producers began using suchdrugs in animals because of the growth promoting capability. Antibiotics routinelyused for the treatment of human infections are also used for animals, for therapy,prophylactic reasons and growth promotion. In the case of growth promotion, subtherapeutic doses of antibiotics usually have been used, and this has contributed topromoting resistance. Research indicates that 70-80 % of the drugs used inaquaculture end up in the environment (Hernández, 2005).Unfortunately, this practice has led to problems, and global concern is nowcentred on treatment failures, environmental impacts and risks to human health.Treatments may fail for several reasons, but probably the most consistent andfundamental cause of their failure is the emergence of resistant bacteria (Inglis, 1996).2.4.2 Antimicrobial resistanceResistance takes two forms, one is inherent or intrinsic resistance, here thespecies is not normally susceptible to a particular drug. This may be due to theinability of the antibacterial agent to enter the bacterial cell and reach its target site, orto a lack of affinity between the antibacterial and its target (site of action), or anabsence of the target in the cell. The other is the acquired resistance, where the

29species is normally susceptible to a particular drug but certain strains express drugresistance (Hernández, 2005).2.4.2.1 Emergence of antimicrobial resistant bacterial strainsIn general, individual bacteria differ in their susceptibility to antibiotics. Whenmost of the bacteria are killed by a particular antibiotic, a few may survive andreproduce. When a bacterium, which is normally susceptible to an antibiotic, is ableto grow in the presence of antibiotic levels which would normally suppress growth orkill susceptible organisms is called resistance.There are two distinct stages in the emergence of antibiotic resistance bacteria.One is genetic change, by mutation (change in the DNA sequence of the relevant genein the chromosome that affects the uptake or action of the antibiotic) or by geneacquisition (an existing antibiotic resistance gene is transferred from a resistantbacterium to another bacterium). The other is the enrichment by selection, as theantibiotic continues to be used the resistance bacteria will make up a greater andgreater percentage of the population because the susceptible bacteria keep dying andthe resistant ones keep reproducing.Antibiotic resistance passes on from parent to offspring and it can also be passedon from one bacterial cell to another because of physical contact. Acquisition of theresistant determinants from one bacterium to another happens mostly via the transferof a small chromosome, either a plasmid or a conjugative transposon. Such transfersoccur most frequently between bacteria of the same strain and species, although crossspecies transfers can occur (JETACAR, 1999).2.4.2.2 Mechanisms of antimicrobial resistance in bacteria• Alteration of the targeted site of action

30This is typically accomplished by subtly changing the “targeted site ofaction” in such a way that the target can perform its function (although perhapsnot quite as well) but is not as sensitive to antimicrobial therapy; e.g. resistance tobacterial cell wall inhibitors such as penicillins.• Overproduction of the targetInstead of altering the sequence (nucleic acid or protein) of the antimicrobialtarget, it is possible to alter (increase) the copy number of the target so that theeffective drug concentration required to inhibit a specific process is increased; e.g.resistance of sulfonamides in case of para-aminobenzoic acid (PABA) conversionto dihydropteric acid and over expression of the enzyme dihydropteroate synthase.• Production of a new enzyme to bypass the targeted site of actionSome bacteria such as members of Enterobacteriaceae and Staphylococcusaureus can synthesize entirely new enzymes, which can carry out the PABA todihydropteroate conversion. These enzymes decrease the affinity for trimethoprimand sulfonamides.• Limiting access of the antibiotic to the targeted site of actionThis mechanism of action plays a significant role in bacterial resistance,particularly Gram negative bacteria. Changes in the lipid composition of the outermembrane may significantly contribute to resistance e.g. resistance toerythromycin. The expression of specific transporters is another way that bacterialimit access of an antimicrobial agent to its site of action eg. resistance totetracycline.• Modification of the antimicrobial agent

31Modifying the chemical structure of the antimicrobial can be accomplishedby a chemical break down (hydrolysis) of the antibiotic; bacteria which produce β-lactamases can hydrolyse the β-lactam ring which is essential for penicillin andcephalosporin antimicrobials or by chemical substitution of the antibiotic which isachieved by bacteria producing the enzyme chloramphenicol acetyltransferase,leading to the acetylation of chloramphenicol (Khan, 1999).2.4.2.3 Incidences of antimicrobial resistance in aquacultureIn aquaculture this antimicrobial resistance was experienced due to a horizontalgene transfer as a consequence of antimicrobial exposure. The included examples areAeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, Citrobacterfreundii, Lactococcus garviae, Yersinia ruckeri, Photobacterium psychrophilum andPseudomonas fluorescens. Sulfonamide resistance in Aeromonas salmonicida wasreported in1985 in the USA and, in the 1960’s; multi-resistant strains were observedin Japan. Transferable resistance plasmids are detected in the strains (SØrum, 2006).The development of resistance by shrimp pathogens such as Vibrio harveyi due toexposure to antimicrobials has been reported by Karunasagar et al., 1994.The transferability of resistance plasmids from fish pathogens and aquaticbacteria shows that these bacteria can act as reservoirs of antimicrobial resistancegenes that can be further disseminated and ultimately reach human pathogens, andthereby add to the burden of antimicrobial resistance in human medicine (SØrum,2006). Vibrio spp. are marine bacteria of which some can be pathogenic to humans.Resistance in V. cholerae and V. parahaemolyticus can be a result of human usage ofantimicrobials, but may also be a consequence of the use of antimicrobials inaquaculture (Hernández, 2005). Kruse et al., 1995, reported a large multi-resistanceplasmid conferring resistance to six antimicrobials was shown to be transferable from

32V. cholerae O1 to A. salmonicida, A. hydrophila, V. parahaemolyticus, V. cholerae, V.anguillarum, Shigella spp., E. coli and Salmonella.2.4.3 Antimicrobial resistance in SalmonellaThe emergence of antimicrobial-resistant Salmonella is associated with the useof antibiotics in animals raised for food; resistant bacteria can be transmitted tohumans through foods, particularly those from meat animals and aquaculture (Whiteet al., 2001; Doyle et al., 2001). A study into ready-to-eat shrimps which aredesigned for consumption without further heat treatment revealed the presence ofantibiotic resistance salmonellae which may result in consumer exposure to antibioticresistantbacteria (Marshal and Durán, 2005). The major public health concern is thatSalmonella will become resistant to antibiotics used in human medicine, therebygreatly reducing therapeutic options and threatening the lives of infected individuals(Doyle et al., 2001). In the last two decades, the emergence and spread ofantimicrobial-resistant pathogens, among them Salmonella, has become a serioushealth hazard worldwide. The routine practice of giving antimicrobial agents todomestic livestock as a means of preventing and treating diseases, as well aspromoting growth, is an important factor in the emergence of antibiotic-resistantbacteria that are subsequently transferred to humans via the food chain (Miko et al.,2005).Non-typhoidal Salmonella strains are a frequent cause of food-borne diseaseoutbreaks in the United States; they account for about 13 % of outbreaks reported tothe Center for Disease Control and Prevention (CDC) from 1993 to 1997 (Olsen,2000). Antimicrobial resistance is common among salmonellae and has beenincreasing, particularly in Salmonella enterica serotype Typhimurium, the mostcommon Salmonella serotype that most threatens food safety. In the 1990’s, a strainof S. Typhimurium that resistant to ampicillin, chloramphenicol, sulfonamides,tetracycline and streptomycin/spectinomycin, emerged in the United States andEurope; most of these isolates were phage definitive type 104 (Glynn et al., 1998;Miko et al., 2005). This microbe has two properties, antibiotic resistance and

33hypervirulence, which separates it from other Salmonella. DT 104 is the reigningmulti-resistant Salmonella, although other phage types, such as DT 208, may be theSalmonella that occupies the DT 104 niche in the future. Alternatively, another S.enterica serotype, such as Agona, may be elevated to the status of DT 104. Otherthan this penta group of antimicrobials, scientists are vigilant about the ceftriaxone,ciprofloxacin and fluoroquinolones resistance in DT 104 since ceftriaxone is thecurrent drug of choice against DT 104 in children and ciprofloxacin andfluoroquinolones are considered to be the last line of defence against DT 104 in adults(Carlson, et al., 2003). Moreover, Miriagou et al., 2002, reported the emergence ofnon-typhoid Salmonella enterica serotype Typhimurium strains that are resistant toexpanded-spectrum cephalosporins from a pediatric population in Iasi, Romania.2.5 Public health perspectives2.5.1 Seafood safety and microbiological standardsFood-borne infections and intoxications were common in ancient times. Ancientpeople based their food laws on religious beliefs. Trial and error were the main toolsfor food safety, and taboos were the primary tool for prevention of food-borneinfections. Certain foods were described as “unhealthy” and unfit for humanconsumption, particularly those of animal origin. Some animals were defined as“unclean” and were therefore unsuitable for consumption. The consumption of theblood of slaughtered animals was totally prohibited since blood was considered to bethe soul of the animal. Only when the study of food microbiology began to develop atthe end of the 19 th century could the pattern of food-borne infections be understood.Every product has a typical spectrum of microbial contaminants reflected by theexposure pattern of the product. There is no demand that a product intended forhuman consumption should be free of micro-organisms. As a result, microbiologicalstandards are recommended for most products (Klinger, 2001).The proper handling of fish between capture and its delivery to the consumer is acrucial element in assuring the final quality of the product. Standards of sanitation,the method of handling and the time/temperature of holding fish are all significant

34quality factors. Apart from a few exceptions, fish are considered to be free ofpathogenic bacteria of public health significance when first caught. The presence ofbacteria harmful to man is generally an indication of poor sanitation while handlingand processing the fish and the contamination is almost always of human or animalorigin (Sciortino and Ravikumar, 1999). Salmonella and E. coli strains, includingresistant strains, can be found in aquatic environments as a result of contaminationwith such bacteria from human, animal or agricultural environments. Sewagecontamination or run-off from agricultural areas with grazing animals to aquacultureoperations can result in the presence of resistant Salmonella and E. coli in productsfrom aquaculture (FAO/OIE/WHO, 2006).A number of microbiological tests of fish and fish products are used byauthorities to check that the microbiological status is satisfactory. The purpose ofthese tests is to detect pathogenic bacteria, indicator organisms or other types ofgeneral contamination or poor handling practices. When unsatisfactory aerobiccolony counts are encountered microbiologists should attempt to identify the microorganismsthat predominate. From these results, and additional detailed informationabout the food sample, it should be possible to provide a more helpful interpretationof high aerobic colony counts (Gilbert et al., 2000).In international trade, the most common reason for import refusal is "filthy"which describes that the product appears to consist in whole or in part of a filthy,putrid or decomposed substance. A major reason for refusal is microbial spoilage.Second in terms of reasons for rejection is the detection of Salmonella. Both cooked,ready-to-eat products and raw, frozen products are rejected if Salmonella is detected(Huss, 2003).The presence of Salmonella and a high bacterial count in the cooked and peeledfrozen shrimps exported from India has meant many problems in international trade inthe past. The local contribution for setting up standards came from the CentralInstitute of Fisheries Technology (CIFT) and Export Inspection Agency (EIA) inIndia (Hatha et al., 2003).

35The increase in imports of seafood from developing countries by the developedeconomies also resulted in the adoption of international guidelines for foodprocessing, such as Hazard Analysis Critical Control Point (HACCP) and EuropeanUnion (EU) guidelines. In an effort to control the microbial contamination of foods,the National Advisory Committee on Microbiological Criteria for Foods (NACMCF)and the International Commission on Microbiological Specification for Foods(ICMSF) have recommended microbiological criteria as a means of assessing theeffectiveness of the HACCP programme (Hatha et al., 2003).In Sri Lanka, the export of fish products (any aquatic organism whether piscineor not, and includes any shell fish, crustacean, pearl oyster, mollusc, holothurians andits young fry, egg or spawn) is governed by the Fish Products (Export) Regulations1998 framed by the Fisheries and Aquatic Resources Act No.2 of 1996. A processorwho wishes to export his products must register with the Department of Fisheries andAquatic Resources, the Competent Authority. A basic requirement to be met in orderto obtain registration is that the establishment shall establish and maintain a HACCPsystem to cover the products and processes concerned. The registered establishmentsare monitored for continual maintenance of the system. These inspections are carriedout by the Sri Lanka Standards Institution under the powers delegated by theCompetent Authority (SLS, 2007).In general, food safety criteria between countries differ and it may causedifficulty to compare protection given to a food product in international trade. Wherepractices in one country differ from the practices in the other country and still bothpractices provide safe products, comparison of the relative level of protection mayscale on a scientific basis. In this context, the International Commission on theMicrobiological Specifications for Foods (ICMSF) has contributed by recommendingpreventive, stepwise approach for the management of microbiological hazards infoods in international trade. The concept of the Food Safety Objectives (FSOs) hasbeen proposed by the ICMSF to communicate the level of hazards of foods in order tofacilitate the acceptance of different but equivalent processes (Stewart et al., 2002).

362.5.2 Impact of food-borne hazards on health and the economy in developed countries2.5.2.1 Biological hazardsIn industrialized countries, the percentage of the population suffering from foodbornediseases each year has been reported to be up to 30 % (WHO, 2007). Thegastro enteric disease is the most frequent disease associated with food-bornetransmission (Bremer et al., 2003). In the USA alone, the annual burden of foodborneinfection has been estimated at 76 million cases, 323,000 hospital admissions,and 5,000 deaths. The three most common identifiable causes of food-borne illness inthe USA in 1997 were Norwalk-like virus (9,200,000 cases), campylobacter(1,963,000 cases) and non-typhoidal Salmonella (1,342,000 cases). In the UK, therewere more than 18,000 notified cases of salmonellosis and almost 63,000 ofcampylobacteriosis in 2001. France reported 16,523 cases of salmonellosis in 1998and the Czech Republic reported more than 49,000. These figures revealed that thediseases caused high morbidity and relatively low mortality. However, the economicburden resulting from this level of illness is huge (Lancet, 2002). About 2-3 % ofcases of food borne diseases lead to long-term ill health, which is far more damagingto human health and the economy than the initial disease (WHO, 1999). A seafoodimplication in the case of food-borne diseases is estimated at 10-19 % out of 76million cases in the USA (Butt et al., 2004).Each year in the United States of America, food-borne diseases cost billions ofdollars. Government sources estimate the cost of human illnesses of seven foodbornepathogens to be between US$ 5.6 to 9.4 billion. The cost of salmonellosis inEngland and Wales in 1992 was estimated at between US$ 560 and 800 million. Over70 % of costs were directly associated with the treatment and investigation of casesand sickness-related absences from work (WHO, 1999).Emerging food-borne problems will have implications for the health status andthe economies of individual countries, as well as affecting international trade and the

37agreements that govern it (Venter, 2000). Food safety in international trade isgoverned by the World Trade Organization (WTO)/Sanitary and Phytosanitory (SPS)Agreement, which recognizes that governments have the right to reject importedfoods when the health of its people is, endangered (Stewart, 2002). With this legalobligation, unsafe food products are rejected in importing countries on healthgrounds, with obvious economic consequences. As an example, when cholera brokeout in Peru in 1991, more than US$ 700 million were lost in exports of fish and fishproducts. The human toll in Peru and its neighbouring countries, where more thanhalf a million people fell ill, amounted to more than 19,000 deaths (WHO, 1999).Among seafood consumers, the group at greatest risk appears to be consumers ofraw molluscs because of environmental contamination and naturally occurringvibrios. Seafood-borne illness reported by the CDC in the 10-year period 1978-1987totalled 558 outbreaks involving 5,980 cases. However, fish and shellfish constituteonly 10.5 % of all outbreaks and 3.6 % of all cases when food-borne illnesses from allfoods are considered. The number of people made ill from beef (4 %) and turkey (3.7%) exceeds the seafood total, whereas pork (2.7 %) and chicken (2.6 %) are slightlylower. If shellfish (2.3 %) and fish (1.2 %) are considered separately, the number ofreported cases from each is lower than for any animal meat category. Natural seafoodtoxins–mainly ciguatera and scombroid poisoning and, to a lesser extent, paralyticshellfish poisoning–were responsible for 62.5 % of all seafood-borne outbreaks ofillness.Fish-borne incidents due to causes other than natural toxins were only 9 % of alloutbreaks and 8 % of all cases. They resulted mainly from bacteria, includingcommon food-borne disease organisms, and from unknown etiology, suspected to beprimarily an enteric viruses or recontaminant vibrios (Ahmed, 1991).2.5.2.2 Antibiotic resistanceAntibiotic resistance is a serious clinical and public health problem of globalproportions. Drug choices for the treatment of common infectious diseases arebecoming increasingly limited, expensive, and, in some cases, useless, due to the

38emergence of drug resistance in bacteria and fungi. This loss of treatment options isthreatening to reverse much of the medical progress of the past 50 years becauseessential life-saving antibiotics are becoming less effective and there are feweralternatives available for treatment (Hernández, 2005; Marshall and Durán, 2005).Development of antimicrobial resistance causes enormous costs: the costs to bringout a new drug onto the market are estimated at a minimum of US$ 300 million(Byarugaba, 2004).2.5.3 Impact of food-borne hazards on health and the economy in developingcountries2.5.3.1 Biological hazardsIn developing countries, where financial resources are scarce, food control issuesusually receive low priority in public health programs. Food-borne illnesses areperceived as mild, self-limiting diseases and their health and economic consequencesare often overlooked (Venter, 2000).While it is recognised that the prevalence of food-borne illness in developingcountries is considerable, in most there is limited data through which the incidences ofparticular diseases and trends over time can be assessed. In many cases, high rates offood-borne illnesses are associated with low levels of general economic developmentand, more specifically, limited capacity to control the safety of the food supply.Furthermore, there are close inter-relationships between food safety issues and otherelements of environmental health, for example sanitation, water quality and housingconditions.Indeed, the high prevalence of diarrhoeal diseases in many developing countriessuggests major underlying food safety problems. In the early 1990’s there wereapproximately 1.5 billion episodes of diarrhoea annually, of which around 70 percentwere associated with contaminated food. In addition, it is estimated that 2.1 millionpeople died from diarrhoeal diseases in 2000 (Henson, 2003) as shown in Table 7.

39Such diseases take a heavy toll in human life and suffering, particularly among infantsand children, the elderly and other susceptible groups. They also create an enormoussocial, cultural and economic burden on communities and their health systems(Venter, 2000; Käferstein, 2003).Table 7: Estimated occurrence of bacterial infections and intoxications in selectedregions (Henson, 2003)Disease Africa Central &SouthSouthEast AsiaWesternPacificAmericaBacillus cereus gastroenteritis +++ +++ +++ +++Botulism + + + +Brucellosis +/++ ++ +/++ +/++Campylobacteriosis +++ +++ +++ +++Cholera +/++ +/++ + +Clostridium perfringens enteritis +++ +++ +++ +++Escherichia coli disease +++ +++ +++ +++Listeriosis + + + +Typhoid and Paratyphoid fever ++ ++ ++ ++Salmonellosis +++ +++ +++ +++Shigellosis +++ +++ +++ +++Staphylococcus aureus intoxication +++ +++ +++ +++Vibrio parahaemolyticus enteritis ++ ++Vibrio vulnificus septicaemia ++Note:-: Absent; +: Occasional or rare; ++: Frequent; +++: Very frequentCholera, dysentery and typhoid fever are largely related to poverty. At present,developed countries are confronted with the importance of emerging food-bornepathogens such as Salmonella, Campylobacter, Enterohaemorrhagic Escherichia coliand antibiotic resistant pathogens, but information in developing countries is veryscarce (Käferstein, 2003).

40Hart and Kariuki, 1998, reported that diarrhoeal disease is a major cause ofmorbidity and mortality in developing countries. Although rotavirus infections are animportant cause of the disease in infants, bacteria are the most important pathogens inolder children and adults.Poor health due to diarrhoeal diseases and acute respiratory diseases areimportant causes of morbidity and mortality among infants and children in Sri Lanka.The Registrar General’s mortality data for 1996 (Registrar General’s Department,1996) indicate that deaths due to diarrhoeal diseases among children under 5 years ofage accounted for 2 % of deaths from all causes in this age group. During the past 10years, hospitalization due to diarrhoeal diseases has increased. Furthermore, in 1998,intestinal infections ranked as the fifth leading cause of hospitalization (Jayasekara,2001). Medical Research Institute (MRI) Sri Lanka diarrhoeal pathogen profile in2004 illustrates the distribution according to the importance of pathogenic bacteria:59 % Shigella sonnei, 16 % Shigella flexnerii, 9 % Campylobacter, 9 % Enteropathogenic Escherichia coli, 6 % Salmonella and 1 % Shigella dysenteriae (MRI,2004).The dramatic rise in global travel has huge implications for the spread of foodborneinfections. In addition to its significance for international health, tourism isalso of great economic importance, especially to developing countries. Diarrhoea andvomiting caused by contaminated foods may damage the reputation of a country as atourist destination (WHO, 1999).2.5.3.2 Antibiotic resistanceThe emergence of antimicrobial resistance in developing countries is a complexproblem driven by numerous interconnected factors, many of which are linked to theuse of antimicrobials in animals, plants and humans. Extreme poverty in mostdeveloping countries leads to poor sanitation, hunger, starvation and malnutrition,poor access to drugs and poor health care delivery, all of which may precipitate

41antimicrobial resistance. The emergence of multi-drug-resistant isolates intuberculosis, acute respiratory infections and diarrhoea, often referred to as diseasesof poverty, has taken its greatest toll in developing countries (Byarugaba, 2004).Increased access to antibiotics in developing countries, without controls on overthe-counteruse, has led to some of the highest rates of resistance in the world, as wasseen with penicillin resistance in Vietnam. Relatively wealthy countries such as theRepublic of Korea and Japan also have lax controls and even greater access to fundsto purchase antibiotics (Song et al., 1999). Antibiotics which are widely available atrelatively affordable prices, even in semi-rural and rural populations, may beresponsible for the emergence of fluoroquinolone resistance to Salmonella Typhi inIndia which has great public health importance. Controlling the deadliest infectiousdiseases in the world-diarrhoeal diseases, respiratory tract infections, sexuallytransmitted infections, meningitis, pneumonia, and hospital acquired infections ismore difficult today because of the emergence of antimicrobial drug resistance(Laxminarayan et al., 2006).In 1996, in Matlab and Dhaka, Bangladesh, more than 95 % of Shigelladysenteriae isolates were resistant to ampicillin, co-trimoxazole, and nalidixic acid,and 14-40 % were resistant to mecillinam. In a cholera epidemic in 1985-6 inSomalia there was a case fatality rate of 13 % because the initially sensitive V.cholerae quickly acquired plasmid encoded resistance to ampicillin, kanamycin,streptomycin, sulphonamides, and tetracycline (Hart and Kariuki, 1998).Although no estimates of disease burden are currently available that are specificto drug resistance, the contribution of drug resistance to the burden of infectiousdiseases is believed to be large. Resistance has emerged in malaria, tuberculosis(TB), and other bacterial infections that together constitute a significant proportion ofthe burden of disease in developing countries (Laxminarayan et al., 2006).In Sri Lanka, the Salmonella surveillance carried out by the MRI during theyears 2004, 2005 and 2006 revealed the isolates that are sensitive to commonly used

42antibiotics in human therapeutics and the percentage of sensitivity respectively, 88.9% for amoxycillin, 84.4 % for nalidixic acid, 83.3 % for furazolidone, 72.7 % forcotrimaxazole, 96.4 % for mecillinam, 93.5 % for ceptazidime, 62.5 % forcefuroxime, 100.0 % for ciprofloxacin, 100.0 % for chloramphenicol and 100.0% forcefriaxone (MRI, 2007).2.5.4 Prevention and control of seafood-borne diseasesThe number of cases of outbreaks of food-borne diseases caused by seafood isgenerally small when compared with those caused by poultry, dairy and meatproducts (Huss et al., 2000). However, as with any type of food, it is important tohandle seafood safely in order to reduce the risk of food-borne illness (FDA, 2006).Some pathogenic bacteria are naturally present in the aquatic and the generalenvironment. Those pathogens may therefore be found in living fish or fish rawmaterial. This would then be called pre-harvest contamination. The presence of theseorganisms is normally not a safety concern since their numbers are too insignificant tocause disease, with the exception of molluscan shellfish, which because of filterfeeding accumulates high numbers of Vibrio spp. It is habitually eaten raw whichconstitutes an additional health concern (Huss et al., 2000).Moreover, Kumar et al., 2005, reported that pre-harvest and post-harvestcontamination is higher in densely populated countries such as India because theestuaries and coastal water bodies are soiled with human activities and the landingcentres and open fish markets only have poor sanitation. Millions of bacteria arepresent in the surface slime, on the gills, and in the guts of living seafood species(UCD, 2006). The hazards related to contamination and recontamination or rather thesurvival of biological hazards during processing can be controlled by applying GoodHygiene Practice (GHP), Good Manufacturing Practice (GMP) and a well designedHACCP- programme (Huss et al., 2000).

43Therefore, the (FDA, 2006) guidelines for general consumers, stated these basicfood safety tips for buying, storing, preparing, serving, and eating raw fish andshellfish,• Buying seafood- buying from a retailer who follows proper handling practiceshelps assure that the seafood purchase is safe. Only buy fish that isrefrigerated or properly iced. All fresh seafood should be held at 32°F (0 o C),fish should be displayed on a thick bed of fresh ice that is not melting, andpreferably in a case or under some type of cover. See whether the seller ispracticing proper food handling techniques. For health safety reasons, it isimportant to look for freshness when choosing seafood.• Storing seafood- if seafood is to be used within two days after purchasing,store in the refrigerator. If seafood is not to be used within two days ofpurchasing, wrap tightly in moisture-proof freezer paper or foil to protect itfrom air leaks, and store it in the freezer.• Preparing seafood- thaw frozen seafood gradually by placing it in therefrigerator overnight. If it should thaw quickly, either seal it in a plastic bagand immerse it in cold water, or- if the food will be cooked immediatelythereafter- microwave it on the “defrost setting and stop the defrost cyclewhile the fish is still icy but pliable. Prevent cross-contamination, avoid crosscontamination between raw and cooked foods. Cook properly. Most seafoodshould be cooked to an internal temperature of 145 °F (63°C) for 15 seconds.• Serving seafood- do not cross-contaminate• Eating raw seafood- special health warnings for susceptible groups such as:pregnant woman, young children, the elderly, immune compromised personsand persons who have decreased stomach acidity, if they choose to eat rawfish anyway, one rule of thumb is to eat fish that has previously been frozen.Some species of fish can contain parasites, and freezing will kill any parasites

44that may be present. However, freezing does not kill all harmful microorganisms.That is why the safest way is to cook seafood.

3. MATERIALS AND METHODS3.1 Study designThe objective of the present study was to assess the bacteriological status ofPenaeus monodon shrimps available at the local sale locations which were purchasedat different times of the day in order to determine the prevalence, the serotypes andthe antimicrobial resistance of Salmonella and the microbiological contamination interms of Aerobic Plate Count, E. coli, Enterobacteriaceae and Total Halophilic Platecount. Therefore, a cross sectional survey was carried out to fulfil the objectives.3.2 Study method3.2.1 Study areaPenaeus monodon shrimps both cultured in farms and captured from the lagoonswere taken from the local sale points of the North Western Province (Kurunegala andPuttalam districts) in Sri Lanka where most of the shrimp farming is carried out. Thesamples were collected between November 2006 and March 2007.3.2.2 PopulationShrimps sold in the local sale locations in North Western Province fromNovember 2006 to March 2007.

453.2.3 SampleOne sample consisted of at least 2 whole Penaeus monodon shrimps. It was acomposite sample pooled from tissues of shrimp including shell and head to produce a35g/sample, 10 g for enumeration and 25 g for Salmonella isolation.Figure 1: Penaeus monodon shrimp3.2.4 Sample sizeAssumptions made concerning the prevalence of Salmonella;1. Expected prevalence 12.5 % Karnataka coast India (Bhaskar et al., 1995) wastaken because no data available in Sri Lanka regarding the prevalence ofSalmonella.2. Shrimps sold per month at the local market in the North Western Province, whichis estimated to be approximately 20,000 batches per year3. Error level 5 %4. Confidence level 95 %The sample size was taken from the statistical program Winepiscope 2.0Sample size = 168 samples, rounded to 180If the prevalence turns out to be the expected value, the prevalence would be between7.94 % and 17.06 %.

46Figure 2: Map of India with Karnataka(www.journeymart.com/.../India/images/map_new.gif)3.2.5 Sampling planSampling was done according to convenient stratified proportional sampling.The two strata represented in this study are the Puttalam and Kurunegala districtswhich form the North Western Province in Sri Lanka. Two thirds of the 180 sampleswere taken from the Puttalam district, 60 from capture and 60 from culture, and onethird were taken from the Kurunegala district, 30 from culture and 30 from capture.Local sale locations were selected according to convenient sampling. Eligiblelocations were defined as having at least 7 vendors selling shrimps. Therefore, fromthe Puttalam district 9 locations were selected to acquire 120 samples from 60vendors and from the Kurunegala district 5 locations were selected to acquire 60samples from 30 vendors. Shrimp samples were randomly collected by the sellerfrom the basket of the fisherman and each time two parallel samples were taken fromeach shop at different times of the day. Samples were collected into sample bagswhich were labelled and transported in an ice container to the laboratory forprocessing. Each sample contained at least two shrimps in order to create a 35 g/test

47sample. Out of 35g of the test sample 10 g were used for enumeration of AerobicPlate Count, Enterobacteriaceae Count, E. coli Count, Total Halophilic Plate Countand 25 g used for Salmonella isolation.North WesternProvinceFigure 3: Map of Sri Lanka with North Western Province(http://www.c-r.org/our-work/accord/sri-lanka/images/map1.gif)

48PuttalamPuttalam DistrictNo. Divisions31 Anamaduwa32 Pallama33 Mahakumbukkadawala34 Karuwalagaswewa35 Wanathawilluwa36 Puttalam37 Kalpitiya38 Mundal39 Arachchikattuwa40 Chilaw41 Madampe42 Mahawewa43 Nattandiya44 Wennappuwa45 DankotuwaKurunegala18Figure 4: Map of the Puttalam and Kurunegala Districts(http://www.agridept.gov.lk/Extension/images/NW_map.jpg)Kurunegala DistrictNo.Divisions1 Pannala2 Narammala3 Alawwa4 Polgahawela5 Kurunegala6 Mallawapitiya7 Mawathagama8 Ridigama9 Ibbagamuwa10 Ganewatta11 Wariyapola12 Maspotha13 Weerambugedara14 Katupotha15 Kuliyapitiya-west16 Kuliyapitiya-East17 Udubaddawa18 Bingiriya19 Hettipola20 Kobeigane21 Nikaweratiya22 Rasnayakapura23 Kotawehera24 Ambanpola25 Maho26 Polpithigama27 Galgamuwa28 Ehatuwewa29 Giribawa30 Nawagaththegama

493.2.6 QuestionnaireA questionnaire was administered to each shop owner to evaluate the routinepractices of the local shrimp sale locations.3.3 Laboratory proceduresFresh shrimps belonging to the species Penaeus monodon obtained from localsale locations were analysed to determine their microbiological quality. Each samplecontained the date, the sale location identity and the district. According to thestandard methods, shrimp samples were analysed and the bacteriological parameterswere Salmonella isolation, serotyping and antimicrobial susceptibility for isolatedSalmonella. Enumeration was carried out in terms of Aerobic Plate Count,Enterobacteriaceae, Escherichia coli and Total Halophilic Plate Count. Most of thetime 10 samples (batch) were analysed per week during the field work for theisolation of Salmonella.3.3.1 Aerobic Plate CountTests were carried out according to the International Standards of ISO4833:2003. Two poured plates were prepared using a specified culture medium and aspecified quantity of the test sample.3.3.1.1 Materials:1. Maximum Recovery Diluent (MRD)2. Plate Count Agar (PCA): 15 ml per plate.

503.3.1.2 Sample preparation (initial dilution):A 10 g shrimp sample was required for testing. Test sample of shrimp wasadded to a stomacher bag containing 90 ml of maximum recovery diluent (MRD) andstomached for 1 minute.3.3.1.3 Procedures:1. Perform decimal dilutions from the shrimp homogenateUsing a fresh sterile pipette, 1ml of the initial inoculum was transferred into 9 mlof MRD. The procedure was repeated for as many decimal dilutions as required.Dilutions were mixed using a vortex mixer for 5 to 10 seconds.2. Inoculation1 ml of the each required dilution was aseptically inoculated into a labelled Petridish. Full compliance with ISO 4833:2003 required duplicate plates for each dilution.Tempered (46+/-1 o C) Plate Count Agar (PCA) was added in 15 ml volume to eachPetri dish. Carefully mixed by swirling six times clockwise, six times left to right, sixtimes anticlockwise and six times up and down then allowed to set. (The timeelapsing between the preparation of the initial suspension and contact with the agardid not exceed 45 minutes).3. IncubationInverted Petri dishes were transferred to an incubator and incubated at 30 +/-1 o Cfor 72 hours.4. Counting of coloniesThe number of microorganisms per gram of sample was calculated from thenumber of colonies obtained on selected plates.

515 . CalculationNumber of micro-organisms = Σ c(n 1 + 0.1 x n 2 ) dΣ c = The sum of characteristic colonies counted after identificationn 1 = The number of dishes retained in the 1st dilutionn 2 = The number of dishes retained in the 2nd dilutiond = The dilution factor corresponding to the 1st dilution3.3.2 Enumeration of EnterobacteriaceaeThe test was carried out based on the ISO 7402:1985 Standard Protocol forenumeration of Enterobacteriaceae.3.3.2.1 Materials:1. Maximum Recovery Diluent (MRD)2. Violet Red Bile Glucose Agar (VRBGA): 30 ml per plate.3.3.2.2 Sample preparation (initial dilution):Preparation of initial dilution was as same as 3.3.1.23.3.2.3 Procedures:1. Perform decimal dilutions from the shrimp homogenateThe decimal dilutions preparation were the same as 3.3.1.32. Inoculation1 ml of each required dilution was aseptically inoculated into labelled petridishes. Full compliance with ISO 7402:1985 required duplicate plates for eachdilution.

523. Pour plate methodTempered (46+/-1 o C) violet red bile glucose agar (VRBGA) in 15 ml were addedto each Petri dish. The inoculum was carefully mixed with the VRBGA and allowedto set. (The time elapsing between the addition of MRD and contact with VRBGAdid not exceed 15 minutes). When the agar was completely set, a further 10-15 ml oftempered (46 +/-1 o C) VRBGA was added onto the surface of the inoculated plate andallowed to solidify.4. IncubationInverted Petri dishes were transferred to an incubator at 37+/-1 o C for 24 hours.5. Biochemical confirmationPlates containing typical colonies were selected. Typical colonies were pinkto red, with or without precipitation haloes or colourless mucoid colonies, with adiameter of 0.5 mm or more. Typical colonies were bio-chemically confirmed. Theprocedure entailed the random selection of five such colonies were streaked onNutrient Agar plates. These plates were incubated at 37 o C for 24+/-2 h. Then anoxidase test was performed using a platinum loop to streak a portion of each wellisolated colony on a filter paper moistened with oxidase reagent. If filter paper didnot change into dark within 10 seconds, the colony was considered as oxidasenegative.6. Counting of coloniesColonies of the suitable two consecutive dilutions were counted in duplicateplates.7. CalculationThe calculations were performed using the same formula at 3.3.1.3 procedure.

533.3.3 Rapid method to enumerate Escherichia coliAs a rapid method 3M TM Petrifilm TM E. coli/Coliform Count Plates (3M-EC)(Petrifilm TM EC, 2000) was used to enumerate E. coli following manufacturesguidelines. The 3M Petrifilm EC/Coliform Count Plate is a ready-made-culturemediumtechnique. It contains Violet Red Bile (VRB) nutrients, a cold-water-solublegelling agent, an indicator of glucuronidase activity, 5bromo-4-chloro-3indolyl-β-Dglucuronide(BCIG), and a tetrazolium indicator which facilitates colony counting(Sasithorn and Sirirat, 2002; Mattelet, 2005). Petrifilm EC Plates are formulated todifferentiate non-coliform, coliform, and E. coli colonies. Most E. coli (about 97%)produce β-glucuronidase which in turn produces a blue precipitate associated with thecolony and about 95% of E. coli produces gas. Therefore, it is indicated by blue tored-blue colonies associated with entrapped gas on the Petrifilm EC plate. Coliformsproduce acid and gas from lactose during metabolic fermentation and these coloniesgrowing on EC plates produce acid which causes the pH indicator to make the gelcolour darker red. Gas trapped around red coliforms colonies indicates confirmedcoliforms (Mattelet, 2005).Manufacturer guidelines for storage:The unopened packages are stored at ≤8 o C (≤46 o F). In areas of high humidity, itis recommended to allow packages to reach room temperature before opening to avoidcondensation in the package.To seal opened package, the end has to be folded over with tape. It is better tokeep released package at ≤25 o C (≤77 o C), ≤50 o Relative Humidity (RH) and avoidrefrigeration of the opened package. Use petrifilm within one month after opening.3.3.3.1 Materials:1. Maximum Recovery Diluent (MRD)2. 3M-EC Plates

543. Spreader3.3.3.2 Procedures:1. InoculationAfter preparation of the initial dilution at step 3.3.1.2 and the required serialdilutions at step 3.3.1.3, the shrimp suspension (1 ml) was pipetted at proper dilutiononto the center of bottom media surface of a 3M Petrifilm TM plate which werelabelled properly and placed on a level surface.Figure 5: Pipetting inoculum onto the 3M-EC plate(http://www.carolina.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)Then the plate was gently closed with film.Figure 6: Closing with the film(http://www.carolina.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)Next, gentle pressure was applied on the spreader in order to distribute the inoculumover a circular area without twisting or sliding the spreader.

55Figure 7: Spreading of the inoculum using spreader(http://www.carolina.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)Spreader was lifted. Gel was allowed to solidify at least 1 minute.Figure 8: Gel solidification(http://www.carolina.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)2. IncubationThen the plates were incubated at 35 o C for 24 h. Plates were kept clear side up instacks of no more than 20.3. InterpretationPetrifilm EC Plates contain an indicator dye in the medium that stains bacterialcolonies red. The red colour helps to distinguish them from dust particles or otherenvironmental contaminants. The EC Plate growth medium contains lactose, and thetop film of the plate’s traps gas produced by coliforms. Finally, EC Plates contain anindicator of beta-glucuronidase activity, 5bromo-4-chloro-3indolyl-β-D-glucuronide(BCIG). The enzyme beta-glucuronidase cleaves BCIG to produce a blue productthat precipates, causing a beta-glucuronidase-producing bacterial colony (E. coli) to

56appear blue. Non-coliform colonies growing on Petrifilm EC plates appear red withno gas bubbles, coliform colonies appear red with gas bubbles, and E. coli coloniesappear blue with gas bubbles.(www.carolina.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000). Triplicate trials were performed per dilution; using this method, plates with15–150 colonies are recommended for counting.4. CalculationThe calculations were performed using the same formula at 3.3.1.3 procedure.Plates which were given at 10 -1 dilution with no colonies, counted as

573.3.4.3 Procedures:1. Perform decimal dilutions from shrimp homogenateThe decimal dilutions preparation was the same as in 3.3.1.3 for enumeration ofthe Total Halophilic Plate Count.2. Inoculation1 ml of the each required dilution was aseptically inoculated into a labelled petridish. For each dilution there were duplicate plates (ISO 4833:2003). Tempered(46+/-1 o C) PCA agar containing 3 % NaCl in 15 ml were added to each Petri dish.Carefully mixed by swirling six times clockwise, six times left to right, six timesanticlockwise and six times up and down. They were allowed to set. (The timeelapsing between the preparation of the initial suspension and contact with the agardid not exceed 45 minutes).3. IncubationInverted Petri dishes were transferred to an incubator and incubated at 30 +/-1 o Cfor 72 hours.4. Biochemical confirmationThe random selection of five such colonies was streaked on Nutrient Agar plates.These plates were incubated at 37 o C for 24+/-2 h. Then an oxidase test wasperformed using a platinum loop to streak a portion of each well isolated colony on afilter paper moistened with oxidase reagent. If filter paper changes into dark within10 seconds, the colony was considered as oxidase positive.5. Counting of coloniesColonies of the suitable two consecutive dilutions were counted in duplicateplates.6. CalculationThe calculations were performed using the same formula at 3.3.1.3 procedure.

583.3.5 Conventional culture method to detect SalmonellaThe study was conducted utilizing the conventional methods for the detection ofSalmonella following the ISO 6579 (2002) standard guidelines; microbiology of foodand animal feeding stuffs-horizontal method for the detection of Salmonella spp. Theprotocol generally has four distinct phases or steps:Step 1. Non-selective pre-enrichment: The sample of 25 grams of shrimp wasblended with 225ml of buffered peptone water in a stomacher bag and incubated at37 o C±1 o C for 18± 2 hours to allow resuscitation of any stressed organism and growthof all organisms as well.Step 2. Selective enrichment: Allowed growth of the organism under investigation,while reducing the numbers of accompanying organisms in the broth. Two types ofselective enrichment media for Salmonella are recommended, Muller-KauffmannTetrathionate novobiocin broth (MKTTn broth) and Rappaport- Vassiliadis mediumwith soya (RVS broth). The pre-enrichment broth after incubation was mixed and 0.1ml of the broth was transferred into a tube containing 10 ml of RVS broth. Another 1ml of the pre-enrichment broth was transferred into a tube containing 10 ml ofMKTTn broth. The inoculated RVS broth was incubated at 41.5 o C± 1 o C for 24h±3hours and the the inoculated MKTTn broth at 37 o C± 1 o C for 24h±3 hours.Step 3. Isolation: After incubation for 24h±3 hours, a loop-full of material from theRVS broth and MKTTn was streaked on selective solid agars. There were 2 selectivesolid agars used in this study: Brilliant-green Phenol-red Lactose-Sucrose agar(BPLS agar) and Xylose-Lysine-Deoxycholate agar (XLD agar) separately. The plateswere incubated in an inverted position at 37 o C± 1 o C for 24h±3 hours. The propertiesof Salmonella colonies are described in Table 8.Step 4. Confirmation: Characteristic colonies on the plates were submitted forbiochemical testing and seroagglutination testing to confirm that the isolates weremembers of the species Salmonella.

59Table 8: Typical growth of Salmonella colonies on selective solid mediaBPLSXLDMediaColony appearanceReddish colour and translucent colonyBlack centered with a lightly transparentzone of reddish colour due to the colourchange of indicator.Salmonella H 2 S negative variants, e.g. S.Paratyphi A, pink with a darker pink centre.Lactose-positive Salmonella, yellow with orwithout blackening.3.3.5.1 Selection of colonies for confirmationFor confirmation, five typical colonies or suspect per plate on the selectivemedium (XLD and BPLS) were transferred and tested for biochemical tests. Theselected colonies were streaked onto the pre-dried nutrient agar plates and incubatedat 37 o C+/-1 o C for 24 h +/-3 h. For the biochemical tests Salmonella colony wasreceived from NA.3.3.5.2 Biochemical confirmation3.3.5.2.1 Triple Sugar Iron (TSI) agarStreaked the agar slant surface and stab the butt with a colony received from NAby just touching the top of the colony. Incubated at 37 o C+/-1 o C for 24 h +/-3 h, thechanges in medium were interpreted as shown in Table 9.

60Sample 25 gPREENRICHMENT225 ml Buffered peptone waterat ambient temperatureIncubation for 18h± 2h at 37 o C±1 o C…………………………………………………………………………………SELECTIVEENRICHMENT0.1 ml of culture+10 ml of RVS brothIncubation for24h±3h at 41.5 o C± 1 o C1 ml of culture+10 ml of MKTTn brothIncubation for24h±3h at 37 o C± 1 o C…………………………………………………………………………………ISOLATIONLoop-full of suspect coloniesXLD medium and BPLS agarIncubation for24h±3h at 37 o C± 1 o C………………………………………………………………………………….From each plate test a characteristic colony.If negative, test the other four marked coloniesCONFIRMATIONBiochemicalconfirmationNutrient agar, incubation for24h±3h at 37 o C± 1 o CSerologicalconfirmationFigure 9: Flow chart of isolation of Salmonella conventional culture method (ISO6579, 2002)

61Table 9: Biochemical reactions in TSI agarArea of reaction ResultMeaningButt Yellow Glucose positive (glucose used)Red or unchangedBlackGlucose negative (glucose notused)Formation of hydrogen sulfideBubbles or cracks Gas formation from glucoseSlant surface Yellow Lactose and/or sucrose positiveRed or unchanged(lactose and/or sucrose used)Lactose and sucrose negative(neither lactose nor sucrose used)Typical Salmonella cultures show alkaline (red) slant and acid (yellow) buttswith gas formation (bubble) and (in about 90 % of the c ases) formation of hydrogensulfide (blackening of the agar). When lactose-positive Salm onella is isolated the TSIagar slant is yellow. Therefore, results were not based on only the TSI agar test.3.3.5.2.2 Urea agarThe agar slant surface was streaked with the colony. Incubated at 37+/-1 o C for24 h +/-3 h. and examined at intervals. If the reaction is positive, splitting of urealiberates ammonia, which changes the colour of phenol red to rose-pink and later todeep cerise, a moderate red, the reaction is often apparent after 2 h to 4 h.3.3.5.2.3 L-lysine decarboxylation mediumThe culture was inoculated just below the surface of the liquid medium andincubated at 37+/-1 o C for 24 h +/- 3 hours. Turbidity and a purple colour afterincubationindicate a positive reaction. A yellow colour indicates a negative reaction.

623.3.5.2.4 Detection of β-galactosidaseA loopful of the suspected colony was suspended in a tube containing 0.25 ml ofthe saline solution. One drop of toluene was added and the tube shaken. The tubewas then placed in a water bath set at 37 o C and left for several minutes(approximately 5 min). 0.25 ml of the β-galactosidase reagent for detection of β -galactosidase was added and mixed. The tube was replaced in the water bath set at37 o C and left for 24 h+/-3 h, and examined at intervals. A yellow colour indicates apositive reaction, the reaction is often apparent after 20 min.3.3.5.2.4 Voges-Proskauer (VP) reactionA loopful of the suspected colony was suspended in a sterile tube containing 3ml of the VP medium. Then the tube was incubated at 37+/-1 o C for 24+/-3 h. Afterincubation, two drops of creatine solution, three drops of ethanolic solution of 1-naphthol and then two drops of the potassium hydroxide solution were added. Afterthe addition of each reagent the tube was shaken. The formation of a pink to brightred colour within 15 min indicates a positive reaction.3.3.5.2.5 Indole reactionA tube containing 5 ml of the tryptone/ tryptophan medium was inoculated withthe suspected colony and then incubated at 37+/-1 o C for 24+/-3 h. After incubation,1ml of the Kovacs reagent was added. The formation of red ring indicates a positivereaction. A yellow-brown ring indicates a negative reaction.

63Table 10: Interpretation of biochemical tests results of Salmonella (Quinn et al.,1994)TestReactionIndole production -Methyl Red +Voges-Proskauer -Citrate +Urease -Phenylalanine deaminase -Hydrogen sulphide +Lysine decarboxylase +Ornithine decarboxylase +Motility (36 o C) +Gelatin liquefaction -Growth in KCN broth-ONPG (beta-galactosidase)-Acid Fro mDulcitol +InositoldLactose -Maltose +Mannitol +Mannose +Rhamnose +Sorbitol +Sucrose -Xylose +Red pigment -Swarming (blood agar)-Mucoid colonies-d = 26-75% positive

643.3.5.3 SerotypingAll iso lates were serotyped by agglutination according to the Kauffmann-Whitescheme using Salmonella Polyvalent I (A-E), Salmonella Polyvalent II (F-67) andSalmonella antiserum specific to the individual group after eliminating autoagglutinablestrains by mixing a drop of NaCl and a part of the colony on a glassslide. Then the following process stages took place (the procedure was based on themanufacturer Sifin, Germany).1. The selected colonies were tested with Salmonella polyvalent I (A-E), if theresu lt was positive (+), the selected colonies possessed the antigen to thisgroup; colonies were regarded as a member of Salmonella group A-E.2. Test negative (-) result colonies from the first step were subjected toagglutination with Salmonella polyvalent II (F-67), if the result was positive(+), those colonies possessed the antigen of this group; colonies were regardedas a member of Salmonella Group F-67.3. Serotyping of Somatic (O) antigens in order to determine Salmonella maingroups (A (O2), B (O4, 5, 27), C (O6, 7, 8, 20), D (O9, 27, 46, Vi), E (O3, 10,15, 19, 34)) by using a sequence of somatic antigen sera.4.Determination of the capsular antigen:Pathogenic strains of Salmonella Typhi carry an additional antigen, "Vi",which is associated with a bacterial capsule and determined using the reagentanti-Salmonella Vi.5.Determination of flagella antigens:Having found the "O" antigen group, the"H" antigen was determined. The"H" antigens are proteins associated with the bacterial flagella. Salmonellasexist in two phases; a motile phase and a non-motile phase. These are alsoreferred to as the specific and non-specific phases.

656. “H” antigen detection by induction:After transferring of the isolate to the motility agar agglutination for flagellaantigen phase 1 and phase 2 were performed. One of the phases of theantigens was blocked by the particular H antiserum to force the strain todevelop the other phase. Then agglutination was performed with the phasecome up from the hidden condition.Salmonella Polyvalent I (A-E) 1 dropPositiveTest with * Negative ControlNegativeDetermination of Somatic(O) antigens(for groupA, B,C, D, E)SalmonellaPolyvalent II (F-67)Determination of flagellaantigens (Phase 1, Phase 2)PositiveNegativeDefine into Salmonellagroup F-67(No more further step)Non-Salmonella* The negative control used was NaCl solutionFigure 10: Salmonella serotypingflow chart

663.3.6 Antimicrobial sensitivity test for SalmonellaThe procedure for performing the Disk Diffusion Test was carried out accordingto the National Committee of Clinical Laboratory Standards (NCCLS, 2000), theinstitute is recognised as Clinical Laboratory Standards Institute (CLSI) from 1 stJanuary 2005.3.3.6.1 Inoculum preparation3.3.6.1.1 Growth methodThe growth method was performed as follows:1. At least three to five well-isolated colonies of the same morphological type wereselected from an agar plate culture. The top of each colony was touched with a loop,and the growth was transferred into a tube containing 4 to 5 ml of a suitable brothmedium, such as tryptic soy broth.2. The broth culture was incubated at 35 o C until it achieves the turbidity of the 0.5McFarland standards (usually 2 to 6 hours).3. The turbidity of the actively growing broth culture was adjusted with sterile salineor broth to obtain turbidity optically comparable to that of the 0.5 McFarlandstandards. A BaSO 4 0.5 McFarland standard was prepared by adding a 0.5 mlaliquot of 0.048 mol/L BaCl 2 (1.175% w/v BaCl 2 .H 2 O) to 99.5 ml of 0.18 mol/LH 2 SO 4 (1% v/v) with constant stirring to maintain a suspension. Then it wasdispensed into 4-6 ml screw capped vials and stored in the dark at room temperature.The turbidity standard always agitated before use.

673.3.6.2 Inoculation of test plates1. Optimally, within 15 minutes after adjusting the turbidity of the inoculumsuspension, a sterile cotton swab was dipped into the adjusted suspension. The swabwas rotated several times and pressed firmly on the inside wall of the tube above thefluid level. This removes excess inoculum from the swab.2. The dried surface of a Mueller-Hinton agar plate was inoculated by streaking theswab over the entire sterile agar surface. This procedure was repeated by streakingtwo more times, rotating the plate approximately 60 o each time to ensure an evendistribution of the inoculum. As a final step, the rim of the agar was swabbed.3. The lid was left from the agar plate for 3 to 5 minutes, but not more than 15minutes, to allow for any excess surface moisture to be absorbed before applying thedrug impregnated disks.3.3.6.3 Application of disks to inoculated agar plates1. Antimicrobial disks were dispensed onto the surface of the inoculated Mueller-Hinton agar plates. For each Salmonella isolate used 3 Petri dishes of 100 mm indiameter to dispense 14 different kinds of antibiotics such as nalidixic acid 30 µg,amoxycillin 10 µg, ampicillin 10 µg, gentamycin 10 µg, erythromycin 15 µg,chloramphenicol 30 µg, kanamycin 30 µg, ciprofloxacin 5 µg, trimethoprim 5 µg,tetracycline 30 µg, streptomycin 10 µg, sulphamethoxazole/trimethoprim 25 µg,compound sulphonamides 300 µg, and doxycycline hydrochloride 30 µg.2. The plates were inverted and placed in an incubator set to 35 o C within 15 minutesof applying the disks.

683.3.6.4 Reading plates and interpreting results1. After 16 to 18 hours of incubation, each plate was examined. The resulting zonesof inhibition were uniformly circular and there was a confluent lawn of growth. Thediameters of the zones of complete inhibition were measured, including the diameterof the disk. Zones were measured to the nearest whole millimeter, using a ruler,which was held on the back of the inverted Petri dish. The Petri dish was held a fewinches above a black, non reflecting background and illuminated with reflected light.2. The sizes of the zones of inhibition were interpreted by referring to the table andthe organism was reported as susceptible, intermediate, or resistant to the agents thathad been tested.3.4 Questionnaire surveyA questionnaire was used to evaluate routine practices at the local sale points.The factors observed are listed in Table 11.Figure 11: Practice of using iceFigure 12: Handling of shrimpswhile on sale

69Table 11: List of observed factorsNo. Factors Level1 Status/seller HighMediumLow2 Receive Middle manFarmer3 Transport/Vehicle Freezer truckMotor bicycleBicycleTruck4 Transport/Duration < 1 Hour1-2 Hours>2-3 Hours> 3 Hours5 Transport/Condition Freezer truckWith iceWithout ice6 Transport/Mix with fish YesNo78GradeHandlingNumber1Number 2Number 3Open handWith gloves9 How oftenWash handsEvery timeSome times1011ContainerCleaning/Container1. Wood2. Tile3. Rigiform4. Cool box5. Freezer6. Other7. Plastic boxEnd of the dayNot every end of the day12 Sales/day1-30 Kg>30-60 Kg>60-90 Kg13 Remains/dayYesNoRarely

703.5 Data management and statistical analysisLaboratory and questionnaire dat a were entered and stored in the databasemanagement software MS Excel 2003.Descriptive statistics:Chart, percentage and median were used to describe prevalence, antimicrobialresistance and microbiological contamination in terms of Aerobic Plate Count,Enterobacteriaceae count, E. coli count and Total Halophilic Plate Count.Interferential statistics:Chi square or Fisher’s exact test was used where appropriate to find significantdifference of factors relating to qualitative variables. The logistic regression test wasused to identify risk factors through odds ratio.Nonparametric statistical analysis was carried out, the Wilcoxon test to identifythe defference between two factors and the Kruskal-Wallis test to identify thedefference among more than two factors relating to quantity of microbiologicalcontamination.Tables of new percentage Confidence Intervals (CI), binomial distribution(Bunke, 1959) was used to get small 95 % CI for prevalences. Since the binomialdistributions may be very asymmetric and have small sample size (n), used the tablesfor n ≤34 and normal approximation Epicalc 2000 version 1.02 for n >34.The statistical package used for these analyses were the Statistical Package forSocial Sciences (SPSS) version 11.5, and Epicalc 2000 version 1.02. All statisticalanalyseswere interpreted at the 5 % level of significance.

4. RESULTS4.1 Source of the sampleThe total number of samples analyzed in this study to determine prevalence,serotypes and antimicrobial resistance of Salmonella in marketed shrimps (Penaeusmonodon) in local markets of North Western Province in Sri Lanka was 180 (120samples from the Puttalam District and 60 samples from the Kurunegala District)samples. Shrimps capture from lagoon and culture from farms were equally dividedin each district. In the Puttalam District there were 9 different locations where shrimpswere sold at retail sale and there were 5 different locations in the Kurunegala District.Table 12: Number of samples of shrimps for Salmonella analysisDistrictTypeNumber of samplesPuttalam (9 locations) Capture from lagoon 60Culture fr om farm60Kurunegala (5 locations) Capture from lagoon30Cul ture from farm30Total 1804.2 Prevalence of Salmonella in marketed shrimps4.2.1 Overall prevalence of Salmonella in shrimpsThe prevalence of Salmonella in marketed Penaeus monodon shrimps (brackishwatercultured and captured) purchased from retail sale points were analysed and the

72results are shown in Table 13. The overall prevalence of Salmonella in marketedPenaeus monodon shrimps in North Western Province was 12.78 % (it lays within the95 % CI, 8.44 % to 18.76 %) and overall prevalence in shrimps from farm culturesand from lagoon captures was 11.11 % (95 % CI, 5.75, 19.92) and 14.44 % (95 % CI,8.22, 23.8) respectively. The prevalences of Salmonella in shrimps by the type is notstatistically significant (p=0.66). An odds ratio (OR) of 1.35 (95 % CI, 0.56, 3.26)would implies that the 1.35 fold greater odds of Salmonella for capture shrimps thanfor culture shrimps.The prevalence of Salmonella in the Puttalam District 13.33 % (95 % CI, 8.05,21.04) and in the Kurunegala District it was 11.67 % (95 % CI, 5.21, 23.18) as shownin Table 13. An odds ratio of 1.16 (95 % CI, 0.45, 3.01) implies that the 1.16 foldgreater odds of Salmonella for the Puttalam district than for the Kurunegala district.Table 13: Prevalence of Salmonella in marketed shrimpsDescription Type of shrimps No. ofsamplesNo. of positives PrevalenceProportion (95 % CI*)Puttalam Capture 60 9 15.00 (7.50, 27.08)Culture 60 7 11.67 (5.21, 23.18)Puttalam overall120 16 13.33 (8.05, 21.04)prevalenceKurunegala Capture 30 4 13.33 (4. 36, 31.64)Culture 30 3 10.00 (2.62, 27.68)Kurunegala 60 7 11.67 (5.21, 23.18)overallprevalenceNWP Capture 90 13 14.44 (8.22, 23.80)Culture 90 10 11.11 (5.75, 19.92)23 12.78 ( 8.44, 18.76)NWP 180overallprevalenceNWP = North Western Province, * Confidence Interval

73The preval ences of Salmonella in shrimps from capture and from culture were15.00 % (7.50, 27.08) and 11.67 % (5.21, 23.18) in the Puttalam district. An odds ratioof 1.34 (95 % CI, 0.46, 3.86) revealed that the occurrence of Salmonella in captureshrimps is more likely than culture shrimps. When compared the same in theKurunegala district an odds ratio of 1.16 (95 % CI, 0.45, 3.01) implies that the 1.38fold greater odds of Salmonella for capture shrimps than culture shrimps.4.2.2 Prevalence of Salmonella in shrimps sold at 14 different retail sale locationsIn this study the samples were purchased from 14 different retail selling locations(9 in the Puttalam District and 5 in the Kurunegala District). The prevalence ofSalmonella in shrimpssold at different retail sale locations varied and it ranged from0 % to 60 % (as shown in Figure 13). The percentage of sale location specificSalmonella contamination was significantly different (p

74Table 14: Prevalence of Salmonella in shrimps sold at the different retail salelocationsRetail saleLocationNo. ofsamplesexaminedNo. ofsamplespositivePrevalence 95 % Confidence Interval(%) Lower limit Upper limit1 10 0 0.00 0.00 26.82 12 0 0.00 0.00 23.43 10 3 30.00 10.8 62.04 10 0 0.00 0.00 26.85 10 4 40.00 17.5 70.96 10 0 0.00 0.00 26.87 20 2 10.00 2.5 29.38 10 6 60.00 29.1 82.59 28 0 0.00 0.00 10.710 10 4 40.00 17.5 70.911 10 0 0.00 0.00 26.812 16 3 18.75 6.5 43.313 14 0 0.00 0.00 20.0014 10 1 10.00 1.0 40.44.3 Salmonella serogroups and serotypes in Penaeus monodon shrimpsThe total number of serovars isolated in this study was 23 from 180 samples. Themajor serogroups of the isolated serovars were serogroup C (56.52 %), E (26.08 %)and B (8.69 %). The serogroups A and D were not found in this study. Thepercentage of strains belonging to Salmonella F-67 was 8.69 % as shown in Figure14.

75E26.08 %C56.52 %B8.69 %F-678.69 %Figure 14: Percentage prevalence of major Salmonella groupsThe retail shrimp selling locations 3, 5, 10 and 14 were positive for Salmonellain capture type of shrimps, locations 8 and 12 were positive for Salomonella in culturetype of shrimps and only the location 7 was positive for both capture and culture typeof shrimps. From capture shrimps have been isolated all 3 major serogroups (C, Eand B) as well as F-67 but from culture type C and F-67 serogroups were isolated(Table15).

76Table 15: Number and percentage of Salmonella serogroups isolated from theshrimps by different locations.Locations Type Serogroup TotalC E B F-673 Capture 1 2 - - 3(4.35 %) (8.69 %) (13.04 %)5 Capture 2 2 - - 4(8.69 %) (8.69 %) (17.39 %)7 Capture 1 - - 1(4.35 %) (4.35 %)Culture 1 - -1(4.35 %) (4.35 %)8 Culture 6 - - 6(26.09 %) (26.09 %)10 Capture - 1 2 1 4(4.35 %) (8.69 %) (4.35 %) (17.39 %)12 Culture 2 1 3(8.69 %) (4.35 %) (13.04 %)14 Capture 1 - - - 1(4.35 %) (4.35 %)Capture 4 6 2 1 13(17.39 %) (26.09%) (8.69 %) (4.35 %) (56.52 %)Culture 9 - - 1 10(39.13 %) - - (4.35 %) (43.48 %)Total 13 6 2 2 23(%) (56.52) (26.09) (8.69) (8.69) (100.00)From the 23 serovars of this study 10 originated from culture and 13 fromcapture (Table 16). Out of 13 culture strains there were 7 serotypes which belong toS. Newport, and each serotype of following were belong to S. Mbandaka, F-67 and C7(not typable). In capture strains there were 4 serotypes which belong to S. Newport,amongthe serotypes that not typable were included 3 serotypes from serogroup E

77(E10 and E15) and 2 serotypes (B4) from serogroup B. F-67 was found only one incapture shrimps.Table 16: Sero vars of Salmonella isolated from shrimpsNo of isolateType of shrimpSerovar1 Capture S. Newport2 CaptureE10*3 CaptureB4*4 Capture B4*5 CaptureE10*6 Capture F-677 Capture E15*8 Capture E15*9 Capture S. Newport10 Capture S. Newport11 CaptureS. Newport12 Capture S. Weltevreden13CaptureS. Weltevreden14CultureS. Newport15 C ulture S. Newport16 CultureS. Newport17 Culture S. Newport18 Culture S. Newport19 Culture C7*20 Culture S. Mbandaka21 Culture F-6722 Culture S. Newport23 Cultu re S. Newport*not typableThe percentage distribution of different serotypes is described in Figure 15. S.Newport is the most frequently found serovar ( 47.83 %) follow ed by S. W eltevreden8.69 %, B4 (not typable) 8.69 %, E15 (not typable) 8.69 %, E10 (not typable) 8.69 %,F-67 (Salmonella Poly II) 8.69 %, C7 (not typable) 4.35 %, S. Mbandaka 4.35 %respectively.

78F-678.69 %C7* 4.35 %E10* 8.69 %E15*8.69 %S. Newport47.83 %B4*8.69 %S. Mbandaka4.35 %S. Weltevreden8.69 %* not typableFigure 15: Distribution of Salmonella serovars isolated from shrimps4.4 Antimicrobial resistance of Salmo nella in marketed shrimpsThe number of Salmonella isolates tested for antimicrobial resistance in thisstudy was 23. There were 14 different kinds of antibiotics nalidixic acid 30 µg,amoxycillin 10 µg, ampicillin 10 µg, gentamycin 10 µg, erythromycin 15 µg,sulphamethoxazole/trimethoprim 25 µg, chloramphenicol 30 µg, kanamycin 30 µg,compound sulphonamides 300 µg, ciprofloxacin 5 µg, trimethoprim 5 µg, tetracycline30 µg, streptomycin 10 µg and doxycycline hydrochloride 30 µg used for theAntibiotic Disc Diffusion Test.

794.4.1 Antimicrobial resistance of SalmonellaThe percentage resistance to any antimicrobial was 95.65 % (95 % CI, 80.2,99.5). The percentage of antimicrobial resistance of Salmonella isolated from cultureshrimps was 100 % (73.2, 100.0) and capture shrimps 92.31 % (67.5, 99.2). However,the difference of the percentage of antimicrobial resistance among culture and captureshrimps was not statistically significant (p>0.05) (Table 17).Table 17: Percentage of antimicrobial resistance SalmonellaType ofshrimpNo. of isolatestestedNo. notresistantNo.resistantPercentage resistance(95 % CI*)Capture 13 1 12 92.31 (67.5, 99.2)Culture 10 0 10 100.00 (73.2, 100.00)Total 23 1 22 95.65 (80.2, 99.5)* Confidence IntervalPvalue1.00Table 18 describes the percentage of isolates resistant, intermediate andsusceptible to 14 different kinds of antibiotics. 100 % of all the 23 isolates ofSalmonella were observed susceptible for nalidixic acid, ampicillin, gentamycin,sulphamethoxazole/ trimethoprim, chloramphenicol, ciprofloxacin, trimethoprim andtetracycline. The next highest susceptibility was shown for the antimicrobialamoxycillin and it was 95.65 %. 21 Salmonella strains were susceptible and 2 wereintermediate for doxycycline hydrochloride. 82.61 %. 19 Salmonella strains weresusceptible and 4 isolates were intermediate for kanamycin. Most of the isolates (16)were intermediate for streptomycin and 7 were susceptible for it. It is important tonote that out of all the 14 kinds of antimicrobials tested, resistance was found only forthree kinds of antimicrobials erythromycin, amoxycillin and sulphonamidesrespectively.

Table 18: Number and percentage of Salmonell a strains resist ant, intermediate and susceptible to antimicrobials tested (n= 30)Type of antibioticResistantIntermediateSusceptibleNo. % 95 % CI* No. % 95 % CI* No. % 95 % CI*Gentamycin 0 0 (0.00, 12.8) 0 0 (0.00, 12. 8) 23 100.00 (87.2, 100. 00)Kanamycin 0 0 (0.00, 12.8) 4 17.39 (7.2, 36.8) 19 82.61 (63.2, 92.8)Streptomycin 0 0 (0.00, 12.8) 16 69.57 (48.9, 85.6) 7 30.43 (14.4, 51.1)Erythromycin 22 95.65 (80.2, 99.5) 1 4.35 (0.5, 19.7) 0 0 (0.00, 12.8)Amoxycillin 1 4.35 (0.5, 19.7) 0 0 (0.00, 12.8) 22 95.65 (80.2, 99.5)Ampicillin 0 0 (0.00, 10.00) 0 0 ( 0 .00, 12. 8) 23 100.00 (87.2, 100. 00)Chloramphenicol 0 0 (0.00, 10.00) 0 0 (0.00, 12. 8) 23 100.00 (87.2, 100. 00)Ciprofloxacin 0 0 (0.00, 10.00) 0 0 (0.00, 12. 8) 23 100.00 (87.2, 100. 00)Nalidixic acid 0 0 (0.00, 10.00) 0 0 (0.00, 12.8) 23 100.00 (87.2, 100. 00)Sulphonamides 22 95.65 (80.2, 99.5) 1 4.35 (0.5, 19.7) 0 0 (0.00, 12.8)Sulpha/Trimethoprim 0 0 (0.00, 12.8) 0 0 (0.00, 12. 8) 23 100.00 (87.2, 100. 00)Trimethoprim 0 0 (0.00, 12.8) 0 0 (0.00, 12. 8) 23 100.00 (87.2, 100. 00)Doxycycline HCl 0 0 (0.00, 12.8) 2 8.70 (2.2, 25.9) 21 91.30 (74.1, 97.8)Tetracycline 0 0 (0.00, 12.8) 0 0 ( 0 .00, 12. 8) 23 100.00 (87.2, 100. 00)* Confidence Interval80

Figure 16 describes the percentage of Salmonella strains resistant to differentkinds of antimicrobials. 4.35 % of Salmonellastrains were not resistant to any kind ofantimicrobial. No Salmonella isolate has been resistant to only one antibiotic.However, there were 91.3 % of Salmonella isolates resistant for 2 kinds ofantimicro bials and 4.35% resistant for 3 kinds of antimicrobials.% Salmonella resistance100908070605040302010091.34.35 0 4.35sulphonamidesulphonamideerythromycinerythromycinamoxycillinNot resistant 1 Resistant 2 Resistant 3 ResistantNo. ofantimicrobialsFigure 16:Percentage of Salmonella isolates resistant to antimicrobialsThe antimicrobial resistance pattern by the type of shrimp is shown in Table 19.Out of 12 capture isolatesall 12 showed the sameresistant pattern that combinationwas erythromycin and sulphonamide.Salmonella isolates from culture shrimpsshow ed 2 resista nt patternsand outof 10 isolatestested there were 9 isolates hadcombination of erythromycin and sulphonamides and 1 isolate had combination oferythromycin, sulphonam ide and amoxycil lin.

82Table 19: Antimicrobial resistance pattern by type of shrimpType No. of Patterns Antimicrobials No. of isolates Percentage(95 % CI*)Capture 1 E, S3 12(n=12)100.00(76.6, 100.00)Culture 2(n=10)* Confidence IntervalE=ErythromycinS3=SulphonamideAML=AmoxycillinE, S3 9 90 (59.6, 99.0)E, S3, AML 1 10 (1.0, 40.4)The antimicrobial resistant pattern by serotype is shown in Table 20. The most ofthe serotypes have the same resistance pattern for the combination of erythromycinand sulphonamide except one of the S. Newport which has the resistance pattern oferythromycin, sulphonamide and amoxycillin.Table 20: Antimicrobial resistance pattern by serotypeSerotype Resistance pattern No. of isolates PercentageS. Newport (n=11)S. Weltevreden(n=2)E, S3 1 0 90.91E, S3, AML 1 9.09E, S3 2 100S. Mbandaka (n=1) E, S3 1 100E15* (n=2) E, S3 2 100B4* (n=2) E, S3 2 100F-67 (n=2) E, S3 2 100E10* (n=1) E, S3 1 100C7* (n=1) E, S3 1 100* not typable

83Table 21 describes the percentage of resistant serotypes. Out of 22 resistantserotypes S. Newport showed the highest resistance (50.00 %). Followed by S.Weltevreden, E15*, B4*, F-67 each (9.09 %). The lowest was observed for S.Mbandaka, E10* and C7* each (4.55 %).Table 21: Percentage of resistant serotypes (n=22)Serotype No. of isolates % isolates 95% CI**S. Newport 11 50.00 29.8, 70.2S. Weltevreden 2 9.09 2.3, 26.9S. Mbandaka 1 4.55 0.5, 20.5E15* 2 9.09 2.3, 26.9B4* 2 9.09 2.3, 26.9F-67 2 9.09 2.3, 26.9E10* 1 4.55 0.5, 20.5C7* 1 4.55 0.5, 20.5* not typable, ** Confidence Interval4.5 Overall microbiological analysis of shrim ps purchased from local sale pointsMicrobiological contamination of shrimps was analysed only for limitednumber of samples for Aerobic Pl ate Count 27, Enterobacteriaceae 34, E . coli 56 andTotal Halophilic Plate Count 20 respectively.4.5.1 Bacteriological profileThe overall bacteriological profile of shrimps purchased (Table 22) describes thelevel of microbial contamination in terms of log Colony Forming Unit/g. AerobicPlate Count, Enterobacteriaceae, E. coli and Total Halophilic Plate Count were up to10.22, 7.68, 6.89 and 9.24 respectively. The minimum levels were 5.94 for Aerobic

84Plate Count, 4.28 for Enterobacteriaceae, 1.96 for E. coli and 6.18 for TotalHalophilic Plate Count respectively.Table 22: Bacteriological profile of shrimps tested in terms of log CFU/gParameter No. of samples Minimum Median MaximumexaminedAPC 27 5.94 7.18 10.22Enterobacteriaceae 34 4.28 6.09 7.68Escherichia coli 56 1.96 1.99 6.89THPC 20 6.18 6.699.24APC = Aerobic Plate CountTHPC = Total Halophilic Plate Count4.5.2 Mi crobiological quality evaluation of shrimpsIn case of quality determination of shrimps sold at local sale locations, the testedmicrobiological parameters of shrimps were compared with the available standards ofraw fish recommended by German society for Hygiene and Microbiology (DeutscheGesellschaft für Hygiene und Mikrobiologie). Enterobacteriaceae ≤ 10 5 CFU/g, E.coli ≤ 10 2 CFU/g were considered as standard limits for the comparison (DGHM,2007). For APC ≤ 10 7 CFU/g was used as standard (ICMSF, 1986).The microbiological contamination of the raw shrimps represented by thebacteriological parameters was high and nearly half of the samples met the standards(Table 23). There were thirteen samples met ≤ 10 7 CFU/g for Aerobic Plate Count(APC). Eleven samples tested met the standards of Enterobacteriaceae. Out of 50samples 28 samples met ≤ 10 2 Escherichia coli counts. Total Halophilic Plate Count(THPC) was not compared since there is no existed standard.

85Table 23: Bacteriological quality of marketed shrimpsParameter No. of samples No. of samples Percentage 95 % CI**Examined met standardsAPC 27 13 48.1 30.8- 66.7Enterobacteriaceae 34 11 32.4 18.3- 49.3Escherichia coli 56 28 50 36.50- 63.50THPC* 20 - --* No existed standards to compare** Confidence Interval4.6 Results of the questionnaireThe questionnaire survey was conducted at 90 different sale points in order toevaluate general contamination through the routine practices of shrimp handling atlocal level. Univariate analysis was carried out and compared proportions using thechi-square test. Numerical data were analysed using non-parametric tests such asKruskal-Wallis test and Wilcoxan test.There are different factors contribute for the level of microbiologicalcontamination. Some factors are favourable and some factors are non favourable andat the same time more than one factor affect for the quality deterioration. The presentstudy revealed represent microbiological contamination is the sum of the favourableand non favourable factors for the studied micro-organisms. Therefore, local salelocation as a last check point where the microbiological contamination can bemeasured, represent the microbiological quality of the shrimp as last check point ofthe commercial operation.

864.6.1 Potential factors and the levels for Salmonella contaminationThe percentages of Salmonella in raw shrimps and the potential factors areshown in Table 24. It shows that th ere are several major potential factors to the level(p0.05). However, higher percentagepositives were observed in the Puttalam district, capture shrimps, receiving via middleman, vehicle type of motor bicycle, duration of transport 2-3 hours, condition oftransport with ice, handling with gloves, wash hands-some times, tile table use ascontainerwhile on sale and sales per day 1-30 Kg.

87Table 24: Summary results of potential factors and the levels for Salmonellacontamination in shrimps (univariate analysis) (n=180)Factors Level No. of samplesPositivePlacePuttalam16/120Kurunegala 7/60Shrimp type Culture10/90Capture13/90Status/seller High2/16Medium6/96Low15/68Receive Middle man 19/132Transport/VehicleTransport/DurationTransport/ConditionTransport/Mix with fishGrade%Positive95 %CI*P-value13.33 8.05-21.04 0.81711.67 5.21-23.1811.11 5.75-19.92 0.65614.44 8.22-23.8012.5 3.2-35.5 0.0126.25 2.57-13.6422.06 13.26-34.0614.39 9.11-21.82 0.326Farmer4/488.33 2.70-20.87Freezer truck 1/147.14 0.8-30.8 0.095Motor bicycle 8/3423.53 11.5-39.9Bicycle0/180.00 0.00-16.0Truck 14/11412.28 7.12-20.07< 1 Hour 2/26 7.69 1.9-23.2 0.8151-2 Hours2-3 Hours> 3 HoursFreezer truckWith iceWithout iceYesNoNumber1Number 2Number 34/3417/1180/21/1419/1343/3215/728/108-6/10017/80Handling Open handWith gloves22/1761/4How often Every time 0/14Wash hands Some times 23/166Container Wood1/6Tile4/14Rigiform9/80Cool box0/12Freezer0/6Other9/48Plastic box 0/14Cleaning/ContainerSales/dayEnd of the dayNot every end ofthe day1-30 Kg>30-60 Kg>60-90 Kg18/1725/815/1126/482/207/307/969/5411.7614.410.007.1414.189.3820.837.41-6.0021.254.8-26.08.86-22.35**0.8-30.88.97-21.513.2-23.512.50-32.333.49-14.51-2.46-13.1213.21-32.110.7380.0120.00312.50 8.17-18.52 0.42425.00 **0.00 0.00-20.00 0.22113.86 9.16-20.2716.67 1.7-59.4 0.15028.57 12.2-55.611.25 5.59-20.760.00 0.00-23.40 0.00-40.618.75 9.44-33.100.00 0.00-20.0010.4762.513.3912.5010.0023.337.2916.67Remains/day YesNoRarely* Confidence Interval, **Sample size too small to calculate CI6.49-16.2727.8-86.37.94-21.445.19-25.942.5-29.310.8-40.83.23-14.948.36-29.790.0010.9140.042

88The low socio-economic status of the vendor, mix with other fish species duringtransport, number 3 grade of the shrimp, container cleaning not every end of the daywere the risk factors associated with Salmonella contamination at local sale locations.Medium socio-economic status of the seller and shrimps remain at the end of the daywere trends to be the risk factors.Table 25: Logistic regression of the risk factors associated with SalmonellacontaminationFactors Level P-value OR 95 % CI*Status/seller High-10Medium0.3991. 981 0.405 – 9 .702Low0.0054. 245 1.553 – 11.606Transport/ No-10Mix with Y es0.0113. 29 1.31 – 8.24fishGrade Number 2-10N umber 30.0044. 228 1.580 – 11.308Cleaning/ End of the day-10Container N ot every end of the day 0.00114.2503.143 – 64.698Remains/day Rarely-10Yes0.4581. 522 0.502 – 4.609No0. 082 0. 393 0.138 – 1. 125* Confidence Interval4.6.2 Potential factors and the levels for APC in shrimpsThe microbial load (APC) in the raw shrimp s and the most likely potentialfactors are shown in Table 26. Since the data are not normally distributed to measurethe central tendency use the median. Statistically significant potential factors to thelevel (p

89Table 26: Summary results of potential factors and the levels for APC (log CFU/g)in shrimps (univariate analysis). (n=27)Factors Level No. of samples % Median P-valuetestedSamplestestedPlacePuttalam 1451.85 6.45 0.438Kurunegala 1348.15 9.07Shrimp type Culture1451.85 9.05 0.073Capture1348.15 6.44Status/seller High414.81 10.17 0.006MediumLow121144.4440.748.516.49Receive Middle man 2281.48 6. 64 0. 021Farmer5Transport/ Freezer truck 4Vehicle Motor bicycle7Bicycle3Truck13Transport/ < 1 Hour 7Duration1-2 Hours3>2-3 Hours17> 3 Hours -Transport/ Freezer truck 4Condition With ice 14Without ice 9Transport/ Yes 17Mix with fish No10Grade Number1 -Number 2 12Number 3 1518.5214.8125.9311.1148.1525.9311.1162.96-14.8151.8533.3362.9637.04-44.4455.55Handling Open handWith gloves25292.597.416.6510.21How often Every time - - -wash hands Some times 27 100.00 7.18Container Wood27.41 6.64Tile---Rigiform 1555.55 6.45Cool box ---Freezer414.81 9.99Other622.22 7.25Plastic box ---Cleaning/ContainerSales/dayRemains/dayEnd of the dayNot every end ofthe day1-30 Kg>30-60 Kg>60-90 KgYesNoRarely2431944812788.8911.1170.3714.8114.8129.6344.4410.1410.170.0026. 236. 359. 076. 12 0.0046.499. 86-10.170.000399. 056. 237. 33 0.8806. 39-0.9226.819.048.186.646.459.9910.179.976.2425.93 10.130.0210.3220.440.00050.004

904.6.3 Potential factors and the levels for Enterobacteriaceae contamination in shrimpsThe level of Enterobacteriaceae contamination in raw shrimps sold at local salepoints is indicative of the handling practices which are statistically significant (Table27). The socio-economic status of the vendor, the type of transport vehicle, theduration of transport, the conditions of transport, the type of shrimp container usedand remain of the shrimp at the end of the day show potential for Enterobacteriaceaeco ntamination to the le vel (p

91Table 27: Summary results of potential factors and the levels for Enterobacteriaceae(log CFU/g) in shrimps (univariate analysis)(n=34)Factors Level No.of samplestestedPlaceShrimp typeStatus/sellerReceiveTransport/VehicleTransport/DurationTransport/ConditionTransport/Mix with fishGradeHandlingHow oftenwash handsContainerCleaning/ContainerSales/dayRemains/dayPuttalamKurunegala1915Culture15Capture19High3Medium 15Low 16Middle man 28Farmer6Freezer truck 3Motor bicycle 9Bicycle4Truck18< 1 Hour1-2 Hours>2-3 Hours> 3 HoursFreezer truckWith iceWithout iceYesNoNumber1Number 2Number 3Open handWith glovesEvery timeSome timesWoodTileRigiformCool boxFreezerOtherPlastic boxEnd of the dayNot every end ofthe day1-30 Kg>30-60 Kg>60-90 KgYesNoRarely7621-32381519-16183223311-136113--3425816226%samplestested55.8844.1244.1255.888.8244.1247.0682.3517.658.8226.4711.7652.9420.5917.6561.76-8.8267.6523.5344.1255.88-47.0652.9494.125.888.8291.182.94-38.2417.652.9438.24--100.0073.5323.532.9417.6564.7117.65Median5.127.326.375.124.636.395.666.245.254.625.124.737.325.085.397.31-4.637.215.107.325.15-5.297.326.244.854.636.37--4.936.397.33--6.096.066.39-7.495.017.27P-value0.0140.2310.0310.8570.0100.0470.0410.2820.1670.1640.0560.0290.6930.001

924.6.4 Potential factors and the levels for E. coli contamination in shrimpsThe occurrence of E. coli in raw shrimps sold at different sale locations whichshow a potential connec tion to the factors of general handling are shown in Tale 28.Results of the univariate analysis describe so me of the factors are statisticallysignificant (P

93Table 28: Summary results of potential factors and the levels for Escherichia coli(log CFU/g) in shrimps (univariate analysis) (n=56)Factors Level No.of samplestestedPlaceShrimp typeStatus/sellerReceiveTransport/VehicleTransport/DurationTransport/ConditionTransport/Mix with fishGradeHandlingHow oftenwash handsContainerCleaning/ContainerSales/dayRemains/dayPuttalamKurunegalaCultureCaptureHighMediumLowMiddle manFarmerFreezer truckMotor bicycleBicycleTruck< 1 Hour1-2 Hours>2-3 Hours> 3 HoursFreezer truckWith iceWithout iceYesNoNumber1Number 2Number 3Open handWith glovesEvery timeSome timesWoodTileRigiformCool boxFreezerOtherPlastic boxEnd of the dayNot every end ofthe day1-30 Kg>30-60 Kg>60-90 KgYesNoRarely243229270371956--1224251338--4792828-223456--5622352-15-5424610-11414%samplestested42.8657.1451.7948.210.0066.0733.93100.00--21.433.5775.008.9323.2167.86--83.9316.0750.0050.00-39.2960.71100.00--100.003.573.5762.53.57-26.79-96.433.5782.1417.86-19.6473.217.14Median3.381.961.962.7601.9631.99--3.56-1.963.123.501.96--1.963.121.962.38-1.963.011.99--1.99--2.76--3-2.38-2.381.96-3.891.96-P-value0.0080.9160.3650.00030.1230.9150.6930.0430.2230.1860.8910.001

944.6.5 Potential factors and the levels for halophilic bacterial contamination in shrimpsRaw shrimps examined for total halophilic bacteria and indicative of generalhandling practices which show significant associati ons are listed in Table 29. Theassociated factors to the level (p

95Table 29: Summary results of potential factors and the levels for Total HalophilicPlate Count (log CFU/g) in shrimps (univariate analysis) (n=20)Factors Level No. of samplesPositivePlacePuttalam13Kurunegala 7Shrimp type Culture7Capture13Status/seller High2Medium8Low10Receive Middle man 14Farmer6Transport/ Freezer truck 0Vehicle Motor bicycleBicycleTruck5411Transport/DurationTransport/ConditionTransport/Mix with fishGradeHandlingHow oftenwash handsContainerCleaning/ContainerSales/dayRemains/day< 1 Hour1-2 Hours>2-3 Hours> 3 HoursFreezer truckWith iceWithout iceYesNoNumber1Number 2Number 3Open handWith glovesEvery timeSome timesWoodTileRigiformCool boxFreezerOtherPlastic boxEnd of the dayNot every end ofthe day1-30 Kg>30-60 Kg>60-90 KgYesNoRarely947--119911-12820--204-9--7-1461622-128%PositiveMedian P-value65.00 6.5135.00 8.7735.00 8.77 0.000365.00 6.5110.00 6.22 0.22140.00 6.6950.00 6.5170.00 6.77 0.04830.00 6.2500.02325.00 6.6520.00 6.3955.00 6.8845.0020.0035.00--55.0045.0045.0055.00-6040100.00--100.0020.00-45.00--35.00-70.0030.0080.0010.0010.00-60.0040.006.516.468.77--6.886.517.536.51-6.587.836.69--6.699.01-6.65--6.51-6.678.786.696.696.22-6.697.490.0010.0070.0250.0640.010.1870.3180.7518

5. DISCUSSIONS AND CONCLUSION5.1 Discussions5.1.1 Prevalence, serotypes and antimicrobial resistance of Salmonella in marketedshrimps (Penaeus monodon)5.1.1.1 Prevalence and serotypes of Salmonella in marketed shrimpsSeafood quality is one of the major issues in the seafood industry and freshlyharvested seafood-whether caught in the wild or aqua-cultured-has the potential todeteriorate in quality due to many factors including technological problems,infrastructure inadequacy, cultural practices or socio-economic factors (Anderson andAnderson, 1991). The United States Food and Drug Administration showed that theprevalence of Salmonella in imported and domestic seafood were 7.2 % and 1.3 %,respectively, from 1990 to 1998, with raw seafood showing the highest contaminationrates: 10 % import and 2.8 % domestic (Heinitz et al., 2000). Contamination canoccur at multiple steps along the food chain including production, processing,distribution, retail marketing, and handling/preparation (Zhao et al., 2002). Shrimpspurchased from wholesale markets and retail sale points in Hyderabad city in Indiahas shown samples to have had high levels of filth and bacteria due to variousunhygienic practices followed during the chain of commercial operation. Theprevalence of Salmonella in retail markets was higher (11 %) than in whole salemarkets (5 %) (Jonnalagadda and Bhat, 2004). In contrast to these findings Hatha etal., 2003, reported that the shrimp products for export trade maintain superiorbacteriological quality by exerting good process control to meet the internationalstandards of the import country. During their study they have found only one samplepositive for Salmonella from a total of 846 samples of raw, peeled and deveined tail-

97on tiger shrimp (Penaeus monodon). Fonseka, 1994, reported higher Salmonellacontamination rate (8 isolates) of shrimps after harvest and a lower rate (2 isolates) forprocessed products from a total of 295 samples. Arumugaswamy et al., 1995observed that the prevalence of Salmonella in raw foods (32 %) were higher than inready-to-eat cooked foods (17 %) in Malaysia. Among the raw foods the proportionof Salmonella in chicken pieces (39 %), liver (35 %), gizzard (44 %), oysters (86 %)were higher than in prawns (25 %).Minimum growth temperature of Salmonella is 6-7 o C, maximum growthtemperature in the range of 45-47 o C (Bremer et al., 2003). Because of thismesophilic nature Salmonella is considered to be having a longer shelf life in ice(Surendran et al., 1994). Pond cultured shrimps showed faster deterioration (12 h)when shrimps were held at ambient temperature than in melting ice (15 days)(Fonseka and Ranjini, 1994). Thus the presence of Salmonella (12.78 %) in marketedshrimps in this study might be due to inadequate hygienic standards and the inabilityto maintain the cold chain during post harvest. The overall prevalence of Salmonellain culture shrimps was found to be 11.11 % and in capture shrimps 14.44 % and thislow prevalence of Salmonella in culture shrimps than capture shrimps was observedeven in the Puttalam and in the Kurunegala districts. The low prevalence in cultureshrimps than in capture shrimps was due to the better farm management practicesunder the close supervision that is not apply in capture shrimp fishery. However, bothtypes of shrimps are grown in brackish water and when compared to other raw foodproducts there is benefit for low levels of Salmonella due to bactericidal nature of thebrackish water; it is reported that salmonellae are unable to tolerate high brine above 9% (Jay, 2000). When concern the place the high prevalence of Salmonella wasobserved in the Puttalam district than the Kurunegala district which might be due tocontamination through bare hands, high remains of shrimps at the end of the day, highnumber of low status of the vendors, high middle man involvement and the inabilityto maintain the cold chain during transportation.There were statistically significant differences observed in the levels ofprevalence of Salmonella among the market locations even though they originate

98from common sources. It might be due to different hygiene practices applied in these14 different selling locations where shrimps were purchased. Conversely, a studycarried out by Adesiyun et al., 2006, detected there were no statistically significantdifferences of prevalence of antimicrobial resistance amongst bacteria from three saleoutlets where table eggs sampled originated from common layer farms. Thesefindings support the theory of the possibility of microbiological contaminationoccurring during the sale at local market locations even though they have a commonorigin.Reilly and Twiddy (1992) reported that the predominant serovar occurring inshrimp farms in South East Asia is Salmonella Weltevreden. The serotypes,Salmonella Anatum, Salmonella Wandsworth and Salmonella Potsdam are some ofthe other serovars reported to occur to a lesser extent. Shabarinath et al., 2007, notedthat the principal serovar prevalent in seafood (fourteen out of nineteen isolates) is S.Weltevreden and that they are genetically diverse which supports the findings ofReilly and Twiddy, from 1992. A study in the Chiangmai and Lampoon provinces ofnorthern Thailand indicated that S. Weltevreden is the most common serotypeinvolved in human infections (Padungtod and Kaneene, 2006). The World HealthOrganization (WHO) National Salmonella and Shigella Centre in Bangkok revealedthat during 1993-2002 the most common serovars accounting for human infectionswere S. Weltevreden, S. Enteritidis, S. Anatum, S. Derby, Salmonella (1,4,5,12:i), S.Typhimurium, S. Rissen, S. Stanley, S. Panama and S. Agona. As well as the mostcommon serovar isolated among the 1,007 isolates from seafood was S. Weltevreden,and it was the most common serovar isolated from humans in Thailand(Bangtrakulnonth et al., 2004).Fish and other seafood associated with Salmonella serovars were reported byNational Salmonella Centre in India as the major Salmonella serovars of zoonoticsignificance, S. Typhimurium, S. Newport, S. Weltevreden, S. Saintpaul and S.Enteritidis (Singh, 2007). Infection due to Salmonella Weltevreden in humans inIndia was quite uncommon before 1970 and it accounted for less than 4 % of totalhuman salmonellosis. The incidence of infection increased considerably after 1970

99and in 1972 this serotype constituted 29.1 % of total human salmonellosis in India(Basu and Sood, 1975).From those serovars commonly involved in food-borne illness in the UnitedStates the most frequent serotypes in import seafood were Salmonella Weltevreden.The other top 20 important serovars are Salmonella Senftenberg, SalmonellaLexington, Salmonella Paratyphi-B, Salmonella Enteritidis, Salmonella Newport,Salmonella Thompson, Salmonella Typhimurium and Salmonella Anatum, butSalmonella Enteritidis is the most common Salmonella serotype causing infection anddeath in people, followed by Salmonella Typhimurium (Heinitz et al., 2000).The present study carried out in North Western province, Sri Lanka, revealed thepredominant Salmonella serovars in marketed Penaeus monodon shrimps to be S.Newport (47.83 %), S. Weltevereden (8.69 %), B4 (not typable) 8.69 %, E15 (nottypable) 8.69 %, E10 (not typable) 8.69 %, F-67 (Salmonella Poly II) 8.69 %, C7 (nottypable) 4.35 %, S. Mbandaka 4.35 %. S. Typhimurium is the serotype of Salmonellawhich has the most ubiquitous host range (Busani et al., 2004). Though S.Weltevreden has been reported by several studies to be a frequently found Salmonellaserovar, it is found in shrimps to a lesser extent than S. Newport and S. Typhimuriumwas not found in the current study. The exhibition of higher percentage of S. Newportwas similar to that of another source which reported 12 out of 30 samples from retailturkey samples in Fargo, North Dakota, USA (Fakhr et al., 2006).5.1.1.2 Antimicrobial resistance in Salmonella isolated from shrimpsThere is still a debate on the relationship between animal use of antibiotics andresistance in human pathogens because human use of antibiotics has undoubtedlycaused the resistance epidemic all over the world (Beović, 2006). There is necessity incoordination between human, veterinarian and environmental sectors to clarify theoccurrence of resistant pathogens in humans, animals and in the environment (Anon,2002). A study carried out by Zhao et al., 2002, in the USA indicated thatantimicrobial resistant Salmonella are present in imported foods, primarily of seafood

100origin. Thirty fish species of capture and aquaculture origin were found to containmulti-resistant strains of Salmonella. Findings of the present study showed that therewas no statistically significant difference in the percentage resistance amongSalmonella obtained from culture shrimps and capture shrimps. This might be due toantibiotic usage in shrimp farming and release of shrimp pond effluent to estuarineecosystems or post harvest contamination of shrimps with the antibiotic resistantSalmonella through the environment and human handling. Moreover the mostprevalent serotypes among these Salmonella were S. Newport but it was reported inSouth East Asia the most prevalent is S. Weltevreden (Reilly and Twiddy, 1992).Therefore, this observation was more supported with the post harvest contaminationof shrimps with antibiotic resistant Salmonella.On the other hand it is important to carry out further investigation on emergingantimicrobial resistance in capture shrimp ecology which was reported by severalstudies. This has been attributed to the rapid expansion of the shrimp farmingindustry, resulting in increased pressure on wild shrimp resources. There are potentialadverse environmental effects on estuarine eco-systems through shrimp pondeffluents, "biological pollution" of native shrimp stocks through escapement ofaquaculture stocks, water use and entrainment of estuarine biota, and the impacts ofshrimp farm chemicals on estuarine systems (Hopkins et al., 1995). A higherpercentage of antibiotic resistance in aquaculture shrimps has been evidenced by Le etal., 2005, from their study carried out in Vietnam. They reported a wide spread use ofantibiotics in aquaculture, which may result in residues of antibiotics in water andmud, and subsequently, the development of antibiotic resistance in bacteria in theenvironment.An overall percentage of 91.3 % for resistance to the antimicrobial agents’sulphonamide and erythromycin was detected among Salmonella isolates fromshrimps in this study. Akinbowale et al., 2006, reported that resistance to ampicillin,amoxycillin, cephalexin and erythromycin was wide spread in aquaculture sources inAustralia and found off label use since no antibiotics registered for use in aquaculture.Resistance to sulfonamides was common, similar to this present study. Moreover,

101isolates from aquaculture sources were resistant to oxytetracycline, tetracycline andnalidixic acid in contrast to this present study. Results of the present study showedthat all the Salmonella isolates are susceptible to other antimicrobials such as nalidixicacid, amoxycillin (except one strain), ampicillin, gentamycin, chloramphenicol,kanamycin, sulphamethoxazole/trimethoprim, ciprofloxacin, trimethoprim,tetracycline, streptomycin and doxycycline hydrochloride that were tested in thisstudy. Fonseka, 1994, observed Salmonella isolated from farm shrimps in Sri Lankawere not resistant to any kind of antibiotic tested such as ampicillin 10 (µg),cephaloridine 5 (µg), colistin sulphate 25 (µg), gentamicin 10 (µg), streptomycin 10(µg), sulphatriad 200 (µg), tetracycline 25 (µg), and cotrimoxazole 25 10 (µg). Thisdetection of highest resistance to erythromycin in Salmonella isolates has beensupported by the findings of erythromycin resistance for all the isolates of Vibriospecies namely Vibrio harveyi, V. parahaemolyticus, V. alginolyticus, V. anguilliarumand V. vulnificus from Penaeus monodon shrimps and water in Sri Lanka attributed tothe wide spread use (Panawala et al., 2005). However, wide use of macrolides inhuman medicine and increasing resistance to macrolides in human pathogens has beenreported (Phillips et al., 2004). Sulphonamide is used as an antibiotic treatment forhuman as well as animal diseases. Therefore, the application of this antibiotic is high.Moreover, it was reported that mostly the fate of antibiotics in the environment hasbeen primarily in surface waters and concentrations of sulphonamides are high inwater wells in Idaho, USA (Angela et al., 2006). This environmental contaminationof antibiotics can lead to emergence of sulphonamide resistance in Salmonella sinceshrimps are associated with water or this might be due to overuse of these two drugsin aquaculture practice.In general bacteria can develop resistance for antibiotics which share similarstructures (Angela et al., 2006). Though some of the antibiotics tested belong to thesame class such as amoxycillin, ampicillin and sulphonamides,sulphamethoxazole/trimethoprim showed resistance in Salmonella only foramoxycillin and sulphonamides due to structural differences of the antibiotics.Amoxycillin, nalidixic acid and ciprofloxacin are used in human medicine in SriLanka, the same antibiotics were used in this study noted percentage susceptibility of

102Salmonella isolates from shrimps are: 95.65 % for amoxycillin, 100 % for nalidixicacid and 100 % for ciprofloxacin. Furthermore, Salmonella isolated from human casesshows they are susceptible to these antimicrobials and percentage susceptibility is:amoxycillin 88.9 %, nalidixic acid 84.4 %, ciprofloxacin 100 %. Other antimicrobialscommonly use for human treatments are observed Salmonella is susceptible and areidentified as furazolidone 83.3 %, cotrimaxazole 72.7 %, mecillinam 96.4 %,ceptazidime 93.5 %, cefuroxime 62.5 %, chloramphenicol 100 %, ceftriaxone 100 %by (MRI, 2005).S. Typhimurium is a globally important serotype regarding the antimicrobialresistance. The antimicrobial resistance of 809 Salmonella Typhimurium isolatescollected from humans in Norway between 1975 and 1998 described the largestincrease in resistance to be in 1996 when 35 % of the isolates were multi-resistant.The first multi-resistant isolate acquired in Norway appeared in 1994, but already in1998, 23 % of the isolates domestically acquired were multi-resistant, and a majoritywere S. Typhimurium DT 104. There was no ciprofloxacin resistance in domesticallyacquired isolates (Leegard et al., 2000). In this study S. Typhimurium was not foundand all the isolated Salmonella serotypes showed 100 % susceptibility tociprofloxacin. Ciprofloxacin and fluoroquinolones are very important because theyare considered to be the last line defence against DT 104 in adults (Carlson, et al.,2003).When compared with S. Typhimurium resistance to penta drugs ampicillin,chloramphenicol, streptomycin, sulphonamide and tetracycline, the present isolatedSalmonella serotypes showed resistance only to sulphonamide. Busani et al., 2004,reported that among S. Typhimurium, multi-resistance was more common in bovine,poultry and rabbit strains, it was lower in swine strains and in the case of pigeons itwas rare. They observed that resistance to trimethoprim–sulphamethoxazole wasmainly in isolates of swine and human origin and this shows rare possibility to occurS. Typhimurium in shrimps.In 1996, Salmonella Newport caused 5 % of Salmonella infections in humans inthe United States; by 2003, it represented 12 % of all reported Salmonella infections.

103Emergence of antibiotics resistant Salmonella Newport was reported by Varma et al.,2006 as multi-drug resistant (MDR) strain of Salmonella Newport, Newport-MDRAmpC, which showed wide dissemination and infection was acquired throughthe US food supply, most likely from bovine and poultry sources. This serotype wasreported in India from frozen duck (3.7 %) (Bangtrakulnonth et al., 2004). In thisstudy, the percentage of S. Newport isolated from shrimps was 47.83 % and all theisolates were resistant to erythromycin and sulphonamide. Conversely, Newport-MDRAmpC strain exhibits resistance to ceftriaxone, ampicillin,amoxycillin/clavulanic acid, cephalothin, cefoxitin, chloramphenicol, streptomycin,sulfamethoxazole, and tetracycline used to treat humans and ceftiofur which is used inveterinary medicine in the USA (Gupta et al., 2003).5.1.2 Microbiological contamination in terms of Aerobic Plate Count,Enterobacteriaceae count, E. coli and Total Halophilic Plate CountFood safety and quality aspects in trade became important because fresh food ismore prone to certain microbiological contamination. The Aerobic Plate Count(APC) indicates the level of micro-organisms in a product. As expected, the presentstudy revealed shrimps were higher in APC than the existing standards of raw fish atthe point of sale recommended by German Society for Hygiene and Microbiology(Deutsche Gesellschaft für Hygiene und Mikrobiologie). Thirteen samples out of 27samples met the standards. This was to be expected since fresh fish and fisheryproducts often have an APC of 10 4 -10 5 /g, although there are examples of seafoodswith an APC of 10 6 -10 8 /g without objectionable quality changes (Nickelson andFinne, 1992). A study undertaken at farm level reported APC of entire shrimp,shrimp surface and guts of the shrimp to be 8.40x10 5 , 4.60x10 5 / cm 2 and 1.26x10 6 /g(Nayyarahamed et al., 1994). This was supported by the evidence that such countsare usual in shrimps cultured in South East Asia (Fonseka, 1990). In the present studyshrimp represent post-harvest contamination which can be attributed to the landingcentres and unhygienic handling at fish markets.

104Enterobacteriaceae are useful indicators of hygiene, however, Surendran et al.,1994, have found that it is a natural contaminant in the case of cultured fishes orprawns. With regards to the cultured species it was reported that cultured prawnswere found to carry greater numbers of Enterobacteriaceae. This study found there tobe a higher number of Enterobacteriaceae count in twenty-three raw shrimp samples.It is reported that Enterobacteriaceae are widely distributed and to be found in thesoil, in water, vegetation and in the intestinal tract of animals (Blood and Curtis,1995) which might lead to cross-contamination. Conversely, Beckers et al., 1981,reported that the microbiological quality of frozen precooked and peeled shrimps forexport were usually low in numbers of Enterobacteriaceae (lesser than 2x10 2 per g) inSouth East Asia and North sea shrimps due to differences in processing.Out of 56 samples tested for E. coli 50 % met the standards. Kumar et al., 2005,reported that the contamination of E. coli was higher in the seafood samples collectedfrom the landing centres and from fresh fish markets than from frozen shrimp samplescollected from processing plants. Eleftheriadou et al., 2002 wrote down the criteria ofa microbiological profile with E. coli as an indicator organism considered significantwhen levels exceed 10 2 CFU/g. They found higher levels than the pathogens they hadstudied, S. aureus and B. cereus indicating the necessity of increasing simple hygienepractices.Samples tested for the Total Halophilic Plate Count (indigenous bacteria) rangedbetween minimum to maximum 6.18 log CFU/g to 9.24 log CFU/g and median was6.69 log CFU/g. The distribution of various bacterial genera on cultured fish andprawn were studied by Surendran et al., 1994, and results with >20% were Vibriowhich was higher than Enterobacteriaceae (10 5 -10 6 cells) for the pathogenic Vibrio spp. as reported by (Twedt, 1989). By contrast,levels from 10 3 to 10 4 per gram in raw seafoods should be considered asunsatisfactory due to inadequate temperature control regarding V. parahaemolyticus

105and limits

106handling and inadequate processing. Transportation was the most commonly affectedsituation for all these microbiological parameters: the type of vehicle, the duration oftransport, maintaining the cold chain (transport condition) during transport weresignificantly different. This is supported by the fact that microbiological changesoccur when shrimps are insufficiently iced and improperly stored at highertemperatures (Antony et al., 2002). Contamination also occurs during post processhandling and transportation (Kumar et al., 2005).Exposure to other fish species during transport was significant for positiveSalmonella and higher Total Halophilic bacterial contamination due to crosscontamination.Moreover, the low socio-economic status of the vendor, number 3grade of the shrimp, container cleaning not every end of the day were the risk factorsassociated with Salmonella contamination at local sale locations. However shrimpsremain at the end of the day and medium socio-economic status of the vendor weretrends to be the risk factors.The storage types of containers were significantly different in the case ofEnterobacteriaceae and Total Halophilic bacteria. The storage containers are better tobe replaced by suitable alternative. As regards to personal hygiene, though most ofthe vendors handle the shrimp with their bare hands and in between hand washing dosometime, both of them were not significant (p>0.05) in the case of Salmonella; andgenerally speaking for other microbiological parameters these higher numbers ofmicrobiological parameters might be due to potential cross-contamination betweenthe environment and the shrimps. Therefore, the chain of commercial operationshould be studied thoroughly in order to evaluate market failures to produce higherquality products.

1075.2 ConclusionIn general retail level is the last check point where contaminated end product canbe identified and the microbiological examination of shrimps plays an important rolein assuring the safety and quality. Salmonella was found in 23 out of 180 shrimpsamples from retail sale locations in North Western Province, Sri Lanka. The isolatedSalmonella serotypes represented environmental contamination of food of animalorigin. Higher levels of antimicrobial resistance were observed for erythromycin andsulphonamide (91.3 %). Salmonella were susceptible for rest of the antibiotics testedrespectively, nalidixic acid, ampicillin, gentamycin, sulphamethoxazole/trimethoprim,chloramphenicol, kanamycin, ciprofloxacin, trimethoprim, tetracycline, streptomycin,doxycycline hydrochloride, amoxycillin (only one isolate resistant). The antimicrobial(erythromycin and sulphonamide) resistant Salmonella isolates from capture shrimpsmust be taken into consideration due to the aquaculture practices can lead to theemergence of resistant strains in the environment which will have a long term effect.The results found in this study explain microbiological contamination of shrimpsrelated mostly to a lack of food hygiene requirements and potential environmentalcontamination where facilitate contamination, due to reckless handling. The riskfactors associated with Salmonella contamination were low status of the seller, mixshrimps with other fish species during transportation, third grade of the shrimp andcleaning of the container not every end of the day. However medium status of theseller and remain of the shrimps at the end of the day were trends to be the riskfactors.Furthermore, it is taken into consideration that it is virtually impossible toexclude all microbiota from food-related surfaces except if such surfaces aresterilized. This would be unpractical and unnecessary as well. However, sea food is ahighly perishable product, its quality deteriorate more rapidly than other proteinsources such as chicken and red meat. Therefore, the evaluation of the chain ofcommercial market failures in the industry would improve seafood distribution, withbetter alternatives achieving higher quality products as with other fresh products in

108terms of appearance, odour, flavour, texture and thus eliminating food-bornepathogens and thereby ensuring food safety.As in many other developing countries, Sri Lanka has little information availableon the occurrence of food contamination with regard to biological and chemicalcontamination, especially in domestic products. Without such information the healthof the people may be threatened. This information of biological contamination oflocal shrimps will help to improve the safety of the food supply and thus reduce theincidences of food-borne diseases.Improving food safety along western standards, however, may incur considerablecosts and as such put it out of the reach of the poor. Therefore, the introduction ofnew food safety tools and rules need to be affordable and build on local seafoodmanagement customs rather than simply imposing western standards that areexpensive to monitor.5.3 Recommendations1. Huss et al., 2000, reported that the safety of various seafood products variesconsiderably and is influenced by a number of factors such as the origin of the fish,microbiological ecology of the product, handling and processing practices andtraditional preparation before consumption. Considering all these factors seafood isranked according to the risk from the highest to the lowest.1. Molluscs, including fresh and frozen mussels, clam, oysters in shell orshucked and raw fish to be eaten without any cooking.2. Fresh/frozen fish and crustaceans-to be eaten after proper cooking.3. Lightly preserved fish products (i.e., NaCl5.0). This group includes salted, marinated, fermented, cold smoked andgravid fish.

1094. Semi-preserved fish i.e., NaCl>6 % (w/w) in water phase, or pH

1106. Improvement of the hygiene of raw seafood at the local sale location level isrecommended which can be achieved by increasing both the education of the seafoodhandlers and consumers.7. Present low contributions of consumer representatives for constructivecommunication with the government and local fishery markets is a factor of societywhich needs to be changed since consumers are responsible for food safety.

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APPENDICESAPPENDIX A: MEDIA AND REAGENTSEnumeration: Aerobic Plate Count1. Maximum Recovery Diluent2. Plate Count AgarEnumeration: Enterobacteriaceae1. Maximum Recovery Diluent2. Violet Red Bile Glucose Agar3. Oxidase reagent4. Nutrient agarEnumeration: Escherichia coli1. Maximum Recovery Diluent2. 3M TM Petrifilm TM E. coli/Coliform Count Plates3. SpreaderEnumeration: Total Halophilic Plate Count1. Maximum Recovery Diluent2. Plate Count Agar3. NaCl

132Isolation and Identification of Salmonella1. Non- selective pre- enrichment medium: Buffered Peptone Water2. Selective enrichment medium:2.1 Rappaport-Vassiliadis medium with soya2.2 Muller-Kauffmann Tetrathionate novobiocin broth3. Solid selective plating out medium:3.1 Brilliant-green Phenol-red Lactose Sucrose agar3.2 Xylose-Lysine-Deoxycholate agar4. Nutrient Agar5. Triple Sugar Iron agar6. Urea agar7. L-Lysine decarboxylation medium8. Reagent for detection of β –galactosidase9. Reagent for detection Voges-Proskauer (VP) reaction10. Reagent for detection indole reaction11. Reagent for detection Methyl Red (MR) reaction12. Semi solid nutrient agar13. Sterile distilled water14. Physiological saline solution, 0.85 %15. Salmonella O Polyvalent A-E16. Salmonella O Polyvalent F-6717. Salmonella O Group A18. Salmonella O Group B19. Salmonella O Group C20. Salmonella O Group D21. Salmonella O Group E22. Salmonella O723. Salmonella O824. Salmonella O6 125. Salmonella O1026. Salmonella O1927. Salmonella O4

13328. Salmonella O529. Salmonella O2730. Flagellar antiserum for SalmonellaAntimicrobial sensitivity test for isolated Salmonella1. Nutrient agar2. Tryptic soy broth3. 0.5 McFarland standards3.1) 0.048 mol/L BaCl 23.2) 0.18 mol/L H 2 SO 44. Mueller-Hinton agar5. Antimicrobial disks5.1 Nalidixic acid 30 µg5.2 Amoxycillin 10 µg5.3 Ampicillin 10 µg5.4 Gentamycin 10 µg5.5 Erythromycin 15 µg5.6 Sulphamethoxazole/Trimethoprim 25 µg5.7 Chloramphenicol 30 µg5.8 Kanamycin 30µg5.9 Compound Sulphonamides 300µg5.10 Ciprofloxacin 5µg5.11 Trimethoprim 5µg5.12 Tetracycline 30µg5.13 Streptomycin 10µg5.14 Doxycycline Hydrochloride 30µg

134APPENDIX B: QUESTIONNAIRE FORMATMicrobiological quality of marketed Penaeus monodon shrimps in NorthWestern Province, Sri Lanka(Questionnaire for the evaluation of routine practice at the local shrimp markets)Date…………………..1. Name of the Province North Western Province2. Name of the District PuttalamKurunegala3. Name of the market ………………………………4. Name of the shop owner ………………………………5. Social Economic status of the ownerHighMediumLow6. How do you receive shrimps Through middle manDirectly from farmer7. Transportationa. Method Type of vehicle ……………b. Duration How many hours ………….c. What condition ……….................................d. Mix with other fish species yesNo8. Grade of the shrimps which sold Number 1Number 2Number 39. Nature of the shrimp which sold DeheadedWhole shrimp

13510. Source of ice use for shrimp ………………………...11. How do you handle shrimps during selling Open handWith gloves12. How often wash hands during selling Every timeSometimes13. Type of container keep shrimps during selling ………………14. How often clean the container End of the dayNot every end of the day15. Do you use disinfectants to clean YesNo16. Water quality treated YesNo17. Sales per daya. How much sold 1-30 Kg>30-60 Kg>60-90 Kgb. Whether remain at the end of the day YesNoRarely

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137CURRICULUM VITAENameDr. (Ms.) Kamalika Harshini UbeyratneJanrabelgeDate of birth September 15, 1971Work positionVeterinary SurgeonWork addressVeterinary Research InstituteDepartment of Animal Production and HealthGannoruwaPeradeniyaSri LankaE-mail addresskamalikau@yahoo.comHome addressNo. 11, St. Thomas Road, Matara, Sri LankaMarital statusSingleCitizenSri LankanReligionBuddhismSecond Language EnglishEducational BackgroundPrimary Education1978-1982 Sudharshana Adharsha Vidyalaya, Welegoda,MataraSecondary Education1983-1991 Sujatha Balika Maha Vidyalaya, Matara

138Tertiary Education1994-1999 Bachelor of Veterinary ScienceUniversity of PeradeniyaPeradeniyaSri LankaPostgraduate Studies2003-2005 Master Degree in Dairy and Meat ProductTechnologyPostgraduate Institute of AgricultureUniversity of PeradeniyaPeradeniyaSri Lanka2005-presentCandidate of Master of Veterinary Public HealthFreie Universität, Berlin, Germany and ChiangMai University, ThailandProfessional Carrier2002- To date Head Office, Department of Animal Productionand Health: Poultry Disease MonitoringVeterinary Research Institute, Department ofAnimal Production and Health: Poultry VaccineProduction, Disease Surveillance and LaboratoryDiagnosisProfessional TrainingLaboratory Practical in food microbiology at theInstitute of Meat Hygiene, Meat Technology andFood Science, University of Veterinary MedicineVienna, Austria (2 May 2006 to 19 May 2006)

139PublicationsUbeyratne, J.K.H., Dematawewa, C.M.B.,Cyril, H.W. (2004): Use of cactus fruit juice as anatural colouring agent in preparation of chickensausages. 56 th Annual Convention of the SriLanka Veterinary AssociationHettiarachchi, R., Ubeyratne, J.K.H.,Kodituwakku, S.N. (2001): A review of theanimal disease situation in Sri Lanka. The SriLanka Veterinary Journal 48.AwardsThe best poster presentationUbeyratne, J.K.H., Dematawewa, C.M.B.,Cyril, H.W. (2004): Use of cactus fruit juice as anatural colouring agent in preparation of chickensausages. 56 th Annual Convention of the SriLanka Veterinary Association.Scholarships2005-2007 German Academic Exchange Service (DeutscherAkademischer Austauschdienst) scholarship bythe Government of Germany