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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 Pr<strong>of</strong>essor Dr. Peter Paulsen from the Institute <strong>of</strong> MeatHygiene, Meat Technology and Food Science, University <strong>of</strong> Veter<strong>in</strong>ary Medic<strong>in</strong>eVienna who <strong>of</strong>fered me his guidance dur<strong>in</strong>g the laboratory practicals.I would like to express my s<strong>in</strong>cere gratitude to Ms. Iseult Rea for hercontributions to the English language edition <strong>in</strong> my thesis.I would personally like to thank all the scientists, pr<strong>of</strong>essors and all the lecturersassociated with the Master <strong>of</strong> Veter<strong>in</strong>ary Public Health Degree Programme at theFreie Universität Berl<strong>in</strong>, Germany, the University <strong>of</strong> Veter<strong>in</strong>ary Medic<strong>in</strong>e Vienna,Austria and Chiang Mai University, Thailand who have contributed immensely byprovid<strong>in</strong>g useful <strong>in</strong>formation, advice and skills. Besides, I would like to thank Ms.Juliane Fischer, Ms. Louise Le Bel and staff <strong>of</strong> the Veter<strong>in</strong>ary Public Health Centerfor Asia Pasific who supported me throughout this study.In addition, I am greatly <strong>in</strong>debted to many <strong>of</strong> my former teachers: Dr. (Ms.)Niranjala De Silva, Pr<strong>of</strong>essor P. Abeynayake, Dr. (Ms.) Thula Wijewardana, Dr.K.F.S.T. Silva, and Pr<strong>of</strong>essor H.W. Cyril for serv<strong>in</strong>g as referees and motivat<strong>in</strong>g me toparticipate <strong>in</strong> this International Programme.Let me also say ‘thank you’ to the follow<strong>in</strong>g people: Dr. Udeni Ediris<strong>in</strong>ghe at theFaculty <strong>of</strong> Agriculture, University <strong>of</strong> Peradeniya, Sri Lanka, for his guidance and<strong>in</strong>sightful comments, Dr. S.K.R. Amarasekara, Dr. J. Dharmawardana, Dr. A.D.N.Chandrasiri, Dr. M. Somaratne and laboratory staff <strong>of</strong> the Veter<strong>in</strong>ary ResearchInstitute, Department <strong>of</strong> Animal Production and Health, Sri Lanka, for provid<strong>in</strong>g mewith the facilities to carry out the laboratory analysis with a great support. Thank youalso to Pr<strong>of</strong>essor P. Abeynayake and staff, Department <strong>of</strong> Pharmacology and PublicHealth, Faculty <strong>of</strong> Veter<strong>in</strong>ary Medic<strong>in</strong>e and Animal Science, University <strong>of</strong>Peradeniya, Sri Lanka, for their cont<strong>in</strong>uous support <strong>in</strong> sample process<strong>in</strong>g throughoutthe research period.I would like to express my deep appreciation for Dr. K.J. Cooray, ConsultantMicrobiologist, Medical Research Institute, Colombo, Sri Lanka, for provid<strong>in</strong>g me
vavailable data <strong>of</strong> Salmonella surveillance <strong>in</strong> Sri Lanka. I am also thankful to Ms.Lashanthi, Statistical Unit, M<strong>in</strong>istry <strong>of</strong> Fisheries and Aquatic Resources, Sri Lanka,for furnish<strong>in</strong>g me with available statistical data on fisheries. I would like to thank Dr.Iddya Karunasagar, Director <strong>of</strong> Research, Karnataka Veter<strong>in</strong>ary, Animal and FisheriesScience University, India for provid<strong>in</strong>g me valuable journal articles.I am very grateful to my friends who participated from different countries <strong>of</strong>South East Asia with aesthetic beauty; we developed a long friendship throughoutthese two years. I hope we will cont<strong>in</strong>ue our friendship.Tr<strong>in</strong> Utsahachant “Wat” from Thailand deserves a special mention because shemade th<strong>in</strong>gs easy for me by help<strong>in</strong>g me on most occasions as do Mr. and Ms.Nawaratne for their encouragement dur<strong>in</strong>g my research study.Last, but not least, I wish to thank my family: my parents, for giv<strong>in</strong>g me life <strong>in</strong>the first place, for educat<strong>in</strong>g me <strong>in</strong> 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 listen<strong>in</strong>g to my compla<strong>in</strong>ts and for<strong>of</strong>fer<strong>in</strong>g guidance and help <strong>in</strong> difficult moments. To my immediate sister, for shar<strong>in</strong>gher experiences, bless<strong>in</strong>gs and for <strong>in</strong>spir<strong>in</strong>g me. To my brother-<strong>in</strong>-laws and sister-<strong>in</strong>lawfor <strong>in</strong>stigat<strong>in</strong>g me to build on my <strong>in</strong>tr<strong>in</strong>sic capabilities and to their small childrenfor giv<strong>in</strong>g me pleasure throughout my life.Thanks to Lord Buddha for giv<strong>in</strong>g me the perseverance and the ability toaccomplish the task.Kamalika Harsh<strong>in</strong>i Ubeyratne Janrabelge
viThesis TitleMicrobiological Quality <strong>of</strong> Marketed Penaeus <strong>monodon</strong>Shrimps <strong>in</strong> North Western Prov<strong>in</strong>ce, Sri LankaAuthorMs. Kamalika Harsh<strong>in</strong>i Ubeyratne JanrabelgeDegreeMaster <strong>of</strong> Veter<strong>in</strong>ary Public HealthThesis Advisory Committee Pr<strong>of</strong>. Dr. Goetz HildebrandtChairperson (FU-Berl<strong>in</strong>)Asst. Pr<strong>of</strong>. Dr. Rutch Khattiya Chairperson (CMU)ABSTRACTThe <strong>microbiological</strong> <strong>quality</strong> <strong>of</strong> <strong>marketed</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong> wasanalysed <strong>in</strong> terms <strong>of</strong> prevalence <strong>of</strong> Salmonella (<strong>in</strong>clud<strong>in</strong>g serotypes and antimicrobialresistance), Aerobic Plate Count (APC), Enterobacteriaceae, Escherichia coli andTotal Halophilic Plate Count. Samples were collected from the retail sale po<strong>in</strong>ts <strong>in</strong>North Western Prov<strong>in</strong>ce <strong>in</strong> Sri Lanka. A total <strong>of</strong> 180 samples both from captured andcultured <strong>shrimps</strong> were <strong>in</strong>volved.The overall prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> was 12.78 %. The prevalence <strong>of</strong>Salmonella <strong>in</strong> captured <strong>shrimps</strong> and cultured <strong>shrimps</strong> was 14.44 % and 11.11 %respectively and differences <strong>in</strong> prevalences were not statistically significant (p=0.66).However, the prevalences <strong>of</strong> Salmonella among 14 different retail sale po<strong>in</strong>ts differedand the difference was statistically significant (p
viiAll 23 stra<strong>in</strong>s isolated from <strong>shrimps</strong> were tested for antimicrobial resistanceus<strong>in</strong>g 14 different antimicrobials <strong>in</strong>clud<strong>in</strong>g nalidixic acid, amoxycill<strong>in</strong>, ampicill<strong>in</strong>,gentamyc<strong>in</strong>, erythromyc<strong>in</strong>, chloramphenicol, cipr<strong>of</strong>loxac<strong>in</strong>, kanamyc<strong>in</strong>, trimethoprim,sulphamethoxazole/trimethoprim, sulphonamides, tetracycl<strong>in</strong>e, streptomyc<strong>in</strong> anddoxycycl<strong>in</strong>e hydrochloride. 100 % <strong>of</strong> all the 23 isolates <strong>of</strong> Salmonella were observedsusceptible for nalidixic acid, ampicill<strong>in</strong>, gentamyc<strong>in</strong>, sulphamethoxazole/trimethoprim, chloramphenicol, cipr<strong>of</strong>loxac<strong>in</strong>, trimethoprim and tetracycl<strong>in</strong>e. Onestra<strong>in</strong> was susceptible to all 14 antimicrobials tested. Twenty-two stra<strong>in</strong>s weredetected for resistance and all these stra<strong>in</strong>s were resistant to two antimicrobials(erythromyc<strong>in</strong> and sulphonamides) <strong>of</strong> which one was resistant to three antimicrobials(erythromyc<strong>in</strong>, sulphonamides and amoxycill<strong>in</strong>). Out <strong>of</strong> 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 <strong>shrimps</strong> washigher than from capture <strong>shrimps</strong> without be<strong>in</strong>g statistically significant (p>0.05).Raw <strong>shrimps</strong> which have not undergone any further process<strong>in</strong>g showed a lowernumber <strong>of</strong> samples meet<strong>in</strong>g the standards. 48.1 % <strong>of</strong> samples tested met the standards<strong>of</strong> ≤10 7 CFU/g for Aerobic Plate Count (APC). 32.4 % <strong>of</strong> samples tested met thestandards <strong>of</strong> ≤10 5 CFU/g for Enterobacteriaceae count. 50 % <strong>of</strong> 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 between<strong>microbiological</strong> contam<strong>in</strong>ation and the rout<strong>in</strong>e commercial operation at different retailsale locations.
viiiชื่อเรื่องวิทยานิพนธ คุณภาพทางจุลชีววิทยาของกุงกุลาดําที่จําหนายในจังหวัดตะวันตก เฉียงเหนือ ศรีลังกาผูเขียน นางสาว คามาลิกา ฮาชินิ อุเบรัด จันราเบลปริญญาสัตวแพทยสาธารณสุขศาสตรมหาบัณฑิตคณะกรรมการที่ปรึกษาวิทยานิพนธ ศ.ดร.กอซ ฮิลเดอบรานด ประธานกรรมการ (FU-Berl<strong>in</strong>)ผศ.ดร. รัชต ขัตติยะ ประธานกรรมการ (CMU)บทคัดยอการวิเคราะหคุณภาพทางจุลชีววิทยากุงกุลาดํา (Penaeus <strong>monodon</strong>) 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-<strong>in</strong>digenous bacteria 102.2.1 Salmonella 102.2.1.1 Historical aspects 102.2.1.2 Taxonomy and nomenclature 112.2.1.3 Serotyp<strong>in</strong>g 122.2.1.4 Biochemical characteristics 142.2.1.5 Growth parameters 162.2.1.6 Incidences <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> 162.2.1.7 Food-borne <strong>in</strong>fections 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 <strong>of</strong> E. coli <strong>in</strong> <strong>shrimps</strong> 19
xi2.2.2.5 Food-borne <strong>in</strong>fections 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 <strong>of</strong> Vibrio <strong>in</strong> <strong>shrimps</strong> 242.3.1.6 Food-borne <strong>in</strong>fections 252.4 Antimicrobial use <strong>in</strong> aquaculture 272.4.1 Historical aspects 272.4.2 Antimicrobial resistance 282.4.2.1 Emergence <strong>of</strong> antimicrobial resistant 28bacterial stra<strong>in</strong>s2.4.2.2 Mechanisms <strong>of</strong> antimicrobial resistance 29<strong>in</strong> bacteria2.4.2.3 Incidences <strong>of</strong> antimicrobial resistance <strong>in</strong> 31aquaculture2.4.3 Antimicrobial resistance <strong>in</strong> Salmonella 312.5 Public health perspectives 332.5.1 Seafood safety and <strong>microbiological</strong> standards 332.5.2 Impact <strong>of</strong> food-borne hazards on health and the 35economy <strong>in</strong> developed countries2.5.2.1 Biological hazards 352.5.2.2 Antibiotic resistance 372.5.3 Impact <strong>of</strong> food-borne hazards on health and the 38economy <strong>in</strong> develop<strong>in</strong>g countries2.5.3.1 Biological hazards 382.5.3.2 Antibiotic resistance 402.5.4 Prevention and control <strong>of</strong> 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 Sampl<strong>in</strong>g plan 463.2.6 Questionnaire 493.3 Laboratory procedures 493.3.1 Aerobic Plate Count 493.3.2 Enumeration <strong>of</strong> 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 <strong>of</strong> colonies for confirmation 593.3.5.2 Biochemical confirmation 593.3.5.3 Serotyp<strong>in</strong>g 643.3.6 Antimicrobial sensitivity test for Salmonella 663.4 Questionnaire survey 683.5 Data management and statistical analysis 704. RESULTS 714.1 Source <strong>of</strong> the sample 714.2 Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong> 714.2.1 Overall prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> 714.2.2 Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> sold at 14 73different retail sale locations4.3 Salmonella serogroups and serotypes <strong>in</strong> Penaeus <strong>monodon</strong> 74<strong>shrimps</strong>4.4 Antimicrobial resistance <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong> 784.4.1 Antimicrobial resistance <strong>of</strong> Salmonella 79
xivREFERENCES 111APPENDICES 131APPENDIX A 131APPENDIX B 134DECLARATION 136CURRICULUM VITAE 137
xvLIST OF TABLESTablePage1. Examples <strong>of</strong> Salmonella nomenclature currently used <strong>in</strong> the11literature2. Biochemical and serological reactions <strong>of</strong> Salmonella 153. Growth parameters <strong>of</strong> Salmonella 164. Growth parameters <strong>of</strong> Escherichia coli 195. Biochemical characteristics <strong>of</strong> human pathogenic Vibrionaceae 23commonly encountered <strong>in</strong> seafood6. Growth parameters <strong>of</strong> the most important Vibrio spp. 247. Estimated occurrence <strong>of</strong> bacterial <strong>in</strong>fections and <strong>in</strong>toxications <strong>in</strong> 39selected regions8. Typical growth <strong>of</strong> Salmonella colonies on selective solid media 599. Biochemical reactions <strong>in</strong> TSI agar 6110. Interpretation <strong>of</strong> biochemical tests results <strong>of</strong> Salmonella 6311. List <strong>of</strong> observed factors 6912. Number <strong>of</strong> samples <strong>of</strong> <strong>shrimps</strong> for Salmonella analysis 7113. Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong> 7214. Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> sold at the different retail sale 74locations15. Number and percentage <strong>of</strong> Salmonella serogroups isolated from the 76<strong>shrimps</strong> by different locations16. Serovars <strong>of</strong> Salmonella isolated from <strong>shrimps</strong> 7717. Percentage <strong>of</strong> antimicrobial resistance Salmonella 7918. Number and percentage <strong>of</strong> Salmonella stra<strong>in</strong>s resistant, <strong>in</strong>termediate 80and susceptible to antimicrobials tested
xviTablePage19. Antimicrobial resistance pattern by type <strong>of</strong> shrimp 8220. Antimicrobial resistance pattern by serotype 8221. Percentage <strong>of</strong> resistant serotypes 8322. Bacteriological pr<strong>of</strong>ile <strong>of</strong> <strong>shrimps</strong> tested <strong>in</strong> terms <strong>of</strong> log CFU/g 8423. Bacteriological <strong>quality</strong> <strong>of</strong> <strong>marketed</strong> <strong>shrimps</strong> 8524. Summary results <strong>of</strong> potential factors and the levels for Salmonella 87contam<strong>in</strong>ation <strong>in</strong> <strong>shrimps</strong> (univariate analysis)25. Logistic regression <strong>of</strong> the risk factor associated with Salmonella 88contam<strong>in</strong>ation26. Summary results <strong>of</strong> potential factors and the levels for APC (log 89CFU/g) <strong>in</strong> <strong>shrimps</strong> (univariate analysis)27. Summary results <strong>of</strong> potential factors and the levels for91Enterobacteriaceae (log CFU/g) <strong>in</strong> <strong>shrimps</strong> (univariate analysis)28. Summary results <strong>of</strong> potential factors and the levels for Escherichia 93coli (log CFU/g) <strong>in</strong> <strong>shrimps</strong> (univariate analysis)29. Summary results <strong>of</strong> potential factors and the levels for Total 95Halophilic Plate Count (log CFU/g) <strong>in</strong> <strong>shrimps</strong> (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 Form<strong>in</strong>g Units per gramConfidence IntervalCentral Institute <strong>of</strong> Fisheries TechnologyCl<strong>in</strong>ical and Laboratory Standards InstituteCholera Tox<strong>in</strong>Deutsche Gesellschaft für Hygiene und MikrobiologieDeoxyribo Nucleic AcidDeputy Director <strong>of</strong> 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+LIALPSMDRm<strong>in</strong>MKTTnmlmmMRDMRINACANACMCFNCCLSNWPOIEORFood Safety ObjectivesFresh Watergram(s)Gross Domestic ProductionGood Hygiene PracticeGood Manufactur<strong>in</strong>g Practicehour(s)Hazard Analysis Critical Control Po<strong>in</strong>tHaemorrhagic ColitisHaemolytic Uraemic SyndromeInternational Commission on MicrobiologicalSpecification for FoodsInternational Organization for StandardizationJo<strong>in</strong>t Expert Advisory Committee on Antibiotic ResistanceKilogram(s)Kanagawa phenomenon positiveLys<strong>in</strong>e Iron AgarLipopolysaccharidesMulti Drug Resistantm<strong>in</strong>ute(s)Muller-Kauffmann Tetra Thionate novobioc<strong>in</strong>millilitre(s)millimetre(s)Maximum Recovery DiluentMedical Research InstituteNetwork <strong>of</strong> Aquaculture Centres <strong>in</strong> Asia-PacificNational Advisory Committee on Microbiological Criteriafor FoodsNational Committee <strong>of</strong> Cl<strong>in</strong>ical Laboratory StandardsNorth Western Prov<strong>in</strong>ceWorld Organization for Animal HealthOdds Ratio
xxpPABAPCARHRVSSLFARASPSSPSSSTECSUMFARSWtTDHTRHTSIUCDUSFDAVRBGAWHOWTOXLDProbability valuePara-Am<strong>in</strong>o Benzoic AcidPlate Count AgarRelative HumidityRappaport-Vassiliadis medium with SoyaSri Lanka Fisheries and Aquatic Resources ActSanitary and PhytosanitaryStatistical Package for Social SciencesShiga Tox<strong>in</strong> produc<strong>in</strong>g E. coliStatistical Unit <strong>of</strong> M<strong>in</strong>istry <strong>of</strong> Fisheries and Aquatic ResourcesSea WaterMetric tonnesThermostable Direct Haemolys<strong>in</strong>TDH-related hemolys<strong>in</strong>Triple Sugar IronUniversity <strong>of</strong> California DavisUnited States Food and Drug Adm<strong>in</strong>istrationViolet Red Bile Glucose AgarWorld Health OrganizationWorld Trade OrganizationXylose-Lys<strong>in</strong>e-Deoxycholate
1. INTRODUCTION AND OBJECTIVES1.1 IntroductionIn Asia, Penaeus <strong>monodon</strong> <strong>shrimps</strong> have been cultivated <strong>in</strong> traditional systemsfor centuries, with low productivity aimed for the domestic markets. Shrimpaquaculture which is oriented for export market is a fairly recent <strong>in</strong>dustry that took <strong>of</strong>f<strong>in</strong> the mid 1970’s. With improved technologies and the <strong>in</strong>troduction <strong>of</strong> formulatedfeeds, the <strong>in</strong>dustry boomed <strong>in</strong> the follow<strong>in</strong>g decade. In 1975, the shrimp aquaculture<strong>in</strong>dustry supplied to 2.5 % <strong>of</strong> total shrimp production, progressively <strong>in</strong>creased toaround 30 % <strong>in</strong> the 1990’s. Today shrimp farm<strong>in</strong>g comprise only 3-4 % <strong>of</strong> globalaquaculture production <strong>in</strong> terms <strong>of</strong> weight, but almost 15 % <strong>in</strong> terms <strong>of</strong> value.Shrimps are one <strong>of</strong> the most important commodities <strong>of</strong> the global fishery trade(Bhaskar et al., 1995). Participation for the global shrimp trade by develop<strong>in</strong>gcountries are greater and about 75 % <strong>of</strong> the shrimp catches worldwide, whether fromculture or capture, orig<strong>in</strong>ate from the develop<strong>in</strong>g countries (Bhasker et al., 1998).Around 80 percent <strong>of</strong> cultured <strong>shrimps</strong> come from Asia, with Thailand, Ch<strong>in</strong>a,Indonesia and India as the top producers. In the Western hemisphere, Ecuador is themajor shrimp produc<strong>in</strong>g country. The giant tiger or black tiger shrimp–Penaeus<strong>monodon</strong>–accounts for more than half <strong>of</strong> the total shrimp aquaculture output(Rönnbäck, 2001).However, <strong>in</strong> Sri Lanka where there has not been a tradition <strong>of</strong> aquaculture,shrimp farm<strong>in</strong>g was slower to take hold. Interest <strong>in</strong> fish culture first appeared dur<strong>in</strong>gthe 1950’s. Shrimp culture was <strong>in</strong>itiated dur<strong>in</strong>g the last decade and before this timeshrimp production <strong>in</strong> Sri Lanka was based solely on capture fisheries. Shrimp culturestarted <strong>in</strong> the early 1980’s and the first commercial production entered the market <strong>in</strong>1984 <strong>in</strong> very small quantities. Dur<strong>in</strong>g 1992, the total production <strong>of</strong> cultured <strong>shrimps</strong>was around 1,200 tonnes (FAO/NACA, 1995). At present, <strong>shrimps</strong> account for a
2significant portion <strong>of</strong> export revenue, <strong>in</strong> the agricultural and fisheries sector onlysecond to tea <strong>in</strong> 1998 (Cattermoul and Devendra, 2002). The percentage contribution<strong>of</strong> <strong>shrimps</strong> to the total value <strong>of</strong> all aquatic products exported varied between 48.5 %and 70.3 % between 1985 and 1992. The Statistical Unit <strong>of</strong> the M<strong>in</strong>istry <strong>of</strong> Fisheriesand Aquatic Resources reported annual fish production, which <strong>in</strong>cludes mar<strong>in</strong>e fishcatch, <strong>in</strong>land and aquaculture, at 286,370 Metric tonnes (t) <strong>in</strong> 2004 (SUMFAR,2004a). That same year prawn production, both aquaculture and wild capture, lay at2,390 and 10,730 t respectively. Export quantities <strong>of</strong> fish and fishery productsamounted to 13,681 t, these <strong>in</strong>cluded prawns, lobsters, crabs, Beche de Mer,ornamental fish, chank and shells, shark f<strong>in</strong>s, molluscs, fish maws, fish and others.The contribution <strong>of</strong> prawns was 30 % <strong>in</strong> quantity and 26 % <strong>in</strong> value (SUMFAR,2004b). The fishery sector is an important part <strong>of</strong> Sri Lanka’s economy and itcontributed around 1.8 % to the Gross Domestic Production (GDP) <strong>in</strong> 2004(SUMFAR, 2004c). Of the total fish land<strong>in</strong>gs <strong>of</strong> the country, about 95 % is handledby the private sector. About 70 % <strong>of</strong> land<strong>in</strong>gs <strong>of</strong> 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 <strong>of</strong> animal prote<strong>in</strong> consumed <strong>in</strong> the country, with about 60 % <strong>of</strong> the populationdepend<strong>in</strong>g solely on fish for their prote<strong>in</strong> requirement, and account<strong>in</strong>g for nearly 65 %<strong>of</strong> the total animal prote<strong>in</strong> consumed (Wijegoonawardena and Siriwardena, 1996a).Per capita consumption <strong>of</strong> fish (Kg/year/person) was 9.39 <strong>in</strong> 2003. The proportion <strong>of</strong>prawns <strong>in</strong> the average household fish consumption/month was 3 % that the same yearaccord<strong>in</strong>g to the M<strong>in</strong>istry <strong>of</strong> Fisheries and Aquatic Resources (SUMFAR, 2003).As a highly prized seafood delicacy, <strong>shrimps</strong> and prawns are cash crop grownma<strong>in</strong>ly for the affluent export (and urban) markets. From farms <strong>in</strong> Southeast Asia,East Asia, South Asia and South America, <strong>shrimps</strong> are exported to the major markets<strong>in</strong> Japan, the USA and Europe; while domestic consumption accounts for only 5-20%, foreign markets absorb 80-95 % <strong>of</strong> 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 <strong>in</strong>crease <strong>in</strong> awareness about the nutritional value and healthbenefits <strong>of</strong> fish consumption <strong>in</strong> the last two decades. On the other hand, seafood isalso known to have been responsible for a significant percentage <strong>of</strong> food-bornediseases (Karunasagar et al., 2005; Wallace et al., 1999).In seafood related outbreaks a wide variety <strong>of</strong> viruses, bacteria, and parasiteshave been implicated, which are reported worldwide. Consumption <strong>of</strong> raw orundercooked seafood is the factor most commonly associated with <strong>in</strong>fection (Butt etal., 2004). There is ample epidemiological evidence, particularly from Japan, thatconsumption <strong>of</strong> raw fish is <strong>in</strong>deed the cause <strong>of</strong> many outbreaks <strong>of</strong> food-bornediseases. Biotox<strong>in</strong>s and histam<strong>in</strong>es make up a large proportion <strong>of</strong> these outbreaks(Huss, 1997). Though viruses are the most common cause <strong>of</strong> seafood related<strong>in</strong>fections, most <strong>of</strong> the hospitalisation and deaths are due to bacterial agents (Butt etal., 2004). As a consequence, food safety and <strong>quality</strong> aspects <strong>in</strong> trade becameimportant, s<strong>in</strong>ce fresh food is more prone to certa<strong>in</strong> <strong>microbiological</strong> contam<strong>in</strong>ation(Sawhney, 2005). Incidences <strong>of</strong> Salmonella <strong>in</strong> different shrimp products have beenreported by a number <strong>of</strong> <strong>in</strong>vestigators <strong>in</strong> India (Kumar et al., 2003). The sources <strong>of</strong>contam<strong>in</strong>ation have been described by different studies <strong>in</strong> numerous ways. Mølbak etal., 2006 have described that it is well established that the about 2500 non-typhoidSalmonella serotypes are widely distributed <strong>in</strong> nature, <strong>in</strong>clud<strong>in</strong>g <strong>in</strong> the gastro<strong>in</strong>test<strong>in</strong>altracts <strong>of</strong> mammals, reptiles, birds and <strong>in</strong>sects. Therefore, Salmonella reaches aquaticenvironments through faecal contam<strong>in</strong>ation. Increas<strong>in</strong>g evidence that certa<strong>in</strong>Salmonella types may also be part <strong>of</strong> the <strong>in</strong>digenous micro-flora <strong>in</strong> tropicalaquaculture has been reported (Huss et al., 2000). Furthermore, contam<strong>in</strong>ation <strong>of</strong>shrimp products with Salmonella may be caused by poor hygiene standards dur<strong>in</strong>gprocess<strong>in</strong>g rather than orig<strong>in</strong>at<strong>in</strong>g from shrimp ponds accord<strong>in</strong>g to (Dalsgaard et al.,1995). Hence any presence <strong>of</strong> Salmonella <strong>in</strong> raw frozen <strong>shrimps</strong> may constitute apotential health hazard due to the food habit <strong>of</strong> consum<strong>in</strong>g raw or slightly cooked<strong>shrimps</strong> (Dalsgaard et al., 1995).Moreover, bacteria <strong>of</strong> the genus Salmonella are widespread and an importantcause <strong>of</strong> food-borne <strong>in</strong>fections <strong>in</strong> humans. They are the most frequent etiologic
4bacterial agents <strong>of</strong> food-borne disease outbreaks. Thus, Salmonella has an immensevalue as a specific pathogen <strong>in</strong>ternationally. The global Salmonella survey,conducted <strong>in</strong> 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 % <strong>of</strong> all Salmonella isolates reported <strong>in</strong>1995. Enteritidis was the most frequently isolated serotype <strong>in</strong> 35 countries, followedby Typhi (12 countries) and Typhimurium (8 countries). The global pandemic <strong>of</strong>Salmonella Enteritidis cont<strong>in</strong>ued to spread as data revealed a rise globally from 25.6% <strong>in</strong> 1990 to 36.3 % <strong>in</strong> 1995. This <strong>in</strong>crease was observed <strong>in</strong> most regions: American(11.3 % <strong>in</strong> 1990 and 42.6 % <strong>in</strong> 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 <strong>in</strong> the Southeast Asian region (11.9 %/2.6 %).The trends <strong>in</strong> the proportion <strong>of</strong> isolates that were Typhimurium were morevariable. This decreased <strong>in</strong> the American (20.8 %/8.0 %) and European region (20.2%/17.6 %), and <strong>in</strong>creased <strong>in</strong> 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 <strong>of</strong> isolates that were Typhidecreased from 15.3 % <strong>in</strong> 1990 to 12.1 % <strong>in</strong> 1995, and sharp decreases were observed<strong>in</strong> 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 ma<strong>in</strong> reservoir, and entericfever (typhoid and paratyphoid fever) as their most important cl<strong>in</strong>ical manifestation.Enteric fever cont<strong>in</strong>ues to be an important cause <strong>of</strong> morbidity and mortality <strong>in</strong>develop<strong>in</strong>g countries. In Sri Lanka, enteric fever is endemic. Many <strong>of</strong> the DeputyDirector <strong>of</strong> Health Services (DPDHS) Divisions show a low prevalence. Although
6human and animal pathogens may threaten the viability <strong>of</strong> export markets. SomeEuropean countries found residues <strong>of</strong> chloramphenicol <strong>in</strong> tiger <strong>shrimps</strong> imported fromCh<strong>in</strong>a, Indonesia, and Vietnam (Holmström et al., 2003).In Sri Lanka, the prevail<strong>in</strong>g 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). Dur<strong>in</strong>g 1988-1990 and <strong>in</strong>1996 Sri Lanka experienced two major disease outbreaks due to viral <strong>in</strong>fection(Jayas<strong>in</strong>ghe and Mac<strong>in</strong>tosh, 1993). Moreover, it was reported that some localisedoutbreaks <strong>of</strong> disease <strong>in</strong> farms occurred, ma<strong>in</strong>ly related to bacterial <strong>in</strong>fections(Wijegoonawardena and Siriwardena, 1996a). In 2001 Senarath and Visvanathanreported that disease outbreaks were due to the rapid expansion <strong>of</strong> the shrimp <strong>in</strong>dustry<strong>in</strong> North Western Prov<strong>in</strong>ce. There is little <strong>in</strong>formation on the amounts <strong>of</strong> differentchemotherapeutants used <strong>in</strong> the shrimp farm<strong>in</strong>g <strong>in</strong>dustry.It was stipulated by the Fisheries and Aquatic Resources Act, No.2 <strong>of</strong> 1996 thatuse or adm<strong>in</strong>istration <strong>of</strong> substances such as chloramphenicol, nitr<strong>of</strong>urans (<strong>in</strong>clud<strong>in</strong>gfurazolidone) and malachite green is prohibited; fish on a regime <strong>of</strong> antibiotics mustnot be harvested for placement on the market for human consumption until fifty daysafter completion <strong>of</strong> the regime and the supervision <strong>of</strong> the use <strong>of</strong> antibiotics <strong>in</strong> fish feed(SLFARA, 1996).The centre <strong>of</strong> shrimp farm<strong>in</strong>g has developed <strong>in</strong> the North Western Prov<strong>in</strong>cecoastal area <strong>of</strong> Sri Lanka because the coast <strong>of</strong> Sri Lanka is endowed with a number <strong>of</strong>lagoons which are ideal for the establishment <strong>of</strong> shrimp farms (Cattermoul andDevendra, 2002). Therefore, it is one <strong>of</strong> the most important fish<strong>in</strong>g grounds <strong>in</strong> SriLanka. The coastl<strong>in</strong>e <strong>of</strong> the North Western Prov<strong>in</strong>ce is approximately 120 Km long.The major brackish water bodies <strong>in</strong> this prov<strong>in</strong>ce are Puttalam lagoon, Mundal lagoonand Chilaw lagoon (FAO/NACA, 1995). North Western Prov<strong>in</strong>ce consists <strong>of</strong> twodistricts, Kurunegala and Puttalam <strong>of</strong> which the major fish<strong>in</strong>g towns are Chilaw andPuttalam. In 2005, <strong>in</strong>land and aquaculture fish production <strong>in</strong> the districts Kurunegala
7and Puttalam amounted to 2140 and 7210 t respectively (SUMFAR, 2005). There aredifferent commercial groups <strong>of</strong> fish catch available at the local markets <strong>in</strong> NorthWestern Prov<strong>in</strong>ce and various hygienic practices followed <strong>in</strong> the cha<strong>in</strong> <strong>of</strong> commercialoperation. There is no monitor<strong>in</strong>g system adopted for the <strong>quality</strong> assurance <strong>of</strong> localconsumption.1.1.1 Significance <strong>of</strong> this studyThe <strong>microbiological</strong> <strong>quality</strong> <strong>of</strong> <strong>marketed</strong> <strong>shrimps</strong> is an important study areabecause <strong>of</strong> the/<strong>in</strong> order:1. Lack <strong>of</strong> <strong>in</strong>formation on micro-flora <strong>of</strong> <strong>shrimps</strong> sold at retail sale locations.2. To assure the safety and <strong>quality</strong> <strong>of</strong> <strong>shrimps</strong> as demanded by the <strong>in</strong>creas<strong>in</strong>gconsumer awareness.3. To provide the <strong>in</strong>formation to Veter<strong>in</strong>ary Public Health Authorities forestablish<strong>in</strong>g safety guidel<strong>in</strong>es for shrimp products.In this present study, the <strong>microbiological</strong> <strong>quality</strong> <strong>of</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong>purchased from retail sail po<strong>in</strong>ts at the time <strong>of</strong> sale was <strong>in</strong>vestigated.1.2 Objectives1.2.1 Ma<strong>in</strong> objectives1 . To determ<strong>in</strong>e the prevalence, the serotypes and the antimicrobial resistance<strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong> (Penaeus <strong>monodon</strong>) <strong>in</strong> local markets <strong>of</strong> NorthWestern Prov<strong>in</strong>ce <strong>in</strong> Sri Lanka.2 . To f<strong>in</strong>d out the associated risk factors for the Salmonella contam<strong>in</strong>ation.
81.2.2 Secondary objectivesTo determ<strong>in</strong>e the <strong>microbiological</strong> contam<strong>in</strong>ation and hygienic status by1.2.2.1 Escherichia coli count <strong>in</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong>1.2.2.2 Microbial load <strong>in</strong> retail <strong>shrimps</strong> <strong>in</strong> terms <strong>of</strong> Aerobic Plate Count1.2.2.3 Enterobacteriaceae count <strong>in</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong>1.2.2.4 Total Halophilic Plate Count <strong>in</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong> at differentlocations and times <strong>of</strong> the day.
2. LITERATURE REVIEW2.1 IntroductionThe epidemiology <strong>of</strong> food-borne diseases is chang<strong>in</strong>g. New pathogens haveemerged, and some have spread worldwide. The threats <strong>of</strong> new food-borne diseasesare occurr<strong>in</strong>g for a number <strong>of</strong> reasons, through the globalization <strong>of</strong> the food supply,the <strong>in</strong>advertent <strong>in</strong>troduction <strong>of</strong> pathogens <strong>in</strong>to new geographic areas, throughtravellers, refugees, and immigrants exposed to unfamiliar food-borne hazards whileabroad, through changes <strong>in</strong> micro-organisms, through changes <strong>in</strong> the humanpopulation (the number <strong>of</strong> highly susceptible persons is expand<strong>in</strong>g worldwide because<strong>of</strong> age<strong>in</strong>g, malnutrition, HIV <strong>in</strong>fections and other underly<strong>in</strong>g medical conditions), andchanges <strong>in</strong> lifestyle. It is well documented that raw or under-processed seafoodprovides important epidemiological pathways for food-borne disease transmission(WHO, 2002).Consumption <strong>of</strong> seafood has <strong>in</strong>creased over the last decade. This trend isexpected to cont<strong>in</strong>ue both for prepared and for fresh or frozen varieties (Ahmed,1991). The likelihood <strong>of</strong> contam<strong>in</strong>ation <strong>of</strong> raw material and/or foods by hazardousagents is equally applicable to seafood when compared to any other food. Changes <strong>in</strong>the level or frequency <strong>of</strong> the hazard over time depend on process<strong>in</strong>g, preservationparameters and storage conditions (Huss, 2003).Seafood may be contam<strong>in</strong>ated by numerous means from the surround<strong>in</strong>g water.Some food-borne agents are permanent residents <strong>of</strong> the mar<strong>in</strong>e environment (Kl<strong>in</strong>ger,2001). The water temperature is naturally hav<strong>in</strong>g a selective effect. Thus morepsychrotrophic organisms (C. botul<strong>in</strong>um and Listeria) are common <strong>in</strong> Arctic andcolder climates, while the more mesophilic types (V. cholerae, V. parahaemolyticus)represent part <strong>of</strong> the natural flora on fish from coastal and estuar<strong>in</strong>e environments <strong>of</strong>
10temperate and warm tropical zones (Huss, 1994). Another way contam<strong>in</strong>ants ga<strong>in</strong>access to products is through contam<strong>in</strong>ation <strong>of</strong> the mar<strong>in</strong>e environments by urbaneffluents. Therefore, bacteria present <strong>in</strong> seafood ma<strong>in</strong>ly comprise two groups,<strong>in</strong>digenous and non-<strong>in</strong>digenous (Kl<strong>in</strong>ger, 2001; Huss, 1994).2.2 Non-<strong>in</strong>digenous bacteria2.2.1 Salmonella2.2.1.1 Historical aspectsThe genus Salmonella was created <strong>in</strong> 1900 by Ligniēres and named <strong>in</strong> honour <strong>of</strong>D.E. Salmon, the American veter<strong>in</strong>ary pathologist who first described Salmonellacholerae-suis (Adams and Moss, 2000). Salmonella, belongs to the family <strong>of</strong>Enterobacteriaceae. They are Gram-negative, non-spore-form<strong>in</strong>g 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 Gall<strong>in</strong>arum (Doyle et al., 2001; Adams and Moss, 2000). The genusSalmonella consists <strong>of</strong> over 2500 serovars, as determ<strong>in</strong>ed by its somatic (O) andflagellar (H) antigens (Cai et al., 2005). The serotypes are closely related, many <strong>of</strong>which are potentially pathogenic for humans and/or animals (Yan et al., 2003). Theantigenic formulae <strong>of</strong> Salmonella serotypes are def<strong>in</strong>ed and ma<strong>in</strong>ta<strong>in</strong>ed by the WHOCollaborat<strong>in</strong>g Centre for Reference and Research on Salmonella at the PasteurInstitute, Paris, France, and new serotypes are listed <strong>in</strong> annual updates <strong>of</strong> theKauffman-White scheme (Brenner et al., 2000). An antigenic scheme for theclassification <strong>of</strong> salmonellae was first proposed by White <strong>in</strong> 1926 and subsequentlyexpanded by Kauffmann <strong>in</strong> 1941 <strong>in</strong>to the Kauffmann-White scheme (Doyle et al.,2001).
112.2.1.2 Taxonomy and nomenclatureThe taxonomy and nomenclature <strong>of</strong> 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 <strong>of</strong>ficial nomenclature the follow<strong>in</strong>gscheme (Brenner et al., 2000). The genus Salmonella conta<strong>in</strong>s two species, each <strong>of</strong>which conta<strong>in</strong> multiple serotypes. The two species are S. enterica and S. bongori. Thespecies S. enterica is divided <strong>in</strong>to 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. <strong>in</strong>dica) (Pop<strong>of</strong>f, 2001). Subspecies V isnow considered to be a separate species and moved <strong>in</strong>to 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 <strong>in</strong>clud<strong>in</strong>g humans (Yan et al., 2003).The most recent proposal to <strong>in</strong>troduce 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 <strong>of</strong> Salmonella nomenclature currently used <strong>in</strong> 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.Mar<strong>in</strong>aS. enterica subsp. <strong>in</strong>dica ser.Sr<strong>in</strong>nagar“Arizona h<strong>in</strong>shawii” ser.7a,7b:1,2,5S. IIIb 60:k:z “A. h<strong>in</strong>shawii” ser.24:29:31S. ser. Mar<strong>in</strong>a S. IV 48:g,z 51:-, S. mar<strong>in</strong>aS. ser.Sr<strong>in</strong>agarS. bongori ser. Brookfield S. ser.BrookfieldS. VI 11:b:en,x, S.sr<strong>in</strong>agarS. V 66:z 41:-, S. brookfield2.2.1.3 Serotyp<strong>in</strong>g1. Conventional serotyp<strong>in</strong>gThe biochemical identification <strong>of</strong> food-borne and cl<strong>in</strong>ical Salmonella isolates isgenerally coupled to serological confirmation (Doyle et al., 2001). The slideagglut<strong>in</strong>ation test is commonly used <strong>in</strong> serotyp<strong>in</strong>g for Salmonella based on theKauffman-White scheme which <strong>in</strong>volves more than 250 antisera (Cai et al., 2005): acomplex and labor-<strong>in</strong>tensive technique <strong>in</strong>volv<strong>in</strong>g the agglut<strong>in</strong>ation <strong>of</strong> bacterial surfaceantigens with Salmonella-specific antibodies. These antigens <strong>in</strong>clude somatic (O)lipopolysaccharides (LPS) on the external surface <strong>of</strong> the bacterial outer membrane,flagellar (H) antigens associated with the peritrichous flagella, and the capsular (Vi)antigen, which is a superficial antigen overly<strong>in</strong>g the O antigen and occurs only <strong>in</strong>Salmonella serovars Typhi, Paratyphi C, and Dubl<strong>in</strong> (Gianella, 1982; Adams andMoss, 2000; Doyle et al., 2001). Many Salmonella show diphasic production <strong>of</strong>flagellar antigens and each stra<strong>in</strong> can spontaneously and reversibly vary betweenthese two phases with different sets <strong>of</strong> H antigens, <strong>in</strong> phase 1 or the specific phase, thedifferent antigens are designated by small letters, and <strong>in</strong> 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 obta<strong>in</strong> a presumptive diagnosis, and the def<strong>in</strong>itiveantigenic formula was determ<strong>in</strong>ed by us<strong>in</strong>g monovalent antisera. Because antigen O
13or H is not always expressed, sometimes no agglut<strong>in</strong>ation was observed for someisolates (approximately 1 % <strong>of</strong> isolates from the environment).1.1 Antigenic structure <strong>of</strong> Salmonella1.1.1 O (somatic) antigensThese somatic antigens represent the side cha<strong>in</strong>s <strong>of</strong> repeat<strong>in</strong>g sugar unitsproject<strong>in</strong>g from the outer lipopolysaccharide layer <strong>of</strong> 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 mak<strong>in</strong>g thelatter unavailable for agglut<strong>in</strong>ation with the correspond<strong>in</strong>g O antibodies. The capsularantigens are heat labile and follow<strong>in</strong>g their destruction by boil<strong>in</strong>g, the heat stable Oantigens are available for antibody agglut<strong>in</strong>ation (Todar, 2006).1.1.2 H (flagellar) antigensThe H antigens located <strong>in</strong> the flagella <strong>of</strong> salmonellae are typically prote<strong>in</strong>aceous<strong>in</strong> 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 stra<strong>in</strong> <strong>of</strong>Salmonella may conta<strong>in</strong> one (monophasic) or both (biphasic) forms (Defigueiredo andSplittstoesser, 1980).1.1.3 Vi (capsular) antigensAlmost all stra<strong>in</strong>s <strong>of</strong> S. Typhi form the Vi-antigen as a cover<strong>in</strong>g layer outsidetheir cell wall. The Vi-antigen is found mostly <strong>in</strong> freshly isolated stra<strong>in</strong>s which mustbe destroyed before this organism will react with group D somatic antisera(Defigueiredo and Splittstoesser, 1980).2. Phage typ<strong>in</strong>g
14Phage typ<strong>in</strong>g has proven to be epidemiologically valuable <strong>in</strong> stra<strong>in</strong>differentiation with<strong>in</strong> a particular Salmonella serotype. Phage typ<strong>in</strong>g utilizes theselective ability <strong>of</strong> bacteriophages to <strong>in</strong>fect stra<strong>in</strong>s <strong>of</strong> Salmonella and thus enabl<strong>in</strong>gthe user to differentiate unique isolates. This has been used to describe pandemicclones <strong>of</strong> Salmonella, such as S. enterica serovar Typhimurium def<strong>in</strong>itive type 104(DT 104) that causes severe gastro<strong>in</strong>test<strong>in</strong>al illness and is typically resistant tomultiple antimicrobials. Although phage typ<strong>in</strong>g 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 typ<strong>in</strong>gMolecular typ<strong>in</strong>g techniques are favoured for stra<strong>in</strong> discrim<strong>in</strong>ation <strong>in</strong> non-phagetypableserotypes <strong>of</strong> Salmonella (Chisholm et al., 1999). These typ<strong>in</strong>g methodsutilize restriction endonuclease digestion, nucleic acid amplification, or nucleotidesequenc<strong>in</strong>g techniques (Yan et al., 2003).2.2.1.4 Biochemical characteristicsSalmonellae <strong>in</strong> general produce H 2 S, produce acid from glucose, maltose,mannitol and sorbitol, utilize citrate, but do not ferment salic<strong>in</strong>, sucrose and lactose.They are catalase positive, oxidase negative, urease negative, <strong>in</strong>dole negative andreduce nitrate to nitrite (Defigueiredo and Splittstoesser, 1980).There are some exceptions regard<strong>in</strong>g the typical biochemical reactions <strong>of</strong>Salmonella. Indole positive Salmonella serotypes have been reported by Wuthe et al.,1989; Pan et al., 1989. The Department <strong>of</strong> Bacteriology, National Institute <strong>of</strong>Infectious Bacteriology and the Yamaguchi Prefectural Research Institute <strong>of</strong> PublicHealth have reported the presence <strong>of</strong> lys<strong>in</strong>e decarboxylase-negative stra<strong>in</strong>s <strong>of</strong>Salmonella enterica serovar Enteritidis (Masatomo et al., 2006). A stra<strong>in</strong> wasidentified by Farmer et al., 1975 as Salmonella cubana was typical <strong>in</strong> all biochemical,serological, and bacteriophage reactions, except that it produced urease strongly.
15Table 2: Biochemical and serological reactions <strong>of</strong> Salmonella ( Andrews andHammack, 2006).Test or SubstrateResultSalmonellaPositiveNegativereaction (a)1. Glucose (TSI) yellow butt red butt +2. Lys<strong>in</strong>e decarboxylasepurple butt yellow butt +(LIA)3. H 2 Sblacken<strong>in</strong>g no blacken<strong>in</strong>g +(TSI and LIA)4. Urease purple-red color no color change -5. Lys<strong>in</strong>e 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 flagellaragglut<strong>in</strong>ation no agglut<strong>in</strong>ation +test11. Polyvalent somatictestagglut<strong>in</strong>ation no agglut<strong>in</strong>ation +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 p<strong>in</strong>k-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 <strong>in</strong> 1 or 2 days; -, 90 % or more negative <strong>in</strong> 1 or 2 days; v,variableb, Majority <strong>of</strong> S. arizonae cultures are negativec, Majority <strong>of</strong> S. arizonae cultures are positive2.2.1.5 Growth parametersTable 3: Growth parameters <strong>of</strong> Salmonella (Bremer et al., 2003)M<strong>in</strong>imumwaterM<strong>in</strong>pHMaxpHMaxpercentageM<strong>in</strong>TemperatureMaxTemperatureOxygenrequirementactivity a w<strong>of</strong> NaCl (°C) 1 (°C) 10.92-0.93 4.0 9.5 8 6-7 45-47 Facultativeanaerobe 21 . M ost salmonellae <strong>in</strong> foods.2 . G rows with or without oxygen.3. M<strong>in</strong>=M<strong>in</strong>imum4. Max=Maximum2.2.1.6 Incidences <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong>Incidences <strong>of</strong> Salmonella <strong>in</strong> seafoods from India have been reported by a number<strong>of</strong> <strong>in</strong>vestigators. Hatha and Lakshmanaperumalsamy (1997) reported 14.25 % <strong>of</strong> thefish samples and 17.39 % <strong>of</strong> crustacean samples were contam<strong>in</strong>ated with Salmonella.The percentage prevalence reported varied among the different products: frozenpeeled and deve<strong>in</strong>ed <strong>shrimps</strong> accounted for 7.46 %, peeled and deve<strong>in</strong>ed <strong>shrimps</strong> for12 %, headless shell-on <strong>shrimps</strong> for 10 %, peeled and undeve<strong>in</strong>ed <strong>shrimps</strong> for 14 %
17(Kumar et al., 2003). Fonseka (1994) has reported the presence <strong>of</strong> Salmonella <strong>in</strong> farm<strong>shrimps</strong> <strong>in</strong> Sri Lanka. Results <strong>of</strong> the <strong>quality</strong> <strong>of</strong> <strong>shrimps</strong> sold <strong>in</strong> the markets <strong>of</strong>Hyderabad, India showed 11 % <strong>of</strong> the shrimp samples purchased were contam<strong>in</strong>atedwith Salmonella (Jonnalagadda and Bhat, 2004).The United States Food and Drug Adm<strong>in</strong>istration (USFDA) noted an overall<strong>in</strong>cidence <strong>of</strong> 7.2 % <strong>in</strong> imported and 1.3 % <strong>in</strong> domestic seafood conta<strong>in</strong><strong>in</strong>g Salmonelladur<strong>in</strong>g a 9-year study (1990–1998) <strong>of</strong> 11,312 imported and 768 domestic seafoodsamples (He<strong>in</strong>itz et al., 2000). In 1989, more than 8300 tonnes <strong>of</strong> frozen raw prawnswere deta<strong>in</strong>ed by the USFDA due to contam<strong>in</strong>ation with Salmonella, contrary to theJapanese who have accepted Salmonella <strong>in</strong> raw frozen <strong>shrimps</strong> (Reilly and Twiddy,1992).2.2.1.7 Food-borne <strong>in</strong>fectionsSalmonellosis is one <strong>of</strong> the most common and widely distributed food-bornediseases. It constitutes a major public health burden and represents a significant cost<strong>in</strong> many countries (WHO, 2005). Early reports on the pathogenicity <strong>of</strong> Salmonella<strong>in</strong>dicated that <strong>in</strong>gestion <strong>of</strong> high numbers (10 5 to 10 7 ) <strong>of</strong> food-borne salmonellae was aprerequisite for human illness but more recent evidence suggests that a s<strong>in</strong>gleSalmonella cell may constitute a human <strong>in</strong>fectious dose (D’Aoust, 2000). The cl<strong>in</strong>icalcourse <strong>of</strong> human salmonellosis is usually characterized by abdom<strong>in</strong>al pa<strong>in</strong>, diarrhoea,nausea and sometimes vomit<strong>in</strong>g (WHO, 2005). It is generally agreed that the foodcha<strong>in</strong> is the major source <strong>of</strong> Salmonella <strong>in</strong>fection for humans (Bezanson et al., 1981).Many factors <strong>in</strong>clud<strong>in</strong>g <strong>in</strong>adequate supplies <strong>of</strong> clean water, <strong>in</strong>adequate sanitarymeasures, lack <strong>of</strong> food hygiene and food safety measures have been responsible for<strong>in</strong>creased <strong>in</strong>cidences <strong>of</strong> food-borne salmonellosis (Miko et al., 2005).Apart from specific genu<strong>in</strong>e salmonellosis (typhoid and paratyphoid fever), theSalmonella <strong>in</strong>fections <strong>of</strong> humans mostly account for food poison<strong>in</strong>g, caused by nontyphoidalSalmonella such as S. Typhimurium, S. Enteritidis, S. Cholerasuis, S. Hader,S. Virchow, S. Dubl<strong>in</strong> etc. (Sakai and Chalermchaikit, 1996).
18Typhoid and the paratyphoid fevers which are the most severe <strong>of</strong> all diseasescaused by salmonellae <strong>in</strong>clude S. Typhi, S. Paratyphi A, S. Paratyphi B and S.Paratyphi C. Typhoid fever has the longest <strong>in</strong>cubation period, produces the highestbody temperature, and the highest mortality rate. S. Typhi may be isolated from theblood and sometimes from the stool and ur<strong>in</strong>e <strong>of</strong> 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 <strong>of</strong> an<strong>in</strong>fected person (Jay, 2000).Symptoms <strong>of</strong> salmonellosis can range from self-limit<strong>in</strong>g diarrhoea to entericfever. Gastroenteritis generally appears 6 to 24 hours after <strong>in</strong>gestion <strong>of</strong> thecontam<strong>in</strong>ated food. Symptoms resolve <strong>in</strong> 24 to 48 hours, but can last as long as oneweek. Abdom<strong>in</strong>al pa<strong>in</strong>, diarrhoea, vomit<strong>in</strong>g, headaches and dehydration are typicalsymptoms and severity ranges from mild pa<strong>in</strong> and little diarrhoea to extreme pa<strong>in</strong> andbloody, severe diarrhoea. Secondary disease syndrome can be chronic and <strong>in</strong>cludeendocarditis, men<strong>in</strong>gitis, pneumonia and arthritis (Patterson and Isaacson, 2003).2.2.2 Escherichia coli2.2.2.1 Historical aspectsEscherichia coli, orig<strong>in</strong>ally known as Bacterium coli commune, was identified <strong>in</strong>1885 by the German paediatrician, Theodor Escherich (Peter et al., 2002). Stra<strong>in</strong>s <strong>of</strong>Escherichia coli are a common part <strong>of</strong> normal microbial flora <strong>of</strong> animals, <strong>in</strong>clud<strong>in</strong>ghumans. Most stra<strong>in</strong>s are harmless, but some cause diarrhoea. Stra<strong>in</strong>s carry<strong>in</strong>gparticularly virulent properties have emerged as a serious hazard, with theconsumption <strong>of</strong> even low numbers <strong>of</strong> these organisms bear<strong>in</strong>g the risk for life–threaten<strong>in</strong>g illness (ICMSF, 2002).2.2.2.2 Biochemical characteristics
19Escherichia coli is <strong>in</strong> the family Enterobacteriaceae. The organism is a Gramnegative,non-spore-form<strong>in</strong>g, straight rod (1.1 µm x 2.0-6.0 µm) arranged <strong>in</strong> pairs ors<strong>in</strong>gly; it is motile by means <strong>of</strong> peritrichous flagella or may be non-motile and mayhave capsules or microcapsules. It is a facultatively anaerobic micro-organism withan optimum growth temperature <strong>of</strong> 37 o C. Escherichia coli is oxidase-negative,catalase- positive, fermentative (glucose, lactose, D-mannitol, D-sorbitol, arab<strong>in</strong>ose,maltose), reduces nitrate and is β-galactosidase-positive. Approximately 95% <strong>of</strong> thestra<strong>in</strong>s are <strong>in</strong>dole and methyl red positive (Fratamico and Smith, 2006). All stra<strong>in</strong>s <strong>of</strong>E. coli are negative <strong>in</strong> the Voges-Proskauer test. Most stra<strong>in</strong>s do not hydrolyse ureaor produce H 2 S <strong>in</strong> 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 <strong>of</strong> Escherichia coli (Bell and Kyriakides, 1998)M<strong>in</strong>imumwaterM<strong>in</strong>pHMaxpHMaxpercentageM<strong>in</strong>TemperatureMaxTemperatureOxygenrequirementactivity a w<strong>of</strong> NaCl (°C)(°C)0.95 4.0 9.5 8.5 7 46 Facultativeanaerobe1. M<strong>in</strong>=M<strong>in</strong>imum2. Max=Maximum2.2.2.4 Incidences <strong>of</strong> E.coli <strong>in</strong> <strong>shrimps</strong>Despite be<strong>in</strong>g part <strong>of</strong> the normal <strong>in</strong>test<strong>in</strong>al flora, E. coli is found <strong>in</strong> aquaticecosystems. The stra<strong>in</strong>s isolated from aquatic environments <strong>of</strong> 13 districts <strong>in</strong>Bangladesh revealed O161 to be the predom<strong>in</strong>ant serotype (19 %) followed by O55and O44 (12 %) and 11 % untypable. Serotype based-pathotyp<strong>in</strong>g <strong>of</strong> the E. colistra<strong>in</strong>s 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 <strong>of</strong> seafood <strong>in</strong> India,are <strong>of</strong>ten contam<strong>in</strong>ated by human activities and are associated with the widespreadoccurrence <strong>of</strong> E. oli <strong>in</strong> seafood. Food-borne outbreaks associated with Shiga tox<strong>in</strong>produc<strong>in</strong>g E. coli (STEC) have been well documented worldwide. The occurrence <strong>of</strong>the STEC has been reported <strong>in</strong> a number <strong>of</strong> food products such as beef, pork, lamb,poultry and fish, and Kumar et al., reported <strong>in</strong> 2001 that STEC is prevalent <strong>in</strong>seafoods <strong>in</strong> India. Dalwara et al., 1987, reported that <strong>shrimps</strong> dest<strong>in</strong>ed for import andexport <strong>in</strong>dustry tested positive for E. coli; these were 14 for fresh water (FW) shellon;5 for FW peeled deve<strong>in</strong>ed; 3 for Sea Water (SW) shell-on; 3 for SW peeleddeve<strong>in</strong>ed and 3 for seawater cooked <strong>shrimps</strong> <strong>in</strong> S<strong>in</strong>gapore respectively.2.2.2.5 Food-borne <strong>in</strong>fectionsEscherichia coli is <strong>of</strong>ten used as an <strong>in</strong>dicator for faecal contam<strong>in</strong>ation; however,this perspective has changed with the identification <strong>of</strong> the ability <strong>of</strong> E. coli to causehuman illness. Some stra<strong>in</strong>s <strong>of</strong> E. coli are capable <strong>of</strong> caus<strong>in</strong>g food-borne disease,rang<strong>in</strong>g from mild enteritis to serious illness and death. E. coli stra<strong>in</strong>s can cause avariety <strong>of</strong> diseases, <strong>in</strong>clud<strong>in</strong>g diarrhoea, dysentery, hemolytic uremic syndrome, andbladder and kidney <strong>in</strong>fections. Different stra<strong>in</strong>s are usually associated with differentdiseases; this versatility <strong>of</strong> E. coli stra<strong>in</strong>s is due to the fact that different stra<strong>in</strong>s haveacquired different sets <strong>of</strong> virulence genes (Teophilo et al., 2002). EnterohemorrhagicE. coli (EHEC) or shiga tox<strong>in</strong>-produc<strong>in</strong>g E. coli (STEC), which cause hemorrhagiccolitis and hemolytic uremic syndrome, enterotoxigenic E. coli (ETEC), which <strong>in</strong>ducetraveller’s diarrhoea, enteropathogenic E. coli (EPEC), which cause a persistentdiarrhoea <strong>in</strong> children liv<strong>in</strong>g <strong>in</strong> develop<strong>in</strong>g countries, enteroadherent E. coli (EAEC),which provoke diarrhoea <strong>in</strong> children, entero<strong>in</strong>vasive E. coli (EIEC) that arebiochemically and genetically related to Shigella species and can <strong>in</strong>duce diarrhoea,diffusely adherent E. coli, which cause diarrhoea and are dist<strong>in</strong>guished by acharacteristic type <strong>of</strong> adherence to mammalian cells (Smith and Fratamico, 2005).
222.3.1.2 VibrioThe genus Vibrio is a member <strong>of</strong> the family Vibrionaceae and consists <strong>of</strong> at least34 recognized species which <strong>in</strong>clude harmless aquatic stra<strong>in</strong>s as well as stra<strong>in</strong>scapable <strong>of</strong> caus<strong>in</strong>g epidemics <strong>of</strong> cholera (Sechi, 2000). Vibrio is found <strong>in</strong> aquatichabitats with a wide range <strong>of</strong> sal<strong>in</strong>ities. It is very common <strong>in</strong> mar<strong>in</strong>e and estuar<strong>in</strong>eenvironments and on the surfaces and <strong>in</strong> the <strong>in</strong>test<strong>in</strong>al contents <strong>of</strong> mar<strong>in</strong>e animals.There is evidence <strong>of</strong> some species <strong>in</strong> fresh water. Several species are pathogenic formar<strong>in</strong>e vertebrates and <strong>in</strong>vertebrates (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 with<strong>in</strong> the genus, such as V. alg<strong>in</strong>olyticus,V. fluvialis, V. furnissii, V. metschnikovii, and V. hollisae are occasional humanpathogens. Due to the ubiquitous nature <strong>of</strong> Vibrio spp. <strong>in</strong> mar<strong>in</strong>e aquaticenvironments, the presence <strong>of</strong> Vibrio spp. <strong>in</strong> mar<strong>in</strong>e and brackish water aquaculture isto be expected and water <strong>quality</strong> control measures have not made a substantialcontribution <strong>in</strong> this context (Dalsgaard, 1998). Vibrio species account for a significantproportion <strong>of</strong> human <strong>in</strong>fections from the consumption <strong>of</strong> raw or undercooked shellfish(Kaysner and DePaola, 2004).2.3.1.3 Biochemical characteristicsMembers <strong>of</strong> the genus Vibrio are def<strong>in</strong>ed as Gram-negative rods that are straightor have a s<strong>in</strong>gle, rigid curve. They are motile; most have a s<strong>in</strong>gle polar flagellum,when grown <strong>in</strong> a liquid medium. Flagella are enclosed <strong>in</strong> a sheath cont<strong>in</strong>uous withthe outer membrane <strong>of</strong> the cell wall. They are facultatively anaerobic andchemoorganotrophic, hav<strong>in</strong>g both a respiratory and a fermentative type <strong>of</strong>metabolism. 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 production<strong>of</strong> acid, but not gas (except for V. furnissii, V. gazogenes, and some stra<strong>in</strong>s <strong>of</strong> 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 <strong>of</strong> human pathogenic Vibrionaceae commonlyencountered <strong>in</strong> 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 + + + + + - + + + + +Arg<strong>in</strong><strong>in</strong>e - - + + - + - - - + +dihydrolaseOrnith<strong>in</strong>e + + - - - - + + + - +decarboxylaseLys<strong>in</strong>e + + - - - + + + + V +decarboxylase0 % NaCl + + + +Growth<strong>in</strong> (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 -Arab<strong>in</strong>ose - - + + + - - + - 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 SGelat<strong>in</strong>ase + + + + - + + + + + -Urease - - - - - - - V - - -(1= V. alg<strong>in</strong>olyticus; 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-polymyx<strong>in</strong> B-colist<strong>in</strong>; AGS = arg<strong>in</strong><strong>in</strong>e-glucoseslant; Y = yellow; NG = no or poor growth; S = susceptible; nd = not done; G = green; V= variable among stra<strong>in</strong>s; R = resistant; P = purple; V = variable; KK = Slant alkal<strong>in</strong>e/Buttalkal<strong>in</strong>e; KA = Slant alkal<strong>in</strong>e /Butt acidic; Ka = Slant alkal<strong>in</strong>e/Butt slightly acidic)2.3.1.4 Growth parametersTable 6: Growth parameters <strong>of</strong> the most important Vibrio spp. (Berl<strong>in</strong> 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 <strong>of</strong> Vibrio <strong>in</strong> <strong>shrimps</strong>Studies describ<strong>in</strong>g the presence <strong>of</strong> vibrions <strong>in</strong> seafood <strong>in</strong> temperate regions and<strong>in</strong> Asia are quite common, and a few <strong>in</strong>stances <strong>of</strong> vibrion-related disease, apart fromVibrio cholerae 01, have been reported <strong>in</strong> tropical countries (Nascimento et al., 2001).Wong et al., 1999, reported the presence <strong>of</strong> Vibrio parahaemolyticus <strong>in</strong> seafoodimported from Hong Kong, Indonesia, Thailand and Vietnam. The contam<strong>in</strong>ationrates <strong>in</strong> <strong>shrimps</strong>, 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 <strong>of</strong> the isolatespossessed the hemolys<strong>in</strong> genes (tdh, trh).
25Oysters, clams and mussels (filter feeders) are reservoirs <strong>of</strong> these microorganismsand the <strong>in</strong>gestion <strong>of</strong> raw or undercooked shellfish could cause an <strong>in</strong>fection(Parvathi et al., 2004). Lhafi and Kühne, 2007, revealed <strong>in</strong> their study on bluemussels from the German Wadden sea, that among Vibrio isolates, Vibrioalg<strong>in</strong>olyticus was the species most frequently detected (51.2 %), followed by Vibrioparahaemolyticus (39.5 %). Vibrio vulnificus was detected <strong>in</strong> 3.5 % <strong>of</strong> the samples.V. parahaemolyticus and V. vulnificus were not found <strong>in</strong> samples collected at lowwater temperatures.Hosse<strong>in</strong>i et al., 2004, reported that the presence <strong>of</strong> V. parahaemolyticus, V.damsela, V. alg<strong>in</strong>olyticus and V. fluvialis <strong>in</strong> <strong>shrimps</strong> <strong>in</strong> Iran and mentioned thatVibrios such as V. damsela, V. alg<strong>in</strong>olyticus and V. fluvialis are <strong>in</strong>digenous to themar<strong>in</strong>e environment and <strong>shrimps</strong>. Warm water <strong>shrimps</strong> imported <strong>in</strong>to Denmark <strong>in</strong>1995 were contam<strong>in</strong>ated with V. vulnificus <strong>in</strong> 3 <strong>of</strong> 46 frozen, raw shrimp samples butnot <strong>in</strong> any <strong>of</strong> the cooked products. The low prevalence <strong>in</strong> frozen raw <strong>shrimps</strong> wasassociated with poor resistance to the cold (Dalsgaard, 1998). Vibrioparahaemolyticus and Vibrio cholerae were exam<strong>in</strong>ed only <strong>in</strong> seafood samples,ma<strong>in</strong>ly imported fish and shellfish. It was reported that both organisms were foundexclusively <strong>in</strong> imported, raw, frozen prawns and <strong>shrimps</strong> <strong>in</strong> the Republic <strong>of</strong> Cyprus(Eleftheriadou et al., 2002).2.3.1.6 Food-borne <strong>in</strong>fections2.3.1.6.1 V. choleraeV. cholerae was first isolated <strong>in</strong> a pure culture <strong>in</strong> 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 alkal<strong>in</strong>e 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 <strong>in</strong>oculation, then only 10 4 were required
26(Fix, 2007). However, Vibrio cholerae serogroups O1 and O139 cause cholerathrough the production <strong>of</strong> an enterotox<strong>in</strong> cholera tox<strong>in</strong> (CT) characterised by severewatery diarrhoea (Faruque et al., 1998). Vibrio cholerae non-O1 <strong>in</strong>fects only humansand other primates and has genetic differences from the "O1" stra<strong>in</strong>. Non-O1 stra<strong>in</strong>scause a less severe disease. Pathogenic and non-pathogenic stra<strong>in</strong>s <strong>of</strong> the organismare found <strong>in</strong> mar<strong>in</strong>e and estuar<strong>in</strong>e environments (Berl<strong>in</strong> et al., 1999).2.3.1.6.2 V. parahaemolyticusV. parahaemolyticus was first isolated <strong>in</strong> Japan <strong>in</strong> 1950 as a causative bacterialagent <strong>of</strong> food poison<strong>in</strong>g named ‘Shirasu (a half dried fish product) food poison<strong>in</strong>g’.This organism has been recognized as a major cause <strong>of</strong> gastroenteritis throughout theworld (Dalsgaard, 1998). Environmental stra<strong>in</strong>s <strong>of</strong> V. parahaemolyticus are typicallynot human pathogens. However, these stra<strong>in</strong>s cause disease <strong>in</strong> <strong>shrimps</strong>, oysters,mussels and other mar<strong>in</strong>e <strong>in</strong>vertebrates. Most cl<strong>in</strong>ical stra<strong>in</strong>s <strong>of</strong> V. parahaemolyticusproduce a major virulence factor, the thermostable direct hemolys<strong>in</strong> (TDH), and aredesignated as Kanagawa phenomenon positive (KP+). Another virulence factor, theTDH-related hemolys<strong>in</strong> (TRH) is generally associated with the Kanagawaphenomenon negative (KP-) stra<strong>in</strong>s or with urease positive stra<strong>in</strong>s <strong>of</strong> V.parahaemolyticus (Nascimento et al., 2001). When <strong>in</strong>gested, V. parahaemolyticuscauses watery diarrhoea <strong>of</strong>ten with abdom<strong>in</strong>al cramp<strong>in</strong>g, nausea, vomit<strong>in</strong>g, fever andchills. Usually these symptoms occur with<strong>in</strong> 24 hours <strong>of</strong> <strong>in</strong>gestion. Illness is usuallyself-limited and lasts three days. Severe disease is rare and occurs more commonly <strong>in</strong>persons with weakened immune systems. V. parahaemolyticus can also cause an<strong>in</strong>fection <strong>of</strong> the sk<strong>in</strong> 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 <strong>of</strong> disease, it can cause disease <strong>in</strong> 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 <strong>of</strong> V. vulnificus. Among healthy people, <strong>in</strong>gestion <strong>of</strong> V. vulnificus cancause vomit<strong>in</strong>g, diarrhoea, and abdom<strong>in</strong>al pa<strong>in</strong>. In immunocompromised persons,particularly those with chronic liver disease, V. vulnificus can <strong>in</strong>fect the bloodstream,caus<strong>in</strong>g a severe and life-threaten<strong>in</strong>g illness characterized by fever and chills,decreased blood pressure (septic shock), and blister<strong>in</strong>g sk<strong>in</strong> lesions. V. vulnificusbloodstream <strong>in</strong>fections are fatal <strong>in</strong> about 50 % <strong>of</strong> cases. V. vulnificus can also causean <strong>in</strong>fection <strong>of</strong> the sk<strong>in</strong> when open wounds are exposed to warm sea water; these<strong>in</strong>fections may lead to sk<strong>in</strong> breakdown and ulceration (CDC, 2005a).2.4 Antimicrobial use <strong>in</strong> aquaculture2.4.1 Historical aspectsAntibacterial chemotherapy has been applied <strong>in</strong> aquaculture for more than 50years, with early attempts to use sulphonamides <strong>in</strong> the treatment <strong>of</strong> furunculosis <strong>in</strong>trout and the tetracycl<strong>in</strong>es aga<strong>in</strong>st a range <strong>of</strong> Gram-negative pathogens. However,they didn't come <strong>in</strong>to general use until the 1970’s when the sulphonamides were used,potentiated with trimethoprim. S<strong>in</strong>ce then, their use has grown both <strong>in</strong> numbers andquantity, as the problem <strong>of</strong> bacterial disease has <strong>in</strong>creased (Inglis, 1996). The risk <strong>of</strong>disease <strong>in</strong> shrimp farm<strong>in</strong>g <strong>of</strong>ten <strong>in</strong>creases with the culture <strong>in</strong>tensity and high stock<strong>in</strong>gdensities. High pond densities facilitate the spread <strong>of</strong> pathogens between ponds. Ashortage <strong>of</strong> clean water supply and <strong>in</strong>sufficient waste removal lead to an overload <strong>of</strong>metabolites, <strong>shrimps</strong> became stressed by bad water <strong>quality</strong>, and are ultimately moreprone to becom<strong>in</strong>g affected by disease (Kautsky et al., 2000). Otta et al., 1999,reported that aquatic animals such as fish and shrimp are <strong>in</strong> direct contact with themicro flora <strong>in</strong> the environment and most diseases are aroused by opportunisticpathogens which are already present <strong>in</strong> the environment and attack theimmunocompromised host.
28The potential <strong>of</strong> most veter<strong>in</strong>ary antibacterials for use <strong>in</strong> aquaculture has beenconsidered, and now countries vary widely <strong>in</strong> the drugs they use <strong>in</strong> their aquaculturesystems (Inglis, 1996). A wide range <strong>of</strong> chemicals and drugs are be<strong>in</strong>g used, both forprophylactic treatment and to prevent or control parasitic, fungal and bacterialdiseases <strong>in</strong> shrimp hatcheries <strong>in</strong> Sri Lanka (Wijegoonawardena and Siriwardena,1996b).Antibiotic usage <strong>in</strong> aquaculture is predom<strong>in</strong>antly applied with three methods <strong>of</strong>adm<strong>in</strong>istration, namely: oral therapy (<strong>in</strong> feed), immersion therapy (bath, dip, flow orflush) or <strong>in</strong>jection (Inglis, 1996). Other than fight<strong>in</strong>g capabilities <strong>of</strong> antibioticsaga<strong>in</strong>st bacterial diseases, fish farmers and livestock producers began us<strong>in</strong>g suchdrugs <strong>in</strong> animals because <strong>of</strong> the growth promot<strong>in</strong>g capability. Antibiotics rout<strong>in</strong>elyused for the treatment <strong>of</strong> human <strong>in</strong>fections are also used for animals, for therapy,prophylactic reasons and growth promotion. In the case <strong>of</strong> growth promotion, subtherapeutic doses <strong>of</strong> antibiotics usually have been used, and this has contributed topromot<strong>in</strong>g resistance. Research <strong>in</strong>dicates that 70-80 % <strong>of</strong> the drugs used <strong>in</strong>aquaculture end up <strong>in</strong> 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 <strong>of</strong> their failure is the emergence <strong>of</strong> resistant bacteria (Inglis, 1996).2.4.2 Antimicrobial resistanceResistance takes two forms, one is <strong>in</strong>herent or <strong>in</strong>tr<strong>in</strong>sic resistance, here thespecies is not normally susceptible to a particular drug. This may be due to the<strong>in</strong>ability <strong>of</strong> the antibacterial agent to enter the bacterial cell and reach its target site, orto a lack <strong>of</strong> aff<strong>in</strong>ity between the antibacterial and its target (site <strong>of</strong> action), or anabsence <strong>of</strong> the target <strong>in</strong> the cell. The other is the acquired resistance, where the
29species is normally susceptible to a particular drug but certa<strong>in</strong> stra<strong>in</strong>s express drugresistance (Hernández, 2005).2.4.2.1 Emergence <strong>of</strong> antimicrobial resistant bacterial stra<strong>in</strong>sIn general, <strong>in</strong>dividual bacteria differ <strong>in</strong> their susceptibility to antibiotics. Whenmost <strong>of</strong> 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 <strong>in</strong> the presence <strong>of</strong> antibiotic levels which would normally suppress growth orkill susceptible organisms is called resistance.There are two dist<strong>in</strong>ct stages <strong>in</strong> the emergence <strong>of</strong> antibiotic resistance bacteria.One is genetic change, by mutation (change <strong>in</strong> the DNA sequence <strong>of</strong> the relevant gene<strong>in</strong> the chromosome that affects the uptake or action <strong>of</strong> the antibiotic) or by geneacquisition (an exist<strong>in</strong>g antibiotic resistance gene is transferred from a resistantbacterium to another bacterium). The other is the enrichment by selection, as theantibiotic cont<strong>in</strong>ues to be used the resistance bacteria will make up a greater andgreater percentage <strong>of</strong> the population because the susceptible bacteria keep dy<strong>in</strong>g andthe resistant ones keep reproduc<strong>in</strong>g.Antibiotic resistance passes on from parent to <strong>of</strong>fspr<strong>in</strong>g and it can also be passedon from one bacterial cell to another because <strong>of</strong> physical contact. Acquisition <strong>of</strong> theresistant determ<strong>in</strong>ants from one bacterium to another happens mostly via the transfer<strong>of</strong> a small chromosome, either a plasmid or a conjugative transposon. Such transfersoccur most frequently between bacteria <strong>of</strong> the same stra<strong>in</strong> and species, although crossspecies transfers can occur (JETACAR, 1999).2.4.2.2 Mechanisms <strong>of</strong> antimicrobial resistance <strong>in</strong> bacteria• Alteration <strong>of</strong> the targeted site <strong>of</strong> action
30This is typically accomplished by subtly chang<strong>in</strong>g the “targeted site <strong>of</strong>action” <strong>in</strong> 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 <strong>in</strong>hibitors such as penicill<strong>in</strong>s.• Overproduction <strong>of</strong> the targetInstead <strong>of</strong> alter<strong>in</strong>g the sequence (nucleic acid or prote<strong>in</strong>) <strong>of</strong> the antimicrobialtarget, it is possible to alter (<strong>in</strong>crease) the copy number <strong>of</strong> the target so that theeffective drug concentration required to <strong>in</strong>hibit a specific process is <strong>in</strong>creased; e.g.resistance <strong>of</strong> sulfonamides <strong>in</strong> case <strong>of</strong> para-am<strong>in</strong>obenzoic acid (PABA) conversionto dihydropteric acid and over expression <strong>of</strong> the enzyme dihydropteroate synthase.• Production <strong>of</strong> a new enzyme to bypass the targeted site <strong>of</strong> actionSome bacteria such as members <strong>of</strong> Enterobacteriaceae and Staphylococcusaureus can synthesize entirely new enzymes, which can carry out the PABA todihydropteroate conversion. These enzymes decrease the aff<strong>in</strong>ity for trimethoprimand sulfonamides.• Limit<strong>in</strong>g access <strong>of</strong> the antibiotic to the targeted site <strong>of</strong> actionThis mechanism <strong>of</strong> action plays a significant role <strong>in</strong> bacterial resistance,particularly Gram negative bacteria. Changes <strong>in</strong> the lipid composition <strong>of</strong> the outermembrane may significantly contribute to resistance e.g. resistance toerythromyc<strong>in</strong>. The expression <strong>of</strong> specific transporters is another way that bacterialimit access <strong>of</strong> an antimicrobial agent to its site <strong>of</strong> action eg. resistance totetracycl<strong>in</strong>e.• Modification <strong>of</strong> the antimicrobial agent
31Modify<strong>in</strong>g the chemical structure <strong>of</strong> the antimicrobial can be accomplishedby a chemical break down (hydrolysis) <strong>of</strong> the antibiotic; bacteria which produce β-lactamases can hydrolyse the β-lactam r<strong>in</strong>g which is essential for penicill<strong>in</strong> andcephalospor<strong>in</strong> antimicrobials or by chemical substitution <strong>of</strong> the antibiotic which isachieved by bacteria produc<strong>in</strong>g the enzyme chloramphenicol acetyltransferase,lead<strong>in</strong>g to the acetylation <strong>of</strong> chloramphenicol (Khan, 1999).2.4.2.3 Incidences <strong>of</strong> antimicrobial resistance <strong>in</strong> aquacultureIn aquaculture this antimicrobial resistance was experienced due to a horizontalgene transfer as a consequence <strong>of</strong> antimicrobial exposure. The <strong>in</strong>cluded examples areAeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, Citrobacterfreundii, Lactococcus garviae, Yers<strong>in</strong>ia ruckeri, Photobacterium psychrophilum andPseudomonas fluorescens. Sulfonamide resistance <strong>in</strong> Aeromonas salmonicida wasreported <strong>in</strong>1985 <strong>in</strong> the USA and, <strong>in</strong> the 1960’s; multi-resistant stra<strong>in</strong>s were observed<strong>in</strong> Japan. Transferable resistance plasmids are detected <strong>in</strong> the stra<strong>in</strong>s (SØrum, 2006).The development <strong>of</strong> resistance by shrimp pathogens such as Vibrio harveyi due toexposure to antimicrobials has been reported by Karunasagar et al., 1994.The transferability <strong>of</strong> resistance plasmids from fish pathogens and aquaticbacteria shows that these bacteria can act as reservoirs <strong>of</strong> antimicrobial resistancegenes that can be further dissem<strong>in</strong>ated and ultimately reach human pathogens, andthereby add to the burden <strong>of</strong> antimicrobial resistance <strong>in</strong> human medic<strong>in</strong>e (SØrum,2006). Vibrio spp. are mar<strong>in</strong>e bacteria <strong>of</strong> which some can be pathogenic to humans.Resistance <strong>in</strong> V. cholerae and V. parahaemolyticus can be a result <strong>of</strong> human usage <strong>of</strong>antimicrobials, but may also be a consequence <strong>of</strong> the use <strong>of</strong> antimicrobials <strong>in</strong>aquaculture (Hernández, 2005). Kruse et al., 1995, reported a large multi-resistanceplasmid conferr<strong>in</strong>g 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 <strong>in</strong> SalmonellaThe emergence <strong>of</strong> antimicrobial-resistant Salmonella is associated with the use<strong>of</strong> antibiotics <strong>in</strong> 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 <strong>in</strong>to ready-to-eat <strong>shrimps</strong> which aredesigned for consumption without further heat treatment revealed the presence <strong>of</strong>antibiotic resistance salmonellae which may result <strong>in</strong> consumer exposure to antibioticresistantbacteria (Marshal and Durán, 2005). The major public health concern is thatSalmonella will become resistant to antibiotics used <strong>in</strong> human medic<strong>in</strong>e, therebygreatly reduc<strong>in</strong>g therapeutic options and threaten<strong>in</strong>g the lives <strong>of</strong> <strong>in</strong>fected <strong>in</strong>dividuals(Doyle et al., 2001). In the last two decades, the emergence and spread <strong>of</strong>antimicrobial-resistant pathogens, among them Salmonella, has become a serioushealth hazard worldwide. The rout<strong>in</strong>e practice <strong>of</strong> giv<strong>in</strong>g antimicrobial agents todomestic livestock as a means <strong>of</strong> prevent<strong>in</strong>g and treat<strong>in</strong>g diseases, as well aspromot<strong>in</strong>g growth, is an important factor <strong>in</strong> the emergence <strong>of</strong> antibiotic-resistantbacteria that are subsequently transferred to humans via the food cha<strong>in</strong> (Miko et al.,2005).Non-typhoidal Salmonella stra<strong>in</strong>s are a frequent cause <strong>of</strong> food-borne diseaseoutbreaks <strong>in</strong> the United States; they account for about 13 % <strong>of</strong> outbreaks reported tothe Center for Disease Control and Prevention (CDC) from 1993 to 1997 (Olsen,2000). Antimicrobial resistance is common among salmonellae and has been<strong>in</strong>creas<strong>in</strong>g, particularly <strong>in</strong> Salmonella enterica serotype Typhimurium, the mostcommon Salmonella serotype that most threatens food safety. In the 1990’s, a stra<strong>in</strong><strong>of</strong> S. Typhimurium that resistant to ampicill<strong>in</strong>, chloramphenicol, sulfonamides,tetracycl<strong>in</strong>e and streptomyc<strong>in</strong>/spect<strong>in</strong>omyc<strong>in</strong>, emerged <strong>in</strong> the United States andEurope; most <strong>of</strong> these isolates were phage def<strong>in</strong>itive 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 reign<strong>in</strong>gmulti-resistant Salmonella, although other phage types, such as DT 208, may be theSalmonella that occupies the DT 104 niche <strong>in</strong> the future. Alternatively, another S.enterica serotype, such as Agona, may be elevated to the status <strong>of</strong> DT 104. Otherthan this penta group <strong>of</strong> antimicrobials, scientists are vigilant about the ceftriaxone,cipr<strong>of</strong>loxac<strong>in</strong> and fluoroqu<strong>in</strong>olones resistance <strong>in</strong> DT 104 s<strong>in</strong>ce ceftriaxone is thecurrent drug <strong>of</strong> choice aga<strong>in</strong>st DT 104 <strong>in</strong> children and cipr<strong>of</strong>loxac<strong>in</strong> andfluoroqu<strong>in</strong>olones are considered to be the last l<strong>in</strong>e <strong>of</strong> defence aga<strong>in</strong>st DT 104 <strong>in</strong> adults(Carlson, et al., 2003). Moreover, Miriagou et al., 2002, reported the emergence <strong>of</strong>non-typhoid Salmonella enterica serotype Typhimurium stra<strong>in</strong>s that are resistant toexpanded-spectrum cephalospor<strong>in</strong>s from a pediatric population <strong>in</strong> Iasi, Romania.2.5 Public health perspectives2.5.1 Seafood safety and <strong>microbiological</strong> standardsFood-borne <strong>in</strong>fections and <strong>in</strong>toxications were common <strong>in</strong> ancient times. Ancientpeople based their food laws on religious beliefs. Trial and error were the ma<strong>in</strong> toolsfor food safety, and taboos were the primary tool for prevention <strong>of</strong> food-borne<strong>in</strong>fections. Certa<strong>in</strong> foods were described as “unhealthy” and unfit for humanconsumption, particularly those <strong>of</strong> animal orig<strong>in</strong>. Some animals were def<strong>in</strong>ed as“unclean” and were therefore unsuitable for consumption. The consumption <strong>of</strong> theblood <strong>of</strong> slaughtered animals was totally prohibited s<strong>in</strong>ce blood was considered to bethe soul <strong>of</strong> the animal. Only when the study <strong>of</strong> food microbiology began to develop atthe end <strong>of</strong> the 19 th century could the pattern <strong>of</strong> food-borne <strong>in</strong>fections be understood.Every product has a typical spectrum <strong>of</strong> microbial contam<strong>in</strong>ants reflected by theexposure pattern <strong>of</strong> the product. There is no demand that a product <strong>in</strong>tended forhuman consumption should be free <strong>of</strong> micro-organisms. As a result, <strong>microbiological</strong>standards are recommended for most products (Kl<strong>in</strong>ger, 2001).The proper handl<strong>in</strong>g <strong>of</strong> fish between capture and its delivery to the consumer is acrucial element <strong>in</strong> assur<strong>in</strong>g the f<strong>in</strong>al <strong>quality</strong> <strong>of</strong> the product. Standards <strong>of</strong> sanitation,the method <strong>of</strong> handl<strong>in</strong>g and the time/temperature <strong>of</strong> hold<strong>in</strong>g fish are all significant
34<strong>quality</strong> factors. Apart from a few exceptions, fish are considered to be free <strong>of</strong>pathogenic bacteria <strong>of</strong> public health significance when first caught. The presence <strong>of</strong>bacteria harmful to man is generally an <strong>in</strong>dication <strong>of</strong> poor sanitation while handl<strong>in</strong>gand process<strong>in</strong>g the fish and the contam<strong>in</strong>ation is almost always <strong>of</strong> human or animalorig<strong>in</strong> (Sciort<strong>in</strong>o and Ravikumar, 1999). Salmonella and E. coli stra<strong>in</strong>s, <strong>in</strong>clud<strong>in</strong>gresistant stra<strong>in</strong>s, can be found <strong>in</strong> aquatic environments as a result <strong>of</strong> contam<strong>in</strong>ationwith such bacteria from human, animal or agricultural environments. Sewagecontam<strong>in</strong>ation or run-<strong>of</strong>f from agricultural areas with graz<strong>in</strong>g animals to aquacultureoperations can result <strong>in</strong> the presence <strong>of</strong> resistant Salmonella and E. coli <strong>in</strong> productsfrom aquaculture (FAO/OIE/WHO, 2006).A number <strong>of</strong> <strong>microbiological</strong> tests <strong>of</strong> fish and fish products are used byauthorities to check that the <strong>microbiological</strong> status is satisfactory. The purpose <strong>of</strong>these tests is to detect pathogenic bacteria, <strong>in</strong>dicator organisms or other types <strong>of</strong>general contam<strong>in</strong>ation or poor handl<strong>in</strong>g practices. When unsatisfactory aerobiccolony counts are encountered microbiologists should attempt to identify the microorganismsthat predom<strong>in</strong>ate. From these results, and additional detailed <strong>in</strong>formationabout the food sample, it should be possible to provide a more helpful <strong>in</strong>terpretation<strong>of</strong> high aerobic colony counts (Gilbert et al., 2000).In <strong>in</strong>ternational trade, the most common reason for import refusal is "filthy"which describes that the product appears to consist <strong>in</strong> whole or <strong>in</strong> part <strong>of</strong> a filthy,putrid or decomposed substance. A major reason for refusal is microbial spoilage.Second <strong>in</strong> terms <strong>of</strong> reasons for rejection is the detection <strong>of</strong> Salmonella. Both cooked,ready-to-eat products and raw, frozen products are rejected if Salmonella is detected(Huss, 2003).The presence <strong>of</strong> Salmonella and a high bacterial count <strong>in</strong> the cooked and peeledfrozen <strong>shrimps</strong> exported from India has meant many problems <strong>in</strong> <strong>in</strong>ternational trade <strong>in</strong>the past. The local contribution for sett<strong>in</strong>g up standards came from the CentralInstitute <strong>of</strong> Fisheries Technology (CIFT) and Export Inspection Agency (EIA) <strong>in</strong>India (Hatha et al., 2003).
35The <strong>in</strong>crease <strong>in</strong> imports <strong>of</strong> seafood from develop<strong>in</strong>g countries by the developedeconomies also resulted <strong>in</strong> the adoption <strong>of</strong> <strong>in</strong>ternational guidel<strong>in</strong>es for foodprocess<strong>in</strong>g, such as Hazard Analysis Critical Control Po<strong>in</strong>t (HACCP) and EuropeanUnion (EU) guidel<strong>in</strong>es. In an effort to control the microbial contam<strong>in</strong>ation <strong>of</strong> foods,the National Advisory Committee on Microbiological Criteria for Foods (NACMCF)and the International Commission on Microbiological Specification for Foods(ICMSF) have recommended <strong>microbiological</strong> criteria as a means <strong>of</strong> assess<strong>in</strong>g theeffectiveness <strong>of</strong> the HACCP programme (Hatha et al., 2003).In Sri Lanka, the export <strong>of</strong> fish products (any aquatic organism whether pisc<strong>in</strong>eor not, and <strong>in</strong>cludes 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 <strong>of</strong> 1996. A processorwho wishes to export his products must register with the Department <strong>of</strong> Fisheries andAquatic Resources, the Competent Authority. A basic requirement to be met <strong>in</strong> orderto obta<strong>in</strong> registration is that the establishment shall establish and ma<strong>in</strong>ta<strong>in</strong> a HACCPsystem to cover the products and processes concerned. The registered establishmentsare monitored for cont<strong>in</strong>ual ma<strong>in</strong>tenance <strong>of</strong> the system. These <strong>in</strong>spections 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 <strong>in</strong> <strong>in</strong>ternational trade. Wherepractices <strong>in</strong> one country differ from the practices <strong>in</strong> the other country and still bothpractices provide safe products, comparison <strong>of</strong> the relative level <strong>of</strong> protection mayscale on a scientific basis. In this context, the International Commission on theMicrobiological Specifications for Foods (ICMSF) has contributed by recommend<strong>in</strong>gpreventive, stepwise approach for the management <strong>of</strong> <strong>microbiological</strong> hazards <strong>in</strong>foods <strong>in</strong> <strong>in</strong>ternational trade. The concept <strong>of</strong> the Food Safety Objectives (FSOs) hasbeen proposed by the ICMSF to communicate the level <strong>of</strong> hazards <strong>of</strong> foods <strong>in</strong> order t<strong>of</strong>acilitate the acceptance <strong>of</strong> different but equivalent processes (Stewart et al., 2002).
362.5.2 Impact <strong>of</strong> food-borne hazards on health and the economy <strong>in</strong> developed countries2.5.2.1 Biological hazardsIn <strong>in</strong>dustrialized countries, the percentage <strong>of</strong> the population suffer<strong>in</strong>g 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 <strong>of</strong> foodborne<strong>in</strong>fection has been estimated at 76 million cases, 323,000 hospital admissions,and 5,000 deaths. The three most common identifiable causes <strong>of</strong> food-borne illness <strong>in</strong>the USA <strong>in</strong> 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 <strong>of</strong> salmonellosis and almost 63,000 <strong>of</strong>campylobacteriosis <strong>in</strong> 2001. France reported 16,523 cases <strong>of</strong> salmonellosis <strong>in</strong> 1998and the Czech Republic reported more than 49,000. These figures revealed that thediseases caused high morbidity and relatively low mortality. However, the economicburden result<strong>in</strong>g from this level <strong>of</strong> illness is huge (Lancet, 2002). About 2-3 % <strong>of</strong>cases <strong>of</strong> food borne diseases lead to long-term ill health, which is far more damag<strong>in</strong>gto human health and the economy than the <strong>in</strong>itial disease (WHO, 1999). A seafoodimplication <strong>in</strong> the case <strong>of</strong> food-borne diseases is estimated at 10-19 % out <strong>of</strong> 76million cases <strong>in</strong> the USA (Butt et al., 2004).Each year <strong>in</strong> the United States <strong>of</strong> America, food-borne diseases cost billions <strong>of</strong>dollars. Government sources estimate the cost <strong>of</strong> human illnesses <strong>of</strong> seven foodbornepathogens to be between US$ 5.6 to 9.4 billion. The cost <strong>of</strong> salmonellosis <strong>in</strong>England and Wales <strong>in</strong> 1992 was estimated at between US$ 560 and 800 million. Over70 % <strong>of</strong> costs were directly associated with the treatment and <strong>in</strong>vestigation <strong>of</strong> casesand sickness-related absences from work (WHO, 1999).Emerg<strong>in</strong>g food-borne problems will have implications for the health status andthe economies <strong>of</strong> <strong>in</strong>dividual countries, as well as affect<strong>in</strong>g <strong>in</strong>ternational trade and the
37agreements that govern it (Venter, 2000). Food safety <strong>in</strong> <strong>in</strong>ternational 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 <strong>of</strong> its people is, endangered (Stewart, 2002). With this legalobligation, unsafe food products are rejected <strong>in</strong> import<strong>in</strong>g countries on healthgrounds, with obvious economic consequences. As an example, when cholera brokeout <strong>in</strong> Peru <strong>in</strong> 1991, more than US$ 700 million were lost <strong>in</strong> exports <strong>of</strong> fish and fishproducts. The human toll <strong>in</strong> Peru and its neighbour<strong>in</strong>g 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 <strong>of</strong>raw molluscs because <strong>of</strong> environmental contam<strong>in</strong>ation and naturally occurr<strong>in</strong>gvibrios. Seafood-borne illness reported by the CDC <strong>in</strong> the 10-year period 1978-1987totalled 558 outbreaks <strong>in</strong>volv<strong>in</strong>g 5,980 cases. However, fish and shellfish constituteonly 10.5 % <strong>of</strong> all outbreaks and 3.6 % <strong>of</strong> all cases when food-borne illnesses from allfoods are considered. The number <strong>of</strong> 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 <strong>of</strong>reported cases from each is lower than for any animal meat category. Natural seafoodtox<strong>in</strong>s–ma<strong>in</strong>ly ciguatera and scombroid poison<strong>in</strong>g and, to a lesser extent, paralyticshellfish poison<strong>in</strong>g–were responsible for 62.5 % <strong>of</strong> all seafood-borne outbreaks <strong>of</strong>illness.Fish-borne <strong>in</strong>cidents due to causes other than natural tox<strong>in</strong>s were only 9 % <strong>of</strong> alloutbreaks and 8 % <strong>of</strong> all cases. They resulted ma<strong>in</strong>ly from bacteria, <strong>in</strong>clud<strong>in</strong>gcommon food-borne disease organisms, and from unknown etiology, suspected to beprimarily an enteric viruses or recontam<strong>in</strong>ant vibrios (Ahmed, 1991).2.5.2.2 Antibiotic resistanceAntibiotic resistance is a serious cl<strong>in</strong>ical and public health problem <strong>of</strong> globalproportions. Drug choices for the treatment <strong>of</strong> common <strong>in</strong>fectious diseases arebecom<strong>in</strong>g <strong>in</strong>creas<strong>in</strong>gly limited, expensive, and, <strong>in</strong> some cases, useless, due to the
38emergence <strong>of</strong> drug resistance <strong>in</strong> bacteria and fungi. This loss <strong>of</strong> treatment options isthreaten<strong>in</strong>g to reverse much <strong>of</strong> the medical progress <strong>of</strong> the past 50 years becauseessential life-sav<strong>in</strong>g antibiotics are becom<strong>in</strong>g less effective and there are feweralternatives available for treatment (Hernández, 2005; Marshall and Durán, 2005).Development <strong>of</strong> antimicrobial resistance causes enormous costs: the costs to br<strong>in</strong>gout a new drug onto the market are estimated at a m<strong>in</strong>imum <strong>of</strong> US$ 300 million(Byarugaba, 2004).2.5.3 Impact <strong>of</strong> food-borne hazards on health and the economy <strong>in</strong> develop<strong>in</strong>gcountries2.5.3.1 Biological hazardsIn develop<strong>in</strong>g countries, where f<strong>in</strong>ancial resources are scarce, food control issuesusually receive low priority <strong>in</strong> public health programs. Food-borne illnesses areperceived as mild, self-limit<strong>in</strong>g diseases and their health and economic consequencesare <strong>of</strong>ten overlooked (Venter, 2000).While it is recognised that the prevalence <strong>of</strong> food-borne illness <strong>in</strong> develop<strong>in</strong>gcountries is considerable, <strong>in</strong> most there is limited data through which the <strong>in</strong>cidences <strong>of</strong>particular diseases and trends over time can be assessed. In many cases, high rates <strong>of</strong>food-borne illnesses are associated with low levels <strong>of</strong> general economic developmentand, more specifically, limited capacity to control the safety <strong>of</strong> the food supply.Furthermore, there are close <strong>in</strong>ter-relationships between food safety issues and otherelements <strong>of</strong> environmental health, for example sanitation, water <strong>quality</strong> and hous<strong>in</strong>gconditions.Indeed, the high prevalence <strong>of</strong> diarrhoeal diseases <strong>in</strong> many develop<strong>in</strong>g countriessuggests major underly<strong>in</strong>g food safety problems. In the early 1990’s there wereapproximately 1.5 billion episodes <strong>of</strong> diarrhoea annually, <strong>of</strong> which around 70 percentwere associated with contam<strong>in</strong>ated food. In addition, it is estimated that 2.1 millionpeople died from diarrhoeal diseases <strong>in</strong> 2000 (Henson, 2003) as shown <strong>in</strong> Table 7.
39Such diseases take a heavy toll <strong>in</strong> human life and suffer<strong>in</strong>g, particularly among <strong>in</strong>fantsand children, the elderly and other susceptible groups. They also create an enormoussocial, cultural and economic burden on communities and their health systems(Venter, 2000; Käferste<strong>in</strong>, 2003).Table 7: Estimated occurrence <strong>of</strong> bacterial <strong>in</strong>fections and <strong>in</strong>toxications <strong>in</strong> selectedregions (Henson, 2003)Disease Africa Central &SouthSouthEast AsiaWesternPacificAmericaBacillus cereus gastroenteritis +++ +++ +++ +++Botulism + + + +Brucellosis +/++ ++ +/++ +/++Campylobacteriosis +++ +++ +++ +++Cholera +/++ +/++ + +Clostridium perfr<strong>in</strong>gens enteritis +++ +++ +++ +++Escherichia coli disease +++ +++ +++ +++Listeriosis + + + +Typhoid and Paratyphoid fever ++ ++ ++ ++Salmonellosis +++ +++ +++ +++Shigellosis +++ +++ +++ +++Staphylococcus aureus <strong>in</strong>toxication +++ +++ +++ +++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 <strong>of</strong> emerg<strong>in</strong>g food-bornepathogens such as Salmonella, Campylobacter, Enterohaemorrhagic Escherichia coliand antibiotic resistant pathogens, but <strong>in</strong>formation <strong>in</strong> develop<strong>in</strong>g countries is veryscarce (Käferste<strong>in</strong>, 2003).
40Hart and Kariuki, 1998, reported that diarrhoeal disease is a major cause <strong>of</strong>morbidity and mortality <strong>in</strong> develop<strong>in</strong>g countries. Although rotavirus <strong>in</strong>fections are animportant cause <strong>of</strong> the disease <strong>in</strong> <strong>in</strong>fants, bacteria are the most important pathogens <strong>in</strong>older children and adults.Poor health due to diarrhoeal diseases and acute respiratory diseases areimportant causes <strong>of</strong> morbidity and mortality among <strong>in</strong>fants and children <strong>in</strong> Sri Lanka.The Registrar General’s mortality data for 1996 (Registrar General’s Department,1996) <strong>in</strong>dicate that deaths due to diarrhoeal diseases among children under 5 years <strong>of</strong>age accounted for 2 % <strong>of</strong> deaths from all causes <strong>in</strong> this age group. Dur<strong>in</strong>g the past 10years, hospitalization due to diarrhoeal diseases has <strong>in</strong>creased. Furthermore, <strong>in</strong> 1998,<strong>in</strong>test<strong>in</strong>al <strong>in</strong>fections ranked as the fifth lead<strong>in</strong>g cause <strong>of</strong> hospitalization (Jayasekara,2001). Medical Research Institute (MRI) Sri Lanka diarrhoeal pathogen pr<strong>of</strong>ile <strong>in</strong>2004 illustrates the distribution accord<strong>in</strong>g to the importance <strong>of</strong> 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 <strong>in</strong> global travel has huge implications for the spread <strong>of</strong> foodborne<strong>in</strong>fections. In addition to its significance for <strong>in</strong>ternational health, tourism isalso <strong>of</strong> great economic importance, especially to develop<strong>in</strong>g countries. Diarrhoea andvomit<strong>in</strong>g caused by contam<strong>in</strong>ated foods may damage the reputation <strong>of</strong> a country as atourist dest<strong>in</strong>ation (WHO, 1999).2.5.3.2 Antibiotic resistanceThe emergence <strong>of</strong> antimicrobial resistance <strong>in</strong> develop<strong>in</strong>g countries is a complexproblem driven by numerous <strong>in</strong>terconnected factors, many <strong>of</strong> which are l<strong>in</strong>ked to theuse <strong>of</strong> antimicrobials <strong>in</strong> animals, plants and humans. Extreme poverty <strong>in</strong> mostdevelop<strong>in</strong>g countries leads to poor sanitation, hunger, starvation and malnutrition,poor access to drugs and poor health care delivery, all <strong>of</strong> which may precipitate
41antimicrobial resistance. The emergence <strong>of</strong> multi-drug-resistant isolates <strong>in</strong>tuberculosis, acute respiratory <strong>in</strong>fections and diarrhoea, <strong>of</strong>ten referred to as diseases<strong>of</strong> poverty, has taken its greatest toll <strong>in</strong> develop<strong>in</strong>g countries (Byarugaba, 2004).Increased access to antibiotics <strong>in</strong> develop<strong>in</strong>g countries, without controls on overthe-counteruse, has led to some <strong>of</strong> the highest rates <strong>of</strong> resistance <strong>in</strong> the world, as wasseen with penicill<strong>in</strong> resistance <strong>in</strong> Vietnam. Relatively wealthy countries such as theRepublic <strong>of</strong> 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 <strong>in</strong> semi-rural and rural populations, may beresponsible for the emergence <strong>of</strong> fluoroqu<strong>in</strong>olone resistance to Salmonella Typhi <strong>in</strong>India which has great public health importance. Controll<strong>in</strong>g the deadliest <strong>in</strong>fectiousdiseases <strong>in</strong> the world-diarrhoeal diseases, respiratory tract <strong>in</strong>fections, sexuallytransmitted <strong>in</strong>fections, men<strong>in</strong>gitis, pneumonia, and hospital acquired <strong>in</strong>fections ismore difficult today because <strong>of</strong> the emergence <strong>of</strong> antimicrobial drug resistance(Laxm<strong>in</strong>arayan et al., 2006).In 1996, <strong>in</strong> Matlab and Dhaka, Bangladesh, more than 95 % <strong>of</strong> Shigelladysenteriae isolates were resistant to ampicill<strong>in</strong>, co-trimoxazole, and nalidixic acid,and 14-40 % were resistant to mecill<strong>in</strong>am. In a cholera epidemic <strong>in</strong> 1985-6 <strong>in</strong>Somalia there was a case fatality rate <strong>of</strong> 13 % because the <strong>in</strong>itially sensitive V.cholerae quickly acquired plasmid encoded resistance to ampicill<strong>in</strong>, kanamyc<strong>in</strong>,streptomyc<strong>in</strong>, sulphonamides, and tetracycl<strong>in</strong>e (Hart and Kariuki, 1998).Although no estimates <strong>of</strong> disease burden are currently available that are specificto drug resistance, the contribution <strong>of</strong> drug resistance to the burden <strong>of</strong> <strong>in</strong>fectiousdiseases is believed to be large. Resistance has emerged <strong>in</strong> malaria, tuberculosis(TB), and other bacterial <strong>in</strong>fections that together constitute a significant proportion <strong>of</strong>the burden <strong>of</strong> disease <strong>in</strong> develop<strong>in</strong>g countries (Laxm<strong>in</strong>arayan et al., 2006).In Sri Lanka, the Salmonella surveillance carried out by the MRI dur<strong>in</strong>g theyears 2004, 2005 and 2006 revealed the isolates that are sensitive to commonly used
42antibiotics <strong>in</strong> human therapeutics and the percentage <strong>of</strong> sensitivity respectively, 88.9% for amoxycill<strong>in</strong>, 84.4 % for nalidixic acid, 83.3 % for furazolidone, 72.7 % forcotrimaxazole, 96.4 % for mecill<strong>in</strong>am, 93.5 % for ceptazidime, 62.5 % forcefuroxime, 100.0 % for cipr<strong>of</strong>loxac<strong>in</strong>, 100.0 % for chloramphenicol and 100.0% forcefriaxone (MRI, 2007).2.5.4 Prevention and control <strong>of</strong> seafood-borne diseasesThe number <strong>of</strong> cases <strong>of</strong> outbreaks <strong>of</strong> 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 <strong>of</strong> food, it is important tohandle seafood safely <strong>in</strong> order to reduce the risk <strong>of</strong> food-borne illness (FDA, 2006).Some pathogenic bacteria are naturally present <strong>in</strong> the aquatic and the generalenvironment. Those pathogens may therefore be found <strong>in</strong> liv<strong>in</strong>g fish or fish rawmaterial. This would then be called pre-harvest contam<strong>in</strong>ation. The presence <strong>of</strong> theseorganisms is normally not a safety concern s<strong>in</strong>ce their numbers are too <strong>in</strong>significant tocause disease, with the exception <strong>of</strong> molluscan shellfish, which because <strong>of</strong> filterfeed<strong>in</strong>g accumulates high numbers <strong>of</strong> 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-harvestcontam<strong>in</strong>ation is higher <strong>in</strong> densely populated countries such as India because theestuaries and coastal water bodies are soiled with human activities and the land<strong>in</strong>gcentres and open fish markets only have poor sanitation. Millions <strong>of</strong> bacteria arepresent <strong>in</strong> the surface slime, on the gills, and <strong>in</strong> the guts <strong>of</strong> liv<strong>in</strong>g seafood species(UCD, 2006). The hazards related to contam<strong>in</strong>ation and recontam<strong>in</strong>ation or rather thesurvival <strong>of</strong> biological hazards dur<strong>in</strong>g process<strong>in</strong>g can be controlled by apply<strong>in</strong>g GoodHygiene Practice (GHP), Good Manufactur<strong>in</strong>g Practice (GMP) and a well designedHACCP- programme (Huss et al., 2000).
43Therefore, the (FDA, 2006) guidel<strong>in</strong>es for general consumers, stated these basicfood safety tips for buy<strong>in</strong>g, stor<strong>in</strong>g, prepar<strong>in</strong>g, serv<strong>in</strong>g, and eat<strong>in</strong>g raw fish andshellfish,• Buy<strong>in</strong>g seafood- buy<strong>in</strong>g from a retailer who follows proper handl<strong>in</strong>g 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 <strong>of</strong> fresh ice that is not melt<strong>in</strong>g, andpreferably <strong>in</strong> a case or under some type <strong>of</strong> cover. See whether the seller ispractic<strong>in</strong>g proper food handl<strong>in</strong>g techniques. For health safety reasons, it isimportant to look for freshness when choos<strong>in</strong>g seafood.• Stor<strong>in</strong>g seafood- if seafood is to be used with<strong>in</strong> two days after purchas<strong>in</strong>g,store <strong>in</strong> the refrigerator. If seafood is not to be used with<strong>in</strong> two days <strong>of</strong>purchas<strong>in</strong>g, wrap tightly <strong>in</strong> moisture-pro<strong>of</strong> freezer paper or foil to protect itfrom air leaks, and store it <strong>in</strong> the freezer.• Prepar<strong>in</strong>g seafood- thaw frozen seafood gradually by plac<strong>in</strong>g it <strong>in</strong> therefrigerator overnight. If it should thaw quickly, either seal it <strong>in</strong> a plastic bagand immerse it <strong>in</strong> cold water, or- if the food will be cooked immediatelythereafter- microwave it on the “defrost sett<strong>in</strong>g and stop the defrost cyclewhile the fish is still icy but pliable. Prevent cross-contam<strong>in</strong>ation, avoid crosscontam<strong>in</strong>ation between raw and cooked foods. Cook properly. Most seafoodshould be cooked to an <strong>in</strong>ternal temperature <strong>of</strong> 145 °F (63°C) for 15 seconds.• Serv<strong>in</strong>g seafood- do not cross-contam<strong>in</strong>ate• Eat<strong>in</strong>g raw seafood- special health warn<strong>in</strong>gs 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 <strong>of</strong> thumb is to eat fish that has previously been frozen.Some species <strong>of</strong> fish can conta<strong>in</strong> parasites, and freez<strong>in</strong>g will kill any parasites
44that may be present. However, freez<strong>in</strong>g does not kill all harmful microorganisms.That is why the safest way is to cook seafood.
3. MATERIALS AND METHODS3.1 Study designThe objective <strong>of</strong> the present study was to assess the bacteriological status <strong>of</strong>Penaeus <strong>monodon</strong> <strong>shrimps</strong> available at the local sale locations which were purchasedat different times <strong>of</strong> the day <strong>in</strong> order to determ<strong>in</strong>e the prevalence, the serotypes andthe antimicrobial resistance <strong>of</strong> Salmonella and the <strong>microbiological</strong> contam<strong>in</strong>ation <strong>in</strong>terms <strong>of</strong> 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 <strong>monodon</strong> <strong>shrimps</strong> both cultured <strong>in</strong> farms and captured from the lagoonswere taken from the local sale po<strong>in</strong>ts <strong>of</strong> the North Western Prov<strong>in</strong>ce (Kurunegala andPuttalam districts) <strong>in</strong> Sri Lanka where most <strong>of</strong> the shrimp farm<strong>in</strong>g is carried out. Thesamples were collected between November 2006 and March 2007.3.2.2 PopulationShrimps sold <strong>in</strong> the local sale locations <strong>in</strong> North Western Prov<strong>in</strong>ce fromNovember 2006 to March 2007.
453.2.3 SampleOne sample consisted <strong>of</strong> at least 2 whole Penaeus <strong>monodon</strong> <strong>shrimps</strong>. It was acomposite sample pooled from tissues <strong>of</strong> shrimp <strong>in</strong>clud<strong>in</strong>g shell and head to produce a35g/sample, 10 g for enumeration and 25 g for Salmonella isolation.Figure 1: Penaeus <strong>monodon</strong> shrimp3.2.4 Sample sizeAssumptions made concern<strong>in</strong>g the prevalence <strong>of</strong> Salmonella;1. Expected prevalence 12.5 % Karnataka coast India (Bhaskar et al., 1995) wastaken because no data available <strong>in</strong> Sri Lanka regard<strong>in</strong>g the prevalence <strong>of</strong>Salmonella.2. Shrimps sold per month at the local market <strong>in</strong> the North Western Prov<strong>in</strong>ce, 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 W<strong>in</strong>episcope 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 <strong>of</strong> India with Karnataka(www.journeymart.com/.../India/images/map_new.gif)3.2.5 Sampl<strong>in</strong>g planSampl<strong>in</strong>g was done accord<strong>in</strong>g to convenient stratified proportional sampl<strong>in</strong>g.The two strata represented <strong>in</strong> this study are the Puttalam and Kurunegala districtswhich form the North Western Prov<strong>in</strong>ce <strong>in</strong> Sri Lanka. Two thirds <strong>of</strong> 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 accord<strong>in</strong>g to convenient sampl<strong>in</strong>g. Eligiblelocations were def<strong>in</strong>ed as hav<strong>in</strong>g at least 7 vendors sell<strong>in</strong>g <strong>shrimps</strong>. 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 <strong>of</strong> the fisherman and each time two parallel samples were taken fromeach shop at different times <strong>of</strong> the day. Samples were collected <strong>in</strong>to sample bagswhich were labelled and transported <strong>in</strong> an ice conta<strong>in</strong>er to the laboratory forprocess<strong>in</strong>g. Each sample conta<strong>in</strong>ed at least two <strong>shrimps</strong> <strong>in</strong> order to create a 35 g/test
47sample. Out <strong>of</strong> 35g <strong>of</strong> the test sample 10 g were used for enumeration <strong>of</strong> AerobicPlate Count, Enterobacteriaceae Count, E. coli Count, Total Halophilic Plate Countand 25 g used for Salmonella isolation.North WesternProv<strong>in</strong>ceFigure 3: Map <strong>of</strong> Sri Lanka with North Western Prov<strong>in</strong>ce(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 <strong>of</strong> 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 B<strong>in</strong>giriya19 Hettipola20 Kobeigane21 Nikaweratiya22 Rasnayakapura23 Kotawehera24 Ambanpola25 Maho26 Polpithigama27 Galgamuwa28 Ehatuwewa29 Giribawa30 Nawagaththegama
493.2.6 QuestionnaireA questionnaire was adm<strong>in</strong>istered to each shop owner to evaluate the rout<strong>in</strong>epractices <strong>of</strong> the local shrimp sale locations.3.3 Laboratory proceduresFresh <strong>shrimps</strong> belong<strong>in</strong>g to the species Penaeus <strong>monodon</strong> obta<strong>in</strong>ed from localsale locations were analysed to determ<strong>in</strong>e their <strong>microbiological</strong> <strong>quality</strong>. Each sampleconta<strong>in</strong>ed the date, the sale location identity and the district. Accord<strong>in</strong>g to thestandard methods, shrimp samples were analysed and the bacteriological parameterswere Salmonella isolation, serotyp<strong>in</strong>g and antimicrobial susceptibility for isolatedSalmonella. Enumeration was carried out <strong>in</strong> terms <strong>of</strong> Aerobic Plate Count,Enterobacteriaceae, Escherichia coli and Total Halophilic Plate Count. Most <strong>of</strong> thetime 10 samples (batch) were analysed per week dur<strong>in</strong>g the field work for theisolation <strong>of</strong> Salmonella.3.3.1 Aerobic Plate CountTests were carried out accord<strong>in</strong>g to the International Standards <strong>of</strong> ISO4833:2003. Two poured plates were prepared us<strong>in</strong>g a specified culture medium and aspecified quantity <strong>of</strong> 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 (<strong>in</strong>itial dilution):A 10 g shrimp sample was required for test<strong>in</strong>g. Test sample <strong>of</strong> shrimp wasadded to a stomacher bag conta<strong>in</strong><strong>in</strong>g 90 ml <strong>of</strong> maximum recovery diluent (MRD) andstomached for 1 m<strong>in</strong>ute.3.3.1.3 Procedures:1. Perform decimal dilutions from the shrimp homogenateUs<strong>in</strong>g a fresh sterile pipette, 1ml <strong>of</strong> the <strong>in</strong>itial <strong>in</strong>oculum was transferred <strong>in</strong>to 9 ml<strong>of</strong> MRD. The procedure was repeated for as many decimal dilutions as required.Dilutions were mixed us<strong>in</strong>g a vortex mixer for 5 to 10 seconds.2. Inoculation1 ml <strong>of</strong> the each required dilution was aseptically <strong>in</strong>oculated <strong>in</strong>to 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 <strong>in</strong> 15 ml volume to eachPetri dish. Carefully mixed by swirl<strong>in</strong>g six times clockwise, six times left to right, sixtimes anticlockwise and six times up and down then allowed to set. (The timeelaps<strong>in</strong>g between the preparation <strong>of</strong> the <strong>in</strong>itial suspension and contact with the agardid not exceed 45 m<strong>in</strong>utes).3. IncubationInverted Petri dishes were transferred to an <strong>in</strong>cubator and <strong>in</strong>cubated at 30 +/-1 o Cfor 72 hours.4. Count<strong>in</strong>g <strong>of</strong> coloniesThe number <strong>of</strong> microorganisms per gram <strong>of</strong> sample was calculated from thenumber <strong>of</strong> colonies obta<strong>in</strong>ed on selected plates.
515 . CalculationNumber <strong>of</strong> micro-organisms = Σ c(n 1 + 0.1 x n 2 ) dΣ c = The sum <strong>of</strong> characteristic colonies counted after identificationn 1 = The number <strong>of</strong> dishes reta<strong>in</strong>ed <strong>in</strong> the 1st dilutionn 2 = The number <strong>of</strong> dishes reta<strong>in</strong>ed <strong>in</strong> the 2nd dilutiond = The dilution factor correspond<strong>in</strong>g to the 1st dilution3.3.2 Enumeration <strong>of</strong> EnterobacteriaceaeThe test was carried out based on the ISO 7402:1985 Standard Protocol forenumeration <strong>of</strong> 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 (<strong>in</strong>itial dilution):Preparation <strong>of</strong> <strong>in</strong>itial 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 <strong>of</strong> each required dilution was aseptically <strong>in</strong>oculated <strong>in</strong>to 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) <strong>in</strong> 15 ml were addedto each Petri dish. The <strong>in</strong>oculum was carefully mixed with the VRBGA and allowedto set. (The time elaps<strong>in</strong>g between the addition <strong>of</strong> MRD and contact with VRBGAdid not exceed 15 m<strong>in</strong>utes). When the agar was completely set, a further 10-15 ml <strong>of</strong>tempered (46 +/-1 o C) VRBGA was added onto the surface <strong>of</strong> the <strong>in</strong>oculated plate andallowed to solidify.4. IncubationInverted Petri dishes were transferred to an <strong>in</strong>cubator at 37+/-1 o C for 24 hours.5. Biochemical confirmationPlates conta<strong>in</strong><strong>in</strong>g typical colonies were selected. Typical colonies were p<strong>in</strong>kto red, with or without precipitation haloes or colourless mucoid colonies, with adiameter <strong>of</strong> 0.5 mm or more. Typical colonies were bio-chemically confirmed. Theprocedure entailed the random selection <strong>of</strong> five such colonies were streaked onNutrient Agar plates. These plates were <strong>in</strong>cubated at 37 o C for 24+/-2 h. Then anoxidase test was performed us<strong>in</strong>g a plat<strong>in</strong>um loop to streak a portion <strong>of</strong> each wellisolated colony on a filter paper moistened with oxidase reagent. If filter paper didnot change <strong>in</strong>to dark with<strong>in</strong> 10 seconds, the colony was considered as oxidasenegative.6. Count<strong>in</strong>g <strong>of</strong> coloniesColonies <strong>of</strong> the suitable two consecutive dilutions were counted <strong>in</strong> duplicateplates.7. CalculationThe calculations were performed us<strong>in</strong>g 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 follow<strong>in</strong>g manufacturesguidel<strong>in</strong>es. The 3M Petrifilm EC/Coliform Count Plate is a ready-made-culturemediumtechnique. It conta<strong>in</strong>s Violet Red Bile (VRB) nutrients, a cold-water-solublegell<strong>in</strong>g agent, an <strong>in</strong>dicator <strong>of</strong> glucuronidase activity, 5bromo-4-chloro-3<strong>in</strong>dolyl-β-Dglucuronide(BCIG), and a tetrazolium <strong>in</strong>dicator which facilitates colony count<strong>in</strong>g(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 <strong>in</strong> turn produces a blue precipitate associated with thecolony and about 95% <strong>of</strong> E. coli produces gas. Therefore, it is <strong>in</strong>dicated by blue tored-blue colonies associated with entrapped gas on the Petrifilm EC plate. Coliformsproduce acid and gas from lactose dur<strong>in</strong>g metabolic fermentation and these coloniesgrow<strong>in</strong>g on EC plates produce acid which causes the pH <strong>in</strong>dicator to make the gelcolour darker red. Gas trapped around red coliforms colonies <strong>in</strong>dicates confirmedcoliforms (Mattelet, 2005).Manufacturer guidel<strong>in</strong>es for storage:The unopened packages are stored at ≤8 o C (≤46 o F). In areas <strong>of</strong> high humidity, itis recommended to allow packages to reach room temperature before open<strong>in</strong>g to avoidcondensation <strong>in</strong> 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 <strong>of</strong> the opened package. Use petrifilm with<strong>in</strong> one month after open<strong>in</strong>g.3.3.3.1 Materials:1. Maximum Recovery Diluent (MRD)2. 3M-EC Plates
543. Spreader3.3.3.2 Procedures:1. InoculationAfter preparation <strong>of</strong> the <strong>in</strong>itial 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 <strong>of</strong> bottom media surface <strong>of</strong> a 3M Petrifilm TM plate which werelabelled properly and placed on a level surface.Figure 5: Pipett<strong>in</strong>g <strong>in</strong>oculum onto the 3M-EC plate(http://www.carol<strong>in</strong>a.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)Then the plate was gently closed with film.Figure 6: Clos<strong>in</strong>g with the film(http://www.carol<strong>in</strong>a.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)Next, gentle pressure was applied on the spreader <strong>in</strong> order to distribute the <strong>in</strong>oculumover a circular area without twist<strong>in</strong>g or slid<strong>in</strong>g the spreader.
55Figure 7: Spread<strong>in</strong>g <strong>of</strong> the <strong>in</strong>oculum us<strong>in</strong>g spreader(http://www.carol<strong>in</strong>a.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)Spreader was lifted. Gel was allowed to solidify at least 1 m<strong>in</strong>ute.Figure 8: Gel solidification(http://www.carol<strong>in</strong>a.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000)2. IncubationThen the plates were <strong>in</strong>cubated at 35 o C for 24 h. Plates were kept clear side up <strong>in</strong>stacks <strong>of</strong> no more than 20.3. InterpretationPetrifilm EC Plates conta<strong>in</strong> an <strong>in</strong>dicator dye <strong>in</strong> the medium that sta<strong>in</strong>s bacterialcolonies red. The red colour helps to dist<strong>in</strong>guish them from dust particles or otherenvironmental contam<strong>in</strong>ants. The EC Plate growth medium conta<strong>in</strong>s lactose, and thetop film <strong>of</strong> the plate’s traps gas produced by coliforms. F<strong>in</strong>ally, EC Plates conta<strong>in</strong> an<strong>in</strong>dicator <strong>of</strong> beta-glucuronidase activity, 5bromo-4-chloro-3<strong>in</strong>dolyl-β-D-glucuronide(BCIG). The enzyme beta-glucuronidase cleaves BCIG to produce a blue productthat precipates, caus<strong>in</strong>g a beta-glucuronidase-produc<strong>in</strong>g bacterial colony (E. coli) to
56appear blue. Non-coliform colonies grow<strong>in</strong>g 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.carol<strong>in</strong>a.com/manuals/manuals9/3M%20Petrifilm%20Ecoli%20Coli.pdf,2000). Triplicate trials were performed per dilution; us<strong>in</strong>g this method, plates with15–150 colonies are recommended for count<strong>in</strong>g.4. CalculationThe calculations were performed us<strong>in</strong>g 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 <strong>in</strong> 3.3.1.3 for enumeration <strong>of</strong>the Total Halophilic Plate Count.2. Inoculation1 ml <strong>of</strong> the each required dilution was aseptically <strong>in</strong>oculated <strong>in</strong>to a labelled petridish. For each dilution there were duplicate plates (ISO 4833:2003). Tempered(46+/-1 o C) PCA agar conta<strong>in</strong><strong>in</strong>g 3 % NaCl <strong>in</strong> 15 ml were added to each Petri dish.Carefully mixed by swirl<strong>in</strong>g six times clockwise, six times left to right, six timesanticlockwise and six times up and down. They were allowed to set. (The timeelaps<strong>in</strong>g between the preparation <strong>of</strong> the <strong>in</strong>itial suspension and contact with the agardid not exceed 45 m<strong>in</strong>utes).3. IncubationInverted Petri dishes were transferred to an <strong>in</strong>cubator and <strong>in</strong>cubated at 30 +/-1 o Cfor 72 hours.4. Biochemical confirmationThe random selection <strong>of</strong> five such colonies was streaked on Nutrient Agar plates.These plates were <strong>in</strong>cubated at 37 o C for 24+/-2 h. Then an oxidase test wasperformed us<strong>in</strong>g a plat<strong>in</strong>um loop to streak a portion <strong>of</strong> each well isolated colony on afilter paper moistened with oxidase reagent. If filter paper changes <strong>in</strong>to dark with<strong>in</strong>10 seconds, the colony was considered as oxidase positive.5. Count<strong>in</strong>g <strong>of</strong> coloniesColonies <strong>of</strong> the suitable two consecutive dilutions were counted <strong>in</strong> duplicateplates.6. CalculationThe calculations were performed us<strong>in</strong>g the same formula at 3.3.1.3 procedure.
583.3.5 Conventional culture method to detect SalmonellaThe study was conducted utiliz<strong>in</strong>g the conventional methods for the detection <strong>of</strong>Salmonella follow<strong>in</strong>g the ISO 6579 (2002) standard guidel<strong>in</strong>es; microbiology <strong>of</strong> foodand animal feed<strong>in</strong>g stuffs-horizontal method for the detection <strong>of</strong> Salmonella spp. Theprotocol generally has four dist<strong>in</strong>ct phases or steps:Step 1. Non-selective pre-enrichment: The sample <strong>of</strong> 25 grams <strong>of</strong> shrimp wasblended with 225ml <strong>of</strong> buffered peptone water <strong>in</strong> a stomacher bag and <strong>in</strong>cubated at37 o C±1 o C for 18± 2 hours to allow resuscitation <strong>of</strong> any stressed organism and growth<strong>of</strong> all organisms as well.Step 2. Selective enrichment: Allowed growth <strong>of</strong> the organism under <strong>in</strong>vestigation,while reduc<strong>in</strong>g the numbers <strong>of</strong> accompany<strong>in</strong>g organisms <strong>in</strong> the broth. Two types <strong>of</strong>selective enrichment media for Salmonella are recommended, Muller-KauffmannTetrathionate novobioc<strong>in</strong> broth (MKTTn broth) and Rappaport- Vassiliadis mediumwith soya (RVS broth). The pre-enrichment broth after <strong>in</strong>cubation was mixed and 0.1ml <strong>of</strong> the broth was transferred <strong>in</strong>to a tube conta<strong>in</strong><strong>in</strong>g 10 ml <strong>of</strong> RVS broth. Another 1ml <strong>of</strong> the pre-enrichment broth was transferred <strong>in</strong>to a tube conta<strong>in</strong><strong>in</strong>g 10 ml <strong>of</strong>MKTTn broth. The <strong>in</strong>oculated RVS broth was <strong>in</strong>cubated at 41.5 o C± 1 o C for 24h±3hours and the the <strong>in</strong>oculated MKTTn broth at 37 o C± 1 o C for 24h±3 hours.Step 3. Isolation: After <strong>in</strong>cubation for 24h±3 hours, a loop-full <strong>of</strong> material from theRVS broth and MKTTn was streaked on selective solid agars. There were 2 selectivesolid agars used <strong>in</strong> this study: Brilliant-green Phenol-red Lactose-Sucrose agar(BPLS agar) and Xylose-Lys<strong>in</strong>e-Deoxycholate agar (XLD agar) separately. The plateswere <strong>in</strong>cubated <strong>in</strong> an <strong>in</strong>verted position at 37 o C± 1 o C for 24h±3 hours. The properties<strong>of</strong> Salmonella colonies are described <strong>in</strong> Table 8.Step 4. Confirmation: Characteristic colonies on the plates were submitted forbiochemical test<strong>in</strong>g and seroagglut<strong>in</strong>ation test<strong>in</strong>g to confirm that the isolates weremembers <strong>of</strong> the species Salmonella.
59Table 8: Typical growth <strong>of</strong> Salmonella colonies on selective solid mediaBPLSXLDMediaColony appearanceReddish colour and translucent colonyBlack centered with a lightly transparentzone <strong>of</strong> reddish colour due to the colourchange <strong>of</strong> <strong>in</strong>dicator.Salmonella H 2 S negative variants, e.g. S.Paratyphi A, p<strong>in</strong>k with a darker p<strong>in</strong>k centre.Lactose-positive Salmonella, yellow with orwithout blacken<strong>in</strong>g.3.3.5.1 Selection <strong>of</strong> 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 <strong>in</strong>cubatedat 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 touch<strong>in</strong>g the top <strong>of</strong> the colony. Incubated at 37 o C+/-1 o C for 24 h +/-3 h, thechanges <strong>in</strong> medium were <strong>in</strong>terpreted as shown <strong>in</strong> Table 9.
60Sample 25 gPREENRICHMENT225 ml Buffered peptone waterat ambient temperatureIncubation for 18h± 2h at 37 o C±1 o C…………………………………………………………………………………SELECTIVEENRICHMENT0.1 ml <strong>of</strong> culture+10 ml <strong>of</strong> RVS brothIncubation for24h±3h at 41.5 o C± 1 o C1 ml <strong>of</strong> culture+10 ml <strong>of</strong> MKTTn brothIncubation for24h±3h at 37 o C± 1 o C…………………………………………………………………………………ISOLATIONLoop-full <strong>of</strong> 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, <strong>in</strong>cubation for24h±3h at 37 o C± 1 o CSerologicalconfirmationFigure 9: Flow chart <strong>of</strong> isolation <strong>of</strong> Salmonella conventional culture method (ISO6579, 2002)
61Table 9: Biochemical reactions <strong>in</strong> TSI agarArea <strong>of</strong> reaction ResultMean<strong>in</strong>gButt Yellow Glucose positive (glucose used)Red or unchangedBlackGlucose negative (glucose notused)Formation <strong>of</strong> 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 alkal<strong>in</strong>e (red) slant and acid (yellow) buttswith gas formation (bubble) and (<strong>in</strong> about 90 % <strong>of</strong> the c ases) formation <strong>of</strong> hydrogensulfide (blacken<strong>in</strong>g <strong>of</strong> 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 exam<strong>in</strong>ed at <strong>in</strong>tervals. If the reaction is positive, splitt<strong>in</strong>g <strong>of</strong> urealiberates ammonia, which changes the colour <strong>of</strong> phenol red to rose-p<strong>in</strong>k and later todeep cerise, a moderate red, the reaction is <strong>of</strong>ten apparent after 2 h to 4 h.3.3.5.2.3 L-lys<strong>in</strong>e decarboxylation mediumThe culture was <strong>in</strong>oculated just below the surface <strong>of</strong> the liquid medium and<strong>in</strong>cubated at 37+/-1 o C for 24 h +/- 3 hours. Turbidity and a purple colour after<strong>in</strong>cubation<strong>in</strong>dicate a positive reaction. A yellow colour <strong>in</strong>dicates a negative reaction.
623.3.5.2.4 Detection <strong>of</strong> β-galactosidaseA loopful <strong>of</strong> the suspected colony was suspended <strong>in</strong> a tube conta<strong>in</strong><strong>in</strong>g 0.25 ml <strong>of</strong>the sal<strong>in</strong>e solution. One drop <strong>of</strong> toluene was added and the tube shaken. The tubewas then placed <strong>in</strong> a water bath set at 37 o C and left for several m<strong>in</strong>utes(approximately 5 m<strong>in</strong>). 0.25 ml <strong>of</strong> the β-galactosidase reagent for detection <strong>of</strong> β -galactosidase was added and mixed. The tube was replaced <strong>in</strong> the water bath set at37 o C and left for 24 h+/-3 h, and exam<strong>in</strong>ed at <strong>in</strong>tervals. A yellow colour <strong>in</strong>dicates apositive reaction, the reaction is <strong>of</strong>ten apparent after 20 m<strong>in</strong>.3.3.5.2.4 Voges-Proskauer (VP) reactionA loopful <strong>of</strong> the suspected colony was suspended <strong>in</strong> a sterile tube conta<strong>in</strong><strong>in</strong>g 3ml <strong>of</strong> the VP medium. Then the tube was <strong>in</strong>cubated at 37+/-1 o C for 24+/-3 h. After<strong>in</strong>cubation, two drops <strong>of</strong> creat<strong>in</strong>e solution, three drops <strong>of</strong> ethanolic solution <strong>of</strong> 1-naphthol and then two drops <strong>of</strong> the potassium hydroxide solution were added. Afterthe addition <strong>of</strong> each reagent the tube was shaken. The formation <strong>of</strong> a p<strong>in</strong>k to brightred colour with<strong>in</strong> 15 m<strong>in</strong> <strong>in</strong>dicates a positive reaction.3.3.5.2.5 Indole reactionA tube conta<strong>in</strong><strong>in</strong>g 5 ml <strong>of</strong> the tryptone/ tryptophan medium was <strong>in</strong>oculated withthe suspected colony and then <strong>in</strong>cubated at 37+/-1 o C for 24+/-3 h. After <strong>in</strong>cubation,1ml <strong>of</strong> the Kovacs reagent was added. The formation <strong>of</strong> red r<strong>in</strong>g <strong>in</strong>dicates a positivereaction. A yellow-brown r<strong>in</strong>g <strong>in</strong>dicates a negative reaction.
63Table 10: Interpretation <strong>of</strong> biochemical tests results <strong>of</strong> Salmonella (Qu<strong>in</strong>n et al.,1994)TestReactionIndole production -Methyl Red +Voges-Proskauer -Citrate +Urease -Phenylalan<strong>in</strong>e deam<strong>in</strong>ase -Hydrogen sulphide +Lys<strong>in</strong>e decarboxylase +Ornith<strong>in</strong>e decarboxylase +Motility (36 o C) +Gelat<strong>in</strong> liquefaction -Growth <strong>in</strong> KCN broth-ONPG (beta-galactosidase)-Acid Fro mDulcitol +InositoldLactose -Maltose +Mannitol +Mannose +Rhamnose +Sorbitol +Sucrose -Xylose +Red pigment -Swarm<strong>in</strong>g (blood agar)-Mucoid colonies-d = 26-75% positive
643.3.5.3 Serotyp<strong>in</strong>gAll iso lates were serotyped by agglut<strong>in</strong>ation accord<strong>in</strong>g to the Kauffmann-Whitescheme us<strong>in</strong>g Salmonella Polyvalent I (A-E), Salmonella Polyvalent II (F-67) andSalmonella antiserum specific to the <strong>in</strong>dividual group after elim<strong>in</strong>at<strong>in</strong>g autoagglut<strong>in</strong>ablestra<strong>in</strong>s by mix<strong>in</strong>g a drop <strong>of</strong> NaCl and a part <strong>of</strong> the colony on a glassslide. Then the follow<strong>in</strong>g process stages took place (the procedure was based on themanufacturer Sif<strong>in</strong>, 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 <strong>of</strong> Salmonella group A-E.2. Test negative (-) result colonies from the first step were subjected toagglut<strong>in</strong>ation with Salmonella polyvalent II (F-67), if the result was positive(+), those colonies possessed the antigen <strong>of</strong> this group; colonies were regardedas a member <strong>of</strong> Salmonella Group F-67.3. Serotyp<strong>in</strong>g <strong>of</strong> Somatic (O) antigens <strong>in</strong> order to determ<strong>in</strong>e Salmonella ma<strong>in</strong>groups (A (O2), B (O4, 5, 27), C (O6, 7, 8, 20), D (O9, 27, 46, Vi), E (O3, 10,15, 19, 34)) by us<strong>in</strong>g a sequence <strong>of</strong> somatic antigen sera.4.Determ<strong>in</strong>ation <strong>of</strong> the capsular antigen:Pathogenic stra<strong>in</strong>s <strong>of</strong> Salmonella Typhi carry an additional antigen, "Vi",which is associated with a bacterial capsule and determ<strong>in</strong>ed us<strong>in</strong>g the reagentanti-Salmonella Vi.5.Determ<strong>in</strong>ation <strong>of</strong> flagella antigens:Hav<strong>in</strong>g found the "O" antigen group, the"H" antigen was determ<strong>in</strong>ed. The"H" antigens are prote<strong>in</strong>s associated with the bacterial flagella. Salmonellasexist <strong>in</strong> 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 <strong>in</strong>duction:After transferr<strong>in</strong>g <strong>of</strong> the isolate to the motility agar agglut<strong>in</strong>ation for flagellaantigen phase 1 and phase 2 were performed. One <strong>of</strong> the phases <strong>of</strong> theantigens was blocked by the particular H antiserum to force the stra<strong>in</strong> todevelop the other phase. Then agglut<strong>in</strong>ation was performed with the phasecome up from the hidden condition.Salmonella Polyvalent I (A-E) 1 dropPositiveTest with * Negative ControlNegativeDeterm<strong>in</strong>ation <strong>of</strong> Somatic(O) antigens(for groupA, B,C, D, E)SalmonellaPolyvalent II (F-67)Determ<strong>in</strong>ation <strong>of</strong> flagellaantigens (Phase 1, Phase 2)PositiveNegativeDef<strong>in</strong>e <strong>in</strong>to Salmonellagroup F-67(No more further step)Non-Salmonella* The negative control used was NaCl solutionFigure 10: Salmonella serotyp<strong>in</strong>gflow chart
663.3.6 Antimicrobial sensitivity test for SalmonellaThe procedure for perform<strong>in</strong>g the Disk Diffusion Test was carried out accord<strong>in</strong>gto the National Committee <strong>of</strong> Cl<strong>in</strong>ical Laboratory Standards (NCCLS, 2000), the<strong>in</strong>stitute is recognised as Cl<strong>in</strong>ical 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 <strong>of</strong> the same morphological type wereselected from an agar plate culture. The top <strong>of</strong> each colony was touched with a loop,and the growth was transferred <strong>in</strong>to a tube conta<strong>in</strong><strong>in</strong>g 4 to 5 ml <strong>of</strong> a suitable brothmedium, such as tryptic soy broth.2. The broth culture was <strong>in</strong>cubated at 35 o C until it achieves the turbidity <strong>of</strong> the 0.5McFarland standards (usually 2 to 6 hours).3. The turbidity <strong>of</strong> the actively grow<strong>in</strong>g broth culture was adjusted with sterile sal<strong>in</strong>eor broth to obta<strong>in</strong> turbidity optically comparable to that <strong>of</strong> the 0.5 McFarlandstandards. A BaSO 4 0.5 McFarland standard was prepared by add<strong>in</strong>g a 0.5 mlaliquot <strong>of</strong> 0.048 mol/L BaCl 2 (1.175% w/v BaCl 2 .H 2 O) to 99.5 ml <strong>of</strong> 0.18 mol/LH 2 SO 4 (1% v/v) with constant stirr<strong>in</strong>g to ma<strong>in</strong>ta<strong>in</strong> a suspension. Then it wasdispensed <strong>in</strong>to 4-6 ml screw capped vials and stored <strong>in</strong> the dark at room temperature.The turbidity standard always agitated before use.
673.3.6.2 Inoculation <strong>of</strong> test plates1. Optimally, with<strong>in</strong> 15 m<strong>in</strong>utes after adjust<strong>in</strong>g the turbidity <strong>of</strong> the <strong>in</strong>oculumsuspension, a sterile cotton swab was dipped <strong>in</strong>to the adjusted suspension. The swabwas rotated several times and pressed firmly on the <strong>in</strong>side wall <strong>of</strong> the tube above thefluid level. This removes excess <strong>in</strong>oculum from the swab.2. The dried surface <strong>of</strong> a Mueller-H<strong>in</strong>ton agar plate was <strong>in</strong>oculated by streak<strong>in</strong>g theswab over the entire sterile agar surface. This procedure was repeated by streak<strong>in</strong>gtwo more times, rotat<strong>in</strong>g the plate approximately 60 o each time to ensure an evendistribution <strong>of</strong> the <strong>in</strong>oculum. As a f<strong>in</strong>al step, the rim <strong>of</strong> the agar was swabbed.3. The lid was left from the agar plate for 3 to 5 m<strong>in</strong>utes, but not more than 15m<strong>in</strong>utes, to allow for any excess surface moisture to be absorbed before apply<strong>in</strong>g thedrug impregnated disks.3.3.6.3 Application <strong>of</strong> disks to <strong>in</strong>oculated agar plates1. Antimicrobial disks were dispensed onto the surface <strong>of</strong> the <strong>in</strong>oculated Mueller-H<strong>in</strong>ton agar plates. For each Salmonella isolate used 3 Petri dishes <strong>of</strong> 100 mm <strong>in</strong>diameter to dispense 14 different k<strong>in</strong>ds <strong>of</strong> antibiotics such as nalidixic acid 30 µg,amoxycill<strong>in</strong> 10 µg, ampicill<strong>in</strong> 10 µg, gentamyc<strong>in</strong> 10 µg, erythromyc<strong>in</strong> 15 µg,chloramphenicol 30 µg, kanamyc<strong>in</strong> 30 µg, cipr<strong>of</strong>loxac<strong>in</strong> 5 µg, trimethoprim 5 µg,tetracycl<strong>in</strong>e 30 µg, streptomyc<strong>in</strong> 10 µg, sulphamethoxazole/trimethoprim 25 µg,compound sulphonamides 300 µg, and doxycycl<strong>in</strong>e hydrochloride 30 µg.2. The plates were <strong>in</strong>verted and placed <strong>in</strong> an <strong>in</strong>cubator set to 35 o C with<strong>in</strong> 15 m<strong>in</strong>utes<strong>of</strong> apply<strong>in</strong>g the disks.
683.3.6.4 Read<strong>in</strong>g plates and <strong>in</strong>terpret<strong>in</strong>g results1. After 16 to 18 hours <strong>of</strong> <strong>in</strong>cubation, each plate was exam<strong>in</strong>ed. The result<strong>in</strong>g zones<strong>of</strong> <strong>in</strong>hibition were uniformly circular and there was a confluent lawn <strong>of</strong> growth. Thediameters <strong>of</strong> the zones <strong>of</strong> complete <strong>in</strong>hibition were measured, <strong>in</strong>clud<strong>in</strong>g the diameter<strong>of</strong> the disk. Zones were measured to the nearest whole millimeter, us<strong>in</strong>g a ruler,which was held on the back <strong>of</strong> the <strong>in</strong>verted Petri dish. The Petri dish was held a few<strong>in</strong>ches above a black, non reflect<strong>in</strong>g background and illum<strong>in</strong>ated with reflected light.2. The sizes <strong>of</strong> the zones <strong>of</strong> <strong>in</strong>hibition were <strong>in</strong>terpreted by referr<strong>in</strong>g to the table andthe organism was reported as susceptible, <strong>in</strong>termediate, or resistant to the agents thathad been tested.3.4 Questionnaire surveyA questionnaire was used to evaluate rout<strong>in</strong>e practices at the local sale po<strong>in</strong>ts.The factors observed are listed <strong>in</strong> Table 11.Figure 11: Practice <strong>of</strong> us<strong>in</strong>g iceFigure 12: Handl<strong>in</strong>g <strong>of</strong> <strong>shrimps</strong>while on sale
69Table 11: List <strong>of</strong> 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 YesNo78GradeHandl<strong>in</strong>gNumber1Number 2Number 3Open handWith gloves9 How <strong>of</strong>tenWash handsEvery timeSome times1011Conta<strong>in</strong>erClean<strong>in</strong>g/Conta<strong>in</strong>er1. Wood2. Tile3. Rigiform4. Cool box5. Freezer6. Other7. Plastic boxEnd <strong>of</strong> the dayNot every end <strong>of</strong> the day12 Sales/day1-30 Kg>30-60 Kg>60-90 Kg13 Rema<strong>in</strong>s/dayYesNoRarely
703.5 Data management and statistical analysisLaboratory and questionnaire dat a were entered and stored <strong>in</strong> the databasemanagement s<strong>of</strong>tware MS Excel 2003.Descriptive statistics:Chart, percentage and median were used to describe prevalence, antimicrobialresistance and <strong>microbiological</strong> contam<strong>in</strong>ation <strong>in</strong> terms <strong>of</strong> 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 f<strong>in</strong>d significantdifference <strong>of</strong> factors relat<strong>in</strong>g 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 relat<strong>in</strong>g to quantity <strong>of</strong> <strong>microbiological</strong>contam<strong>in</strong>ation.Tables <strong>of</strong> new percentage Confidence Intervals (CI), b<strong>in</strong>omial distribution(Bunke, 1959) was used to get small 95 % CI for prevalences. S<strong>in</strong>ce the b<strong>in</strong>omialdistributions 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 <strong>in</strong>terpreted at the 5 % level <strong>of</strong> significance.
4. RESULTS4.1 Source <strong>of</strong> the sampleThe total number <strong>of</strong> samples analyzed <strong>in</strong> this study to determ<strong>in</strong>e prevalence,serotypes and antimicrobial resistance <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong> (Penaeus<strong>monodon</strong>) <strong>in</strong> local markets <strong>of</strong> North Western Prov<strong>in</strong>ce <strong>in</strong> 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 divided<strong>in</strong> each district. In the Puttalam District there were 9 different locations where <strong>shrimps</strong>were sold at retail sale and there were 5 different locations <strong>in</strong> the Kurunegala District.Table 12: Number <strong>of</strong> samples <strong>of</strong> <strong>shrimps</strong> for Salmonella analysisDistrictTypeNumber <strong>of</strong> samplesPuttalam (9 locations) Capture from lagoon 60Culture fr om farm60Kurunegala (5 locations) Capture from lagoon30Cul ture from farm30Total 1804.2 Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong>4.2.1 Overall prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong>The prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong> (brackishwatercultured and captured) purchased from retail sale po<strong>in</strong>ts were analysed and the
72results are shown <strong>in</strong> Table 13. The overall prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong>Penaeus <strong>monodon</strong> <strong>shrimps</strong> <strong>in</strong> North Western Prov<strong>in</strong>ce was 12.78 % (it lays with<strong>in</strong> the95 % CI, 8.44 % to 18.76 %) and overall prevalence <strong>in</strong> <strong>shrimps</strong> 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 <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> by the type is notstatistically significant (p=0.66). An odds ratio (OR) <strong>of</strong> 1.35 (95 % CI, 0.56, 3.26)would implies that the 1.35 fold greater odds <strong>of</strong> Salmonella for capture <strong>shrimps</strong> thanfor culture <strong>shrimps</strong>.The prevalence <strong>of</strong> Salmonella <strong>in</strong> the Puttalam District 13.33 % (95 % CI, 8.05,21.04) and <strong>in</strong> the Kurunegala District it was 11.67 % (95 % CI, 5.21, 23.18) as shown<strong>in</strong> Table 13. An odds ratio <strong>of</strong> 1.16 (95 % CI, 0.45, 3.01) implies that the 1.16 foldgreater odds <strong>of</strong> Salmonella for the Puttalam district than for the Kurunegala district.Table 13: Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong>Description Type <strong>of</strong> <strong>shrimps</strong> No. <strong>of</strong>samplesNo. <strong>of</strong> 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 Prov<strong>in</strong>ce, * Confidence Interval
73The preval ences <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> from capture and from culture were15.00 % (7.50, 27.08) and 11.67 % (5.21, 23.18) <strong>in</strong> the Puttalam district. An odds ratio<strong>of</strong> 1.34 (95 % CI, 0.46, 3.86) revealed that the occurrence <strong>of</strong> Salmonella <strong>in</strong> capture<strong>shrimps</strong> is more likely than culture <strong>shrimps</strong>. When compared the same <strong>in</strong> theKurunegala district an odds ratio <strong>of</strong> 1.16 (95 % CI, 0.45, 3.01) implies that the 1.38fold greater odds <strong>of</strong> Salmonella for capture <strong>shrimps</strong> than culture <strong>shrimps</strong>.4.2.2 Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> sold at 14 different retail sale locationsIn this study the samples were purchased from 14 different retail sell<strong>in</strong>g locations(9 <strong>in</strong> the Puttalam District and 5 <strong>in</strong> the Kurunegala District). The prevalence <strong>of</strong>Salmonella <strong>in</strong> <strong>shrimps</strong>sold at different retail sale locations varied and it ranged from0 % to 60 % (as shown <strong>in</strong> Figure 13). The percentage <strong>of</strong> sale location specificSalmonella contam<strong>in</strong>ation was significantly different (p
74Table 14: Prevalence <strong>of</strong> Salmonella <strong>in</strong> <strong>shrimps</strong> sold at the different retail salelocationsRetail saleLocationNo. <strong>of</strong>samplesexam<strong>in</strong>edNo. <strong>of</strong>samplespositivePrevalence 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 <strong>in</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong>The total number <strong>of</strong> serovars isolated <strong>in</strong> this study was 23 from 180 samples. Themajor serogroups <strong>of</strong> the isolated serovars were serogroup C (56.52 %), E (26.08 %)and B (8.69 %). The serogroups A and D were not found <strong>in</strong> this study. Thepercentage <strong>of</strong> stra<strong>in</strong>s belong<strong>in</strong>g to Salmonella F-67 was 8.69 % as shown <strong>in</strong> Figure14.
75E26.08 %C56.52 %B8.69 %F-678.69 %Figure 14: Percentage prevalence <strong>of</strong> major Salmonella groupsThe retail shrimp sell<strong>in</strong>g locations 3, 5, 10 and 14 were positive for Salmonella<strong>in</strong> capture type <strong>of</strong> <strong>shrimps</strong>, locations 8 and 12 were positive for Salomonella <strong>in</strong> culturetype <strong>of</strong> <strong>shrimps</strong> and only the location 7 was positive for both capture and culture type<strong>of</strong> <strong>shrimps</strong>. From capture <strong>shrimps</strong> 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 <strong>of</strong> Salmonella serogroups isolated from the<strong>shrimps</strong> 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 <strong>of</strong> this study 10 orig<strong>in</strong>ated from culture and 13 fromcapture (Table 16). Out <strong>of</strong> 13 culture stra<strong>in</strong>s there were 7 serotypes which belong toS. Newport, and each serotype <strong>of</strong> follow<strong>in</strong>g were belong to S. Mbandaka, F-67 and C7(not typable). In capture stra<strong>in</strong>s there were 4 serotypes which belong to S. Newport,amongthe serotypes that not typable were <strong>in</strong>cluded 3 serotypes from serogroup E
77(E10 and E15) and 2 serotypes (B4) from serogroup B. F-67 was found only one <strong>in</strong>capture <strong>shrimps</strong>.Table 16: Sero vars <strong>of</strong> Salmonella isolated from <strong>shrimps</strong>No <strong>of</strong> isolateType <strong>of</strong> 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 <strong>of</strong> different serotypes is described <strong>in</strong> 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 <strong>of</strong> Salmonella serovars isolated from <strong>shrimps</strong>4.4 Antimicrobial resistance <strong>of</strong> Salmo nella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong>The number <strong>of</strong> Salmonella isolates tested for antimicrobial resistance <strong>in</strong> thisstudy was 23. There were 14 different k<strong>in</strong>ds <strong>of</strong> antibiotics nalidixic acid 30 µg,amoxycill<strong>in</strong> 10 µg, ampicill<strong>in</strong> 10 µg, gentamyc<strong>in</strong> 10 µg, erythromyc<strong>in</strong> 15 µg,sulphamethoxazole/trimethoprim 25 µg, chloramphenicol 30 µg, kanamyc<strong>in</strong> 30 µg,compound sulphonamides 300 µg, cipr<strong>of</strong>loxac<strong>in</strong> 5 µg, trimethoprim 5 µg, tetracycl<strong>in</strong>e30 µg, streptomyc<strong>in</strong> 10 µg and doxycycl<strong>in</strong>e hydrochloride 30 µg used for theAntibiotic Disc Diffusion Test.
794.4.1 Antimicrobial resistance <strong>of</strong> SalmonellaThe percentage resistance to any antimicrobial was 95.65 % (95 % CI, 80.2,99.5). The percentage <strong>of</strong> antimicrobial resistance <strong>of</strong> Salmonella isolated from culture<strong>shrimps</strong> was 100 % (73.2, 100.0) and capture <strong>shrimps</strong> 92.31 % (67.5, 99.2). However,the difference <strong>of</strong> the percentage <strong>of</strong> antimicrobial resistance among culture and capture<strong>shrimps</strong> was not statistically significant (p>0.05) (Table 17).Table 17: Percentage <strong>of</strong> antimicrobial resistance SalmonellaType <strong>of</strong>shrimpNo. <strong>of</strong> 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 <strong>of</strong> isolates resistant, <strong>in</strong>termediate andsusceptible to 14 different k<strong>in</strong>ds <strong>of</strong> antibiotics. 100 % <strong>of</strong> all the 23 isolates <strong>of</strong>Salmonella were observed susceptible for nalidixic acid, ampicill<strong>in</strong>, gentamyc<strong>in</strong>,sulphamethoxazole/ trimethoprim, chloramphenicol, cipr<strong>of</strong>loxac<strong>in</strong>, trimethoprim andtetracycl<strong>in</strong>e. The next highest susceptibility was shown for the antimicrobialamoxycill<strong>in</strong> and it was 95.65 %. 21 Salmonella stra<strong>in</strong>s were susceptible and 2 were<strong>in</strong>termediate for doxycycl<strong>in</strong>e hydrochloride. 82.61 %. 19 Salmonella stra<strong>in</strong>s weresusceptible and 4 isolates were <strong>in</strong>termediate for kanamyc<strong>in</strong>. Most <strong>of</strong> the isolates (16)were <strong>in</strong>termediate for streptomyc<strong>in</strong> and 7 were susceptible for it. It is important tonote that out <strong>of</strong> all the 14 k<strong>in</strong>ds <strong>of</strong> antimicrobials tested, resistance was found only forthree k<strong>in</strong>ds <strong>of</strong> antimicrobials erythromyc<strong>in</strong>, amoxycill<strong>in</strong> and sulphonamidesrespectively.
Table 18: Number and percentage <strong>of</strong> Salmonell a stra<strong>in</strong>s resist ant, <strong>in</strong>termediate and susceptible to antimicrobials tested (n= 30)Type <strong>of</strong> antibioticResistantIntermediateSusceptibleNo. % 95 % CI* No. % 95 % CI* No. % 95 % CI*Gentamyc<strong>in</strong> 0 0 (0.00, 12.8) 0 0 (0.00, 12. 8) 23 100.00 (87.2, 100. 00)Kanamyc<strong>in</strong> 0 0 (0.00, 12.8) 4 17.39 (7.2, 36.8) 19 82.61 (63.2, 92.8)Streptomyc<strong>in</strong> 0 0 (0.00, 12.8) 16 69.57 (48.9, 85.6) 7 30.43 (14.4, 51.1)Erythromyc<strong>in</strong> 22 95.65 (80.2, 99.5) 1 4.35 (0.5, 19.7) 0 0 (0.00, 12.8)Amoxycill<strong>in</strong> 1 4.35 (0.5, 19.7) 0 0 (0.00, 12.8) 22 95.65 (80.2, 99.5)Ampicill<strong>in</strong> 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)Cipr<strong>of</strong>loxac<strong>in</strong> 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)Doxycycl<strong>in</strong>e HCl 0 0 (0.00, 12.8) 2 8.70 (2.2, 25.9) 21 91.30 (74.1, 97.8)Tetracycl<strong>in</strong>e 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 <strong>of</strong> Salmonella stra<strong>in</strong>s resistant to differentk<strong>in</strong>ds <strong>of</strong> antimicrobials. 4.35 % <strong>of</strong> Salmonellastra<strong>in</strong>s were not resistant to any k<strong>in</strong>d <strong>of</strong>antimicrobial. No Salmonella isolate has been resistant to only one antibiotic.However, there were 91.3 % <strong>of</strong> Salmonella isolates resistant for 2 k<strong>in</strong>ds <strong>of</strong>antimicro bials and 4.35% resistant for 3 k<strong>in</strong>ds <strong>of</strong> antimicrobials.% Salmonella resistance100908070605040302010091.34.35 0 4.35sulphonamidesulphonamideerythromyc<strong>in</strong>erythromyc<strong>in</strong>amoxycill<strong>in</strong>Not resistant 1 Resistant 2 Resistant 3 ResistantNo. <strong>of</strong>antimicrobialsFigure 16:Percentage <strong>of</strong> Salmonella isolates resistant to antimicrobialsThe antimicrobial resistance pattern by the type <strong>of</strong> shrimp is shown <strong>in</strong> Table 19.Out <strong>of</strong> 12 capture isolatesall 12 showed the sameresistant pattern that comb<strong>in</strong>ationwas erythromyc<strong>in</strong> and sulphonamide.Salmonella isolates from culture <strong>shrimps</strong>show ed 2 resista nt patternsand out<strong>of</strong> 10 isolatestested there were 9 isolates hadcomb<strong>in</strong>ation <strong>of</strong> erythromyc<strong>in</strong> and sulphonamides and 1 isolate had comb<strong>in</strong>ation <strong>of</strong>erythromyc<strong>in</strong>, sulphonam ide and amoxycil l<strong>in</strong>.
82Table 19: Antimicrobial resistance pattern by type <strong>of</strong> shrimpType No. <strong>of</strong> Patterns Antimicrobials No. <strong>of</strong> isolates Percentage(95 % CI*)Capture 1 E, S3 12(n=12)100.00(76.6, 100.00)Culture 2(n=10)* Confidence IntervalE=Erythromyc<strong>in</strong>S3=SulphonamideAML=Amoxycill<strong>in</strong>E, S3 9 90 (59.6, 99.0)E, S3, AML 1 10 (1.0, 40.4)The antimicrobial resistant pattern by serotype is shown <strong>in</strong> Table 20. The most <strong>of</strong>the serotypes have the same resistance pattern for the comb<strong>in</strong>ation <strong>of</strong> erythromyc<strong>in</strong>and sulphonamide except one <strong>of</strong> the S. Newport which has the resistance pattern <strong>of</strong>erythromyc<strong>in</strong>, sulphonamide and amoxycill<strong>in</strong>.Table 20: Antimicrobial resistance pattern by serotypeSerotype Resistance pattern No. <strong>of</strong> 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 <strong>of</strong> resistant serotypes. Out <strong>of</strong> 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 <strong>of</strong> resistant serotypes (n=22)Serotype No. <strong>of</strong> 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 <strong>microbiological</strong> analysis <strong>of</strong> shrim ps purchased from local sale po<strong>in</strong>tsMicrobiological contam<strong>in</strong>ation <strong>of</strong> <strong>shrimps</strong> was analysed only for limitednumber <strong>of</strong> samples for Aerobic Pl ate Count 27, Enterobacteriaceae 34, E . coli 56 andTotal Halophilic Plate Count 20 respectively.4.5.1 Bacteriological pr<strong>of</strong>ileThe overall bacteriological pr<strong>of</strong>ile <strong>of</strong> <strong>shrimps</strong> purchased (Table 22) describes thelevel <strong>of</strong> microbial contam<strong>in</strong>ation <strong>in</strong> terms <strong>of</strong> log Colony Form<strong>in</strong>g 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 m<strong>in</strong>imum 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 pr<strong>of</strong>ile <strong>of</strong> <strong>shrimps</strong> tested <strong>in</strong> terms <strong>of</strong> log CFU/gParameter No. <strong>of</strong> samples M<strong>in</strong>imum Median Maximumexam<strong>in</strong>edAPC 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 <strong>quality</strong> evaluation <strong>of</strong> <strong>shrimps</strong>In case <strong>of</strong> <strong>quality</strong> determ<strong>in</strong>ation <strong>of</strong> <strong>shrimps</strong> sold at local sale locations, the tested<strong>microbiological</strong> parameters <strong>of</strong> <strong>shrimps</strong> were compared with the available standards <strong>of</strong>raw 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 <strong>microbiological</strong> contam<strong>in</strong>ation <strong>of</strong> the raw <strong>shrimps</strong> represented by thebacteriological parameters was high and nearly half <strong>of</strong> 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 <strong>of</strong> Enterobacteriaceae. Out <strong>of</strong> 50samples 28 samples met ≤ 10 2 Escherichia coli counts. Total Halophilic Plate Count(THPC) was not compared s<strong>in</strong>ce there is no existed standard.
85Table 23: Bacteriological <strong>quality</strong> <strong>of</strong> <strong>marketed</strong> <strong>shrimps</strong>Parameter No. <strong>of</strong> samples No. <strong>of</strong> samples Percentage 95 % CI**Exam<strong>in</strong>ed 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 <strong>of</strong> the questionnaireThe questionnaire survey was conducted at 90 different sale po<strong>in</strong>ts <strong>in</strong> order toevaluate general contam<strong>in</strong>ation through the rout<strong>in</strong>e practices <strong>of</strong> shrimp handl<strong>in</strong>g atlocal level. Univariate analysis was carried out and compared proportions us<strong>in</strong>g thechi-square test. Numerical data were analysed us<strong>in</strong>g non-parametric tests such asKruskal-Wallis test and Wilcoxan test.There are different factors contribute for the level <strong>of</strong> <strong>microbiological</strong>contam<strong>in</strong>ation. Some factors are favourable and some factors are non favourable andat the same time more than one factor affect for the <strong>quality</strong> deterioration. The presentstudy revealed represent <strong>microbiological</strong> contam<strong>in</strong>ation is the sum <strong>of</strong> the favourableand non favourable factors for the studied micro-organisms. Therefore, local salelocation as a last check po<strong>in</strong>t where the <strong>microbiological</strong> contam<strong>in</strong>ation can bemeasured, represent the <strong>microbiological</strong> <strong>quality</strong> <strong>of</strong> the shrimp as last check po<strong>in</strong>t <strong>of</strong>the commercial operation.
864.6.1 Potential factors and the levels for Salmonella contam<strong>in</strong>ationThe percentages <strong>of</strong> Salmonella <strong>in</strong> raw <strong>shrimps</strong> and the potential factors areshown <strong>in</strong> Table 24. It shows that th ere are several major potential factors to the level(p0.05). However, higher percentagepositives were observed <strong>in</strong> the Puttalam district, capture <strong>shrimps</strong>, receiv<strong>in</strong>g via middleman, vehicle type <strong>of</strong> motor bicycle, duration <strong>of</strong> transport 2-3 hours, condition <strong>of</strong>transport with ice, handl<strong>in</strong>g with gloves, wash hands-some times, tile table use asconta<strong>in</strong>erwhile on sale and sales per day 1-30 Kg.
87Table 24: Summary results <strong>of</strong> potential factors and the levels for Salmonellacontam<strong>in</strong>ation <strong>in</strong> <strong>shrimps</strong> (univariate analysis) (n=180)Factors Level No. <strong>of</strong> 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 34/3417/1180/21/1419/1343/3215/728/108-6/10017/80Handl<strong>in</strong>g Open handWith gloves22/1761/4How <strong>of</strong>ten Every time 0/14Wash hands Some times 23/166Conta<strong>in</strong>er Wood1/6Tile4/14Rigiform9/80Cool box0/12Freezer0/6Other9/48Plastic box 0/14Clean<strong>in</strong>g/Conta<strong>in</strong>erSales/dayEnd <strong>of</strong> the dayNot every end <strong>of</strong>the 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 0.00-20.0010.4762.513.3912.5010.0023.337.2916.67Rema<strong>in</strong>s/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 <strong>of</strong> the vendor, mix with other fish species dur<strong>in</strong>gtransport, number 3 grade <strong>of</strong> the shrimp, conta<strong>in</strong>er clean<strong>in</strong>g not every end <strong>of</strong> the daywere the risk factors associated with Salmonella contam<strong>in</strong>ation at local sale locations.Medium socio-economic status <strong>of</strong> the seller and <strong>shrimps</strong> rema<strong>in</strong> at the end <strong>of</strong> the daywere trends to be the risk factors.Table 25: Logistic regression <strong>of</strong> the risk factors associated with Salmonellacontam<strong>in</strong>ationFactors 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 – 11.308Clean<strong>in</strong>g/ End <strong>of</strong> the day-10Conta<strong>in</strong>er N ot every end <strong>of</strong> the day 0.00114.2503.143 – 64.698Rema<strong>in</strong>s/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 <strong>in</strong> <strong>shrimps</strong>The microbial load (APC) <strong>in</strong> the raw shrimp s and the most likely potentialfactors are shown <strong>in</strong> Table 26. S<strong>in</strong>ce the data are not normally distributed to measurethe central tendency use the median. Statistically significant potential factors to thelevel (p
89Table 26: Summary results <strong>of</strong> potential factors and the levels for APC (log CFU/g)<strong>in</strong> <strong>shrimps</strong> (univariate analysis). (n=27)Factors Level No. <strong>of</strong> 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 1518.5214.8125.9311.1148.1525.9311.1162.96-14.8151.8533.3362.9637.04-44.4455.55Handl<strong>in</strong>g Open handWith gloves25292.597.416.6510.21How <strong>of</strong>ten Every time - - -wash hands Some times 27 100.00 7.18Conta<strong>in</strong>er Wood27.41 6.64Tile---Rigiform 1555.55 6.45Cool box ---Freezer414.81 9.99Other622.22 7.25Plastic box ---Clean<strong>in</strong>g/Conta<strong>in</strong>erSales/dayRema<strong>in</strong>s/dayEnd <strong>of</strong> the dayNot every end <strong>of</strong>the 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 contam<strong>in</strong>ation <strong>in</strong> <strong>shrimps</strong>The level <strong>of</strong> Enterobacteriaceae contam<strong>in</strong>ation <strong>in</strong> raw <strong>shrimps</strong> sold at local salepo<strong>in</strong>ts is <strong>in</strong>dicative <strong>of</strong> the handl<strong>in</strong>g practices which are statistically significant (Table27). The socio-economic status <strong>of</strong> the vendor, the type <strong>of</strong> transport vehicle, theduration <strong>of</strong> transport, the conditions <strong>of</strong> transport, the type <strong>of</strong> shrimp conta<strong>in</strong>er usedand rema<strong>in</strong> <strong>of</strong> the shrimp at the end <strong>of</strong> the day show potential for Enterobacteriaceaeco ntam<strong>in</strong>ation to the le vel (p
91Table 27: Summary results <strong>of</strong> potential factors and the levels for Enterobacteriaceae(log CFU/g) <strong>in</strong> <strong>shrimps</strong> (univariate analysis)(n=34)Factors Level No.<strong>of</strong> samplestestedPlaceShrimp typeStatus/sellerReceiveTransport/VehicleTransport/DurationTransport/ConditionTransport/Mix with fishGradeHandl<strong>in</strong>gHow <strong>of</strong>tenwash handsConta<strong>in</strong>erClean<strong>in</strong>g/Conta<strong>in</strong>erSales/dayRema<strong>in</strong>s/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 <strong>of</strong> the dayNot every end <strong>of</strong>the 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 contam<strong>in</strong>ation <strong>in</strong> <strong>shrimps</strong>The occurrence <strong>of</strong> E. coli <strong>in</strong> raw <strong>shrimps</strong> sold at different sale locations whichshow a potential connec tion to the factors <strong>of</strong> general handl<strong>in</strong>g are shown <strong>in</strong> Tale 28.Results <strong>of</strong> the univariate analysis describe so me <strong>of</strong> the factors are statisticallysignificant (P
93Table 28: Summary results <strong>of</strong> potential factors and the levels for Escherichia coli(log CFU/g) <strong>in</strong> <strong>shrimps</strong> (univariate analysis) (n=56)Factors Level No.<strong>of</strong> samplestestedPlaceShrimp typeStatus/sellerReceiveTransport/VehicleTransport/DurationTransport/ConditionTransport/Mix with fishGradeHandl<strong>in</strong>gHow <strong>of</strong>tenwash handsConta<strong>in</strong>erClean<strong>in</strong>g/Conta<strong>in</strong>erSales/dayRema<strong>in</strong>s/dayPuttalamKurunegalaCultureCaptureHighMediumLowMiddle manFarmerFreezer truckMotor bicycleBicycleTruck< 1 Hour1-2 Hours>2-3 Hours> 3 HoursFreezer truckWith iceWithout iceYesNoNumber1Number 2Number 3Open handWith glovesEvery timeSome timesWoodTileRigiformCool boxFreezerOtherPlastic boxEnd <strong>of</strong> the dayNot every end <strong>of</strong>the 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 contam<strong>in</strong>ation <strong>in</strong> <strong>shrimps</strong>Raw <strong>shrimps</strong> exam<strong>in</strong>ed for total halophilic bacteria and <strong>in</strong>dicative <strong>of</strong> generalhandl<strong>in</strong>g practices which show significant associati ons are listed <strong>in</strong> Table 29. Theassociated factors to the level (p
95Table 29: Summary results <strong>of</strong> potential factors and the levels for Total HalophilicPlate Count (log CFU/g) <strong>in</strong> <strong>shrimps</strong> (univariate analysis) (n=20)Factors Level No. <strong>of</strong> samplesPositivePlacePuttalam13Kurunegala 7Shrimp type Culture7Capture13Status/seller High2Medium8Low10Receive Middle man 14Farmer6Transport/ Freezer truck 0Vehicle Motor bicycleBicycleTruck5411Transport/DurationTransport/ConditionTransport/Mix with fishGradeHandl<strong>in</strong>gHow <strong>of</strong>tenwash handsConta<strong>in</strong>erClean<strong>in</strong>g/Conta<strong>in</strong>erSales/dayRema<strong>in</strong>s/day< 1 Hour1-2 Hours>2-3 Hours> 3 HoursFreezer truckWith iceWithout iceYesNoNumber1Number 2Number 3Open handWith glovesEvery timeSome timesWoodTileRigiformCool boxFreezerOtherPlastic boxEnd <strong>of</strong> the dayNot every end <strong>of</strong>the 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 <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong><strong>shrimps</strong> (Penaeus <strong>monodon</strong>)5.1.1.1 Prevalence and serotypes <strong>of</strong> Salmonella <strong>in</strong> <strong>marketed</strong> <strong>shrimps</strong>Seafood <strong>quality</strong> is one <strong>of</strong> the major issues <strong>in</strong> the seafood <strong>in</strong>dustry and freshlyharvested seafood-whether caught <strong>in</strong> the wild or aqua-cultured-has the potential todeteriorate <strong>in</strong> <strong>quality</strong> due to many factors <strong>in</strong>clud<strong>in</strong>g technological problems,<strong>in</strong>frastructure <strong>in</strong>adequacy, cultural practices or socio-economic factors (Anderson andAnderson, 1991). The United States Food and Drug Adm<strong>in</strong>istration showed that theprevalence <strong>of</strong> Salmonella <strong>in</strong> imported and domestic seafood were 7.2 % and 1.3 %,respectively, from 1990 to 1998, with raw seafood show<strong>in</strong>g the highest contam<strong>in</strong>ationrates: 10 % import and 2.8 % domestic (He<strong>in</strong>itz et al., 2000). Contam<strong>in</strong>ation canoccur at multiple steps along the food cha<strong>in</strong> <strong>in</strong>clud<strong>in</strong>g production, process<strong>in</strong>g,distribution, retail market<strong>in</strong>g, and handl<strong>in</strong>g/preparation (Zhao et al., 2002). Shrimpspurchased from wholesale markets and retail sale po<strong>in</strong>ts <strong>in</strong> Hyderabad city <strong>in</strong> Indiahas shown samples to have had high levels <strong>of</strong> filth and bacteria due to variousunhygienic practices followed dur<strong>in</strong>g the cha<strong>in</strong> <strong>of</strong> commercial operation. Theprevalence <strong>of</strong> Salmonella <strong>in</strong> retail markets was higher (11 %) than <strong>in</strong> whole salemarkets (5 %) (Jonnalagadda and Bhat, 2004). In contrast to these f<strong>in</strong>d<strong>in</strong>gs Hatha etal., 2003, reported that the shrimp products for export trade ma<strong>in</strong>ta<strong>in</strong> superiorbacteriological <strong>quality</strong> by exert<strong>in</strong>g good process control to meet the <strong>in</strong>ternationalstandards <strong>of</strong> the import country. Dur<strong>in</strong>g their study they have found only one samplepositive for Salmonella from a total <strong>of</strong> 846 samples <strong>of</strong> raw, peeled and deve<strong>in</strong>ed tail-
97on tiger shrimp (Penaeus <strong>monodon</strong>). Fonseka, 1994, reported higher Salmonellacontam<strong>in</strong>ation rate (8 isolates) <strong>of</strong> <strong>shrimps</strong> after harvest and a lower rate (2 isolates) forprocessed products from a total <strong>of</strong> 295 samples. Arumugaswamy et al., 1995observed that the prevalence <strong>of</strong> Salmonella <strong>in</strong> raw foods (32 %) were higher than <strong>in</strong>ready-to-eat cooked foods (17 %) <strong>in</strong> Malaysia. Among the raw foods the proportion<strong>of</strong> Salmonella <strong>in</strong> chicken pieces (39 %), liver (35 %), gizzard (44 %), oysters (86 %)were higher than <strong>in</strong> prawns (25 %).M<strong>in</strong>imum growth temperature <strong>of</strong> Salmonella is 6-7 o C, maximum growthtemperature <strong>in</strong> the range <strong>of</strong> 45-47 o C (Bremer et al., 2003). Because <strong>of</strong> thismesophilic nature Salmonella is considered to be hav<strong>in</strong>g a longer shelf life <strong>in</strong> ice(Surendran et al., 1994). Pond cultured <strong>shrimps</strong> showed faster deterioration (12 h)when <strong>shrimps</strong> were held at ambient temperature than <strong>in</strong> melt<strong>in</strong>g ice (15 days)(Fonseka and Ranj<strong>in</strong>i, 1994). Thus the presence <strong>of</strong> Salmonella (12.78 %) <strong>in</strong> <strong>marketed</strong><strong>shrimps</strong> <strong>in</strong> this study might be due to <strong>in</strong>adequate hygienic standards and the <strong>in</strong>abilityto ma<strong>in</strong>ta<strong>in</strong> the cold cha<strong>in</strong> dur<strong>in</strong>g post harvest. The overall prevalence <strong>of</strong> Salmonella<strong>in</strong> culture <strong>shrimps</strong> was found to be 11.11 % and <strong>in</strong> capture <strong>shrimps</strong> 14.44 % and thislow prevalence <strong>of</strong> Salmonella <strong>in</strong> culture <strong>shrimps</strong> than capture <strong>shrimps</strong> was observedeven <strong>in</strong> the Puttalam and <strong>in</strong> the Kurunegala districts. The low prevalence <strong>in</strong> culture<strong>shrimps</strong> than <strong>in</strong> capture <strong>shrimps</strong> was due to the better farm management practicesunder the close supervision that is not apply <strong>in</strong> capture shrimp fishery. However, bothtypes <strong>of</strong> <strong>shrimps</strong> are grown <strong>in</strong> brackish water and when compared to other raw foodproducts there is benefit for low levels <strong>of</strong> Salmonella due to bactericidal nature <strong>of</strong> thebrackish water; it is reported that salmonellae are unable to tolerate high br<strong>in</strong>e above 9% (Jay, 2000). When concern the place the high prevalence <strong>of</strong> Salmonella wasobserved <strong>in</strong> the Puttalam district than the Kurunegala district which might be due tocontam<strong>in</strong>ation through bare hands, high rema<strong>in</strong>s <strong>of</strong> <strong>shrimps</strong> at the end <strong>of</strong> the day, highnumber <strong>of</strong> low status <strong>of</strong> the vendors, high middle man <strong>in</strong>volvement and the <strong>in</strong>abilityto ma<strong>in</strong>ta<strong>in</strong> the cold cha<strong>in</strong> dur<strong>in</strong>g transportation.There were statistically significant differences observed <strong>in</strong> the levels <strong>of</strong>prevalence <strong>of</strong> Salmonella among the market locations even though they orig<strong>in</strong>ate
98from common sources. It might be due to different hygiene practices applied <strong>in</strong> these14 different sell<strong>in</strong>g locations where <strong>shrimps</strong> were purchased. Conversely, a studycarried out by Adesiyun et al., 2006, detected there were no statistically significantdifferences <strong>of</strong> prevalence <strong>of</strong> antimicrobial resistance amongst bacteria from three saleoutlets where table eggs sampled orig<strong>in</strong>ated from common layer farms. Thesef<strong>in</strong>d<strong>in</strong>gs support the theory <strong>of</strong> the possibility <strong>of</strong> <strong>microbiological</strong> contam<strong>in</strong>ationoccurr<strong>in</strong>g dur<strong>in</strong>g the sale at local market locations even though they have a commonorig<strong>in</strong>.Reilly and Twiddy (1992) reported that the predom<strong>in</strong>ant serovar occurr<strong>in</strong>g <strong>in</strong>shrimp farms <strong>in</strong> South East Asia is Salmonella Weltevreden. The serotypes,Salmonella Anatum, Salmonella Wandsworth and Salmonella Potsdam are some <strong>of</strong>the other serovars reported to occur to a lesser extent. Shabar<strong>in</strong>ath et al., 2007, notedthat the pr<strong>in</strong>cipal serovar prevalent <strong>in</strong> seafood (fourteen out <strong>of</strong> n<strong>in</strong>eteen isolates) is S.Weltevreden and that they are genetically diverse which supports the f<strong>in</strong>d<strong>in</strong>gs <strong>of</strong>Reilly and Twiddy, from 1992. A study <strong>in</strong> the Chiangmai and Lampoon prov<strong>in</strong>ces <strong>of</strong>northern Thailand <strong>in</strong>dicated that S. Weltevreden is the most common serotype<strong>in</strong>volved <strong>in</strong> human <strong>in</strong>fections (Padungtod and Kaneene, 2006). The World HealthOrganization (WHO) National Salmonella and Shigella Centre <strong>in</strong> Bangkok revealedthat dur<strong>in</strong>g 1993-2002 the most common serovars account<strong>in</strong>g for human <strong>in</strong>fectionswere 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 <strong>in</strong> Thailand(Bangtrakulnonth et al., 2004).Fish and other seafood associated with Salmonella serovars were reported byNational Salmonella Centre <strong>in</strong> India as the major Salmonella serovars <strong>of</strong> zoonoticsignificance, S. Typhimurium, S. Newport, S. Weltevreden, S. Sa<strong>in</strong>tpaul and S.Enteritidis (S<strong>in</strong>gh, 2007). Infection due to Salmonella Weltevreden <strong>in</strong> humans <strong>in</strong>India was quite uncommon before 1970 and it accounted for less than 4 % <strong>of</strong> totalhuman salmonellosis. The <strong>in</strong>cidence <strong>of</strong> <strong>in</strong>fection <strong>in</strong>creased considerably after 1970
99and <strong>in</strong> 1972 this serotype constituted 29.1 % <strong>of</strong> total human salmonellosis <strong>in</strong> India(Basu and Sood, 1975).From those serovars commonly <strong>in</strong>volved <strong>in</strong> food-borne illness <strong>in</strong> the UnitedStates the most frequent serotypes <strong>in</strong> import seafood were Salmonella Weltevreden.The other top 20 important serovars are Salmonella Senftenberg, SalmonellaLex<strong>in</strong>gton, Salmonella Paratyphi-B, Salmonella Enteritidis, Salmonella Newport,Salmonella Thompson, Salmonella Typhimurium and Salmonella Anatum, butSalmonella Enteritidis is the most common Salmonella serotype caus<strong>in</strong>g <strong>in</strong>fection anddeath <strong>in</strong> people, followed by Salmonella Typhimurium (He<strong>in</strong>itz et al., 2000).The present study carried out <strong>in</strong> North Western prov<strong>in</strong>ce, Sri Lanka, revealed thepredom<strong>in</strong>ant Salmonella serovars <strong>in</strong> <strong>marketed</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong> 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 <strong>of</strong> 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 <strong>in</strong> <strong>shrimps</strong> to a lesser extent than S. Newport and S. Typhimuriumwas not found <strong>in</strong> the current study. The exhibition <strong>of</strong> higher percentage <strong>of</strong> S. Newportwas similar to that <strong>of</strong> another source which reported 12 out <strong>of</strong> 30 samples from retailturkey samples <strong>in</strong> Fargo, North Dakota, USA (Fakhr et al., 2006).5.1.1.2 Antimicrobial resistance <strong>in</strong> Salmonella isolated from <strong>shrimps</strong>There is still a debate on the relationship between animal use <strong>of</strong> antibiotics andresistance <strong>in</strong> human pathogens because human use <strong>of</strong> antibiotics has undoubtedlycaused the resistance epidemic all over the world (Beović, 2006). There is necessity <strong>in</strong>coord<strong>in</strong>ation between human, veter<strong>in</strong>arian and environmental sectors to clarify theoccurrence <strong>of</strong> resistant pathogens <strong>in</strong> humans, animals and <strong>in</strong> the environment (Anon,2002). A study carried out by Zhao et al., 2002, <strong>in</strong> the USA <strong>in</strong>dicated thatantimicrobial resistant Salmonella are present <strong>in</strong> imported foods, primarily <strong>of</strong> seafood
100orig<strong>in</strong>. Thirty fish species <strong>of</strong> capture and aquaculture orig<strong>in</strong> were found to conta<strong>in</strong>multi-resistant stra<strong>in</strong>s <strong>of</strong> Salmonella. F<strong>in</strong>d<strong>in</strong>gs <strong>of</strong> the present study showed that therewas no statistically significant difference <strong>in</strong> the percentage resistance amongSalmonella obta<strong>in</strong>ed from culture <strong>shrimps</strong> and capture <strong>shrimps</strong>. This might be due toantibiotic usage <strong>in</strong> shrimp farm<strong>in</strong>g and release <strong>of</strong> shrimp pond effluent to estuar<strong>in</strong>eecosystems or post harvest contam<strong>in</strong>ation <strong>of</strong> <strong>shrimps</strong> with the antibiotic resistantSalmonella through the environment and human handl<strong>in</strong>g. Moreover the mostprevalent serotypes among these Salmonella were S. Newport but it was reported <strong>in</strong>South East Asia the most prevalent is S. Weltevreden (Reilly and Twiddy, 1992).Therefore, this observation was more supported with the post harvest contam<strong>in</strong>ation<strong>of</strong> <strong>shrimps</strong> with antibiotic resistant Salmonella.On the other hand it is important to carry out further <strong>in</strong>vestigation on emerg<strong>in</strong>gantimicrobial resistance <strong>in</strong> capture shrimp ecology which was reported by severalstudies. This has been attributed to the rapid expansion <strong>of</strong> the shrimp farm<strong>in</strong>g<strong>in</strong>dustry, result<strong>in</strong>g <strong>in</strong> <strong>in</strong>creased pressure on wild shrimp resources. There are potentialadverse environmental effects on estuar<strong>in</strong>e eco-systems through shrimp pondeffluents, "biological pollution" <strong>of</strong> native shrimp stocks through escapement <strong>of</strong>aquaculture stocks, water use and entra<strong>in</strong>ment <strong>of</strong> estuar<strong>in</strong>e biota, and the impacts <strong>of</strong>shrimp farm chemicals on estuar<strong>in</strong>e systems (Hopk<strong>in</strong>s et al., 1995). A higherpercentage <strong>of</strong> antibiotic resistance <strong>in</strong> aquaculture <strong>shrimps</strong> has been evidenced by Le etal., 2005, from their study carried out <strong>in</strong> Vietnam. They reported a wide spread use <strong>of</strong>antibiotics <strong>in</strong> aquaculture, which may result <strong>in</strong> residues <strong>of</strong> antibiotics <strong>in</strong> water andmud, and subsequently, the development <strong>of</strong> antibiotic resistance <strong>in</strong> bacteria <strong>in</strong> theenvironment.An overall percentage <strong>of</strong> 91.3 % for resistance to the antimicrobial agents’sulphonamide and erythromyc<strong>in</strong> was detected among Salmonella isolates from<strong>shrimps</strong> <strong>in</strong> this study. Ak<strong>in</strong>bowale et al., 2006, reported that resistance to ampicill<strong>in</strong>,amoxycill<strong>in</strong>, cephalex<strong>in</strong> and erythromyc<strong>in</strong> was wide spread <strong>in</strong> aquaculture sources <strong>in</strong>Australia and found <strong>of</strong>f label use s<strong>in</strong>ce no antibiotics registered for use <strong>in</strong> aquaculture.Resistance to sulfonamides was common, similar to this present study. Moreover,
101isolates from aquaculture sources were resistant to oxytetracycl<strong>in</strong>e, tetracycl<strong>in</strong>e andnalidixic acid <strong>in</strong> contrast to this present study. Results <strong>of</strong> the present study showedthat all the Salmonella isolates are susceptible to other antimicrobials such as nalidixicacid, amoxycill<strong>in</strong> (except one stra<strong>in</strong>), ampicill<strong>in</strong>, gentamyc<strong>in</strong>, chloramphenicol,kanamyc<strong>in</strong>, sulphamethoxazole/trimethoprim, cipr<strong>of</strong>loxac<strong>in</strong>, trimethoprim,tetracycl<strong>in</strong>e, streptomyc<strong>in</strong> and doxycycl<strong>in</strong>e hydrochloride that were tested <strong>in</strong> thisstudy. Fonseka, 1994, observed Salmonella isolated from farm <strong>shrimps</strong> <strong>in</strong> Sri Lankawere not resistant to any k<strong>in</strong>d <strong>of</strong> antibiotic tested such as ampicill<strong>in</strong> 10 (µg),cephalorid<strong>in</strong>e 5 (µg), colist<strong>in</strong> sulphate 25 (µg), gentamic<strong>in</strong> 10 (µg), streptomyc<strong>in</strong> 10(µg), sulphatriad 200 (µg), tetracycl<strong>in</strong>e 25 (µg), and cotrimoxazole 25 10 (µg). Thisdetection <strong>of</strong> highest resistance to erythromyc<strong>in</strong> <strong>in</strong> Salmonella isolates has beensupported by the f<strong>in</strong>d<strong>in</strong>gs <strong>of</strong> erythromyc<strong>in</strong> resistance for all the isolates <strong>of</strong> Vibriospecies namely Vibrio harveyi, V. parahaemolyticus, V. alg<strong>in</strong>olyticus, V. anguilliarumand V. vulnificus from Penaeus <strong>monodon</strong> <strong>shrimps</strong> and water <strong>in</strong> Sri Lanka attributed tothe wide spread use (Panawala et al., 2005). However, wide use <strong>of</strong> macrolides <strong>in</strong>human medic<strong>in</strong>e and <strong>in</strong>creas<strong>in</strong>g resistance to macrolides <strong>in</strong> human pathogens has beenreported (Phillips et al., 2004). Sulphonamide is used as an antibiotic treatment forhuman as well as animal diseases. Therefore, the application <strong>of</strong> this antibiotic is high.Moreover, it was reported that mostly the fate <strong>of</strong> antibiotics <strong>in</strong> the environment hasbeen primarily <strong>in</strong> surface waters and concentrations <strong>of</strong> sulphonamides are high <strong>in</strong>water wells <strong>in</strong> Idaho, USA (Angela et al., 2006). This environmental contam<strong>in</strong>ation<strong>of</strong> antibiotics can lead to emergence <strong>of</strong> sulphonamide resistance <strong>in</strong> Salmonella s<strong>in</strong>ce<strong>shrimps</strong> are associated with water or this might be due to overuse <strong>of</strong> these two drugs<strong>in</strong> aquaculture practice.In general bacteria can develop resistance for antibiotics which share similarstructures (Angela et al., 2006). Though some <strong>of</strong> the antibiotics tested belong to thesame class such as amoxycill<strong>in</strong>, ampicill<strong>in</strong> and sulphonamides,sulphamethoxazole/trimethoprim showed resistance <strong>in</strong> Salmonella only foramoxycill<strong>in</strong> and sulphonamides due to structural differences <strong>of</strong> the antibiotics.Amoxycill<strong>in</strong>, nalidixic acid and cipr<strong>of</strong>loxac<strong>in</strong> are used <strong>in</strong> human medic<strong>in</strong>e <strong>in</strong> SriLanka, the same antibiotics were used <strong>in</strong> this study noted percentage susceptibility <strong>of</strong>
102Salmonella isolates from <strong>shrimps</strong> are: 95.65 % for amoxycill<strong>in</strong>, 100 % for nalidixicacid and 100 % for cipr<strong>of</strong>loxac<strong>in</strong>. Furthermore, Salmonella isolated from human casesshows they are susceptible to these antimicrobials and percentage susceptibility is:amoxycill<strong>in</strong> 88.9 %, nalidixic acid 84.4 %, cipr<strong>of</strong>loxac<strong>in</strong> 100 %. Other antimicrobialscommonly use for human treatments are observed Salmonella is susceptible and areidentified as furazolidone 83.3 %, cotrimaxazole 72.7 %, mecill<strong>in</strong>am 96.4 %,ceptazidime 93.5 %, cefuroxime 62.5 %, chloramphenicol 100 %, ceftriaxone 100 %by (MRI, 2005).S. Typhimurium is a globally important serotype regard<strong>in</strong>g the antimicrobialresistance. The antimicrobial resistance <strong>of</strong> 809 Salmonella Typhimurium isolatescollected from humans <strong>in</strong> Norway between 1975 and 1998 described the largest<strong>in</strong>crease <strong>in</strong> resistance to be <strong>in</strong> 1996 when 35 % <strong>of</strong> the isolates were multi-resistant.The first multi-resistant isolate acquired <strong>in</strong> Norway appeared <strong>in</strong> 1994, but already <strong>in</strong>1998, 23 % <strong>of</strong> the isolates domestically acquired were multi-resistant, and a majoritywere S. Typhimurium DT 104. There was no cipr<strong>of</strong>loxac<strong>in</strong> resistance <strong>in</strong> domesticallyacquired isolates (Leegard et al., 2000). In this study S. Typhimurium was not foundand all the isolated Salmonella serotypes showed 100 % susceptibility tocipr<strong>of</strong>loxac<strong>in</strong>. Cipr<strong>of</strong>loxac<strong>in</strong> and fluoroqu<strong>in</strong>olones are very important because theyare considered to be the last l<strong>in</strong>e defence aga<strong>in</strong>st DT 104 <strong>in</strong> adults (Carlson, et al.,2003).When compared with S. Typhimurium resistance to penta drugs ampicill<strong>in</strong>,chloramphenicol, streptomyc<strong>in</strong>, sulphonamide and tetracycl<strong>in</strong>e, the present isolatedSalmonella serotypes showed resistance only to sulphonamide. Busani et al., 2004,reported that among S. Typhimurium, multi-resistance was more common <strong>in</strong> bov<strong>in</strong>e,poultry and rabbit stra<strong>in</strong>s, it was lower <strong>in</strong> sw<strong>in</strong>e stra<strong>in</strong>s and <strong>in</strong> the case <strong>of</strong> pigeons itwas rare. They observed that resistance to trimethoprim–sulphamethoxazole wasma<strong>in</strong>ly <strong>in</strong> isolates <strong>of</strong> sw<strong>in</strong>e and human orig<strong>in</strong> and this shows rare possibility to occurS. Typhimurium <strong>in</strong> <strong>shrimps</strong>.In 1996, Salmonella Newport caused 5 % <strong>of</strong> Salmonella <strong>in</strong>fections <strong>in</strong> humans <strong>in</strong>the United States; by 2003, it represented 12 % <strong>of</strong> all reported Salmonella <strong>in</strong>fections.
103Emergence <strong>of</strong> antibiotics resistant Salmonella Newport was reported by Varma et al.,2006 as multi-drug resistant (MDR) stra<strong>in</strong> <strong>of</strong> Salmonella Newport, Newport-MDRAmpC, which showed wide dissem<strong>in</strong>ation and <strong>in</strong>fection was acquired throughthe US food supply, most likely from bov<strong>in</strong>e and poultry sources. This serotype wasreported <strong>in</strong> India from frozen duck (3.7 %) (Bangtrakulnonth et al., 2004). In thisstudy, the percentage <strong>of</strong> S. Newport isolated from <strong>shrimps</strong> was 47.83 % and all theisolates were resistant to erythromyc<strong>in</strong> and sulphonamide. Conversely, Newport-MDRAmpC stra<strong>in</strong> exhibits resistance to ceftriaxone, ampicill<strong>in</strong>,amoxycill<strong>in</strong>/clavulanic acid, cephaloth<strong>in</strong>, cefoxit<strong>in</strong>, chloramphenicol, streptomyc<strong>in</strong>,sulfamethoxazole, and tetracycl<strong>in</strong>e used to treat humans and cefti<strong>of</strong>ur which is used <strong>in</strong>veter<strong>in</strong>ary medic<strong>in</strong>e <strong>in</strong> the USA (Gupta et al., 2003).5.1.2 Microbiological contam<strong>in</strong>ation <strong>in</strong> terms <strong>of</strong> Aerobic Plate Count,Enterobacteriaceae count, E. coli and Total Halophilic Plate CountFood safety and <strong>quality</strong> aspects <strong>in</strong> trade became important because fresh food ismore prone to certa<strong>in</strong> <strong>microbiological</strong> contam<strong>in</strong>ation. The Aerobic Plate Count(APC) <strong>in</strong>dicates the level <strong>of</strong> micro-organisms <strong>in</strong> a product. As expected, the presentstudy revealed <strong>shrimps</strong> were higher <strong>in</strong> APC than the exist<strong>in</strong>g standards <strong>of</strong> raw fish atthe po<strong>in</strong>t <strong>of</strong> sale recommended by German Society for Hygiene and Microbiology(Deutsche Gesellschaft für Hygiene und Mikrobiologie). Thirteen samples out <strong>of</strong> 27samples met the standards. This was to be expected s<strong>in</strong>ce fresh fish and fisheryproducts <strong>of</strong>ten have an APC <strong>of</strong> 10 4 -10 5 /g, although there are examples <strong>of</strong> seafoodswith an APC <strong>of</strong> 10 6 -10 8 /g without objectionable <strong>quality</strong> changes (Nickelson andF<strong>in</strong>ne, 1992). A study undertaken at farm level reported APC <strong>of</strong> entire shrimp,shrimp surface and guts <strong>of</strong> 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 <strong>in</strong> <strong>shrimps</strong> cultured <strong>in</strong> South East Asia (Fonseka, 1990). In the present studyshrimp represent post-harvest contam<strong>in</strong>ation which can be attributed to the land<strong>in</strong>gcentres and unhygienic handl<strong>in</strong>g at fish markets.
104Enterobacteriaceae are useful <strong>in</strong>dicators <strong>of</strong> hygiene, however, Surendran et al.,1994, have found that it is a natural contam<strong>in</strong>ant <strong>in</strong> the case <strong>of</strong> cultured fishes orprawns. With regards to the cultured species it was reported that cultured prawnswere found to carry greater numbers <strong>of</strong> Enterobacteriaceae. This study found there tobe a higher number <strong>of</strong> Enterobacteriaceae count <strong>in</strong> twenty-three raw shrimp samples.It is reported that Enterobacteriaceae are widely distributed and to be found <strong>in</strong> thesoil, <strong>in</strong> water, vegetation and <strong>in</strong> the <strong>in</strong>test<strong>in</strong>al tract <strong>of</strong> animals (Blood and Curtis,1995) which might lead to cross-contam<strong>in</strong>ation. Conversely, Beckers et al., 1981,reported that the <strong>microbiological</strong> <strong>quality</strong> <strong>of</strong> frozen precooked and peeled <strong>shrimps</strong> forexport were usually low <strong>in</strong> numbers <strong>of</strong> Enterobacteriaceae (lesser than 2x10 2 per g) <strong>in</strong>South East Asia and North sea <strong>shrimps</strong> due to differences <strong>in</strong> process<strong>in</strong>g.Out <strong>of</strong> 56 samples tested for E. coli 50 % met the standards. Kumar et al., 2005,reported that the contam<strong>in</strong>ation <strong>of</strong> E. coli was higher <strong>in</strong> the seafood samples collectedfrom the land<strong>in</strong>g centres and from fresh fish markets than from frozen shrimp samplescollected from process<strong>in</strong>g plants. Eleftheriadou et al., 2002 wrote down the criteria <strong>of</strong>a <strong>microbiological</strong> pr<strong>of</strong>ile with E. coli as an <strong>in</strong>dicator organism considered significantwhen levels exceed 10 2 CFU/g. They found higher levels than the pathogens they hadstudied, S. aureus and B. cereus <strong>in</strong>dicat<strong>in</strong>g the necessity <strong>of</strong> <strong>in</strong>creas<strong>in</strong>g simple hygienepractices.Samples tested for the Total Halophilic Plate Count (<strong>in</strong>digenous bacteria) rangedbetween m<strong>in</strong>imum to maximum 6.18 log CFU/g to 9.24 log CFU/g and median was6.69 log CFU/g. The distribution <strong>of</strong> 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 <strong>in</strong> raw seafoods should be considered asunsatisfactory due to <strong>in</strong>adequate temperature control regard<strong>in</strong>g V. parahaemolyticus
105and limits
106handl<strong>in</strong>g and <strong>in</strong>adequate process<strong>in</strong>g. Transportation was the most commonly affectedsituation for all these <strong>microbiological</strong> parameters: the type <strong>of</strong> vehicle, the duration <strong>of</strong>transport, ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g the cold cha<strong>in</strong> (transport condition) dur<strong>in</strong>g transport weresignificantly different. This is supported by the fact that <strong>microbiological</strong> changesoccur when <strong>shrimps</strong> are <strong>in</strong>sufficiently iced and improperly stored at highertemperatures (Antony et al., 2002). Contam<strong>in</strong>ation also occurs dur<strong>in</strong>g post processhandl<strong>in</strong>g and transportation (Kumar et al., 2005).Exposure to other fish species dur<strong>in</strong>g transport was significant for positiveSalmonella and higher Total Halophilic bacterial contam<strong>in</strong>ation due to crosscontam<strong>in</strong>ation.Moreover, the low socio-economic status <strong>of</strong> the vendor, number 3grade <strong>of</strong> the shrimp, conta<strong>in</strong>er clean<strong>in</strong>g not every end <strong>of</strong> the day were the risk factorsassociated with Salmonella contam<strong>in</strong>ation at local sale locations. However <strong>shrimps</strong>rema<strong>in</strong> at the end <strong>of</strong> the day and medium socio-economic status <strong>of</strong> the vendor weretrends to be the risk factors.The storage types <strong>of</strong> conta<strong>in</strong>ers were significantly different <strong>in</strong> the case <strong>of</strong>Enterobacteriaceae and Total Halophilic bacteria. The storage conta<strong>in</strong>ers are better tobe replaced by suitable alternative. As regards to personal hygiene, though most <strong>of</strong>the vendors handle the shrimp with their bare hands and <strong>in</strong> between hand wash<strong>in</strong>g dosometime, both <strong>of</strong> them were not significant (p>0.05) <strong>in</strong> the case <strong>of</strong> Salmonella; andgenerally speak<strong>in</strong>g for other <strong>microbiological</strong> parameters these higher numbers <strong>of</strong><strong>microbiological</strong> parameters might be due to potential cross-contam<strong>in</strong>ation betweenthe environment and the <strong>shrimps</strong>. Therefore, the cha<strong>in</strong> <strong>of</strong> commercial operationshould be studied thoroughly <strong>in</strong> order to evaluate market failures to produce higher<strong>quality</strong> products.
1075.2 ConclusionIn general retail level is the last check po<strong>in</strong>t where contam<strong>in</strong>ated end product canbe identified and the <strong>microbiological</strong> exam<strong>in</strong>ation <strong>of</strong> <strong>shrimps</strong> plays an important role<strong>in</strong> assur<strong>in</strong>g the safety and <strong>quality</strong>. Salmonella was found <strong>in</strong> 23 out <strong>of</strong> 180 <strong>shrimps</strong>amples from retail sale locations <strong>in</strong> North Western Prov<strong>in</strong>ce, Sri Lanka. The isolatedSalmonella serotypes represented environmental contam<strong>in</strong>ation <strong>of</strong> food <strong>of</strong> animalorig<strong>in</strong>. Higher levels <strong>of</strong> antimicrobial resistance were observed for erythromyc<strong>in</strong> andsulphonamide (91.3 %). Salmonella were susceptible for rest <strong>of</strong> the antibiotics testedrespectively, nalidixic acid, ampicill<strong>in</strong>, gentamyc<strong>in</strong>, sulphamethoxazole/trimethoprim,chloramphenicol, kanamyc<strong>in</strong>, cipr<strong>of</strong>loxac<strong>in</strong>, trimethoprim, tetracycl<strong>in</strong>e, streptomyc<strong>in</strong>,doxycycl<strong>in</strong>e hydrochloride, amoxycill<strong>in</strong> (only one isolate resistant). The antimicrobial(erythromyc<strong>in</strong> and sulphonamide) resistant Salmonella isolates from capture <strong>shrimps</strong>must be taken <strong>in</strong>to consideration due to the aquaculture practices can lead to theemergence <strong>of</strong> resistant stra<strong>in</strong>s <strong>in</strong> the environment which will have a long term effect.The results found <strong>in</strong> this study expla<strong>in</strong> <strong>microbiological</strong> contam<strong>in</strong>ation <strong>of</strong> <strong>shrimps</strong>related mostly to a lack <strong>of</strong> food hygiene requirements and potential environmentalcontam<strong>in</strong>ation where facilitate contam<strong>in</strong>ation, due to reckless handl<strong>in</strong>g. The riskfactors associated with Salmonella contam<strong>in</strong>ation were low status <strong>of</strong> the seller, mix<strong>shrimps</strong> with other fish species dur<strong>in</strong>g transportation, third grade <strong>of</strong> the shrimp andclean<strong>in</strong>g <strong>of</strong> the conta<strong>in</strong>er not every end <strong>of</strong> the day. However medium status <strong>of</strong> theseller and rema<strong>in</strong> <strong>of</strong> the <strong>shrimps</strong> at the end <strong>of</strong> the day were trends to be the riskfactors.Furthermore, it is taken <strong>in</strong>to 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 <strong>quality</strong> deteriorate more rapidly than other prote<strong>in</strong>sources such as chicken and red meat. Therefore, the evaluation <strong>of</strong> the cha<strong>in</strong> <strong>of</strong>commercial market failures <strong>in</strong> the <strong>in</strong>dustry would improve seafood distribution, withbetter alternatives achiev<strong>in</strong>g higher <strong>quality</strong> products as with other fresh products <strong>in</strong>
108terms <strong>of</strong> appearance, odour, flavour, texture and thus elim<strong>in</strong>at<strong>in</strong>g food-bornepathogens and thereby ensur<strong>in</strong>g food safety.As <strong>in</strong> many other develop<strong>in</strong>g countries, Sri Lanka has little <strong>in</strong>formation availableon the occurrence <strong>of</strong> food contam<strong>in</strong>ation with regard to biological and chemicalcontam<strong>in</strong>ation, especially <strong>in</strong> domestic products. Without such <strong>in</strong>formation the health<strong>of</strong> the people may be threatened. This <strong>in</strong>formation <strong>of</strong> biological contam<strong>in</strong>ation <strong>of</strong>local <strong>shrimps</strong> will help to improve the safety <strong>of</strong> the food supply and thus reduce the<strong>in</strong>cidences <strong>of</strong> food-borne diseases.Improv<strong>in</strong>g food safety along western standards, however, may <strong>in</strong>cur considerablecosts and as such put it out <strong>of</strong> the reach <strong>of</strong> the poor. Therefore, the <strong>in</strong>troduction <strong>of</strong>new food safety tools and rules need to be affordable and build on local seafoodmanagement customs rather than simply impos<strong>in</strong>g western standards that areexpensive to monitor.5.3 Recommendations1. Huss et al., 2000, reported that the safety <strong>of</strong> various seafood products variesconsiderably and is <strong>in</strong>fluenced by a number <strong>of</strong> factors such as the orig<strong>in</strong> <strong>of</strong> the fish,<strong>microbiological</strong> ecology <strong>of</strong> the product, handl<strong>in</strong>g and process<strong>in</strong>g practices andtraditional preparation before consumption. Consider<strong>in</strong>g all these factors seafood isranked accord<strong>in</strong>g to the risk from the highest to the lowest.1. Molluscs, <strong>in</strong>clud<strong>in</strong>g fresh and frozen mussels, clam, oysters <strong>in</strong> shell orshucked and raw fish to be eaten without any cook<strong>in</strong>g.2. Fresh/frozen fish and crustaceans-to be eaten after proper cook<strong>in</strong>g.3. Lightly preserved fish products (i.e., NaCl5.0). This group <strong>in</strong>cludes salted, mar<strong>in</strong>ated, fermented, cold smoked andgravid fish.
1094. Semi-preserved fish i.e., NaCl>6 % (w/w) <strong>in</strong> water phase, or pH
1106. Improvement <strong>of</strong> the hygiene <strong>of</strong> raw seafood at the local sale location level isrecommended which can be achieved by <strong>in</strong>creas<strong>in</strong>g both the education <strong>of</strong> the seafoodhandlers and consumers.7. Present low contributions <strong>of</strong> consumer representatives for constructivecommunication with the government and local fishery markets is a factor <strong>of</strong> societywhich needs to be changed s<strong>in</strong>ce 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 <strong>of</strong> Salmonella1. Non- selective pre- enrichment medium: Buffered Peptone Water2. Selective enrichment medium:2.1 Rappaport-Vassiliadis medium with soya2.2 Muller-Kauffmann Tetrathionate novobioc<strong>in</strong> broth3. Solid selective plat<strong>in</strong>g out medium:3.1 Brilliant-green Phenol-red Lactose Sucrose agar3.2 Xylose-Lys<strong>in</strong>e-Deoxycholate agar4. Nutrient Agar5. Triple Sugar Iron agar6. Urea agar7. L-Lys<strong>in</strong>e decarboxylation medium8. Reagent for detection <strong>of</strong> β –galactosidase9. Reagent for detection Voges-Proskauer (VP) reaction10. Reagent for detection <strong>in</strong>dole reaction11. Reagent for detection Methyl Red (MR) reaction12. Semi solid nutrient agar13. Sterile distilled water14. Physiological sal<strong>in</strong>e 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-H<strong>in</strong>ton agar5. Antimicrobial disks5.1 Nalidixic acid 30 µg5.2 Amoxycill<strong>in</strong> 10 µg5.3 Ampicill<strong>in</strong> 10 µg5.4 Gentamyc<strong>in</strong> 10 µg5.5 Erythromyc<strong>in</strong> 15 µg5.6 Sulphamethoxazole/Trimethoprim 25 µg5.7 Chloramphenicol 30 µg5.8 Kanamyc<strong>in</strong> 30µg5.9 Compound Sulphonamides 300µg5.10 Cipr<strong>of</strong>loxac<strong>in</strong> 5µg5.11 Trimethoprim 5µg5.12 Tetracycl<strong>in</strong>e 30µg5.13 Streptomyc<strong>in</strong> 10µg5.14 Doxycycl<strong>in</strong>e Hydrochloride 30µg
134APPENDIX B: QUESTIONNAIRE FORMATMicrobiological <strong>quality</strong> <strong>of</strong> <strong>marketed</strong> Penaeus <strong>monodon</strong> <strong>shrimps</strong> <strong>in</strong> NorthWestern Prov<strong>in</strong>ce, Sri Lanka(Questionnaire for the evaluation <strong>of</strong> rout<strong>in</strong>e practice at the local shrimp markets)Date…………………..1. Name <strong>of</strong> the Prov<strong>in</strong>ce North Western Prov<strong>in</strong>ce2. Name <strong>of</strong> the District PuttalamKurunegala3. Name <strong>of</strong> the market ………………………………4. Name <strong>of</strong> the shop owner ………………………………5. Social Economic status <strong>of</strong> the ownerHighMediumLow6. How do you receive <strong>shrimps</strong> Through middle manDirectly from farmer7. Transportationa. Method Type <strong>of</strong> vehicle ……………b. Duration How many hours ………….c. What condition ……….................................d. Mix with other fish species yesNo8. Grade <strong>of</strong> the <strong>shrimps</strong> which sold Number 1Number 2Number 39. Nature <strong>of</strong> the shrimp which sold DeheadedWhole shrimp
13510. Source <strong>of</strong> ice use for shrimp ………………………...11. How do you handle <strong>shrimps</strong> dur<strong>in</strong>g sell<strong>in</strong>g Open handWith gloves12. How <strong>of</strong>ten wash hands dur<strong>in</strong>g sell<strong>in</strong>g Every timeSometimes13. Type <strong>of</strong> conta<strong>in</strong>er keep <strong>shrimps</strong> dur<strong>in</strong>g sell<strong>in</strong>g ………………14. How <strong>of</strong>ten clean the conta<strong>in</strong>er End <strong>of</strong> the dayNot every end <strong>of</strong> the day15. Do you use dis<strong>in</strong>fectants to clean YesNo16. Water <strong>quality</strong> treated YesNo17. Sales per daya. How much sold 1-30 Kg>30-60 Kg>60-90 Kgb. Whether rema<strong>in</strong> at the end <strong>of</strong> the day YesNoRarely
136
137CURRICULUM VITAENameDr. (Ms.) Kamalika Harsh<strong>in</strong>i UbeyratneJanrabelgeDate <strong>of</strong> birth September 15, 1971Work positionVeter<strong>in</strong>ary SurgeonWork addressVeter<strong>in</strong>ary Research InstituteDepartment <strong>of</strong> Animal Production and HealthGannoruwaPeradeniyaSri LankaE-mail addresskamalikau@yahoo.comHome addressNo. 11, St. Thomas Road, Matara, Sri LankaMarital statusS<strong>in</strong>gleCitizenSri LankanReligionBuddhismSecond Language EnglishEducational BackgroundPrimary Education1978-1982 Sudharshana Adharsha Vidyalaya, Welegoda,MataraSecondary Education1983-1991 Sujatha Balika Maha Vidyalaya, Matara
138Tertiary Education1994-1999 Bachelor <strong>of</strong> Veter<strong>in</strong>ary ScienceUniversity <strong>of</strong> PeradeniyaPeradeniyaSri LankaPostgraduate Studies2003-2005 Master Degree <strong>in</strong> Dairy and Meat ProductTechnologyPostgraduate Institute <strong>of</strong> AgricultureUniversity <strong>of</strong> PeradeniyaPeradeniyaSri Lanka2005-presentCandidate <strong>of</strong> Master <strong>of</strong> Veter<strong>in</strong>ary Public HealthFreie Universität, Berl<strong>in</strong>, Germany and ChiangMai University, ThailandPr<strong>of</strong>essional Carrier2002- To date Head Office, Department <strong>of</strong> Animal Productionand Health: Poultry Disease Monitor<strong>in</strong>gVeter<strong>in</strong>ary Research Institute, Department <strong>of</strong>Animal Production and Health: Poultry Vacc<strong>in</strong>eProduction, Disease Surveillance and LaboratoryDiagnosisPr<strong>of</strong>essional Tra<strong>in</strong><strong>in</strong>gLaboratory Practical <strong>in</strong> food microbiology at theInstitute <strong>of</strong> Meat Hygiene, Meat Technology andFood Science, University <strong>of</strong> Veter<strong>in</strong>ary Medic<strong>in</strong>eVienna, Austria (2 May 2006 to 19 May 2006)
139PublicationsUbeyratne, J.K.H., Dematawewa, C.M.B.,Cyril, H.W. (2004): Use <strong>of</strong> cactus fruit juice as anatural colour<strong>in</strong>g agent <strong>in</strong> preparation <strong>of</strong> chickensausages. 56 th Annual Convention <strong>of</strong> the SriLanka Veter<strong>in</strong>ary AssociationHettiarachchi, R., Ubeyratne, J.K.H.,Kodituwakku, S.N. (2001): A review <strong>of</strong> theanimal disease situation <strong>in</strong> Sri Lanka. The SriLanka Veter<strong>in</strong>ary Journal 48.AwardsThe best poster presentationUbeyratne, J.K.H., Dematawewa, C.M.B.,Cyril, H.W. (2004): Use <strong>of</strong> cactus fruit juice as anatural colour<strong>in</strong>g agent <strong>in</strong> preparation <strong>of</strong> chickensausages. 56 th Annual Convention <strong>of</strong> the SriLanka Veter<strong>in</strong>ary Association.Scholarships2005-2007 German Academic Exchange Service (DeutscherAkademischer Austauschdienst) scholarship bythe Government <strong>of</strong> Germany