Evaluating the Importance of Zoonotic Bacteria

EVALUATING THE IMPORTANCE OF ZOONOTIC BACTERIA,

ANTIMICROBIAL USE AND RESISTANCE IN AQUACULTURE AND SEAFOOD

A Thesis

Presented to

The Faculty of Graduate Studies

The University of Guelph

NATASA TUSEVLJAK

In partial fulfillment of requirements

for the degree of

Master of Science

April, 2011

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I*: Canada ABSTRACT

EVALUATING THE IMPORTANCE OF ZOONOTIC BACTERIA,

ANTIMICROBIAL USE AND RESISTANCE IN AQUACULTURE AND

SEAFOOD

Natasa Tusevljak Co-Advisors: University of Guelph, 2011 Dr. Andrijana Rajic Dr. Scott McEwen

This thesis employs research synthesis methods (scoping study (ScS) and systematic review-meta-analysis (SR-MA)) to characterize and evaluate the existing primary research on antimicrobial use (AMU), antimicrobial resistance (AMR), selected zoonotic bacteria in aquaculture. It also describes an elicitation of global expert opinion on these topics. Evidence maps from the ScS identified knowledge gaps (AMU and the association between AMU-AMR), and strengths (prevalence/interventions for Vibrio and

Aeromonas at the farm level). SR revealed consistent flaws in methodology/reporting of research on prevalence/concentration of Aeromonas, E. coli. Salmonella and Vibrio in clams, mussels, oysters, salmon, shrimp (including prawn), and tilapia from processing to retail. Retail level MA revealed significant heterogeneity across the studies. The questionnaire achieved a response rate of 32.9% (n=199) and was most representative of

North Americans (n=152) involved in clinical work (n=91). Respondents reported quinolone use in North America, Europe and Asia; frequent tetracycline use and resistance across aquatic species. ACKNOWLEDGEMENTS

I would like to extend my sincerest gratitude to my advisory committee, particularly Drs. Andrijana Rajic, Lucie Dutil and Scott McEwen who have provided constant guidance and support throughout my journey over the past two years. Your dedication to the field of Epidemiology and your students is truly inspiring. I would also like to thank my coworkers at PHAC who dedicated time in support of this project as well as PHAC for funding.

To the many reviewers, pre-testers and procurers that were involved throughout the development and progression, this project was truly a team effort and my deepest thanks for your unwavering dedication. To my fellow graduate students who maintained their free spirits and lightheartedness, thank you for knowing just how to break up a busy day.

To my parents, Dragomir and Vesna, and my sister, Dejana, I stand in awe of your strength and unyielding support in my attempts to create new paths. Thank you for the freedom in allowing me to do so. LIST OF TABLES

Table 1.1. Comparisons between traditional literature reviews, scoping studies and systematic reviews 28 Table 2.1. Demographic characteristics of respondents to a questionnaire soliciting information on AMU and AMR administered globally to aquaculture professionals 49 Table 2.2. Reported respondent level of experience with various aquatic species 51 Table 2.3. Reported antimicrobial use and observation of resistance to antimicrobial drug classes for various aquatic species 52 Table 2.4. Reporting of antimicrobial usage per region for all aquatic species presented for all respondents and excluding those responding only for ornamental fish 55 Table 2.5. Reported observed bacterial resistance for selected bacteria and antimicrobial drug classes ...58 Table 3.1. Number of relevant quality-assessed studies submitted to data extraction by aquatic species, bacteria and point in food chain 88 Table 3.2. A summary of various subsets of data reporting the prevalence of four selected bacteria in four aquatic species-seafood categories at retail (including RTE) and sub-grouped by region of sampling to improve interpretation and decrease heterogeneity 90 Table 3.3. A summary of various subsets of data reporting the concentration of three selected bacteria in selected aquatic species-seafood categories at retail (including RTE) and sub-grouped by region of sampling to improve interpretation and decrease heterogeneity 93

ii LIST OF FIGURES

Figure 1.1. A stepwise depiction of the study goals and components: scoping study- systematic review and expert questionnaire 29 Figure 3.1. Scoping study and systematic review process flow-chart 94 Figure 3.2. Evidence mapping. Distribution of primary research according to type of study, topic, pathogen, aquatic species and point in food chain 96 Figure 3.3. Random effects meta-analysis of the prevalence (expressed as proportion) of Aeromonas, E. coli and Salmonella on salmon 97

iii TABLE OF CONTENTS

ACKNOWLEDGEMENTS I

LIST OF TABLES II

LIST OF FIGURES Ill

TABLE OF CONTENTS IV

CHAPTER ONE

INTRODUCTION, LITERATURE REVIEW AND OBJECTIVES .1

INTRODUCTION 1

LITERARATURE REVIEW 2 Overview of the Global Aquaculture Industry 2 Overview of Aquaculture and Seafood Industries in Canada 4 Food Safety Risks Associated With Aquaculture and Seafood 5 Zoonotic Bacteria in Aquaculture and Seafood 6 Antimicrobial Use (AMU) in Aquaculture 8 Antimicrobial Resistance (AMR) in Aquaculture , 9 Monitoring of AMR and AMU and Control of AMR 10 Current Legislative Framework for Production of Safe Seafood in Canada 11 Research Synthesis Methods and Their Application in Zoonotic Public Health 11 Scoping Studies as Tools for Evidence Mapping 12 Systematic Review -Methodology and Comparison to Scoping Study 13 The Meta-Analysis Approach to Understanding Global Evidence 14 The Use of Questionnaires in Epidemiological Research 14

STUDY RATIONALE AND OBJECTIVES 16

REFERENCES 19

CHAPTER TWO

ANTIMICROBIAL USE AND RESIT ANCE IN AQUACULTURE: FINDINGS OF A GLOBALLY ADMINSITERED SURVEY OF AQUACULTURE-ALLIED PROFESSIONALS 30

iv SUMMARY 30

INTRODUCTION 31

MATERIALS AND METHODS 32 Questionnaire design description 32 Database of aquaculture-allied professionals 33 Survey administration 34 Data analysis 34

RESULTS 35 Respondent demographic characteristics and non-response bias evaluation 35 Overview of respondent professional experience 35 Opinions on Frequency of AMU and AMR in various aquatic species ..36 AMR in selected bacteria 37 Knowledge of AMR monitoring and surveillance 38

DISCUSSION 38

ACKNOWLEDGEMENTS 42

REFERENCES 43

CHAPTER THREE

PREVALENCE OF SELECTED ZOONOTIC BACTERIA IN SELECTED AQUATIC SPECIES AND SEAFOOD: A SCOPING STUDY, SYSTEMATIC REVIEW AND META-ANALYSIS OF PUBLISHEDRESEARCH .61

ABSTRACT 61

INTRODUCTION 62

MATERIALS AND METHODS 64 ScS Study: Defining its broad scope 64 Search Strategy and Execution 65 Relevance Screening and Exclusion Criteria 66 Development of Focused Question for SR 67 SR; Inclusion of Studies, Methodological Assessment and Data Extraction 67 Management of ScS and SR i 69 Meta-analysis 69

RESULTS 71 ScS: Research Themes, Study Characteristics and the Focused Question 71 SR Database and Meta-Analysis 72

V DISCUSSION 73 SR-MA highlights 73 Knowledge gaps 75 Recommendations for future seafood surveys 77 Limitations and strengths of the study 78 Relevance in the Canadian context 79

CONCLUSION 80

ACKNOWLEDGEMENTS 81

REFERENCES 82

CHAPTER FOUR

SUMMARY DISCUSSION AND CONCLUCIONS 98

SUMMARY DISCUSSION 98

CONCLUSION 104

REFERENCES 106

APPENDIX 2. QUESTIONNAIRE: SURVEY ON ANTIMICROBIAL USE AND RESISTANCE IN ZOONOTIC BACTERIA IN AQUACULTURE, SEAFOOD AND ORNAMENTAL/PET FISH 109

APPENDIX 2.1. Questionnaire-English Version 109

APPENDIX 2.2. Questionnaire-Spanish Version 123

APPENDIX 2.3. Descriptive Statistics Report 141

APPENDIX 3. PREVALENCE OF SELECTED ZOONOTIC BACTERIA IN SELECTED AQUATIC SPCOES AND SEAFOOD: A SCOPING STUDY, SYSTEMATIC REVIEW AND META-ANALYSIS OF PUBLISHED RESEARCH 170

APPENDIX 3.1. Project protocol, methodology and decisions 170

APPENDIX 3.2. Search algorithms as they were put into the databases 186

APPENDIX 3.3. List of search verification references 187

APPENDIX 3.4. Definitions for Reviewers 193

vi APPENDIX 3.5. Relevance Screening Tool 1 201

APPENDIX 3.6. Relevance Screening Tool II 205

APPENDIX 3.7. Assessment of methodological quality and reporting 214

APPENDIX 3.8. Data extraction tool for prevalence studies 234

APPENDIX 3.9. Bibliography of studies included in the SR 239

APPENDIX 3.10. Extracted data from 72 studies included in SR 245

vii CHAPTER ONE

INTRODUCTION, LITERATURE REVIEW AND OBJECTIVES

INTRODUCTION

The global and domestic aquaculture industries have grown considerably over the past two decades due to increased consumer demand. This has positioned aquaculture

(farmed or cultivated fish/shellfish) and seafood production (derived from aquaculture or wild-caught fish/shellfish) as important parts of the food production sector. This growth has, however, been accompanied by several environmental, food safety and other public health issues. Besides many environmental concerns, which are beyond the scope of this thesis, the main food safety and public health issues range from zoonotic pathogens, food-bome disease associated with certain types of seafood, microbial safety of imported/exported products, antimicrobial use (AMU), and two related public health issues, antimicrobial resistance (AMR) and drug residues. Some topics have been studied less thoroughly (e.g., antimicrobial use) than others (e.g., paralytic shellfish poisoning) for which an extensive literature has already accumulated. In addition, knowledge gaps may frequently exist in the literature and until they can be addressed through additional published research, emphasis is sometimes placed on expert opinion when action or decision is needed.

Specifically, the issues of AMU, AMR and zoonotic bacteria in aquaculture and seafood production have not received the same attention as they have in other food production sectors. Though this may not be a reflection on the importance of these issues to aquaculture and seafood production, this information remains to be elucidated. To

1 date, the collective body of knowledge surrounding these issues, composed of both

primary research and expert opinion, has not been synthesized; a process crucial for fully

informed decision- making by stakeholders.

This thesis characterizes and evaluates the existing primary research on AMU,

AMR and prevalence of selected zoonotic bacteria in aquaculture, and describes an elicitation of global expert opinion on AMU and AMR in order to fill some important data gaps. It is intended that this information will be useful for Canadian decision-makers regarding identification of surveillance needs in aquaculture and seafood and knowledge gaps in need of future research in these fields.

Given that research synthesis methods are investigated in this thesis, as well as their application in the aquaculture and seafood industry, technical and contextual background information, including rationale for this study and specific research objectives are provided in lieu of a traditional literature review. Additional subject- specific literature is extensively reviewed in subsequent chapters. The literature review in this chapter includes: an overview of the aquaculture industry (both global and domestic); hazards associated with seafood (chemical, physical, with a focus on biologic, specifically bacterial pathogens); AMU and AMR in aquaculture; current surveillance initiatives and regulations associated with aquaculture and seafood products; and application of research synthesis methods and questionnaires in epidemiological research.

LITERARATURE REVIEW

Overview of the Global Aquaculture Industry

2 The aquaculture industry is the fastest growing food-animal production sector in the world (FAO, 2008). Comprising both capture fisheries and aquaculture operations,

the seafood industry produced a total of 146.3 tonnes of products worldwide in 2008;

110.4 million tormes was fish and shellfish were intended for public consumption and the remainder was used for the production of fishmeal and fish oil (State of World Fisheries

and Aquaculture, 2008). About 47% of the total seafood harvest was produced in

aquaculture operations; the rest was catch from both inland and marine fisheries (State of

World Fisheries and Aquaculture, 2008). Globally, fish/shellfish provided 2.9 billion

people with 15% or more of their average per capita animal protein intake (State of

World Fisheries and Aquaculture, 2008). Ranked by quantity produced in 2008, the

major aquaculture species groups included freshwater fish (54%), mollusks (27%),

crustaceans (9%), diadromous fish (6%), and marine fish (3%) (FAO, 2008).

Approximately 90% of aquaculture production comes from the Asia-Pacific

region and the rest is contributed by Europe (4.2%), Latin America and the Caribbean

(3.0%), Africa (1.5%) and North America (1.2%) (FAO, 2(X)8). The dominance of the

Asia-Pacific region is largely attributable to China, which comprises 67% and 49% of

global production and global value, respectively (FAO, 2008). The other large

aquaculture-producing countries in this region (ranked by production volume) include

India, Vietnam, Thailand, Indonesia, Bangladesh, Japan, and the Philippines (FAO,

2008).

The main regions of production vary with fish/seafood species. For example, the

Asia-Pacific region produces 98 % of carp, 95% of oyster and 88% of shrimp (including

prawns throughout this thesis) production worldwide (FAO, 2008). Furthermore, the

3 largest producers of salmon are Norway and Chile, accounting for 33% and 31% of the

total worldwide production, respectively (FAO, 2008). The production of salmon in

South America and the Caribbean has over-taken shrimp production, previously the

primary product of these regions (FAO, 2008).

Overview of Aquaculture and Seafood Industries in Canada

Canadian aquaculture spans from the East to the West coast, producing a wide

range of fish and shellfish species. All Canadian provinces are involved in aquaculture

production with British Columbia, New Brunswick and Prince Edward Island being the

leading provinces (Canadian Aquaculture Industry Alliance, 2008). In 2008, the

Canadian aquaculture industry was valued at approximately $5 billion and provided

130,000 jobs (Canada Food Statistics, 2009). The four main categories of species by

volume were salmon (66.7%), mussels (15.8%), oysters (8.7%) and trout (3.4%). Other

species included clams, tilapia, Arctic char, Atlantic cod, white sturgeon and scallops,

with spotted wolffish, American eels, abalone, sea cucujtnbers, sea urchins, cockles and

marine plants as emerging species in the industry (Canadian Aquaculture Industry

Alliance, 2008).

Canada ranks as the seventh leading exporter of aquaculture products in the world

and the thirteenth largest importer (Fish and Seafood Overview, 2009) (by value). The

export market largely involves salmon, blue mussels and oysters shipped to the United

States, the United Kingdom, France, Denmark, Japan and China (Fisheries and Oceans

Canada, 2009). Although salmon is the top aquaculture species in Canadian production,

Canada's contribution to the global market does not place it among those dominating the

4 species group (Canadian Aquaculture Industry Alliance, 2008). The major import product both in terms of quantity and value is shrimp followed by lobster and tuna from the

United States, China, Thailand and Vietnam (in that order) (Fisheries and Oceans

Canada, 2009b).

Increasingly, seafood and aquaculture products have become a substantial part of the average Canadian diet. In 2009, the average Canadian consumed approximately 8 kg of seafood (Fisheries and Oceans Canada, 2009a); compared to the rest of Canada, citizens residing in the Maritime provinces consumed more seafood and those in the

Prairie provinces consumed less, likely due to cultural and geographical factors

(Agriculture and Agri-Food Canada, 2005). An increase in the domestic consumption of seafood and aquaculture products is projected in the next few decades, coinciding with global trends, and influencing further growth of the industry (Agriculture and Agri-Food

Canada, 2005; FAO, 2008).

Food Safety Risks Associated With Aquaculture and Seafood

Consumption of seafood is associated with potential exposure to a number of chemical, physical and biological hazards (Huss et al., 2000). Chemical hazards include a variety of industrial chemical residues that may be present in aquaculture products and seafood due to human activity, including veterinary drug residues from fish production, heavy metals, persistent organic pollutants and pesticides. Some of these hazards can cause severe and chronic human illness due to toxicity (e.g., mercury) (Crinnion, 2000) and numerous systemic, respiratory and topical illnesses (California Department of

Pesticide Regulation, Pesticide Illness Surveillance Program, 2008). In contrast, effects

5 from physical hazards can range from bites, stings and cuts from fish spines (CDC,

2005). Other hazards are biological in nature, including viral (e.g., Norovirus, Hepatitis

A), bacterial (e.g., Aeromonas hydrophila. Vibrio vulnificus) and parasitic infections

(e.g., flukes, nematodes, cestodes, protozoa) (CDC, 2005) that can cause a wide range of symptoms, infections and diseases.

Human exposure to the microbiological hazards may occur during handling, processing, preparation and consumption of the fish and other seafood, or from leisure activities, such as fishing. In the case of bacteria, factors other than the carrier state or primary contamination of the live animals may play a role in exposure; recontamination or cross-contamination at the processing level and beyond (e.g., retail, ready to eat, restaurant, home preparation) may also be important (CDC, 2005). In other cases, for example viruses or saxitoxin in raw shellfish, contamination generally occurs prior to the processing level; during feeding where the filter feeding mechanism utilized enables bioaccumulation of pathogens or toxins (Butt et al., 2004; Feldhusen, 2000).

Antimicrobial resistance in zoonotic bacteria is of major interest in this thesis and therefore the subsequent sections focus on human bacterial pathogens and the important issues surrounding their role in seafood, aquaculture and public health.

Zoonotic Bacteria in Aquaculture and Seafood

Bacterial contamination of seafood can be classified as follows: bacteria indigenous to the aquatic environment (e.g.. Vibrio spp., Aeromonas spp.), bacteria from the terrestrial or aquatic environment (e.g.. Listeria monocytogenes), and bacteria from human or animal reservoirs (e.g., Salmonella spp., E. coli. Staphylococcus spp.. Shigella

6 spp.) (Huss et al, 2000). Exposure to these pathogens may cause a range of adverse effects on human health including gastroenteritis, septicemia, wound infection, dysentery, fever, and vomiting (Feldhusen, 2000). The incubation periods vary depending on the genus/species of bacteria (assuming ingestion of the minimal infectious dose), but can range between hours (e.g., Vibrio parahemolyticus) to days (e.g.. Yersinia enterocolitica)

(Butt et al., 2004).

Globally, the incidence of seafood-related diseases is unknown (Huss et al., 2000)

because countries are not obligated to report illness outbreaks and many lack any relevant foodbome disease recording or surveillance system (CDC, 2005). Moreover, even in countries which have incorporated these systems, such as the United States, Canada and the Netherlands, under-reporting of foodbome diseases, and the fact that surveillance systems often fail in identifying the source of infection biases the seafood-specific disease data (CDC, 2005). Since 2005, globally, 126 reported outbreaks of seafood-

related illness have occurred, 3 of which were in Canada; 73 have been caused by

bacterial pathogens including 8 caused by Staphylococcus, 23 caused by Salmonella and

26 caused by Vibrio (personal communication with Judy Greig, Global Foodbome

Outbreak Database, Laboratory for Foodbome Zoonoses, Guelph, Canada). The

Salmonella and Vibrio outbreaks were largely due to consumption of oysters and salmon

{Salmonella), and shellfish and shrimp (Vibrio). Vibrio has been implicated in seafood-

related outbreaks more frequently than any other bacteria (Kaysner et al., 2001; Liu et al.,

2004). It is a leading cause of seafood-bome disease in the United States (Kaysner et al,

2001) and Japan, and accounted for about a third of foodbome outbreaks in China (Liu et

al., 2004). Vibrio infections from seafood were reported rarely in Europe, but occasional

7 outbreaks have been reported in Spain (Lozano-Leon et al., 2003) and France (Martinez-

Urtaza et al., 2005). As with most foodbome diseases, the actual number of cases and outbreaks is thought to be higher due to underreporting. Underreporting can be caused by small size in the case of outbreaks, inability to identify pathogen (particularly in sporadic illness cases), mild illness so individuals do not seek medical care, and failure to report illness to public health authorities (DeWaal et at., 2009).

Antimicrobial Use (AMU) in Aquaculture

For the purposes of this review and thesis, the term 'antimicrobial' is restricted to the discussion of antibacterials (versus antivirals, antiparasitics, etc.). Globally, only a few antimicrobials are approved for use in aquaculture. In Canada, these drugs are restricted in labelling in salmonids and include: florfenicol, oxytetracycline, and the combinations of ormetropim and sulfadimethoxine, and of trimethoprim and sulfadiazine

(Health Canada, 2010). Tetracyclines are approved for many aquaculture indications in many countries, including Canada (CFIA, 2005), the United States (Benbrook, 2002),

India, Norway, Indonesia, Japan and People's Republic of China (FAO, 2005). In contrast, the approval of quinolones for aquaculture use is less widespread; they are not approved in Canada or the United States but are approved in certain European and Asiatic countries (FAO/OIEAVHO, 2006).

As the aquaculture industry continues to grow, selective pressure from increased utilization of antimicrobials may lead to the development of antimicrobial resistance

(AMR). Although the overall risk of AMU in aquaculture is unknown, it is estimated that

70-80% of antimicrobials end up in the environment allowing for the potential

8 development of resistant bacterial strains in environmental organisms (Hernandez, 2005).

In order to protect their therapeutic efficacy and limit antimicrobial resistance, some countries tightly regulate or have prohibited the use of certain antimicrobials in aquaculture. However, other countries which are leading producers and exporters are believed to have lax regulations concerning antimicrobial use (FAO, 2005).

Antimicrobial resistance does not respect international borders; therefore trade is a potential mechanism for international spread of resistant bacteria (DuraN and Marshall,

2005).

Antimicrobial Resistance (AMR) in Aquaculture

Antimicrobial resistance (AMR) has been documented in bacterial species from aquaculture and is an emerging issue with regards to public health. For example, the association between the use of tetracycline and resistance to this antimicrobial has been confirmed in numerous studies (Kerry et al, 1994, Tendencia et al, 2000; Le et al., 2005).

Public health may be adversely affected by the direct spread of resistant bacteria that are human pathogens or indirectly, by spread of resistance determinants from pathogens, non-pathogens or opportunistic pathogens to human pathogens (Hernandez, 2005). For example, tetracycline resistant genes (e.g., Tnl721) can be transferred from fish pathogens (Aeromonas salmonicida) to human pathogens (Aeromonas hydrophila,

Aeromonas cavia and E. coli) (Rhodes et al, 2000). Similarly, Salmonella enterica, the cause of non-typhoidal salmonellosis in humans, has been found to carry the resistance gene for florfenicol, an antibiotic used in aquaculture (Bolton et al., 1999; Angulo et al,

2004). This implies that even if there is no direct human exposure to aquaculture or

9 CHAPTER ONE

INTRODUCTION, LITERATURE REVIEW AND OBJECTIVES

INTRODUCTION

The global and domestic aquaculture industries have grown considerably over the past two decades due to increased consumer demand. This has positioned aquaculture

(farmed or cultivated fish/shellfish) and seafood production (derived from aquaculture or wild-caught fish/shellfish) as important parts of the food production sector. This growth has, however, been accompanied by several environmental, food safety and other public health issues. Besides many environmental concerns, which are beyond the scope of this thesis, the main food safety and public health issues range from zoonotic pathogens, food-borne disease associated with certain types of seafood, microbial safety of imported/exported products, antimicrobial use (AMU), and two related public health issues, antimicrobial resistance (AMR) and drug residues. Some topics have been studied less thoroughly (e.g., antimicrobial use) than others (e.g., paralytic shellfish poisoning) for which an extensive literature has already accumulated. In addition, knowledge gaps may frequently exist in the literature and until they can be addressed through additional published research, emphasis is sometimes placed on expert opinion when action or decision is needed.

1 date, the collective body of knowledge surrounding these issues, composed of both primary research and expert opinion, has not been synthesized; a process crucial for fully informed decision- making by stakeholders.

This thesis characterizes and evaluates the existing primary research on AMU,

LITERARATURE REVIEW

Overview of the Global Aquaculture Industry

2 The aquaculture industry is the fastest growing food-animal production sector in the world (FAO, 2008). Comprising both capture fisheries and aquaculture operations, the seafood industry produced a total of 146.3 tonnes of products worldwide in 2008;

110.4 million tonnes was fish and shellfish were intended for public consumption and the remainder was used for the production of fishmeal and fish oil (State of World Fisheries and Aquaculture, 2008). About 47% of the total seafood harvest was produced in aquaculture operations; the rest was catch from both inland and marine fisheries (State of

World Fisheries and Aquaculture, 2008). Globally, fish/shellfish provided 2.9 billion people with 15% or more of their average per capita animal protein intake (State of

World Fisheries and Aquaculture, 2008). Ranked by quantity produced in 2008, the major aquaculture species groups included freshwater fish (54%), mollusks (27%), crustaceans (9%), diadromous fish (6%), and marine fish (3%) (FAO, 2008).

Approximately 90% of aquaculture production comes from the Asia-Pacific region and the rest is contributed by Europe (4.2%), Latin America and the Caribbean

(3.0%), Africa (1.5%) and North America (1.2%) (FAO, 2008). The dominance of the

Asia-Pacific region is largely attributable to China, which comprises 67% and 49% of global production and global value, respectively (FAO, 2008). The other large aquaculture-producing countries in this region (ranked by production volume) include

India, Vietnam, Thailand, Indonesia, Bangladesh, Japan, and the Philippines (FAO,

2008).

The main regions of production vary with fish/seafood species. For example, the

Asia-Pacific region produces 98 % of carp, 95% of oyster and 88% of shrimp (including prawns throughout this thesis) production worldwide (FAO, 2008). Furthermore, the

3 largest producers of salmon are Norway and Chile, accounting for 33% and 31% of the total worldwide production, respectively (FAO, 2008). The production of salmon in

South America and the Caribbean has over-taken shrimp production, previously the primary product of these regions (FAO, 2008).

Overview of Aquaculture and Seafood Industries in Canada

Canadian aquaculture spans from the East to the West coast, producing a wide range of fish and shellfish species. All Canadian provinces are involved in aquaculture production with British Columbia, New Brunswick and Prince Edward Island being the leading provinces (Canadian Aquaculture Industry Alliance, 2008). In 2008, the

Canadian aquaculture industry was valued at approximately $5 billion and provided

130,000 jobs (Canada Food Statistics, 2009). The four main categories of species by volume were salmon (66.7%), mussels (15.8%), oysters (8.7%) and trout (3.4%). Other species included clams, tilapia, Arctic char, Atlantic cod, white sturgeon and scallops, with spotted wolffish, American eels, abalone, sea cucumbers, sea urchins, cockles and marine plants as emerging species in the industry (Canadian Aquaculture Industry

Alliance, 2008).

Canada ranks as the seventh leading exporter of aquaculture products in the world and the thirteenth largest importer (Fish and Seafood Overview, 2009) (by value). The export market largely involves salmon, blue mussels and oysters shipped to the United

States, the United Kingdom, France, Denmark, Japan and China (Fisheries and Oceans

Canada, 2009). Although salmon is the top aquaculture species in Canadian production,

Canada's contribution to the global market does not place it among those dominating the

4 species group (Canadian Aquaculture Industry Alliance, 2008). The major import product both in terms of quantity and value is shrimp followed by lobster and tuna from the

United States, China, Thailand and Vietnam (in that order) (Fisheries and Oceans

Canada, 2009b).

Prairie provinces consumed less, likely due to cultural and geographical factors

Canada, 2005; FAO, 2008).

Food Safety Risks Associated With Aquaculture and Seafood

Pesticide Regulation, Pesticide Illness Surveillance Program, 2008). In contrast, effects

5 from physical hazards can range from bites, stings and cuts from fish spines (CDC,

2005). Other hazards are biological in nature, including viral (e.g., Norovirus, Hepatitis

A), bacterial (e.g., Aeromonas hydrophila. Vibrio vulnificus) and parasitic infections

(e.g., flukes, nematodes, cestodes, protozoa) (CDC, 2005) that can cause a wide range of symptoms, infections and diseases.

Zoonotic Bacteria in Aquaculture and Seafood

Bacterial contamination of seafood can be classified as follows: bacteria indigenous to the aquatic environment (e.g.. Vibrio spp., Aeromonas spp.), bacteria fi-om the terrestrial or aquatic environment (e.g.. Listeria monocytogenes), and bacteria from human or animal reservoirs (e.g.. Salmonella spp., E. coli. Staphylococcus spp.. Shigella

6 spp.) (Huss et al, 2000). Exposure to these pathogens may cause a range of adverse effects on human health including gastroenteritis, septicemia, wound infection, dysentery, fever, and vomiting (Feldhusen, 2000). The incubation periods vary depending on the genus/species of bacteria (assuming ingestion of the minimal infectious dose), but can range between hours (e.g.. Vibrio parahemolyticus) to days (e.g.. Yersinia enterocolitica)

(Butt et al., 2004).

Globally, the incidence of seafood-related diseases is unknown (Huss et al., 2000) because countries are not obligated to report illness outbreaks and many lack any relevant foodbome disease recording or surveillance system (CDC, 2005). Moreover, even in countries which have incorporated these systems, such as the United States, Canada and the Netherlands, under-reporting of foodbome diseases, and the fact that surveillance systems often fail in identifying the source of infection biases the seafood-specific disease data (CDC, 2005). Since 2005, globally, 126 reported outbreaks of seafood- related illness have occurred, 3 of which were in Canada; 73 have been caused by bacterial pathogens including 8 caused by Staphylococcus, 23 caused by Salmonella and

26 caused by Vibrio (personal communication with Judy Greig, Global Foodbome

Outbreak Database, Laboratory for Foodbome Zoonoses, Guelph, Canada). The

Salmonella and Vibrio outbreaks were largely due to consumption of oysters and salmon

{Salmonella ), and shellfish and shrimp {Vibrio). Vibrio has been implicated in seafood- related outbreaks more fi-equently than any other bacteria (Kaysner et al., 2001; Liu et al.,

2004). It is a leading cause of seafood-bome disease in the United States (Kaysner et al,

2001) and Japan, and accounted for about a third of foodbome outbreaks in China (Liu et al., 2004). Vibrio infections fi-om seafood were reported rarely in Europe, but occasional

7 outbreaks have been reported in Spain (Lozano-Leon et al., 2003) and France (Martinez-

Antimicrobial Use (AMU) in Aquaculture

(Health Canada, 2010). Tetracyclines are approved for many aquaculture indications in many countries, including Canada (CFIA, 2005), the United States (Benbrook, 2002),

As the aquaculture industry continues to grow, selective pressure from increased utilization of antimicrobials may lead to the development of antimicrobial resistance

(AMR). Although the overall risk of AMU in aquaculture is unknown, it is estimated that

70-80% of antimicrobials end up in the environment allowing for the potential

8 development of resistant bacterial strains in environmental organisms (Hernandez, 2005).

Antimicrobial resistance does not respect international borders; therefore trade is a potential mechanism for international spread of resistant bacteria (DuraN and Marshall,

2005).

Antimicrobial Resistance (AMR) in Aquaculture

Public health may be adversely affected by the direct spread of resistant bacteria that are human pathogens or indirectly, by spread of resistance determinants firom pathogens, non-pathogens or opportunistic pathogens to human pathogens (Hernandez, 2005). For example, tetracycline resistant genes (e.g., Tnl721) can be transferred from fish pathogens (Aeromonas salmonicida) to human pathogens (Aeromonas hydrophila,

2004). This implies that even if there is no direct human exposure to aquaculture or

9 products of aquaculture, certain resistance determinants found in human pathogens may indirectly stem from antimicrobial use in aquaculture. In other words, antimicrobial use in aquaculture can contribute to resistance in bacteria in different environmental or host niches (Cabello, 2006).

Monitoring of AMR and AMU and Control of AMR

Surveillance programs for AMR in bacteria from animals, food and humans have been developed in several countries; AMU monitoring is less well established in most countries, with the exception of Scandanavia. Well-established programs include the

Danish Integrated Antimicrobial Resistance Monitoring and Research Programme in

Denmark (DANMAP, 2008), the National Antimicrobial Resistance Monitoring System in the United States of America (NARMS, 2010), Swedish Veterinary Antimicrobial

Resistance Monitoring (SVARM, 2008), Usage of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Norway (NORM-VET, 2008), Monitoring of

Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands (MARAN,

2008) and the Canadian Integrated Program for Antimicrobial Resistance Surveillance in

Canada (CIPARS, 2009). These programs traditionally focused on the main food-animal commodities such as pork, beef and poultry, but most, with the exception of DANMAP,

SVARM and NORM-VET, do not include aquaculture. This omission constitutes a gap in important information because national surveillance is an essential source of information about exposure of humans to resistant bacteria through an important part of the food supply. Thus, to date, much of the information pertaining to AMR and patterns

10 of AMU in aquacuiture remains fragmented and largely unsynthesized, particularly with respect to AMU (Benbrook, 2002).

Current Legislative Framework for Production of Safe Seafood in Canada

In Canada, the Canadian Food Inspection Agency (CFIA) is responsible for inspecting the safety of all fish/shellfish products processed in federally registered plants or imported into the country. CFIA duties include registration of federal fish processing establishments, inspection of imported products, development/maintenance of agreements with countries with an inspection program, enforcement of regulations and residue testing (CFIA Fish and Seafood, 2009). Products intended for international and / or interprovincial trade are subject to the CFIA's Quality Management Program (QMP).

The QMP relies on a preventative approach to food safety rather than relying of inspection of the final product (Quality Management Program, 2010). Regarding imported products, the CFIA uses a target of 5% of imported lots to test for biological and chemical hazards. If an importer fails, they are immediately placed on a Mandatory

Inspection List (MIL), meaning that all subsequent products are inspected until four consecutive shipments are cleared (Regulation, 2010). The Enhanced Inspection List

(EIL) includes products that may be unsafe as per CFIA standards (e.g., fi-om product recall, review of MIL). With regard to exporting, all products must be derived &om registered fish processing establishments.

Research Synthesis Methods and Their Application in Zoonotic Public Health

11 Decision makers need to have access to high quality research evidence to make informed decisions regarding policies in public health. However, only rarely can a decision be made based purely on the empirical evidence from one study. Traditionally, review articles have been used to summarize large amounts of information regarding a topic. Despite their common use, they have often been criticized for their lack of reproducibility and selection bias with respect to study inclusion (Table 1.1) (Arksey and

O'Malley, 2005). New synthesis research methods, such as scoping studies and systematic reviews (discussed below) do not face the same challenges and have been used primarily in the healthcare sector (Arksey and O'Malley, 2005). Recently, they have been utilized in several initiatives in the agri-food industries including swine, poultry and dairy (Sanchez et al., 2007; Sargeant et al., 2007; Waddell et al., 2009; Wilhelm et al.,

2009; Young et al., 2009).

Scoping Studies as Tools for Evidence Mapping

Scoping studies are the product of a relatively new methodology for synthesizing literature in a given field. Sometimes referred to as evidence mapping (Arksey and

O'Malley, 2005), scoping studies may be used to assess the breadth of literature available, rapidly identify concepts in a broad area of research, the type of evidence available, and research gaps (Arksey and O'Malley, 2005). To date, the existing literature provides little guidance for undertaking a scoping study and when its use is most appropriate

(Arksey and O'Malley, 2005). Moreover, the method / approach is still in development and its application is not yet standardized across industry sectors (Davis et al., 2009).

Additionally, there is a lack of consensus on whether the scoping study is a study design

12 in its own right, or a method of providing preliminary groundwork for additional research, such as systematic review (Arksey and O'Malley, 2005). Compelling arguments have been raised on both sides regarding this dual nature (Arksey and

O'Malley, 2005; Mays et al, 2001); some stress that a goal of a scoping study is to determine whether it can be considered complete by itself (Mays et al, 2001). For example, if its purpose is to identify knowledge gaps or summarize research findings, it may be able to accomplish these in its own right. However, if it is necessary to assess the literature as part of an on-going processes or to provide directives for another process

(e.g., a systematic review (see below)), then it likely cannot stand alone (Arksey and

O'Malley, 2005; Mays et al. 2001).

Systematic Review -Methodology and Comparison to Scoping Study

In comparison to the scoping study, the methodology for systematic reviews is well developed and has long been utilized in clinical health sectors as a means of research synthesis. Systematic reviews, in contrast to traditional literature reviews, use a transparent fi-amework to identify, evaluate and summarize primary research in a given field (Table 1.1) (Sargeant, 2006; Cook et al., 1997). In contrast to scoping studies, systematic reviews focus on a far more precise topic or question (Arksey and O'Malley,

2005; Cook et al., 1997). They often involve a far narrower range of studies (Anderson et al., 2008) and, more often than not, entail assessment of study quality and extraction of specific data, whereas scoping studies may not (Arksey and O'Malley, 2005). Other than that, the actual execution of systematic reviews and scoping studies is fundamentally the same (Table 1.1).

13 The Meta-Analysis Approach to Understanding Global Evidence

The completion of a high quality systematic review can identify studies which can be used for meta-analysis. Meta-analysis is a statistical technique that allows the results from multiple independent studies to be weighed and combined into a more precise, pooled estimate of effect (Crombie and Davies, 2009). In order to achieve this, studies must measure the same effect and have low heterogeneity (measured by Cochran's Q statistic) (Crombie and Davies, 2009). Heterogeneity among studies can exist due to individual study characteristics, such as sampled populations, settings or methods used

(Crombie and Davies, 2009). If heterogeneity is large (p-value for Q statistic <0.1), the pooled estimate should not be reported, and other means to explain the heterogeneity should be investigated (e.g., stratified meta-analysis by region) (Sutton et al., 2000).

Frequently, meta-analysis cannot be conducted due to a low number of studies included in the review and large heterogeneity that exists between them (Crombie and Davies,

2009). Consequently, most systematic reviews that have been published in the agri-food industry have been qualitative in nature (Sargeant et al., 2007; Wilhelm et al., 2009).

The Use of Questionnaires in Epidemiological Research

Questionnaires are valuable tools for data collection and have long been used in epidemiological research. Their benefits include a relatively rapid and cost-effective way of gathering information from large geographical locations or large populations and are generally familiar to most people, reducing apprehensiveness (Willem and Gallhofer,

2007). Their drawbacks include the limited scope and type of data that are collectable

14 using this method and the potential for low response rate and associated non-response bias (Willem and Gallhofer, 2007). There are several stages to questionnaire design and administration: determining which information is needed, identifying potential respondents (sample population), designing, pre-testing and debugging, implementation, data recording and data analysis (Bradbum et al., 2004).

Question design is vitally important for collecting the necessary data. Questions can be structured in a number of closed formats including multiple-choice, select one/all that apply, rating/ranking (e.g., 5 point Likert scale, 'not important' to 'very important') or open ended questions (Dillman et al., 2009; Rea et al., 2005). Some suggestions for proper questionnaire design include; avoiding leading questions (e.g.. Wouldn't you agree that antimicrobial resistance is an important issue in aquaculture?), double-barreled questions (e.g., Do you have professional experience with salmon and tilapia?) or including double-negatives (e.g., Should producers not avoid using antimicrobials?)

Another point to stress is that options should be mutually exclusive (e.g.. How many years have you worked in aquaculture? a. <5, b.5-10, c. 10-15, d. 15-20) (Rea et al.,

2005). Pre-testing of the questionnaire on a sample group of respondents is essential to ensure questions are clear, the questionnaire is kept short and all necessary options are provided (Dillman et al., 2009; Rea et al., 2005; Bradbum et al., 2004).

Administration methods include self-administration by mail, e-mail/electronic management software (e.g., Survey Monkey, QuestionPro), or interview by telephone or face-to-face meetings (Dillman et al., 2009). Methods to increase response rate should be explored and may include sending a pre-contact letter notifying the participant that a questionnaire will shortly follow, administering scheduled reminders and providing

15 incentives (Bradbum et al., 2004). A database of coded responses is typically constructed with either manual input of results (for postal questionnaires and interviews) or downloading a database from the survey management software.

The approach for data analysis depends on the overall goal of the questionnaire and can be descriptive (e.g., frequency distributions, cross-tabulations), normative tests

(e.g., t-tests, F-tests, Chi-square), or cause-and-effect measures (e.g., regression analysis)

(Heeringa et al., 2010). In order to strengthen findings, it is recommended that non- response bias be investigated by sampling a small group of non- responders, providing generally demographic question and reason(s) for declining participation (Heeringa et al.,

2010). Other things to consider in data analysis are examining the internal validity (how correctly the questiormaire measures the sampled population) and external validity (how well the data obtained reflects the general population) (Heeringa et al., 2010).

STUDY RATIONALE AND OBJECTIVES

In Canada, to date, aquaculture has not been included in national surveillance of

AMU and AMR (with the exception of AMU in British Columnbia (Ministry of

Agriculture and Lands, 2008)). Similarly, there is a lack of information concerning the potential exposure of Canadians to zoonotic bacteria from seafood commodities. It is important that the breadth of the existing primary research evidence in this area is comprehensively and critically summarized and evaluated to provide insights into these issues. A scoping study-systematic review approach offers a unique way of navigating through and synthesizing literature in fields where such a task seems daunting due to the quantity of research present. This approach is ideal for industries such as the aquaculture

16 industry with its diversity of species, farming methods, processing methods and potential food-borne hazards; all features that contribute to the complexity in synthesizing the relevant research. The transparent, replicable and evidence-based nature of this approach allows decision makers to have unbiased information when addressing policies, which is not offered by traditional literature review methods. Equally as important is elicitation of professional insights on these critical issues from global experts in the various fields related to aquaculture and seafood production. Experts are often an un-tapped resource for knowledge that cannot be found in published documents and their opinions can be used to bridge gaps in the published literature.

To the best of our knowledge, the application of a research synthesis-expert questionnaire approach to investigate these topics (Figure 1.1) has not been used in aquaculture and seafood production. The simultaneous utilization of these two components provides a more comprehensive view than either alone. The collated findings are discussed in the context of their applicability as evidence-based inputs for aquaculture stakeholder consideration in two main regards: potential AMR and AMU monitoring/surveillance in Canada and the direction of future research.

The specific objectives of this thesis were:

1) Assess the overall quantity, distribution, characteristics and knowledge gaps in existing global primary research investigating: the prevalence, risk factors and interventions for selected zoonotic bacteria, AMU and AMR in aquaculture and seafood production, using a scoping study.

17 2) Evaluate and appraise, using a rigorous systematic review-meta analysis, the prevalence of Aeromonas, Vibrio, Salmonella, E. coli at the processing, retail, ready-to- eat, or import/export level in clam, mussel, oyster, salmon, shrimp (including prawn) and tilapia.

3) Elicit the opinions of aquaculture-allied experts on importance of AMR and AMU in aquaculture; AMU/AMR in selected bacteria/aquatic species, AMU in aquatic species, and AMR surveillance and laboratory testing.

This thesis is written using a paper style format; Chapters 2 and 3 are formatted according to the style of the journal to which the manuscripts will be submitted (noted at the top of the title page for each paper). The last Chapter is a summarized discussion of the findings.

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27 Table 1.1. Comparisons between traditional literature reviews, scoping studies and systematic reviews.

Step/Characteristic Traditional Literature Scoping Study Systematic Review Review

1. Topic/Question of Broad topic. Broad topic. Specific topic. Interest 2. Conducting a Search Generally unspecified, Explicitly specified and documented. Explicitly specified and documented. Strategy un-documented and not replicable. 3. Selection of Studies Criteria for relevance Relevance criteria Relevance criteria determination of (inclusion/exclusion) is explicitly (inclusion/exclusion) is explicitly studies is generally not stated. Can involve more than one stated. Can involve more than one specified. round of screening studies for round of screening studies for relevance. relevance. 4. Appraisal of Studies Can be variable. Can be variable. Less likely to be Rigorous appraisal of studies. Criteria Criteria used for conducted due to a large number of explicitly reported. Typically smaller appraisal may not be studies included at Step 3. number of studies included than in reported. scoping reviews.

5. Study Synthesis Often qualitative Often qualitative. Likely includes Extraction of relevant data from summary provided. 'charting' of data by key issues and studies. Can be qualitative or themes. quantitative. 6. Likely not evidence- Usually evidence-based. Typically Usually evidence-based. Aim to weigh Inferences/Conclusions based. does not weigh evidence in support of evidence in one direction if one view. scientifically valid. (Adapted from Cook et al., 1997; Arksey and O'Malley, 2005)

28 Scoping Study Questionnaire Protocol development Desiga/prc-testing Deterniine (the breadth) scope Global identification of targeted participants/ Replicable search strategy construction of database Survey platform and Abstract relevance screening management (by two reviewers) Administration and reminders Evidence mapping/question prioritization for SR-MA Non-response evaluation Systematic Review- Meta-analysis Methodological assessment (two reviewers) Data extraction (two reviewers)

Summary or synthesis (MA) Basehne information

Evidence and empirkal inputs for potential surveillance initiatives in Canadian aquaculture and seafood

Figure 1.1. A stepwise depiction of the study goals and components: scoping study- systematic review and expert questionnaire.

29 CHAPTER TWO

ANTIMICROBIAL USE AND RESIT ANCE IN AQUACULTURE: FINDINGS OF

A GLOBALLY ADMINSITERED SURVEY OF AQUACULTURE-ALLIED

PROFESSIONALS'

SUMMARY

There is limited published information regarding antimicrobial use (AMU) and antimicrobial resistance (AMR) in aquaculture. Our objective was to determine the opinions of aquaculture-allied professionals around the world on the frequency of AMU and AMR in common aquatic species. The study questionnaire included five sections: respondent demographics, extent of AMU in aquaculture, frequency of observations of

AMR in aquaculture, AMR monitoring and surveillance, and antimicrobial susceptibility testing in various jurisdictions. It was administered in English and Spanish to 604 professionals in 25 countries and with varying expertise in aquaculture. The response rate was 32.9 % (n=199) Over half of the participants had >10 years of experience in aquaculture; 70.4 % (n=140) were involved in fish health/clinical work and their primary experience was with salmon, tilapia, trout, shrimp (including prawn) and/or catfish.

Tetracycline use was reported by 28.0,46.5,17.6, 37.2 and 9.4%, of respondents working with catfish salmon, tilapia, trout and shrimp, respectively. Resistance to tetracycline in one or more species of bacteria was reported as 'frequent-to-almost always' for the same species by 38.9,27.8,16.7, 52.4, and 36.4% of respondents, respectively). Quinolone use was reported in North America where there is no quinolone product approved for aquaculture, and where extra-label fluoroquinolone use is either prohibited (USA) or

' Prepared in the style of Zoonoses and Public Health. 30 discouraged (Canada). Quinolone use was reported as 'frequent-to-almost always' by some respondents in Europe and Asia where labeled indications exist. This baseline information can be used to prioritize research or surveillance for AMU and AMR in aquaculture.

INTRODUCTION

Antimicrobials are widely used to treat and prevent infections in humans and animals. However, antimicrobial use (AMU) can select for resistance in bacteria and has become a concern in veterinary medicine and public health (WHO, 2006). Antimicrobial resistance (AMR) can result in ineffective treatment and higher costs for development and use of new antibiotics (Report of the ASM Taskforce on Antibiotic Resistance,

1995).

Antimicrobial resistance (AMR) has been well documented in food-animal and human bacterial isolates (McEwen et al., 2008; EFSA, 2009) and concerns arising from this led to many AMR monitoring/surveillance and research initiatives, primarily in Europe and

North America (DANMAP, 2009; NARMS, 2007; CIPARS, 2007). These programs monitor trends in patterns of AMR in targeted pathogen and commensal bacteria and identify emerging AMR problems within targeted populations. Their primary focus has traditionally been the main food-animal commodities such as pork, beef and chicken

(McEwen et al., 2006).

Nowadays, consumers are increasingly relying on seafood produced by the aquaculture industry as traditional wild-caught fisheries are depleted. In the past two decades, this industry has grown four-fold (Naylor et al., 2003). Canada ranks 20th in

31 terms of aquaculture production in the world (FAO Fisheries and Aquaculture, 2008) and is actively involved in importing and exporting aquaculture products (Fisheries and

Oceans Canada, 2004).

In the aquaculture industry, there is limited and often incomplete AMU and AMR information (Sapkota et al., 2008). Apart from AMU monitoring results from British

Columbia providing total grams of antimicrobial use in aquaculture regardless of drug class (Fish Health Program Annual Report, 2008), products from Canadian aquaculture or imported seafood are currently not incorporated in routine surveillance for AMU or

AMR. In light of Canada's contribution to both domestic and international aquaculture industries, and increased quantities of imported seafood consumed by Canadians

(Fisheries and Oceans Canada, 2004), AMU and AMR information in this sector is required to assess potential needs for surveillance and research in this field. The objective of this study was to elicit the opinions of aquaculture-allied professionals from around the globe regarding the frequency of AMU and AMR in aquaculture through an online questiormaire.

MATERIALS AND METHODS

Questionnaire design description

A core research team (Drs. Lucie Dutil, Carl F. Uhland, Andrijana Rajic) developed a draft questiormaire and identified aquatic zoonotic pathogens, aquatic species, and antimicrobial drugs of interest. The draft was discussed with three individuals with specialized aquaculture expertise from Canada and was pre-tested by five professionals from Canada and the USA with a broad range of expertise in

32 aquaculture. The questionnaire included five sections: respondent demographics, extent of AMU in aquaculture, frequency of observations of AMR in aquaculture, AMR monitoring and surveillance, and laboratory antimicrobial susceptibility methods (results about laboratory methods are reported elsewhere (Appendix 2.3). The final questionnaire was composed of 26 questions; 23 closed and 3 open (to gather supplemental information). Closed question formats included multiple choice (8 questions), check all that apply (5 questions), yes/no (5 questions) and rating using a five-point ordinal scale (5 questions) (e.g., "Never" to "always used"). Open format questions were used to capture contact information as well as any additional respondent comments.

The questiormaire was prepared in English then translated into Spanish for administration in Spanish-speaking regions (South and Central America and Caribbean).

In addition, a brief questiormaire was designed for non-responders to the main questiormaire to aid in the assessment of non-response bias. It consisted of five questions; four were demographic in nature and the fifth elicited reasons for non-participation in the questionnaire.

Database of aquaculture-alliedprofessionals

Contacts on an initial list of aquaculture-allied professionals (consisting of 95 individuals provided by a project member) were asked to provide names of other colleagues and professionals with expertise in aquaculture, selected zoonotic bacteria and

AMU/AMR. Contacts were also gathered from various aquaculture-related mailing address lists or publications. In addition, a blog on Aquavetmed E-news was used to advertise the project and resulted in 3 additional potential participants.

33 Survey administration

The questionnaire and four reminders were administered using Survey Monkey, a web-based application (Survey Monkey, Portland, OR). Two weeks prior to initial administration, a letter was sent by e-mail to 715 individuals inviting them to consider participation in the questionnaire. The initial questionnaire was administered on June

12th 2009 with four reminders sent at two-week intervals for both the English and

Spanish versions. Participants could choose to refuse and be removed from the mailing list.

The non-response evaluation was sent through e-mail to randomly-selected non- responders (n=70 of 388) to the English language survey. Attempts were also made on five separate occasions to contact Spanish-speaking non-responders (n=17) by telephone call from a Spanish-speaking interviewer. Prior to administration of the questionnaire, ethical approval was received from the University of Guelph Review Ethics Board

(protocol # 09MY010) for participation of human subjects.

Data analysis

Questionnaire and non-response data were exported separately to spreadsheets

(Microsoft Excel, Microsoft Corporation, Redmond, WA), cleaned and imported into

Stata 10 for statistical analysis (Stata Corporation, College Station, TX). For the rating questions in the AMR and AMU sections, categories were collapsed from five to three for the purposes of results presentation ('Rarely' and 'Occasionally' collapsed into

Rarely', and 'Frequently' and 'Almost always' collapsed into 'Frequently'). Frequency

34 tabulations were performed and basic descriptive statistics were generated. Fisher's exact test (p<0.05) was used to detect significant differences between responders and non- responders.

RESULTS

Respondent demographic characteristics and non-response bias evaluation

The initial database included 715 contacts but 99 email addresses bounced back,

12 individuals opted out, leaving 604 potential respondents. Questionnaires were completed by 199/604 (32.9%) globally; 30.2% (147/482) in North America, 50.0%

(18/36) in Asia, 51.1% (22/43) in Europe, 36.8% (8/19) in Oceania, 20.0% (1/20) in

South/Central America and Caribbean, 2 of 3 in the Middle East, and 1 of 1 in Africa

(regions determined firom IP addresses). Out of 70 English- and 17 Spanish-speaking randomly sampled non-responders, 23 and 0 answered the non-response form, respectively. No differences were detected in the demographic information between

English responders and non-responders (p>0.05). The main reasons for non-participation were "I don't believe I can contribute as it is not relevant to my professional experience" as a reason for refusal in participation (14/23) followed by "I don't have time" (5/23).

Overview of respondent professional experience

The main expertise and work setting characteristics of the respondents are summarized in Table 2.1. A large majority had more than 10 years experience in aquaculture (n=110) with about half (n=86) claiming that aquaculture, seafood or ornamental fish comprised more than half of their work. Respondents had most

35 experience with trout, salmon, tilapia and catfish, and the lowest experience corresponded to crawfish, lobster and clams (Table 2.2).

Opinions on Frequency of AMU and AMR in various aquatic species

Responses to AMU or AMR questions by species for were tabulated when >10 respondents provided answers (Table 2.3). A large number of respondents indicated they had experience with AMU (n=130). However, out of these individuals, only 105 professionals answered AMU questions, and the most common relevant expertise was in clinical medicine (n=91), microbiology (n=28), and outbreak investigation (n=23).

Overall, tetracyclines were the antimicrobial drugs most often reported as being used (Table 2.3) across all species and around the world (Table 2.4), followed by potentiated sulfonamides, penicillins and phenicols. Quinolones were more often reported as being used 'Never' or 'Rarely'. However 24%, 21%, 16%, 14%, and 14%, of respondents reported using quinolones 'Frequently' in catfish, shrimp, trout, salmon, and tilapia, respectively (Table 2.3). The use of quinolones was reported in all regions, with the USA most fi-equently reporting 'Never' or 'Rarely' used (Table 2.4). Eighty-eight percent of respondents who provided information regarding quinolones use indicated having fish clinical expertise (Asia: 70%; Europe: 80%; USA: 96%; Canada: 83%;

Multiple: 84%). Quinolone use in Canada was mainly reported in salmon and trout (n=6 and 5, respectively); in the USA, in catfish, salmon, trout and tilapia (n=10, 9, 6, 3 respondents, respectively); in Asia, in shrimp (n=8), and in Europe, in trout and salmon

(n=5 and 4, respectively).

36 Of 110 individuals reporting experience with AMR, only 74 provided answers to the questions in this section (North America, n=53; Europe, n=8, Asia, n=7; Oceania,

South and Central America, each n=2; Africa and Middle East, each n=l), and the majority had experience in clinical medicine (n=64). Overall, resistance to tetracyclines was most frequently reported across aquatic species, generally followed by potentiated sulfonamides (Table 2.3). Shrimp were the species where respondents most often reported resistance across all antimicrobials. Resistance to quinolones was reported mainly as 'Never' or 'Rarely' across species, shrimp and tilapia being the species in which quinolones resistance was most often reported as 'Frequent'.

For clams, crawfish, lobster, mussel and oyster species, the number of responses for the AMU section was >10, but <4 average responses were reported for observations of AMR and results were not tabulated. Overall, there was rare to no resistance reported for these species.

AMR in selected bacteria

The frequencies of reportedly observed AMR in six bacterial species are shown in

Table 2.5. Approximately half (61/110) of respondents reporting experience with AMR provided information pertaining to AMR (North America, n=39; Europe, n=10, Asia, n=7; Oceania, n=2; Africa, Middle East, South and Central America, each n=l). All respondents provided answers for more than one pathogen, and respondents did not rate similarly across the bacterial genus/species.

Most of the responses were foxAeromonas (n=37) and Vibrio (n=20). Across all bacteria listed, AMR was most often reported for tetracycline. Resistance in Aeromonas

37 spp. was 'Frequently' observed by 20% or more of respondents for 5 out of 9 antimicrobials surveyed, and only 20% and 23% of respondents reported never observing resistance to macrolides and quinolones, respectively. In the case of Vibrio, 20% or more of respondents reported observing resistance 'Frequently' for 3 out of 9 drugs, and only

21% and 35% of respondents reported never observing resistance in macrolides and quinolones, respectively.

Knowledge of AMR monitoring and surveillance

When asked how important various practices were in the development of AMR, participants (n= 165) identified "inappropriate duration of treatment" (n=l 19),

"utilization of sub-therapeutic dosages" (n=117), "absence of accurate diagnosis"

(n=115) and "use of antimicrobials in place of improving husbandry" (n=72). When asked about knowledge gaps, (open question) 12 of 66 respondents listed "potential runoff of antimicrobials into aquaculture settings from other facilities" (e.g.,, hospitals, large animal production) and "risk of AMR from aquaculture to human health".

DISCUSSION

This questionnaire-based study provides semi-quantitative basic data reflecting the opinions of professionals working in aquaculture on AMU and AMR in aquaculture around the world. In the absence of integrated international surveillance data on these important topics, the results of this survey provide some useful insights into issues that warrant further investigation, particularly with regard to monitoring of imported fish and seafood.

38 Most participants reported 'Never' or 'Rarely' when describing use of and resistance to various classes of antimicrobials. This is a positive finding from the point of view of antimicrobial selection pressure. Tetracycline was the antimicrobial class most often reported as being used 'Frequently' across aquatic species. This is not surprising as tetracyclines are one of the few antimicrobials approved for use in aquaculture with multiple indications in many countries, including Canada (CFIA, 2005), United States

(Benbrook, 2002), India, Norway, Indonesia, Japan, and the People's Republic of China

(FAO, 2005), and tetracyclines are generally inexpensive antimicrobials (Schwarz et al,

2001; US General Accounting Office, 1999). However, tetracycline has recently been classified as "highly important" to human health by the World Health Organization

(Collignon et al., 2009). When antimicrobial use is necessary, it is prudent to choose from among those that are approved, effective and are of lower importance to human health.

Quinolones were most often reported as 'Rarely' used, which is in accordance with regulatory measures in many countries for restricted or prohibited use of quinolones because of the critical importance of this class to human health (WHO, 2006; Sorum,

2006); however the questionnaire did not distinguish between fluoroquinolones and other quinolones (generally being considered a more serious issue). The reported use

(sometimes ft^equent even when ornamental fish respondents were excluded) of quinolones in the USA and Canada where this drug class is not labeled in aquaculture raises questions. In the US, the extra-label use of fluoroquinolones is prohibited (FDA,

2003). In Canada, fluoroquinolone labels clearly warn against extra-label use; however, extra-label use is legal under provincial legislation (Veterinary Drug Directorate, 2004) in the context of a valid veterinary-client-patient relationship and fluoroquinolone use in aquaculture is technically possible. Use of oxolinic acid or lower class quinolones may also be occurring.

Quinolones, including fluoroquinolones, are labeled for use in aquaculture in certain European and Asiatic countries (FAO/OIE/WHO, 2006). In Denmark and

Norway, most of the use indiciates only oxilinic acid, a lower class of quinolones

(DANMAP, 2009; NORM-Vet, 2009). The 'Frequently' reported use of the quinolones drug class in those regions is of concern because of the possibility of rapid development of resistance (Ruiz, 2003). In Canada, shrimp are the main imported products both, by quantity and value (Fisheries and Oceans Canada, 2009), and our results show that AMR to quinolones was most frequently reported in shrimp. Despite restrictions around the use of quinolones in aquaculture in certain countries, there is no restriction in Canada on products imported from countries where the drugs are used. This highlights the importance of international food trade as a potential mechanism for transmission of resistance determinants (DuraN and Marshall, 2005). Further study is warranted to determine more precisely which quinolones are being used in aquaculture in the various regions and under what conditions. Quinolones are among WHO's "critically important" antimicrobials for human health (WHO, 2007); any use in food species is of concern and should be discouraged, and if illegal use is taking place, it should be prevented.

Respondents most frequently implicated "inappropriate duration of treatment"

(119/161) as a suspected contributor to the development of AMR. Use of antimicrobials in place of improving husbandry practices was selected less often (72/165). The recommendation that improving husbandry practices such as reducing fish populations and isolating infected fish/pens, is discussed in numerous studies and reports aimed at

40 reducing antimicrobial resistance through reductions in use for disease control

(Benbrook, 2002; Anthony et al., 2001; WHO, 2006;).

This study is probably more representative of the opinions of professionals from

North America since most respondents were from this region (n=149). Even though the response rate was comparatively low, the absence of detected non-response bias (i.e., lack of significant difference in responses among responders and non-responders) in encouraging. A less successful result was achieved in South and Central America, and the

Caribbean. These regions were clearly under-represented in our study (n=2), despite repeated efforts to increase response and the importance of this region in terms of aquaculture production (FAO, 2008). Future global surveys should consider use of an incentive (e.g., money, gift) to increase response rate in certain region or use of qualitative approaches to address regional differences in responses and ways to overcome them. Additionally, involvement of local collaborators to encourage participation within countries should be considered.

The results of this study represent professional opinion based on empirical experience and, as such, are susceptible to bias. Respondents were not asked to consult any records/databases in reporting of their answers, rather to answer according to the best of their abilities. Participants were given the option to answer only questions relavant to their experience, which should have improved the quality of answers; however, this also decreased the response rate for some questions below the 32.9 % overall mean, especially for the AMR ones. As not all potential respondents were veterinary practitioners, participants were not asked to provide use information for their own practice, but rather to provide answers to the best of their knowledge regarding listed aquatic species. It is

41 therefore not excluded that a few respondents did report "illegal" or "not recommended" use in the industry from their region.

There is very little published aquaculture-related information for AMU and AMR.

This study has generated initial basic information on the perceived frequency of antimicrobial use and antimicrobial resistance in bacteria from various aquaculture species and can be used to guide primary research or plan surveillance for AMU and

AMR in aquaculture. These findings should be interpreted with an understanding of the nature of the data. The AMU information generated by this study highlights the need for better government monitoring of antibiotic use in many countries. Targeted primary research investigating resistance to selected antimicrobials in selected aquatic and bacterial species, e.g.,, sampling of shrimp and AMR testing of Aeromonas, Vibrio and E. coli, is needed to further build on our findings.

ACKNOWLEDGEMENTS

The authors would like to thank Drs Victoria Alday-Sanz and Roy Yanong, who generously dedicated their time in pre-testing the questionnaire, Dr. America Mederos for her translation of the questionnaire and help with Spanish non-response administration, as well as the Public Health Agency of Canada for fimding and support.

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48 Table 2.1. Demographic characteristics of respondents to a questionnaire soliciting information on AMU and AMR administered globally to aquaculture professionals

Responses per Category Nof Characteristic Respondents N Field of Expertise 199 Microbiology 47 24 Epidemiology 25 13 Fish health/Clinical Medicine 140 70 Food Safety 25 13 Outbreak Investigation 33 17 Molecular Biology/Genetics 25 13 Acquisition of Expertise 199 Government 60 30 Academia 118 59 Industry 48 24 Primary Activities 199 Research 81 41 Clinical/Field Work 116 58 Laboratory Work 72 36 Administrative Tasks 57 29 Animal Health Surveillance 61 31 Public Health Surveillance 11 6 Location of Experience 199 Aquisition Africa 5 3 Asia 29 15 Europe 34 17 North America 148 74 Oceania 7 4 South and Central America 7 4 and Caribbean Years of Experience 176 <3 years 13 7 3-10 years 53 30 >10 years 110 63 Aquaculture or seafood or 176 ornamental fish as proportion of

49 daily activities <25% 67 38 26-50% 23 13 51-75% 24 14 76-100 62 35 ® Percentages were calculated per question and do not sum to 100%.

50 Table 2.2. Reported respondent level of experience with various aquatic species

N of Responses in each category No Experience Low-Medium Fairly High-High Aquatic N of Species Respondents N % N % N % Catfish 165 49 30 88 53 28 17 Clams 161 86 53 66 41 9 5 Crawfish 160 92 59 64 40 4 2 Lobster 156 79 51 69 43 8 5 Mussels 162 77 48 73 45 12 7 Oysters 158 71 45 73 46 14 9 Salmon 158 38 24 77 49 43 27 Shrimp 164 54 33 86 52 24 15 Tilapia 161 44 27 88 55 29 18 Trout 159 36 23 71 45 52 33

51 Table 2.3. Reported antimicrobial use and observation of resistance to antimicrobial drug classes for various aquatic species

Reported Observed Antimicrobial Reported Antimicrobial Use Resistance N (%) of Respondents N (%) of Respondents in in each category each category Frequen Frequentl Never Rarely Never Rarely „c Nof Resp Nof Aquatic Antimicrobial onde Respond Drug Class nts N % N % N % ents N % N % N % Catfish Aminoglycosides 47 31 66 13 28 3 6 15 5 33 8 53 2 13 Tetracyclines 50 13 26 23 46 14 28 18 1 6 10 56 7 39 Macrolides 37 26 70 10 27 1 3 13 5 39 8 62 0 0 Nitrofurans 40 29 73 9 23 2 5 13 4 31 7 54 2 15 Penicillins 41 28 68 12 29 1 2 13 3 23 7 54 3 23 Phenicols 41 19 46 16 39 6 15 15 5 33 8 53 2 13 Potentiated Sulfonamides 43 12 28 23 54 8 19 17 3 18 11 65 3 18 Quinolones 42 21 50 11 26 10 24 15 6 40 8 53 1 7 Sulfonamides 39 23 59 14 36 2 5 15 4 27 10 67 1 7 Salmon Aminoglycosides 41 31 78 10 25 0 0 9 5 56 4 44 0 0 Tetracyclines 43 12 28 11 26 20 47 18 1 6 12 67 5 28 Macrolides 37 18 49 13 35 6 16 12 4 33 8 67 0 0 52 Nitrofurans 37 31 84 4 11 2 5 8 4 50 3 38 1 13 Penicillins 37 26 70 11 30 0 0 11 3 27 5 46 3 27 Phenicols 40 17 43 14 35 9 23 13 4 31 9 69 0 0 Potentiated Sulfonamides 41 16 39 14 34 11 27 14 3 21 9 64 2 14 Quinolones 43 18 42 19 44 6 14 11 4 36 6 55 1 9 Sulfonamides 39 24 62 14 36 I 3 11 3 27 5 46 3 27 Tilapia Aminoglycosides 30 22 73 8 27 0 0 10 6 60 4 40 0 0 Tetracyclines 34 10 29 18 53 6 18 12 2 17 8 67 2 17 Macrolides 26 19 73 7 27 0 0 9 3 33 6 67 0 0 Nitrofurans 27 22 82 5 19 0 0 9 4 44 4 44 1 11 Penicillins 26 16 62 10 39 0 0 10 3 30 3 30 4 40 Phenicols 29 18 62 11 38 0 0 12 7 58 4 33 1 8 Potentiated Sulfonamides 28 13 46 13 46 2 7 11 5 46 4 36 2 18 Quinolones 29 18 62 7 24 4 14 10 3 30 6 60 1 10 Sulfonamides 28 21 75 6 21 1 4 11 5 46 4 36 2 18 Trout Aminoglycosides 37 28 76 9 24 0 0 10 5 50 5 50 0 0 Tetracyclines 43 9 21 18 42 16 37 21 1 5 9 43 11 52 Macrolides 36 22 61 9 25 5 14 11 4 36 7 64 0 0 Nitrofurans 38 30 79 7 18 1 3 9 3 33 6 67 0 0 Penicillins 34 26 77 7 21 1 3 11 3 27 5 46 3 27 Phenicols 40 21 53 9 23 10 25 19 6 32 13 68 0 0 Potentiated Sulfonamides 40 15 38 18 45 7 18 17 3 18 9 53 5 29 Quinolones 37 16 43 15 41 6 16 15 6 40 8 53 1 7 i3 Sulfonamides 37 25 68 10 27 2 5 13 3 23 7 54 3 23

Aminoglycosides 29 23 79 5 17 1 3 9 5 56 3 33 1 11 Tetracyclines 34 12 35 12 35 10 29 11 2 18 5 46 4 36 Macrolides 26 21 81 4 15 1 4 9 3 33 2 22 4 44 Nitrofurans 28 21 75 6 21 1 4 9 3 33 4 44 2 22 Penicillins 26 17 65 9 35 0 0 9 2 22 4 44 3 33 Phenicols 30 18 60 8 27 4 13 9 3 33 4 44 2 22 Potentiated Sulfonamides 26 16 62 6 23 4 15 9 3 33 2 22 4 44 Quinolones 28 13 46 9 32 6 21 10 3 30 4 40 3 30 Sulfonamides 25 19 76 3 12 3 12 9 2 22 . 3 33 4 44 a Only species with average of >10 observations for AMR are shown, see text for results of additional species investigated. b Category "Rarely" includes "Rarely used/observed" and "Occasionally used/observed". c Categories "Frequently" includes "Frequently used/observed" and "Always used/observed".

54 Table 2.4. Reporting of antimicrobial usage per region for all aquatic species presented for all respondents and excluding responses regarding use in ornamental fish.

All respondents Without ornamental pet fish N (%) of Responses in each N (%) of Responses in each category category

Rarely Freque Freque Never nlty" Never Rarely" nltyT N Nof Nof of Antimicro Resp Nof Resp Res bial Drug Contine onde Respon onde pon Class nt' nts ses*" N % N % N % nts ses*" N % N % N % Aminoglico sides Asia 9 40 29 73 9 23 2 5 9 35 28 80 5 14 2 6 Europe 10 32 24 75 5 16 3 9 9 30 24 80 5 17 1 3 USA 43 221 162 73 52 24 7 3 32 184 159 86 24 13 1 1 Canada 12 40 30 75 8 20 2 5 12 36 29 81 6 17 1 3 Multiple 18 62 42 68 15 24 5 8 16 52 41 79 11 21 0 0 Macrolides Asia 9 39 29 74 8 21 2 5 9 34 28 82 5 15 1 3 Europe 8 23 16 70 6 26 1 4 7 20 15 75 4 20 1 5 USA 39 191 152 80 28 15 11 6 28 159 138 87 12 8 9 6 Canada 12 40 19 48 18 45 3 8 12 36 18 50 16 44 2 6 Multiple 16 47 32 68 15 32 0 0 14 37 27 73 10 27 0 0 Nitrofurans Asia 11 41 33 80 4 10 4 10 10 35 31 89 3 9 1 3 Europe 8 29 22 76 6 21 1 3 8 26 20 77 0 USA 41 198 141 71 37 19 20 10 30 164 135 82 5 Canada 11 34 24 71 9 26 1 3 10 30 24 80 0 Multiple 18 55 46 84 8 15 1 2 17 44 41 93 0 Asia 9 40 26 65 12 30 2 5 9 35 25 71 3 Europe 8 25 17 68 7 28 1 4 8 22 16 73 5 USA 40 191 160 84 30 16 1 1 30 160 141 88 0 Canada 12 35 23 66 12 34 • 0 0 11 31 23 74 0 Multiple 17 50 33 66 15 30 2 4 16 40 27 68 0 Asia 10 40 27 68 11 28 2 5 9 34 26 76 3 Europe 9 29 18 62 7 24 4 14 9 26 17 65 12

USA 41 196 129 66 53 27 14 7 30 164 118 72 7 Canada 12 38 19 50 9 24 10 26 12 34 19 56 21 Multiple 19 59 30 51 20 34 9 15 18 47 26 55 15

Asia 9 41 25 61 10 24 6 15 9 35 24 69 14 Europe 10 31 17 55 9 29 5 16 10 29 16 55 17

USA 42 196 125 64 56 29 15 8 32 164 117 71 6

Canada 12 34 15 44 7 21 12 35 11 30 15 50 33 Multiple 18 58 27 47 28 48 3 5 17 47 25 53 4 Asia 10 41 20 49 12 29 9 22 9 35 20 57 23 Europe 10 30 13 43 10 33 7 23 10 27 12 44 22 USA 46 204 127 62 45 22 32 16 30 163 127 78 4 Canada 12 34 16 47 10 29 8 24 11 30 16 53 8 27 6 20 Multiple 19 60 30 50 20 33 10 17 18 48 26 54 16 33 6 13 Sulfonamid es Asia 8 39 25 64 8 21 6 15 8 34 24 71 5 15 5 15 Europe 9 27 17 63 9 33 1 4 9 24 16 67 8 33 0 0 USA 40 198 164 83 31 16 3 2 30 165 147 89 18 11 0 0 Canada 10 32 17 53 10 31 5 16 10 29 17 59 8 28 4 14 Multiple 18 51 35 69 15 29 1 2 16 39 29 74 10 26 0 0 Tetracyclin 1 es Asia 11 49 18 37 13 27 18 37 11 42 18 43 12 29 2 29 Europe 11 35 16 46 11 31 8 23 11 33 16 48 10 30 7 21 z USA 45 208 113 54 56 27 39 19 35 173 109 63 37 21 1 16 1 Canada 13 40 9 23 14 35 17 43 13 36 9 25 14 39 3 36 1 Multiple 18 67 19 28 35 52 13 19 17 56 18 32 28 50 0 18 ® Category "Rarely" includes "Rarely used" and "Occasionally used". ^ Categories "Frequently" includes "Frequently used" and "Always used". Multiple category includes respondents indicating experience on more than one continent. Respondents from Africa and South America are included under this category because they had indicated experience in other continents as well. One respondent from Oceania and one from Mexico were merged under 'Multiple' due to low number of responses per region and even if they had experience only in that continent/country. '' Each respondent could answer for multiple fish species.

57 Table 2.5. Reported observed bacterial resistance for selected bacteria and antimicrobial drug classes

N (%) of respondents in each category Never Rarely" Frequently'' Bacteria Antimicrobial N of Species Drug Class Respondents N % N % N ^ Aeromonas spp. Aminoglycosides 32 8 25 24 75 0 0.0 Tetracyclines 48 3 6 22 46 23 48 Macrolides 25 5 20 17 68 3 12 Nitrofurans 21 5 24 11 52 5 24 Penicillins 32 3 9 15 47 14 44 Phenicols 40 8 20 29 73 3 8 Potentiated Sulfonamides 43 7 16 25 58 11 26 Quinolones 39 9 23 24 62 6 15 Sulfonamides 29 5 17 18 62 6 21

Aminoglycosides 14 7 50 7 50 0 0 Tetracyclines 13 2 15 6 46 5 39 Macrolides 9 3 33 6 67 0 0 Nitrofurans 10 4 40 5 50 1 10 Penicillins 12 2 17 4 33 6 50 Phenicols 12 2 17 8 67 2 17 Potentiated Sulfonamides 12 4 33 7 58 1 8 Quinolones 14 3 21 9 64 2 14 Sulfonamides 11 4 36 5 46 2 18 Vibrio spp. Aminoglycosides 24 7 29 13 54 4 17 Tetracyclines 28 2 7 18 64 8 29 Macrolides 19 4 21 12 63 3 16 Nitrofurans 17 5 29 9 53 3 18 Penicillins 19 5 26 8 42 6 32 Phenicols 20 7 35 11 55 2 10 Potentiated Sulfonamides 25 5 20 15 60 5 20 Quinolones 26 9 35 15 58 2 8 Sulfonamides 16 4 25 9 56 3 19 Salmonella spp. Aminoglycosides 12 3 25 8 67 1 8 Tetracyclines 9 2 22 4 44 3 33 Macrolides 8 3 38 5 63 0 0 Nitrofurans 9 3 33 6 67 0 0 Penicillins 10 2 20 6 60 2 20 Phenicols 10 2 20 8 80 0 0 Potentiated Sulfonamides 9 4 44 4 44 1 11 Quinolones 11 5 46 5 46 1 9 Sulfonamides 9 3 33 4 44 2 22 Edwardsiella spp. Aminoglycosides 14 8 57 6 43 0 0 Tetracyclines 13 2 15 7 54 4 31 Macrolides 13 6 46 7 54 0 0 Nitrofurans 10 4 40 6 60 0 0

59 Penicillins 12 3 25 5 42 4 33 Phenicols 12 4 33 8 67 0 0 Potentiated Sulfonamides 12 4 33 7 58 1 8 Quinolones 14 6 43 8 57 0 0 Sulfonamides 12 3 25 7 58 2 17 Streptococcus spp. Aminoglycosides 16 4 25 10 63 2 13 Tetracyclines 18 2 11 11 61 5 28 Macrolides 15 3 20 10 67 2 13 Nitrofurans 13 5 39 7 54 1 8 Penicillins 17 5 29 10 59 2 12 Phenicols 17 5 29 11 65 1 6 Potentiated Sulfonamides 17 6 35 8 47 3 18 Quinolones 17 5 29 11 65 1 6 Sulfonamides 15 6 40 7 47 2 13 Category "Rarely" includes "Rarely observed " and "Occasionally observed ". '' Categories "Frequently" includes "Frequently observed" and "Always observed'

60 CHAPTER THREE

PREVALENCE OF SELECTED ZOONOTIC BACTERIA IN SELECTED

AQUATIC SPECIES AND SEAFOOD: A SCOPING STUDY, SYSTEMATIC

REVIEW AND META-ANALYSIS OF PUBLISHEDRESEARCH^

ABSTRACT

A scoping study (ScS) was conducted to characterize research investigating zoonotic bacteria and other public health topics in various aquatic species and seafood.

The most frequently investigated themes in the literature were prevalence and interventions for Vibrio and Aeromonas at the farm level. Antimicrobial use (AMU) and the association between AMU and antimicrobial resistance (AMR) were rarely investigated. Systematic review-meta-analysis (SR-MA) was used to analyze prevalence and concentration from surveys of Aeromonas, generic Escherichia coli, Salmonella and

Vibrio genera in clams, mussels, oysters, salmon, shrimp (including prawn) and tilapia from processing-to-retail. MA was conducted on prevalence data only at the retail level;

Aeromonas, generic E. coli and Salmonella species in salmon at retail resulted in homogenous (p>0.1) summary estimates of 13% (6-27%), 2% (0.1-11%) and 1% (0-5%), respectively. Many subsets of data had significant (p<0.1) heterogeneity for pathogen- seafood-region combinations in which case prevalence or concentration median/range are reported. Consistent flaws in reporting prevalence results were observed, precluding the use of many studies for MA. Attempts to summarize the reported prevalence estimates revealed many gaps in knowledge about the microbial contamination of many common seafood products. The synthesized data and insights into study quality provided in this paper can be used to prioritize and design future baseline bacteriological surveys on

^ Prepared in style of Foodborne Pathogens and Disease. seafood, for inputs into risk assessments, and in the development of aquaculture and seafood monitoring and surveillance programs.

INTRODUCTION

Increased consumption and demand for seafood has resulted in rapid growth of the global aquaculture industry (FAQ, 2010; Naylor et al., 2003) and concurrently, public health concerns have arisen, as with other foods of animal origin, regarding bacterial contamination of seafood (Lalitha et al., 2006). Between 2005 and 2010, globally, 126 outbreaks of seafood-related illness were reported in the Global Foodbome Outbreak

Database; 73 were associated with bacterial pathogens; 23 Salmonella outbreaks were related to consumption of oysters and salmon, and 26 Vibrio outbreaks to shellfish and shrimp (personal communication with Judy Greig, Global Foodbome Outbreak Database,

Laboratory for Foodbome Zoonoses, Guelph, Canada). However, these are likely very conservative estimates due to under-reporting (FAO, 1998). Other issues relevant to the safety of seafood include antimicrobial use (AMU) (Heuer et al., 2009), antimicrobial resistance (AMR) (MacMillian et al., 2001; Sapkota et al., 2008), dmg residues (Sapkota et al., 2008), and use of dyes, the latter being, among agri-food industries, mainly an aquaculture-specific issue (Zhai et al., 2007; Lin et al., 2008).

Intemationally, stakeholders at all levels of aquaculture production have started addressing a number of food safety issues (FAO/OIE/WHO, 2006; FAO, 2010). As in other agri-food sectors, research reviews and expert opinions are frequently used to inform and guide the policy development processes (Sapkota et al., 2008; Cole et al.,

2009; Liao and Chao, 2009). Although narrative reviews provide useful issue overviews,

62 their lack of methodological transparency in terms of study selection and appraisal, possible bias in interpretation, and limited utility to the policy and decision making process are frequently noted (Cook et al, 1997, Sargeant 2006; Waddell, 2009).

Systematic reviews (SR) offer a transparent and replicable way to identify, appraise and synthesize primary research on specific questions regarding intervention efficacy, etiology, diagnostic test accuracy and summarizing prevalence of disease estimates that are often needed to inform surveillance, risk assessment, policy and decision making, and to prioritize fixture research (Sargeant, 2006; Ruzante et al., 2010).

Data synthesis is preferably conducted using meta-analysis (MA), a statistical method for pooling the results from multiple, similar and homogenous studies resulting in more precise and robust summary estimates of the outcome (Borenstein et al., 2009). The use of these methods has a long tradition in health, environment and other sectors (Arksey et al., 2005) and more recent application in zoonotic public health (Sanchez et al., 2007;

Sargeant et al., 2007; Waddell et al., 2009; Wilhelm et al., 2009; Young et al., 2009).

By nature, policy-driven issues or questions in food safety and zoonotic public health are usually broad in scope and heterogeneous in the research that addresses the issue. A synthesis method, the scoping study (ScS), that can be used to summarize and contextualize evidence on a broad issue is extensively used in the healthcare sector to rapidly identify and characterize the evidence on a broader scope, identify research gaps and to assist with framing questions for rigorous SRs and MA (Davis et al., 2009;

Anderson et al., 2008; Arksey et al., 2005). The main difference between the SR and ScS methods is in the appraisal of methodological soundness and quantitative synthesis of data; these are critical steps of SR, and rarely part of a ScS (Arksey et al., 2005). For the purpose of this study, the role of the ScS was to map out all published evidence pertaining to aquatic species and seafood from farm to fork that examined eight selected zoonotic bacteria, antimicrobial use, antimicrobial resistance, drug residues, and the use of dyes. The ScS categorized and characterized the evidence while addressing the volume of research, where knowledge gaps were apparent and what areas of research had sufficient evidence to consider prioritizing for the application of SR-MA. The latter was applied to published research reporting prevalence or concentration of Aeromonas, generic E. coli. Salmonella and Vibrio species in six major aquatic species/seafood products, from processing to retail, to generate evidence-based inputs for surveillance, risk assessment and design of future baseline bacteriological surveys.

MATERIALS AND METHODS

A ScS and SR-MA protocol that includes details of the study methodology, definitions and samples of all forms used in this project can be accessed on line

(http://www.liebertpub.com/products/product.aspx?pid=108) or as a PDF from the corresponding author. An expert committee that included members of the Canadian

Integrated Program for Antimicrobial Resistance Surveillance Division (CIPARS) of the

Public Health Agency of Canada and individual academic experts from Canada, the

United-States of America and Europe was consulted throughout the study to ensure that it met general and specific surveillance, risk assessment, research and policy needs of various stakeholders associated with aquaculture and seafood safety.

ScS Study: Defining its broad scope

64 The research team comprised three members from the expert committee with considerable expertise in aquaculture and seafood production in addition to six individuals with expertise in epidemiology, microbiology, food safety, zoonotic diseases and synthesis research methods. The research team identified the scope of the study and guided its implementation. The scope of the ScS included any research investigating the prevalence, risk factors and/or interventions for eight selected zoonotic bacteria (e.g.,

Vibrio, Salmonella) (Figure 3.2), AMR, AMU, antimicrobial drug residues and dye use in various aquatic species, aquaculture and seafood products or seafood related disease in humans, seafood related consumption, production, or import/export reports.

Search Strategy and Execution

The search strategy was developed to capture all research on the broad scope of the ScS and was composed of groups of search terms: 21 population terms (e.g., fish and aquaculture terms), 11 bacteria terms (e.g., coli" or Escherichia) and 21 general antimicrobial, 55 specific antimicrobial and 4 dye terms that were combined into four search strings of population and bacteria, general antimicrobial, specific antimicrobial or dye terms. The expert committee indicated that the most relevant research should have been published over the past two decades, thus, the search was limited to research published after 1990. No language restriction or other filters were imposed at this stage.

The search was conducted in October 2008 and updated in November 2010 using six electronic bibliographic databases: PubMed (1990-current), AFSA 1 Biological

Sciences and Living Resources (1971- current), AFSA 3 Aquatic Pollution and

Environmental Quality (1990- current), AFSA Aquaculture abstracts (1984- current). Ecology Abstracts (1982- current), and ZoologicalRecordPlus (2008). The search was verified by hand searching reference lists from five literature reviews (Heuer et al., 2009;

Kummerer, 2009; Lin et al., 2008; Sapkota, 2008; Toranzo et al., 2005) on a variety of topics selected to cover the breadth of the ScS. Any identified citation not captured through the electronic search was manually added to the electronic database and included in the screening process.

Relevance Screening and Exclusion Criteria

Two levels of relevance screening were conducted as part of the ScS without imposing language restrictions. An initial, abstract-based relevance screening 1 (RSI) was conducted to rapidly exclude research irrelevant to the scope of the study (e.g., diagnostic tests, non-primary research). Article-based, relevance screening 2 (RS2) was applied to confirm article relevance, apply specific exclusion criteria and characterize the relevant research according to type of bacteria, location of sampling, antimicrobial drug/dye investigated, sampling point in the production / processing chain, type of fish/shellfish sampled, study design and research focus (e.g., prevalence). At this stage studies were excluded: if only sediment, water or diseased fish or shellfish (studies reporting primarily morbidity and mortality) were sampled; if laboratory-based studies reported only genotyping results; and if the study setting was 'outside of field conditions'. The latter included studies that employed strictly controlled experimental conditions or used samples that did not originate from wild populations or actual aquaculture operations.

66 Development of Focused Question for SR

The ScS results (RS 1-2) were summarized into context-based evidence maps and summary tables so the research team could evaluate the distribution and characteristics of research underpinning the main themes/topics, identify the knowledge gaps, and prioritize questions where the evidence seemed potentially suitable for SR-MA. Specific question formulation for in-depth SR-MA was guided by the findings of the ScS, current global food safety and zoonotic public health trends, and the potential relevance of the

SR-MA findings to surveillance and monitoring programs, risk assessments or development of future research within both the global and Canadian contexts, including the overall food safety, fish/seafood consumption and trade importance for Canada.

The SR-MA review question prioritized for this study was: what is the reported prevalence (or concentration) of Aeromonas, generic E. coli, Salmonella and Vibrio species in clams, mussels, oysters, salmon, shrimp (including prawn), and tilapia from processing to the retail level (including ready-to-eat (RTE)). Generic E. coli was kept because it is an opportunistic pathogen, and a reservoir for AMR, and because of the need to access prevalence of the bacteria before assessing its susceptibility profile. Using SR-

MA, the evidence pertaining to this question was synthesized and summarized to provide estimates for future risk assessments and design of bacteriological prevalence surveys or on-going surveillance.

SR: Inclusion of Studies, Methodological Assessment and Data Extraction

Studies not published in English, French, Spanish, Portuguese or in one of the

Slavic languages were excluded due to a lack of resources to translate additional

67 languages. Assessment of methodological soundness and reporting (MSR) included two steps conducted in parallel. First, studies were checked to confirm their relevance to the prioritized question and that they met the minimum inclusion criteria for the SR-MA

(simply to preclude wasting manpower on MSR and data extraction). Studies were included if they reported: the numbers of specimens sampled and tested positive in raw or unadjusted form; adjusted data (if applicable) or measures of association/effect, and at least some measures of variability (standard error, standard deviation, confidence intervals, or p-value). Second, all studies that were confirmed to have appropriate extractable data were fiirther evaluated for MSR and categorized with respect to: type of study design, type of subject selection, sample size/strategy, type of operation sampled

(e.g., commercial farm, supermarket) and sufficient reporting of laboratory methods for bacterial culture and/or PCR to allow their replication. Aside fi"om sample size, none of the above was used for exclusion, but rather to evaluate the overall robustness of the existing scientific evidence. Given the nature of the research question, the research team decided a priori to exclude studies with less than a total of 30 samples in the entire study; studies with less than 30 samples were considered not representative of the study target population and thus of low utility.

Data extraction was conducted on all studies that passed the above mentioned exclusion criteria and included: 1) general information (author, journal, publication type);

2) population investigated (e.g., fish/shellfish species); 3) sampling characteristics (e.g., sample type, sample weight), outcome measured (e.g., type of test and cut-off threshold, and 4) reported results (e.g., number of samples positive/total samples). For studies reporting multiple results, for example, if the author sampled multiple years, seasons,

68 aquatic species or isolated different bacterial genera and reported the results for each subset separately, the data were extracted accordingly. Thus, some studies would have many lines of data available for meta-analysis (termed 'trials' for our purposes).

Management of ScS and SR

The citations obtained from the search were managed and de-duplicated using

Procite 5.0 (Thomson ResearchSoft, Philadephia, PA) and imported into an electronic SR management program, SRS 4.0 (Trialstat! Corporation, Ottawa, Canada). Following the completion of RSI, the data were transferred to another electronic SR management program, DistillerSR (Evidence Partners, Ottawa, Canada) that was used for the remainder of the ScS (RS2) and completion of SR. Every level of the ScS and SR

(relevance screening, assessment of methodological soundness and reporting, and data extraction) was developed and tested prior to initiation of the study. Evaluation and refinement of each tool took place during the testing period including development of numerous definitions and guidelines to standardize interpretation among eight reviewers.

All stages of the review were conducted by two independent reviewers and disagreements were resolved by the reviewing pair or group consensus, if required.

Meta-analysis

Extracted raw data were imported from DistillerSR into excel (Microsoft Excel,

Microsoft Corporation, Redmond, WA) where the data were cleaned, double-checked for correctness and consistency, and prepared for MA. Concentration data that were not already presented on the natural logarithm scale was transformed into log cfu/g to meet

69 the MA assumption of normally distributed data. Clean data were exported to

Comprehensive meta-analysis software V2 (Borenstein M, Hedges L, Higgins J,

Rothstein H. Comprehensive Meta-analysis Version 2, Biostat, Englewood NJ (2005)).

Datasets were created of studies measuring prevalence or concentration on the same seafood product with the same target bacteria, and were sub-grouped by the region where the study was conducted (e.g.. North America, Europe, Asia, Caribbean and South

America) to accommodate the variation by region and to increase utility for fixture use.

Based on this there were 55 subsets of data of which 14 only had 1 line of data, and random effects MA was used to analyze 41 datasets of >2 lines of data.

Given the a priori assumption of significant heterogeneity, random-effects MA models were constructed. Heterogeneity was assessed via a %2 test using the Q-statistic and quantified on a relative scale using the 12 value (Higgins et al., 2003; Borenstein et al., 2009). Due to low power, %2 tests for heterogeneity were considered significant if p

<0.10 (Borenstein et al., 2009; Higgins and Green, 2009). Between trial and study variance was estimated using the DerSimonian and Laird method; it was not possible to control for studies given the study/trial ratio (i.e., insufficient number of studies)

(DerSimonian and Kacker, 2007; Borenstein et al., 2009). When heterogeneity statistics were not significant (p-value>0.10 or 12 <50%), MA pooled estimates of prevalence or concentration and the upper and lower 95% confidence intervals were reported, otherwise the median and range was calculated from the individual studies within the subset of data.

An a priori decision was made to evaluate the potential for publication bias through

Begg's adjusted rank correlation and Egger's regression asymmetry tests only for homogeneous datasets with >10 unique observations (Begg and Mazumdar, 1994; Egger, et al., 1997; loannidis et al., 2007) with publication bias considered present if either

Begg's or Egger's tests resulted in a p-value <0.05.

RESULTS

ScS: Research Themes, Study Characteristics and the Focused Question

The citation flow through various stages of the ScS is shown in Figure 3.1 and the evidence map (distribution of primary research by the main characteristics of research themes and topics within the ScS scope) is displayed in Figure 3.2. In total, 1,760 research studies fell within the a priori determined broad scope (Figure 3.2). All of these were captured by the electronic search; no additional potentially relevant citations were identified during search verification. Investigation of the prevalence or intervention pertaining to the eight selected zoonotic bacteria was the focus of 80% (n=1,405 studies) of the relevant studies that were captured. Studies investigating AMR in aquaculture had the second highest amount of relevant research accounting for 10% (n=l 82) of the relevant studies; however, for brevity reasons, the AMR findings will be presented in another paper. In addition, a relatively low to moderate amount of research was found that investigated prevalence and food safety or public health aspects of dyes and drug residues (n=47). The knowledge gaps (least often investigated topics) included AMU in aquaculture (n=12), association between AMU and AMR (n=4), and AMU or AMR as a risk factor for human illness (n=l). Knowledge strengths included investigation of Vibrio across all research themes, followed by Aeromonas, generic E. coli and Salmonella. The majority of studies targeted the pre-harvest (farm/production) level followed by a

71 moderate number at the retail level for prevalence of selected zoonotic bacteria (Figure

3.2).

Aside from the formulated question selected for our SR-MA, other topics may include the prevalence of AMR in various bacteria found in aquaculture and in seafood, and to a lesser extent (due to a relatively small number of published studies), prevalence of drug and dye residues in seafood (Figure 3.1).

SR Database and Meta-Analysis

From 146 relevant studies, 79% (n=l 14) were of transversal prevalence survey design; the remainder were of longitudinal prevalence design. Over 80% of the studies

(n=121) reported a total sample size of >30; 14 and 11 studies had respective sample sizes of 11-29, and <10 samples, and were excluded. Sampling mostly occurred at local or specialty markets (n=78) followed by commercial processing plant and supermarket

(each n=34) and commercial farm (n=22). Laboratory methods were adequately reported in 89% (n=130) of studies; 76% (n=l 11) reported media type, time and temperature of incubation and enrichment steps, and the remaining studies reported used of methods described in another paper (n=19). Due to language restriction, 6 potentially relevant foreign articles were excluded at this stage. Steps in the SR process, complementary to the ScS, including confirmation of relevance to the SR question, and the reasons for study exclusion at the MSR stage is depicted in Figure 3.1. The SR database contained 72 relevant prevalence studies that passed the exclusion criteria of MSR assessment (Figure

3.1). The distribution of studies by aquatic species, bacteria and point in food chain is presented in Table 3.1. Over 70% of studies were published in 2000 or later and all were published in English except 2 published in Spanish (Pereira et al., 2007, Torres Vitela et al., 1993). The overwhelming majority of all studies reported prevalence outcomes

(n=66) as opposed to concentration (n=6 studies). The SR database included sample collection and test results from 23 countries. The most frequent sampling locations included India (n=18 studies) followed by the United States (7), China (7), Brazil (6) and

Japan (4). Others included Italy, Spain, Taiwan (each n=3), Bangladesh, Malaysia,

Mexico, Thailand and Vietnam (each n=2), and Chile, Germany, Indonesia, Nigeria,

Peru, Philippines, Portugal, Switzerland, Trinidad and Tobago, Turkey and the United

Kingdom (each n=l).

The MA was restricted to studies (n=56 studies/252 unique data observations) conducted at the retail and ready to eat (RTE) settings (Figure 3.1) due to the close proximity of this setting to the point of consumption and low numbers of studies/considerable heterogeneity across studies at other levels (Table 3.1). Region-level

MA prevalence, concentration (if available) summary estimates and respective confidence intervals (given insignificant heterogeneity (p-value >0.10)) were reported; median and range in the presence of significant heterogeneity (p<0.10) for various bacteria-seafood combinations are presented in Tables 3.2 and 3.3. The subgroup analysis forest plots showing trends in the prevalence of Aeromonas, E. coli and Salmonella in salmon by country is presented in Figure 3.3. None of the MA conducted met the criteria for evaluating potential publication bias.

DISCUSSION

SR-MA highlights

73 Through the ScS, we were able to identify several important characteristics of the body of published research underpinning our broadly defined scope of interest in aquaculture, including the numbers of studies published, their distribution by various categories of interest, the main study characteristics, and we also identified knowledge strengths and gaps. Evaluating the research underpinning various themes, topics and sub topics was useful in prioritizing SR-MA focused questions where suitable research exists.

Within the context of our SR-specific question, the most often investigated bacterial pathogen across the selected shellfish/crustaceans species (oysters, mussels, clams, shrimp) was Vibrio. Vibrio (e.g.. Vibrio parahemolyticus. Vibrio vulnificus) infection in humans is most often associated with consumption of raw shellfish and is a leading cause of seafood-borne disease in the United States (Kaysner et al., 2001) and Japan (IDSC,

1999), and accounts for about a third of all foodbome outbreaks in China (Liu et al.,

2004). Although heterogeneity of the data precluded the computation of pooled estimates, the broad range of prevalence of Vibrio observed across and within specific species- bacteria-region in shellfish/crustaceans at retail (often reaching prevalence above 90%) is of particular interest. This variability in prevalence was observed even in Europe and

North America where retail store conditions may be more homogenous than in other regions.

Only 11 relevant prevalence studies of bacterial contaminants were conducted in selected finfish populations (salmon and tilapia) with only 2-3 studies investigating

Aeromonas, Salmonella, E. coli and Vibrio (each). Random-effect MA highlighted that

Aeromonas and generic E. coli, two bacteria generally considered as commensal but which can also be pathogenic, tend to be more fi*equently isolated than Salmonella

74 (reported overall prevalence below 5%) in retail salmon, and an insufficient number of publications on Vibrio precluded prevalence estimates. Aeromonas is common in fmfish

(Yucel et al, 2010; Herrera et al., 2006) resulting in contamination likely during processing (e.g., smoking) or retail packaging (Butt et al., 2004).

The results from the MA indicate that significant heterogeneity existed across the studies included in the SR. Although some of the heterogeneity might be the result of geographic differences in study locations, other sources could include inadequate randomization at sampling, type of tests used for measuring prevalence (e.g., culture media), sample type (e.g., homogenized meat), and country of origin of the product (an information rarely available). Wide ranges in prevalence may also be due to the fact that most seafood is purchased in only lightly processed forms so improper harvesting or processing can still harbor microbiological agents, potentially putting consumers at risk

(Canadian Aquaculture Industry Alliance, 2008).

There was a relatively large amount of intervention research on Vibrio and

Aeromonas that may be further explored through SR-MA, in the event that more research

(with an acceptable level of homogeneity) becomes available to allow for more robust

MA. The SR format has been traditionally used on questions assessing intervention efficacy, although it is also usefiil for answering other types of policy-driven questions

(Sanchez et al., 2007; Sargeant et al., 2007; Waddell et al., 2009; Young et al., 2009;

Wilhelm, 2009). To the best of our knowledge, SR-MA addressing these topics in aquaculture does not currently exist.

Knowledge gaps

75 The ScS identified several knowledge gaps (as indicated by a deficiency of published studies) including AMU in aquaculture, associations between AMU and AMR, and the human health impact of AMU/AMR from aquaculture. AMU data are publicly available on an annual basis from a few European surveillance programs (DANMAP,

NORM-VET) but no quantitative information could be retrieved &om regions other than

Europe. In Canada, the province of British Columbia recently collected AMU information and published the report online (Ministry of Agriculture and Lands, 2008).

AMR and AMU are internationally recognized as priorities for public health

(WHO/OIE/FAO, 2007) and as such are in need of more primary research. Existing narrative reviews have discussed general food safety / human health concerns about

AMU and AMR (MacMillian 2001; Cabello, 2006; Sapkota, 2008) and also reported that the published evidence to support or refiite these concerns is very limited.

Although Aeromonas and Vibrio are important aquatic and human pathogens, the overall number of relevant (and of sufficient quality) studies investigating their prevalence in the 6 aquatic/seafood species was low iox Aeromonas (1-8 studies/ species) and low-moderate for Vibrio (1-21 studies/ species). Salmonella and generic E. coli were less studied across the species (generic E. coli: 1-8 studies/ species; Salmonella: 1-14 studies/species) and investigated the most for shrimp. Given that between 2005-2010, most seafood-bome outbreaks of Salmonella were attributed to consumption of oysters and salmon, perhaps fixture studies and surveillance programs should focus more on

Salmonella, in oyster/salmon as well as other species. Given its importance as human pathogen and reports of sometimes very high prevalence at retail, there is also a need for

76 adequate representative surveys investigating the prevalence of Vibrio, particularly in shellfish/crustaceans.

Lastly, a relatively low volume of research investigating Campylobacter,

Edwarsdiella tarda and ictaluri, Erysipelothrix rhusiopathie and Streptococcus iniae was found. This might be explained by their perceived lack of relevance to seafood safety and/or public health, their higher relevance to other types of food-animal production (e.g.,

Campylobacter in poultry) or recent emergence in the aquaculture sector.

The overall scarcity of information for many species-bacteria-region combinations can be partly explained by small and sporadic sampling, or low quality studies, as indicated by the number of studies dropped between "Confirmation of relevance" and "Data extraction". The assessment of methodological soundness of studies included in the SR consistently indicated study design and reporting deficiencies.

Over 21% (n=30) that were relevant and were based on more than 30 samples were excluded due to poor reporting (Figure 3.1). The sample size or sampling frame was not justified in any studies and only 26 studies used more than 100 retail or RTE samples.

This relatively small, unjustified sampling limits the precision and generalisability of individual study estimates (Borenstein, 2009); small studies have more variation around the true estimate and unjustified sampling may not be representative of the country/region or of the product. Unfortunately, the effect of this on the MA is unpredictable as there is no way to characterize the direction of the impact on the effect estimate (Borenstein,

2009).

Recommendations for future seafood surveys

77 To avoid study exclusion and to inform the overall SR-MA process with better designed and reported data, researchers in food safety, including seafood safety, should follow the STROBE (Strengthening the Reporting of Observational Studies in

Epidemiology) and REFLECT (Reporting Guidelines for Randomized Controlled Trials for Livestock and Food Safety) guidelines. These guidelines provide suggestions for reporting observational research in general (Vandenbroucke et al., 2007) and intervention research in food safety in particular (Sargeant, 2010) with the aim to standardize the quality and utility of published data. Implementation of these guidelines, already adopted by many relevant journals in this area, would considerably improve the overall utility of

SR-MA, an otherwise powerful methodology.

Limitations and strengths of the study

The ScS used a comprehensive search and search verification strategy to identify published literature; however, there appear to be limitations in the work. Firstly, there may be a significant amount of data in aquaculture that is unpublished or cannot be captured via electronic searching of bibliographic databases. We did not contact any international or Canadian researchers working in this area to find out about relevant on going projects or in-press publications. Secondly, our efforts to reduce language bias (a form of publication bias) included efforts to translate only select languages (French,

Portuguese, Spanish and Slavic languages) that resulted in exclusion of 6 articles, all in one of the Asian languages. Given that Asia includes leading aquaculture coimtries (FAO

SOFIA, 2010), potentially important data may have been omitted with the exclusion of

78 these articles. Perhaps international collaboration can be used to further reduce publication bias in SRs encompassing foreign literature.

The initial ScS-SR process as a whole is very demanding on all resource fronts

(e.g., time, manpower, expertise required). The main advantage of this process is its transparent synthesis of research that can be up-dated at any time using the same protocol as the initial review. Up-dated searches typically capture far fewer citations than the initial reviews and can therefore be conducted relatively quickly. As new research becomes available, this process allows for continual evaluation of the distribution and quality of research and can provide current pooled outcome estimates. The results can be used as transparent, evidence-based inputs for decision models or information for policy makers when addressing complex microbial food safety issues. The data generated though our MA can be used as inputs for risk assessments.

Relevance in the Canadian context

The six species selected for the SR-MA were based on our impression of their importance in Canada as imported (e.g., shrimp) and domestically produced products

(e.g., salmon), yet, our SR has clearly indicated a lack of any published or on-line accessible prevalence research regarding domestic production or products imported in a

Canadian context. In Canada, there is a growing interest in aquaculture food safety.

Salmon is the top aquaculture species in Canadian seafood production (Canadian

Aquaculture Industry Alliance, 2008) and 487 million kilograms of seafood is imported each year (DFO, 2009). Trade of aquaculture products occurs worldwide between regions with different production practices and government regulations. Surveillance of domestic

79 and imported products and review of regulations and food safety programs in exporting countries are important for reducing the probability of importing contaminated foods, including seafood (DuraN and Marshall, 2005). About 5% of imported seafood products are investigated for both chemical (e.g., drug residues, pesticides) and biological contaminants (e.g., bacterial pathogens, viruses, parasites) by the Canadian Food

Inspection Agency (CFIA, 2009). To address part of the knowledge gaps, the Public

Health Agency of Canada is currently piloting a study investigating seafood contamination and antimicrobial resistance at retail with a number of bacteria-seafood- matrix protocols (personal communication with Brent Avery, PHAC). Canadian regulatory agencies in charge of policy development and their implementation could consider using the findings fi-om our SR-MA along with available proprietary data on domestic or imported products (not included in our review), and data generated through on-going and future bacteriological surveys to develop and prioritize risk-based surveillance (Stark et al., 2006) for both domestic and imported aquaculture and seafood products.

CONCLUSION

Although the methodology for ScS is still being developed and its use in the agri- food industry is relatively recent, we have found it to be a useful tool in assessing the breadth of evidence investigating various aquatic species, zoonotic bacteria, public health topics and main knowledge gaps. From the evidence maps that were created based on the results of the ScS, the research team was able to prioritize questions for rigorous SR on selected pathogens, aquatic species and point in food chain that were significant to the

80 global and Canadian contexts. We have found that a small number of studies investigated the prioritized question, passed methodological assessment, there were consistent issues with reporting results and our meta-analysis revealed significant study heterogeneity for all species aside from salmon.

ACKNOWLEDGEMENTS

The authors would like to thank Drs. Sophie St-Hilaire and Carol McClure for their guidance; Drs. Ian Young and America Mederos Silveira, Maqorie Bercier, Rebecca de Parent, Karine Forget for their help in reviewing papers; Janet Harris, Nanky Rai,

Kyle Burgers, Malcolm Weir for their help in procurement of papers; as well as the funding support of the Public Health Agency of Canada.

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87 Table 3.1. Number of relevant quality-assessed studies submitted to data extraction by aquatic species, bacteria and point in food chain. Point in Food Chain Number Bacterial Ready Aquatic Total of Genera/ Total Processi -to- Restau Imported/ Species Studies Trials Species Studies ng Retail Eat rant Exported Salmon 6 13 Aeromonas 3 0 3 0 0 2 E. coli 1 0 1 0 0 0 Salmonella 2 0 1 1 0 0 Vibrio 0 0 0 0 0 0 Tilapia 5 12 Aeromonas 3 0 3 0 0 0 E. coli 2 0 2 0 0 0 Salmonella 1 0 1 0 0 0 Vibrio 2 0 2 0 0 0 Oyster 27 88 Aeromonas 3 0 1 0 0 0 E. coli 4 0 3 1 0 0 Salmonella 6 1 3 1 0 1 Vibrio 17 2 12 1 1 0 Mussel 21 42 Aeromonas 2 0 1 0 0 0 E. coli 1 0 1 0 0 0 Salmonella 5 2 4 0 0 0 Vibrio 16 3 10 1 0 0 Clam 23 57 Aeromonas 0 0 0 0 0 0 E. coli 3 0 3 0 0 0 88 Salmonella 3 0 3 0 0 0 Vibrio 18 2 12 0 0 0 Shrimp 43 115 Aeromonas 8 0 9 0 0 0 E. coli 8 1 7 0 0 0 Salmonella 14 5 8 0 0 1 Vibrio 21 7 16 0 0 1

89 Table 3.2. A summary of various subsets of data reporting the prevalence of four selected bacteria in four aquatic species-seafood categories at retail (including RTE) and sub-grouped by region of sampling to improve interpretation and decrease heterogeneity. Prevalence Number Heterogeneity^ Bacterial (proportion) Aquatic Sampling of Genera/ Region Pooled Estimate Species Level' Studies (P-value) Species (CI)' (Trials)^ Median (Range)® Tilapia Retail Asia/ME 2(2) No (0.55) 0.74 (0.60 - 0.85)"^ Carib/SA 1(2) No (0.40) 0.35 (0.30 - 0.39)^ Retail Asia/ME 1(1) NA 0.27 (0.10-0.53)^ N. Amer 1(2) No (0.38) 0.06 (0.02-0.16)^ Retail N. Amer 1(2) No (0.95) 0.01 (0.00-0.09)^ Vibrio Retail Asia/ME 2(3) Yes (0.06) 0.08 (0.08 - 0.60)^ Salmon Retail Europe 3(3) No (0.52) 0.13 (0.06-0.27)"^ E. coll Retail N. Amer 1(2) No (0.70) 0.02(0.00-0.11)'^ N. Amer & Retail 2(3) No (0.99) 0.01 (0.00 - 0.05)"* Europe Shrimp Retail Asia/ME 4(4) Yes (0.00) 0.16(0.05-0.36)^ E. coli Retail Asia/ME 3(9) Yes (0.00) 0.13 (0.03-0.98)^ Carib./SA 1(2) No (0.59) 0.37 (0.26 - 0.50)^ Europe 1(1) NA 0.03 (0.02 - 0.05)^ Retail Asia/ME 7(15) Yes (0.00) 0.04 (0.00 - 0.59)^ Europe 1(1) NA 0.00 (0.00 - 0.02)^ N. Amer 1(1) NA 0.08 (0.08 - 0.09)^ Retail Africa 1(3) Yes (0.00) 0.15(0.01-0.43)' Asia/ME 11(35) Yes (0.00) 0.25 (0.01 -0.94)' Carib/SA 3(6) Yes (0.00) 0.04(0.01 -0.35)' 90 Europe 1(1) NA 0.00 (0.00 - 0.02)^ Clams Aeromonas Retail Europe 1(1) NA 0.75 (0.24 - 0.91 f E. coli Retail Asia/ME 3(5) Yes (0.00) 0.82 (0.06 - 0.94)^ Salmonella Retail Asia/ME 2(4) No (0.27) 0.23 (0.13-0.37)^ Europe 1(1) NA 0.01 (0.00-0.12)^ Vibrio Retail Asia/ME 7(16) Yes (0.00) 0.26 (0.01 -0.95)^ Europe 1(4) No (0.15) 0.40 (0.29 - 0.52)^ N. Amer 2(15) Yes (0.00) 0.42 (0.06 - 0.90)^ Mussels E. coli Retail Europe 1(1) NA 0.04 (0.02 - 0.05)^ Salmonella Retail Asia/ME 1(1) NA 0.16(0.08-0.29)^ Europe 2(3) No (0.26) 0.00 (0.00-0.01)^ Vibrio Retail Asia/ME 4(8) No (0.95) 0.24 (0.16-0.34)^ Carib./SA 1(1) NA 0.02 (0.00- 0.13)^ Europe 3(5) Yes (0.00) 0.29 (0.03 - 0.44)^ N. Amer 2(6) Yes (0.09) 0.45 (0.24 - 0.92)^ RTE Asia/ME 1(2) Yes (0.00) 0.48 (0.12-0.84)^ Oysters Aeromonas Retail Asia/ME 1(1) NA 0.50 (0.30 - 0.70)^ E. coli Retail Asia/ME 1(1) NA 0.95 (0.55- 1.00)^ Carib/SA 1(4) No (0.19) 0.76 (0.70 - 0.82)^ RTE Carib/SA 1(4) No (0.69) 0.77 (0.70 - 0.82)^ Salmonella Retail Asia/ME 1(2) Yes (0.03) 0.19(0.07-0.30)^ Carib/SA 1(4) No (0.13) 0.05 (0.03-0.11)^ Europe 1(1) NA 0.01 (0.03-0.02)^ N. Amer 1(1) NA 0.03 (0.02 -0.05)^ RTE Carib/SA 1(4) No (0.95) 0.02 (0.01 -0.05)^ Vibrio Retail Asia/ME 6(11) Yes (0.00) 0.13 (0.03-0.80)' Europe 2(2) No (0.44) 0.56 (0.42 - 0.69)'* 91 N.Amer 4(25) Yes (0.00) 0.71 (0.14-0.97)^ RTE N.Amer 1(2) Yes (0.00) 0.68(0.63-0.73)^ ' Samples were reported as retail or RTE products. ^ Trial is a unique combination of aquatic and bacteria species using a specific outcome and sample type. ^ Heterogeneity is the differences in the estimates of effects due to variability between studies. Pooled prevalence estimate is presented only in absence of heterogeneity (p > 0.10). ^ Prevalence median (range) is reported using reported prevalence from individual studies representing a unique subset of data. ^ Only a single trial was available in this subset of data, the trial data is presented in table. ^ ME=Middle East; SA=South America.

92 Table 3.3. A summary of various subsets of data reporting the concentration of three selected bacteria in selected aquatic species-seafood categories at retail (including RTE) and sub-grouped by region of sampling to improve interpretation and decrease heterogeneity. Aquatic Bacterial Sampling Region Number of Heterogeneity^ Concentration cfu/g Species Genera/Species Level* Studies (P-value) Pooled Estimate (CI)'* (Trials)^ Median (Range)® Salmon Aeromoms Retail Europe 1(4) Yes (0.00) 2.99 (2.27-3.28)^ Shrimp Aeromonas Retail Asia/ME 1(5) No (0.27) 5.34 (4.39 -6.29)^ Oysters E. coli Retail Carib/SA 1(4) No (0.31) 5.97 (5.03-6.91)"^ RTE Carib/SA 1(4) Yes (0.01) 4.68 (3.18-6.94)^ Shrimp E. coli Retail Asia/ME 1(2) No (1.00) 5.08 (3.62-6.52)"^ Clams Vibrio Retail Asia/ME 1(2) Yes (0.02) 4.71 (4.00-5.42)^ Asia/ME Oyster Vibrio Retail &N. 2(20) Yes (0.00) 1.76(0.40-5.67)^ Amer Shrimp Vibrio Retail Africa 1(2) No (0.42) 4.51 (3.64-5.39)^ ' Samples were reported as retail or RTE products. ^ Trial is a unique combination of aquatic and bacteria species using a specific outcome and sample type. ^ Heterogeneity is the differences in the estimates of effects due to variability between studies. Pooled prevalence estimate is presented only in absence of heterogeneity (p > 0.10). ^ Prevalence median (range) is reported using reported prevalence from individual studies representing a unique subset of data. ^ Only a single trial was available in this subset of data, the trial data is presented in table. ^ ME=Middle East; SA=South America.

93 Search Execution 18123

Excluded 3673: Duplicates First Round of Relevance Screening 14450

Excluded 12159: Irrelevant to broad scope

Second Round of Relevance Screening 2391

Excluded 631: Irrelevant to broad scope i Evidence Mapping 1760

Prevalence for Aeromonas, Vibrio, Salmonella, E.coli at the processing, Prioritization of One Topic for SR-MA \ retail, ready-to-eat, or import/export level in salmon, tilapia, shrimp/prawn Excluded (with filter in review and oyster, mussel, clam. management software) 1511: Irrelevant to SR-MA topic Potentially Relevant Articles 249

Confirmation of Relevance to Topic 249

Excluded 103: Irrelevant to SR-MA topic Quality Assessment 146

Excluded Excluded 6: Foreign article not in accepted Quality Assessment Criteria: 39: Raw/unadjusted data or Data Extraction measures of association/effect 72 not provided Excluded 4: No reporting of measures of 25: Studies with total samples < 30 variability provided for adjusted data

Excluded from MA 3: samples of mixed species 2: no usable data /^eta-Analysis of Studies at the Rretaih 5: only processing data Level (including ready-to-eat) 6: data from live fish sold at retail V 56 J

Figure 3.1. Scoping study and systematic review process flow-chart. Square boxes depict scoping study; rounded boxes indicate systematic review.

94 Aeromonus E.coU Salmonella Campylohacler Edwardsiella Erysipeluthrix Streptococcus Selected Zoonotic 543 283 181 179 16 30 2 16 Bacteria Finfish 197 187 89 74 5 26 0 12 853 Mollusk 203 36 71 65 8 0 1 0 Crustacean 171 50 35 51 1 0 1 1 Harvest 718 127 148 151 37 28 137 49 40 42 28 0 3 4 0 0 25 0 0 1 0 11 AMR 14 26 1 Processing 53 10 9 8 0 7 6 3 8 4 7 0 0 0 0 0 0 0 0 0 0 182 2 10 1 0 Retail 199 31 39 32 9 6 25 13 20 35 16 13 30 0 5 1 0 0 1 0 1 0 0 0 1 Ready-to-Eat 17 2 2 2 1 1 3 4 4 5 5 6 8 0 0 0 0 0 0 0 0 0 Q 0 0 Antimicrobial/Dye Import/Export 12 5 2 1 2 0 0 2 1 0 4 3 2 0 0 0 0 0 0 0 0 0 6 0 0 Residue Prevalence/Risk 47 Factor Studies

1166 AMU in Commercial Aeromonas E.coli Salmonella Campylobacter Edwardsiella Erysipelothrix Streptococcus Setting 105 91 21 25 1 20 0 3 12 Finfish 37 58 8 12 0 7 0 1 Mollusk 15 6 4 4 1 0 0 0 AMU-AMR Association Crustacean 36 8 8 12 0 0 0 0 4 Haivest 194 23 11 32 6 4 46 4 2 6 6 0 7 0 0 0 0 0 7 0 0 0 0 0 1 Processing 5 0 0 0 0 0 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Retail 41 6 3 3 2 0 4 4 2 2 5 3 2 0 1 0 0 0 0 0 0 0 0 0 0 Ready-to-Eat 3 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 AMU/AMR as Risk Import/Export 5 1 0 0 0 0 0 1 0 0 2 1 2 0 0 0 0 0 0 0 0 0 0 0 0 Factor for Human Illness 1

Selected Zoonotic Vibrio Aeromonas E.coli Salmonella Campylobacter Edwardsiella Erysipelothrix Streptococcus Bacteria 283 180 35 21 1 82 1 22 552 Intervention Finfish 123 158 13 6 0 57 0 15 Studies Mollusk 61 4 14 8 0 0 0 0 Crustacean 87 8 9 7 0 0 0 560 0 AMU/Dye Residue Harvest 379 54 102 87 2 1 102 0 5 2 1 1 0 0 0 0 0 0 57 0 0 0 0 0 15 Processing 67 4 7 2 2 3 7 3 6 3 5 4 4 0 0 0 0 0 0 0 0 0 0 0 0 Retail 30 5 1 3 1 0 1 8 1 3 3 1 1 0 0 0 0 0 0 0 0 0 0 0 0 Ready-to-Eat 1 0 0 1 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Import/Export 2 0 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 Human Illness 1 229

Seafood Consumption

Other Vibrio Aeromonas E.coli Salmonella Campylobacter Edwardsiella Erysipelothrix Streptococcus 241 161 7 22 39 8 6 4 7 Finfish 33 2 8 13 1 4 3 5 Import/Export Mollusk 77 0 7 12 5 1 0 0 4 Crustacean 17 1 Tl T]

95 Figure 3.2. Evidence mapping. Distribution of primary research according to type of study, topic, pathogen, aquatic species and point in food chain. Edwardsiella is exclusive to E. tarda, E. ictaluri. Erysipelothrix is exclusive to E. rhusiopathiae. Streptococcus is exclusive to S. iniae.

96 P

Figure 3.3. Random effects meta-analysis of the prevalence (expressed as proportion) of Aeromonas, E. coli and Salmonella on Salmon. Study prevalences were homogenous, 12 statistics of 0%. No publication bias was detected in subgroups with 3 or more lines. Pooled prevalence: yieromowai' 0.131(0.058-0.271), E. coli 0.024 (0.005-0.109), Salmonella 0.013 (0.003-0.051).

97 CHAPTER FOUR

SUMMARY DISCUSSION AND CONCLUCIONS

SUMMARY DISCUSSION

Globally, the aquaculture industry has, in the last two decades, been the fastest growing food production sector (Naylor et al., 2003) and increased consumption of seafood raises important issues regarding public health. Concerns about the safety of seafood include contamination with pathogens of animal, human or environmental origin, including those that are antimicrobial resistant (AMR), the safety of import/exported products, and contamination with various chemical residues, including those arising from natural or industrial pollution (e.g., mercury), antimicrobial use (AMU), as well as use of other drugs and dyes in aquaculture production. The wide-ranging nature of these issues, compounded by the diverse nature of the industry itself, provides challenges to the identification, summarization and prioritization of issues for decision-makers in charge of protecting public health. For these decision or policy-makers to make effective and responsible decisions regarding necessary public health initiatives (e.g., potential surveillance activities) it is imperative that unbiased, evidence-based, complete and synthesized information is provided to guide this process.

A research synthesis-expert questionnaire approach was used in the research described in this thesis to characterize and evaluate the existing primary research in aquaculture and seafood production and elicit global expert opinion on these topics. The questionnaire, described in Chapter 2, elicited opinions of aquaculture-allied professionals on topics related mainly to AMR and AMU, and was carried out primarily to attempt to bridge knowledge gaps present in the published literature. The research

98 synthesis component was described in Chapter 3 and included both a scoping study (ScS) and a systematic review-meta-analysis (SR-MA), offering a transparent and replicable process for the review and synthesis of all the available primary research, identifying research strengths, weaknesses, knowledge gaps, syntheses of data, and future research needs (Sargeant, 2006; Borenstein et al., 2009). Firstly, a ScS, which has recently been proposed for reviewing and characterizing a diverse body of research, (Arksey and

O'Malley, 2005; Anderson et al., 2008; Davis et al., 2009), was used to identify, evaluate and synthesize the existing primary research on the prevalence, risk factors and interventions for selected zoonotic bacteria, AMU and AMR in aquaculture and seafood production. Secondly, based on the results of the ScS and relevance to current food safety and public health issues, a topic was prioritized (prevalence and/or concentration of selected bacteria) for the application of SR-MA; methods traditionally applied on narrow and focused questions. These methoids have long been used in many sectors of public health, including recent applications in addressing various issues regarding food safety (Sanchez et al., 2007; Sargeant et al., 2007; O'Connor et al., 2008; Waddell et al.,

2009; Wilhelm et al., 2009; Young et al., 2009).

Evidence maps were created from the scoping study, and revealed several knowledge strengths and gaps in the current body of published research. A large volume of literature investigated interventions (n=560) mainly on the reduction of Vibrio and

Aeromonas at harvest, prevalence of selected zoonotic bacteria (n=853), human illness associated with seafood (n=229) and prevalence of AMR (n=182). In contrast, far fewer studies investigated prevalence of drug and dye residues in seafood (n=47) and imported/exported products (n=4), indicating knowledge gaps within the literature. Similarly, our study also identified a lack of information on AMU (n=12), the potential association between AMU and AMR (n=4), and the role of AMU/AMR (n=l) from aquaculture in disease in humans. Regarding AMU, the results of the questionnaire suggested that although there is overall limited use of some antimicrobials, indicated by the frequent selection of 'Never' used category by respondents, some classes of antimicrobials important to human health may be used frequently (e.g., quinolones).

Because the responses of experts were based on opinion, rather than direct empirical evidence, the results should be interpreted with caution and cannot be used to entirely fill the gap in the literature. In order to draw more accurate conclusions regarding AMU in this sector, more on-farm studies and surveys should be conducted aimed at measuring

AMU, and monitoring and publication of AMU in aquaculture is needed at regional/national/ international levels. The aquaculture industry is predicted to continue expanding in the next decade (Agriculture and Agri-Food Canada, 2005), making it more important that these data gaps are adequately assessed in order to protect public health.

In Canada, salmon is the top aquaculture species in terms of seafood production

(Canadian Aquaculture Industry Alliance, 2008) and a lack of food safety research in this species, globally and in the Canadian context, was observed throughout the ScS and SR-

MA. In addition, the results of the questionnaire suggest that while there is at least occasional use of antimicrobial drugs in this species, information pertaining to AMR in bacteria from salmon is an important knowledge gap due to a very low number of responses. We suggest that additional studies are required and we recommend more attention be given to this species in food safety and antimicrobial resistance monitoring programs.

100 A lack of published studies investigating samples from imported seafood was also observed and although such data might exist with regulatory authorities in this area, it was not accessible online. In Canada, 487 million kilograms of seafood is imported each year (Fisheries and Oceans Canada, 2009) and 5% of imported seafood products are investigated by the CFIA for both chemical (e.g., drug residues, pesticides) and biological contaminants (e.g., bacterial pathogens, viruses, parasites). Of the imported seafood, shrimp are the main products both by quantity and value (Fisheries and Oceans Canada,

2009). The results from our questionnaire show that resistance to quinolones, which are antibiotics not approved for use in Canadian aquaculture (Veterinary Drug Directorate,

2004) but are used in other countries (FAO, 2005), was most frequently reported in shrimp. Trade of aquaculture products occurs worldwide between regions with different production practices and government regulations. Surveillance of domestic and imported products and review of regulations and food safety programs in exporting countries are important for reducing the probability of importing contaminated foods, including seafood (DuraN et al, 2005). Thus, we believe there is merit in including testing of susceptibility to quinolones and other critically important antimicrobial classes in food safety monitoring of imported shrimp.

The evidence maps were useful aids in the prioritization of a question for the SR-

MA component of the research. It was also important that the question was relevant to current global food safety, public health trends, potential monitoring, risk assessment and research needs within global and Canadian contexts. The following question was identified: What is the reported prevalence (or concentration) of Aeromonas, E. coli

(generic/pathogenic). Salmonella and Vibrio species in clams, mussels oysters, salmon,

101 shrimp (including prawn), and tilapia from processing to the retail level (including RTE)?

The SR-MA revealed wide range in the prevalence of important zoonotic bacteria in seafood at the retail level. The sometimes high prevalence reported (e.g., Vibrio,

Aeromonas) may be due to the fact that most seafood is purchased in only lightly processed forms and, due to improper harvesting or processing, can still harbor microbiological threats, putting consumers at risk (Canadian Aquaculture Industry

Alliance, 2008). However, overall, inadequate reporting of study conduct and results was observed across the studies included in the SR-MA, precluding their use in the analysis.

This finding supports the need and implementation of guidelines, such as STROBE

(Strengthening the Reporting of Observational Studies in Epidemiology) and REFLECT

(Reporting Guidelines for Randomized Controlled Trials for Livestock and Food Safety) aimed at standardizing the quality and utility of published data (Vandenbroucke, 2007;

Sargeant et al., 2010). Also, small and unjustified sampling was used throughout the studies, questioning the overall generalizability of study estimates. Larger representative sampling of fish/shellfish is needed to increase representativeness and utility of the MA methodology in evaluating pathogenic prevalence in retail seafood.

Efforts were made to maximize the benefits of the research synthesis and questionnaire methodology used in this study. The work was guided by an expert committee with extensive areas of expertise relevant to the topics and methodology and efforts were made to improve the overall applicability of the research to the industry and relevant stake-holders. Specifically, for the research synthesis component, a comprehensive search and search verification strategy was used in order to capture all published literature on the topics of interest and paired reviewers ensured accurate

102 selection of relevant articles. Though this process is very resource consuming, its main benefit is that it can be up-dated relatively quickly in order to provide up-to-date effect measures and conclusions as new research is published. As for the questionnaire, strategies were utilized to increase the response rate including sending a pre-notification letter, administering scheduled reminders, a Spanish version for relevant regions and allowing participants to skip sections irrelevant to their expertise. We believe that these actions may have aided in the respectable response rate that was achieved (32.9%) and may also be beneficial to other studies using the questionnaire methodology.

Despite out best efforts, there are some limitations of the work. Both components of the study revealed an underrepresentation of regions. A very low response rate (15% compared to 32.9% for the English version) was achieved from the Spanish speaking regions (mainly South and Central America and the Caribbean), and, for the synthesis research component, a language restriction imposed by strained resources led to the exclusion of 6 articles in one of the Asian languages. Given the importance of these regions to the aquaculture and seafood production industry, important information may have been omitted. Perhaps international collaboration with researchers in these regions may have produced different results from both components as collaborators may locally encourage participation within countries and aid in translating articles.

Through our study, we have identified several areas that were beyond the scope of this thesis and require some additional work, but may be of interest to domestic and international aquaculture stake-holders. Examples include intervention research on Vibrio and Aeromonas, prevalence of AMR, and research into human illness associated with consumption of seafood. The relevant articles have already been collected and classified

103 in our database and may be considered for future SR-MA projects. Canadian regulatory agencies in charge of policy development and implementation could consider using the findings from our SR-MA along with potentially available proprietary data, and data generated through on-going and future bacteriological surveys to develop and prioritize risk-based surveillance (Stark et al., 2006) for both domestic and imported aquaculture and seafood products. Also, future studies should consider administering questionnaires to producers in aquaculture. The target population of our questionnaire was professionals with a wide range of expertise and the responses to the questionnaire were most representative of those professionals with clinical experience and not, as would be expected, of producers. While veterinarians with clinical experience remains the preferred target audience when investigating antimicrobial drug use, producers may be the only source of information in regions where drugs can be administered without the supervision of a veterinarian. As well, solicitation of opinion and other data fi"om producers concerning foodbome hazards in aquaculture would be a useful complement to any future on-farm research in this area.

CONCLUSION

This study has contributed to the current state of knowledge regarding foodbome human health hazards in aquaculture and seafood production and overall, we have found the research synthesis-expert questionnaire as a useful approach in completing this task.

It would be useful to make other researchers more aware of the issues that were identified in this work regarding study conduct and reporting to improve the quality of published literature in this field. The initial baseline information captured in our questionnaire and

104 the results from our synthesis research can be used by government and funding agencies to guide future primary research addressing the various knowledge gaps, potential monitoring and surveillance for pathogens, products, AMU and AMR, and as inputs in risk-assessment analysis.

105 REFERENCES

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108 APPENDIX 2. Questionnaire: Survey on Antimicrobial Use and Resistance in

Zoonotic Bacteria in Aquaculture, Seafood and Ornamental/Pet Fish

APPENDIX 2.1. Questionnaire-English Version

Survey on Antimicrobial Use and Resistance in Zoonotic Bacteria in Aquaculture, Seafood and Ornamental/Pet Fish'

1: Definitions: Aquaculture: The farming of marine and freshwater fish and animals for human consumption. Seafood: Any fish and shellfish (including mollusks and crustaceans, excluding seaweed) that is served as food or is suitable for eating. Ornamental Fish: Any small fish/shellfish that are kept in aquariums intended to be kept as pets and not for consumption.

Section 1:Respondent demographics (Applicable to all respondents) 1. My main area(s) of expertise is: {Please check all that apply) Microbiology Epidemiology Fish health / Clinical medicine Food safety Outbreak investigation Molecular biology / Genetics Other, please specify:

2. My expertise was primarily acquired in: (Please check all that apply) Government Academia Industry Other, Please specify:

3. My primary activity(ies) is (are) in: {Please check all that apply) Research (e.g., new test development) Clinical or field work Laboratory work (e.g., routine diagnostic testing) Administrative tasks (e.g., management) Animal health surveillance Public health surveillance Other, please specify: 109 4. I acquired my expertise primarily in: (Please check all that apply)

_ Africa _ Asia _ Europe _ North America _ Oceania _ South and Central America and Caribbean _ Other, Please specify region:

5. Do you have experience with any of the following OR other aquatic species: catfish, clams, crawfish, lobster, mussels, oysters, salmon, shrimps/prawns, tilapia, trout, wild fish populations and/or ornamental/pet fish?

• Yes, I have experience with one or more of the listed OR other aquatic species. • No, I do not have experience with any of the listed OR other aquatic species (Please proceed to Section 3. Do not complete the remaining questions in this section).

6. Please rate your level of experience with the following types of fish or seafood. (Please rate all that apply.)

Please use the scale 1 to 5 or check "No experience" where appropriate.

Catfish Clams Crawfish Lobster Mussels Oysters

110 Salmon Shrimps / prawns Tilapia Trout Ornamental/pet fish Wild fish populations Other If you selected ornamental/pet fish and/or wild fish populations and/or other, please specify:

7. How many years of expertise have you accumulated with aquacuiture and/or seafood and/or ornamental fish?

• <3 years • 3-10 years • >10 years

8. Aquacuiture and/or seafood and/or ornamental fish represents (Please choose one of the following.)

• < than 25 % of my day-to-day work activities. • 26 to 50% of my day-to-day work activities. • 51 to 75% of my day-to-day work activities. • 76 to 100 of my day-to-day work activities.

Ill Section 2: Antimicrobial drug usage in aquacuiture (including ornamental/pet fish production)

9. Do you have expertise with antimicrobial drug usage in aquacuiture?

• Yes • No (Please proceed to Section 3. Do not complete the remaining questions in this section.)

10. To the best of your knowledge, please approximate how frequently each antimicrobial drug class was used for the listed aquatic species?

Please use the scale 1 to 5, choosing the one that best applies. Leave blank if you do not know.

Aminoglycosi des (e.g., 1 gentamicin, streptomycin.) Tetracyclines (e.g., chlortetracyclin 2 6, oxytetracycline ) Macrolides (e-9; erythromycin, spiramycin) Nitrofurans (e.g., 1 furazolidone) Penicillins 5 (e.g.,

112 u ampicillin, amoxicillin) Phenicols (eg- chloramphenic 1 ol, florphenicol, tiamphenicol) Potentiated Sulfonamides (eg- ormetoprim- sulphamethoxa zole) Quinolones (e.g., ciprofloxacin, enrofloxacin, norfloxacin, 3 oxolinic acid, perfloxacin, flumequine, sarafloxacin) Sulfonamides (eg-, sulfamerazine, 1 sulfadimidine, sulfonamides) Other, please specify:

11. To the best of your knowledge, please approximate what proportion of the listed species' production was treated with antimicrobial drugs in each production phase.

113 Please leave blank if you are NOT involved in a given production phase.

Iggllgliiyggi^gggij^gj WiiHMiMi Example: Fish 75% 50% We do not produce or hatch Species 1 eggs Example: Fish 0% 0% 0% No antimicrobial used in Species 2 that species Catfish Clams Crawfish Lobster Mussels Oysters Salmon Shrimp/prawns Tilapia Trout Ornamental/pet fish Other, please specify:

12. To the best of your knowledge, please approximate the proportion of the total volume of antimicrobial drugs used in each species for the purposes listed.

Leave blank if you do not know.

Growth promotio

114 Preventiv e 80 treatment 2 % Therapeut ic 15 treatment % 3 Total 100 100 100 100 100 100 100 100 100 100 100 100 100% % % % % % % % % % % % % 1: used primarily to increase feed efficiency, not to control or treat diseases 2; antimicrobial treatment initiated before the onset of clinical signs/disease 3: antimicrobial treatment initiated after observation of clinical signs/disease in any part of the treated population

Section 3: Antimicrobial resistance in aquaculture, including seafood and ornamental/pet fish.

13. in your opinion, how important are the following practices for the development of antimicrobial resistance in aquaculture?

Please use the scale 1 to 5, choosing the one that best applies or select "Don't know" where appropriate.

Absence of accurate diagnosis Inadequate storage of antimicrobials Use of antimicrobials of unsure/unproven quality

115 Lack of information concerning fish cohort (ex. biomass) Utilisation of sub-therapeutic dosages inappropriate duration of treatment Lack of approved medications (few products available) Use of antimicrobials in place of improving husbandry

14. Have you ever submitted samples for testing or tested resistance to antimicrobials in bacteria recovered from aquaculture, seafood or ornamental/pet fish?

• Yes • No (Please proceed to Section 4. Do not complete the remaining questions in this section.)

15. To the best of your knowledge, please approximate how frequently antimicrobial resistance was observed for each antimicrobial drug class and aquatic species listed.

Please use scale 1 to 5, choosing the one that best applies. Leave blank if you do not know or not tested.

Aminoglycosid es (e.g., 1 gentamicin, streptomycin.) Tetracyclines 5 {e.g.. 116 chlortetracycline oxytetracycllne) Macrolides (e.g., erythromycin, 2 spiramycin) Nitrofurans (e.g., 1 furazolidone) Penicillins (e.g., ampicillin, 4 amoxicillin) Phenicols (e.g., chloramphenico 1 1, florphenicof, tiamphenicol) Potentiated Sulfonamides (eg. 3 ormetoprim- sulphamethoxa zole) Quinolones (eg. ciprofloxacin, enrofloxacin, norfloxacin, 2 oxolinic acid, perfloxacin, flumequine, sarafloxacin) Sulfonamides 1 (e.g., sulfamerazine, sulfadimidine, sulfonamides) Other, please specify:

—

117 16. To the best of your knowledge, please approximate how frequently antimicrobial resistance was observed for each antimicrobial drug class and bacteria listed.

Please use scale 1 to 5, choosing the one that best applies. Leave blank if you do not know or not tested.

Aminoglycosides (e.g., gentamicin, streptomycin.) Tetracyclines (e.g., chlortetracycline, 1 oxytetracycline) Macrolides (e.g., erythromycin, spiramycin) Nitrofurans (e.g., 2 furazolidone)

Penicillins (e.g., 4 ampicillin, amoxicillin) Phenicols (e.g., chloramphenicol, 1 florphenicol, tiamphenicol) Potentiated Sulfonamides (e.g., 2 ormetoprim- sulphamethoxazole) Quinolones (e.g., ciprofloxacin, 1 enrofloxacin, norfloxacin, oxolinic acid, perfloxacin.

118 flumequine, sarafloxacin) Sulfonamides (e.g., suifamerazine, 1 sulfadimidine, sulfonamides) Other, please specify:

Please use this space for additional relevant comments pertaining to the previous questions if needed.

Section 4: Antimicrobial resistance monitoring and surveillance. 17. To the best of your knowledge, which of the following practices or measures for controlling antimicrobial resistance are applied in aquaculture in your jurisdiction:

Please check all that apply.

None Rotation of antibiotic class usage Ban the use of certain antimicrobial drugs (please specify: ) Restricted use of certain antimicrobial drugs (e.g., authorization required) (please specify: ) Adoption of general prudent use guidelines In aquaculture Depopulation of a fish farming site when antimicrobial resistant strains of animal health or public health significance are identified 119 strict biosecurity measures to avoid pathogen strain introduction Other (please specify: )

18. Are you aware of a jurisdiction-specific government or industry program for monitoring or surveillance of antimicrobial drug usage and /or antimicrobial resistance data in aquaculture?

• Yes (Please specify in the space below the name of the program and the country of origin. If possible, please also provide contact name and information. Thank-You!) • No

19. Please identify what you feel are important knowledge gaps in evaluating the risks of antimicrobial resistant aquatic bacteria on human health.

20. Are you aware of other professionals that would potentially be interested in completing this questionnaire and whose input would be valuable for the success of our project? If so, please list the name and available contact information: 120 Name: Organisation: Address:

Phone: Email:

Name: Organisation: Address:

Phone: Email:

Name: Organisation: Address:

Phone: Email:

Section 5: Antimicrobial susceptibility testing - laboratory information

21. Do you have expertise in microbiology?

• Yes • No (Do not complete the remaining questions in this section. Please proceed to question 26.)

22. For antimicrobial susceptibility testing in your laboratory, what guidelines/criteria are used for interpretation of susceptibility and resistance of aquatic bacteria?

Example: y CLSI M31-A3

121 CLSI (Clinical and Laboratory Standards Institute) EUCAST (European Committee on Antimicrobial Susceptibility Testing) Breakpoints developed In-house (or locally) Other guidelines, please specify:

23. What temperature is routinely used in your laboratory to incubate bacterial isolates for susceptibility testing?

• 15°C • 22°C • 28°C • 37T Other

24. Do criteria for resistance interpretation vary among different bacterial genera?

• All aquatic bacteria are evaluated with the same criteria • Different genera of bacteria may be evaluated differently • Don't know

25. Would it be possible to contact you for more specific information about the susceptibility methods or breakpoints used in your laboratory?

• Yes (If yes, fill in your contact information below.) • No

Name: Organisation: Address:

Phone: Email:

26. Thank you for your participation! If you have any additional comments, please use the space below.

122 123 APPENDIX 2.2. Questionnaire-Spanish Version

Relevamiento del Uso y Resistencia de Antimicrobianos en Bacterias zoonoticas en Acuicultura, Mariscos y Feces Ornamentales/Mascotas^

1: Definiciones:

Acuicultura: Produccion de peces marines o de agua dulce y animates para consumo humano. Mariscos; Cualquier pez o marisco (incluyendo moluscos y crustaceos, excluyendo algas marinas) que son servidos como comida o son apropiados para el consumo. Pez ornamental: Cualquier pez pequeno/marisco que es mantenido en acuarios como mascotas y no para el consumo

Seccion 1 iDemografia del Encuestado (Aplicable a todos los encuestados)

1. Mi(s) area(s) principal(es) de conocimiento es (son); (Por favor marque todas las opciones que correspondan) Microbiologia Epidemiologia Salud de peces / Medicina clinica Seguridad alimentaria Investigacion de brotes Biologia molecular/Genetica Otras, por favor especifique;

2. Mis conocimientos fueron primariamente adquiridos en: (Por favor marque todas las opciones que correspondan) Gobierno Academia Industria Otros, por favor especifique:

3. Mi(s) actividad(es) principal(es) es (son) en: (Por favor marque todas las opciones que correspondan) Investigacion (ej., desarrollo de nuevas pruebas de diagnostico) Trabajo de campo o cli'nico Trabajo de laboratorio (ej., pruebas de diagnostico de rutina) Tareas administrativas (ej., gerencia) Vigilancia en salud animal Vigilancia en salud publica Otras, por favor especifique;

124 4. Mis conocimientos los he adquirido primariamente en: (Por favor marque todas las opciones que correspondan)

Africa Asia Europa America del Norte Oceania America del Sur, Central y el Caribe Otras, por favor especifique la region:

5. ^Tlene usted experiencia con alguna de las siguientes especies acuaticas U otras: bagre, almeja, cangrejo, langosta, mejillon, ostra, salmon, camaron/langostino, tilapia, trucha, poblaclones de peces silvestres y/o peces ornamentales/mascotas?

• Si, tengo experiencia con una o mas de las especies acuaticas mencionadas U otras • No, no tengo experiencia con ninguna de las especies acuaticas mencionadas U otras (Por favor continue en la Seccion 3. No complete el resto de las preguntas en esta seccion)

6. Por favor claslflque su nivel de experiencia con las siguientes clases de peces o mariscos. (Por favor marque todas opciones que correspondan)

Por favor use la escala de 1 a 5 o de ser necesario marque "Sin experiencia".

Bagre Almeja Cangrejo Langosta

125 Mejilldn Ostra Salmbn Camar6n/langostino Tilapia Trucha Peces ornamentales/mascotas Poblaciones de peces silvestres Otras Si marco ornamentales/mascotas y/o peces silvestres y/u otras, por favor especifique la especie:

7. ^Cuantos anos de experiencia ha acumulado usted en acuicuitura y/o mariscos y/o peces ornamentales? • <3 anos • 3-10 anos • >10 anos

8. La acuicuitura y/o mariscos y/o peces ornamentales representan (Por favor seleccione una de las siguientes opciones)

• < de 25% de mis actividades diarias. • 26 a 50% de mis actividades diarias. • 51 a 75% de mis actividades diarias. • 76 a 100% de mis actividades diarias.

126 Seccion 2: Uso de drogas antimicrobianas en acuicultura (incluyendo produccion de peces ornamentales/mascotas)

9. ^Tiene usted experiencia con el uso de antimicrobianos en acuicultura?

• Si • No (Per favor continue en la Seccion 3. No complete el resto de las preguntas en esta seccion)

10. De acuerdo a su conocimiento, indique con que frecuencia fueron usadas cada una de las siguientes clases de drogas antimicrobianas en las especies acuaticas enumeradas.

Por favor use la escala de 1 a 5, seleccionando el valor que mejor corresponda. Deje en bianco si no sabe.

// //. I / ' /j/j // ; / / y^/ # 'y/wE ^ / #

Per favor marque esta celda si NINGUNA DROGA ANTIMIC ROBIANA FUE USADA PARA NADA

127 Aminoglu cosidos (ej; 1 gentamici na, estreptomi cina.) Tetracicli nas (ey., clorotetrac 2 iciina, • oxitetracici ina) Macrolido s (ej., eritromicin a, espiramici na) Nitrofura nos (ej., 1 furazolido na) Penicilina (ej; 5 ampicilina, amoxicilin a) Fenicoles (ej; cloranfeni 1 col, florfenicol, tianfenicol ) Sulfonam Idas

128 Potencia das (ej., trimetopri m- sulfameto xazol) Quinolon as (ej., ciprofloxa cina, enrofloxac ina, norfloxaci 3 na, acido oxollnico, pefloxacin 3, flumequin a, sarafloxac ina) Sulfonam Idas (sulfamera zina, 1 sulfadimidi na, , sulfonami das) Otras, por favor especifiq ue:

129 11. De acuerdo a su conocimiento, por favor indique aproximadamente que proporcion de la produccion de las especies enumeradas, fueron tratados con drogas antimicrobianas en cada una de las eta pas de produccion.

Por favor deje en bianco si usted NO esta involucrado en ninguna de las etapas de produccion mencionadas.

111^^

Ejempio: Especie de X 75% 50% No producimos ni pez 1 cultivamos ovas Ejempio: Especie de 0% 0% 0% Ningiin pez 2 antimicrobiano fue usado en aquella especie Bag re Almeja Cangrejo Langosta Mejillon Ostra Salmon Camaron/langostino Tiiapia

Trucha

Pez Omamental/Mascota Otras, por favor especifique:

12. De acuerdo a su conocimiento, por favor indique aproximadamente que proporcion del volumen total de drogas antimicrobianas fue usado en cada una de las especies para los motivos enumerados.

Deje en bianco si no sabe.

130 #- "## 0^:& ;##/. #'"""#g 'i#'#':5## #^3# Promoci on del 5% crecimie nto^ Tratamie nto 80 preventi % vo^ Tratamie 15 nto % curativo^ Total 100 100 100 100 100 100 100 100 100 100 100 100 100% % % % % % % % % % % % %

1: utilizado primariamente para aumentar la eficiencia de conversion alimenticia, no para controlar o tratar enfermedades

2: tratamiento antimicrobiano iniciado antes del comienzo de signos clinicos/enfermedad

3: tratamiento antimicrobiano iniciado despues del comienzo de signos clinicos/enfemnedad en cualquier parte de la poblacion tratada

131 Seccion 3: Resistencia antimicrobiana en acuicultura, incluyendo mariscos y peces ornamentales/mascotas.

13. En su opinion, ^cuan importante son las siguientes practicas en el desarrollo de resistencia antimicrobiana en acuicultura?

Por favor use la escala de 1 a 5, seleccionando el valor que mejor corresponda o marque "No lo se" cuando sea necesario.

Ausencia de diagnostico preciso

Almacenamiento inadecuado de los

antimicrobianos

Uso de antimicrobianos de calidad dudosa/no

probada

Carencia de informacion acerca del cohorte de

peces (ej. biomasa)

Utilizaci6n de dosis subterapeuticas

Duracion inapropiada del tratamlento

Carencia de medicamentos aprobadas (pocos

productos disponibles)

Uso de antimicrobianos en lugar de mejorar las

prActicas de manejo en la produccion

132 14. ^Ha enviado usted aiguna vez muestras para analizar o ha realizado pruebas de resistencia antimicrobiana en bacterias aisladas de acuicultura, mariscos o peces ornamentales/mascotas?

• Si • No (Por favor continue en la Seccion 4. No complete el resto de las preguntas en esta seccion)

15. De acuerdo a su conocimiento, por favor indique aproximadamente con que frecuencia ha observado resistencia antimicrobiana en cada una de las siguientes clases de drogas antimicrobianas y especies acuaticas enumeradas.

Por favor use la escala de 1 a 5 seleccionando el valor que mejor corresponda. Deje en bianco si no sabe o no analizado.

Nunea Raramente Ocasionalmente Frecuentemente Casisiempre observada observada f;/'; observada observada 2 5

Aminoglu cosidos (ej; gentamicin estreptomi cine) Tetracicll nas (ej., clortetraci clina, oxitetracici ina)

133 Macrolido s (ej., eritromicin 2 a, espiramici na) Nitrofuran OS (ej, 1 furazolido na) Penicilina (ej; 4 ampicilina, amoxicilin a) Fenicoles (ej. cloranfenic 1 ol, florfenicol, tianfenicol) Sulfonami das Potenciad as (ej, 3 trimetopri m- sulfametox azol) Quinolon as (ej, ciprofloxac 2 ina, enrofloxaci na. 134 norfloxacin a, acido oxolinico, 11 pefloxacin a, flumequin 3, sarafloxaci 1 na) Sulfonami 1 das (ej., sulfamera zina, sulfadimidi na, sulfonamid as) Otras, por favor especifiq ue:

16. De acuerdo a su conocimiento, por favor indique aproximadamente con que frecuencia ha observado resistencia antimicrobiana en cada una de las siguientes clases de drogas antimicrobianas y bacterias enumeradas

Por favor use la escala de 1 a 5 seleccionando el valor que mejor corresponda. Deje en bianco si no sabe o no analizado.

135 Nunca Raramente Ocasionalmente Frecuentemente Casi sianpre observada observada observada 1 3 4 i , j",' '

Aminoglucosido

^(ej; gentamicina, estreptomicina)

Tetraciclinas (ey., clortetraciclina, 1 oxitetraciclina^

Macrolidos (ey., eritromicina, espiramicina)

Nitrofuranos (ey., 2 furazoUdona)

Penicilinas (ey., ampicilina, 4 amoxicilina)

Fenicoles (ej., 1 cloranfenicol,

136 florfenicol, tiamfenicol)

Sulfonamidas

Potenciadas

(ej., trimetoprim- sulfametoxazol)

Quinolonas (ej., ciprofloxacina, enrofloxacina, norfloxacina, ^cido oxoUnico, pefloxacina, flumequina,saraflo xacina)

Sulfonamidas

(ej., sulfamerazina, sulfadimidina, sulfonamidas)

Otras, por favor

137 especifique:

For favor use este espacio para agregar comentarios relevantes relacionados a las preguntas anteriores en caso de considerarlo necesario.

Seccion 4:Monitoreo y vigilancia epidemiologica de resistencia antimicrobiana.

138 17. De acuerdo a su conocimiento, ^cual de las siguientes practicas o tnedidas para controlar la resistencia antimicrobiana son aplicadas en acuicultura en su jurisdiccion?

Por favor marque todas las opciones que correspondan.

Ninguna Rotacion del use de clase de antibioticos Prohibicion del use de ciertas drogas antimicroblanas (por favor especlfique; ) Uso restringido de ciertas drogas antimicroblanas (ej., requerimlento de autorlzaclon) (por favor especifique: ) Adopclon de directives generates de uso prudente en acuicultura Despoblacion del sitio productor pesquero cuando cepas antimicroblanas resistentes de significancia para la salud animal o publica son identificadas Medidas de bioseguridad estrlctas para evitar la introduccion de cepas patogenas _ Otro (por favor especifique: )

18. ^Conoce programas de gobierno o de la Industria especificos de la jurisdiccion, para el monitoreo o vigilancia epidemiologica de uso de drogas antimicroblanas y/o datos de resistencia antimicrobiana en acuicultura?

• Si (Por favor especifique en el espacio de abajo el nombre del programa y el pals de origen. De ser posible, por favor provea el nombre e informacion de contacto. jMuchas gracias!) • No

139 19. For favor identifique en lo que usted considers hay brechas importantes en el conocimiento acerca de la evaluacion de riesgos de resistencia antimicrobiana en bacterias acuaticas o en la salud humana.

20. tConoce a otros profesionales que potenciaimente podrian estar interesados en completar este cuestionario y cuyo aporte seria de gran valor para el exito de nuestro proyecto? De ser asi, por favor escriba el nombre y la informacion de contacto:

Nombre: Organizacion: Direccion:

Telefono: Correo electronico;

Nombre: Organizacion: Direccion:

Telefono: Correo electronico:

Nombre: Organizacion: Direccion:

Telefono: Correo electronico:

140 Seccion 5: Analisis de susceptibilidad antlmicrobiana - informacion de laboratorio

21. ^Tiene experiencia en microbiologia? • Si • No (For favor continue en la Seccion 6. No complete el resto de las preguntas en esta seccion)

22. Para analisis de susceptibilidad antlmicrobiana, &que pautas/criterios utiliza en su laboratorio para la interpretacion de susceptibilidad y resistencia de bacterias acuaticas?

Ejempio: 1 y lECL M31-A3

lECL (Institute de Estandares Clinicos y de Laboratorio) EUCAST (Comision Europea de Evaluacion de Susceptibilidad Antlmicrobiana) Puntos de corte desarrollados internamente (localmente) Otras directivas, por favor especifique:

23. ^Que temperature es utilizada rutinariamente en su laboratorio para incubar aislamientos bacterlanos para analisis de susceptibilidad?

• 15°C • 22°C • 28°C • 37°C Otra

24. ^Varian los criterios de interpretacion de resistencia entre los diferentes generos bacterlanos?

• Todas las bacterias acuaticas son evaluadas bajo los mismos criterios • Diferentes generos bacterlanos pueden ser evaluados de diferente forma • No lo se

141 25. ^Sena posible contactarlo para obtener mas informacion acerca de los metodos de susceptibilidad o los puntos de corte empleados en su laboratorio?

• Si (si la respuesta es si, complete su informacion de contacto) • No

Nombre: Organizacion: Direccion:

Telefono: Correo electronico:

26. jMuchas gracias por su participacion! Si tiene comentarios adicionales, por favor use el espacio provisto a continuacion.

142 APPENDIX 2.3. Descriptive Statistics Repori;

Antimicrobial use and antimicrobial resistance in selected zoonotic bacteria in aquaculture: A survey of aquaculture-allied professionals with various types of expertise - Preliminary findings

Table 1. Country profile of respondents to English version of questionnaire Total Respondents=196 (out of a possible 564) Number of Bounce Opt- Number that % Country Individuals s Outs Responded Responded Australia 23 2 0 6 3.1 Belgium 4 0 0 0 0.0 Canada 56 5 1 26 13.3 China 2 0 0 0 0.0 Denmark 4 0 0 2 1.0 Egypt 1 0 0 1 0.5 France 3 0 0 2 1.0 Germany 2 1 0 0 0.0 Greece 16 1 0 7 3.6 India 5 2 0 1 0.5 Iran 2 0 0 0 0.0 Israel 1 0 0 2 1.0 Italy 11 1 0 4 2.0 Japan 9 1 0 1 0.5 Malaysia 2 1 0 1 0.5 Netherlands 2 0 0 1 0.5 Norway 5 1 0 3 1.5 Philippines 8 1 0 4 2.0 Singapore 1 0 0 0 0.0 Sri Lanka 3 0 0 1 0.5 Switzerland 1 0 0 1 0.5 Thailand 11 2 0 6 3.1 United Kingdom 3 2 0 1 0.5 United States 495 58 11 126 64.3 Vietnam 4 0 0 0 0.0 Total 674 78 12 196 33.6

Table 2. Country profile of respondents to Spanish version of questionnaire Total Respondents=3 (out of a possible 20) Country Number of Individuals Bounces Opt-Outs Number Responded % Responded Argentina 2 2 0 0 0.0 Brazil 3 0 0 0 0.0 Chile 6 3 0 1 5.0 143 Cuba 2 2 0 0 0.0 Ecuador 3 2 0 0 0.0 El Salvador 2 2 0 0 0.0 Guatemala 1 4 0 0 0.0 Honduras 5 0 0 0 0.0 Mexico 6 2 0 2 10.0 Nicaragua 1 0 0 0 0.0 Peru 2 2 0 0 0.0 Uruguay 1 0 0 0 0.0 Venezuela 7 2 0 0 0.0 Total 41 21 0 3 15

Table 3. Questionnaire administration schedule and number of responses after initial administration and subsequent reminders Date N % June 12th/2009 104 52.3 R1 (June 29th) 40 20.1 R2 (July 15 th) 31 15.6 R3 (July 28th) 18 9.0 R4 (Aug 12th) 6 3.0

Table 4. Response proportions for Spanish and English questionnaire versions Response Proportion N % Overall (199/604) 32.9 English (196/584) 33.6 Spanish (3/20) 15.0

Table 5. Respondent area of expertise (Ql) Total Respondents=199 Area of Expertise: N % of Respondents Microbiology 47 23.6 Epidemiology 25 12.6 Fish health/Clinical Medicine 140 70.4 Food Safety 25 12.6 Outbreak investigation 33 16.6 Molecular biology/Genetics 25 12.6 Other^ 57 29.6 Responses under other: pathology (n=21), fish behavior (n=l), veterinary medicine (n=12), ecology (n=2), virology (n=3), immunology (n=5), antimicrobials (n=2), public health (n=l), no specialization (n=2), pharmacology/toxicology (n=l), anesthesia/analgesia (n=2), biosecurity (n=l), bovine medicine (n=l), molecular diagnostics (n=l), experimental modeling (n=l), forensics (n=l).

Table 6. Field in which experience was acquired (Q2) Total Respondents=198 Field N % of Respondents Government 60 30.8 144 Academia 118 59.2 Industry 48 24.1 Other' 2 1.0 ' Responses under other: specialized training (n=2).

Table 7. Primary activities (Q3) Total Respondents^199 Primary Activities N % of Respondents Research 81 40.1 Clinical/Field work 116 58.3 Laboratory work 72 36.1 Administrative tasks 57 28.6 Animal health surveillance 61 30.7 Public health surveillance 11 5.5 Other" 11 5.5 'Responses under other: education (n=9), sales (n=l), aquaculture production (n=l).

Table 8. Location of work/training (Q4) Total Respondents^199 Country N % of Respondents Africa 5 2.5 Asia 29 14.6 Europe 34 17.1 North America 148 74.4 Oceania 7 3.5 South and Central America and 7 3.5 Caribbean Other 0 0

145 Table 9. Experience with catfish, clams, crawfish, lobster, mussels, oysters, salmon, shrimp/prawn, tilapia, trout, ornamental fish, wild fish populations, or other aquatic species(Q5) T otal Respondents^199

Option N %

Yes 185 93.0

No 14 7.0

Table 10. Rating of professional experience for listed species (Q6) Total Respondents=176 Tot No Medium- Average al Experienc Low- High Rating Species e Low Medium Medium N High N N N N % N % % % % % 16 2 16. 18. 3 18. 1 Catfish 49 29.7 30 13 7.9 9.1 1.9 5 7 4 2 1 8 5 16 3 22. 14. Clams 87 53.0 23 8 4.9 7 4.3 2 1.2 0.9 4 7 6 0 Crawfis 16 3 22. 12. 94 57.7 21 7 4.3 3 1.8 1 0.6 0.7 h 3 7 7 9 15 3 18. 15. 1 Lobster 82 51.6 24 9.4 6 3.8 2 1.3 1.0 9 0 9 1 5 16 3 22. 16. 1 Mussels 78 47.9 27 6.1 9 5.5 3 1.8 1.0 3 6 1 6 0 16 3 24. 11. 1 Oysters 72 44.7 19 9.9 9 5.6 6 3.7 1.2 1 9 2 8 6 16 2 14. 15. 3 18. 2 18. Salmon 39 24.2 25 15 9.3 2.3 1 3 3 5 0 6 9 0 Shrimp/ 16 4 24. 12. 2 15. 1 55 32.9 21 9 5.4 9.6 1.6 Prawn 7 0 0 6 5 0 6 16 2 16. 22. 2 15. 1 Tilapia 45 27.4 36 19 11.6 6.7 1.9 4 7 5 0 6 9 1 16 2 13. 15. 2 16. 2 17. Trout 37 22.8 25 24 14.8 2.4 2 1 0 4 6 0 9 9 Omame 16 1 10. 4 25. 4 26. 21 12.5 15 8.9 28 16.7 3.0 ntal fish 8 8 7 2 0 4 2 Wild fish 16 2 14. 16. 2 15. 1 10. 41 25.3 27 29 17.9 2.2 populati 2 3 2 7 5 4 7 5 ons 2 25. Other' 98 39 39.8 8 8.2 9 9.2 6 6.1 11 11.2 2.2 5 5 ' Responses under other included various ornamental, wild fish populations and other species.

146 Table 11. Years of experience in aquaculture/seafood and/or ornamental fish (Q7) Total Respondents^ 176 Years of Experience N ^ <3 years 13 7.4 3-10 years 53 30.1 >10 years 110 62.5

Table 12. Aquaculture/seafood/omamental fish as proportion of daily activities (Q8) Total Respondents=176 Proportion of day-to-day activities N <25% 67 38.1 26-50% 23 13.1 51-75% 24 13.6 76-100 62 35.2

147 Table 13. Experience with AMU (Q9)

Total Respondents^ 176 Option N % Yes 130 73.9 No 46 26.1

' Table 14. Frequency of AMU per species (QIO)

N %

Total Responded „ (Out of a possible 130)

Catfish

. . . , . , ^ Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 31 66.0 7 14.9 6 12.8 3 6.4 0 0.0 47 Tetracyclines 13 26.0 9 18.0 14 28.0 11 22.0 3 6.0 50 Macrolides 26 70.3 8 21.6 2 5.4 1 2.7 0 0.0 37 Nitrofurans 29 72.5 3 7.5 6 15.0 2 5.0 0 0.0 40 Penicillins 28 68.3 7 17.1 5 12.2 1 2.4 0 0.0 41 Phenicols 19 46.3 5 12.2 11 26.8 6 14.6 0 0.0 41 Potentiated Sulfonamides 12 27.9 10 23.3 13 30.2 6 14.0 2 4.7 43 Quinolones 21 50.0 3 7.1 8 19.0 8 19.0 2 4.8 42 Sulfonamides 23 59.0 10 25.6 4 10.3 1 2.6 1 2.6 39 Other' 14 82.4 1 5.9 1 5.9 0 0.0 1 5.9 17

148 Clams

Antimicrobial Drug Never Rarely Occasionally Frequently Almost Always Total Class N % N % N % N % N % Aminoglycosides 31 96.9 0 0.0 0 0.0 0 0.0 1 3.1 32 Tetracyclines 25 92.6 2 7.4 0 0.0 0 0.0 0 0.0 27 Macrolides 23 95.8 1 4.2 0 0.0 0 0.0 0 0.0 24 Nitrofurans 24 92.3 1 3.8 0 0.0 0 0.0 I 3.8 26 Penicillins 25 100.0 0 0.0 0 0.0 0 0.0 0 0.0 25 Phenicols 23 92.0 2 8.0 0 0.0 0 0.0 0 0.0 25 Potentiated 25 100.0 0 0.0 0 0.0 0 0.0 0 0.0 25 Sulfonamides Quinolones 22 91.7 2 8.3 0 0.0 0 0.0 0 0.0 24 Sulfonamides 24 100.0 0 0.0 0 0.0 0 0.0 0 0.0 24 Other® 14 93.3 1 6.7 0 0.0 0 0.0 0 0.0 15

Crawfish

Antimicrobial Drug Never Rarely Occasionally Frequently Almost Always Total Class N % N % N % N % N % Aminoglycosides 25 92.6 1 3.7 1 3.7 0 0.0 0 0.0 27 Tetracyclines 18 78.3 3 13.0 1 4.3 1 4.3 0 0.0 23 Macrolides 21 95.5 0 0.0 1 4.5 0 0.0 0 0.0 22 Nitrofurans 20 87.0 1 4.3 1 4.3 1 4.3 0 0.0 23 Penicillins 20 90.9 1 4.5 1 4.5 0 0.0 0 0.0 22 Phenicols 19 86.4 2 9.1 1 4.5 0 0.0 0 0.0 22 Potentiated C1 ll 1 21 95.5 0 0.0 1 4.5 0 0.0 0 0.0 22

149 Quinolones 20 90.9 1 4.5 1 4.5 0 0.0 0 0.0 22 Sulfonamides 21 95.5 0 0.0 1 4.5 0 0.0 0 0.0 22 Other^ 16 100.0 0 0.0 0 0.0 0 0.0 0 0.0 16

Lobster

. . . Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 25 89.3 2 7.1 1 3.6 0 0.0 0 0.0 28 Tetracyclines 16 57.1 3 10.7 7 25.0 2 7.1 0 0.0 28 Macrolides 21 95.5 0 0.0 1 4.5 0 0.0 0 0.0 22 Nitrofurans 20 83.3 2 8.3 1 4.2 1 4.2 0 0.0 24 Penicillins 19 86.4 2 9.1 1 4.5 0 0.0 0 0.0 22 Phenicols 18 81.8 3 13.6 1 4.5 0 0.0 0 0.0 22 Potentiated Sulfonamides 21 95.5 0 0.0 1 4.5 0 0.0 0 0.0 22 Quinolones 20 90.9 1 4.5 1 4.5 0 0.0 0 0.0 22 Sulfonamides 20 95.2 0 0.0 1 4.8 0 0.0 0 0.0 21 Other 16 100.0 0 0.0 0 0.0 0 0.0 0 0.0 16

Mussels

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 33 100.0 0 0.0 0 0.0 0 0.0 0 0.0 33 Tetracyclines 28 96.6 1 3.4 0 0.0 0 0.0 0 0.0 29 Macrolides 28 100.0 0 0.0 0 0.0 0 0.0 0 0.0 28 Nitrofurans 27 96.4 1 3.6 0 0.0 0 0.0 0 0.0 28

150 Penicillins 28 100.0 0 0.0 0 0.0 0 0.0 0 0.0 28 Phenicols 27 96.4 1 3.6 0 0.0 0 0.0 0 0.0 28 Potentiated Sulfonamides 29 100.0 0 0.0 0 0.0 0 0.0 0 0.0 29 Quinolones 27 96.4 1 3.6 0 0.0 0 0.0 0 0.0 28 Sulfonamides 28 100.0 0 0.0 0 0.0 0 0.0 0 0.0 28 Other' 18 100.0 0 0.0 0 0.0 0 0.0 0 0.0 18

Oysters

. . . , . , „ Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Glass J J ^ J N % N % N % N % N % Aminoglycosides 33 97.1 1 2.9 0 0.0 0 0.0 0 0.0 34 Tetracyclines 27 93.1 2 6.9 0 0.0 0 0.0 0 0.0 29 Macrolides 27 96.4 1 3.6 0 0.0 0 0.0 0 0.0 28 Nitrofurans 27 96.4 1 3.6 0 0.0 0 0.0 0 0.0 28 Penicillins 27 100.0 0 0.0 0 0.0 0 0.0 0 0.0 27 Phenicols 26 92.9 2 7.1 0 0.0 0 0.0 0 0.0 28 Potentiated Sulfonamides 29 100.0 0 0.0 0 0.0 0 0.0 0 0.0 29 Quinolones 26 92.9 2 7.1 0 0.0 0 0.0 0 0.0 28 Sulfonamides 28 100.0 0 0.0 0 0.0 0 0.0 0 0.0 28 Other' 16 100.0 0 0.0 0 0.0 0 0.0 0 0.0 16

Salmon

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 31 77.5 9 22.5 1 2.5 0 0.0 0 0.0 40 Tetracyclines 12 27.9 2 4.7 9 20.9 14 32.6 6 14.0 43

151 Macrolides 18 48.6 8 21.6 5 13.5 5 13.5 1 2.7 37 Nitrofurans 31 83.8 4 10.8 0 0.0 2 5.4 0 0.0 37 Penicillins 26 70.3 8 21.6 3 8.1 0 0.0 0 0.0 37 Phenicols 17 42.5 8 20.0 6 15.0 8 20.0 1 2.5 40 Potentiated Sulfonamides 16 39.0 4 9.8 10 24.4 11 26.8 0 0.0 41 Quinolones 18 41.9 9 20.9 10 23.3 4 9.3 2 4.7 43 Sulfonamides 24 61.5 5 12.8 9 23.1 1 2.6 0 0.0 39 Other" 11 100.0 0 0.0 0 0.0 0 0.0 0 0.0 11

Shrimps/Prawns

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 23 79.3 4 13.8 1 3.4 1 3.4 0 0.0 29 Tetracyclines 12 35.3 4 11.8 8 23.5 6 17.6 4 11.8 34 Macrolides 21 80.8 2 7.7 2 7.7 1 3.8 0 0.0 26 Nitrofurans 21 75.0 2 7.1 4 14.3 1 3.6 0 0.0 28 Penicillins 17 65.4 7 26.9 2 7.7 0 0.0 0 0.0 26 Phenicols 18 60.0 4 13.3 4 13.3 4 13.3 0 0.0 30 Potentiated Sulfonamides 16 61.5 5 19.2 1 3.8 3 11.5 1 3.8 26 Quinolones 13 46.4 8 28.6 1 3.6 5 17.9 1 3.6 28 Sulfonamides 19 76.0 1 4.0 2 8.0 2 8.0 1 4.0 25 Other" 9 100.0 0 0.0 0 0.0 0 0.0 0 0.0 9

Tilapia

152 Never Rarely Occasionally Frequently Almost Always Total

N % N % N % N % N % 22 73.3 7 23.3 1 3.3 0 0.0 0 0.0 30 10 29.4 4 11.8 14 41.2 4 11.8 2 5.9 34 19 73.1 5 19.2 2 7.7 0 0.0 0 0.0 26 22 81.5 4 14.8 1 3.7 0 0.0 0 0.0 27 16 61.5 6 23.1 4 15.4 0 0.0 0 0.0 26 18 62.1 7 24.1 4 13.8 0 0.0 0 0.0 29 13 46.4 9 32.1 4 14.3 1 3.6 1 3.6 28 18 62.1 3 10.3 4 13.8 4 13.8 0 0.0 29 21 75.0 4 14.3 2 7.1 0 0.0 1 3.6 28 7 87.5 1 12.5 0 0.0 0 0.0 0 0.0 8

Never Rarely Occasionally Frequently Almost Always Total

N % N % N % N % N % 28 75.7 9 24.3 0 0.0 0 0.0 0 0.0 37 9 20.9 5 11.6 13 30.2 12 27.9 4 9.3 43 22 61.1 5 13.9 4 11.1 4 11.1 1 2.8 36 30 78.9 5 13.2 2 5.3 1 2.6 0 0.0 38 26 76.5 5 14.7 2 5.9 1 2.9 0 0.0 34 21 52.5 5 12.5 4 10.0 9 22.5 1 2.5 40 15 37.5 6 15.0 12 30.0 6 15.0 1 2.5 40 16 43.2 7 18.9 8 21.6 4 10.8 2 5.4 37 25 67.6 3 8.1 7 18.9 1 2.7 1 2.7 37 9 90.0 1 10.0 0 0.0 0 0.0 0 0.0 10

153 Omamental/Pet Fish

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 6 10.3 19 32.8 19 32.8 14 24.1 0 0.0 58 Tetracyclines 5 8.5 11 18.6 17 28.8 21 35.6 5 8.5 59 Macrolides 22 40.7 16 29.6 12 22.2 4 7.4 0 0.0 54 Nitrofurans 15 25.9 10 17.2 15 25.9 18 31.0 0 0.0 58 Penicillins 27 50.9 6 11.3 16 30.2 4 7.5 0 0.0 53 Phenicols 17 29.8 17 29.8 13 22.8 10 17.5 0 0.0 57 Potentiated Sulfonamides 12 21.8 13 23.6 21 38.2 6 10.9 3 5.5 55 Quinolones 5 7.6 5 7.6 22 33.3 25 37.9 9 13.6 66 Sulfonamides 25 44.6 15 26,8 9 16.1 5 8.9 2 3.6 56 Other' 5 33.3 2 13.3 5 33.3 3 20.0 0 0.0 15

Other

. . . , . , ^ Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 15 71.4 2 9.5 3 14.3 1 4.8 0 0.0 21 Tetracyclines 8 33.3 3 12.5 8 33.3 3 12.5 2 8.3 24 Macrolides 15 71.4 1 4.8 4 19.0 1 4.8 0 0.0 21 Nitrofurans 14 73.7 2 10.5 1 5.3 2 10.5 0 0.0 19 Penicillins 14 63.6 5 22.7 2 9.1 0 0.0 1 4.5 22 Phenicols 13 61.9 4 19.0 3 14.3 1 4.8 0 0.0 21 154 Potentiated Sulfonamides 9 39.1 3 13.0 6 26.1 3 13.0 2 8.7 23 Quinolones 10 40.0 2 8.0 6 24.0 4 16.0 3 12.0 25 Sulfonamides 12 57.1 4 19.0 5 23.8 0 0.0 0 0.0 21 Other'' 8 88.9 0 0.0 0 0.0 1 11.1 0 0.0 9 ®No additional AMs were specified for the specific species. ^ Under other, only 25 answers were specified including just species and species-AM combination. Just species included sea bass, sea bream, eel, koi, abalone, rockfish, walleye, yellow perch, game fish. Species-AM combination: metronidazole (ornamental fish), cephalosporin-ceftazidime(omamental fish), koi(fortaz), metronidazoke (all ornamental fish), abalone (no AM used).

Table 15. Proportion of AMU per species and production phase (Q11)

N % Total Responded ^ . (Out of a possible 130) ^

Hatchery (Eggs/Larvae)

Species 0-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71-80% 81-90% Total 155 N % N % N % N % N % N % N % N % N % N % Catfish 10 71.4 1 7.1 0 0.0 0 0.0 1 7.1 0 0.0 0 0.0 0 0.0 0 0.0 2 14.3 14 Clams 4 80.0 1 20.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 5 Crawfish 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Lobster 1 50.0 0 0.0 1 50.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 Mussels 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Oysters 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Salmon 11 78.6 1 7.1 1 7.1 1 7.1 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 14 Shrimps/Prawns 2 16.7 1 8.3 1 8.3 2 16.7 1 8.3 1 8.3 1 8.3 1 8.3 0 0.0 2 16.7 12 Tilapia 9 64.3 2 14.3 2 14.3 0 0.0 1 7.1 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 14 Trout 9 69.2 1 7.7 1 7.7 0 0.0 0 0.0 1 7.7 0 0.0 0 0.0 1 7.7 0 0.0 13 Omamental/Pet 13 56.5 2 8.7 2 8.7 3 13.0 0 0.0 1 4.3 1 4.3 1 4.3 0 0.0 0 0.0 23 Fish Other 6 46.2 1 7.7 1 7.7 1 7.7 0 0.0 0 0.0 0 0.0 2 15.4 1 7.7 1 7.7 13

Fingerlings 0-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71-80% 81-90% 91-100% Species Total N % N % N % N % N % N % N % N % N % N % Catfish 7 46.7 3 20.0 0 0.0 1 6.7 0 0.0 1 6.7 1 6.7 0 0.0 0 0.0 2 13.3 15 Clams 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Crawfish 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Lobster 2 66.7 1 33.3 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Mussels 2 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 Oysters 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Salmon 7 36.8 1 5.3 3 15.8 1 5.3 2 10.5 2 10.5 0 0.0 2 10.5 0 0.0 1 5.3 19 Shrimps/Prawns 5 55.6 2 22.2 0 0.0 0 0.0 0 0.0 0 0.0 1 11.1 0 0.0 0 0.0 1 11.1 9 Tilapia 7 50.0 3 21.4 2 14.3 1 7.1 0 0.0 1 7.1 0 0.0 0 0.0 0 0.0 0 0.0 14 Trout 4 20.0 3 15.0 5 25.0 1 5.0 0 0.0 3 15.0 1 5.0 2 10.0 1 5.0 0 0.0 20 Omamental/Pet 7 26.9 9 34.6 1 3.8 3 11.5 1 3.8 1 3.8 1 3.8 1 3.8 0 0.0 2 7.7 26 Fish Other 5 38.5 1 7.7 0 0.0 3 23.1 0 0.0 0 0.0 1 7.7 2 15.4 0 0.0 1 7.7 13 156 0-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71-80% 81-90% 91-100% Total N % N % N % N % N % N % N % N % N % N % 7 50.0 3 21.4 0 0.0 1 7.1 1 7.1 1 7.1 0 0.0 0 0.0 0 0.0 1 7.1 14 5 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 5 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 0 O.O 0 0.0 2 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 1 50.0 1 50.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 6 33.3 1 5.6 0 0.0 2 11.1 0 0.0 3 16.7 1 5.6 2 11.1 1 5.6 2 11.1 18 3 30.0 4 40.0 0 0.0 1 10.0 1 10.0 1 10.0 0 0.0 0 0.0 0 0.0 0 0.0 10 7 46.7 4 26.7 2 13.3 0 0.0 1 6.7 0 0.0 0 0.0 1 6.7 0 0.0 0 0.0 15 7 41.2 3 17.6 3 17.6 0 0.0 0 0.0 3 17.6 1 5.9 0 0.0 0 0.0 0 0.0 17 3 8.3 5 13.9 7 19.4 5 13.9 1 2.8 2 5.6 2 5.6 2 5.6 3 8.3 6 16.7 36 6 42.9 0 0.0 2 14.3 1 7.1 2 14.3 1 7.1 1 7.1 0 0.0 0 0.0 1 7.1 14

157 Table 16. Proportion of AMU per species and purpose (Q12) N % Total Responded (Out of a possible 130)

Growth Promotion 0-10% 11 -20% 21 -30% 31 -40% 41-50% 51-60% 61-70% 71-80% 81-90% 91-100% Species N % N % N % N % N%N%N%N%N%N% Catfish 10 90.9 1 9.1 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 11 Clams 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Crawfish 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Lobster 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1

Mussels 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0

Oysters 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 Salmon 9 90.0 1 10.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 10 Shrimps/Prawns 4 57.1 1 14.3 2 28.6 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 7 Tilapia 8 88.9 0 0.0 0 0.0 1 11.1 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 9 Trout 8 88.9 1 11.1 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 9 Omamental/Pet Fish 14 77.8 3 16.7 0 0.0 1 5.6 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 18 Other 4 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 4

158 Preventative Treatment 0-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71-80% 81-90% 91-100% Species Total N % N % N % N % N % N % N % N % N % N % Catfish 7 50.0 4 28.6 2 14.3 0 0.0 0 0.0 1 7.1 0 0.0 0 0.0 0 0.0 0 0.0 14 Clams 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Crawfish 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Lobster 1 25.0 1 25.0 0 0.0 0 0.0 1 25.0 0 0.0 0 0.0 0 0.0 1 25.0 0 0.0 4

Mussels 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0

Oysters 0 - 0 - 0 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 Salmon 7 41.2 3 17.6 3 17.6 0 0.0 0 0.0 1 5.9 1 5.9 0 0.0 0 0.0 2 11.8 17 Shrimps/Prawns 2 16.7 1 8.3 1 8.3 1 8.3 3 25.0 0 0.0 1 8.3 1 8.3 1 8.3 1 8.3 12 Tilapia 6 54.5 2 18.2 3 2T3 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 11 Trout 7 41.2 4 23j 3 17.6 1 5.9 1 5.9 0 0.0 1 5.9 0 0.0 0 0.0 0 0.0 17 Omamental/Pet Fish 6 20.0 7 23J 5 16.7 1 3.3 3 10.0 2 6.7 1 3.3 1 3.3 3 10.0 1 3.3 30 Other 3 42.9 2 28.6 0 0.0 0 0.0 1 14.3 0 0.0 1 14.3 0 0.0 0 0.0 0 0.0 7

Therapeutic Treatment 0-10% 11-20% 21-30% 31-40% 41 -50% 51-60% 61-70% 71-80% 81-90% 91-100% Total N % N % N % N % N % N % N % N % N % N % Catfish 2 9.1 2 9.1 1 4.5 0 0.0 2 9.1 0 0.0 1 4.5 1 4.5 3 13.6 10 45.5 22 Clams 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Crawfish 1 33J 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 66.7 3 Lobster 1 11.1 0 0.0 0 0.0 0 0.0 1 11.1 0 0.0 0 0.0 0 0.0 1 11.1 6 66 7 9

Mussels 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0

Oysters 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 - 0 Salmon 3 12.5 2 8.3 1 4.2 1 4.2 0 0.0 0 0.0 3 12.5 1 4.2 4 16.7 9 37.5 24 Shrimps/Prawns 1 7.1 2 14.3 1 7.1 2 14.3 2 14.3 0 0.0 0 0.0 0 0.0 2 14.3 4 28.6 14 Tilapia 2 13.3 1 6.7 0 0.0 2 13.3 0 0.0 1 6.7 0 0.0 2 13.3 2 13.3 5 33J 15 Trout 2 7.1 1 3.6 1 3.6 0 0.0 1 3.6 0 0.0 4 14.3 2 7.1 5 17.9 12 42.9 28 Omamental/Pet Fish 1 2.0 6 11.8 1 2.0 2 3.9 3 5.9 1 2.0 3 5.9 4 7.8 6 11.8 24 47.1 51 Other 1 7.7 0 0.0 1 7.7 2 15.4 1 7.7 0 0.0 0 0.0 0 0.0 2 15.4 6 46.2 13

159 Table 17. Importance of practice for development of AMR (Q13) Total Responded =165 Don't know Not at all Slightly Fairly Quite Very Total Answer Options N % N % N % N % N % N % Absence of accurate diagnosis 13 8.1 6 3.7 9 5.6 18 11.2 32 19.9 83 51.6 161 Inadequate storage of antimicrobials 43 26.5 15 9.3 35 21.6 22 13.6 29 17.9 18 11.1 162 Use of antimicrobials of unsure/unproven quality 25 15.4 16 9.9 14 8.6 35 21.6 40 24.7 32 19.8 162 Lack of information concerning fish cohort (ex. biomass) 29 18.5 13 8.3 17 10.8 30 19.1 44 28.0 24 15.3 157 Utilization of sub-therapeutic dosages 13 7.9 2 1.2 14 8.5 18 11.0 45 27.4 72 43.9 164 Inappropriate duration of treatment 13 8.1 5 3.1 8 5.0 16 9.9 42 26.1 77 47.8 161 Lack of approved medications (few products available) 21 12.7 12 7.3 13 7.9 26 15.8 39 23.6 54 32.7 165 Use of antimicrobials in place of improving husbandry 14 8.5 6 3.7 11 6.7 26 15.9 45 27.4 62 37.8 164

Table 18. Submitting of samples for AMR testing (Q14) Total Respondents= 167 Option N % Yes 110 65.9 No 57 34.1

160 Table 19. Frequency of AMR for aquatic species and antimicrobial drug class (Q15) Total Responded=74 (Out of a possible 110) Catfish .T o 1 ^ . ,1 Frequent Almost Tot Antimicrobial Drug Never Rarely Occasionally Always al N % N % N % N % N % 26. 13. 5 333 4 4 26.7 2 0 0.0 15 Aminoglycosides 7 3 33. 27. 1 5.6 6 4 2Z2 5 2 11.1 18 Tetracyclines 3 8 30. 5 38 5 4 4 30.8 0 0.0 0 0.0 13 Macrolides 8 23. 4 30.8 3 4 30.8 1 7.7 1 7.7 13 Nitrofurans 1 30. 15. 3 23.1 4 3 23.1 2 1 7.7 13 Penicillins 8 4 40. 5 33J 6 2 13.3 1 6.7 1 6.7 15 Phenicols 0 Potentiated 23. 11. 3 17.6 4 7 41.2 2 1 5.9 17 Sulfonamides 5 8 33. 6 40.0 5 3 20.0 0 0.0 1 6.7 15 Quinolones 3 33. 4 26.7 5 5 333 0 0.0 1 6.7 15 Sulfonamides 3

Other^ 0 - 0 - 0 - 0 - 0 - 0

Clams Frequent Almost Tot Never Rarely Occasionally Antimicrobial Drug ly Always al Class N % N % N % N % N %

Aminoglycosides 3 75.0 0 0.0 1 25.0 0 0.0 0 0.0 4 25. 3 75.0 0 0.0 0 0.0 1 0 0.0 4 Tetracyclines 0 Macrolides 3 75.0 0 0.0 1 25.0 0 0.0 0 0.0 4 25. 3 75.0 1 0 0.0 0 0.0 0 0.0 4 Nitrofurans 0 Penicillins 3 75 0 0 0.0 0 0.0 0 0.0 1 25.0 4 25. 3 75.0 1 0 0.0 0 0.0 0 0.0 4 Phenicols 0 Potentiated 3 75.0 0 0.0 0 0.0 0 0.0 1 25.0 4 Sulfonamides 25. 3 75.0 1 0 0.0 0 0.0 0 0.0 4 Quinolones 0 Sulfonamides 3 75.0 0 0.0 0 0.0 0 0.0 1 25.0 4

Other" 0 - 0 - 0 - 0 - 0 - 0

Crawfish o..Wo.»„y ™

161 N%N% N %N%N%

Aminoglycosides 4 80.0 0 0.0 1 20.0 0 0.0 0 0.0 5 25. 3 75.0 1 0 0.0 0 0.0 0 0.0 4 Tetracyclines 0 Macrolides 4 100.0 0 0.0 0 0.0 0 0.0 0 0.0 4 Nitrofiirans 4 100.0 0 0.0 0 0.0 0 0.0 0 0.0 4 20. 3 60.0 1 0 0.0 0 0.0 1 20.0 5 Penicillins 0 Phenicols 4 100.0 0 0.0 0 0.0 0 0.0 0 0.0 4 Potentiated 20. 4 80.0 0 0.0 0 0.0 1 0 0.0 5 Sulfonamides 0 Quinolones 4 100.0 0 0.0 0 0.0 0 0.0 0 0.0 4 Sulfonamides 4 100.0 0 0.0 0 0.0 0 0.0 0 0.0 4

Other' 0 - 0 - 0 - 0 - 0 - 0

Lobster Never Rarely Occasionally Frequent Almost Tot Antimicrobial Drug ^ Always al Class N % N % N % N % N %

Aminoglycosides 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 50. 1 50.0 1 0 0.0 0 0.0 0 0.0 2 Tetracyclines 0 Macrolides 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Nitrofiirans 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Penicillins 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Phenicols 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Potentiated 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Sulfonamides Quinolones 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Sulfonamides 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1

Other' 0 0 - 0 - 0 - 0 - 0

Mussels Never Rarely Occasionally frequent Almost Tot Antimicrobial Drug Always al Class N % N % N % N % N %

Aminoglycosides 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Tetracyclines 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Macrolides 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Nitrofiirans 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Penicillins 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Phenicols 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Potentiated 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Sulfonamides Quinolones 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3

162 Sulfonamides 3 100.0 0 0.0 0 0.0 0.0 0.0 3 Other' 0 0 0 0

Oysters

Never Rarely Occasionally ^ Antimicrobial Drug Class N % N % N

Aminoglycosides 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Tetracyclines 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Macrolides 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Nitrofurans 3 100.0 0 p.o 0 0.0 0 0.0 0 0.0 3 Penicillins 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Phenicols 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Potentiated 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Sulfonamides Quinolones 3 100.0 0 0.0 0 0.0 0 0.0 0 0.0 3 Sulfonamides 2 100.0 0 0.0 0 0.0 0 0.0 0 0.0 2 Other" 0 - 0 - 0 - 0 - 0 - 0 Salmon Frequent Almost Tot Never Rarely Occasionally Antimicrobial Drug Ay Always al Class N % N % N % N % N %

33. 5 55.6 3 1 11.1 0 0.0 0 0.0 9 Aminoglycosides 3 16. 22. 1 5.6 3 9 50.0 4 1 5.6 18 Tetracyclines 7 . 2 25. 4 333 3 5 41.7 0 0.0 0 0.0 12 Macrolides 0 12. 12. 4 50.0 1 2 25.0 1 0 0.0 8 Nitrofurans 5 5 36. 27. 3 2T3 4 1 9.1 3 0 0.0 11 Penicillins 4 3 61. 4 30.8 8 1 7.7 0 0.0 0 0.0 13 Phenicols 5 Potentiated 14. 14. 3 21.4 2 7 50.0 2 0 0.0 14 Sulfonamides 3 3 45. 4 36.4 •5 1 9.1 0 0.0 1 9.1 11 Quinolones 5 18. 3 2T3 1 9.1 4 36.4 2 1 9.1 11 Sulfonamides 2 Other" 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1

Shrimps/Prawns Antimicrobial Drug Frequent Almost Tot Never Rarely Occasionally Class ly Always al

163 N % N % N % N % N %

11. 55.6 0 0.0 3 333 1 0.0 9 Aminoglycosides 1 27. 18.2 0 0.0 5 45.5 3 9.1 11 Tetracyclines 3 22. 44. 33J 2 0 0.0 4 0.0 9 Macrolides 2 4 22: 11. 33J 2 22.2 1 11.1 9 Nitrofurans 2 1 33. 11. 222 1 11.1 1 2Z2 9 Penicillins 3 1 22. 33J 2 22.2 0 0.0 2Z2 9 Phenicols 2 Potentiated 11. 22. 113 1 1 11.1 2 222 9 Sulfonamides 1 2 20. 10. 30.0 2 20.0 1 20.0 10 Quinolones 0 0 22. 33. 222 1 11.1 3 11.1 Sulfonamides 2 3 Other' 100.0 0 0.0 0 0.0 0 0.0 0.0

Tilapia Frequent Almost Tot Never Rarely Occasionally Antimicrobial Drug ly Always al Class N % N % N % N % N %

40. 6 60.0 4 0 0.0 0 0.0 0 0.0 10 Aminoglycosides 0 41. 2 16.7 5 3 25.0 1 8.3 1 8.3 12 Tetracyclines 7 55. 3 333 5 1 11.1 0 0.0 0 0.0 9 Macrolides 6 33. 4 44.4 3 1 11.1 0 0.0 1 11.1 9 Nitrofiirans 3 20. 20. 3 30.0 2 1 10.0 2 2 20.0 10 Penicillins 0 0 25. 7 5&3 3 1 8.3 0 0.0 1 8.3 12 Phenicols 0 Potentiated 18. 5 45.5 2 2 18.2 1 9.1 1 9.1 11 Sulfonamides 2 50. 3 30.0 5 1 10.0 0 0.0 1 10.0 10 Quinolones 0 18. 5 45.5 2 2 18.2 1 9.1 1 9.1 11 Sulfonamides 2 Other" 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Trout Frequent Almost Tot Never Rarely Occasionally Antimicrobial Drug ly Always al Class N % N % N % N % N % Aminoglycosides 5 50.0 5 50. 0 0.0 0 0.0 0 0.0 10 164 0 38. 1 4.8 1 4.8 8 38.1 8 3 14.3 21 Tetracyclines 1 36. 4 36.4 4 3 27.3 0 0.0 0 0.0 11 Macrolides 4 33. 3 33.3 3 3 33.3 0 0.0 0 0.0 9 Nitrofurans 3 27. 27. 3 27.3 3 2 18.2 3 0 0.0 11 Penicillins 3 3 1 52. 6 31.6 3 15.8 0 0.0 0 0.0 19 Phenicols 0 6 Potentiated Q 11. 23. J 17.6 Z 7/ 41.2 4A 1 5.9 17 Sulfonamides 8 5 40. 6 40.0 6 2 13.3 1 6.7 0 0.0 15 Quinolones 0 15. 23. 3 23.1 2 5 38.5 3 0 0.0 13 Sulfonamides 4 1 Other" 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1

Omamental/Pet Fish Frequent Almost Tot Never Rarely Occasionally Antimicrobial Drug ly Always al Class N % N % N % N % N % 1 56. 5 16.7 6 20.0 2 6.7 0 0.0 30 Aminoglycosides 7 7 1 45. 2 5.7 3 8.6 10 28.6 4 11.4 35 Tetracyclines 6 7 22. 6 22.2 6 11 40.7 2 7.4 2 7.4 27 Macrolides 2 25. 16. 7 29.2 6 5 20.8 4 2 8.3 24 Nitrofurans 0 7 19. 22. 4 12.9 6 8 25.8 7 6 19.4 31 . Penicillins 4 6 1 40. 7 23.3 9 30.0 1 3.3 1 3.3 30 Phenicols 2 0 Potentiated 21. 18. 5 15.2 7 14 42.4 6 1 3.0 33 Sulfonamides 2 2 1 27. 13. 10 27.8 10 27.8 5 1 2.8 36 Quinolones 0 8 9 24. 5 20.0 6 11 44.0 2 8.0 1 4.0 25 Sulfonamides 0 Other" 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1

Other Frequent Almost Tot Never Rarely Occasionally Antimicrobial Drug ly Always al Class N % N % N % N % N % 16. 16. 3 50.0 1 1 16.7 1 0 0.0 6 Aminoglycosides 7 7 23. 3 23.1 1 7.7 6 46.2 0 0.0 13 Tetracyclines 1 Macrolides 4 80.0 0 0.0 1 20.0 0 0.0 0 0.0 5 165 Nitrofurans 4 80.0 0 0.0 1 20.0 0 0.0 0 0.0 5 20. 4 80.0 0 0.0 0 0.0 1 0 0.0 5 Penicillins 0 28. 4 57.1 2 1 14.3 0 0.0 0 0.0 7 Phenicols 6 Potentiated 16. 5 41.7 2 4 313 1 8.3 0 0.0 12 Sulfonamides 7 15. 6 46.2 2 4 30.8 1 7.7 0 0.0 13 Quinolones 4 16. 3 50.0 1 2 33J 0 0.0 0 0.0 6 Sulfonamides 7 33. 2 66.7 1 0 0.0 0 0.0 0 0.0 3 Other^ 3 ^ Responses under other are incomplete as no antimicrobials were specified. ^ Only species specified: Dicentrarchus labrax, Sparus aurata, Diplodus sargus, Pagellus erythrinus, Puntazzo puntazzo, Argyrostomus regius, eel, percids, walleye and yellow perch

Table 20. Frequency of AMR in selected bacteria (Q16) Total Respondents=61 (Out of a possible 110) Aeromonas spp. Almost Tota Never Rarely Occasionally Frequently Antimicrobial Always 1 Drug Class N % N % N % N % N %

Aminoglycoside 25. 8 14 43.8 10 31.3 0 0.0 0 0.0 32 s 0 Tetracyclines 3 6.3 4 8.3 18 37.5 20 41.7 3 6.3 48 20. 5 9 36.0 8 32.0 2 8.0 1 4.0 25 Macrolides 0

5 5 3 14.3 8 38.1 3 14.3 2 9.5 21

Nitrofurans 00 18. 3 9.4 8 25.0 7 21.9 8 25.0 6 32 Penicillins 8 20. 8 14 35.0 15 37.5 2 5.0 1 2.5 40 Phenicols 0 Potentiated 16. 7 11 25.6 14 32.6 10 23.3 1 2.3 43 Sulfonamides 3 23. 9 10 25.6 14 35.9 4 10.3 2 5.1 39 Quinolones 1 17. 5 7 24.1 11 37.9 5 17.2 1 3.4 29 Sulfonamides 2

Other 0 - 0 - 0 - 0 - 0 - 0

Edwardsiella spp. Antimicrobial Almost Tota Never Rarely Occasionally Frequently Drug Class Always 1

166 N % N % N % N % N %

Aminoglycoside 57. 8 2 14.3 4 28.6 0 0.0 0 0.0 14 s 1 15. 2 3 23.1 4 30.8 4 30.8 0 0.0 13 Tetracyclines 4 46. 6 3 23.1 4 30.8 0 0.0 0 0.0 13 Macrolides 2 40. 4 3 30.0 3 30.0 0 0.0 0 0.0 10 Nitrofiirans 0 25. 3 3 25.0 2 16.7 4 33J 0 0.0 12 Penicillins 0 33. 4 5 41.7 3 25.0 0 0.0 0 0.0 12 Phenicols 3 Potentiated 33. 4 4 333 3 25.0 1 &3 0 0.0 12 Sulfonamides 3 42. 6 4 28.6 4 28.6 0 0.0 0 0.0 14 Quinolones 9 25. 3 2 16.7 5 41.7 2 16.7 0 0.0 12 Sulfonamides 0

Other 0 - 0 - 0 - 0 - 0 - 0

167 Escherchia coli Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N % Aminoglycosides 7 50.0 4 28.6 3 21.4 0 0.0 0 0.0 14 Tetracyclines 2 15.4 2 15.4 4 30.8 5 3&5 0 0.0 13 Macrolides 3 333 1 11.1 5 55.6 0 0.0 0 0.0 9 Nitrofiirans 4 40.0 3 30.0 2 20.0 0 0.0 1 10.0 10 Penicillins 2 16.7 0 0.0 4 333 3 25.0 3 25.0 12 Phenicols 2 16.7 5 41.7 3 25.0 2 16.7 0 0.0 12 Potentiated Sulfonamides 4 333 3 25.0 4 333 1 83 0 0.0 12 Quinolones 3 21.4 7 50.0 2 143 1 7.1 1 7.1 14 Sulfonamides 4 36.4 2 18.2 3 273 2 18.2 0 0.0 11

Other 0 - 0 - 0 - 0 - 0 - 0

Salmonella spp. Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N %

Aminoglycosides 3 25.0 6 50.0 2 16.7 0 0.0 1 83 12 Tetracyclines 2 222 2 2Z2 2 22.2 3 333 0 0.0 9 Macrolides 3 37.5 3 37.5 2 25.0 0 0.0 0 0.0 8 Nitrofiirans 3 333 4 44.4 2 22.2 0 0.0 0 0.0 9 Penicillins 2 20.0 4 40.0 2 20.0 I 10 0 1 10.0 10 Phenicols 2 20.0 7 70.0 1 10.0 0 0.0 0 0.0 10 Potentiated Sulfonamides 4 44.4 I 11.1 3 333 0 0.0 1 11.1 9 Quinolones 5 45.5 2 18.2 3 27.3 0 0.0 1 9.1 11 Sulfonamides 3 333 2 222 2 222 2 22.2 0 0.0 9

Other 0 - 0 - 0 - 0 - 0 - 0

Streptococcus spp.

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N %

Aminoglycosides 4 25.0 7 43.8 3 18.8 2 12.5 0 0.0 16 Tetracyclines 2 11.1 6 333 5 27.8 4 22.2 1 5.6 18 Macrolides 3 20.0 7 46.7 3 20.0 2 133 0 0.0 15 Nitrofiirans 5 38.5 5 385 2 15.4 0 0.0 I 7.7 13 Penicillins 5 29.4 1 5.9 9 52.9 1 5.9 1 5.9 17 Phenicols 5 29.4 6 353 5 29.4 0 0.0 1 5.9 17 Potentiated Sulfonamides 6 353 2 11.8 6 353 2 11.8 1 5.9 17 Quinolones 5 29.4 6 353 5 29.4 0 0.0 I 5.9 17 Sulfonamides 6 40.0 5 333 2 133 1 6.7 1 6.7 15

Other 0 - 1 - 0 - 0 - 0 - 0

168 Vibrio spp.

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N %

Aminoglycosides 7 29.2 7 292 6 25.0 3 12.5 1 4.2 24 Tetracyclines 2 7.1 6 21.4 12 42.9 7 25.0 1 3.6 28 Macrolides 4 21.1 5 263 7 36.8 3 15.8 0 0.0 19 Nitrofurans 5 29.4 4 23j 5 29.4 1 5.9 2 11.8 17 Penicillins 5 2&3 2 10.5 6 31.6 5 26J 1 5.3 19 Phenicols 7 35.0 4 20.0 7 35.0 1 5.0 1 5.0 20 Potentiated Sulfonamides 5 20.0 8 3Z0 7 28.0 3 12.0 2 8.0 25 Quinolones 9 34.6 7 269 8 30.8 1 3.8 1 3 8 26 Sulfonamides 4 25.0 4 25.0 5 31.3 2 12.5 1 6.3 16

Other 0 - 0 - 0 - 0 - 0 - 0

Other

Never Rarely Occasionally Frequently Almost Always Total Antimicrobial Drug Class N % N % N % N % N %

Aminoglycosides 1 50.0 0 0.0 0 0.0 0 0.0 1 50.0 2 Tetracyclines 2 22.2 2 2Z2 1 11.1 3 33J 1 11.1 9 Macrolides 2 66.7 0 0.0 1 333 0 0.0 0 0.0 3 Nitrofurans 1 100.0 0 0.0 0 0.0 0 0.0 0 0.0 1 Penicillins 3 75.0 0 0.0 0 0.0 0 0.0 1 25.0 4 Phenicols 3 60.0 1 20.0 0 0.0 0 0.0 1 20.0 5 Potentiated Sulfonamides 3 42.9 0 0.0 0 0.0 2 28.6 2 28.6 7 Quinolones 1 25.0 1 25.0 1 25.0 1 25.0 0 0.0 4 Sulfonamides 2 50.0 0 0.0 0 0.0 1 25.0 1 25.0 4

Other 0 - 0 - 0 - 0 - 0 - 0

169 Table 21. Control measure for antimicrobial resistance (Q17) Total Respondents^ 131 Practice N % Rotation of antibiotic class usage 34 26.0 Ban the use of certain antimicrobial drugs^ 33 25.2 Restricted use of certain antimicrobial drugs'' 38 29.0 Adoption of general prudent use guidelines in aquaculture 71 54.2 Depopulation of a fish farming site when antimicrobial resistant strains of animal health or public health 18 13.7 significance are identified Strict biosecurity measures to avoid pathogen strain 46 35.1 introduction None 30 22.9 Other' 9 6.9 ^ Antimicrobials drugs that were listed as banned included: chorophenicol, nitrofiirans, quinolone, amoxicillin and penicillin, oxytetracycline ^ Restricted use included drugs that obtained only through prescriptions after diagnosis, VFD (veterinary feed directive), using drugs that were only FDA approved, use after written MUM form, vaccination where possible, following, USD A food fish restrictions, use after Emergency Drug Release forms no antimicrobials used at all manipulate farming properties (e.g., density of fish)

Table 22. Jurisdiction-specific government/industry program for AMR surveillance and monitoring (Q18) Total Respondents=139 Knowledge of Program N % Yes" 33 No 106 763 ^ Programs specified: British Columbia Fish Health Auditing and Surveillance Program, Bureau of Fisheries and Aquatic Resources (BFAR) Monitoring Program for Antibiotic Residues(Phillippines), Australia's National Residue Survey, Defi*a Antimicrobial Resistance Coordination (DARC) Group (UK), FDA, FDA-APHIS, FDA CVM, AMDUCA, Ministry of Agriculture, Forestry and Fishery (Japan), Antimicrobial Resistance Monitoring Programme (Thailand), Bureau of Fisheries and Aquatic Resources, Dr Diana Chee, Aquatic Animal Health Laboratory, AgriFood & Veterinary Authority of (Singapore), U.S. Fish and Wildlife Service, FDA Seafood Safety HACCP program.

170 Table 23. Identification of knowledge gaps (Q19) Total Respondents=66 N 18.2 12 Runoff of AM into aquaculture from other facilities (e.g., human health, large animal production) Risk of aquatic AMR on human health 12 18.2 Unregulated use of AM/AM use surveillance 8 12.1 Evaluation of user's knowledge of AM, AMR/lack of knowledge on user's part 7 10.6 Unnecessarily or relaxed use of AMs 7 10.6 Imported products (e.g., seafood, ornamental fish) 2 3.0 Link AMU-AMR 5 7.6 AMR surveillance/lack of proper diagnosis 7 10.6 Pharmacokinetics of drugs and drug residues 3 4.5 Other: plasmid characterization, shellfish bacteria, breakpoint therapeutic values 5 7.6 ^ The absolute frequency of the comments are provided as participants offered more than comment. For this reason, the percentage does not sum to 100.

Table 24. Expertise in microbiology (Q21) Total Respondents= 152 Option N % Yes 70 46.1 No 82 53.9

Table 25. Use of guidelines for interpretation of susceptibility/resistance (Q22) Total Respondents=48 Guideline N % CLSI (Clinical and Laboratory 58.3% 28 Standards Institute) EUCAST (European Committee on Antimicrobial Susceptibility 12.5% 6 Testing) Breakpoints developed In-house 31.3% 15 (or locally) Other guidelines^ 16.7% 8 ® Names of guidelines provided: Performance Standards for Antimicrobial Disc Susceptibility Tests NCCLS 1981; Model Comprehensive Fish Health Protection Program, Pacific Northwest Fish Health Protection Committee, September 1989; ADF&G Fish Pathology Section Laboratory Manual; M42-P Vol 25, No. 21 from clinical and Laboratory Standards Institute, European Union, a Standard Operating Procedure (SOP/H/3043), AFS Bluebook, Japanese Society of Antimicrobials for Animals.

171 Table 26. Incubation temperature of bacterial isolates (Q23) Total Respondents=51 Temperature N % 15 degrees Celsius 2 3.9 22 degrees Celsius 12 23.5 28 degrees Celsius 13 25.5 37 degrees Celsius 5 9.8 Other' 19 37.3 ^ Other temperatures specified: 'depends on bacteria', 25, 20 degrees, 15 for seawater isolates 22 for freshwater, 15 for Flavobacterium psychrophilum, 22C for typical salmonid isolate 28C for catfish, bass, tilapia isolates, 25, 30, 37, 22 C for cold-water fish and 28 C for warm-water fish, 24 degrees.

Table 27. Interpretation of resistance across bacterial species (Q24) Total Respondents=46 Options Total N % All aquatic bacteria are evaluated with the same criteria 17 37.0% Different genera of bacteria may be evaluated differently 24 52.2% Don't know 5 10.9%

172 APPENDIX 3. Prevalence of Selected Zoonotic Bacteria in Selected Aquatic Spcoes and Seafood: A Scoping Study, Systematic Review and Meta-Analysis of Published

Research

APPENDIX 3.1. Project protocol, methodology and decisions

Protocol for the Scoping Studv-Svstematic Review: Development of a Transparent /Structured Global Knowledge-Base on Prevalence, Risk Factors and Interventions Related to Selected Zoonotic Bacteria, Antimicrobial Use, Antimicrobial Resistance in Bacteria, and Selected Drug and Dye Residues in Aquaculture and Seafood

Date: March 10, 2011

Project Core Team: Andrijana Rajic, Project Lead Lucie Dutil, Reviewer/Co-Lead Lisa Waddell, SR Manager/Team co-ordinator Barbara Wilhelm, Reviewer Judy Greig, Reviewer Brent Avery, Reviewer Carl Uhland, Reviewer Natasa Tusevljak, SR Manager/Reviewer

173 Rationale for the Scoping Study and Systematic Review:

Development of Transparent/Structured Global Knowledge-Base for Prevalence, Risk Factors and Interventions Related to Selected Zoonotic Bacteria, Antimicrobial Use, Antimicrobial Resistance in Bacteria, and Selected Residues in Aquaculture and Ornamental Fish

Systematic Review: Use and Methodology A systematic review is a formal process methodology for conducting a rigorous literature review of all the available information on the topic or question of interest. This process is designed that so that the same level of rigour originally used to conduct the primary research is maintained in reviewing the scientific evidence (Davies et al., 2000). The process of conducting a systematic review offers a replicable, structured and transparent approach to evaluating the existing evidence that minimizes bias: a fundamental problem with traditional literature reviews (Davies et al., 2000; Sargeant et al., 2006). Unlike traditional literature reviews, a systematic review approaches questions to evaluate all existing information without biasing results due to preconceived topic bias, incomplete literature search strategies and unstructured methods of assessing study relevance and quality (Davies et al., 2000). Some additional advantages to conducting systematic reviews include increased power of available results, enhanced generalisability, increased robustness and transferability of the data (Khan et al., 2001). The results can be analyzed using meta-analysis (MA), a statistical method for pooling the results fi-om multiple, similar and homogenous studies resulting in more precise and robust summary estimates of the outcome (Borenstein et al., 2009). Systematic reviews are also good for identifying gaps in the scientific evidence, which helps to focus further research in these areas (Davies et al., 2000; Sargeant et al., 2006).

Systematic reviews have primarily evaluated therapeutic health questions where a large body of literature with a substantial amount of uncertainty exists (Davies et al., 2000). They are used by clinicians, consumers and policy makers to enable that informed decisions be made regarding health care, treatments and drug interventions (Khan et al., 2001, Clark et al., 2004). The systematic review process has been used to a lesser extent in veterinary science, food safety and topics related to the agri-food public health (Sargeant et al., 2006).

Scoping Studies: Use and Methodology The nature of policy driven issues or questions in food safety and zoonotic public health are usually broad in scope. As with systematic reviews, scoping studies are extensively used in the healthcare sector to rapidly identify and characterize the evidence on a broader scope, identify research gaps and to assist with framing questions for rigorous SRs and MA (Davis et al., 2009; Anderson et al., 2008; Arksey et al., 2005). The main difference between the two methods is in the appraisal of methodological soundness and quantitative synthesis of data; these are critical steps of SR, and rarely part of a ScS 174 (Arksey et al., 2005). To date, not much literature exists in undertaking a scoping study and when its use is most appropriate (Arksey and O'Malley, 2005). This problem is compounded by the fact that the processes is still in development and much regarding it, such as the interpretation or methods, is not yet standardized so that its application is comprehensive across sectors (Davis et al., 2009).

A scoping study-systematic review approach offers a unique way of navigating and synthesizing literature in fields where such a task seems daunting due to the quantity of research present. This approach is ideal for industries such as aquaculture industry with its diversity of species, farming methods, processing methods and potential food-borne hazards; all variations that contribute to the complexity in synthesizing the relevant research. This approach will be applied to investigate prevalence, risk factors and interventions related to selected zoonotic bacteria, antimicrobial use, antimicrobial resistance in bacteria, and selected drug and dye residues in aquaculture.

REFERENCES

Anderson, S., P. Allen, S. Peckham, and N. 2008.Goodwin. Asking the right questions: Scoping studies in the commissioning of research on the organisation and delivery of health services. Health Res Policy Syst. 6:7.

Arksey H, and O'Malley L. 2005. Scoping studies: towards a methodological framework, Int J of Soc Res Meth. 8: 1, 19-32.

Borenstein, M., Hedges, L.V., Higgins, J.P.T., Rothstein, H.R., 2009. Introduction to Meta-Analysis. John Wiley & Sons, Chichester; Hoboken.

Clarke M.; Oxman A. Cochrane Reviewers' Handbook 4.2.0. [Web Page] Mar 2004; http://www.cochrane.org/resources/handbook/handbook.pdf [Accessed 7 Jan 2005].

Davis K, Drey N, Gould D. 2009. What are scoping studies? A review of the nursing literature. Int. J. Nursing Studies, 46:1386-1400.

Davies H.T.O. CIK. 2000. What is a Systematic Review. Evidence Based Medicine 1:1- 6. Khan K.S.; Riet G.; Glanville J., et al., editors. 2001. Undertaking Systematic Reviews of Research and Effectiveness CRD's Guidance for those Carrying Out or Commissioning Reviews. 2nd edition ed. Publications Office, NHS Center for Reveiws and Dissemination, University of York, York: York Publishing Services Ltd. Sargeant J, Rajic A, Read S, Ohlsson A. 2006. The process of systematic review and its application in agri-food public-health. Preventative Veterinary Medicine. 75:141-51.

175 Systematic Review Team

Project Core Team Role Andrijana Rajic Project Lead/ SR expert

Lucie Dutil Co-Lead /Reviewer Lisa Waddell SR Manager/ Team coordinator/ SR expert/ QA reviewer Brent Avery SR design/ Reviewer Judy Greig SR design/ Reviewer Juliana Ruzante SR Manager/Reviewer Natasa Tusevljak * SR design/ Reviewer/ future SR manager graduate project UoG Carl Uhland SR design/ Reviewer / Aquaculture Expert Barbara Wilhelm SR design/ Reviewer Other Reviewers America Mederos Rl/2 Review (contract) Rebecca De Auburn Parent Rl/2 Review FSWEP I Maijorie Bercier FSWEP R1 Review Karine Forget FSWEP R1 Review Kyle Burgers FSWEP R1 Review Sarah Totton R2/Quality Assessment/Data extraction Natalia Cemicchiaro Foreign article review Additional Expert Advisory Committee Members: Carol McClure Topic Expert Richard Read-Smith Question Prioritization Craig Stephen Topic Expert Sophie St. Hillarie Topic Expert

176 SR Project Implementation Timeline

November 2008 - December 2010

What? Who? When? Finalize/Implement (electronic LW/NT/BW Before Dec. 15 databases)/Document search strategy/download into SRS Finalize relevance tools 1 and Pre-test AR/BW/LW Feb 15,2009 Finalize relevance tools 2 and Pre-test AR/LW/NT Apr 30, 2009 Complete relevance screening Reviewers June, 2009 Develop and pre-test QA form AR/NT March, 2010 Develop and pre-test DE tool AR/NT April, 2010 Develop/Implement search verification NT April, 2010 strategy Complete QA Reviewers June, 2010 Complete DE Reviewers August, 2010 Specific project question refinement / AR/RRS/BW March, 2010 assignment of specific graduate project /Team components NT/Malcolm Weir/Co-op students Individual component analysis/summary, NT/ students September-December, synthesis, manuscript writing 2010 Up-dated search/SR process NT/All November, 2010

177 Systematic Review Ouestion(s)

Broad Question: Systematic Review on Prevalence, Risk Factors and Interventions Related to Selected Zoonotic Bacteria, Antimicrobial Use, Antimicrobial Resistance in Bacteria, and Selected Drug and Dye Residues in Aquaculture and Ornamental Fish

Specific Questions: What are the prevalence, risk factors and interventions use to control selected zoonotic bacteria {Aeromonas (any species), Campylobacter spp, Edwardsiella (tarda, ictaluri), Erysipelothrix rhusiopathie, Escherichia coli (any serotype including generic or non-type specific), Salmonella (any serovar), Streptococcus (iniae). Vibrio spp.) in fish and seafood?

What are the prevalence, risk factors or interventions for antimicrobial/ antibiotic resistance for any bacteria in fish and seafood?

What are the prevalence, risk factors or interventions for antimicrobial/ antibiotic use for any bacteria in fish and seafood?

What are the prevalence, risk factors or interventions for antimicrobial/ antibiotic residues for any bacteria in fish and seafood?

What are the prevalence, risk factors or interventions for dye residues (Malachite green, leucomalachite green, acriflavine, methylene blue) in fish and seafood?

What reports or literature is there of outbreaks or sporadic disease in humans related to aquaculture through occupation, consumption, or leisure? Specific to Canada?

What reports or literature is there of human consumption patterns of fish and seafood? Specific to Canada?

What reports or literature is there of importation or exportation of fish and seafood from countries globally? Specific to Canada?

Inclusion/ Exclusion Criteria: Restricted the search to papers published from 1990 to the present is acceptable. Our expert panel agreed that while some literature exists prior to 1990 the aquaculture industry has changes a lot since then and the few studies that would be excluded would likely not impact the conclusions of the review.(Oct 2008)

Selected bacteria were chosen because there is a history of human illness due to those bacteria. Some bacteria e.g., E. coli we did not limit species, while others like Streptococcus we limited to species that have been known to cause human illness, not just production issues. (Oct 2008)

178 Selected bacteria do not include Listeria, Clostridium botulinum, and Mycobacterium spp. because they are more closely associated with processing, than aquaculture and its products (Nov 17,2008).

Parasites, heavy metal residues and sea lice have been excluded from the topic of this review (Oct 1, 2008).

Studies that only have environmental samples were excluded. We are interested in product state and potential to affect human health (Nov 2008).

This SR is limited to field studies, all small laboratory investigations are not considered relevant because they do not reflect the situation or impact of an intervention under field conditions. It has been shown that while laboratory results are a first step in understanding a hypothesis, the application and impact under field conditions is ultimately what we are interested in (Jan 2009).

We are not interested in studies evaluating diagnostic tests (Jan 2009).

Included are studies fi"om the larval stage to consumption of a seafood product. (Jan 2009)

Gene studies, immunology studies (insulin factor/ IL+ response etc) are not relevant. (Jan 2009)

Search strategy

Electronic Search

All potentially relevant peer-reviewed primary studies will be identified using the predetermined search algorithms within the electronic databases listed below. Abstracts of all potentially relevant studies will be downloaded into a bibliographical manager database, Procite 5.0 (10). Appendix 1 has information regarding the filters corresponding to each database for the purposes of uploading into Procite. Procite has a duplicate filter that will be applied to identify records that are exact duplicates. The filtered Procite database will then be uploaded into the SRS system and filtered again using a less strict filter; all additional duplicates will be manually reviewed and confirmed by a member of the review team (14). An update search will be conducted to identify any relevant papers published between the initial search and the end of the quality assessment phase, any additional citations will be uploaded into SRS for screening.

Databases (6): PubMed, and on the Biological Sciences platform at University of Guelph: AFSA 1 Biological Sciences and Living Resources (1971- current), AFSA 3 Aquatic Pollution and Envirormiental Quality (1990- current), AFSA 179 Aquaculture abstracts (1984- current). Ecology Abstracts (1982- current), ZoologicalRecordPlus (2008).

Search Limits: date range 1990- October 28, 2008.

Executed by: Lisa Waddell on October 28,2008

The components to the search algorithms: Acronym Search String FISH (fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or salmon or salmonid or salmonids or finfish or lobster or crab) PATH (Aeromonas or Campylobacter or campylobacteriosis or Salmonella or salmonellosis or Vibrio* or coll" or escherichia or edwardsiella OR erysipelothrix OR streptococcus) AMR (resistance or resistant or residue or residues or use or usage) AND (drug or antimicrobial* or "anti microbial" or "anti microbials" or anti-microbial* or antibiotic* or "anti biotic" or "anti biotics" or anti-biotic* or antibacterial* or "anti bacterial" or "anti bacterials" or anti-bacterial* or biosecurity or effluent) AMs (resistance or resistant or residue or residues or use or usage or treatment*) AND (sulphonamide OR sulfadiazine OR trimethoprim OR sulfamethoxazole OR sulfisoxazole OR sulfamerazine OR sulfamethazine OR sulfadimethoxine OR sulfamonomethoxine OR chloramphenicol OR chloramine OR novobiocin OR "nalidixic acid" OR ormetoprim OR spiramycin OR fiorfenicol OR tetracycline OR oxytetracycline OR doxycycline OR chlortetracycline OR penicillin OR ampicillin OR amoxicillin OR fluoroquinolone OR enrofloxacin OR "oxalinic acid" OR flumequine OR nitrofuran OR nifurpirinol OR nitrofurazone OR furazolidone OR virginiamycin OR trifluralin OR neomycin OR macrolides OR erythromycin OR tylosin OR streptomycin OR dihydrostreptomycin OR nystatin OR antifungal OR cephalosporins OR rifampicin OR aminoglycosides OR gentamicin OR kanamycin OR isoniazid OR griseofulvin) DYE (malachite green or leucomalachite green or acriflavine or methylene blue) Final FISH and PATH, FISH and AMR, FISH and AMs, FISH and DYE Strings

Results of the search by search algorithm and Database. Biological Sciences- AFSA 1,3, AFSA BioSci- Search Strings (see aquaculture and ecology Zoological below) PubMed abstracts Records Plus 180 FISH and PATH 5388 6709 FISH and AMR 2972 2365 FISH and DYE 152 803 + 561 FISH and AM 1215 225 8325 ASFA 1: 1747 ASFA 3: 5007 ASFA Aquaculture 277 Ecology Abstracts FISH and (PATH or AMR or DYE or Ams) 7963 8926 198 Total deduplicated 14540

Background on Search Development: Databases on the health Canada and University of Guelph library sites were evaluated for coverage of aquaculture and a list was provided to the systematic review team to evaluate. The goal was to choose 5 or more databases that together covered all disciplines and research related aquaculture. The five databases are listed at the beginning of this section. The results of the electronic search will be coupled with a search verification strategy that will evaluate "n" randomly selected literature reviews on each sub-question in the systematic review to identify potentially relevant research that may have been missed by the electronic search which will verify or improve the search sensitivity and the systematic review.

Terms: Search terms were initially brainstormed by the systematic review team to come up with Population terms (fish, aquaculture and human terms), Outcome terms (Pathogens, residues, AMR terms), Intervention/Risk factor terms (dyes, antimicrobials). More terms were added by evaluating a collection of potentially relevant papers. This same collection was used to verify the search being implemented was capturing all identified papers.

Appendix two has a list of terms that were suggested listed under reasons for exclusion. Whole groups of terms and individual terms were identified by our search expert as likely to be removed. Reasons for removal included that the term-was too broad and that there were other terms already capturing that term. All terms relating to humans, and disease in human were removed as they inflated the search by 30k citations and aquaculture + pathogens or AM or AMR or Dye would capture any human related citations that had to do with exposure to aquaculture + outcomes of interest. The next step was to look at terms that may not stay in the search, which required testing each term to see what it uniquely captured and evaluating whether it was capturing relevant literature. Within PubMed each string was tested to make sure the database didn't detect any spelling errors, was mapping terms properly within the database and was interpreting punctuation correctly (quotes, wildcards or phrases).

181 Population terms: initially evaluated with general and specific fish terms, aquaculture terms, aquarium terms and water terms. Upon refinement, water terms and aquatic environment were removed because the hits were not relevant to the topic. AND fishery, finfish, crab and lobster were added due to a very small number of citations that were using only those terms and not our other key words. Pathogen terms: The systematic review team had brainstormed a list of pathogens, some were quite specific and some were general e.g., E. coli. It was decided that Listeria, botulium and mycobacteria would be removed from this list as they were infections of processed products or handler infections. The bacteria left were tested to make sure the general genus names were properly mapping in the database, specific species were not searched for as the difference was only a few hundred hits. - AMR terms: Based on the first algorithm and the objectives of this search, general anti-microbial terms, biosecurity and effluent were included in the search string in combination with residue, resistance and use terms. Without the latter the search was too large for most databases to handle. Antimicrobial terms: Family and specific antimicrobial terms were extensively tested to come up with the final algorithm of those that appeared to be capturing relevant articles. Dye terms: This list was short, direct the terms functioned well within the databases. - Note the MISC terms and the Occupational terms: Not used in search because their hits were being full captured by other algorithms. E.g., FISH AND AMR = FISH AND (AMR OR MISC).

Search Verification

To verify that the search strategy yielded all relevant articles and conference proceedings and to identify any additional articles of potential relevance, the reference lists of several selected literature reviews will be checked. To identify a random sample of review articles covering general and specific aquaculture topics, literature reviews will be grouped by coverage of information and a random selection of each group will be drawn for verification. All references found within the Literature Cited section of the selected papers will be cross-checked with the references in SRS. All new references (ie. not already in the eSR system) will be screened liberally by title for potential relevance. This is accomplished by looking for any of the Aquaculture and/or fish terms used in the original database searches. The citations and abstracts for all potentially relevant articles will be downloaded into Procite, uploaded into eSR and screened using the relevance tool by two reviewers.

Procedure for obtaining articles.

182 Sequence for locating primary literature articles: • The strategy for obtaining primary literature articles starts with searching for a free electronic version of the article. • If unavailable from the Health Canada virtual library, the University of Guelph electronic library will be searched for an electronic version of the article. • If no electronic versions are available then the University of Guelph and the Trellis system will be checked for paper versions of the article. • If still unsuccessfiil, a request will be placed with CISTI, a Canada wide library, to obtain a copy of the article. • The next step is to use RACER, an Ontario wide inter-library program that can obtain a copy of the requested article from any university library in Ontario. Generally this system is free to members of an Ontario university. • Failing this, the citation will be placed in a separate pile for group review. If the article is deemed important by the team, then the author of the article will be contacted for a copy of the paper. • For foreign language papers, we follow the same steps outlined above, but if an English version is not available we will exclude the study from our review due to lack of time and money for translations.

Locating abstracts from conference proceedings • All relevant research located in conference proceeding will be matched to a published paper by searching for the author and subject identifiers with the data contained in the abstract. If a published article is not located, we will accept the conference proceeding citation if the citation is a longer, paper-style report with enough data to be usefiil. • A list of authors to contact will be populated and a letter inquiring about the publication status of the information in the conference abstract will be sent.

Letters, notes and author's responses • These publications are not primary research and should not continue to quality assessment. However, when they are associated with a relevant primary article these letters etc. can be attached to the primary study so the reviewers can examine the comments and notes related to the citation. • Electronically the letters will have the same file name as the primary article (RefID_0230.pdf) with the exception of an additional letter to denote a sub file (RefID_0230B.pdf, RefID_0230C.pdf). • For paper only copies, the comment or note will be attached directly to the back of the original article.

Theses • Many of the theses in our reference list are associated with a number of primary articles also in our database. Thus, when relevant theses are identified a copy is

183 obtained (if possible) and chapters are to be compared to primary articles in the database so that the same information is not reported twice.

Management of the Scoping Studv/Svstematic Review

Electronic Review Systematic reviews generally require considerably more effort than traditional literature reviews. As an example, Hirst et al. (2002) reported that a systematic compilation and classification of the literature on lameness in cattle took over two years from planning to completion (Hirst et al, 2002). It is anticipated that even greater effort is needed for a Cochrane type review (Clarke et al., 2004). Electronic Systematic Review (SRS) (Trialstat! Corporation, Ottawa, Canada), and DistillerSR (Evidence Partners, Ottawa, Canada) are tools, which allows for all steps of systematic review to be conducted and documented electronically(0'Blenis, 2004). The tools operates via web interface, which decreases the amount of administrative work involved in a systematic review as all articles and review updates are available online at all times, allowing the project to run with optimum efficiency (O'Blenis, 2004). Due to the electronic nature of the program, administrative tasks such as photocopying and delivery of articles to reviewers are also obsolete (O'Blenis, 2004; SRS, 2004). Review team members are given different levels of access to a review project as project administrator and/or reviewers. The review progress can be evaluated more effectively and efficiently through the statistical features of the tool that allow the project administrator to monitor the progress of the review and specific reviewer progress in real-time. Here, SRS 4.0 will be used to conduct the scoping study to assess articles for relevance (Level 1 - liberal screening level) and further classify articles (Level 2-classification) to prioritize processing through QA and DE. The systematic review will entail critical appraisal (Level 3 - quality assessment level) and data extraction (Level 4 - data extraction and summary) and will be conducted in Distiller SR because of it enhanced ability to handle complex queries and filter implementation by topic.

References

1. Clarke M.; Oxman A. Cochrane Reviewers' Handbook 4.2.0. [Web Page] Mar 2004; http://www.cochrane.org/resources/handbook/handbook.pdf. [Accessed 7 Jan 2005].

2. Hirst WM, Le Fevre AM, Logue DN, Offer JE, Chaplin SJ, Murray RD, Ward WR, French NP. A systematic compilation and classification of the literature on lameness in cattle. Vet J 2002 Jul;164(l);7-19.

3. O'Blenis P. The Electronic Systematic Review Handbook Practical Concepts and Methods for Electronic Screening and Data Abstraction. 1st edition ed. First TrialStat Corporation Paperback Edition, 2004: 2004 TrialStat Corporation; 2004.paperback.

184 4. SRS 3.0 [Electronic Systematic Review]. O'Blenis P., software development management. VP Product Management. Version 3.0. Ottawa, ON: TrialStat Corporation; 2004 Oct 1; c2001-2004. 44 By Ward Market Square, Suite 270, Ottawa, ON, Canada, KIN 7A2. NOW owned (Feb 2009) by Mobius Analytics at www, mobiusanalvtics. com .

SCOPING STUDY

Relevance Screening

Relevance Screening 1 Relevance tool 1 will be developed to identify primary research investigating the general and refined questions and will be done at the abstract level. This tool will be short and allow us to rapidly screen-in all relevant articles pertaining to the study topic and to screen out all irrelevant articles. Furthermore, we will be able to trace back those irrelevant articles that could be potentially useful for background information with regards to writing papers, such as literature review articles The review team will design and test the tool on a selected number of articles. When the team is satisfied with reviewer's agreement and the tool is screening in all relevant articles. Level 1 screening will commence. All citation loaded into SRS will be screened for relevance by at two reviewers in order to be included or excluded from the review. The reviewing pair will settle conflicts between reviewers and in the event that a consensus cannot be reached, the citation will be presented to the rest of the team for a decision on relevance. All articles passing relevance screening I will enter relevance screening 2.

Relevance Screening 2

Relevance tool 2 will be developed to 1) classify articles based on topics relevant to the scope (Section 4) and 2) further exclude irrelevant articles based on additional criteria. Articles will be classified according to bacteria, point in food chain, antimicrobial drug, antimicrobial dye, type of study, aquatic species investigated, and study focus. All studies not sampling fish/seafood, or sampling diseased fish/seafood, or are conducted under laboratory conditions will be excluded at this level.

The review team will design and test the tool on a selected number of articles. When the team is satisfied with reviewer's agreement and the tool is screening in all relevant articles, Level 2 screening will commence.

All articles passing relevance screening 2 will enter quality assessment.

185 Frequency tabulations will be conducted to assess the quantity and distribution of evidence at this stage to guide the direction and main topic of the systematic review.

SYSTEMATIC REVIEW

Quality Assessment

Due to the broad nature of the relevant questions, several topics will be prioritized for completion of the systematic review based on the distribution of evidence from relevance screening 2. Quality assessment will be designed to first reconfirm relevance according to these prioritized topics and apply basic criteria regarding methodological soundness of studies. The topics selected for prioritization will be listed in the quality assessment tool. A filter in the review management software (DistillerSR) will be used to prioritize the selected topics. The design logic of this filter will be assessed by three epidemiologists along with testing of 25 articles that would pass through to quality assessment and 25 that would not. Studies not published in English, French, Spanish, Portuguese or in one of the Slavic languages will be excluded due to a lack of resources to translate additional languages. The articles passing the relevance reconfirmation stage of the quality assessment tool will be assessed for methodological soundness. Studies will be excluded if they do not report: number sampled and tested positive in raw or unadjusted form; adjusted data (if applicable) or measures of association/effect and at least some measures of variability (SE, SD, CI or p-value). Further evaluated of study quality will be: type of study design, type of subject selection, sample size/strategy, type of operation sampled and sufficient reporting of laboratory methods for bacterial culture and/or PCR to allow their replication. The quality assessment tool will be pre-tested on full articles and refined until reviewer agreement is achieved.

Data Extraction To complete the systematic review, one question will be prioritized for data extraction. The question fi^om the results was identified as: what is the reported prevalence (or concentration) of Aeromonas, E. coli (generic/pathogenic). Salmonella and Vibrio species in clams, mussels oysters, salmon, shrimp/prawn, and tilapia from processing to the retail level (including RTE). A filter in the review management software (DistillerSR) will be used to prioritize the selected topic out of all of those included in quality assessment. The filter will also exclude studies with less than 30 samples (due to the nature of the selected question low power of small studies). The design logic of this filter will be assessed by three

186 epidemiologists along with testing of 25 articles that would pass and 25 that would not. (The filter for data extraction is presented in Appendix 10.) The data will be extracted based on 1) general information (author, journal, publication type); 2) population investigated (e.g., fish/shellfish species); 3) sampling characteristics (e.g., sample type, sample weight), outcome measured (e.g., type of test and cut-off threshold and 4) reported results (e.g., total samples/number of samples positive). The data will be extracted as it is presented in the paper with multiple submissions of the extraction form representing each unique combination of species/bacteria/point in food chain/test used.

Data Summary and Synthesis Extracted raw data will be imported fi-om DistillerSR into excel (Microsoft Excel, Microsoft Corporation, Redmond, WA) where the dataset will be cleaned and coded. Comprehensive meta-analysis software V2 (Borenstein M, Hedges L, Higgins J, Rothstein H. Comprehensive Meta-analysis Version 2, Biostat, Englewood NJ (2005)) will be used for data-analysis. Datasets will be created of studies measuring prevalence or concentration on the same seafood product with the same target bacteria, and will be sub- grouped by the region where the study was conducted (e.g.. North America, Europe, Asia, Caribbean and South America.) to accommodate the variation by region and to increase utility for future use. Random-effects MA models will be performed where appropriate given the a priori assumption of significant heterogeneity. Heterogeneity will be assessed via a test using the Q-statistic and quantified on a relative scale using the value with a significance cut-off if the p-value <0.10 (Borenstein et al., 2009; Higgins and Green, 2009). Between trials variance vdll be estimated using the DerSimonian and Laird method. When heterogeneity statistics are not significant (p-value>0.10 or <50%), MA pooled estimate of prevalence or concentration and the upper and lower 95% confidence intervals will be reported. Otherwise, median and range will calculated from the individual studies within the subset of data. Publication bias will be investigated with the a priori decision using Begg's adjusted rank correlation and Egger's regression asymmetry tests only for homogeneous datasets with >10 unique observations (Begg and Mazumdar, 1994, Egger, et al., 1997)with the cut-off for significance of either test p- value <0.05.

References Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication bias. Biometrics 1994; 50:1088-1101.

Borenstein, M., Hedges, L.V., Higgins, J.P.T., Rothstein, H.R., 2009. Introduction to Meta-Analysis. John Wiley & Sons, Chichester; Hoboken.

Egger M, Davey Smith G, Schneider M, et al. Bias in meta-analysis detected by a simple, graphical test. BMJ 1997; 315:629-634. 187 Higgins JPT, Green S. Cochrane handbook for systematic reviews of interventions: Version 5.0.2 [updated September 2009]. The Cochrane Collaboration 2009.

188 APPENDIX 3.2. Search algorithms as they were put into the databases.

Search Strings- Algorithms as they were put into databases.

PUBMED export to text file and upload all in one go—> import to Procite with PubMed filter. (fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or salmon or salmonid or salmonids or finfish or lobster or crab) AND {{Aeromonas or Campylobacter or campylobacteriosis or Salmonella or sahnonellosis or Vibrio* or co//" or escherichia or edwardsiella OR erysipelothrix OR streptococcus) OR ((resistance or resistant or residue or residues or use or usage) AND (drug or antimicrobial* or "anti microbial" or "anti microbials" or anti-microbial* or antibiotic* or "anti biotic" or "anti biotics" or anti-biotic* or antibacterial* or "anti bacterial" or "anti bacterials" or anti-bacterial* or biosecurity or effluent)) OR ((resistance or resistant or residue or residues or use or usage or treatment*) AND (sulphonamide OR sulfadiazine OR trimethoprim OR sulfamethoxazole OR sulfisoxazole OR sulfamerazine OR sulfamethazine OR sulfadimethoxine OR sulfamonomethoxine OR chloramphenicol OR chloramine OR novobiocin OR "nalidixic acid" OR ormetoprim OR spiramycin OR florfenicol OR tetracycline OR oxytetracycline OR doxycycline OR chlortetracycline OR penicillin OR ampicillin OR amoxicillin OR fluoroquinolone OR enrofloxacin OR "oxalinic acid" OR flumequine OR nitrofuran OR nifurpirinol OR nitrofurazone OR furazolidone OR virginiamycin OR trifluralin OR neomycin OR macrolides OR erythromycin OR tylosin OR streptomycin OR dihydrostreptomycin OR nystatin OR antifungal OR cephalosporins OR rifampicin OR aminoglycosides OR gentamicin OR kanamycin OR isoniazid OR griseofulvin)) OR (malachite green or leucomalachite green or acriflavine or methylene blue)) export to text file (long form- no reference), 500 at a time—> import into Procite using //CSA-Biological_Sci.cfg filter and do zoological abstracts separate with CSA- BIOSCI Zoological_Record.cfg ((fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or salmon or salmonid or salmonids or finfish or lobster or crab) and (Aeromonas or Campylobacter or campylobacteriosis or Salmonella or salmonellosis or Vibrio"^ or "£. coli" or escherichia or edwardsiella OR erysipelothrix OR streptococcus)) or((fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or^^almon or salmonid or salmonids or finfish or lobster or crab) and ((resistance or resistant or residue or residues or use or usage) AND (drug or antimicrobial* or "anti microbial" or "anti microbials" or anti-microbial* or antibiotic* or "anti biotic" or "anti biotics" or anti-biotic* or antibacterial* or "anti bacterial" or "anti bacterials" or anti-bacterial* or biosecurity or effluent))) or((fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or salmon or salmonid or salmonids or finfish or lobster or crab) and ((resistance or resistant or residue or residues or use or usage or treatment*) AND (sulphonamide OR sulfadiazine OR trimethoprim OR sulfamethoxazole OR sulfisoxazole OR sulfamerazine OR sulfamethazine OR sulfadimethoxine OR sulfamonomethoxine OR chloramphenicol OR chloramine OR novobiocin OR "nalidixic acid" OR ormetoprim OR spiramycin OR florfenicol OR tetracycline OR oxytetracycline OR doxycycline OR chlortetracycline OR penicillin OR ampicillin))) or((fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or salmon or salmonid or salmonids or finfish or lobster or crab) and ((resistance or resistant or residue or residues or use or usage or treatment*) AND (amoxicillin OR fluoroquinolone OR enrofloxacin OR "oxalinic acid" OR flumequine OR nitrofuran OR nifiirpirinol OR nitrofurazone OR furazolidone OR virginiamycin OR trifluralin OR neomycin OR macrolides OR erythromycin OR tylosin OR streptomycin OR dihydrostreptomycin OR nystatin OR antifungal OR cephalosporins OR rifampicin OR aminoglycosides OR gentamicin OR kanamycin OR isoniazid OR griseofulvin))) or((fish or aquaculture or aquafarm* or fishfarm* or aquarium* or seafood or shellfish or crustacean* or mollusk* or mollusc* or oyster* or clams or shrimp or mussel* or trout or salmon or salmonid or salmonids or finfish or lobster or crab) and (malachite green or leucomalachite green or acriflavine or methylene blue))

189 APPENDIX 3.3. List of search verification references.

Reference List:

L A.M. Hatha, G. Vivekanandan. 2005. Antibiotic Resistance of Aeromonas Hydrophila from Farmed Fish and Wild Caught Fish. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfb.dist.unige.it/Asiaresist/workshop.htm 2. Abdel-Tawwab, M., Abdel-Rahman, A. M., and Ismael, N. E. M. 2008. Evaluation of commercial live bakers' yeast, Saccharomyces cerevisiae as a growth and immunity promoter for Fry Nile tilapia, Oreochromis niloticus (L.) challenged in situ With Aeromonas hydrophila. Aquaculture. 280:185-189. 3. Akinbowale, O. L., Peng, H., and Barton, M. D. 2007. Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J Appl Microbiol. 103:2016-25. 4. Aoki, T., Takami, K., and Kitao, T. 90. Drug resistance in a non-hemolytic Streptococcus sp. isolated from cultured yellowtail Seriola quinqueradiata.. Diseases of aquatic organisms. 8: 171-177. 5. Apama Chaudhari, M. Viji I. Rakkaiah and S. C. Mukheqee . 2005. Development of a Per Based Diagnostic Probe for the Fish Pathogen Edwardsiella Tarda. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 6. Arias, C. R., Garay, E., and Aznar, R. 95. Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water. Appl Environ Microbiol. 61:3476-8. 7. Atanassova Viktoria, Reich Felix, and Klein Gunter 2008. Research Note Microbiological Quality of Sushi from Sushi Bars and Retailers. Journal of food protection. 71:860-864. 8. Bachrach, G., Zlotkin, A., Hurvitz, A., Evans, D. L., and Eldar, A. 2001. Recovery of Streptococcus iniae from diseased fish previously vaccinated with a streptococcus vaccine. Appl Environ Microbiol. 67:3756-8. 9. Backhaus, T. and Grimme, L. H. 99. The toxicity of antibiotic agents to the luminescent bacterium Vibrio fischeri. Chemosphere. 38:3291-301. 10. Bjoerklund, H. V., Raabergh, C. M. I., and Bylund, G. 91. Residues of oxolinic acid and oxytetracycline in fish and sediments from fish farms.. Aquaculture. 97: 85-96. 1991. 11. Celia R. Lavilla-Pitogo, Leobert D. de la Pena and Milagros R. Paner. 2005. Qualitative and Quantitative Comparison of Bacterial Flora Associated with Hatcheryreared and Wild-caught Shrimp Postlarvae. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 190 12. Channarong Rodkhum, Supawadee Na-pompet Anan Jongthaleong Ikuo Hirono and and Takashi Aoki.2005. Molecular Detection and Confirmation of Fluoroquinolone-resistant mutation in GyrA. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 13. Channarong Rodkhum, Takashi Maki Ikuo Hirono and Takashi Aoki.2005. Nucleotide Sequences of gyrA, gyrB, parC and parE of Vibrio anguillarum. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 14. Chen, M. F., Henry-Ford, D., Kumlin, M. E., Key, M. L., Light, T. S., Cox, W. T., and Modin, J. C. 94. Distribution of Edwardsiella ictaluri in California. Journal of aquatic animal health. 6:234-241. 15. Colom, A., Diamant, A., Eldar, A., Kvitt, H., and Zlotkin, A. 2002. Streptococcus iniae infections in Red Sea cage-cultured and wild fishes. Dis Aquat Organ. 49:165-70. 16. Coyne, R., Hiney, M., O'Connor, B., Kerry, J., Cazabon, D., and Smith, P. 94. Concentration and persistence of oxytetracycline in sediments under a marine salmon farm. Aquaculture.. 123: 31-42. 1994. 17. Dowling, G., Mulder, P. P., Duffy, C., Regan, L., and Smyth, M. R. 2007. Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography-tandem mass spectrometry. Anal Chim Acta. 586:411-9. 18. E.A. Tendencia and C.R. Lavilla-Pitogo. 2005. Antimicrobial Resistance in Bacteria Isolated from Aquaculture Environments in the Philippines. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 19. Eldar, A. and Ghittino, C. 99. Lactococcus garvieae and Streptococcus iniae infections in rainbow trout Oncorhynchus mykiss: similar, but different diseases. Dis Aquat Organ. 36:227-31. 20. Eldar, A., Horovitcz, A., and Bercovier, H. 97. Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout. Vet Immunol Immunopathol. 56:175-83. 21. Eldar, A., Perl, S., Frelier, P. F., and Bercovier, H. 99. Red drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection. Dis Aquat Organ. 36:121-7. 22. Eleonor A. Tendencia. Two methods to investigate antibiotic resistance of bacteria from shrimp ponds. Aquaculture. 195: 193-204 23. Fidalgo, S. G., Wang, Q., and Riley, T. V. 2000. Comparison of methods for detection of Erysipelothrix spp. and their distribution in some Ausfralasian seafoods. Appl Environ Microbiol. 66:2066-70. 24. Furushita, M., Shiba, T., Maeda, T., Yahata, M., Kaneoka, A., Takahashi, Y., Torii, K., Hasegawa, T., and Ohta, M. 2003. Similarity of tetracycline resistance

191 genes isolated from fish farm bacteria to those from clinical isolates. Appl Environ Microbiol. 69:5336-42. 25. Graeslund, S., Holmstroem, K., and Wahlstroem, A. 2003. A field survey of chemicals and biological products used in shrimp farming. Marine pollution bulletin. 46:81-90. 26. Greger, E. and Goodrich, T. 99. Vaccine development for winter ulcer disease. Vibrio viscosus, in Atlantic salmon, Salmo salar L.. Journal of fish diseases. 22:193-199. 27. Guglielmetti, E., Korhonen, J. M., Heikkinen, J., Morelli, L., and von Wright, A. 2009. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments. FEMS Microbiol Lett. 293:28- 34. 28. Ha, T. A. and Pham, T. Y. 2006. Study of Salmonella, Campylobacter, and Escherichia coli contamination in raw food available in factories, schools, and hospital canteens in Hanoi, Vietnam. Ann N Y Acad Sci. 1081:262-5. 29. Hatha, M., Vivekanandhan, A. A, Joice, G. J., and Christol 2005. Antibiotic resistance pattern of motile aeromonads from farm raised fresh water fish. Int J Food Microbiol. 98:131-4. 30. Heinitz, M. L., Ruble, R. D., Wagner, D. E., and Tatini, S. R. 2000. Incidence of Salmonella in fish and seafood. J Food Prot. 63:579-92. 31. Inglis, v., Frerichs, G. N., Millar, S. D., and Richards, R. H. 91. Antibiotic resistance of Aeromonas salmonicida isolated from Atlantic salmon, Salmo salar L., in Scotland. Journal of fish diseases. 14:353-358. 32. K.L. Bartie, G. Huys J. Swings D. T. H. Oanh N. T. Phuong M. Shariff F. M. Yusoff T. and Somsiri, S. Chinabut S. Bertone M. Giacomini A. J. Teale. 2005. Hazard Analysis Of Antimicrobial Resistance Associated With Asian Aquaculture. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 33. K M. Mujeeb Rahiman, Abirosh Chandran M. L. Sreenath M. Manoj and A. M. Hatha. 2005. Antibiotic resistance pattern of heterotrophic bacteria of the water and sediment samples collected from the natural habitat and traditional rice-polder culture environment of Macrobrachium rosenbergii. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 34. Kawanishi, M., Kojima, A., Ishihara, K., Esaki, H., Kijima, M., Takahashi, T., Suzuki, S., and Tamura, Y. 2005. Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, ambeijack and kingfish) in Japan. Lett Appl Microbiol. 40:322-8. 35. Koonse, B., Burkhardt, W. 3rd, Chirtel, S., and Hoskin, G. P. 2005. Salmonella and the sanitary quality of aquacultured shrimp. J Food Prot. 68:2527-32. 36. L. Brandi, A. Fabbretti M. Di Stefano A. Lazzarini M. Abbondi and C. O. Gualerzi. 2005. Mechanism of action of two newly discovered antibiotics.

192 Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 37. L. Grisez . 2005.Alternative Options to Antibiotics. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 38. L. Ruangpan and T. Chaweepack. 2005. Monitory on Drug Resistance of Vibrio Bacteria in Shrimp Farm. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 39. Le, T. X. and Munekage, Y. 2004. Residues of selected antibiotics in water and mud from shrimp ponds in mangrove areas in Viet Nam. Mar Pollut Bull. 49:922- 9. 40. Li, P. and Gatlin, D. M. Ill* 2004. Dietary brewers yeast and the prebiotic Grobiotic registered AE influence growth performance, immune responses and resistance of hybrid striped bass (Morone chrysopsM. saxatilis) to Streptococcus iniae infection. Aquaculture. 231:445-456. 41. Liu, F., Liu, Y., Li, F., Dong, B., and Xiang, J. 2005. Molecular cloning and expression profile of putative antilipopolysaccharide factor in Chinese shrimp(Fenneropenaeus chinensis). Mar Biotechnol (NY). 7:600-8. 42. Lu Tongyan, Yang Yuhui, Xu Lianwei, Zhao Jiwei, and Sun Dajiang 2004. Acute toxicity of danofloxacin in Amur sturgeon and the body residue. Journal of fishery sciences of china/zhongguo shuichan kexue. 11:542-548. 43. M. A. T. Siringan, H. B. Perdigon L. Pagaduan and M. C. A. De Unghia. 2005. Phenotypic and PCR-based detection of Aeromonas hyrophila in Oreochromis niloticus. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 44. Mahapatra, K. D., Gjerde, B., Sahoo, P. K., Saha, J. N., Barat, A., Sahoo, M., Mohanty, B. R., Odegard, J., Rye, M., and Salte, R. 2008. Genetic variations in survival of rohu carp (Labeo rohita, Hamilton) after Aeromonas hydrophila infection in challenge tests. Aquaculture. 279:29-34. 45. Maji, S., Mali, P., and Joardar, S. N. 2006. Diagnostic potentiality of fractionated antigen of Aeromonas hydrophila isolated from gold fish, Carassius auratus (Linn.). Indian journal of fisheries. 53:13-17. 46. Mason, B. W., Williams, N., Salmon, R. L., Lewis, A., Price, J., Johnston, K. M., and Trott, R. M. 2001. Outbreak of Salmonella Indiana associated with egg mayonnaise sandwiches at an acute NHS hospital. Commun Dis Public Health. 4:300-4. 47. Muratori, M., De Oliveira, A., Ribeiro, L., Leite, R., Costa, A., and Da Silva, M. 2000. Edwardsiella tarda isolated in integrated fish farming. Aquaculture research. 31:481-483. 48. Nguyen, H. T. and Kanai, K. 99. Selective agars for the isolation of Streptococcus iniae from Japanese flounder, Paralichthys olivaceus, and its cultural environment. J Appl Microbiol. 86:769-76.

193 49. Nutchamart Tipmongkolsilp , Lila Ruangpan Pischa Lusanandana Janenuj Wongtavatchai. 2005. In Vitro Minimum Inhibitory Concentrations of Florfenicol and Chloramphenicol against Clinical Vibrio Isolates from Black Tiger Shrimp (Penaeus monodon). Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 50. Nutchamart Tipmongkolsilp, Yanin Limpanon Monton Lertworapreecha Janenuj and Wongtavatchai. 2005. Surveillance of Antimicrobial Resistance in Food - borne Vibrios from Black Tiger. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 51. P.T Van. 2005. Current status of aquatic veterinary drugs usage for Aquaculture in Vietnam. Conference Proceeding of the ASIARESIST Project. Available at: http://www.medinfo.dist.unige.it/Asiaresist/workshop.htm. 52. P.V.S. Panawala, T. G. Wijewardana P. Abeynayake. 2005. 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195 APPENDIX 3.4. Definitions for Reviewers

Selected zoonotic bad teria Genus Spp. Aeromonas All Edwardsiella Tarda, Ictaluri Erysipelothrix Rhusiopathiae Escherichia coli All (pathogenic and non-pathogenic) Mycobacteria Marinum, Shottsi, Ulcerans, Peregrinum, Scrofulaceum, Fortuitum and Chelonae Salmonella All Streptococcus Iniae Vibrio All Any bacteria genus/strain for which AMR is reported

Selected Antimicrobials Residue Specific Type Antimicrobi Aditoprim, Amikacin, Ampicillin, Amoxicillin, Aquaflor al/ Bactrim, Baquiloprim Antibiotic Carbenicillin,Cephapirin,Cephalexin,Cefoxitin,Ceftiofur,Chloramp henicol,Chlor-tetracycline,Cloxacillin,Ciprofloxacin Diflunisal, Dihidrostreptomycin, Doxycycline Enrofloxacin, Erythromycin Florfenicol, Fosfomycin, Furazolidone Gentamicin ,Josamycin,Lincomycin, Nalidixic acid,Neomycin, Nifurpirinol, Nitrofurazone, Novobiocin, Nystatin, Ormetoprim, Oxolinic acid,Oxytetracycline,Penicillin G, Phosphomycin, Phosphonomycin Quinupristin/Dalfopristin,Kanamycin,Rifampin,Romet-30, Septra, Spiramycin, Sulfadiazine, Sulfamethazine, Sulfameter, Sulfaquanidine, Sulfaquinoxaline, Sulfapyridine, Sulfadimethoxine, Sulfadoxine, Sulfamethazine, Sulfamerazine, Sulfisoxazole Thiamphenicol,Tobicillin,T ribrissen,T rimethoprim,T rifluraline,T y1 osin Virginiamycin

3. Selected Dyes Residue Type Specific Dyes Malachite green, Leucomalachite green, Acriflavine, Methylene blue

196 4. Definitions for the Aquaculture Systematic Review

Term Definition Active monitoring The performance and analysis of routine measurements, aimed at detecting changes in the environment or health status of populations. The information is gathered according to a predefined sampling strategy. Active surveillance Surveillance is the systematic ongoing collection, collation and analysis of data gathered according to a predefined sampling strateev in coni unction with an action plan that is triggered bv set goals and threshold values. Antimicrobial use for Also sometimes called non-therapeutic use. Use of growth promotion antimicrobials to stimulate the growth of animal or enhance feed conversion. Antimicrobial use to Use of antimicrobials before the onset of a disease, to prevent the prevent disease apparition of clinical signs. Preventive use may be at therapeutic (prophylaxis) or sub therapeutic levels. Antimicrobial use to Use of antimicrobials at therapeutic levels in order to treat treat disease (therapy) diseases. Antimicrobial/antibio When an antimicrobial substance, or agent, is no longer effective tic resistance/AMR in killing or inhibiting the growth of a particular microorganism Antimicrobial/antibio use of antimicrobials tic use Aquaculture The farming of marine and fi-eshwater fish or shellfish for human consumption. Within the context of this SR, aquaculture can also include culture of aquarium or ornamental fish. Aquatic bacteria Bacteria found in the aquatic environment or on aquatic organisms or animals. Aquatic environment Here refers to sediments, fresh water, salt water, drinking water, aquaculture waste water, sedimentation basin. This should include all water samples near farms, hospitals, processing plants, factories, etc. It should also include benthic samples especially under or near aquaculture sites and seaweed. Association When examining two variables, there exists a relationship between the value of one to the value of the other. Bacterial culture Growth of bacteria in selected agar, broth, or other media Consumption of raw Consumption of seafood that was not cooked or not cooked or insufficiently enough to destroy bacteria. cooked seafood Cross-contamination Transfer of bacteria from seafood to non-seafood product with raw seafood through direct or indirect (knives, cutting boards, counter tops) contact.

197 Crustacean shellfish Crustacean shellfish (eg. Crabs, lobsters, shrimps, prawns (freshwater), crayfish) example Homarus americanus (lobster), Pandalus spp., Crangon spp. (Shrimp), Macrobrachium rosenbergii (prawn), Procambarus clarkii (crayfish), Procambarus sp. Disease in humans A pathological condition of a part, organ, or system resulting from various causes, such as infection, genetic defect, or environmental stress, and characterized by an identifiable group of signs or symptoms (www.univadis.ca) Environment Here refers to sediments, fresh water, salt water, drinking water, aquaculture waste water, sedimentation basin. This should include all water samples near farms, hospitals, processing plants, factories, etc. It should also include benthic samples especially under or near aquaculture sites and seaweed. Export Commodities (goods or services) sold to a foreign country (http://www.google.coni/search?hl=en&rls=com.niicrosoft%3Ae n-ca&a=define%3AexDort) Exposure to pet Contact with pet fish, aquarium fish, ornamental fish (ornamental) fish Farmed crustacean Crustacean shellfish (eg. Crabs, lobsters, shrimps, prawns shellfish (freshwater), crayfish) raised in aquaculture settings (cages, tanks, basins, ponds, etc) example Homarus americanus (lobster), Pandalus spp., Crangon spp. (Shrimp), Macrobrachium rosenbergii (prawn), Procambarus clarkii (crayfish), Procambarus sp. Farmed fresh water Examples include; carp, tilapia, catfish, striped bass, sturgeon, fish yellow perch, walleye etc. Example: Mylopharyngodon piceus (carp), Oreochromis spp., Sarotherodon spp., Tilapia spp. (Nile tilapia, Mozambique tilapia. Blue tilapia, St. Peter's fish), Ictalurus punctatus (Farmed catfish, US farmed catfish), Morone chrysops X Morone saxatilis (striped bass) Farmed marine fish Examples include; flounder(winter flounder, Pseuodpleuronectes americanus), Olive flounder (Paralichythys olivaceus),, atlantic cod,( Gadus morhuas), halibut (Atlantic halibut, Hippoglossus hippoglossus), seabass (European Sea ^ass, Dicentrarchus labrax), grouper (Epinephelus spp. Ex.Brown-spptted, Epinephelus tauvina, yellowtailn(Serioloa spp.), perch (ex. Silver perch, (Bidyanus, bidyanus), tuna (Thunnus sp) etc.

198 Farmed molluscs Molluscs (eg. Clams, oysters, scallops, mussels, snails, octopus, shellfish squids, etc.) raised in aquaculture settings (cages, tanks, basins, ponds, etc) example Tapes japonica, Meretrix lusoria, Anadara spp.,Venerupis decussates (cockles, arc shells, Manila clam, short necked clam, hard clam, grooved carpet shell clam), Crassostrea gigas (Pacific oysters), Tiostrea chilensis or Ostrea chilensis or Tiostrea lutaria (Bluff oysters), Argopecten irradians (Bay Scallop), Placopecten magellanicus (giant sea scallop);; Pecten yessoensis (Japanese Scallop), Mytiluse edulis; M. galloprovincialis; Pema viridis; P. canaliculus (Sea mussel. Blue mussel; Mediterranean mussel; Green mussel; New Zealand mussel). Octopus vulgaris , Dosidicus gigas (giant squid) Farmed salmon Salmon raised in aquaculture settings (cages, tanks, basins, ponds, etc) example Salmo salar (Atlantic salmon). Farmed shellfish Includes farmed crustacean s and molluscs Farmed shrimp Shrimp raised in aquaculture settings (cages, tanks, basins, ponds, etc) example Penaeus monodon,, P.vannam, P. Chinensis, , Pandalus spp., Crangon spp. Farmed trout Trout raised in aquaculture settings (cages, tanks, basins, ponds, etc) example Oncorhynchus mykiss (Rainbow trout. Coastal rainbow trout, Kamloops trout, Redband trout, Columbia River redband trout. Eagle Lake trout. Kern River trout, Shasta trout, Sacramento redband trout, Kamchatkan trout, Rainbow trout (FDA approved), Steelhead trout (FDA approved)). Fish Fish are any of various cold-blooded, aquatic vertebrates, having gills, commonly fins, and typically an elongated body covered with scales, (e.g., trout, salmon, bass see examples under farmed fish) Fresh product Product that remains in its original condition (ie; has not been frozen, cool

199 Immunology tests Tests based on immunological response to infection eg serotyping - immunological identification of surface antigens of a pathogen; fluorescent antibody staining, direct ELISA, immunoperoxidase staining, immunohistochemistry, immunocytochemistry etc. Import / Imported Commodities (goods or services) bought from a foreign country products fhttp://www.gooele.com/search?hl=en&rls=com.microsoft:en- ca&defl=en&q=define:import&sa=X&oi=elossarv definition&c t=title) Indicator bacteria Also commensal bacteria. Indicator bacteria are generally not dangerous to health but may be used as an indicator of the level of contamination e.g., fecal coliforms are commonly used. Examples of such bacteria are E. coli. Fecal coliforms (Klebsiella sp., Enterobacter sp. etc.), Enterococcus sp., (httD://en.wikiDedia.or2/wiki/Indicator bacteria) Interventions Intervention is an action taken to reduce prevalence, contamination, or colonisation with a pathogen. For example, an intervention at the farm level could be vaccination; an intervention at the processing level could be lactic acid wash of the carcass. In-vitro In-vitro studies are studies performed in laboratory settings. To qualify for this SR, in-vitro studies must have been conducted on field-type sampling unit such as a sample of fish, a sample of water. We exclude in-vitro studies studying reference strains or any samples not normally found in the field Literature review A survey of publications in a specific field of study or related to a particular line of research. Generally does not have material and methods for how research was identified, evaluared and synthesized. (new.wales.gov.uk/about/aboutresearch/social/glossarv/) Metaphylaxis The timely mass medication of a group of animals to eliminate or minimize an expected outbreak of disease. Can sometimes be included under preventive use. Molecular tests Eg. Nucleic acid hybridization = synthetic nucleic acid probes specific for a pathogen are applied either to prepared clinical specimens, or to extracted genetic material. PCR (polymerase chain reaction). Southern blot. Northern blot, RFLP, PFGE

200 Molluscs shellfish Molluscs (eg. Clams, oysters, scallops, mussels, snails, octopus, squids, etc.) example Tapes japonica, Meretrix lusoria, Anadara spp.,Venerupis decussates (cockles, arc shells, Manila clam, short necked clam, hard clam, grooved carpet shell clam), Crassostrea gigas (Pacific oysters), Tiostrea chilensis or Ostrea chilensis or Tiostrea lutaria (Bluff oysters), Argopecten irradians (Bay Scallop), Placopecten magellanicus (giant sea scallop);; Pecten yessoensis (Japanese Scallop), Mytiluse edulis; M. galloprovincialis; Pema viridis; P. canaliculus (Sea mussel, Blue mussel; Mediterranean mussel; Green mussel; New Zealand mussel). Octopus vulgaris , Dosidicus gigas (giant squid) North America Canada and the United States Occupational Exposure to potentially harmful chemical, physical, or biological exposure agents during the course of work or as a result of one's occupation. Outbreak A disease outbreak is the occurrence of cases of disease in excess of what would normally be expected in a defined community, geographical area or season. An outbreak may occur in a restricted geographical area, or may extend over several countries. It may last for a few days or weeks, or for several years. (WHO, http://www.who.int/topics/disease outbreaks/en/) Ornamental fish Any fish/shellfish (mollusk or crustacean) kept in an aquarium and intended for display rather than consumption. Passive monitoring The analysis of measurements, obtained through voluntary sample submissions or for another purpose, aimed at detecting changes in the environment or health status of populations. The information is NOT gathered according to a predefined sampling strategy. Passive surveillance The analysis of measurements, obtained through voluntary sample submissions or for another purpose, aimed at detecting changes in the environment or health status of populations for which there is a threshold and action plan for control in place. A predefined sampling strategy is not used. Pathogenic bacteria Bacteria that cause infectious diseases. (http://en.wikipedia.org/wiki/Pathogenic_bacteria) Phenotyping The expression of antimicrobial resistance as measured by antimicrobial sensitivity tests (agar dilution, broth dilution, disc diffiision). Pharmacokinetics Pharmacokinetics study is the investigation into the fate of study pharmacological substances in the body, as their absorption, distribution, metabolism, and elimination. Post-cooked improper Eg. Using the same knife of cutting board to cut (or the same food handling plate to serve) cooked and raw meat.

201 Precision The ability of the test to produce consistent results when repeated under the same conditions and interpreted without knowing the first test's results. Commonly used measures of precision are repeatability and reproducibility. (Greiner et al. 2003). Prevalence (or Prevalence refers to the total number of sampling units infected concentrations) with a pathogen, in a population at risk, in a particular geographic area, at one specified time, or during a given period. Primary research Experiments, investigations, or tests carried out to acquire data observed or collected directly from first-hand experience. Processing The act of taking something through an established and usually routine set of procedures to convert it fi*om one form to another, as a manufacturing procedure, such as processing milk into cheese. (Wikipedia) Quarantine A period of time during which a vehicle, person, or material suspected of carrying a contagious disease is detained at a port of entry under enforced isolation to prevent disease from entering a country Ready to eat product Product that can be eaten without being further cooked. Residues A chemical residue is what remains of the chemical at a particular time and can occur as the chemical itself or as breakdown products Resistance genes Genes in a microorganism which confer resistance to antibiotics, (e.g: production of enzymes which destroy the antibiotic, prevent the antibiotic from entering the microorganism by coding for surface proteins, or by mutating the antibiotics target to an unrecognisable form, (http://cancerweb.ncl.ac.uk/cgi- bin/omd?antibiotic+resistance+gene) Resistance transfer The transfer of resistance genes or resistance factors firom one microorganism to another by conjugation, transformation or transduction. Resistant bacteria Bacteria that have acquired or that naturally possesses resistance genes and whose growth is not inhibited when in presence of a given antimicrobial.. Risk factors An exposure that affects the likelihood of a sampling unit experiencing the outcome of interest, eg recycling of excreta Seafood Seafood is any fish or shellfish fi-om the sea used for food Seafood / Retail Seafood is any fish and shellfish (including molluscs and seafood crustaceans) (excluding seaweed) that is served as food or is suitable for eating. In North America the term seafood is also applied to similar animals from fi-esh water and all edible aquatic animals are collectively referred to as seafood. (http://en.wikiDedia.ore/wiki/Seafood) Seaweed are not included in this study.

202 Seafood-aquaculture The act of producing/raising fish or shellfish (including molluscs production / Primary and crustaceans) in a controlled or semi-controlled environment aquaculture (e.g., cages / lagoons etc.). production Selected zoonotic Microorganisms that may be transmitted from animals, both wild pathogens and domestic, to humans or from humans to animals. Within the context of this study, selected pathogens are: Aeromonas sp., Edwardsiella sp., Escherichia coli, Mycobacterium sp.. Salmonella, Streptococcus sp.. Vibrio sp.. Sensitivity The probability of a positive test result given the disease is present. (Greiner et al. 2003). Shellfish Includes crustaceans and molluscs South and Central Antigua and Barbuda, Bahamas , Barbados, Belize, Costa Rica, America Cuba, Dominica, Dominican Republic, El Salvador, Grenada, Guatemala, Haiti, Honduras, Jamaica, Mexico, Nicaragua, Panama, Saint Kitts and Nevis, Saint Lucia, Saint Vincent and the Grenadines, Trinidad and Tobago, Argentina, Aruba, Bolivia, Brazil, Chile, Colombia, Ecuador, Falkland Islands, French Guiana, Guyana, Netherlands Antilles, Paraguay, Peru, Suriname, Uruguay, Venezuela Specificity The probability of a negative test result given the disease is not present. (Greiner et al. 2003). Sporadic disease occurring at irregular intervals or only in a few places; scattered or isolated (http://www.encyclopedia.eom/doc/10999- sporadic.html) Sufficient raw data Raw data provided for 2x2 tables so Se and Sp can be calculated. Transport Moving something or somebody around; usually over long distances Unspecified Use for antimicrobials without specified indication. antimicrobial use Wild caught fish Fish captured by commercial or sport fishing. Wild caught shellfish Shellfish captured by commercial fishing.

203 APPENDIX 3.5. Relevance Screening Tool I

Relevance Tool for Initial Screening of Abstracts

RefID Reviewer: Relevance CrittrisJAbstract Level Please see the 'Definition' document for explanations or examples for certain terms or expressions.

1. Does the study investigate the following under field conditions'? (Please check all that apply) Prevalence^, risk factors^ or interventions'^ for selected zoonotic bacteria *(list provided below). Prevalence^, risk factors^ or interventions'* for antimicrobial/antibiotic resistance in ANY bacteria^ Prevalence^ of antimicrobial/antibiotic use in aquaculture Prevalence^, risk factors^ or interventions'^for antimicrobial/antibiotic drug residues^*(examples provided) Prevalence^, risk factors^ or interventions'^for dye residues^*(examples provided) Aquaculture/Seafood related outbreak^ or sporadic^ disease in humans Seafood related human consumption report Seafood related production volume or import/export report^ '*^ Can't tell, but likely one of above. None of the above (exclude)

2. Does the study investigate seafood'' (fish and shellfish), ornamental fish, or human exposure to aquaculture'^ (including any of the above) through occupation, consumption, or leisure? Yes'^ No (exclude) Can't tell

3. What type of publication is this? (Please check all that apply). Primary research'^ Monitoring or surveillance report^ Other report^ 204 Literature review Can't tell (neutral) None of the above (includes commentaries, news, risk assessments and predictive models etc.) (exclusion)

Relevant Abstract Decision If both reviewers answer Yes to question 1 (any of the options 1-8), and question 2 and question 3 (any of the options 1-3), then the abstract will be included in RS II for relevance confirmation and classification (full article will be obtained). The logic will be incorporated into the SRS system and will happen automatically.

* Selected zoonotic bacteria; Aeromonas (any species), Campylobacter spp, Edwardsiella (tarda, ictaluri), Erysipelothrix rhusiopathie, Escherichia coli (any serotype including generic or non-type specific). Salmonella (any serovar). Streptococcus (iniae). Vibrio spp.

•Examples of antimicrobial/antibiotic drug residues are (vou can include antimicrobials not listed here): Aditoprim, Amikacin, Ampicillin, Amoxicillin, Aquaflor Bactrim, Baquiloprim Carbenicillin,Cephapirin,Cephalexin,Cefoxitin,Ceftiofur,Chloramphenicol,Chlor- tetracycline,Cloxacillin,Ciprofloxacin Diflunisal, Dihidrostreptomycin, Doxycycline Enrofloxacin, Erythromycin Florfenicol, Fosfomycin, Furazolidone Gentamicin, Josamycin, Lincomycin, Nalidixic acid. Neomycin, Nifurpirinol, Nitrofurazone, Novobiocin, Nystatin, Ormetoprim, Oxolinic acid,Oxytetracycline,Penicillin G, Phosphomycin, Phosphonomycin Quinupristin/Dalfopristin, Kanamycin, Rifampin, Romet-30, Sarafloxacin, Septra, Spiramycin, Sulfadiazine, Sulfamethazine, Sulfameter, Sulfaquanidine, Sulfaquinoxaline, Sulfapyridme, Sulfadimethoxine, Sulfadoxine, Sulfamerazine, Sulfamethoxazole, Sulfisoxazole Thiamphenicol, Tobicillin, Tribrissen, Trimethoprim, Trifluraline, Tylosin Virginiamycin

* Selected dyes residues: Malachite green, leucomalachite green, acriflavine, methylene blue

Definitions ^Field Conditions: For the purpose of this review 'field conditions', for 'prevalence and risk factor studies', mean that samples originate from aquatic wild, farmed or ornamental populations (e.g., groups of fish eggs, fish, shellfish or seafood) that are raised or produced under commercial, field conditions (commercial hatcheries, farms, processing plants, retail stores and aquarium for ornamental fish) or under commercial conditions on a research farm (large ponds and tanks). 'Field conditions' for 'intervention studies' mean that interventions were applied on live aquatic wild, farmed and/or ornamental populations under strict commercial conditions, or in large ponds and/or tanks on research farms or in aquariums for ornamental fish. ^Prevalence: any study that reports frequency, occurrence, distribution or presence of selected zoonotic bacteria, antibiotic resistance, antibiotic use, and residues (antibiotic or dies) in aquatic wild, farmed or ornamental population samples collected under 'field conditions'. The sampling might occur at one or multiple stages of fish production (from hatchery to retail). Ideally, the abstract should indicate numerator/denominator, population, and the sampling point (e.g., 1,200 samples of salmon from 120

205 salmon farms; 10% were positive for Vibrio spp) or imply that this information is available in the paper. If it is really not clear, 'can't tell' option can be selected if you think this information may be available in the paper. ^Risk factor: any study that investigates associations between prevalence of selected zoonotic bacteria or antibiotic resistance or antibiotic use or residues (antibiotic and dies) and one or more exposures (potential risk factors) such as farm management (use of certain feeding or bio-security practices). These studies are observational studies and must be conducted under field, commercial conditions or in large ponds and tanks on research farms or for ornamental fish in aquariums. ^Intervention trials: only trials in which interventions are applied on live wild, farmed or ornamental populations that are raised or processed under strict commercial conditions, or in large ponds and tanks on research farms or for ornamental fish in aquariums, should be included. In a challenge trial, the researcher will artificially infect fish or seafood with a bacterium and than measure the effectiveness of interventions. In a controlled trial the effectiveness of intervention is measured using natural course of disease or infection. If intervention is evaluated using bacterial isolates at the petri-dish level, such study should be excluded. ^Residue (antimicrobial and dies) related studies examine the remainder of a substance (AM or dye) in samples of aquatic wild, farmed or ornamental populations (e.g., groups of fish eggs, fish, shellfish or seafood) that are raised or produced under commercial, field conditions (commercial hatcheries, farms, processing plants, retail stores and aquarium for ornamental fish). Pharmacokinetics studies investigating the fate, absorption, distribution, elimination and withdrawal times of pharmacological substances should be excluded. ^Molecular epidemiology studies that measure transfer of antimicrobial resistance within and between aquatic wild, farmed, ornamental populations and humans through the recovery of bacteria fi*om samples collected under field conditions should be included under question I option 3 (prevalence, risk factors or interventions for antibiotic resistance in any bacteria). Studies that only investigate genetic profiles or bacteria strain relatedness of selected zoonotic bacteria, without an indication of necessary prevalence data (please see example under 2) should be excluded. Example, A study was performed on 20 strains of Vibrio and the paper states 80% of these strains were genetically related. However we cannot derive how fi*equently these bacteria were observed in the studied population. This study should be excluded. ^ Disease outbreak is the occurrence of two or more cases of a similar illness with clustering patterns in time and space. Sporadic disease refers to I or more cases of disease that occur in a scattered or unpredictable or unconnectable manner. For this review, these refer to all human studies related to exposure to aquaculture for any reason e.g., occupational, consumption, leisure etc. ^Reports are non-peer reviewed publications that contain original data collected via routine passive or active data collection systems and are typically published by government agencies. ^Human consumption report will likely be a government document related to how much seafood was consumed by a population in a certain period of time. However, such information might be published in the peer reviewed literature as well and should be classified as a consumption report if this happens. Seafood related production reports or import / export reports are often government reports about how much seafood was produced by a geographic area (country, province etc) in a certain period of time or was imported into or exported out of a country in a period of time. We are not interested in production practices under this category; all relevant research should fall into the first 5 options. Seafood is any fM or shellfish from the sea used for food and for the purposes of this review we are interested in studies that examine aquaculture and wild seafood from hatchery to consumption. Fish are any of various cold-blooded, aquatic vertebrates, having gills, commonly fins, and typically an elongated body covered with scales. Shellfish includes crustaceans (eg. crabs, lobsters, shrimps, prawns, crayfish) and molluscs (eg. Clams, oysters, scallops, mussels, snails, octopus, squids, etc) For more examples see definitions.

206 Aquaculture is the farming of marine and freshwater fish or shellfish for human consumption and includes all stages of production from the hatchery to consumption. Within the context of this SR, aquaculture can also include culture of aquarium or ornamental fish. Studies with environmental samples are included in this review ONLY if they ALSO sample either humans OR aquatic animals (fish / seafood). Primary research: any experiments or planned investigation that is carried out to generate new data and information.

207 APPENDIX 3.6. Relevance Screening Tool II

Relevance Tool for Classification of Abstracts or Full papers (Updated-July 7, 2010)

Relevance Screening Tool II - Relevance confirmation and classification -

RefID Reviewer:

Relevance Criteria/Classification for QA-DE Prioritization/Abstract Only select "Can't tell" as a LAST option when truly unknown from abstract.

Note: Please see 'Guideline' document for explanations or examples for certain terms or expressions.

1. What is the study publication date? 1998-present 1990-1997 before 1990

2. Please indicate which bacteria are investigated in this study. (Please check all that apply.)

Aeromonas (any species) Campylobacter spp. Edwardsiella {tarda, ictaluri) Erysipelothrix rhusiopathiae Escherichia spp (any serotype including generic) Salmonella (any serovar) Streptococcus iniae Vibrio spp. AMR investigated in bacteria not listed above, please specify: Can't tell (not specified in abstract) None of the above.

3. If samples were collected by the investigators, where was the sampling conducted? (Please check all that apply.)

208 Africa Asia Australia and New Zealand Europe Middle East North America (USA and Canada) South and Central America (including Mexico and the Caribbean) Multi-location study (be sure to check all applicable above) Can't tell (not specified in abstract)

4. Please indicate which antibiotic/antimicrobial' classes were investigated in this study. If only the antibiotic/antimicrobial name is provided, use the classification document to find the appropriate family. (Please check all that apply.)

Aminoglycosides Antifungals Arsenicals Cephalosporins and cephamycins Diaminopyrimidines Diaminopyrimidine-sulfonamide combinations Lincosamides Macrolides Nitrofurans Novobiocin Penicillins Phenicol group Quinolones Rifamycins Streptogramins Sulfonamides Tetracyclines Can't tell (specific AM of family is not specified in abstract) Other, please specify (name and/or family): . Antimicrobials were not investigated in this study Disinfectants, please specify (common/commercial name):

5. Please indicate which dyes were investigated in this study. (Please check all that apply.)

Malachite green Leucomalachite green Acriflavine Methylene blue

209 Dyes were not investigated in this study Other, please specify: .

6. What point in the food chain does this study investigate? (Please check all that apply.)

Harvest^ (including aquaculture^ and wild caught"^) Processing^ (transport included) Retail/market products^ (seafood^) Ready-To-Eat products^ Imported products^ (select only if specified in abstract) Public preparation of food"' (e.g., restaurant) Home preparation of food'' Human illness related to aquaculture or seafood exposure'^ Ornamental fish or aquarium fish'^ Quarantine'"^ Can't tell (not specified in abstract) Only aquatic environment'^ Only lab experiment not specific to point in chain'^ Other* (e.g., traditionally produced products), please specify: None of the above

(*These studies will be examined for relevance)

7. Please indicate which fish/shellfish was investigated? (Please check all that apply.)

Finfish'^, please, specify: Crustacean'^, please specify: Mollusk'^, please specify: Ornamental fish, please specify: Other, please specify: Can't tell (not specified in abstract) Fish/Shellfish were not investigated (or diseased fish/shellfish were sampled)

8. For the above species, please indicate whether the samples were farmed or wild caught? (Please check all that apply.)

Farmed^® Wild caught Can't tell (not specified in abstract) Not applicable (e.g., fish/shellfish not sampled, likely human illness or outbreak study)

210 9. What is the field study design? (Please check all that apply.) Experimental^': challenge, randomized controlled trial Observational^^: cross-sectional, cohort, case-control, prevalence survey, outbreak investigation, case report Quasi-experimental^^: 'before and after' study Can't tell (not specified in abstract) Descriptive laboratory-based study^"*: e.g., genotyping This is not a field study^^

10. What is the focus of this study? (Please check all that apply.)

Prevalence (or concentrations) and/or risk factors for selected zoonotic bacteria^^ Prevalence and/or risk factors associated with antimicrobial/antibiotic or dye residues Prevalence and/or risk factors of antimicrobial resistance in any bacteria Prevalence of antimicrobial use in field/commercial setting

Intervention for reducing the prevalence of selected zoonotic bacteria Intervention for reducing selected antimicrobial/antibiotic or dye residues. (Note: study must apply intervention under field or field-like conditions to a sample that represents or mimics the normal field state (e.g., if an intervention is applied to live fish in the field, the experiment should be on live fish))

Association between AMU^^ and AMR^^ AMU and/or AMR in aquaculture as risk factors for human AMR

(Note: These studies must be conducted in a field setting, i.e. they do not just investigate transfer mechanisms or pathogenesis, but sample a defined population for both exposure and outcome)

Outbreak, sporadic disease, or case reports^^ of human illness suspected or confirmed to be due to exposure to aquaculture or seafood

Seafood related human consumption report^'' Seafood related production or import/export report^' Can't tell (not specified in abstract)

*Other, please describe:

(*These studies will be examined for relevance)

The goal of relevance screening tool II is to categorize on bacteria, antimicrobial drug/residue or dye, point in food chain, commodity investigated, study design, and whether it is prevalence, risk factor or intervention study.

211 Reviewer Decision

Exclusion Criteria (To Fail)

Ql: before 1990, Q6: Aquatic environment, lab experiment not specific to point in chain, potentially 'other' (analyzed for relevance), none of the above. Q7: fish / seafood not investigated, Q9: descriptive lab study or not a field study

Notes:

If reviewer(s) select "Can't tell (not specified in abstract)" option for any of Q2 and/or Q3 and/or Q4 and/or Q7, and the article is set to pass according to all other criteria, then refid will enter conflict and the article will have to be procured so "can't tells" can be resolved.

All consequential and inconsequential conflicts must be resolved to pass RS II.

Definitions and Examples:

Antibiotic/antimicrobial^: Any substance that is used to either kill or inhibit the growth of bacteria. For our purposes, bacteria include the selected group listed below (selected zoonotic bacteria). Disinfectants are not considered antibiotics/antimicrobials. The classification document for antimicrobials lists the most common ones and any unlisted ones may be recorded under 'other'.

Harvest^: For the purposes of question 6, farm includes both wild caught seafood AND seafood harvested though aquaculture.

Aquaculture^: The farming of marine and freshwater fish and animals for human consumption (excluding seaweed).

Wild caught^: Any seafood (finfish, mollusk or crustacean) captured by commercial or sport fishing intended for consumption.

Processing^: Techniques involved in transforming raw seafood or further modifying the products for consumption (e.g., scaling of fish, preparation of fillets, packaging). This can be conducted in processing plants or on farms depending on its capabilities. Processing includes any transport from farm to processing plants.

Retail/market products (seafood) Any raw, frozen or prepared seafood sold as goods and is typically available to consumers from a market place or grocery store. Typically, these products require additional preparation.

Seafood^: Seafood is any fish and shellfish (including mollusks and crustaceans) (excluding seaweed) that is served as food or is suitable for eating.

Ready-To-Eat products^: Product that can be eaten without being further cooked.

212 Imported products^: Commodities (goods or services) bought from a foreign country (e.g., ornamental fish or any type of seafood).

Public preparation of food^^: Seafood that has been processed or prepared and is suitable for eating by the general public. It is typically intended for immediate consumption and provided at restaurants or fast food establishments.

Home preparation of fbod^\ Seafood that is processed or prepared in the household.

Seafood exposure^^: Seafood is any fish and shellfish (including mollusks and crustaceans) (excluding seaweed) that is served as food or is suitable for eating. Exposure to seafood can occur either through consumption, production or leisure.

Ornamental fish^^: Any fish/shellfish (mollusk or crustacean) kept in an aquarium and intended for display rather than consumption.

Quarantine Any fish/shellfish that has been isolated (likely due to presence of disease).

Only aquatic environment^^: Samples were taken typically from sediment or water but not from fish/shellfish or humans.

Only lab experiment not specified to point in food chain^^: A laboratory experiment on fish/shellfish samples not specific to a point in food chain (e.g., fish from a laboratory aquarium).

Finfish^^: Examples include; carp, tilapia, catfish, striped bass, sturgeon, yellow perch, walleye, eel etc. Species example: Mylopharyngodon piceus (carp), Oreochromis spp., Sarotherodon spp., Tilapia spp. (Nile tilapia, Mozambique tilapia, Blue tilapia, St. Peter's fish), Ictalurus punctatus (Farmed catfish, US farmed catfish), Morone chrysops X Morone saxatilis (striped bass). Examples include; flounder(winter flounder, Pseuodpleuronectes americanus), Olive flounder (Paralichythys olivaceus),, atlantic cod,( Gadus morhuas), halibut (Atlantic halibut, Hippoglossus hippoglossus), seabass (European Sea Bass, Dicentrarchus labrcDc), grouper (Epinephelus spp. Ex.Brown-spotted, Epinephelus tawina, yellowtailn(Serioloa spp.), perch (ex. Silver perch, Bidyanus, bidyanus), tuna (Thunnus sp), etc.

Crustacean^^: Crustacean shellfish: Examples include; crabs, lobsters, shrimps, prawns (freshwater), crayfish) raised in aquaculture settings (cages, tanks, basins, ponds, etc). Species examples: Homarus americanus (lobster), Pandalus spp., Crangon spp. (Shrimp), Macrobrachium rosenbergii (prawn), Procambarus clarkii (crayfish), Procambarus sp., etc.

Mollusk^^: (eg. Clams, oysters, scallops, mussels, snails, octopus, squids, etc.) raised in aquaculture settings (cages, tanks, basins, ponds, etc). Species examples: Tapes japonica, Meretrix lusoria, Anadara spp.,Venerupis decussates (cockles, arc shells, Manila clam, short necked clam, hard clam, grooved carpet shell clam), Crassostrea gigas (Pacific oysters), Tiostrea chilensis or Ostrea chilensis or Tiostrea lutaria (Bluff oysters), Argopecten irradians (Bay Scallop), Placopecten magellanicus (giant sea scallop); Pecten yessoensis (Japanese Scallop), Mytiluse edulis; M. galloprovincialis; Pema viridis; P. canaliculus (Sea mussel. Blue mussel; Mediterranean mussel; Green mussel; New Zealand mussel). Octopus vulgaris, Dosidicus gigas (giant squid), etc.

Farmed^®: Any seafood that has been produced by farming techniques used in aquaculture (e.g., fish tanks, aquaculture farms, oyster beds).

Experimental study^^: These studies will most likely be challenge or randomized_controlled trial testing interventions for the selected zoonotic pathogens, antimicrobial drug residues or antimicrobial resistance. The samples must come from field conditions (i.e. from aquatic wild, farmed or ornamental populations)

213 and the application of the intervention must be conducted under 'field conditions' (e.g., on a research farm, commercial farm or aquarium for ornamental fish). In a challenge trial the researcher will artificially infect fish or seafood with a bacterium and than measure the effectiveness of interventions. In a controlled trial the effectiveness of intervention is measured using natural course of disease or infection. If intervention is evaluated using bacterial isolates at the petri-dish level, such study should be excluded.

Some examples of interventions include: application of antibiotic/antimicrobial drugs, dietary supplements, feed medication, interval bath treatments to eliminate infection, use of probiotics, intraperitoneal vaccination or other vaccination.

Observational study^^: These studies will most likely measure prevalence or risk factor and may be of a cross-sectional, cohort, case-control, prevalence survey, outbreak investigation or case report design. Prevalence studies will report frequency, occurrence, distribution or presence of selected zoonotic bacteria, antibiotic resistance, antibiotic use, and residues (antibiotic or dies) in aquatic wild, farmed or ornamental population samples collected under 'field conditions'. Risk factor studies investigate associations between prevalence of selected zoonotic bacteria or antibiotic resistance or antibiotic use or residues (antibiotic and dyes) and one or more exposures (potential risk factors) such as farm management (use of certain feeding or bio-security practices). These studies are observational studies and must be conducted under field, commercial conditions or in large ponds and tanks on research farms or for ornamental fish in aquariums.

Quasi-experimental^^: These are typically 'before and after' studies and may be intervention, prevalence or risk factor studies where there is no random assignment of subjects into groups (subjects are grouped by nature). They are typically experimental design without randomization.

Descriptive laboratory-based study^"^: These studies are typically done in the laboratory and involve phylogenetics or genotyping (e.g., PCR, DNA sequencing, DNA probes, hybridization) and other microbiological procedures not intended to measure prevalence, risk factors or interventions. Regardless of whether the samples are field-based, they are to be excluded.

Field conditions^^: For the purpose of this review 'field conditions', for 'prevalence and risk factor studies', mean that samples originate from aquatic wild, farmed or ornamental populations (e.g., groups of fish eggs, fish, shellfish or seafood) that are raised or produced under commercial, field conditions (commercial hatcheries, farms, processing plants, retail stores and aquarium for ornamental fish). 'Field conditions' for 'intervention studies' mean that interventions were applied on live aquatic wild, cultured and ornamental populations under strict commercial conditions, or in large ponds and tanks on research farms or for ornamental fish in aquariums. Clinical samples or samples from diseased fish are not considered field conditions.

Selected zoonotic bacteria^^: Aeromonas (any species), Campylobacter spp, Edwardsiella (Tarda, Ictaluri), Erysipelothrix Rusiopathie, Escherichia coli (any serotype including generic or non-type specific). Salmonella (any serovar). Streptococcus (Iniae), Vibrio spp (also called photobacterium in older papers.)

AMU^^: The use of antimicrobials or antimicrobial drugs. For our purposes, disinfectants are not considered antimicrobial drugs.

AMR^^: When an antimicrobial substance, or agent, is no longer effective in killing or inhibiting the growth of a particular microorganism.

Outbreak, sporadic disease, case report^^: The occurrence of two or more cases of a similar illness with clustering patterns in time and space. Sporadic disease refers to I or more cases of disease that occur in a scattered or unpredictable or unassociated manner. For this review, these refer to all human studies related to exposure to aquaculture for any reason e.g., occupational, consumption, leisure etc

214 Seafood related human consumption report^®: Human consumption report will likely be a government document related to how much seafood was consumed by a population in a certain period of time. There is the potential that such information is contained in the peer reviewed literature as well and should be classified as a consumption report if this happens.

Seafood related production or import/export report^ ^: Seafood related production reports or import / export reports are often government reports about how much seafood was produced by a geographic area (country, province etc) in a certain period of time or was imported into or exported out of a country in a period of time. We are not interested in production practices under this category; all relevant research should fall into the first 5 options.

C3ass Class aditoprim (Diaminopyrimidines) ormetoprim (Diaminopyrimidines) amikacin (Aminoglycosides) Oxolinic acid (Quinolone) amoxicillin (Penicillins) oxytetracycline (Tetracyclines) ampicillin (Penicillins) penicillin G (Penicillins) arsanilic acid (Arsenicals) phosphonomycin (Aminoglycosides) (Diaminopyrimidine- sulfonamide quinupristin/dalfopristi Bactrim combinations) n (Streptogramins) baquiloprim (Diaminopyrimidines) rifampin (Rifamycins) (Diaminopyrimidine- sulfonamide carbenicillin (Penicillins) Romet-30 combinations) (Cephalosporins and cefoxitin cephamycins) sarafloxacin (Quinolones) (Diaminopyrimidme- (Cephalosporins and sulfonamide ceftiofur cephamycins) Septra combinations) (Cephalosporins and cephalexin cephamycins) sodium arsinalate (Arsenicals) (Cephalosporins and cephapirin cephamycins) spiramycin (Macrolides) chloramphenic ol (Phenicol) streptomycin (Aminoglycosides) chlortetracycli ne (Tetracyclines) sulfadiazine (Sulfonamides) ciprofloxain (Quinolones) sulfadimethoxine (Sulfonamides) cloxacillin (Penicillins) sulfadoxine (Sulfonamides) dihidrostrepto mycin (Aminoglycosides) sulfamerazine (Sulfonamides) doxycycline (Tetracyclines) sulfameter (Sulfonamides) enrofloxacin (Quinolones) sulfamethazine (Sulfonamides) erythromycin (Macrolides) sulfamethazine (Sulfonamides) florfenicol (aquaflor) (Phenicol) sulfapyridine (Sulfonamides) 215 fosfomycin (Streptogramins) sulfaquanidine (Sulfonamides) furazolidone (Nitrofurans) sulfaquinoxaline (Sulfonamides) gentamicin (Aminoglycosides) sulfisoxazole (Sulfonamides) josamycin (Macrolides) tetracycline (Tetracyclines) kanamycin (Aminoglycosides) thiamphenicol (Phenicol) (Diaminopyrimidine- sulfonamide lincomycin (Lincosamides) Tribrissen combinations) nalidixic acid (Quinolones) trimethoprim (Diaminopyrimidines) (Diaminopyrimidine- trimethoprim- sulfonamide neomycin (Aminoglycosides) sulfadiazine combinations) nifurpirinol (Nitrofurans) tylosin (Macrolides) nitrofurazone (Nitrofurans) virginiamycin (Streptogramins) nystatin (Antifungals)

216 APPENDIX 3.7. Assessment of methodological quality and reporting

QUESTION CODING RESPONSE OPTIONS AND APPLICABLE Exclusion REVIEWER GUIDELINES STUDY DESIGNS Bold indicates question containing exclusion criteria. If you select exclusion criteria at any question in the relevance confirmation section, please st op and submit your answers, The primary quality assessment section must be answered in its entirety for papers passing re evance confirmation. Relevance confirmation 1. What does Please check all that apply Four aquatic bacteria are: All 6. Exclude = this study 1. Yes. • Aeromonas (any species) Exclusion investigate? Prevalence or risk factors or • Salmonella (any serovar) • Vibrio spp. interventions for selected four If yes is aquatic bacteria at processing, • Escherichia spp. (generic or pathogenic) checked for retail, readv-to-eat or import/export any option fish or seafood. See also definition document. from 1-5, study Additional Information for Reviewer's is included. 2. Yes. Responses: Antimicrobial use, or antimicrobial resistance, or association between Response Option 1: antimicrobial use and resistance, or • Includes prevalence, risk factor or interventions for reducing intervention studies for Vibrio, antimicrobial resistance, in the four Aeromonas, Salmonella and selected aquatic bacteria Escherichia • OMLY processing, retail, ready-to- 3.(2b) Yes. Antimicrobial use, or eat products, and imported/exported antimicrobial resistance, or products are relevant association between antimicrobial Response Option 2: use and resistance, or interventions • Must come from fish/seafood derived for reducing antimicrobial 217 resistance, in the four selected from aquaculture systems, NOT from aquatic bacteria but unclear as to wild captures whether fish/seafood is derived from o e.g., grow-out ponds, large aquaculture or wild captures. rearing tanks, raceways, fish cages placed: in lakes, bayous, ponds, rivers or 4. Yes. oceans Human illness cases or outbreaks o if can't tell, please contact suspected or associated with ANY Natasa resistant aquatic bacteria, • Measurement of antimicrobial use at farm level, at ANY point in the 5. Yes. aquaculture systems/operations (from Prevalence or risk factors or hatchery to market size live interventions or antimicrobial use or fish/seafood) is relevant. antimicrobial resistance or human • Measurement of antimicrobial illness cases or outbreaks associated resistance at the farm level (EXCEPT hatchery/egg level) with ANY zoonotic aquatic bacteria and at in ORNAMENTAL FISH processing, retail, ready-to-eat or import/export fish 6. Exclude or seafood is relevant None of above • Includes interventions SOLELY (not for pathogens only) for reducing antimicrobial resistance applied at ANY point of the aquaculture systems/operations (from hatchery to market size live fish/seafood).

Response Option 3 (2b): • This option is selected when it is truly unknown whether fish/seafood is derived from aquaculture systems or from wild captures. (Encompasses all the criteria under Option 2 except the first point.)

218 Response Option 3: (Human Illness) • Suspected or confirmed human illness cases or outbreaks due to resistance to ANY bacteria from fish/seafood {excluding ornamental fish) through consumption, leisure or occupation • Includes human illness studies where resistance was tested for but may or may not have been found

Response Option 4: (Ornamental Fish) • Applies only to studies with ORNAMENTAL FISH • Any studies regarding prevalence, risk factors, interventions or human related cases for ANY zoonotic aquatic bacteria in ornamental fish are to be included under this option • Studies on antimicrobial use or antimicrobial resistance in ANY bacteria in ornamental fish are to be included under this option 2. What Please check all that apply Please select as reported in the article - check All 11. Exclude aquatic 1. Yes. Salmon all that apply: =Exclusion species does 2. Yes. Trout If yes is 3. Yes. Shrimp/Prawn Other; Please specify genus, species and this study common name as provided. checked for investigate? 4. Yes. Catfish any option 5. Yes. Oyster/Mussel/Clam from 1-11, 6. Yes. Tilapia 7. Yes. Lobster study is 8. Yes. Cod included.

219 9. Yes. Carp 10. Not applicable. This study reports on ornamental fish or human illness cases or outbreaks suspected or confirmed to be associated with any resistant aquatic bacteria. 11. Exclude None of above.

220 3. Was this study Please select one 0. No: All 0 No = conducted under field option samples originate from diseased fish/shellfish, or intervention is Exclusio conditions? 0. No applied in the laboratory n sampled experimental fish kept under laboratory as opposed to 1. Yes 'natural' conditions 2. Not If yes is applicable. This checked study involves l.Yes: for 1 or ornamental fish or Prevalence/ Risk factor Studies: 2, study reports cases or o For live, apparently healthy fish/shellfish, or for outbreaks of human processed/packaged fish/shellfish, all samples of the is disease selected four bacteria originate from aquatic wild or included. farmed populations (e.g., fish, shellfish or seafood) that are raised or produced under commercial, field conditions • e.g., farms, processing plants, retail stores o Antimicrobial use at ANY point of the aquaculture systems/operations (from hatchery to market size live fish/seafood) are considered field conditions o Antimicrobial resistance at ANY point of the farm to fork continuum, EXCEPT hatchery/egg/larvae, are considered field conditions Intervention Studies: o For reducing the levels of the four selected aquatic bacteria for retail products/seafood, intervention may be applied at the: • processing or • retail or • ready to eat or • import/export level o Interventions for reducing anti-microbial resistance AT ANY POINT of the of the farm to fork continuum SHOULD be included 221 2. Not applicable: • Studies related to prevalence or risk factors or interventions or antimicrobial use or antimicrobial resistance or human illness cases or outbreaks associated with ANY zoonotic aquatic bacteria in ORNAMENTAL FISH • Human illness cases or outbreaks suspected or associated with the four selected bacteria or ANY resistant aquatic bacteria.

Primary Quality Assessment 4. Is raw/unadjusted Please select one 0. No, please specify: All 0 No = data or measures of option • Results provided included (please select all that apply) for all of Exclusio the components of the study (whether it is prevalence, risk association/effect 0. No, please n factor, intervention, antimicrobial use, antimicrobial resistance provided? specify or human cases) 1. Yes, please a. No reporting of raw results specify b. Just median 2. Not c. Only p-value d. Only denominator applicable. This e. Only numerator Study reports cases f Graphical format from which results could not be extracted or outbreaks of g. Other, please specify human disease.

1.Yes, please specify: • Raw results or measures of association/effect unadjusted by statistical modeling are provided • At least one of the below (options a, b, c) is reported along with its minimum necessary data for at least one of the components of the study (whether it is prevalence or risk factor or intervention or antimicrobial use, antimicrobial resistance or

222 human cases) Minimum necessary data: a. Prevalence/frequency: • Following data must be reported measuring in at least ONE microbe in ONE discrete sample type (eg Vibrio in oysters or AMR) • Numerator and denominator, or • proportion + EITHER numerator or denominator b. Concentration: • Original measured value (usually a mean for a treatment group) cfu/unit or log cfu/unit andxd^N count (n) reported. c. Measures of association/effect: • OR/RR/IR/RD/PAF/ AFe reported and its measure of variability (SE, SD, CI) or P-value is provided and sample size (n) reported

5. Were adjusted Please select one 0. No, please specify: All 0 No = measures of option • Results of the multivariable model (please specify Exclusio association/effect and 0. No, please below) for all of the components of the study (whether n it is prevalence, risk factor, intervention, antimicrobial measures of specify use, antimicrobial resistance or human cases): variability presented? 1. Yes a. Were not provided or 2. Not b. Were not presented in extractable format (graph from which applicable results cannot be extracted) or c. Included measure of association/effect but no measures of variability were presented

1.Yes: • results of the multivariable model are presented for at least one of the components of the study (whether it is prevalence or risk factor or intervention or antimicrobial use or antimicrobial resistance or human cases): a. Included parameter estimates (e.g., OR, RR) and b. Measure of variability (e.g., SE, SD, CI) or f-value are provided 223 after adjusting for other variables, confounders and/or clustering and c. Sample size (n) reported

2. Not applicable: No multivariable model was used.

6. What is the study Please check all 1. Prevalence survey: All design as identified that apply • Study that measures outcome only (prevalence and distribution by the reviewer? 1. Prevalence of microbial hazard on commodity) at a single point in time 2. Longitudinal prevalence: survey • Study that measures outcome (prevalence and distribution of 2. Longitudinal disease only) at multiple points in time on the same population prevalence • Does not evaluate intervention 3. Challenge 3. Challenge trial: trial (ChT) • Planned experiment where subjects are artificially challenged or 4. Controlled exposed to the studied microorganism • Challenge may occur in the lab but the intervention is applied trial (CT) under 'field' conditions 5. Quasi- 4. Controlled trial: experiment (QE) • Planned experiment with natural disease exposure 6. Cohort study • May or may not be randomized 7. Case-control 5. Quasi-experiment: study (C-C) • Before and after trials, including prevalence measures at various points (before and after one or more stages) in the farm to 8. Cross- consumer continuum sectional study 6. Cohort study: (XS) • Group of fish/shellfish exposed to a hypothesized risk factor 9. Other (please (exposure), and a group not exposed to the factor are selected specify) and observed over the study period to record contamination with selected microorganism in each group 7. Case-control study: • Group of patients with a foodbome disease and a group without the disease are selected and compared with respect to the presence of the hypothesized risk factor (exposure) 8. Cross-sectional study: 224 • Study done at a single point in time to investigate the prevalence and distribution of the microbial hazard in fish/shellfish and hypothesized risk factors within the population 9. Other: • Case report on human illness, or • Descriptive and other studies 7. If this is an Please check all 0. No: Controlled 0 No = INTERVENTION that apply • No control group is used, or controls are from a different trial Exclusio study, was an 0.No sampling frame Challenge n appropriate control 1. Yes: 1. Yes • Controls are drawn from same sampling frame and are measured trial group used? 2. Yes, historical in the same timeframe as treatment group Quasi- 3. Before and 2. Yes, historical: experimental after trial • Controls are drawn from same sampling frame measured in an Cohort 4. Not earlier timeframe than the treatment groups and are not the same samples as the freatment groups applicable; this 3. Before and after trial: is not an • The study uses the same samples as its own control from intervention measurements at an earlier point in time study 4. Not applicable: • This is not a Controlled trial. Challenge trial, Quasi-experiment, or Cohort study 8. Was the Please select one 0. No: Case Control 0 No = intervention protocol option • Necessary information is missing Controlled Exclusio described in 0. No (Please see 'yes' option for minimum necessary data reported) trial n 1.Yes: sufficient detail to 1. Yes • Methods are thoroughly described and allow for replication Challenge allow reproduction of 2. Referenced Minimum necessary data reported: trial the experiment? paper - intervention used Quasi- 3. Not - method of application experimental applicable - dosage - duration/frequency of treatment 2. Reference paper: • Methods are referenced in another paper

225 3. Not applicable: • This is not a Controlled trial, Challenge trial or Quasi- experiment

9. Was the challenge Please select one 0. No: Challenge 0 No = protocol sufficiently option • Necessary information is missing trial Exclusio reproduced to allow 0. No (Please see 'yes' option for minimum necessary data reported) n 1.Yes: reproduction of the 1. Yes • Methods are thoroughly described and allow for replication experiment? 2. Referenced Minimum necessary data reported: paper - challenge organism 3. Not inoculation method applicable - concentration and frequency of inoculation 2. Reference paper: • Protocol is referenced in another paper 3. Not applicable: • This is not a Challenge trial 10. What is the total Please select one Samples can be obtained from seafood, live cultivated fish, ornamental All number of samples option fish or human cases. Samples refer to individual fish/seafood as opposed tested in this study? 0. <10 one individual sampled multiple times. 1. 11-29 0. <10: Study investigates interventions or risk factors or prevalence for 2. >30 four selected bacteria using less than or equal to 10 samples. 3. Not 1. 11-29: Study investigates interventions or risk factors or prevalence for applicable. four selected bacteria using 11-29 samples. The 2. >30: Study investigates interventions or risk factors or prevalence for study four selected bacteria using 30 or more samples. investigates: 3. Any antimicrobial resistance or antimicrobial use studies, OR any - Antimicrobial ornamental fish studies, OR studies reporting both prevalence of the 226 resistance or four selected bacteria and AMR or AMU, OR studies reporting human antimicrobial use case/outbreak suspected or associated with ANY resistant bacteria. - Ornamental fish -BOTH prevalence and AMR or AMU - or human case/outbreak suspected or associated with ANY resistant bacteria 11. Which type of Please check all Please answer based on the operation sampled; you may choose more All operation was that apply than one option: sampled in this 0. Wild caught 0. Wild caught: • Study conducted on samples of wild-caught fish/shellfish study? 1. Commercial obtained: farm o outside of commercial production or 2. Research o not raised in an aquaculture setting farm 1. Commercial farm: 3. Commercial • Study conducted on operations rearing fish/shellfish in ponds, nets, cages, raceways for commercial (consumption or display) processing plant purposes 4. Supermarket 2. Research farm: 5. Local or • Study conducted on operations affiliated with universities and/or specialty research organizations markets • Typically mimic field conditions and keep fish/shellfish in large tanks or large scale holding facilities including ponds (e.g., 6. Imported earthen ponds), tanks, farms, raceways, oyster lines, floating products cages 7. Restaurant • Fish/shellfish may be sampled from commercial farm 8. Human 3. Commercial processing plant: case(s) / • Study conducted in operating commercial processing plant outbreak setting 4. Supermarket: 9. Ornamental • Study conducted from samples purchased at a large grocery 227 Fish store belonging to a company chain with numerous locations 10. Other 5. Local or Specialty markets: (please specify) • Study was conducted on samples purchased at a small, typically privately-owned establishment selling fish/shellfish products among other produce • includes specialized fish markets 6. Imported products: • Study conducted on samples that were reported as imported 7. Restaurant: • Study conducted on samples that were obtained from establishments selling prepared meals 8. Human illness/outbreak: • Study conducted on samples that were obtained from human illness/outbreak as a result of exposure to fish/seafood through occupation, leisure or consumption. 9. Ornamental Fish: • Samples derived from source providing ornamental fish such as a retail fish tank or commercial facility selling or distributing fish for the ornamental fish industry 10. Other: Please specify. 12. Were laboratory Please select one 0. No: All methods for aquatic option • Methods are not sufficiently reported bacteria sufficiently 0. No • Study cannot be reproduced without contacting the author (Please view under 'yes' option for minimum necessary data described to allow 1. Yes reported) replication of the 2. Referenced study? paper 1. Yes: 3. Not Minimum necessarv laboratorv protocol data reported: applicable. Media type Laboratory - Time and temperature of incubation Pre-enrichment and enrichment steps (if used) to allow for methods were replication not used or 2. Referenced paper: human illness • Methods are referenced in another paper 3. Not applicable: 228 study. • Laboratory methods were not used in this study

13. Were laboratory Please select one 0. No: methods for option • Methods are not sufficiently reported susceptibility testing 0. No • Study cannot be reproduced without contacting the author (Please view under 'yes' option for minimum necessary data sufficiently described 1. Yes reported) to allow replication 2. Referenced of the study? paper 1. Yes: 3. Not Minimum necessary laboratory protocol data reported; applicable. Susceptibility testing: Susceptibility - Type of method applied: disk diffusion, broth dilution, micro broth dilution, gel dilution, E-test, methods were etc not used or - Media used human illness - Time and temperature of incubation study. 2. Referenced paper: • Methods are referenced in another paper 3. Not applicable: • Susceptibility testing methods were not used in this study 14. If the study Please select one 0. No: All reports antimicrobial option • Methods are not sufficiently reported to be extracted and used resistance, was the 0. No for qualitative or quantitative summary reporting of 1. Yes 1. Yes: antimicrobial 2. Reference Minimum necessary reported data include: resistance done paper - Numerator OR % and denominator for resistant appropriately? 3. Not bacterial isolates AND breakpoints used or Applicable - reference for breakpoints used - Full description of the distribution of minimum study does not inhibitory concentration, (any bacterial species, report AMR breakpoints do not need to be provided) 229 - Full range of dilutions tested if dilution technique is reported (any bacterial species, breakpoints do not need to be provided)

2. Not applicable: • Susceptibility testing was not reported in this study Additional comments Text box (If the reviewer feels there is something that was not captured in the tool but should be acknowledged in the primary QA)

QUESTION DEFINITIONS AND ADDITIONAL INFO FOR REVIEWERS: 1. What does Four aquatic bacteria are: Aeromonas (any serovar), Salmonella (any serovar), Vibrio spp., Escherichia spp. (generic or this study pathogenic) investigate? Definitions:

Prevalence: Prevalence is the reporting of frequency, occurrence, distribution or presence of selected zoonotic bacteria, antibiotic resistance, antibiotic use in aquatic wild, farmed or ornamental population. Risk factors: Risk factor are the associations between prevalence of selected zoonotic bacteria or antibiotic resistance or antibiotic use and one or more exposures (potential risk factors) such as farm management (use of certain feeding or bio-security practices) Interventions: Any technique or procedure applied aimed at reducing/eliminating/preventing the growth of selected aquatic bacteria in wild, farmed or ornamental population samples. Processing: Techniques involved in transforming raw seafood or further modifying the products for consumption (e.g., scaling of fish, preparation of fillets, shucking oysters, packaging). This can be conducted in processing plants or on farms depending on its capabilities. Processing includes any transport from farm to processing plants. Retail: Any raw, frozen or prepared seafood sold as goods and is typically available to consumers from a market place or

230 grocery store. Typically, these products require additional preparation. Ready-to-eat: Product that can be eaten without being further cooked. Fish: Any of numerous cold-blooded aquatic vertebrate characteristically having fins, gills, and a streamlined body. This term may also refer to any products intended for consumption that come from these animals. Seafood: Seafood is any fish and shellfish (including molluscs and crustaceans) (excluding seaweed) that is served as food or is suitable for eating. Ornamental: Any fish/shellfish (mollusc or crustacean) kept in an aquarium and intended for display rather than consumption. 2. What Commonly used terms in literature. If you are not sure, please contact Natasa. aquatic species does Salmon: Atlantic species - genus =Salmo this study Pacific species - genus = Onchorhynchus investigate? ( Common names - Atlantic, cherry, coho, chinook, pink, sockeye ,chum) Trout: 3 genera: Salmo (e.g.. Brown trout = S. trutta) Oncorhynchus (e.g., Rainbow trout = O. mykiss) Salvelinus (e.g., Arctic Char = S. alpinus) Shrimp: genus Penaeus Prawn: genus Macrobrachium Catfish: Ictalurus Ovster/Mussels/Clants: Crassostrea, Mvtilidae, Haliotis. Anv edible bivalves are to be included under this option. Bivalves are marine or freshwater molluscs having a soft body enclosed within two shells hinged together. Tilapia: Genus Tilapia: Genus Sarotherodon: Genus Oreochromis Lobster: numerous genera Cod: genus Gadus Carp: several genera potentially of interest re. aquaculture Genus Ctenopharvn2odon: Grass carp Genus Cvprinus: Common carp Genus Hvpophthalmichthvs: Bishead carp Genus Labeo: Bigmouth carp Genus Hvpophthalmichthvs: Biehead carp. Silver carp Mav also see Indian carp": catla (Gibelion catla). rohu (Labeo rohita) and mrigal (Cirrhinus cirrhosus) 6. What is the Study design choice determines the sections to be answered on this QA tool.

231 study design Prevalence survey: A study that measures outcome only (prevalence and distribution of microbial hazard on commodity) at a as identified single point in time. by the Longitudinal prevalence: A study that measures outcome (prevalence and distribution of disease only) at multiple points in time on the same population. Does not evaluate intervention. reviewer? Challenge trial: A planned experiment where subjects are artificially challenged or exposed to the studied microorganism. Challenge may occur in the lab but the intervention is applied under 'field' conditions. Controlled trial: A planned experiment with natural disease exposure. May or may not be randomized. Quasi-experiment: Before and after trials, including prevalence measures at various points (before and after one or more stages) in the farm to consumer continuum. Cohort study: Group of fish/shellfish exposed to a hypothesized risk factor (exposure), and a group not exposed to the factor are selected and observed over the study period to record contamination with selected microorganism in each group. Case-control study: A group of patients with a foodbome disease and a group without the disease are selected and compared with respect to the presence of the hypothesized risk factor (exposure). Cross-sectional study: A study done at a single point in time to investigate the prevalence and distribution of the microbial hazard in fish/shellfish and hypothesized risk factors within the population. Other: case report on human illness, or descriptive and other studies

232 10. If the Examples of Acceptable and Unacceptable MIC and Diffusion Disk Reporting study reports If minimum inhibitory concentration or diffusion diameters are reported, they must be reported in such a way that it is possible to antimicrobia calculate % resistance or % susceptibility. An example of an acceptable MIC distribution would be a full description of the I resistance number or percent of isolates under each MIC (Table 1). An example of an inappropriate description of the distribution of MICs (AMR), was would be when only the median, MIC90, mean or range is reported (Table 2). This is not acceptable because it is not possible to Minimum derive percent resistance from this type of information. An acceptable description of disk diffusion diameters would be a table Inhibitory with the number or percent of isolates within different diameters ranges, or a histogram graph showing the number or percent of Concentratio isolates within different diameters ranges (Fig. I and 2). n (MIC) or Minimum inliibitory concentration (MIC) definition; Lowest antimicrobial concentration required to inhibit bacterial growth diffusion after an overnight in vitro incubation. The MIC is used to confirm or monitor antimicrobial resistance in bacteria. Resistance is disk said to exist when the MIC is higher than the defined breakpoint of resistance for a given bacterial isolate. diameters Table I. lie of an accec reported in : an acceptable amoxicillin-clavulanic acid 71 1.41 71.8 2.8 5.6 15.51i 2.8 11 1.4 way ceftiofur 71 1 1 1.4 39.4 57.7 1.4 1 1 1.4 1 <=0.25 <=0.25 ceftriaxone 71 0.0 98.6 11 1.4 1 <=0.015 ciprofloxacin 71 0.03 0.0 87.3 12.7 11 amikacin 71 1 2 0.0 16.9 60.6 19.7 1.4 1.4 1 ampicillin 71 <=1 >32 25.4 67.6 5.6 1.4 25.4 cefoxitin 71 2 4 1.4 19.7 43.7 33.8 1.4 1.4 gentamicin 71 <=0.25 0.50 0.0 73.2 23.9 1.4 1.4 II i <=8 kanamycin 71 16 9.9 88.7 1 nalidixic acid 71 4 4 0.0 19.7 76.1 4.2 i streptomycin 71 <=32 >64 43.7 56.31 9.9 : trimethoprim-sulphamethoxazole 71 0.25 1 7.0 40.8 33.8 14.1 2.8 1.4 11 14 5.6 chloramphenicol 71 8 >32 14.1 26.8 59.21i 11 14.1 III sulfisoxazole 71 128 >256 49.3 4.2 25.4 19.7 tetracycline 71 >32 >32 63.4 36.61 11 2.8 4.2 56.3

233 Table 2. Example of an unacceptable reporting of the distribution of MICs Hmakpoim MIC(jjLgfml) cilsirttNiiioft for. Antimicrobial Tesi ranp K vtiln^ctts (« = l5 l ) (>i^nil) K paraftmnHîcm (n = 16S) MICm Miq,, Range Mea# MtC^ MIC^» Range r Amfjicillin 0.03-64 16 "^^32 32 32 05-64 1 1 0.06-8 Cefotaxime 0.03-64 5^8 16-32 3*64' 0:5 05 ^0,03^ -1.85^ 0JZ5 Oj%5 ^0.03^ Ceftazidime 0.03-64 16 2^32 0.25 05 -2.13 0.25 05 fÔ.03-1 Chkiraitiphenicol 1-1,034 16 2=32 1 1 0.06-8 -0.75 1 1 0.06-8 Ciproflcxvdctn 0.03-64 2 ^4 0.12S 05 ^0.03-1 -3jzy f&0.03 0.06 fg0.03-0JZ5 Gentamiein 0.0(M>4 ^4 8 2^16 06 1 0.125-^ -0.49 05 1 ^0.03^ Imipenem 0.03-64 8 ZrZ 16 ôm 0.06 ^0.031-2 0.125 0.25 ^0.03-05 Tetracycline 0.03-64 •^4 8 ^16 05 1 0.06-2 -1J4^ 0.25 05 0.06-2 ^ iireaicpolnis remmnwnded by tlie Clinical and Laboramry Smndards InsiiiitJie In M45* A (H). S, ], and R smnd for susceptible, iniermedtnie, and reg fe^»ctiwly. Mean MICs are e^êssed on a ioga scale. When ihe MIC was ^ 0.(0 ii^ml, -6 was used as the value for mean calciihidon. ^ lndoit«s significafiily different mean MICs (in 1% scale) between K iMtmhamnofyticm and K vuinificm (P < 0.0001). Figure 1. First example of an acceptable presentation of disk diffusion diameters. Although this requires work, we can still assess the percent of resistance using a different breakpoint if needed.

234 Cephafothin

60j

45:.

30:.

1 I i M t » MM * M M M M M 6 8 10 12 14 16 18 20 21 24 26 28 30 32 34 >

Figure 2. Second example of an acceptable presentation of disk diffusion diameters. Although this requires work, we can still assess the percent of resistance using a different breakpoint if needed.

235 36

34 1 32 1 30

28 1 2 26 1

24 1 2 2 3 2 2 22 14 2 2 123 12 1 (Disk) 20 1 13 12 1 1 2 18 1 1 2 1 1 2 16 1 1 1 14 1 12 1 10

5 1

8 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 GEN (Disk)

236 APPENDIX 3.8. Data extraction tool for prevalence studies

AQUACULTURE STUDY DE TOOL

LEAVE BLANK IF QUESTION IS NOT APPLICABLE TO THE STUDY. multiple submissions of the form will be used to capture combinations of pathogens, species, points in food chain, and antimicrobials investigated. if using multiple submissions please record only data that is unique to the combination, extract all information on one combination before cloning the form and extracting on the subsequent combination. General Information Variable Category Explanation and Definitions RefID

Captured in DistillerSR Journal name Author(s) name Publication year Nature of Data. P ease answer for ALL articles. Publication type a. Peer reviewed Please specify type of b. Conference proceeding publication. c. Thesis d. Government or research station report e. Other (please specify)

POPULATION (applicable to all studies) If the population sampled or reported on includes FISH/SHELLFISH please complete the following: For each question, please select only ONE option. (Use multiple submissions if it is possible to extract for combinations of species or age group or point in food chain.) Species Salmon, please specify: Please specify genus and investigated Trout, please specify: species or as reported in the Shrimp/Prawn, please specify: article. Catfish, please specify: Oyster/Mussel/Clam, please specify: Tilapia, please specify: Lobster, please specify: Cod, please specify: Carp, please specify: Unspecified shellfish Ornamental, please specify:

237 Total Farms, please specify: Please specify numbers as number of Hatcheries, please specify: reported in the article. each Pens/pond/tank, please specify: sampled Transport vehicles, please specify: Processing plants, please specify: Batches/lots, please specify: Fillets/pieces, please specify: Other, please specify Not reported Age- Egg, please specify: Please specify numbers as group(s) Fry, please specify: reported in the article. Juvenile, please specify: Adult, please specify: Other, please specify: Not reported

Points in a. Live fi-om farm Live from farm: Fish/shellfish chain in b. Processing was farmed in aquaculture which the c. Retail systems for production. sampling d. Ready to Eat Processing: Techniques occurred e. Restaurant involved in transforming raw e. Imported/exported product seafood or further modifying f. Ornamental fish the products for consumption (e.g., scaling of fish, preparation of fillets, packaging). This can be conducted in processing plants or on farms depending on its capabilities. Retail: Any raw, frozen or prepared seafood sold as goods and is typically available to consumers from a market place or grocery store. Ready-To-Eat products: Product that can be eaten without being further cooked. Imported/exported: Commodities (goods) bought/sold from a foreign country (e.g., ornamental fish or any type of seafood). Ornamental fish: Any fish/shellfish (mollusk or crustacean) kept in an

238 aquarium and intended for display rather than consumption.

Date of Please specify Please specify: Date as sampling Not reported MM/DD/YYYY or range if period given as MM/DD/YYYY- MM/DD/YYYY) Country/area Please specify: Please specify the country in in which Not reported which sampling had taken sampling place. occurred.

Country of Country of import, please specify: Please specify importing import Not reported country. Country of Country of export, please specify: Please specify exporting export Not reported country.

OUTCOME MEASUREMENT Please extract separately for each bacteria investigated. (Use multiple submissions if data can be extracted for more than one bacterium.) Bacteria investigated Please specify Please specify genus and species as reported by the author.

Test(s) used to Bacteriological Please specify as reported by the author. monitor the presence culture or absence bacteria PCR (Polymerase Chain Reaction) Other, please specify Not reported

If bacterial culture, a. Culture media Please specify the culture media (pre- specify the culture b. Described in enrichment, enrichment), supplements media used another paper and commercial name as reported by the c. Not reported author.

For example, pre-enrichment in phosphate buffered peptone water; Rappaport-Bassiliadis broth; tetrathionate broth; BG Sulfa agar

239 plates; modified lysine iron agar plates; Rambach agar. If PGR, please specify PGR Please specify as reported by the author. the reported PGR used Not reported

If reported, specify Sn, please specify: If applicable please indicate the Sn and sensitivity (Sn) and Sp, please specify: Sp of all tests used. specificity (Sp) of the Not reported test(s) used

Gut-off level for a. At least one If applicable indicate the cut off level test(s) used positive used for Pathogen positivity of all b. More than one levels. positive c. Not reported

RESULTS FOR PREVALENGE STUDIES PREVALENGE OF BAGTERIA (if applicable) Please extract separately if results for multiple bacteria are provided. (Use multiple submissions if data can be extracted separately for each bacteria) Bacteria List genus and species of bacteria. Sample type Please specify sample type. Sample weight Please specify sample type (e.g., blood, meat, homogenized shellfish). Total samples The total number of samples tested. Number refers to individual fish/shellfish/seafood samples. Total samples The total number of samples tested that were positive. positive Prevalence Number of positive units divided by the total number of units. Prev 95% GI low 95% confidence interval. Prev_95% GI_high 95% confidence interval. Prev SE/SD Standard error or standard deviation (please indicate which). Goncentration The mean concentration (e.g., cfii) of pathogen/antimicrobial in a sample. Gone 95% GI low 95% confidence interval. Gone 95% 95% confidence interval. GI high Gone SE/SD Standard error or standard deviation (please indicate which). Mean diff The mean concentration (e.g., cfu) of Pathogen in a sample. Goncentration MD Gone 95% 95% confidence interval.

240 CI low MD Cone 95% 95% confidence interval. CI_high MD Cone SE/SD Standard error or standard deviation (please indicate which). Other Open textbox for additional information.

241 APPENDIX 3.9. Bibliography of studies included in the SR

References included in SR-MA

1. Asai, Y., Kaneko, M., Ohtsuka, K., Morita, Y., Kaneko, S., Noda, H., Furukawa, L, Takatori, K., and Hara-Kudo, Y. 2008. Salmonella prevalence in seafood imported into Japan. J Food Prot. 71:1460-4. 2. Ayulo, A. M., Machado, R. A., and Scussel, V. M. 1994. Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from the southern region of Brazil. Int J Food Microbiol. 24:171-8. 3. Bandekar, J. R., Kamat, A. S., Karani, M., Dhokane, V., Shashidhar, R., Kakatkar, A., Ghadge, N., Bhat, A., Venugopal, V., and Warrier, S. B. 2004. Bacteriological quality of farmed freshwater fish and shellfish meant for Export. Fishery technology. Society of fisheries technologists (india). 41:57-62. 4. Buck, J. D. 1998. Potentially Pathogenic Vibrio spp. in Market Seafood and Natural Habitats from Southern New England and Florida. Journal of aquatic food product technology. 7:53-62. 5. Cabedo, L., Barrot LPicart, and Teixido'canelles, A. 2008. Research Note Prevalence of Listeria monocytogenes and Salmonella in Ready-to-Eat Food in Catalonia, Spain. Journal of food protection. 71:855-859. 6. Cai, T., Jiang, L., Yang, C., and Huang, K. 2006. Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China. FEMS Immunol Med Microbiol. 46:180-6. 7. Carvajal, G. H., Sanchez, J., Ayala, M. E., and Hase, A. 1998. Differences among marine and hospital strains of Vibrio cholerae during Peruvian epidemic. J Gen Appl Microbiol. 44:27-33. 8. Castro-Escarpulli, G., Figueras, M. J., Aguilera-Arreola, G, Soler, L., Femandez- Rendon, E., Aparicio, G. O., Guarro, J., and Chacon, M. R. 2003. Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. Int J Food Microbiol. 84:41-9. 9. Chaicumpa, W., Ngren-ngarmlert, W., Kalambaheti, T., Ruangkunapom, Y., Chongsa-nguan, M., Tapchaisri, P., Desakom, V., and Suthienkul, 0.1995. Monoclonal antibody-based dot-blot ELISA for the detection of Salmonella in foods. Asian Pac J Allergy Immunol. 13:159-66. 10. Chigbu, L. N. and Iroegbu, C. U. 2000. Vibrio species from diarrhoeal stools and water environment in Cross River State, Nigeria. International journal of environmental health research. 10:219-228. 11. Cook, D. W., Oleary, P., Hunsucker, J. C., Sloan, E. M., Bowers, J. C., Blodgett, R. J., and Depaola, A. 2002. Vibrio vulnificus and Vibrio parahaemolyticus in U.S. retail shell oysters: a national survey from June 1998 to July 1999. J Food Prot. 65:79-87. 12. dela Cruz A-RG, Santos, L. M., Agudo, F., Dangla, E., and FAO Indo-Pacific

242 Fisheries Comm., Bangkok (Thailand). 1990. Microbiology of prawn processing. Fao fisheries report. No. 401 13. do Nascimento, S. M., dos Femandes Vieira, R. H., Theophilo, G. N., Dos Prazeres Rodrigues, D., and Vieira, G. H. 2001. Vibrio vulnificus as a health hazard for shrimp consumers. Rev Inst Med Trop Sao Paulo. 43:263-6. 14. Dore, W. J., Henshilwood, K., and Lees, D. N. 2000. Evaluation of F-specific RNA bacteriophage as a candidate human enteric virus indicator for bivalve molluscan shellfish. Appl Environ Microbiol. 66:1280-5. 15. Elhadi, N., Radu, S., Chen, C. H., and Nishibuchi, M. 2004. Prevalence of potentially pathogenic Vibrio species in the seafood marketed in Malaysia. J Food Prot. 67:1469-75. 16. Ellison, R. K., Malnati, E., Depaola, A., Bowers, J., and Rodrick, G. E. 2001. Populations of Vibrio parahaemolyticus in retail oysters from Florida using two methods. J Food Prot. 64:682-6. 17. Evangelista-Barreto, N. S., Vieira, R. H., Carvalho, F. C., Torres, R. C., Sant'Anna, E. S., Rodrigues, D. P., and Reis, C. M. 2006. Aeromonas spp. isolated from oysters (Crassostrea rhizophorea) from a natural oyster bed, Ceara, Brazil. Rev Inst Med Trop Sao Paulo. 48:129-33. 18. Fuenzalida, L., Armijo, L., Zabala, B., Hernandez, C., Rioseco, M. L., Riquelme, C., and Espejo, R. T. 2007. Vibrio parahaemolyticus strains isolated during investigation of the summer 2006 seafood related diarrhea outbreaks in two regions of Chile. Int J Food Microbiol. 117:270-5. 19. Fuenzalida, L., Hernandez, C., Toro, J., Rioseco, M. L., Romero, J., and Espejo, R. T. 2006. Vibrio parahaemolyticus in shellfish and clinical samples during two large epidemics of diarrhoea in southern Chile. Environ Microbiol. 8:675-83. 20. Gobat, P. F. and Jemmi, T. 1993. Distribution of mesophilic Aeromonas species in raw and ready-to-eat fish and meat products in Switzerland. Int J Food Microbiol. 20:117-20. 21. Gonzalez-Rodriguez, M. N., Santos, J. A., Otero, A., and Garcia-Lopez, M. L. 2002. PCR detection of potentially pathogenic aeromonads in raw and cold- smoked freshwater fish. J Appl Microbiol. 93:675-80. 22. Gonzalez-Rodriguez, M. N., Sanz, J. J., Santos, J. A., Otero, A., and Garcia- Lopez, M. L. 2002. Numbers and types of microorganisms in vacuum-packed cold-smoked freshwater fish at the retail level. Int J Food Microbiol. 77:161-8. 23. Han, F., Walker, R. D., Janes, M. E., Prinyawiwatkul, W., and Ge, B. 2007. Antimicrobial susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus isolates from Louisiana Gulf and retail raw oysters. Appl Environ Microbiol. 73:7096-8. 24. Hara-Kudo, Y., Sugiyama, K., Nishibuchi, M., Chowdhury, A., Yatsuyanagi, J., Ohtomo, Y., Saito, A., Nagano, H., Nishina, T., Nakagawa, H., Konuma, H., Miyahara, M., and Kumagai, S. 2003. Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus 03:K6 in seafood and the coastal

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247 APPENDIX 3.10. Extracted data from 72 studies included in SR

Table 1. Results of data extraction of studies investigating Salmon and Tilapia presented by point in food chain Numb er of Sampl Prevale Concentrati Point in es nee (if on (if Finfish Food Authors (Sampl applicab applicable) Chain es le) ±SD Positiv

Salmon Ready-To- Cabedo et al. 68 2008 Spain 1998- Salmone meat homogenate 84(1) 0.01 Eat 2004 lla (25 g) Retail Pao et al. 7123 2008 USA 2006, E. coli meat homogenate 32 (0) 0.00 2007 (25 g) E, coli 34(1) 0.03 Salmone 34 (0) 0.00 lla Salmone 32(0) 0.00 lla Ullmann et al. 9590 2005 Germany 2000 Aeromo meat homogenate 11(0) 0.00 nas (25 g) Gonzales- 10996 2002 Spain Aeromo .(lOg) 16(3) 0.19 Rodrigues et al, nas Retail + Gonzales- 11104 2002 Spain Aeromo meat homogenate 30(.) 3.11 ±0.82 Imported Rodrigues et al, nas (50 g) cfu/g 30(.) 3.28 ± 0.67 cfu/g 30 (.) 2.27 ± 0.85 cfu/g 30 (.) 2.7 ± 0 cfu/g 30 (.) 2.87 ± 0.35 cfu/g 248 Gobat et al. 14019 1993 Switzerla Aeromo meat homogenate 19(2) 0.11 nd nas (50 g) Tilapia Retail Pao et al. 7123 2008 USA 2006, E. coli meat homogenate 32(1) 0.03 2007 (25 g) 36(3) 0.08 Salmone 32(0) 0.00 lla 36(0) 0.00 Yano et al. 9931 2004 China 2001, Vibrio 5(0) 0.00 2002 Vibrio 5(0) 0.00 Castro- 10639 2003 Mexico Aeromo meat homogenate 225 0.36 Escarpulli et al. nas (25 g) (82) 250 0.33 (82) Carvajal et al. 10869 2003 Malaysia Aeromo meat homogenate 32 (23) 0.72 nas (25 g) Hassan et al. 13825 1994 Banglade Aeromo surface swab 15(12) 0.80 sh nas E. coli 15(4) 0.27 Vibrio 15(9) 0.60

^Refld is the reference identification number. Blank cells indicate the information is the same as the cell above. Period indicates information was not reported in the study.

249 Table 2. Results of data extraction on studies investigating Shrimp/prawn presented by point in food chain Numbe r of Point Sampl Prevale Concentration Sampli in Pub. Country of Bacteri Sample Type es nee (if (if ReflD' Autliors ng Food Year Sampling a (Sample weight) (Sampl applica applicable)) ± Year Chain es ble) SD Positiv e) Import 11964 Heinitz et al. 2000 USA (worldwide 1990- Salmon meat 4311 0.09 import) 1998 ella homogenate (25 (365) g) 12040 Wong et al. 1999 Taiwan (imported 1996- Vibrio meat 62 (47) 0.76 from Thailand) 1997 homogenate (50 g) Live from farm 1132 Surendraraj et al. 2005 India Salmon 12(5) 0.42 ella Process ing 692 Vongxay 2006 China 2003- Vibrio meat 117(4) 0.03 Khamphouth et 2004 homogenate (25 al. g) 34 (7) 0.21 1497 Bandekar et al. 2004 India E. coli 6(3) 0.50 12(9) 0.75 Salmon 6(2) 0.33 ella 12 (5) 0.42

250 Vibrio 6(0) 0.00 12(0) 0.00 6(0) 0.00 2284 Sanjeev et al. 2002 India Vibrio 100(2) 0.02 68 (6) 0.09 3120 Sanjeev et al. 2000 India Vibrio 100 0.15 (15) 100 0.17 (17) 29 (25) 0.86 29 (29) 1.00 68 (34) 0.50 68 (39) 0.57 11964 Heinitzetal. 2000 USA 1990- Salmon meat 129 (5) 0.039 1998 ella homogenate (25 g) 4997 Prasad et al. 1995 India Salmon meat 60 (0) 0.00 ella homogenate (25 g) 60 (0) 0.00 40 (0) 0.00 40(0) 0.00 6785 dela Cruz et al. 1990 Philippines Salmon meat 15(0) 0.00 ella homogenate (25 g) Vibrio 15(2) 0.13 Processing + Retail 10434 Sanath Kumar et 2003 India Salmon meat 20(1) 0.05 al. ella homogenate (25 g)

251 20(1) 0.05 10862 Carvajal et al. 1998 Peru 1991- Vibrio 42 (0) 0.00 1992 6783 Karunasagar et 1990 India Vibrio meat 71(1) 0.01 al. homogenate (50 g) 71(6) 0.09 71 (20) 0.28 71 (57) 0.80 Retail 7158 Asai et al. 2008 Japan 2005- Salmon meat 104 (0) 0.00 2006 ella homogenate (25 g) 61(0) 0.00 104(0) 0.00 61(0) 0.03 47(2) 0.04 47 (3) 0.06 7214 Luan et al. 2008 China Vibrio liver (0.1-l.Og) 80 (66) 0.83 8072 Sagoo et al. 2007 UK 2003 E. coli meat 372 0.03 homogenate (25 (11) 8) Salmon 372 (0) 0.00 ella Vibrio 372 (0) 0.00 8389 Shabarinath et al. 2007 India Salmon 27 (16) 0.59 ella 27 (5) 0.19 8756 Miwaetal. 2006 Japan 2002- Vibrio 10(3) 0.30 2004 10(5) 0.50

252 10(6) 0.60 10(6) 0.60 625 Sindhu et al. 2006 India 2003- Vibrio meat 19(4) 0.21 2004 homogenate (25 8) 14(5) 0.36 932 Kumar et al. 2005 India E. coli meat 10(0) 0.00 homogenate (.) 15(0) 0.00 8(0) 0.00 5(2) 0.40 15(2) 0.13 15(5) 0.33 1131 Surendraraj et al. 2005 India E. coli 20(20) 1.00 1053 Vivekanandhan 2005 India 1997- Aeromo liquid rinse 278 0.18 et al. 1999 nas (49) 9460 Phan et al. 2005 Vietnam 2000- Salmon 110 0.25 2001 ella (27) 9590 Ullmann et al. 2005 Germany 22/06/1 Aeromo meat 1(0) 0.00 905 nas homogenate (10 g) 9975 Elhadi et al. 2004 Malaysia 1998- Vibrio meat 433 0.59 1999 homogenate (25 (255) g) 50 (24) 0.48 50(41) 0.82 9656 Hwang et al. 2004 Taiwan E. coli 50 (5) 0.10 47 (14) 0.30 9931 Yano et al. 2004 China 2001, Vibrio 9(0) 0.00 2002 9(5) 0.56

253 1.00 8(7) 1.00 10686 Thayumanavan 2003 India 2000- Aeromo surface swab 104 0.36 etal. 2001 nas (37) 11392 do Nascimento et 2001 Brazil Vibrio meat 20(7) 0.35 al. homogenate (25 g) 11396 Sanathetal. 2001 India E. coli meat homogenate (25 g) 2818 Chigbuetal. 2000 Nigeria Vibrio meat 60(0) 0.00 homogenate (2 g) 60(9) 0.15 4.19 ±4.66 cfu/g 60 (26) 0.43 4.91 ±5.16 cfu/g 11790 Neytsetal. 2000 Belgium 1990- Aeromo meat 4(2) 0.5 1998 nas homogengate (25g) 4463 Landgraf et al. 1996 Brazil 1991- Vibrio meat 24 (0) 0.00 1993 homogenate (25 g) 24(1) 0.04 25(1) 0.04 26(1) 0.04 4690 Singh et al. 1996 India Vibrio surface swab 13(0) 0.00 13(0) 0.00 13(0) 0.00 13(0) 0.00 13(0) 0.00 13(0) 0.00

254 13(1) 0.08 13 (2) 0.15

13227 Tsaietal. 1996 Taiwan Aeromo meat 14(2) 0.14 nas homogenate (25 g) 4997 Prasad etal. 1995 India Salmon meat 500 (1) 0.00 ella homogenate (25 g) 500(4) 0.01

5082 Wong etal. 1995 China 1992 Aeromo meat 40(5) 0.13 nas homogenate (25 g) Vibrio 40(1) 0.03 40(2) 0.05 40(3) 0.08 40 (8) 0.20 40 (10) 0.25 40(18) 0.45

13426 Chaicumpa et al. 1995 Thailand Salmon meat 50 (10) 0.20 ella homogenate (10 g) 13738 Ayuloetal. 1994 Brazil E. coli meat 30(10) 0.33 homogenate (25 g) 30 (12) 0.40

13957 Rahim et al. 1994 Bangladesh Aeromo 18 8.9 ±0.81 nas 10^ cfu/g 18 1.1 ±0.11 10^ cfu/g stomach (.) 18 1.5 ±0.16 10^ cfu/g 18 1.1 ±0.21

255 W cfij/g

eggs (.) 18 6.0 ± 1.0 X 10' cfu/g E. coli 18 1.2 ±0.3x 10' cfu/g eggs (.) 18 1.2 ± 0.3 cfu/g 5557 Velammal et al. 1994 India 1989- Vibrio meat 73 (20) 0.27 1990 homogenate (25 g)

6785 dela Cruz et al. 19 Philippines Salmon meat 128 (0) 0.00 90 ella homogenate (25 g) Vibrio meat 128 0.09 homogenate (30 (12) g) Not reported 1409 Torres Vitela et al. 19 Mexico Vibrio meat 28(5) 0.18 0 96 homogenate (20 g) 28(5) 0.18

^Refld is the reference identification number. Blank cells indicate the information is the same as the cell above. Period indicates information was not reported in the study.

256 Table 3. Results of data extraction on studies investigating Oyster presented by point in food chain

Numbe rof Point Sampl Sampli Prevalence in Refl Pub. Country of Bacteri Sample Type es Concentration (if Authors ng (if Food Year Sampling a (Sample weight) (Sampl applicable) ± SD Year applicable) Chain es Positiv e) Import 11964 Heinitz et al. 2000 USA 1990- Salmon meat homogenate (25 1124 1998 g) 0.03 Live from farm 8655 Evangelista- 2006 Brazil 2002 Aeromo meat homogenate (.) 30(13) 0.43 Barreto et al. nas meat homogenate (25 30(15) 0.50 g) meat homogenate (.) 30 (20) 0.67 3772 Buck, J.D. 1998 USA Vibrio liquid rinse 1(1) 1.00 2(2) 1.00 4463 Landgraff et al. 1996 Brazil 1991- Vibrio meat homogenate (25 56(2) 0.04 1993 g) 56 (7) 0.13 56 (29) 0.52 56 (49) 0.88 13177 Motes et al. 1996 USA 1994- Aeromo meat homogenate (.) 600 (.) 1995 nas Processing 11964 Heinitz et al. 2000 USA 1990- Salmon meat homogenate (25 80(1) 0.01 257 1998 ella g) 3120 Sanjeevetal. 2000 India Vibrio 6(5) 0.83 6(5) 0.83 Processing + Retail 6783 Karunasagar et 1990 India Vibrio meat homogenate (50 15(0) 0.00 al. g) 15(0) 0.00 15(6) 0.40 15(12) 0.80 Ready-To-Eat 11316 Cook et al. 2002 USA 1998- Vibrio meat homogenate (.) 345(217) 0.63 1999 347 (253) 0.73 12165 Rampersad et al. 1999 Trinidad and 1997- E. coli surface swab 50 (39) 0.78 Tobago 1998 50(41) 0.82 50 (36) 0.74 50 (38) 0.76 meat homogenate (25 50 (.) 2.9 X 10 mean g) count/g 50(.) 1.3 X 10' mean count/g 50(.) 2.7 X 10^ mean count/g 50 (.) 4.9 X lO' mean count/g Salmon 50(0) 0.00 ella 50(1) 0.02 50(1) 0.02

258 50 (0) 0.00 Restaurant 11610 Ellison et al. 2001 USA 1997- Vibrio meat homogenate (.) 14(14) 1.00 0.79 log 10/ g 1998 14(14) 1.00 0.38 log 10/g 14(10) . 0.71 0.69 log 10/g 14(12) 0.86 0.53 log 10/g 14(4) 0.29 0.62 log 10/ g 14(4) 0.29 0.59 log 10/g 14(8) 0.57 1 log 10/g 14(9) 0.64 1.05 log 10/ g 14(14) 1.00 0.68 log 10/g 14(14) 1.00 0.97 log 10/g 14(14) 1.00 1 log 10/g 14(14) 1.00 0.62 log 10/g 14(13) 0.93 0.59 log 10/g 14(4) 0.29 0.88 log 10/g 14(2) 0.14 0.57 log 10/g 14(9) 0.64 0.85 log 10/ g 14(9) 0.64 0.67 log 10/g 14(14) 1.00 0.37 log 10/g Retail 7848 Han et al. 2007 USA 2005- Vibrio meat homogenate (.) 94 (82) 0.87 2006 8389 Shabarinath et 2007 India Salmon meat homogenate (.) 30(2) 0.07 al. ella 30(9) 0.30 8929 Cai et al. 2006 China 50 (20) 0.40 g) 259 50 (25) 0.50 791 Lu et al. 2006 USA 2001- Vibrio liquid rinse 13(5) 0.39 2002 13(9) 0.69 625 Sindhu et al. 2006 India 2003- Vibrio meat homogenate (25 6(0) 0.00 2004 g) 8399 Vuddhakul et al. 2006 Thailand 2000 Vibrio meat homogenate (.) 2(0) 0.00 932 Kumar et al. 2005 India E. coli meat homogenate (.) 10(10) 1.00 9818 Parisi et al. 2004 Italy 2000- Salmon meat homogenate (25 49 (0) 0.00 2002 ella g) Vibrio 49 (28) 0.57 9931 Yano et al. 2004 China 2001, Vibrio meat homogenate (.) 2(0) 0.00 2002 meat homogenate (.) 2(0) 0.00 10591 Hara-Kudo et al. 2003 Japan 2001 Vibrio meat homogenate (25 80 (7) 0.09 g) 2341 Pereiraetal. 2001 Portugal 1998- Vibrio meat homogenate 3(1) 0.33 1999 (50g + water) 12029 Dore et al. 2000 UK 1995- E. coli meat homogenate (7 13(.) 1997 g) 39 (.) 24 (.) 58 (.) 12165 Rampersad et al. 1999 Trinidad and 1997- E. coli surface swab 50 (44) 0.88 Tobago 1998 50 (37) 0.74 50 (35) 0.70 50 (38) 0.76 meat homogenate (25 50 (.) 1.7 X 10^ mean g) count/g 50(.) 2.7 X 10^8 mean count/g 260 50(.) 6.9 X 10^5 mean count/g 50(.) 2.8 X 10^7 mean count/g Salmon meat homogenate (25 50(5) 0.10 ella g) 50(0) 0.00 50(1) 0.02 50(1) 0.02 3772 Buck, J.D. 1998 USA Vibrio liquid rinse 3(1) 0.33 2(2) 1.00 9(4) 0.44 11(11) 1.00 13227 Tsaietal. 1996 Taiwan Aeromo meat homogenate (25 22(11) 0.50 nas g) Not Reported 14090 Torres Vitela et 1993 Mexico Vibrio meat homogenate (20 25(11) 0.44 al. Refid is the reference identification number. Blank cells indicate the information is the same as the cell above. Period indicates information was not reported in the study.

261 Table 4. Results of data extraction on studies investigating Mussel presented by point in food chain Number Point in Country of Prevalence Pub. Food ReflD* Authors y of Sampling Bacteria Sample Type (Sample weight) Samples (if Chain Sampling (Samples applicable) Positive) Live from farm 8038 Fuenzalida et al. 2007 Chile 2006 Vibrio meat homogenate (50 g) 20(16) 0.80 8564 Ottaviani et al. 2006 Italy 2002-2004 Aeromoms meat homogenate (25 g) 144(32) 0.22 3772 Buck, J.D. 1998 USA Vibrio liquid rinse (.) 2(1) 0.50 10(10) 1.00 4463 Landgraf et al. 1996 Brazil 1991-1993 Vibrio meat homogenate (25 g) 20(2) 0.10 20(5) 0.25 20(10) 0.50 20(1) 0.05 5557 Velammal et al. 1994 India 1998-1990 Vibrio meat homogenate (25 g) 85(17) 0.20 Live from farm + Processing 8063 Pereira et al. 2007 Brazil 2000 Vibrio meat homogenate (25 g) 86(10) 0.12 Processing 10235 Martinez-Urtaza et al. 2004 Spain 1998-2001 Salmonella meat homogenate (1 kg) 3410(574) 0.17 Processing + Retail 10434 Sanath Kumar et al. 2003 India Salmonella meat homogenate (25 g) 6(.) 6(.) 10862 Carcajal et al. 1998 Peru 1991-1992 Vibrio meat homogenate (.) 51 (1) 0.02 6783 Karunasagar et al. 1990 India Vibrio meat homogenate (50 g) 2 (0) 0.00 2 (0) 0.00 2(1) 0.50

262 2(1) 0.50 Ready-To-Eat 91 Kisla Duyga et al. 2008 Turkey Vibrio surface swab 25(3) 0.12 25(21) 0.84 Retail 791 Lu et al. 2006 USA 2001-2002 Vibrio liquid rinse (40 g) 22(7) 0.32 22(9) 0.41 9155 Normanno et al. 2006 Italy 2001-2004 E. coli 600(21) 0.04 Salmonella meat homogenate (25 g) 600(1) 0.00 Vibrio 600(17) 0.03 Vibrio 600 (47) 0.08 625 Sindhu et al. 2006 India 2003-2004 Vibrio meat homogenate (25 g) 1(0) 0.00 6(2) 0.33 8399 Vuddhakul et al. 2006 Thailand 2000-2001 Vibrio meat homogenate (.) 6(1) 0.17 20(4) 0.20 9590 Ullmann et al. 2005 Germany 22/06/1905 Aeromonas meat homogenate (10 g) 4(3) 0.75 9975 Elhadi et al. 2004 Malaysia 1998-1999 Vibrio meat homogenate (25 g) 50(12) 0.24 9818 Parisi et al. 2004 Italy 2000-2002 Salmonella meat homogenate (25 g) 513(0) 0.00 25 (0) 0.00 Vibrio 25(11) 0.44 513(219) 0.43 2341 Pereiraetal. 2001 Portugal 1998-1999 Vibrio meat homogenate (50g + water) 7(2) 0.29 3772 Buck, J.D. 1998 USA Vibrio liquid rinse (.) 2(1) 0.50 17(4) 0.24 5(5) 1.00 8(6) 0.75 13426 Chaicumpa et al. 1995 Thailand Salmonella meat homogenate (10 g) 50(8) 0.16

263 ^Refld is the reference identification number. Blank cells indicate the information is the same as the cell above. Period indicates information was not reported in the study. Table 5. Results of data extraction on studies investigating Clam presented by point in food chain

Number of Point in Country Prevalence Pub. Sampling Samples Food ReflD' Authors of Bacteria Sample Type (Sample weight) (if Year Year (Samples Chain Sampling applicable) Positive) Live from farm 8038 Fuenzalida et al. 2007 Chile 2006 Vibrio meat homogenate (50 g) 20(0) 0.00 8023 Marlina et al. 2007 Indonesia 2003-2005 Vibrio meat homogenate (.) 35(13) 0.37 3772 Buck. J.D. 1998 USA Vibrio liquid rinse (.) 9(2) 0.22 10(9) 0.90 Processing 2284 Sanjeev et al. 2002 India Vibrio 5(0) 0.00 3120 Sanjeev et al. 2000 India Vibrio 4(2) 0.50 4(2) 0.50 5(4) 0.80 5(5) 1.00 Retail 7214 Luan et al. 2008 China Vibrio liver (.) 100(64) 0.64 83 89 Shabarinath et al. 2007 India Salmonella meat homogenate (.) 6(2) 0.33 6(3) 0.50 8929 Cai et al. 2006 China Vibrio meat homogenate (25 g) 50(11) 0.22 50(13) 0.26 625 Khamphouth et al. 2006 India 2003-2004 Vibrio meat homogenate (25 g) 9(5) 0.56 791 Lu et al. 2006 USA 2001-2002 Vibrio liquid rinse (40g) 5(1) 0.20 10(2) 0.20

265 5(2) 0.40 10(4) 0.40 20(5) 0.25 20 (7) 0.35 13(7) 0.54 8756 Miwa et al. 2006 Japan 2002-2004 Vibrio 10(10) 1.00 10(10) 1.00 8399 Vuddhakul et al. 2006 Thailand 2000-2002 Vibrio meat homogenate (.) 38(0) 0.00 5(1) 0.20 135(2) 0.02 17(5) 0.29 932 Kumar et al. 2005 India E. coli meat homogenate (.) 10(7) 0.70 22(18) 0.82 1131 Surendraraj et al. 2005 India E. coli 8(8) 1.00 8(8) 1.00 9818 Parisietal. 2004 Italy 2000-2002 Salmonella meat homogenate (25 g) 57 (0) 0.00 Vibrio 57 (20) 0.35 9931 Yano et al. 2004 China 2001,2002 Vibrio meat homogenate (.) 14(3) 0.21 14 (4) 0.29 10434 Sanath Kumar 2003 India Salmonella meat homogenate (25 g) 20(3) 0.15 20 (3) 0.15 11396 do Nascimento et al. 2001 India E. coli meat homogenate (25 g) 48 (3) 0.06 2341 Pereira et al. 2001 Portugal 1998-1999 Vibrio meat homogenate (50g + water) 1(0) 0.00 7(3) 0.43 6(4) 0.67 4(4) 1.00 3772 Buck.J.D. 1998 USA Vibrio liquid rinse (.) 8(0) 0.00

266 6(3) 0.50 5(3) 0.60 9(7) 0.78 26(11) 0.42 17(14) 0.82 22 (16) 0.73 20(18) 0.90 6783 Karunasagar et al. 1990 India Vibrio meat homogenate (50 g) 26 (0) 0.00 26 (3) 0.12 26(7) 0.27 26(18) 0.69 10862 Carvajal et al. 1998 Peru 1991-1992 Vibrio meat homogenate (.) 17(.) Not reported 10591 Hara-Kudo et al. 2003 Japan 2001 Vibrio meat homogenate (25 g) 171(24) 0.14

Refld is the reference identification number. Blank cells indicate the information is the same as the cell above. Period indicates information was not reported in the study.

267 Table 6. Results of data extraction on studies investigating Miscellaneous species presented by point in food chain Number Point in of Prevalence Food ReflD^ Species Details Authors P"" Bacteria Sample Type (Sample Samples (if Chain Sampling (Samples applicable) Positive) Live from farm 8851 "small mussels, Fuenzalida 2006 Chile 2004, Vibrio meat homogenate (25 g) 204 (108) 0.53 clams, oysters" et al. 2005 Processing

8072 "crustacean Sagoo et 2007 UK 2003 E. coli meat homogenate (25 g) 35 (2) 0.08 shellfish" al. Salmonella meat homogenate (25 g) 35 (0) 0.01 Vibrio meat homogenate (25 g) 35 (0) 0.01 "molluscan E. coli meat homogenate (25 g) 16 (2) 0.13 shellfish" Salmonella meat homogenate (25 g) 16 (0) 0.01 Vibrio meat homogenate (25 g) 16 (2) 0.13 Retail 8072 "crustacean Sagoo et 2007 UK 2003 E. coli 259(27) 0.10 shellfish" al. Salmonella 259 (0) 0.00 Vibrio 259 (4) 0.02 7768 "shellfish" Van et al. 2007 Vietnam 2004 Salmonella 50(9) 0.18

268 9656 "clam, oyster, Hwang et 2004 Taiwan E. coli 79(13) 0.17 green mussel and al. cuttle fish" 78(34) 0.44 14127 "oyster and clam" Yuan et al. 1993 Taiwan Aeromonas meat homogenate (10 g) 23 (19) 0.83

"shrimp and crab" 25(21) 0.84

^Refld is the reference identification number. Blank cells indicate the information is the same as the cell above. Period indicates information was not reported in the study.

269