Shellfish safety and quality edited by sandra e shumway and gary e rodrick

Key words: microbial contamination, shellfish safety, enteric viruses,vibrios, harvest water classification, fecal pollution indicators, microbialsurvival, pollution sources... The most

Shellfish safety and quality

Edited by Sandra E Shumway and Gary E Rodrick

Great Abington, Cambridge CB21 6AH, England

First published 2009, Woodhead Publishing Limited and CRC Press LLC

ß 2009, Woodhead Publishing Limited

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century of dedication to ensuring shellfish quality and safety.

University of New HampshireJackson Estuarine Laboratory

85 Adams Point RoadDurham, NH 03824USA

E-mail: shj@unh.edu

Chapter 2

HeÂleÂne HeÂgaret* andSandra E ShumwayDepartment of Marine SciencesUniversity of Connecticut

1080 Shennecossett RoadGroton, CT 06340USA

E-mail: helene.hegaret@gmail.comsandra.shumway@uconn.eduContributor contact details

Albert Bosch* and Rosa M PintoÂ

Enteric Virus Laboratory

French Research Institute for

Exploitation of the Sea

Syracuse, NY 13210USA

E-mail: glboyer@esf.edu

Chapter 6

Per AndersenOrbicon A/SJens Juuls Vej 16

8260 Viby J

DenmarkE-mail: pea@orbicon.dk

Chapter 7

Mario SengcoSmithsonian Environmental ResearchCenter

PO Box 28

647 Contees Wharf RoadEdgewater, MD 21037-0028USA

E-mail: sengcom@si.edu

Chapter 8

Juan BlancoCentro de InvestigacioÂns MarinÄasApdo 13

Pedras de CoroÂn s/n

36620 Vilanova de ArousaSpain

E-mail: jblanco@cimacoron.org

Department of Biological Sciences

University of New Orleans

Commonwealth Scientific and

Industrial Research Organisation

University of Florida,Gainesville, FL 32611USA

University College CorkCooperage Building, Distillery FieldsNorth Mall

CorkIrelandE-mail: D.Watson@ucc.ieSandra E Shumway and

R B WhitlachDepartment of Marine SciencesUniversity of Connecticut

1080 Shennecossett RoadGroton, CT 06340USA

E-mail: Ldabramo@cfr.msstate.edu

Juan L Silva and Taejo Kim

Department of Food Science,

Nutrition and Health Promotion

Mississippi State University

USAE-mail: smoss@oceanicinstitute.org

Chapter 17

Shaun M Moss* and Dustin R MossOceanic Institute

41-202 Kalanianaole HighwayWaimanalo, HI 96795

USAE-mail: smoss@oceanicinstitute.org

Chapter 18

Clive AskewShellfish Association of Great BritainFishmongers' Hall

London BridgeLondon EC4R 9ELUK

E-mail:

Clive.Askew@btinternet.com;clive@sagb.freeserve.co.uk

Chapter 19

Lorna H MurrayLocal Authority Food LawEnforcement BranchFood Standards Agency Scotland

St Magnus House

25 Guild StreetAberdeen AB11 6NJUK

E-mail: lorna.murray@

foodstandards.gsi.gov.uk

PO Box 110370Gainesville, FL 32611-0370USA

E-mail: gerodrick@mail.ifas.ufl.eduVictor Garrido

Aquatic Food Products LaboratoryFood Science and Human NutritionUniversity of Florida

Gainesville, FL 32611USA

E-mail: vmga@ufl.edu

Contributor contact details xiii

Preface xix

Part I Shellfish safety: an introduction 1 Microbial contamination and shellfish safety 3

S Jones, University of New Hampshire, USA 1.1 Introduction 3

1.2 Major microbial contaminants of shellfish 4

1.3 Impacts of microbial contamination of shellfish on human health 7

1.4 Effects of microbial contamination on the international shellfish industry 10

1.5 Incidence of microbial contamination in shellfish waters 15

1.6 Contamination sources and their identification 20

1.7 Future trends 27

1.8 Sources of further information and advice 28

1.9 References and further reading 28

2 Biotoxin contamination and shellfish safety 43

H HeÂgaret, University of Connecticut, USA, G H Wikfors, NOAA Northeast Fisheries Science Center, USA and S E Shumway, University of Connecticut, USA 2.1 Introduction 43

2.2 Origins of phycotoxins 47 Contents

2.3 Trophic dynamics of phycotoxins in molluscan shellfish 52

2.4 Human health impacts 57

2.5 Management responses 59

2.6 Economic impacts of harmful algal blooms (HABs) 66

2.7 Conclusions 67

2.8 Future trends 67

2.9 References and further reading 68

Part II Improving molluscan shellfish safety and quality 3 Viral contaminants of molluscan shellfish: detection and characterisation 83

A Bosch and R M PintoÂ, University of Barcelona, Spain and F S Le Guyader, Laboratoire de Microbiologie, France 3.1 Introduction: human enteric viruses and their fate in the environment 83

3.2 Shellfish-borne transmission of virus infections 85

3.3 Effects of viral contamination of molluscs on the international shellfish industry 88

3.4 Methods for detecting viruses in molluscan shellfish and associated problems 89

3.5 Improving detection of molluscan shellfish virus contamination using new molecular-based methods 93

3.6 Depuration of viral contaminants in molluscan shellfish 95

3.7 Future trends in virus studies in shellfish 96

3.8 References 98

4 Monitoring viral contamination of molluscan shellfish 108

M Pommepuy, J C Le Saux, D Hervio-Heath and S F Le Guyader, IFREMER, France 4.1 Introduction 108

4.2 Identifying sources of pollution 110

4.3 Identifying the conditions responsible for microbial contamination of shellfish 112

4.4 Potential strategies for reducing microbial contamination in shellfish harvesting areas 114

4.5 Improving risk management strategies for shellfish harvesting areas 118

4.6 Conclusions and future trends 120

4.7 References and further reading 121

5 Algal toxins and their detection 129

G Boyer, State University of New York, USA 5.1 Introduction 129

5.2 Major algal toxins found in shellfish and their sources 130

5.3 Current methods for detection of algal toxins in shellfish 142

5.4 New techniques and future trends 153

5.5 References 154

6 Monitoring of harmful algal blooms 162

P Andersen, Orbicon A/S, Denmark 6.1 Introduction 162

6.2 Action plan design 164

6.3 Regulation of mandatory harmful algal monitoring programmes 166

6.4 Methods and techniques used to forecast and monitor harmful algal blooms 168

6.5 Future trends 170

6.6 Sources of further information and advice 171

6.7 References and further reading 172

7 Mitigation of effects of harmful algal blooms 175

M Sengco, Smithsonian Environmental Research Center, USA 7.1 Introduction 175

7.2 Novel techniques to mitigate the effects of harmful algal blooms 177

7.3 Ethos of harmful algal bloom (HAB) control 190

7.4 Future trends 190

7.5 Sources of further information and advice 191

7.6 References 191

8 Modelling as a mitigation strategy for harmful algal blooms 200

J Blanco, Centro de InvestigacioÂns MarinÄas, Spain 8.1 Introduction 200

8.2 Why model the accumulation of toxins in bivalves? 201

8.3 Historical use and development of toxin/toxicity accumulation models 204

8.4 Models of the kinetics of accumulation and transformation of toxins in shellfish 206

8.5 Applications of modelling for improved shellfish safety and quality 220

8.6 Future trends 221

8.7 Sources of further information and advice 222

8.8 References 223

9 Metals and organic contaminants in bivalve molluscs 228

W.-X Wang, HKUST, Hong Kong 9.1 Introduction 228

9.2 Metal concentrations in bivalve molluscs 229

9.3 Internal speciation of metals in bivalve molluscs 233

9.4 Exposure routes and application of the kinetic model 234

9.5 Uptake and transfer of metals 236

9.6 Safety standards 240

9.7 Detection, management, and risk assessment 241

9.8 Future trends 242

9.9 Acknowledgments 243

9.10 References 243

10 Managing molluscan shellfish-borne microbial diseases 248

T Soniat, University of New Orleans, USA (formerly of Nicholls State University, USA) 10.1 Introduction 248

10.2 Microbial indicators and pollution-associated pathogens 249

10.3 Enteric viruses 252

10.4 Naturally occurring pathogens 254

10.5 Pathogens associated with handling, processing, and distribution 257

10.6 Management of pollution-associated pathogens 258

10.7 Management of naturally occurring pathogens 259

10.8 Management of pathogens associated with handling, processing, and distribution 261

10.9 Future trends 262

10.10 Sources of further information and advice 263

10.11 Acknowledgments 263

10.12 References 263

11 Disease and mollusc quality 270

S Corbeil, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia and F C J Berthe, Animal Health and Welfare Unit, Italy 11.1 Introduction 270

11.2 Major pathogens and diseases of molluscs causing significant economic losses in molluscan aquaculture 271

11.3 Diagnostic methods 279

11.4 Effects of shellfish disease on the international shellfish industry 279

11.5 Reducing disease in molluscan aquaculture 281

11.6 Future trends 283

11.7 Sources of further information and advice 284

11.8 References 285

12 Hazard analysis and critical control point programs for raw oyster processing and handling 295

V Garrido and S Otwell, University of Florida, USA 12.1 Introduction 295

12.2 HACCP for oyster production and safety 298

12.3 HACCP plan for processing of frozen raw oysters 300

12.4 Hazard analysis 301

12.5 Identify the critical control points (CCP) 301

12.6 Definition of critical limits (CL) 306

12.7 Designate monitoring procedures 306

12.8 Corrective action (CA) 309

12.9 Specify verification (and validation) procedures 309

12.10 Specified records 310

12.11 References 310

Appendix: Annex 1 ± examples of HACCP and sanitation records 311

13 Biofouling and the shellfish industry 317

D I Watson, University College Cork, Ireland and S E Shumway and R B Whitlatch, University of Connecticut, USA 13.1 Introduction 317

13.2 Biofouling and shellfish 318

13.3 Problems and benefits of biofouling 320

13.4 Current removal/treatment methods 325

13.5 Future trends 331

13.6 Sources of further information and advice 331

13.7 Acknowledgments 332

13.8 References and further reading 332

Part III Improving crustacean safety and quality 14 Optimization of crustacean quality through husbandry and adherence to post-harvest standards for processing 339

L R D'Abramo, J L Silva and T Kim, Mississippi State University, USA 14.1 Introduction 339

14.2 Land (site) selection 340

14.3 Water: source, conservation, and preservation of quality 341

14.4 Fertilization and semi-intensive systems 342

14.5 Formulated feeds, bio-flocs, and intensive pond culture systems 344

14.6 Substrate 345

14.7 Water quality management 346

14.8 Collection during harvest 348

14.9 Harvest and post-harvest treatment 349

14.10 Safety and quality standards 350

14.11 Conclusions 357

14.12 References 357

15 Development of vaccines and management of viral diseases of

crustaceans 359

M C W van Hulten, Intervet International BV, The Netherlands and A C Barnes and K N Johnson, Queensland University, Australia 15.1 Introduction: disease and the foundations for preventative healthcare in aquaculture 359

15.2 Using the RNA interface to target shrimp viruses 365

15.3 Developing vaccines to manage viral disease in shrimp 369

15.4 Using vaccines as part of health management strategies 372

15.5 Future trends 375

15.6 Sources of further information and advice 376

15.7 References 376

16 Specific pathogen-free shrimp stocks in shrimp farming facilities as a novel method for disease control in crustaceans 384

D V Lightner and R M Redman, University of Arizona, USA and S Arce and S M Moss, The Oceanic Institute, USA 16.1 Introduction 384

16.2 The concept of domesticated SPF shrimp: a historical perspective 386

16.3 Events leading to development of Litopenaeus vannamei as the dominant species in the Americas 388

16.4 Adaptation of the SPF concept to domesticated shrimp stocks 392

16.5 International Principles for Responsible Shrimp Farming 397

16.6 Biosecurity and the culture of wild seed/broodstock 413

16.7 Biosecurity through environmental control and best management practices 414

16.8 Conclusions 415

16.9 Acknowledgments 415

16.10 References and further reading 416

17 Selective breeding of penaeid shrimp 425

S M Moss and D R Moss, Oceanic Institute, USA 17.1 Introduction 426

17.2 Selective breeding 427

17.3 Conclusions 444

17.4 References 445

Part IV Regulation and management of shellfish safety 18 Legislation, regulation and public confidence in shellfish 455

C Askew, Shellfish Association of Great Britain, UK 18.1 Introduction: public confidence in shellfish 455

18.2 Hygiene legislation and public confidence 461

18.3 Environmental legislation for the quality of shellfish growing waters 462

18.4 Limitations of the regulatory approach 464

18.5 Self-regulation and good management practice (GMP) 464

18.6 Dietary and health advisories 465

18.7 Public perception of health benefits and risks associated with shellfish 469

18.8 Future trends 470

18.9 The risk-averse marketplace 471

18.10 References 472

19 Risk management of shellfisheries 474

L H Murray, Food Standards Agency, UK and R J Lee, Cefas Weymouth Laboratory, UK 19.1 Introduction 474

19.2 Interaction between public health controls and industry 476

19.3 Identification of need for improved bases for, and application of, risk management in practice 476

19.4 Optimising risk management 476

19.5 Improved application of risk management to microbiological and biotoxin problems 479

19.6 Official and industry roles in risk management 485

19.7 Future trends 486

19.8 Interaction of research, legislation and risk management 497

19.9 Shared resources and working together 500

19.10 Conclusions 502

19.11 Sources of further information and advice 502

19.12 References and further reading 503

Part V Post-harvest issues 20 Molluscan shellfish depuration 509

K R Schneider, J Cevallos and G E Rodrick, University of Florida, USA 20.1 Introduction 509

20.2 Types of depuration plant 510

20.3 Importance of seawater quality 511

20.4 Types of seawater treatment 515

20.5 Rules and guidelines for controlled purification 519

20.6 Depuration plant location, design, and construction 524

20.7 Source of shellfish to be depurated 526

20.8 Equipment construction and depuration facility design 529

20.9 International depuration 535

20.10 Shellfish relaying 537

20.11 References and further reading 538

21 Slaughter, storage, transport, and packaging of crustaceans 542

G J Flick, L A Granata and L S Marsh, Virginia Tech, USA 21.1 Introduction 542

21.2 Slaughter/cooking 543

21.3 Packaging and preservation 549

21.4 Contaminants 552

21.5 Conclusions 560

21.6 References 561

22 Packaging, storage and transport of molluscan shellfish 568

V Garrido, Institute of Food and Agricultural Sciences, USA and G E Rodrick, University of Florida, USA 22.1 Introduction 568

22.2 Product specification 568

22.3 Packaging formats and materials 569

22.4 Product labeling and tagging 571

22.5 Product size standards 574

22.6 Accepting shellfish shipments 574

22.7 Conclusions 574

22.8 References 575

Index 576

It is estimated that by the year 2050 the world's population will reach 10 billionpeople, and seafood, especially cultured shellfish, will play a major role infeeding these populations Shellfish are a very popular and nutritious foodsource worldwide and their consumption continues to rise globally Because oftheir unique nature as compared with beef and poultry, shellfish have their owndistinct aspects of harvest, processing and handling Guaranteeing shellfishquality and safety is critical for protecting public health as well as for marketingseafood products This collection of review papers discusses issues of currentinterest and reviews steps that can be taken by the shellfish industry to maintainand improve shellfish safety and eating quality.

The United States Senate recently introduced the Commercial SeafoodConsumer Protection Act, legislation to improve the safety of seafood productsimported into the United States, and two recent FAO publications, Huss et al.(2004) and Ababouch et al (2005) provide excellent overviews of internationalmanagement issues associated with seafood safety and international trade Webelieve that Shellfish safety and quality takes these documents a step further,specifically detailing issues related to shellfish

The opening chapters provide an overview of the key issues associated withmicrobial and biotoxin contamination Parts II and III then address in moredetail methods to improve molluscan shellfish and crustacean quality and safety.Chapters focus on detection of algal toxins, monitoring and mitigation of theeffects of harmful algal blooms, metals and organic contaminants, biofouling,disease control and selective breeding Part IV reviews legislation, regulation,public confidence in shellfish and risk management Chapters on post-harvestissues, such as depuration, storage and packaging complete the volume.Preface

Several individuals have helped to make this volume possible LynseyGathercole, Sarah Whitworth and Woodhead Publishing recognized the gap inthe available literature and persuaded us to take on the challenge of filling it Agreat debt is owed to the authors for providing their time and expert contribu-tions and especially for their patience during the inevitable delays Maxprovided countless hours of support and, sadly, will not get to see the finalvolume.

Shellfish safety and quality will be an essential reference for those in theshellfish industry, managers, policymakers and academics in the field

References

ABABOUCH, L., G GANDINI and J RYDER(2005) Causes of Detentions and Rejections inInternational Fish Trade Food and Agriculture Organization of the United NationsFisheries Technical Paper 473, Rome

HUSS, H.H., L ABABOUCH and L GRAM(2004) Assessment and Management of SeafoodSafety and Quality Food and Agriculture Organization of the United NationsFisheries Technical Paper 444, Rome

Part I

Shellfish safety: an introduction

1.1 Introduction

The quality of coastal and estuarine waters throughout the world has becomeadversely impacted by a variety of contaminants, including microorganisms Inmany areas where sewage treatment is inadequate, microbial contamination is

by far the most important contaminant affecting shellfish safety Even in developed areas, however, microbial contamination from nonpoint pollutionremains a critical problem The studies and results presented here reflect pub-lished findings that pertain to the study-specific geographical areas Different

well-1

Microbial contamination and shellfish safety

S Jones, University of New Hampshire, USA

Abstract: Microbial contamination is a challenging and significant issue forthe shellfish industry It is the main public health concern associated withconsuming shellfish, and it often limits shellfish harvesting throughout theworld Enteric viruses, pathogenic Vibrio species, and fecal-borne bacterialpathogens are the main causes of shellfish-borne disease These

microorganisms have widely different properties, sources, virulence factors,and fate in the environment, and the current indicators used to classifyharvest waters have significant limitations A great deal of progress iscurrently being made in the detection of pathogenic microorganisms and inunderstanding their fate in the environment With increasing human

development in coastal areas, emerging diseases, habitat destruction, andglobal climate changes, the challenges associated with managing microbialcontamination and shellfish safety continues to evolve

Key words: microbial contamination, shellfish safety, enteric viruses,vibrios, harvest water classification, fecal pollution indicators, microbialsurvival, pollution sources

findings and phenomena could be expected in other areas of the world because

of differences, however, in environmental conditions and pollutioncharacteristics

The main themes in this chapter include an initial definition of microbialcontaminants and a discussion of those that are the major contaminants inshellfish Human diseases are then presented to emphasize their significance tothe shellfish industry and consuming public Specific impacts of microbialcontamination on the shellfish industry are then discussed, followed by anoverview of their incidence in the natural environment and their sources Thefinal sections include probable future issues and trends in research, and asummary of useful sources for further information

1.2 Major microbial contaminants of shellfish

Microbial contaminants as defined here are pathogenic microorganisms thatcause disease in shellfish-consuming humans Included are fecal-borne viral,bacterial and protozoan pathogens, and naturally occurring bacterial pathogens,but biotoxin-producing algae are excluded as they are covered in other chapters

It is also necessary to include fecal indicator and organisms used in microbialsource tracking (MST) The indicator bacteria used within the shellfish industryare generally fecal (and total) coliforms and Escherichia coli Enterococci,Clostridium perfringens and other indicators are also useful in helping toelucidate the sources and fate of fecal-borne contamination in shellfish waters(Watkins and Burkhardt, 1996) MST methods have effectively used otherorganisms such as Bacteroides spp (Field et al., 2003), Bifidobacterium spp.(Bernhard and Field, 2000) and F-RNA coliphage (Vinje et al., 2004), amongothers, to track sources of fecal contamination Though indicator organisms arenot necessarily pathogens, their universal use for assessing shellfish safetynecessitates their inclusion in this chapter It is also useful to note that many newpathogen detection methods do not require culturing the target organism andfocus instead on the direct detection of species, strain or virulence-specific genesfrom environmental samples The issue of microbial contamination and shellfishsafety can thus be addressed using methods that involve detection of microbialcells, genetic material, or both

The most frequent causes of shellfish-borne disease (CDC, 2006; Cato, 1998;Rippey, 1994) are of greatest concern Most shellfish-borne diseases are likelycaused by enteric viruses, though pathogenic vibrios are emerging as anincreasing threat, and infections from Vibrio vulnificus have the highest fatalityrate of any foodborne infectious agent Fecal-borne bacterial pathogens havebecome less prevalent worldwide (Rippey, 1994) as a result of successes withtheir management, though they still account for a significant fraction ofshellfish-borne disease in some areas (Cato, 1998) Thus, the major microbialcontaminants include viruses, vibrios, and to a lesser extent fecal-borne bacteria

1.2.1 Human pathogenic viruses

Viruses are species-specific intracellular parasites and are the leading cause ofshellfish-borne disease in humans Although human pathogenic viruses arereadily taken up and accumulated, they do not infect or grow in molluscanshellfish They can persist for extensive periods in the marine environment(Gantzer et al., 1998; Callahan et al., 1995) and in shellfish (Formiga-Cruz etal., 2002; Lees, 2000) The main source of human viruses is sewage from septicsystems, wastewater treatment facilities and direct discharges, all humansources Relatively high viral concentrations can exist in wastewater treatmentfacilities (Katayama et al., 2008; Gantzer et al., 1998), and standard treatmentssuch as chlorination are only partially effective in inactivating or removingviruses from effluent (Tree et al., 2003; Tyrrell et al., 1995) Thus, varying yetpotentially significant loading of viruses is discharged to shellfish harvestwaters Levels of viruses vary considerably over predictable cycles, with highestlevels observed in winter months (Katayama et al., 2008; Formiga-Cruz et al.,2002; Burkhardt et al., 2000; Dore et al., 2000) This seasonal variation inenvironmental incidence relates well with the incidence of viral infections inhumans, and is also influenced by environmental factors and the presence ofindividuals carrying pathogenic viruses within the human population

Lees (2000) summarized the many common types of human viruses that havebeen associated with contaminated shellfish, including the rotaviruses, astro-viruses, enteroviruses, adenoviruses, hepatitis A, and the calciviruses Smallround structure viruses are a subset of calciviruses that include Norwalk-likeviruses (NLV) Viruses most commonly associated with infectious diseaseincidents through shellfish consumption are NLV and hepatitis A, though theyproduce different disease symptoms Viruses are comparatively more difficult toremove from shellfish than most bacteria (Schwab et al., 1998) In addition,considerable effort has therefore been focused on their detection in shellfishbecause they are also more difficult to detect (Myrmel et al., 2004; Le Guyader

et al., 2003; Lees, 2000; Green et al., 1998; Henshilwood et al., 1998; Dore andLees, 1995)

1.2.2 Pathogenic vibrios

The genus Vibrio is comprised of bacteria found free-living in marineenvironments and at elevated levels in association with a variety of eukaryotichosts, including shellfish (Thompson et al., 2004) and seaweeds (Mahmud et al.,2007) They play key roles in ecosystem carbon cycling, as an important foodsource for copepods and as light organ symbionts In other cases vibrios arepathogens, primarily of shellfish and fish, though the human pathogens arenotable Of particular concern are Vibrio cholerae (Colwell, 2004), Vibriovulnificus (Gulig et al., 2005; Linkous and Oliver, 1999) and Vibrioparahaemolyticus (Yeung and Boor, 2004), which cause severe diarrhealdisease, gastroenteritis, wound infections, and septicemia (Thompson et al.,2004) Their normal association with shellfish presents a common mechanism

for human infection (Morris, 2003; Potasman et al., 2002) V hollisae and otherpotentially pathogenic species are of less concern at present.

The widespread and consistent detection of V vulnificus and V lyticus in bivalve shellfish suggests shellfish as an important ecological niche forthese vibrios Persistence appears to involve interactions between the vibrios andthe shellfish hemolymph that result in low levels of elimination (Pruzzo et al.,2005) The presence of a protozoan pathogen of oysters, Perkinsus marinus, canlead to greater inefficiency of oyster hemolymph for eliminating V vulnificus(Tall et al., 1999) Levels in oysters may also depend on oyster genotype(Sokolova et al., 2006) The lack of elimination of V vulnificus may lead tosignificant growth that may be shed to overlying waters (Tamplin and Capers,1992) Vibriophage can control pathogenic vibrios in shellfish and can be bothnumerous and diverse in mollusks (DePaola et al., 1997, 1998; Baross et al.,1978) In fact, oysters may be one of the main reservoirs of bacteriophage-controlling vibrios, and this phage population may change seasonally (Comeau

parahaemo-et al., 2005) Vibriophage density and diversity tracks Vibrio abundance inshellfish but not in sediments, where the bacteria are numerous but the phage arenot (Comeau et al., 2005)

The presence of pathogenic vibrios in shellfish and overlying waters is mostpronounced in the warmer waters of tropical and sub-tropical ecosystems(Martinez-Urtaza et al., 2008; Zimmerman et al., 2007; Thompson et al., 2004;DePaola et al., 2003) while they are typically rarely detected (Bauer et al., 2006)

or absent (Wilson and Moore, 1996) in northern temperate and colder climates.LaValley (2005) found varying detection of V vulnificus occurrence in bothfreshly harvested and depurated oysters Recent outbreaks in Alaska(McLaughlin et al., 2005) and the northeast and northwest coasts of the US(DePaola et al., 2000) have, however, heightened concerns of the spread of theseorganisms to colder waters

1.2.3 Fecal-borne bacteria

Fecal-borne bacteria are present in nearly all coastal waters where humanactivities and animals contribute fecal contamination Fecal-borne bacterialspecies have been the cause of significant outbreaks of shellfish-borne diseases(Rippey, 1994) and are used as indicators for classifying shellfish harvestingwaters almost exclusively worldwide

Fecal-borne bacteria are found in the gastrointestinal tracts of a wide range oforganisms, including humans, all livestock and poultry, a wide range of rodents,waterfowl, other birds and other wild animals, and marine fish (Anderson et al.,1997; Silva and Hofer, 1993; Pourcher et al., 1991) Many of these species canalso persist and even grow outside of the host in the natural environment, includ-ing coastal waters, sediments, and seaweed wrack (Jones et al., 2006; Brands etal., 2005; Anderson et al., 1997; Weiskel et al., 1996; Wilson and Moore, 1996;Gonzales et al., 1992) Other species, such as Aeromonas and Plesiomonas spp.can occur either in sewage or in estuarine environments (Rippey, 1994)

There have been a variety of fecal-borne bacteria used as indicators of fecalcontamination, with total and fecal coliforms, enterococci, E coli, fecal strepto-cocci, and Clostridium perfringens being the most commonly used Some ofthese taxa may include virulent strains and species For example, many strains of

E coli are non-virulent, but H7:O157 is a common pathogenic strain Fecalcoliforms include several pathogenic bacterial species, including Salmonella andShigella spp., E coli, Klebsiella spp and Aeromonas hydrophila (APHA, 1995),which are all species that have been causes of shellfish-borne diseases inhumans

1.2.4 Protozoa

Giardia lamblia and Cryptosporidium parvum are relatively common sources ofwaterborne disease outbreaks worldwide Both have been detected in shellfishfrom a wide array of areas worldwide (Graczyk et al., 2007; Schets et al., 2007;Graczyk, 2003; Potasman et al., 2002; Stowell, 2001) Neither has beenimplicated in any major reported shellfish-related outbreak (Butt et al., 2004;Potasman et al., 2002)

1.3 Impacts of microbial contamination of shellfish on human health

The relationship between sewage pollution and shellfish-borne disease incidencehas been recognized for well over a century, though the actual microbial vectorswere largely unknown until their successful identification during the last fewdecades The incidence of shellfish-related infectious diseases is known to beassociated with a few major types of pathogenic microorganisms, norovirus,Vibrio spp., Salmonella spp., Shigella spp., Hepatitis A virus (CDC, 2006; Cato,1998; Rippey, 1994) and to a lesser extent other bacterial species and viruses.Although many studies have reported the presence of Cryptosporidium andGiardia in shellfish, there have been no reported outbreaks of cryptosporidiosis

or giardiasis from human consumption of shellfish (Potasman et al., 2002).The availability of summarized data for shellfish-borne disease outbreaks isspotty and inconsistent, yet useful for general comparisons Data coveringoutbreaks associated with shellfish or seafood in general from as far back as

1898 to as recent as 2002 have been summarized by CDC (2006), Cato (1998),and Rippey (1994) Large differences can be seen in the total number of thedifferent bacterial causes in different areas (Table 1.1) Japan had a much greatincidence of vibrio-borne outbreaks, especially compared with Canada and the

EU, probably as a result of environmental conditions and types of food andcooking, but as well diagnosis and reporting The US reported fewer outbreaksassociated with fecal-borne bacteria than all other areas Sumner and Ross(2002) calculated risk rankings for different hazard/product pairings as part of aseafood safety risk assessment for Australia Viruses in oysters from

uncontaminated waters had a relatively low risk ranking, V vulnificus in oystershad an intermediate ranking, and viruses in oysters from contaminated watershad the highest ranking of shellfish-related hazard/product pairs In the US, therewas an average of 32 annual cases of reported V vulnificus septicemia fromeating raw Gulf of Mexico oysters from 1995 to 2001 (WHO/FAO, 2005).The relationship between the presence of pathogenic microbial species anddisease is complex; species that are pathogenic often have strains that vary fromvirulent to avirulent Using pathogenic vibrios as an example, the infective dosefor humans is dependent in part on the environmental conditions at harvest, thepresence and concentration of pathogenic strains, the physiological state of cells

in the shellfish tissue (Smith and Oliver, 2006), host susceptibility (Gulig et al.,2005; Hlady and Klontz, 1996), the degree to which shellfish are cooked beforebeing consumed, and a variety of other factors (FDA, 2005; WHO/FAO, 2005).Many shellfish-borne infectious diseases are caused by bacterial species that arefor the most part non-pathogenic, including of Escherichia/Shigella, Salmonella,Campylobacter, and Vibrio (Hofreuter et al., 2006; Schaechter et al., 2001;Chatzidaki-Livanis et al., 2006)

Most of the types of human viruses that have been detected in contaminatedshellfish, i.e., the rotaviruses, astroviruses, enteroviruses, adenoviruses, and thecalciviruses cause viral gastroenteritis, but infectious hepatitis from hepatitis Avirus is also prevalent (Lees, 2000) The Norwalk-like viruses (NLV) are themost common, and significant recent progress has been made in detecting NLV

in shellfish (Jothikumar et al., 2005; Loisy et al., 2005; Kageyama et al., 2003).Most fecal-borne bacteria also cause gastroenteritis as shellfish-borne disease.Improved wastewater treatment in many areas of the world has diminished their

Table 1.1 Most recent summaries of seafood or shellfish-borne disease outbreaksreported in different areas worldwide

Causative agent Japan* Canada* EU** USy USz USx Australia{

* Cato (1998) seafood-borne outbreaks: Japan: 1987±96; Canada: 1991±97.

** Cato (1998) fish and shellfish-number times reported: 1983±92.

y CDC (2006) US shellfish-borne outbreaks: 1998±2002.

z Rippey (1994) US shellfish-borne outbreaks: 1898±1990; sewage and/or wastewater.

x Rippey (1994) US shellfish-borne outbreaks: 1967±90; associated with Vibrio genus.

{ Sumner and Ross (2002) seafood-borne outbreaks: 1990±2000.

significance as disease agents in shellfish, but they remain problems (Cato,1998) and pathogenic species are commonly detected in shellfish in many parts

of the world (Brands et al., 2005; Wilson and Moore, 1996)

1.3.1 Pathogenic vibrios

The pathogenic vibrio species of most concern are Vibrio parahaemolyticus(Yeung and Boor, 2004), Vibrio vulnificus (Gulig et al., 2005; Linkous andOliver, 1999), and Vibrio cholerae (Colwell, 2004) All cause diarrhea andgastroenteritis, but V vulnificus, and more rarely V parahaemolyticus, may alsocause septicemia through wound and intestinal infections with tissue damageoccurring with rapid replication (Gulig et al., 2005) Death can occur within 24hours and its case/fataility rate (~50%) is the highest of all foodborne pathogens(WHO/FAO, 2005; Hlady and Klontz, 1996) V parahaemolyticus is one of theleading causes of foodborne illness in countries such as Japan and Taiwan (Pan

et al., 1997; Wong et al., 1999) and is the most frequent cause of foodbornevibrio-associated gastroenteritis in the US (Daniels et al., 2000), with anestimated 2800 cases each year from the consumption of raw oysters (FDA,2005) V cholerae still infects millions annually throughout the world, thoughthe incidence of shellfish-borne disease is relatively low (CDC, 2006; Rippey,1994) Some climate change scenarios suggest significant trends of warming seatemperatures, in which case pathogenic vibrios may soon be found in coastalwaters previously considered to be too cool

Not all environmental strains or vibrios have equal potential to cause disease,and pathogenic and non-pathogenic strains often coexist (Hurley et al., 2006;Deepanjali et al., 2005; Rosche et al., 2005; DePaola et al., 2003, 2000; Louis etal., 2003) Fortunately, pathogenic and non-pathogenic strains can bediscriminated based on the presence or absence of known virulence genes(Nordstrom et al., 2007; Harwood et al., 2004; Panicker et al., 2004) There hasbeen limited research on the heterogeneity of estuarine populations (Zimmerman

et al., 2007; Lin et al., 2003; Jiang et al., 2000) Estuarine V parahaemolyticuspopulations are thought to contain relatively low levels of pathogenic strains andthese are a small fraction of total V parahaemolyticus populations (FDA, 2005).Horizontal transfer and acquisition of genetic material, including virulencefactors, between different bacterial species is likely to occur in shellfish watersand shellfish tissue where bacteria live in large populations as consortia V.vulnificus, V parahaemolyticus, and V cholerae can co-exist within shellfish,and growth of V cholera (Meibom et al., 2005) on chitin oligomers induces astate of natural competence allowing them to uptake foreign DNA Other studieshave shown evidence of horizontal gene acquisition between pathogenic vibriospecies (Gonzalez-Escalona et al., 2006; Nishibuchi et al., 1996)

There have been increases in the number of pathogenic vibrio outbreaks inthe US in recent decades associated with the consumption of raw shellfish,including oysters and clams, and these outbreaks correlate with environmentalconditions that enhance microbial populations (Balter et al., 2006; Underwood

et al., 2006; McLaughlin et al., 2005; Collins, 2003; CDC, 1998) Vibrio diseaseoutbreaks are generally associated with harvested shellfish from areas withwarm water, like the Gulf of Mexico and the waters of Southeast Asia Less isknown about the incidence of virulent strains of V vulnificus and V parahaemo-lyticus in north temperate ecosystems One general assumption is that strainsfrom these areas are not virulent Bauer et al (2006) detected V vulnificus, V.parahaemolyticus, and V cholerae in Norwegian shellfish, but the isolates werenegative for virulence factors In the US, however, several recent outbreak of V.parahaemolyticus-induced gastroenteritis involving consumption of rawAlaskan oysters (McLaughlin et al., 2005), oysters and clams from WashingtonState and British Columbia (Balter et al., 2006), and several cases of V.parahaemolyticus infections in Maine can be traced to shellfish from statewaters (Koufopolous, 2007; Maine CDC, 2005) These suggest virulent strainscan be present in colder waters.

1.4 Effects of microbial contamination on the international shellfish industry

The success of the international shellfish industry is significantly affected withmicrobial contamination To protect public health, shellfish harvesting in mostareas of the world is limited as a result of careful monitoring, especially underconditions where shellfish may be exposed to microbial contaminants Assum-ing humans consume the shellfish raw or partially cooked, microbial con-taminants taken up by shellfish and accumulated in tissue can remain viable andpotentially virulent, and thus cause disease The most effective strategy forlimiting the harvest of contaminated shellfish is to harvest from areas with goodwater quality, i.e., microbial contamination is absent or at minimal levels.Comprehensive risk assessments that take into consideration a balance betweenshellfish harvesting to support the industry and protecting public health areessential tools to help guide how shellfish harvesting relative to microbialcontamination is managed

1.4.1 Major issues

The many issues that are associated with microbial contamination for theinternational shellfish industry can be categorized into three main issues Thefirst is that microbial contamination limits shellfish harvesting The concern byboth the public health community and the industry to prevent disease outbreaksforces reduced harvesting efforts in areas that are contaminated The changinglandscape of scientific study results for effects of pathogens on human health,detection methods for specific pathogens, and emerging infectious diseasesposes a challenge for the management community and industry to improve theirabilities to prevent disease outbreaks and to adjust to new or re-emergent threats.Conflicting uses of coastal resources and other human activities is anotherissue Other uses of coastal resources may conflict with shellfish harvesting if

they result in increased microbial contamination Expanding human ment, especially in critical shoreline and wetland areas, can contribute todegradation of water quality directly through increased pollution, or via loss ofecosystem processes with habitat destruction that serve to mitigate upstreampollution Climate change and the potential for sudden emergence of infectiousdiseases (Hunter, 2003) via insects, birds and other vectors are also humanactivity induced concerns that influence marine microbial communities andpathogen levels (Harvell et al., 1999) The popular press and other socio-economic factors greatly influence the attitudes of potential shellfish consumers.Throughout the world, the relatively infrequent occurrence of shellfish-bornedisease outbreaks often are accompanied by stories in the press that portray allshellfish consumption as dangerous without a balanced effort to put the cause ofthe outbreak into a more general context of safety for most shellfishconsumption.

develop-At the crux of the overall problem of microbial contamination for the national shellfish industry is how to regulate harvesting, in relation to microbialcontamination that successfully strikes a balance between the economic interests

inter-of the industry and the consumer, and the public health risk Basic questionsrelate to whether microbial contamination is present, and whether contaminationthat is present is a threat to public health, either by its composition or itsstrength

Countries and their states and regions have adopted different standards forclassifying shellfish harvesting waters, in most cases based on the likelihood ofthese areas becoming contaminated by human sewage These standards, based

on microbial indicators, have for the most part served both sides of the issue,though outbreaks have occurred in areas thought to be safe for consumption andthere is a wide range of problems with current indicators This topic will becovered in more detail in the next section Beyond these issues, the variety ofstandards used in different areas of the world is in itself an issue for theinternational shellfish industry Countries seeking to export shellfish arerequired to abide by these different standards, and choices are thus requiredwhich classification strategy should be adopted, thus limiting the number ofcountries that can import product Different standards also confuse importation

as the safety of imported product is more difficult to ascertain if harvestingstandards are different from the importing countries food safety standards Thereare efforts underway through the International Conference for MolluscanShellfish Safety to determine how standards may be harmonized and to providethe means for sharing of new detection methods and related research betweenlaboratories

1.4.2 Existing and potential alternative standards for fecal-borne

microbial contamination of shellfish waters

Water quality (US) and shellfish tissue (EU) standards have long beenestablished as a means of determining the level of sewage and microbial

contamination likely to be in harvested shellfish Simultaneous with the term use of these accepted indicators, many studies have identified a wide array

long-of limitations that inform their application and have stimulated further studies onmore effective, alternative indicators

There are many different fecal-borne human pathogens that have beendetected and may be present in shellfish harvesting waters, as well as indigenousmicroorganisms that are also human pathogens Direct monitoring of all poten-tial pathogens is prohibitive for many reasons (Watkins and Burkhardt, 1996),including the cost for routine monitoring at multiple sites, many pathogens aredifficult to detect, some are present rarely and at low concentrations, pathogensare not indexed to each other and infectious doses are not always known,avirulent strains exist, new pathogens are always emerging, and pathogen dataare not predictive Thus, an alternative strategy was obviously needed to addressthis issue

Most countries now have a classification system for shellfish harvesting areasthat is based on the likelihood of these areas becoming contaminated by humansewage, largely because the main risk from shellfish consumption at thebeginning of the twentieth century was sewage contamination The concept ofusing fecal-borne indicator bacteria as surrogates for all fecal-borne pathogenslogically emerged and has served as a relatively effective tool for managingpublic health risks associated with shellfish consumption Fecal coliforms, asubset of the first indicators (total coliforms), are still widely used today and aremade up of many different bacterial species with phenotypic properties thatmatched the defined assay conditions (APHA, 1995) This property wasbeneficial because it captured the most ubiquitous bacteria such as E coli, andall of the targeted pathogens met the conditions Thus, if Salmonella or Shigellaspp strains were present in higher concentrations than the more common E coli,the assay would still be positive Problems also existed as a result of the widearray of non-intestinal and naturally occurring species that met the assayconditions Thus, measured fecal coliform levels could exceed standards eventhough no fecal pollution may be present Other ideal indicator criteria that areviolated by fecal coliforms, E coli, enterococci, and other microbial indicators

of fecal pollution include: growth in the environment; occasional lack ofstatistical relationship to pathogen concentrations; die off sooner than pathogens

in unfavorable environmental conditions and under disinfection; and a tendency

to become viable but non-culturable (Griffin et al., 2001; Watkins andBurkhardt, 1996) With noroviruses and vibrio bacteria being the main concernswith shellfish-borne microbial pathogens, alternative indicators are needed toaddress the full range of potential public health threats

Waters contaminated by sewage may also contain a great variety of humanpathogenic viruses that may be taken up and accumulated by molluscanshellfish The survival of traditional indicator bacteria in environmental waters,and their residence times and depuration kinetics in shellfish are significantlydifferent from those for viruses Consequently, standard bacterial indicatormonitoring does not accurately reflect the presence of these human pathogenic

viruses, especially in shellfish (Formiga-Cruz et al., 2003; Dore et al., 2003,2000; Dore and Lees, 1995; Richards, 1988; Metcalf et al., 1979) In addition,direct enumeration of noroviruses, hepatitis A viruses, and other humanenteroviruses in water and shellfish is again not practical Thus, a reliableindicator that represents the viral quality of waters and shellfish is needed.The standards by which different countries classify shellfish harvesting areasare based on both water quality and the sanitary quality of shellfish tissue.Despite this basic difference, both systems use fecal-borne bacterial indicators

as standards, fecal coliforms for the water quality-based system, and E coli forthe tissue-based standard These are closely related because E coli is a fecalcoliform species Lees (2000) summarized the different legislative standards forharvesting live shellfish in the US and the EU Each classification system hastwo related classifications that require the same treatment of harvested shellfish:approved and restricted classifications in the US correspond to Categories A and

B in the EU, requiring no treatment and depuration or relaying, respectively The

EU includes a Category C that requires protected relaying for >2 months Whenmicrobiological standards in each system are exceeded, then harvesting isprohibited

Fecal coliforms and E coli have been shown to be poor indicators of viruslevels in shellfish (Formiga-Cruz et al., 2003; Dore et al., 2003, 2000; Dore andLees, 1995; Richards, 1988; Metcalf et al., 1979) There are also reports on thepresence of enteric viruses in shellfish otherwise meeting standards (Romalde etal., 2002), and sporadic outbreaks of shellfish-borne illnesses caused by viralpathogens continue to occur periodically around the world (Ng et al., 2006;Gallimore et al., 2005; Cheng et al., 2005) Thus, existing bacterial indicators ofsewage are not reliable indices of risk from sewage-borne viral pathogens As aresult, considerable effort over the past two decades has been focused onidentifying a more reliable assay for virus levels (Lees, 2000) Several viralindicators have been proposed (Muniain-Mujika et al., 2003), but several recentstudies in Europe and the US have identified male specific coliphage as apotentially reliable viral indicator (Dore et al., 2003, 2000; Formiga-Cruz et al.,2003; Muniain-Mujika et al., 2003; Chung et al., 1998; Dore and Lees, 1995;Havelaar et al., 1993)

Male-specific coliphage (MSC) are bacterial viruses (bacteriophage) thatinfect and replicate in E coli cells that have F-pili MSC are RNA or DNAviruses that do not replicate in E coli below about 28 ëC (Dore et al., 1995;Woody and Cliver, 1995), so replication in most shellfish harvestingenvironments is limited Relatively low levels of the MSC are found in freshhuman and animal feces, and high levels occur in sewage (Calci et al., 1998).Enumeration methods for MSC are relatively inexpensive, easy to perform, andrapid, providing results within 24 hours As such, these bacterial viruses arepotentially important as indicators of sewage contamination monitoring the viralquality of shellfish harvesting waters and shellfish tissue In the US, theInterstate Shellfish Sanitation Conference (ISSC) is in the process of evaluatingMSC as a viral indicator (ISSC, 2007) A quantitative relationship between

measurable levels of MSC and the absence of viral pathogens may exist (Dore etal., 2000), though further studies would be needed to verify this in a widergeographical context and under a range of environmental conditions.

1.4.3 Other limitations

Neither fecal-borne bacteria nor viral indicators are useful in managing shellfishharvesting for control of diseases from naturally occurring vibrios (Marino et al.,2005; Koh et al., 1994; Shiaris et al., 1992), though Robertson and Tobin (1983)found V parahaemolyticus concentrations increased with increases in fecalindicator bacteria concentrations in Nova Scotia In the US, the ISSC has formed

a Vibrio Management Committee with subcommittees to develop illness controlmeasures for both V vulnificus and V parahaemolyticus The main strategyrecommended for control of V vulnificus has been to require development andimplementation of a Vibrio vulnificus Management Plan in any state with two ormore V vulnificus illnesses confirmed from shellfish originating from that state.The plan consists of increased educational efforts toward individuals withunderlying health conditions that increase their risk for disease, processes totrack each individual illness and products implicated in illnesses, limited harvestrestriction, reduction in time from harvest to refrigeration, and post-harvesttreatments

For V parahaemolyticus, the main pre-harvest strategy to control illnesses is

an interim control plan The plan involves reacting to illnesses from an identifiedshellfish growing area, notify the shellfish industry and public health agencies ofthe potential for illness at historical times of onset/time of year, conduct anoyster meat sampling and assay program, recommend post-harvest temperaturecontrol, and, in more intensive cases, issue a consumption warning or ban onharvesting (ISSC, 2005; Daniels et al., 2000) Monitoring of shellfish for V.parahaemolyticus would be required in all areas where V parahaemolyticus hasoccurred, the extent to which is dependent on the frequency of outbreaks in theprevious 3 years Growing areas would be closed for harvest if five or more tdh+

V parahaemolyticus colony-forming units were detected per 0.1 g tissue inreplicate samples Reopening these areas would require confirmation throughfurther testing of shellfish containing fewer than this threshold concentration intwo consecutive samples

The Food and Drug Administration (FDA) has recently proposed the MSCassay for use in the National Shellfish Sanitation Program (NSSP) to monitorviral levels in quahogs and oysters as a means to re-opening harvest areasimpacted by emergency closures due to wastewater treatment failure (ISSC,2007) Shellfish that have been subjected to sewage contamination are assumed

to contain viral pathogens and the NSSP presently requires a 21-day closure.Detection of MSC as indicators of pathogenic viruses in shellfish could be used

as the basis for re-opening shellfish harvest areas in as few as 8 days after asewage contamination event Beyond harvest limitations resulting from emerg-ency wastewater treatment failure events, significant areas of shellfish resources

lie within the direct outfall zones of wastewater treatment facilities whereshellfish harvesting is prohibited Partially because of the lack of dataconcerning pathogen levels in shellfish the outfall zones, the FDA has adoptedthe relatively conservative 1 : 1000 dilution line for bed closure Research isneeded to determine how restrictive such closures should be, either to supportthis or a different strategy.

Several other issues remain as significant impacts of microbial contamination

on the international shellfish industry The sources for most microbialcontamination remain unknown as a result of a lack of programs to incorporatemicrobial source tracking methods into harvest area classification strategies.Under these conditions, general closures result even though the actual sources ofelevated levels of indicator bacteria may have little or no public health signifi-cance Outbreaks caused by shellfish from countries with inadequate controlprograms in place, or from illegal product from areas where harvesting isprohibited can be devastating influences on consumer attitudes to shellfishconsumption Public education efforts are key strategies for allaying the effectsand to aid the press in accurate reporting on these incidences

1.5 Incidence of microbial contamination in shellfish waters

The incidence of microbial contamination is a concern at critical steps in theprocess from when shellfish are harvested from coastal waters to the consumer,including post-harvest conditions that may exacerbate contamination risks.Besides post-harvest processes, which are covered in a different chapter, ele-vated temperatures during handling of shellfish immediately after their harvestcan stimulate growth of pathogenic bacteria, especially vibrios, in shellfishtissue that increases their potential for infection Temperature conditions arethus critical during harvest and initial processing, transportation and retailing.One management strategy currently used for controlling vibrios in US shellfishstock is to minimize temperature of freshly harvested oysters to reduceproliferation (ISSC, 2005) DePaola et al (2000) state that any tdh and/or trh-positive strains of V parahaemolyticus in oysters from the environment atconcentrations >10/g tissue would be extraordinary In addition, theconcentrations of total V parahaemolyticus rarely exceed 104/g This may atfirst suggest a minimal public health threat Vibrios are, however, known togrow rapidly to much higher concentrations, frequently >104/g, underinadequately managed post-harvest conditions, i.e., elevated temperatures fromlack of refrigeration (Cook et al., 2002) The needle in the haystack can thusbecome a highly significant strain within the oyster microbial community

1.5.1 Environmental effects on microbial contamination in shellfish watersPathogenic vibrio species

The abundance of vibrios in natural habitats, including bivalve shellfish (Pruzzo

et al., 2005) and seaweeds (Mahmud et al., 2007) is intimately tied to abiotic

factors (Martinez-Urtaza et al., 2008; Randa et al., 2004; Louis et al., 2003;Pfeffer et al., 2003; Lipp et al., 2002; Motes et al., 1998; Jones and Summer-Brason, 1998) Temperature and nutrients strongly correlate with populations ofall Vibrio spp; however, several studies indicate that distinct Vibrio populationsare also associated with specific environmental conditions (Sousa et al., 2006;Eiler et al., 2006; Thompson et al., 2004) An important predictor of bothgeneral and specific populations is salinity Vibrios tolerate a wide range ofsalinity (Farmer et al., 2005), with high levels detected at moderate salinities (5±

25 ppt) and an inverse correlation between salinity and abundance at elevated(>25 ppt) salinities (Martinez-Urtaza et al., 2008; Huq et al., 2005; Castaneda etal., 2005; Parvathi et al., 2004; Randa et al., 2004; Louis et al., 2003; DePaola etal., 2000; Motes et al., 1998; Chowdhury et al., 1992; O'Neill et al 1992; Miller

et al., 1982; Singelton et al 1982; Kaper et al., 1979) Other factors such asdissolved organic carbon (DOC), suspended solids and plankton and copepodsmay also play a role (Gugliandolo et al., 2005; Jones and Summer Brason, 1998;Watkins and Cabelli, 1985) These correlations suggest direct effects of salt andother environmental factors on Vibrio growth and or survival in estuarineecosystems The potential for growth of pathogenic vibrios in harvested oysters

is a highly significant factor relative to the public health risk for these bacteria(FDA, 2005; FAO/WHO, 2005) Surprisingly, despite these correlationsbetween salinity and vibrio abundance or virulence, the direct mechanisticeffect of salt on vibrios has been investigated to only a limited extent For V.cholerae, high salinity induces conversion to the viable but non-culturable(VBNC) state (Thomas et al., 2006), whereas for V vulnificus higher saltconcentrations in sterile water reduced viability (Kaspar and Tamplin, 1993).Increases in total Vibrio populations seasonally or through time stronglycorrelate with increased temperature (Colwell, 2004; Thompson et al., 2004;DePaola et al., 2000), and nutrients (DOC) (Jones and Summer-Brason 1998).The climatic variables of higher temperature and increased flow/lower salinityare predicted conditions associated with global climate change (Louis et al.,2003) Acute rainfall can also result in the discharge of untreated waste intoestuaries leading to increases in nitrogen and DOC

There have been many studies on the effects of environmental factors onpathogenic vibrio species throughout the world (Martinez-Urtaza et al., 2008;Lhafi and Kuhne, 2007; Zimmerman et al., 2007; Normanno et al., 2006; Eiler

et al., 2006; Huq et al., 2005; Binsztein et al., 2004; Parvathi et al., 2004) All ofthese have served to provide useful information to shellfish growers, regulators,and researchers Much less is known about the incidence of pathogenic strains of

V vulnificus and V parahaemolyticus, especially in colder waters like those ofthe northeast US Perhaps the key study on the occurrence of pathogenic V.parahaemolyticus strains in the US was by DePaola et al (2003), in which21.8% of samples of Alabama oysters had detectable pathogenic strains Bauer

et al (2006) found a low frequency of trh+ but no tdh+ V parahaemolyticusstrains, while V vulnificus was detected in only 0.1% of 885 blue musselssamples from the cold waters of Norway DePaola et al (2000) found tlh

positive V parahaemolyticus in oysters collected from Oyster Bay, NY, inOctober following the outbreak from that area in 1998, but no tdh+ strains Thewater temperature in October was low enough to decrease total V parahaemo-lyticus levels to <100/g, with one exception from 15 samples Thus, fewpathogenic strains of V parahaemolyticus have been detected in northern watersand it is unclear at present what conditions may be conducive to their growthand persistence.

The ecological and evolutionary factors that favor the minority pathogenicstrains remain elusive (Zimmerman et al., 2007) Evidence is also building thatthe frequency of epidemics caused by typically non-pathogenic microbes such asvibrios is on the rise as a result of changes in land use and climate (Paz et al.,2007) For example, nutrient enrichment could differentially promote prolifera-tion of microbial species or strains of differing virulence potential and climatevariability can have profound impacts resulting from changes in precipitationand temperature (Louis et al., 2003) Whereas the individual effects of thesephysical variables are documented, their combined or synergistic effects are notunderstood The inherent seasonal and spatial variability of climate (e.g.,rainfall/salinity, pH, temperature) and nutrients (e.g., nitrogen, DOC, dissolvedoxygen, chlorophyll) in shellfish-growing waters makes them ideal sites forgenetic diversity and evolution of resident and pollutant bacterial species thatmay harbor pathogenic strains Evidence for selection of different V vulnificusgenotypes in oysters compared with surrounding waters has recently beenreported in North Carolina and Florida (Warner and Oliver, 2008) and elsewhere

in Florida (Gordon et al., 2008)

Fecal-borne bacteria and viruses

The environmental fate of pathogens and indicator bacteria ultimatelydetermines strategies for management of sources of shellfish contamination.The capacity for survival of microbial species in natural environments dictatestheir potential for exposure and the persistence of public health threat ofpathogens to humans The incidence of fecal contamination has often beenrelated to seasonal and meteorological events Fecal coliforms were detected atelevated levels in the winter when evapotranspiration was lower and runoffhigher over 49 years in the Tchefuncte River, Louisiana (Barbe and Francis,1995) Seasonal differences in wind speed, direction, and gull roosting hadimportant impacts on coliform concentrations in the Quabbin Reservoir inMassachusetts (Garvey et al., 1998) High tributary flows that affect turbidity ofinstream and downstream waters can affect the degree to which sunlight candecrease contaminant concentrations Sunlight is an important factor in survival

of protozoan, bacterial and viral pathogens in surface waters (Johnson et al.,1997), and the greater sunlight during the summer in the US is thought tocontribute to increased die-off during summer months, in addition to increasedtemperatures Increased temperatures can also increase the activities of bio-logical factors such as protozoa that graze on and decrease concentrations ofpathogens and other bacteria (Gonzalez et al., 1992) Thus, the influence of

seasonal changes in a variety of factors can affect the incidence and persistence

of fecal-borne pathogens and indicators

The transport and fate of indicator and pathogenic microorganisms instormwater runoff and other sources can be affected by a variety of physical,chemical, and biological factors Schillinger and Gannon (1985) found >50% offecal coliforms, Klebsiella pneumoniae, and Gram-negative bacteria were notadsorbed to particles > 5.0 m in diameter in natural stormwater in Michigan.This showed that most of the microorganisms of concern were not physicallyremoved from the effluent, but the fact that a significant fraction of themicroorganisms were attached suggests that bacterial attachment is a significantfactor in the fate in stormwater runoff Milne et al (1986) studied the adsorption

of fecal coliforms to both estuarine and sewage effluent suspended solids inlaboratory experiments They found almost immediate adsorption of fecalcoliforms (20%) to estuarine suspended solids and this increased with time,indicating that fecal coliforms are deposited to estuarine sediment bedsdownstream of effluent discharges

Once the bacteria enter the water column, they may be more susceptible toenvironmental conditions Solar radiation has been shown to be a major factor inthe die-off of fecal coliforms and enterococci (Kay et al., 2005; Solic andKrstulovic, 1992) Shiaris et al (1992) found tidal exposure to be a significantfactor associated with disappearance of fecal coliforms, and probablyenterococci, in sediments below a sewage outfall in Massachusetts, probably

as a function of solar radiation Pommepuy et al (1992) showed that Salmonella

sp survived longer in turbid rather than clear marine waters because thesuspended particles helped to protect bacterial cells from sunlight Sorensen(1991) and Gonzalez et al (1992) showed that predation by eucaryoticmicroorganisms was a very significant factor controlling bacteria survival inmarine waters De Vicente et al (1988) reported greater die off of P aeruginosa

in laboratory seawater than in freshwater, but P aeruginosa die off was slowerthan that of fecal and total coliforms in seawater

Studies by Pettibone et al (1987) and Evison (1988) both found enterococcibetter able to survive in the environment compared with E coli Growth of fecal-borne bacteria may also be possible in marine fish that live near sewage outfallsand polluted beaches (Silva and Hofer, 1993) Temperature by itself typicallyincreases die off of bacteria and protozoa (Medema et al., 1997), but other factorscan contribute to die off at higher temperatures There is a balance in some areas

as increased temperature also is accompanied by increases in re-growth of someorganisms Anderson et al (1997) showed summer levels of enterococci inseaweed were elevated compared with winter levels They concluded this reflectseither re-growth or increased contamination by animals and insects Weiskel et

al (1996) also found evidence of increases in fecal coliforms during summer inshoreline deposits of decaying vegetation/wrack in Buttermilk Bay, MA.There are many studies that have shown E coli to be present in shellfish and

in overlying waters, and they come from a variety of sources (McLellan, 2004).Other fecal-borne bacteria also occur in shellfish, including Salmonella and

Campylobacter spp (Brands et al., 2005; Teunis et al., 1997; Wilson andMoore, 1996) Fecal-borne bacteria typically depurate rapidly from shellfishtissue following contamination events (Marino et al., 2005; Croci et al., 2002;Jones et al., 1991), and may have short residence times in shellfish in harvestwaters The situation is different for viruses Enteric viruses and MSC alwaysremain in shellfish for longer periods of time and at higher levels than fecal-borne bacteria (Lees, 2000) Enteric viruses and MSC have been detected inshellfish from many areas in the world (Umesha et al., 2008; Dore et al., 2003)and can persist for extensive periods in the marine environment (Gantzer et al.,1998; Callahan et al., 1995) and in shellfish (Myrmel et al., 2004; Formiga-Cruz

et al., 2002; Lees, 2000)

An interesting and consistently observed phenomenon is the higher levels ofMSC and enteric virus incidence during winter that accompanies an increase indisease incidence compared with other months Several research groups havereported this in many areas of the world, including Norway (Myrmel et al.,2004), England and Wales (Dore et al., 2003), France, and the US (Burkhardtand Calci, 2000) Burkhardt and Calci (2000) suggested this may be associatedwith selective accumulation of viruses by oysters, and potentially other shellfish,during the winter

Catastrophic natural events

Some disasters that result from catastrophic natural events may be unusual yetsignificant sources of microbial contamination to coastal waters, includingshellfish harvesting areas Hurricane Katrina that impacted the US Gulf ofMexico in August 2005 not only resulted in the closure of shellfish harvestingareas well into September 2005, but also caused loss of product that spoiledowing to lack of refrigeration or exposure to flood waters following thehurricane (FDA/CFSAN, 2005) Elevated levels of pathogenic vibrio specieswere detected in post-hurricane floodwaters, (Demcheck et al., 2005) Fivedeaths involving V parahaemolyticus and V vulnificus as wound infectionsoccurred as a result of exposure to contaminated flood waters (CDC, 2005) In asimilar fashion, after 42 consecutive days of rain during March 2006 in theHonolulu area of Hawaii, a sewage main ruptured and untreated wastewaterspilled into a canal (UH-WRRC, 2006) The canal contained ~8  105entero-cocci/100 ml, and there were three related wound infections reported, two notserious, with one of those related to fecal-borne bacteria The other involved a V.vulnificus infection where a man died 2 days after falling into a polluted canal

In many other areas of the world, disasters may displace populations who thenlack sources of clean water and sanitation, creating conditions where cholera andother diseases may thrive

1.5.2 Microbial physiology and survival capacity

There seems to be an obvious ecological purpose for the many bacteria that enter

a VBNC physiological state A variety of conditions can induce this state in

bacteria, including low temperatures and carbon/energy source concentrations,both of which are key ecological conditions that could affect survival in marineecosystems This condition has more significant public health implicationsbecause of the potential for pathogens to be present yet not be detected usingtraditional culture-based methods, and may affect molecular genetic methods(Vora et al., 2005) The pathogenic vibrio species of concern to shellfishconsumers are enteropathogenic and also retain pathogenicity in the VBNC state(Vora et al., 2005; Binsztein et al., 2004; Wong et al., 2004; Nishino et al.,2003; Mizunoe et al., 2000; Whitesides and Oliver, 1997) Many fecal-bornebacteria also exhibit the VBNC state Pommepuy et al (1996) reported VBNC

E coli retained enteropathogenicity even when exposed to seawater andsunlight Enterococcus faecalis can survive for long periods of time without anynutrients (Hartke et al., 1998) and can be detected as VNBC cells usingcompetitive polymerase chain reaction (PCR) (Lleo et al., 1998)

The traditional concept for fecal indicator bacteria is that they are adapted forexistence in gastrointestinal tracts and cannot survive for long in theenvironment, especially in seawater Not only can they survive, but some havebeen shown to grow under favorable conditions (Ishii et al., 2007; Kon et al.,2007; Jones et al., 2006; Hartel et al., 2005; Solo-Gabriele et al., 2000) Inaddition, wastewater disinfection is intended to be lethal for microorganisms,and its effectiveness is essential for preventing shellfish exposure to potentiallyabundant bacterial and viral pathogens in wastewater In many comparativestudies, E coli, Ent faecalis and other fecal-borne bacteria have been shown to

be inactivated by chlorine to a greater degree and more rapidly thanenteroviruses and MSC (Tree et al., 2003, 1997; Tyrrell et al., 1995) Bolster

et al (2005), however, found E coli could not only survive chlorination, butcould also grow in some estuarine water, though they used a recovery methodfor E coli detection that is not commonly used Similar findings were reported

by Blatchley et al (2007) and Knorr and Torella (1995) showed evidence ofSalmonella multiplication in a wastewater depuration pond in Spain UVdisinfection (Blatchley et al., 2007) and ozone (Xu et al., 2002) have beeneffective at inactivating viruses in wastewater, with some preconditions toensure effectiveness

1.6 Contamination sources and their identification

There are several ways to define what may be considered fecal-borne sources ofmicrobial contaminants in shellfish waters One way is to consider marinas,dysfunctional wastewater treatment facilities, septic systems, animal feedlots,and various types of runoff as sources Another way is to consider actual sourcespecies The latter fall into categories that include human versus non-human, or

at a more detailed level, human, pet, livestock, wild birds, and other wildanimals Still another approach would be to consider sources as being related totransport mechanism, i.e., direct deposition or transported via groundwater,

stormwater or surface water (Weiskel et al., 1996) All approaches for definingsources are useful in directing different management actions to eliminatecontamination of shellfish harvesting waters.

The following section discusses different sources of microbial contaminationand strategies used to identify sources Pollution source identification is acritical step in the management of shellfish harvesting waters Strategies thatresult in accurate identification of the most significant source(s) of pollution areinvaluable for focusing the most effective allocation of what are often scarceresources to improve water quality Though naturally occurring pathogens such

as Vibrio spp are significant public health concerns for the shellfish industry,their source is for the most part the natural environment; their incidence relative

to fecal contamination was discussed in the previous discussion It is the tially manageable fecal-borne contamination, from sewage and other sources,that is the focus of this section

poten-The categorization of sources of microbial contamination as they relate toactual source species is inclusive of other ways of categorizing sources At thesimplest level of discrimination, sources can be considered to be of either human

or non-human origin Examples of what constitutes human and non-humansources in shellfish areas in the Northeast US are presented in Table 1.2 Thedelineations could be rearranged depending on perspective, but this schemesuggests different strategies for managing different types of contamination

Table 1.2 Different sources of fecal contamination in

shellfish areas of the northeast US

Human sources

Point sources

Inadequate wastewater treatment facility sewage treatment

Illegal sanitary connections to storm drains

Illegal disposal to storm drains

Combined sewer overflows

Sanitary sewer overflows

Foxes, raccoons, skunks

Livestock and rural wildlife

Cattle, horses, poultry, other livestock

Otters, muskrats, deer, bear

Waterfowl

sources Both types can be significant sources of contamination Accurateidentification of which type of source may be significant is obviously critical forassessing public health risks and resource allocation for eliminating sources.Human pathogenic viruses are from human sources Thus, the prevailingperception is that there is a greater public health risk associated with humansources compared with non-human sources (Sinton et al., 1998; Wiggins, 1996;O'Shea and Field, 1992) The public health significance of non-human sources

of fecal pollution may be less than for human-borne sources, but pathogens mayalso occur in non-human sources (Arnone and Walling, 2007) and can be aconcern when exposure to high concentrations occurs The US EnvironmentProtection Agency (EPA) has recently acknowledged the potential public healthrisks associated with non-human sources of pollution (US EPA, 2007) It isaccepted that pathogens in humans are only present in significant numbers indiseased individuals, while some animal hosts may act as more stable reservoirs

of pathogens There are many reports of human disease occurrence associatedwith livestock animals, but few reported cases associated with wild animals(Field and Samadpour, 2007; Craun et al., 2004) This difference betweendomestic and wild animals is probably a function of both the frequency ofexposure and the number of studies conducted on the different animal groups.Diseases associated with exposure to pet feces are also well documented, sodomestic animal sources are of concern at a level that falls between that forhuman sources and wild animal sources There are a variety of transportmechanisms by which these types of source may contaminate shellfish waters,but little information on their significance is available at present For shellfishgrowing waters, non-human sources of microbial contamination have alwaysbeen a management dilemma because of the competing issues of public healthprotection and opening areas for harvest

1.6.1 Microbial source tracking

Recent adoption of biotechnological techniques for application to water qualityissues has spawned a number of approaches to address identification of sources offecal-borne contamination Many methods use non-microbiological approaches

to identify sewage contamination, while other approaches that utilize organisms are often called `microbial source tracking' (MST) methods MSTmethods have been used successfully for nearly 15 years throughout the US Thevarious methods range from those that determine phenotypic aspects of bacteria

micro-to cultivation and `library'-dependent methods micro-to direct detection of species specific genetic markers that allow for source tracking

source-There has been a series of reviews that have compared the advantages anddisadvantages of a comprehensive list of MST methods The US EPA publishedthe `Microbial Source Tracking Guideline Document' in 2005 (US EPA, 2005) as

a comprehensive review of all aspects of MST up to that time period The generalconsensus conveyed in that and other independent reviews has been that mostMST methods have merit, no one method has been deemed superior to the others