Biosecurity Oata

BIOSECURITY
AND THE
ORNAMENTAL FISH INDUSTRY

FUTURE PROOFING THE INDUSTRY
ORNAMENTAL AQUATIC TRADE ASSOCIATION (OATA)
The notes and recommendations in this report are based on the limited information presented. Any member choosing to act on the information included here must do so on that basis. No liability for loss occasioned to any business or the author or OATA LTD can accept person taking action or refraining from taking action, as a result of any material in this document. OATA 2006.
Ornamental Aquatic Trade Association, Wessex House, 40 Station Road, Westbury, Wiltshire, BA13 3JN, UK Tel: 0870 0434013, Fax: 01373 301236 info@ornamentalfish.org, www.ornamentalfish.org
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CONTENTS
Questionnaire 1 Questionnaire 3 Contents Questionnaire 2 Questionnaire 4 Risk reduction strategies
CONTENTS
CONTENTS INTRODUCTION STRUCTURE OF THE REPORT AIMS OF THE REPORT QUESTIONNAIRES HOW TO USE THIS REPORT WHICH QUESTIONNAIRE TO USE RISK REDUCTION STRATEGIES WHAT THIS REPORT DOES NOT AIM TO DO 1.0 WHAT IS BIOSECURITY? 1.1 WHY DOES BIOSECURITY MATTER? 2.0 RISK ANALYSIS 2.1 HAZARD IDENTIFICATION 2.2 RISK ASSESSMENT UNCERTAINTY IS THE ONLY CERTAINTY! CONSEQUENCES!!! 2.3 RISK MANAGEMENT THREE TRUISMS A PRACTICAL APPROACH 3 STAGE DISEASE CONTROL SYSTEM MANAGEMENT OF PATHOGENS THAT ARE UBIQUITOUS 2.4 RISK COMMUNICATION
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CONTENTS
Questionnaire 1 Questionnaire 3 Contents
Questionnaire 2 Questionnaire 4 Risk reduction strategies
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CONTENTS
2.5 HACCP (HAZARD ANALYSIS AND CRITICAL POINT CONTROL) TAKING RISK ANALYSIS ONE
STEP FURTHER
DECIDING WHAT A CRITICAL CONTROL POINT IS EXAMPLES OF POTENTIAL CRITICAL CONTROL POINTS 3.0 DECIDING WHICH DISEASES YOU WANT TO CONTROL 3.1
ACCEPTABLE LEVEL OF RISK
TOLERANCE OF RISK REAL OR THEORETICAL RISKS 3.2 USING A RISK EVALUATION TABLE RISK TOLERANT OR RISK AVERSE A DECISION YOU MUST MAKE! ICH, KHV AND SVC EXAMPLES OF THE USE OF RISK EVALUATION TABLES REALITY TEST 3.3 DECISION TREE FOR DECIDING WHICH PATHOGENS AND DISEASES ARE OF CONCERN 3.4 TRAFFIC LIGHT SYSTEM 4.0 KEEPING THE BAD BUGS OUT!! CAN WE PROTECT OURSELVES? 4.1 CHOOSING YOUR FISH SUPPLIER THE EU APPROVED FARM MODEL THE LOGIC OF THE EU CONDITIONS 4.2 ACCEPTING RETURNS FROM CUSTOMERS 4.3 SIZE OF IMPORT CONSIGNMENTS OR DELIVERIES 4.4 RISK AND SUPPLY CHAIN CHARACTERISTICS WHY FISH DISEASES BREAK OUT MULTIPLY THE SOURCES - MULTIPLY THE RISK?
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CONTENTS
5.0 STOPPING THE BAD BUGS SPREADING!!! 5.1 ARE MY FISH DISEASE FREE? FREE OF DISEASE SPECIFIC PATHOGEN FREE 5.2
QUARANTINE AND ACCLIMATISATION
CAN FISH BE QUARANTINED? ACCLIMATISATION PREVENTATIVE ACCLIMATISATION 6.0 FUNDAMENTAL ISSUES 6.1 WHAT CAUSES DISEASES IN FISH POPULATIONS? 6.2 IDENTIFYING THE PATHOGEN CAUSING A DISEASE 6.3 DIFFERENT TYPES OF PATHOGEN FOUND EVERYWHERE OR NOWHERE ALWAYS A PATHOGEN? OR JUST ON SPECIAL OCCASIONS 6.4 HOW NEW OR EMERGING DISEASES APPEAR 6.5 CHARACTERISTICS OF NEW DISEASES 6.6 RATES OF PATHOGEN MULTIPLICATION 6.7 TYPES OF FISH DISEASE TRANSMISSION VERTICAL DISEASE TRANSMISSION (BETWEEN GENERATIONS) HORIZONTAL DISEASE TRANSMISSION (BETWEEN INDIVIDUAL FISH) 6.8 CHARACTERISTICS OF PATHOGENS THAT DETERMINE THE LIKELIHOOD OF DISEASE PRESENCE OR ABSENCE OF PATHOGEN HOW EASILY THE DISEASE IS TRANSMITTED POOR HUSBANDRY
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CONTENTS
DEAD AND MORIBUND FISH DISEASE SPECIFICITY INFECTIVE DOSE OR VIRULENCE OF PATHOGEN AVOIDING AN INFECTIVE DOSE PATHOGEN PERSISTENCE 6.9 INFECTION ROUTES DIRECT CONTACT INHALATION INGESTION VECTORS AND FOMITES INTERMEDIATE HOSTS 6.10 DISEASE TRANSMISSION ROUTES WATERBORNE EXAMPLES OF VECTORS AND FOMITES FISH OTHER LIVING ORGANISMS PARASITES FOOD FAECES CLOTHES AND PERSONAL CONTACTS 7. SYSTEM OR FARM MANAGEMENT 7.1 BASIC WORKING PRACTICES 7.2 WATER MANAGEMENT
A. B.
FLOW THROUGH SYSTEMS RECIRCULATION

SYSTEMS
C. INFECTION OR REINFECTION? WHICH IS THE GREATER PROBLEM?

D.
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CONTENTS
ULTRA VIOLET SUNLIGHT CONVERSION FACTORS EFFECTIVE DOSE OF UV
E.
OZONE ADVANTAGES DISADVANTAGES REDOX POTENTIAL EFFECTIVE LEVELS
F. CALCULATION OF TURNOVER TIMES 7.3 STOCKING OF SYSTEMS DRAINING, DRYING AND TREATMENT FALLOWING (EMPTYING SYSTEMS) BETWEEN STOCKINGS CONTINUAL STOCKING 7.4
VENTILATION
DRY SURFACES VS WET SURFACES AS A HARBOUR FOR PATHOGENS 7.5 BIO FILTRATION AND DISEASE PREVENTION HOW BIO FILTERS CAN REDUCE PATHOGEN NUMBERS IN A SYSTEM OR INCREASE THEM!! 8.0 DISINFECTION 8.1 DEFINITIONS 8.2 DISINFECTION OF WET SURFACES DISINFECTANTS DONT DISINFECT DIRT THE EFFECTIVENESS OF DIPPING, WASHING AND SCRUBBING
DISINFECTANTS WITH
8.3 HUMAN SKIN
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CONTENTS
8.4 CLOTHES
BACTERIA ON THE SKIN SKIN CLEANSING AND DISINFECTION BARRIERS FOR THE SKIN
DISINFECTION OF CLOTHES 8.5 FOOTWEAR 8.6 EQUIPMENT 8.7 DISCHARGE WATER 8.8 DISINFECTING SYSTEMS 9.0 IDENTIFYING DISEASES TECHNIQUES FOR DIAGNOSING AND SCREENING FOR SIMPLE ON-SITE INVESTIGATIONS 9.1 WATER QUALITY 9.2 PHYSICAL SIGNS IS THE PROBLEM INFECTIOUS? DISEASE CALLING CARDS CLINICAL SIGNS CLINICAL SIGNS CAN DECEIVE THE EYE MORE COMPLEX ON-SITE INVESTIGATIONS 9. 3 HAND LENS 9.4 AGGLUTINATION TESTS 9.5 MICROBIAL SAMPLING 9.6 BLOOD SAMPLING 9.7 TAKING SAMPLES FOR MICROSCOPY LABORATORY INVESTIGATIONS 9.8 SHOULD YOU HAVE AN ON-SITE LABORATORY?
DISEASES
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IN FAVOUR AGAINST 9.9 MICROSCOPY 9.10 CULTURE OF PATHOGENS 9.11 IMMUNOLOGICAL TECHNIQUES A. ELISA - (ENZYME LINKED IMMUNO-SORBANT ASSAY) CAPTURE ELISA SCREENING ELISA
B. C.
CONTENTS
IHC (IMMUNO-HISTOCHEMISTRY) IFAT - (INDIRECT FLUORESCENT ANTIBODY TEST)
D. ISH (In-situ Hybridisation) and FISH (Fluorescent In-situ Hybridisation) 9.12 MOLECULAR TESTS PCR -POLYMERASE CHAIN REACTION WHAT IS PCR? SINGLE AND NESTED PCR INTERPRETING RESULTS FROM PCR TESTS 9.13 CELL LINE ISOLATION OF VIRUSES 9.14 SUMMARY TABLE OF USES OF ON-SITE AND LABORATORY TESTS 10.0 TREATMENTS 10.1 THE FISHS IMMUNE SYSTEM - A NATURAL DEFENCE AGAINST DISEASES? FISHS BASIC RESPONSE TO PATHOGENS STRESS AND THE FISHS IMMUNE SYSTEM KEY FACTORS AFFECTING THE IMMUNE SYSTEM STRESS LOW
WATER TEMPERATURES
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CONTENTS
10.2 CHEMICALS 10.3 ANTIBIOTICS 10.4 PROBIOTICS 10.5 PREBIOTICS COMPARISON

TYPES OF THE USE AND EFFICACY OF SOME DIFFERENT TREATMENT
10.6 IMMUNOSTIMULANTS 10.7 IMMUNISATION AND VACCINATION HISTORY PROTECTION BY VACCINES SPECIFIC IMMUNITY NON-SPECIFIC IMMUNITY TYPES OF IMMUNITY AND IMMUNISATION NATURAL OR INHERITED NATURAL EXPOSURE KILLED VACCINES LIVE ATTENUATED VACCINES METHODS OF VACCINATION IMMERSION INJECTIONS ORAL BOOSTER
QUESTIONNAIRES
QUESTIONNAIRE QUESTIONNAIRE QUESTIONNAIRE QUESTIONNAIRE 1 2 3 4 YOUR BUSINESS ATTITUDE TO RISK IDENTIFYING PATHOGENS OF CONCERN TO YOU ASSESSMENT OF PATHWAYS BY WHICH PATHOGENS SITE BIOSECURITY APPRAISAL
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CONTENTS
MAY ENTER A SITE
SUMMARY OF RISK REDUCTION STRATEGIES

GENERAL POLICY AND MANAGEMENT GENERAL HUSBANDRY AVOID BRINGING DISEASE ONTO A SITE AVOID MOVING DISEASE OFF A SITE HIGH RISK AREAS , SUCH AS HATCHERIES, NURSERIES AND DISEASE INVESTIGATION AREAS.
APPENDICES
1 OATA WATER QUALITY CRITERIA 2 - EUTHANASIA 3 DISPOSAL OF CLINICAL WASTE 4 - LETHAL DOSE OF UV FOR VARIETY OF ORGANISMS 5 EFFICACY OF OZONE TYPICAL DOSAGE AND REACTION TIMES FOR A VARIETY OF ORGANISMS 6 - VETERINARY MEDICINES 7 - NATIONAL AND INTERNATIONAL LAW 8 - DEFRAS ADVICE ON AVOIDING SVC 9 - EMERGENCE OF INFECTIONS 10 - ZOONOSES 11 - FURTHER HELP BIBLIOGRAPHY (AVAILABLE ON THE OATA MEMBERS LOGIN SITE) WEBLIOGRAPHY (AVAILABLE ON THE OATA MEMBERS LOGIN SITE)
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CONTENTS
INTRODUCTION
The movement of fish, invertebrates and plants across international frontiers and within countries between fish farmers, collectors, wholesalers and retailers is essential for our industry. The fish and plants we move intentionally may also carry viruses, bacteria and larger organisms such as fungi, nematodes and crustacea. History has shown that most of these are apparently harmless. However, recent history has shown that some of these hitchhikers, albeit rarely, can cause serious diseases. The impact KHV has had on the market for Koi in the UK serves as a recent painful reminder of the problems that can arise when there is a loss of confidence among the buying public in the fish we sell. Governments take notice of these diseases, in particular the impact they have had (or might have) on native fish stocks or on food producing aquaculture industries. This document is intended to alert all interest groups to the responsible and pro-active stance that the industry is taking to safeguard both its future and the interests of the wider community. Many ornamental fish are kept in closed systems and so problems are contained and pose negligible risks outside of those systems. This document has been written primarily to help all contributors to the industry make informed decisions on where to buy fish and how their management of those purchases can minimise the chances of either receiving or passing on disease or health problems. Each individual business must decide to what extent, if at all, they apply the information provided. Even if every suggestion included in this document is fully implemented it cannot be guaranteed that problems will never be encountered, however the risks will be considerably reduced. The choice is not between a risky purchase and an absolutely risk free purchase (zero risk does not exist), rather it provides indications on how the risks may be reduced to a level acceptable to your business, consumers and to Government agencies. In December 2001 OATA wrote and distributed the document Koi Herpes Virus-KHV. A number of Governments specifically requested copies, at least one inter-Government agency posted the whole document on their website. It is hoped this document will demonstrate, and further, raise the credibility of the industry. We must reiterate that this document is what we believe to be, at the time of writing, sound information concerning risks and the management practices that may be employed to avoid or manage diseases. Since the information for aquatic animals is incomplete compared to terrestrial vertebrates, it is almost inevitable that advice about best practice will change with time. That said, universally accepted basic husbandry rules will always apply, and will always help prevent disease outbreaks.
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INTRODUCTION
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INTRODUCTION
STRUCTURE OF THE REPORT

This report is a working tool. It can be read straight through in one sitting but it is not designed for that purpose. It is designed so that: Each section can be used for reference separately but cross-referencing to other sections or topics is easy. Information is available in bite size chunks. Diagrams and tables are used for clarity and reinforcement of points made in the text. You can read about a topic at the level relevant to your situation. Thus there are overviews, more detailed descriptions and lists of scientific and other references for each topic. You can choose and easily find what information you need, when you need it. It is accessible. It is available in both hard copy and electronic format enabling rapid access to particular topics and cross-referencing by hyperlinks.
Brief and Practical
Questionnaires
Risk Analysis and HACCP
Deciding which are the bad bugs
Keeping out the bad bugs
Stopping the bad bugs spreading
Disease treatments, Identifying Disease
Site biosecurity and risk reduction strategies
Detailed appendices and a webliography of Internet resources to be available via website Technical and Detailed
AIM OF THE REPORT

A recent report from the UK Government stated, Livestock will always be at risk from new and emerging diseases, even as current problems are overcome. Proactive farm health planning will help the livestock industry as a whole move forward. This report and the suggestions contained in it will contribute to health plans relevant to our industry devised by individual businesses (exporters, farmers, importers, wholesalers and retailers) seeking to ensure, as far as they are able, the health of livestock upon which our industry depends. As the report looks at diseases as a generic issue it is hoped the implementation of some ideas will help future proof the industry.
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INTRODUCTION
QUESTIONNAIRES
The questionnaires can be used to assess how bio-secure your set up and those of your suppliers and customers are. They: Allow estimates of the risks associated with various scenarios to enable you to manage biosecurity issues now and take action, if wished, needed or required, to raise standards by establishing future targets and taking practical steps to achieve them. Establish a guide to good practice in some areas such as sourcing livestock and enable you identify how current practice might be improved. Provide outline background information on topics such as disease identification and vaccination that will help understanding of key issues.
By using the questionnaires, this report can be used to identify where you stand currently on biosecurity issues and if appropriate set future targets. The report is designed to consolidate in one place information that will enable each business to develop plans that might help decide buying policies, sales policies and work patterns to help prevent the introduction of unwanted organisms on to site or to detect them and prevent them spreading around once they are present.
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INTRODUCTION
R eview annually o r as required
HOW TO USE THIS REPORT

Determine yo ur business's attitude t o risk
Decide which pat ho gens or diseases yo u are co ncerned abo ut
Assess o r re-assess how these patho gens might get onto yo ur site
Unacceptable Risk
Review management of bio securit y and implement changes
Acce pta ble
Assess or re-assess t he biosecurity of yo ur suppliers
Unacceptable R isk
Negot iat e changes
or
Change supplier
Acceptable
Assess or re-assess t he bio securit y o n yo ur o wn site
Unacceptable R isk
Review management o f biosecurity on yo ur o wn site and implement changes
Accept able
WHICH QUESTIONNAIRE TO USE
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INTRODUCTION
Questio nnaire 1
Questio nnaire 2
R eview annually o r as required
Questio nnaire 3
Questio nnaire 4
Questio nnaire 4
Have you told everybody about your biosecurity policy?

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INTRODUCTION
RISK REDUCTION STRATEGIES
As an aide memoire and checklist a range of risk reduction strategies are listed. It is impossible to list all possible strategies in this document for all circumstances or confine such lists to those relevant to particular business types or situations. Thus not all will be relevant in al circumstances. The strategies can be used separately from or in conjunction with the rest of the report. Any risk reduction strategies will only be as good as the least effectively applied measure. Pathogens are likely to exploit any lapses or gaps.
WHAT THIS REPORT DOES NOT AIM TO DO

It does not tell you what to do but merely gives you the principles and access to information sources from which you can decide how to manage biosecurity issues to meet the needs of your business. The hazards and hence the risks will vary from site to site and with time on any particular site. Thus each site must address the specific issues relevant to that individual site and any plans must be reviewed regularly. It does not repeat detailed descriptions of even common diseases, their treatment or diagnosis. Such descriptions with excellent photographs are available in the many textbooks, scientific journals and websites. This document does not reinvent the wheel nor is the information new but it does bring together many strands of information that many in the industry might not have had ready access to before.
THE FUTURE
This report will be revised as new information becomes available or gaps are identified. The most up to date information will be posted in the OATA members login area on our website.
CHAPTER 1 WHAT IS BIOSECURITY?

Biosecurity is a general description of the measures you may take to protect your business (or for Governments, their country) by taking actions to prevent the entry of new or unwanted organisms, especially infectious agents. It can also include species that may have an adverse effect on indigenous species or ecosystems e.g. invasive aquatic weeds or pest species such as the Tobacco Whitefly; (Bemesia tabacci) or Colorado beetle (Leptinotarsa decemlineata) from arriving and/or controlling or eradicating those already present. This report will only deal with pathogens and the diseases that they may cause. Any business importing, wholesaling or retailing fish, invertebrates or plants has more or less formally been applying biosecurity measures already. You may think this sounds just like a fancy name for what most of us call disease prevention and you would be right!
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1.0 WHAT IS BIOSECURITY
Disease prevention
Biosecurity
Definitions of biosecurity
Biosecurity has no single definition but a working definition for our purposes could be The sum of all procedures in place to protect ornamental aquatic organisms (fish, invertebrate or plant) from contracting, carrying or spreading disease. The practical steps to achieve this will include: Deciding which diseases you want to control (Which bugs are bad?) then Deciding how to keep the bad bugs out!! Or if that fails Stopping the bad bugs spreading!!!
In this document OATA has tied together information that may help members make better informed decisions on a more formally structured basis. The level of biosecurity given by taking or omitting any of the actions or options mentioned is indicative only.
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WHAT IS BIOSECURITY
1.1
WHY DOES BIOSECURITY MATTER?
IN GENERAL
Biosecurity is attaining the status of the next buzzword and will have a long shelf life in the minds of politicians and the like. Fuelled by the costs associated with recent disease outbreaks they are very sensitised to the issues. For instance it is estimated that the foot and mouth disease outbreak in the UK during 2001 cost of 8 billion ($12 billion). A recent report from the Cabinet Office in the UK estimated that the potential cost of importing from Europe or Scandinavia of Gyrodactylus salaris, a disease of salmon, would be in the region of 1 billion ($1.8 billion) per annum. Increasingly non-native species of plant and animal are being viewed in much the same biosecurity terms as diseases. The information in this report will help address those issues also, though it is not its the primary aim.
COMMERCIALLY - DISEASES ALWAYS COST MONEY!!!! (EVEN IF YOU DONT GET THEM)
Whether in costs of prevention or in dealing with the problems of an outbreak, (direct economic loss, loss of consumer confidence or action taken by Governments) it is an inescapable fact that diseases cost money. Each business has to make a management decision, based on its appraisal of and policy towards risk, and choose where it meets those costs. The choices are to either avoid a disease or cope with its consequences. Inevitably there will be costs. Thus the ideas presented in this document are not and cannot be free of cost in either time or finance, but they are often cost effective compared to an outbreak of disease. An example of the impacts of disease on the industry can be seen by the records of ornamental fish exports from Israel where koi herpes virus came to prominence during the spring of 1998. (Israel exports koi, goldfish and some tropical fish though the precise breakdown between the categories is not known.) Compared to 1997 the quantity of ornamental fish exported (in terms of freight weight) to the UK reduced by 30%, 43% and almost 60% in 1999, 2000 and 2001 respectively. Indications of a recovery only became evident during 2002. This example also demonstrates that the market mechanism works very rapidly to avoid sources of fish that are identified by the industry as posing a risk.
CHAPTER 2 RISK ANALYSIS
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2.0 RISK ANALYSIS
Risk analysis is an everyday occupation for all businessmen. Will we make a profit if we stock fish from this or that supplier? What are the risks of fish losses if we buy from this or that source? In almost all cases decisions are made on the best information available: almost invariably there are gaps in that information about which judgements have to be made. Clearly the more definite and complete the information the more likely you are to be able to make a correct decision. Risk Analysis is usually broken into four interrelated parts which are described in the table below:
The four parts of risk analysis
More practically and bluntly described in everyday English as: Deciding which diseases you want to control. (Which are the bad bugs?)
Hazard identification:
What organisms pose a risk? How can they be identified?
Risk assessment:
Under what conditions will they pose a risk? How great is the risk? What will be the consequences if a disease is:
What happens if we dont keep the bad bugs out?
-introduced on to your site or -is passed from your site to a clients or customers site What happens if they spread?
Risk management:
What can be done to limit the risks? What can be done to prevent the spread of pathogens? How do you keep the bad bugs out and stop them spreading if they get on site?
Risk communication:
Which this document hopes to achieve!
Telling all relevant people including suppliers, staff and customers about the results of the analysis and what is required of them and the benefits to be gained.
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2.1 HAZARD IDENTIFICATION 2.0 RISK ANALYSIS
The three main issues here are to decide: Which pathogens you are concerned about? The ways in which they might arrive or be transferred? How they can be identified practically?
2.2
RISK ASSESSMENT
UNCERTAINTY IS (ALMOST) THE ONLY CERTAINTY

Compared to mammalian diseases relatively little (and in most cases very little) is known about individual fish diseases. The biology of most fish diseases (how they can spread? how infective they are? etc), especially those of non-food fish, is little understood, and thus assessments of and solutions to the problems they can cause must, at this point, be based on an incomplete data set. New diseases can and do emerge. The virus responsible for KHV was only identified a few years ago.
CONSEQUENCES!!!
Any assessment of risk is incomplete without a review of the consequences. It is pointless putting time, effort and money in to protecting your business from an organism that causes no problems ( to the fish, you or the wider environment). Each business will need to balance the risks and outcomes associated with presence of particular pathogens in their stocks before deciding how or if they try to manage them or the disease(s) they may cause. Each business must ultimately make a choice of strategy based on its particular circumstances and attitude to risk and of course what is required by law.
2.3
RISK MANAGEMENT
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2.0 RISK ANALYSIS
The golden rule is that prevention is better than cure.
Three truisms worth remembering:

If a pathogen isnt present on a site it cant cause a disease on that site If a pathogen isnt present on a site it cant be transmitted from that site. If the pathogen is present at a suppliers site then better it remains there: curative treatment to eradicate the pathogen prior to fish leaving that site may be required.
A PRACTICAL APPROACH
Pathogens are invaders. Just as with any invaders a three stage approach is best adopted : Approach Prevention Where to apply it at source or upon arrival Detection of newly arrived unwanted pathogens As soon after arrival as possible Techniques to apply Exclusion from the supply chain by vaccination/health screening, prophylaxis etc. Isolation/quarantine. Screening using appropriate method to ensure early detection. Rapid action, including treatment and/or euthanasia to eliminate problem before it spreads. As for prevention and detection of newly arrived unwanted pathogens.
Long term reduction of the impact and control of infection
On site continual
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2.0 RISK ANALYSIS
Managem ent of pathogens that are not ubiquitous
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2.0 RISK ANALYSIS
Isolation, preventative acclimatization, treatment and management to eliminate or reduce pathogen loading at each "import" along the supply chain. Treatment on an "as needed" basis on in-coming stocks
Exclusion of pathogen. Closed systems eg. using principles of EU approved farm model to prevent pathogen entering supply chain.
Elimination or reduction by treatment or management prior to "export" from each site along the supply chain.
H igher biosecurity
Lower biosecurity
Some organisms that can cause disease such as Ich and Aeromonas hyrophilia are ubiquitous that is they are found everywhere. Others have a more restricted distribution and can potentially be more easily avoided.
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2.0 RISK ANALYSIS
2.4
RISK COMMUNICATION
A risk analysis is of no use at all if it remains in your head or on a piece of paper in the desk. It is vital that you communicate to others: what you think the risks are the risks you are not prepared to accept how you will exclude those risks that you are not prepared to accept the risks you are prepared to accept how you will manage those risks you are prepared to accept (even if that includes doing nothing)
Who should you be telling? fish suppliers and their staff and in turn the producer or collector if possible your staff customers and other visitors to your site
This might be achieved by briefly stating your position on paper and possibly even in contracts of supply. This document is designed to help you reach your own conclusions on a range of issues. The questionnaires may help in crystallising your views.
2.5
HACCP TAKING RISK ANALYSIS ONE STEP FURTHER
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2.0 RISK ANALYSIS
HACCP is the acronym for Hazard Analysis and Critical Control Point. This approach, which first arose in the food industry, seeks to identify hazards. It then identifies critical stages at which controls can most effectively be applied, to minimise or eliminate a risk. These are known as CCPs Critical Control Points. CCPs might include which fish you allow on to your site species, their origin, the supply chain used and the like. On your site critical control points might include isolation of new arrivals, preventative treatments, mixing (or not) of fish from different sources, movement of stock or equipment between different systems. All of these matters you have or could have control over and the controls you put in place and how they are managed can dramatically alter how secure your site and business is from problems caused by fish diseases. To identify CCPs you need to clearly layout all the livestock procedures on your site.
HACCP OVERVIEW
Consider all the potential biosecurity hazards that could occur on your site. Identify and rank significant hazards or critical concerns. Consider how to limit, avoid or eliminate the identified hazards. List the methods used and confirm preventative measures are in place. Consider appropriate action, corrective action or alternative plans. Enable and ensure record keeping is in place to facilitate monitoring of activities and outcomes. Put in place a system to check all the actions are actually taking place.
Of course none of this will work unless the outcomes of any review are communicated effectively to staff and they are trained to put recommendations into action. It may be very valuable to get them involved in the whole process, as they are actively involved in what really happens on a daily basis.
DECIDING WHAT A CRITICAL CONTROL POINT IS
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2.0 RISK ANALYSIS
CC P DECIS ION TREE
Hazard
Modify the step, process or product
Yes
No
Is control necessary?
Are preventative measures in place?

Yes Yes
No
Do the measures reduce the hazard?
No No
Could hazards reach unacceptable levels?
Yes
Yes
Will a subsequent step reduce or eliminate the hazard?
No
NOT A CCP
CCP
* Flow chart taken from "Code of Practice for Bio-security in the Egg Industry" RIRDE Publication No. 01/102 Project No>MS001-02
EXAMPLES OF POTENTIAL CRITICAL CONTROL POINTS

The suggestions below are indicative only. They can only be applied to an appropriate extent for a given site e.g. retailers cannot control the people who come onto the site but a farmer or wholesaler may. 1. What do you allow onto your site? o o o which may be influenced by o o o o o Fish, which fish and where they come from Suppliers biosecurity management policy and practice Prophylactic treatment of fish or plants prior to export or dispatch to your site Your water supply Equipment (vehicles, vets etc) you allow on your site. Entry of animals e.g. birds, rodents, snails Personnel (including clothing, boots etc)
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2.0 RISK ANALYSIS
Staff training in and knowledge of fish health, animal transport and phyto-sanitary legislation
2. Movements of live animals, plants and equipment on site o o which may be influenced by o o o o o o Acclimatisation/quarantine/isolation of new arrivals Staff knowledge and training Policy on the use and movement of equipment e.g. buckets, hoses, nets etc and water between different systems Fish health maintenance and monitoring programmes Water quality management and monitoring programmes Proper maintenance of UV or ozone systems if installed Isolation of any on-site disease investigation area Ventilation
3. What you do when your system fails and you have a disease problem o o o o o Be aware of any local legislation concerning requirements to notify disease Be aware of local legislation on movement of animals or plants after a disease outbreak. Ensure staff are trained to detect and react to disease outbreaks including legal or ethical requirements concerning euthanasia and disposal of mortalities Be aware of diagnosis techniques and treatment (including the rules governing these) Build up a relationship with a local veterinary surgeon.
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2.0 RISK ANALYSIS
CHAPTER 3 DECIDING WHICH DISEASES YOU WANT TO CONTROL. (Which are the bad bugs!)
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3.0 DECIDING WHICH DISEASES YOU WANT TO CONTROL
3.1
ACCEPTABLE LEVEL OF RISK
First you must decide what level of risk or threat you will tolerate? Remember some organisms found on fish can causes disease in humans these are known as zoonoses. (See Appendix 10). Almost inevitably cost and personal attitude will be a driver in deciding what level of risk your business will tolerate (unless of course legislation decides the matter for you). Massive costs controlling a problem that has no impact are unlikely to be acceptable.
Tolerance of risks
Each business must make its own decision on what is an Acceptable Level Of Protection (you will often see this reduced to the acronym - ALOP), for itself and its customers based on experience and their own attitude to risk.
Real or theoretical risks

Before going any further with your list, its worth highlighting what conditions must be met before an organism can cause you a problem: The pathogen must be present on the site from which you buy fish; more specifically it must be present in the population of fish from which those you purchased were obtained. The pathogen must be able to survive for a sufficient time in sufficient numbers to reach your site and infect your stock. Any susceptible stock on your site must be exposed to the pathogen by a route and in conditions that allows infection.
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3.2
USING A RISK EVALUATION TABLE

(to decide which are the bad bugs)
Probability X Consequences = Risk

When evaluating whether a risk is acceptable two issues are usually taken into account: The probability or chance that a certain policy or action will lead to the introduction of a particular virus, bacteria, fungi, parasite or other pathogen and The consequences of the introduction of that organism to the fish and therefore the business.
RISK TOLERANT OR RISK AVERSE?

It is important to decide if you will tolerate the presence of an organism and manage your fish stocks so that you avoid disease, or whether you wish to totally exclude an organism, so it cannot cause disease. Some pathogens can be excluded from a site. Many others cannot be excluded, but can be managed by taking simple basic husbandry measures, so they do not cause unacceptable harm. Either way you may be particularly concerned about this disease or that parasite. Thus as part of your risk evaluation you should make a list of which pathogens are of greatest concern to you and/or your customers. In some circumstances the presence of an organism, even in the complete absence of any clinical signs of disease will trigger stringent Government action e.g. SVC in the UK. You may be happy to tolerate the high probability of introducing a relatively harmless bacteria.; the consequences of which may be negligible. On the other hand you may only be prepared to tolerate a negligible risk of importing a virus such as KHV which will have catastrophic impacts on either you and your clients or both.
A DECISION YOU MUST MAKE!
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In the risk evaluation table (a blank copy of which can be found for your use at questionnaire 2) below an attempt has been made to give an indicative value for the risk of the scenarios of probability and consequences that might be encountered. The higher the score the greater risk is associated with a particular set of circumstances.
Risk evaluation table
Probability
of introduction Negligible or very low Score Minimal or Negligible Very low Low High Very High 1 3 5 7 9 1 1 3 5 7 9
Consequences of introduction
Low Medium High Very High Catastrophic 3 3 9 15 21 27 5 5 15 25 35 45 7 7 21 35 49 63 9 9 27 45 63 81
Using your own experience and the information contained in this report, you may be able to make an informed estimate of the probability of entry, on to your site, for each pathogen. Past experience and reports from others will enable you to predict the most likely consequences of the introduction of a pathogen on to your site, based on site-specific information such as temperature, stocking density, water quality and food used etc. To an extent both the probability of the entry of a pathogen and the consequences of its introduction are in your own hands. By implementing controls and appropriate management to: suppliers on-site activities(eg acclimatisation, isolation and treatment etc)
you can reduce (to zero in some cases)the probability of a pathogen entering your site, an outbreak of disease and in turn the consequences including disruption and cost can be reduced. It must be your decision about the level of risk you are happy for your business to tolerate. In making a decision loss of reputation and business caused by disease outbreaks are maybe just as valid as biological considerations such as the number of fish dying in a disease outbreak.
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EXAMPLES OF THE USE OF RISK EVALUATION TABLES

Low probability of pathogen arriving but catastrophic consequences KHV or SVC Risk evaluation table
Probability
of introduction Negligible or very low Score Minimal or Negligible Very low Low High Very High 1 3 5 7 9 1
Low Medium High Very High Catastrophic 3 5 7 9
Spring Viraemia of Carp (SVC) and Koi Herpes Virus (KHV) SVC has been rarely encountered in the ornamental fish industry in the UK, but as a notifiable disease it must be reported to the relevant authorities. The consequences are that when found all the susceptible species on a site must be slaughtered and the site disinfected appropriately. In countries where SVC is not notifiable the consequences of the disease may be less extreme, the mortalities caused by the disease are rarely 100%, but the option of exporting to countries like the UK is lost. The mere presence of the virus causing SVC can be catastrophic in countries operating stringent controls, whether or not it is causing disease at the time it is found Measures can be put in place to exclude KHV. The success of excluding KHV will depend on the testing undertaken and the quality of the exclusion and isolation techniques used by suppliers. Once on a site when the temperature is in the correct range it is almost inevitable that catastrophic mortalities of koi will follow.
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Very high probability variable consequences Ich Icthyophthirius multifilis Risk evaluation table
Probability
Ich Icthyophthirius multifilis Ich is found a round the globe. Thus there is a chance of it being present in any group of fish. Its population can grow very rapidly and it can cause serious mortalities, particularly among young fish. However, if properly treated it can be controlled and be of very little problems or consequences.
Reality test?
Past performance, though not 100% reliable, acts as a useful indicator of the effectiveness of your current practice. If your style of trading has meant that no problems have occurred either on your premises or those of customers in the onward supply chain or in the local environment, then that practical prior experience indicates that the operation is of low risk. Even so, we would encourage all businesses to review their operations using the concepts in this document. You could have just been lucky so far!!
3.3 DECISION TREE FOR DECIDING WHICH PATHOGENS AND DISEASES ARE OF CONCERN
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Review and list the range of pathogens that the fish you sell might came into contact with
Determine the potential costs and consequences of the introduction of each pathogen on your
own stock customer's stock business reputation
Decide which diseases to
Exclude from your site
Tolerate
Interact actively with your suppliers to ensure management practices to exclude pathogen
Manage
Ignore as inconsequential
Request health guarantees as part of contract of supply
Determine and implement treatment or management regime required
No further action required
3.4
TRAFFIC LIGHT SYSTEM
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You might adopt a traffic light system of prioritisation for action. A green light or low risk - no or little action required - would be given to Ubiquitous organisms, or strains of them, that cause no problems. On the risk evaluation matrix (section 3.2) the arrival of these on site would be regarded as highly probable, but whose impact would be negligible. It may not be cost effective to try to exclude these organisms (but bear in mind there may be pathogenic strains of otherwise benign species which you might want to exclude). Good general husbandry can be used to ensure they do not become a problem. For instance some internal parasites where the life cycle cannot be completed in captivity (eg Diphylopbothrium). An amber light or medium risk - would be given to species or pathogens That are widely distributed or ubiquitous and that while they can cause serious problems you can tolerate them, cant avoid them or exclude them from the supply chain and because management by effective treatments are available e.g. Trichodina, Ich. A red light or high risk - would be given to. Notifiable diseases. In the UK the main disease of relevance for goldfish and carp is SVC. Very infectious diseases causing high mortalities for which treatment is inappropriate, difficult, expensive or not available. Efforts should be taken to exclude the pathogens causing these diseases (which might include strains of otherwise harmless bacteria) from the supply chain.
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CHAPTER 4 KEEPING THE BAD BUGS OUT!
CAN WE PROTECT OURSELVES?
As pointed out earlier a truism is that if a pathogen is not present in a country or region or on an individual fish producers site it cannot be spread to your site. Many countries require that imports are free of specific pathogens. To achieve this, different standards are incorporated in their import laws. There are lessons to be learnt from some of the model conditions established in these laws. The EU specifies the conditions that must be met for a farm to be approved free of a disease. This model and the issues it raises can be used to establish a qualitative idea of the risks of trading with a particular supplier, whether that be an exporter, importer or wholesaler. You may choose to vary the model, for instance by adding more pathogens you want to exclude from the supply chain, or disregard bits of the model that you regard as irrelevant in your particular circumstance. It is likely that as more Governments, become more nervous about biosecurity issues, that they have already introduced or will introduce changes to their import controls with the aim of keeping fish diseases out. In a few instances there might be a relaxation of excessively tight regulation to avoid international action for imposing unfair controls through bodies like the WTO. However, rules on the national and international transfer of live aquatic (or indeed any) animals and plants are more likely to be gradually tightened than relaxed.
4.0 KEEPING THE BAD BUGS OUT
Buy from sites that can prove they have not got the pathogen or pathogens about which you are concerned. Or
Buy from sites that have treated the fish for the pathogens of concern prior to export Or
Treat the fish for the pathogens of concern during the isolation/acclimatisation period on your own site.
Some treatments may kill many more species of parasite than is intended. This may have positive health benefits and be very useful in reducing not only the pathogen of concern but also in reducing total pathogen loading. Some treatments should not be repeated too frequently eg formalin based remedies so it is best to gather as much information as possible about those used on the fish you purchased in the recent past.
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4.1
CHOOSING YOUR FISH SUPPLIER
THE EU APPROVED FARM MODEL

Below is a synthesis of current and proposed conditions that a designated or approved farm that wishes to export live coldwater fish, susceptible to a notifiable disease (this is a disease a country may try to keep out or eradicate for your business this would equate to a disease you wished to exclude or actively manage), to the EU would be required to meet. You may wish to require some or all of these conditions of your suppliers. Water for the farm is supplied directly from a borehole, spring or well or carried through a pipe, channel or natural conduit, which does not constitute a source of infection. The channel must be under the control of the farm or official services Alternatively water may be supplied by a means that completely inactivates or is known to exclude named pathogens The site is protected from flooding Fish are prevented from swimming upstream on to the farm The fish have been inspected for at least two years by the official authorities to ensure the absence of specified pathogens. The site has to be inspected by vets or fish health experts at a time of year a particular disease might be expected to be seen. The farm keeps updated records of the live fish or eggs entering or leaving the site, including both their source and destination. The farm keeps records of mortalities. Sites should report any abnormal* mortalities, or clinical signs of disease, that caused a significant impact* in the six months prior to dispatch. Have not introduced any fish or eggs in the last two years from a farm of lower health status. That there is no abnormal* mortality or other clinical signs of disease on the day of loading. Packing materials used to transport fish are sterile
* you and your supplier would have to agree what abnormal and significant means. For instance you may think it should include only problems for which no explanation can be given or diseases that fish can be treated though they remain carriers or it could mean any problems explained or not of an infectious nature or not.
THE EU APPROVED FARM MODEL
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Water supplied from borehole well or spring
Site free from flooding
Regular fish health checks Good records kept
No fish from lower health status farm introduced
Routine checks and reports No fish allowed to swim in from rivers or lakes
Healthy fish exported or sent out to customers
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THE LOGIC OF THE EU CONDITIONS

The EU has developed a set of minimum standards that must be applied to fish farms wishing to supply live fish to importers. They are applied to temperate species particularly those susceptible to certain serious diseases. Generally the diseases of greatest concern affect salmon and trout; however it is worth remembering that it is the disease that is notifiable, not the species it is in. These standards for the moment are not applied to tropical fish entering the EU. This is because they are kept in closed isolated systems (aquaria) with no discharge to the natural environment and the likelihood of them or any of their pathogens becoming established in native waters is regarded as minimal. However this situation may be different in different countries around the world and thus these or other controls might be applied to any species by individual businesses or Governments of any country.
.. ARE OF SIGNIFICANCE
The logic behind these standards used in the EU could be applied to any transfer in the world to identify those that might be viewed as potentially risky. Such as: Moving temperate species to temperate zones whether the fish come from another temperate zone or from tropical areas (e.g. goldfish which though temperate in origin are produced in the far east). It is the characteristics of the fish species that counts in this assessment, not the point of origin. Moving tropical species to tropical zones. Already NACA (Network of Aquaculture Centres in Asia) a network including the Governments of many far eastern countries has identified the transfer of ornamental (particularly wild caught) specimens as requiring additional attention.
. TO YOUR BUSINESS ANYWHERE IN THE WORLD

Each country or region will apply its own standards based on the local environment, trade types and volumes and maybe most crucially, each Governments appraisal of what constitutes an acceptable risk. Governments make for countries decisions about acceptable levels of risk. Individual farmers, importers, wholesalers and retailers of ornamental fish can also make these decisions for their own business as a matter of general trading policy or on a consignment by consignment basis.
4.2
ACCEPTING RETURNS FROM CUSTOMERS AND OTHER UNUSUAL SOURCES
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No fish or plants should be accepted from any unknown source, including retail customers, and returned to your holding systems, unless full and effective biosecurity measures are in place to prevent any disease infecting the new fish or your stock. You cannot be sure of where these fish have been or what they have been mixed with.
Beware of hitchhikers on any fish entering your site. These might be present as sub-clinical infections of any pathogen e.g. Ich or even latent infections such as KHV.
4.3
SIZE OF IMPORT CONSIGNMENTS OR DELIVERIES
The risk of losses may be reduced by importing smaller consignments more frequently than perhaps has been the custom in the past. However to be effective this strategy will require investment in effective isolation facilities.
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4.4 RISK AND SUPPLY CHAIN CHARACTERISTICS

Every delivery of fish poses a potential infection risk. That risk is dependant, to a large degree, on the characteristics of the supply chain. Key factors determining the risk in a supply chain include the: number of steps (producer, collector, middlemen exporter, importer, retailer) number of producers or collectors level of disease control used by businesses A retailer who uses two importer/wholesalers, who each buy from two exporters, who each in turn purchase their fish from two farmers or collectors is directly or indirectly exposed to fish, pathogens and any lapses in husbandry or biosecurity on 14 other sites.
Retailer
Wholesalers (1) Exporters (3) Exporters (4)
Wholesalers (2) Exporters (5) Farmer/ Collector (9) Farmer/ Collector (13) Exporter (6) Farmer/ Collector (10) Farmer/ collector (14)
Farmer/ Collector (7) Farmer/ Collector (11)
Farmer/ Collector (8) Farmer/ collector (12)
The number of sites that are involved in a chain can rise dramatically as is illustrated by the examples used in the table below. In exemplar supply chains A, B and C the same doubling principle used in the flow chart above is applied. Supply chain Retailer Importer/Wholesaler Exporters Farmers or collectors Total number of sites in supply chain to retailer A 1 2 4 8 14 B 1 4 16 64 84 C 1 8 64 512 584 30 30 D 1
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If a retailer only wants fish that originate from or carry the risk from a certain number of sites (30 sites is used as an example in the tables below), this could be achieved either by: buying directly from farmers or collectors or by rigorous separation of stocks and on-site biosecurity at each step in the chain of supply. The other fish handled by exporters and importers should then be irrelevant to the retailer and the risks may be cut substantially, but they may still be significant if the pathogens are of concern present are found commonly on sites.
Even if very few producers are infected the other businesses in the chain to the retailer may have a significant probability of picking up pathogens from the stock originating on infected producers or sites infected as fish from these sites pass through the supply chain. The latter is particularly likely to happen if there are many stages in the supply chain at which co-mingling of stocks from different sources can or does occur. The risk of the retailer at the end of a supply chain receiving fish with a given pathogen present on or in the fish will depend on at least the: prevalence or infection rate of fish at the production or collection sites number of businesses involved in the supply chain the amount of co-mingling and cross-infection of fish from different producers or other businesses as they move along the supply chain efficiency of screening, treatment, monitoring, isolation and quarantine at the sites along the supply chain
Uncontrolled i.e. screening, no treatments, monitoring or quarantine

Infection rate with pathogen of concern at the production or collection sites 0.1% 1% 10% 1 in 1000 1 in 100 1 in 10 Probability of a retailers stock becoming infected in various supply chains (refer to table 1 above) A 0.8% 7.7% 57.0% B 6.2% 47.4% 99.9% C 40.1% 99.4% 100% D 2.95% 26.0% 95.7%
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With screening and 66.67% effective regime of treatments, monitoring and quarantine
Infection rate with pathogen of concern at the production or collection sites 0.1% 1% 10% 1 in 1,000 1 in 100 1 in 10 Probability of a retailers stock becoming infected in various supply chains (refer to table 1 above) A 0.0% 0.3% 2.9% B 0.2% 2.3% 19.5% C 1.9% 16.8% 75.4% D 0.11% 1.1% 10.5%
Measures such as quarantine that screen out infection can also greatly reduce the risks of infection passing on to a site and contaminating the supply chain. Illustrated is a model in which using disease screening and control techniques which gives a 2/3rd chance of stopping infection at each step. Even then, with many supplier scenarios, there is still a substantial risk of the retailer becoming infected. Rigorous separation of stocks reduces transmission so that combining quarantine and separation of stocks reduces transmission and greatly reduces risks. Different levels of investment in screening reduce disease risks to different degrees. If there is a 5% risk of producers (not 10% as in the scenarios outlined in the tables above) being infected then, without screening, in the 4 supplier scenario (B above) the retailer has 96% chance of picking up infection.
Chance retailer infected % 120 100 80 60 40 20 0 0 10 25 50 66 Efficiency of screening and treatment %
If stocks are screened for disease, the risk can be significantly reduced. The more efficient the screening and treatment of fish the greater the reduction of risk passing along the supply chain. However, minimal screening and treatment has minimal effect, so the process must be taken seriously to be effective. The risk of infection may be magnified through the supply chain so that, uncontrolled, small risks at the producer level can be become greater risks as the fish move towards the retailer. By reducing the mixing of stock from different sources and by applying screening for disease, these risks may be contained or reduced.
SUPPLY CHAINS AND DISEASE RISK (see also Appendix 9) WHY DISEASE BREAKS OUT
The likelihood of a disease breaking out is summed up by the following equation:
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The rate of removal of the pathogen
The ease with which the disease or pathogen spreads between individual fish or sites
X
o o
The number of infectable fish present on site or in the supply chain
o o o o o
Example of factors influencing each part of the equation are listed below with links/reference to more details coverage elsewhere in the report. o o Number of infected consignments arriving on a site Presence or absence of quarantine/preventative acclimatisation Isolation or separation of batches The number of other carriers e.g. birds, vehicles, staff etc. entering the site Inadequate husbandry which stresses the fish and disables their immune system eg poor water quality, rough handling Lower or higher temperatures than are optimum for the fish Conditions that are optimum for pathogen e.g. temperature Emerging or new diseases High stocking density Number or fish present which are susceptible to the disease Some strains or species resistant (naturally or via vaccination) to some diseases Number of sources of fish that are used The number of occasions that fish are co-mingled with those from other sources in the supply chain Removal of dying or dead fish Single use flow of water through system removes pathogen Efficient UV and/or ozone Effective treatment with medicines Good husbandry (especially maintenance of water quality)
o o
o o
o o o o
Relatively Biosecure
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4.0 KEEPING THE BAD BUGS OUT Bioinsecure insecure Bio
An all in all out using a single source
Multiply the sources multiply the risk (unless on each site biosecurity is managed effectively)
Multiple sources or co-mingled batches allows mixing and spread of pathogens.
CHAPTER 5 STOPPING THE BAD BUGS SPREADING 5.1 ARE MY FISH DISEASE FREE?
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5.0 STOPPING THE BAD BUGS SPREADING
There are various definitions of disease free. It would be wise to ensure that you are aware of the difference between them. It would be wise to confirm in writing what you expect of your supplier.
FREE OF DISEASE
Pathogens that can cause disease may be present even if the fish do not show any signs of disease. However, if they are not showing clinical signs they are often regarded as disease free. Such specimens can act as carriers of disease even if they themselves are not affected. Fish may not show any of the clinical signs of ulcer disease or KHV but could still be carriers for either those diseases.
Absence of a particular disease does not mean the absence of the pathogen causing that disease.
SPECIFIC PATHOGEN FREE

Even the official sampling technique of taking 150 fish from a site cant give an absolute certainty of finding a specific pathogen if it is present. If this test is undertaken only once then it just gives the assurance that: On 95 out of 100 times the test will find the pathogen if it is present (and 100% detectable by the technique used) on or in 2% or more of the fish These fish could be regarded as Specific Pathogen Free or SPF. This of course assumes the test will find the pathogen every time it is present. This may not be the case when the: Pathogen cannot be found on every occasion it is present e.g. latent KHV. Tests are not specific to a single pathogen Tests used are not the best available Laboratories do not use appropriate methods and standards when completing the tests.
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5.2
QUARANTINE AND ACCLIMATISATION
CAN FISH BE QUARANTINED?

The term quarantine is defined as isolation imposed on persons or animals that have arrived from elsewhere or been exposed to, and might spread, infectious or contagious disease. It is derived from the Italian quarantina, which means forty days. Quarantine (that is a forty-day period of isolation) can be applied to any animal, but was originally applied to humans and warm blooded animals. The expectation was that forty days (or other period of time stipulated by law) would be longer than the incubation period of serious diseases like small pox or rabies. Thus any infected animal would become identifiably unwell in that period. Warm-blooded animals have a metabolism that keeps their body temperatures stable within a narrow range. Pathogens of these animals have adapted to cause disease within that narrow temperature range. This is why the incubation period, the time between exposure to infection and signs of disease becoming evident, of diseases in warm blooded animals can be predicted with some accuracy. Fish are not warm blooded, and their diseases do not have incubation periods that are similar in all conditions. Fish adopt the temperature of the water that surrounds them. If their environment is temperature stable, then the incubation period of a disease may be predictable. However most fish are subject to quite wide fluctuations in temperature; Koi can be in found in water close to freezing, or up to temperatures of 300C or more, there are wide differences in incubation periods for diseases across this temperature range. To facilitate the detection of disease in cold-blooded animals a series of stress tests has been developed to induce the clinical symptoms of a disease carried by an asymptomatic animal. The judicious use of these tests may be very useful in establishing the health status of a population of animals prior to their importation e.g. holding fish within the temperature range at which the disease occurs. To a larger degree than in many other animals, stress predisposes fish to disease. Stress does not cause these diseases, as the pathogen must be present. Unless the trigger, frequently stress, is present, a disease may not develop even if the pathogen is present.
ACCLIMATISATION
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For fish acclimatisation may be a more accurate description of what is usually described as quarantine. Basically fish are rested and kept in optimal conditions that are as stress free as possible. It could be argued that these are the conditions least likely to precipitate full-blown disease in fish. Thus acclimatisation may achieve the complete opposite of quarantine as used for mammals.
PREVENTATIVE ACCLIMATISATION
However an acclimatisation period may be used to try to determine if the fish are carrying a particular disease. Each batch of fish must be isolated; this is a normal requirement of any good husbandry or stock system. Clearly there are costs associated with meeting this requirement, but these may prove to be less than those incurred during outbreaks of this disease at your establishment and/or that of your customers. No transfer of water between batches must be permitted including that on hands, nets, aerosols (eg the droplets formed by working air stones), wellingtons or buckets. Ideally each batch of fish should be isolated completely. No transfer of fish, water, waste products, gametes or equipment should be permitted. If you require exporters to give assurances as part of the contract of supply, they may require that you provide evidence of an effective isolation policy of new stocks, and its effective practical application. Basic records of water quality, mortalities and observations of fish stocks should be maintained and held for reference. Fish should be subject to a period of preventative acclimatisation/isolation for a suitable period at a temperature, or in conditions in which the pathogen of concern would normally cause disease or otherwise become detectable. The development of many diseases in fish are dependent on temperature, and any period of preventative acclimatization should take account of that fact. For instance in the case of a KHV this period should last at least 14 days, ideally longer at 23 to 28C. Any batch of fish in which signs of illness or disease, should be subject to tests. If these prove positive for a disease of concern, then that source of supply may be considered unsuitable until adequate remedial action has been undertaken. The growth of parasite populations is also at least in part attributable to how quickly they can complete their lifecycle, which is closely linked to water temperature. Batches from major sources of supply might usefully be screened prior to the start of the season. Given the apparent infectious nature of some diseases in closed systems, no chances of cross-contamination should be tolerated if any amount of confidence is to be placed in the results. All water should be discharged via the foul sewerage system never to open or natural waters.
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CHAPTER 6 FUNDAMENTAL ISSUES

6.1 WHAT CAUSES DISEASE IN FISH POPULATIONS?
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6.0 FUNDAMENTALS OF DISEASE TRANSMISSION
Almost anything! Fish in aquariums and ponds are entirely dependent on our management of their environment to maintain their health. Health or disease is a complex balance between pathogens, the fish or other organisms and the environment. It is beyond the scope of this report to look into this balance in detail. Mismanaged environments stressed fish and opportunist organisms may lead to disease. It has been estimated that over 90% of diseases reported by hobby keepers are caused by poor environment, particularly poor water quality. To know what causes a particular disease at a particular time, the more information the better. Disease, per se, is not an entity or an end in itself. Disease is the end product of an interaction between a noxious stimulus and a biological system, and to understand disease is to understand all aspects of the biology of the species. (Mawdesley-Thomas 1972) Many diseases can be eradicated or kept to minimal levels by careful management and a good knowledge of the basic biology and natural history of the species (both the fish and the pathogen) concerned.
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6.2
IDENTIFYING THE PATHOGEN CAUSING A DISEASE
Are we sure that scientists have identified the guilty organism correctly? To decide what causes a disease, scientists use a set of four rules known as Kochs postulates. These are: 1. The organism suspected of causing the disease must be present in all cases of the disease. 2. The organism can then be isolated and grown in the laboratory. 3. The organism isolated can infect animals that then show the same signs of disease. 4. The organism must then be isolated from those animals experimentally infected. Just being present when a disease occurs does not mean that a particular organism is the cause of a disease. Kochs postulates help avoid innocent bystanders being blamed for diseases they didnt cause.
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6.3
DIFFERENT TYPES OF PATHOGEN
FOUND EVERYWHERE OR (ALMOST) NOWHERE Fish pathogens can have geographical distributions that range from the: Ubiquitous - found everywhere or very widely on a wide range of fish species e.g. Aeromonas hydrophilia (a bacteria associated with some ulcers on fish), or Ich (white spot)
to those Confined to particular areas of the world or particular species only e.g. KHV, neon tetra disease.
ALWAYS A PATHOGEN? OR JUST ON SPECIAL OCCASIONS

Diseases causing organisms fall into two categories: OBLIGATE PATHOGENS: always cause disease when able to invade the hosts bodies. They may only cause disease in certain circumstances eg Koi Herpes Virus (KHV) can become latent in the fishes body and generally only causes disease between 18 and 300C. FACULTATIVE PATHOGEN: dont usually cause disease but can when their population explodes due to environmental conditions or for instance because a fishs immune system is depressed, the water quality is diminished or the fish is wounded. Most fish pathogens fit this description and are very opportunistic relatively rarely causing disease in healthy wellmanaged fish stocks. The risks posed and strategies to avoid obligate and facultative pathogens may differ. Indeed the conditions that lead to facultative pathogens causing problems in your facility may not ever exist. Theoretically such organisms pose a risk but in practice may never do so.
Some organisms found on fish can cause disease in man. These diseases are known as zoonoses. They can generally be avoided by good personal hygiene (Appendix 10).
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Obligate Pathogen Faculative pathogen may always be present
Host
A symptomatic carrier "vector" Diseased
Does release infective material
Does not release infective material
Recovers but liable to reinfection e.g. parasites
Recovers resistant to infection but not carrier
Morbid
Recovers resistant to infection: CARRIER
Does not release infective material
Does release infective material
Ma y infect other fish of same species or other species
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6.4
HOW NEW OR EMERGING DISEASES APPEAR
The organisms causing new and emerging diseases are probably not new, but have always been around. Viruses, bacteria and parasites, are opportunistic; normally they are present in small numbers and cause little or no problem. However if conditions change they may take advantage of the situation, for instance: Viruses and bacteria have been known to jump from one species to another, even from a saltwater fish to a freshwater species, if for example, the potential pathogen is brought into close association with a new host, by practices such as feeding raw diets containing marine fish to freshwater fish. Virtually harmless in their original host species, they may become very virulent in the new one. Geographically separate species carry different bacteria, viruses and parasites. The organisms carried harmlessly by one species may be pathogenic to another species if the two are mixed together. Geographically separate strains of fish of the same species may evolve resistance to different pathogens. When distinctive strains of fish of the same species are mixed organisms that cause no problem to one strain may cause serious disease in the other. Changes in husbandry conditions such as rapid rises or falls in temperature, or increased stocking density will change the type and level of stress the fish are subjected to, and may promote susceptibility to diseases caused by organisms that had previously been benign.
6.5
CHARACTERISTICS OF NEW DISEASES
Cross-species transmission is inherently unlikely but if species are mixed in large numbers and in close proximity this unlikely step has many chances to occur. The more chances that are given for such an event to occur the more probable it is. Thus the more species that are mixed, on more occasions in bigger numbers the more likely it is that a pathogen will jump from one target species to another. Pathogens that jump from one species to another may, at first, be very inefficient at transmitting themselves within the new host species. If the new hosts are held at high density, or are stressed, then the pathogen is more likely to survive this period of inefficient transmission, allowing a new disease to emerge. Once the pathogen has successfully jumped from one fish species to another it may: cause a dead end infection. It kills all its new hosts and dies out. Killing all or most of your hosts is not a good strategy for a pathogen, if its host dies it dies too. Often over time these diseases may become less virulent, as that permits their hosts and thus themselves to survive. It kills its new host species, but does not spread to further fish species. Far more seriously it may be able to adapt and transmit to others of the new species; this adaptation may be a slow process with the pathogen improving its ability to transmit with each successful transmission.
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6.6 RATES OF PATHOGEN MULTIPLICATION
In 7 days at 200C one Ich growing on a fish can multiply to 500-1000 At 70C this same growth in population could take 5 weeks or more. In ideal conditions some bacteria can double their population every 20 minutes. At that rate after 24 hours one bacteria can divide and create: 1,000,000,000,000,000,000,000,000 or in words one million billion billion Viruses takeover the cells of the animals they infect. A single cell infected by a virus can expand to 50,000 to 100,000 times its original size because of the new viral particles being produced. The numbers of virus particles produced by each cell are astronomic and each of those viral particles can infect another cell.
The closer to the optimum temperature of the pathogen and/or the lower the fishs immunity the faster the pathogens population will grow and the quicker a disease may become apparent.
Optimum temperature
Sub-optimum temperatures
Number of pathogens
Time
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6.7
TYPES OF FISH DISEASE TRANSMISSION
VERTICAL TRANSMISSION (BETWEEN GENERATIONS)
Diseases pass down the generations
Vertical transmission can be avoided by taking great care in selecting, or treating broodstock so they are free of a particular pathogen before they breed. This can be achieved by: testing the parents gametes (eggs, ovarian fluid and milt or sperm) for pathogens and the eggs/ offspring destroyed if they are positive for these pathogens or by treating the brood stock prophylactically with antibiotics or immuno-stimulants to reduce the risk of infection or by only breeding from certified pathogen or disease free offspring
There is also the potential that non-destructive tests (that is not requiring the fish to be killed) will, for some diseases become available shortly so potentially an individual fish can be tested for disease on an annual basis Some parasites match their reproductive cycle to that of the host so there are plenty of young infective stages available to infect the newly hatched fish or re-infect their parents. This strategy is common in parasites where the animals live solitary lives and only come together for reproduction.
HORIZONTAL TRANSMISSION - (BETWEEN INDIVIDUAL FISH)
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Intermediate hosts
Transmission by direct contact
Transmission by human activity Infected aerosols or water
Equipment including any solid object
Nets The interior or exterior of vehicles
Personal clothing Unwashed hands
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6.8
CHARACTERISTICS OF PATHOGENS THAT DETERMINE LIKELIHOOD OF DISEASE
Listed below are some of the characteristics of disease that it would be useful to be aware of when working out a management strategy.
PRESENCE OR ABSENCE OF THE PATHOGEN If the pathogen isnt present it cant cause a disease.
HOW EASILY THE DISEASE IS TRANSMITTED

Some diseases can be transmitted by direct contact between fish, others can be transmitted via the water, yet others require the presence of another host e.g. a snail before they can cause problems. The easier they can be transmitted the more likely they are to cause problems.
POOR HUSBANDRY
Failure to maintain good water quality and hygienic conditions can directly eg via dead and dying fish or indirectly e.g. by causing stress and thus loss of immune response, create conditions in which pathogens may cause disease.
DEAD AND DYING FISH CAN BE DEADLY!!

Pathogens tend to leave dying and newly dead fish as they are no longer any use as hosts! A research study using salmon of 25 gms weight (a 3-4 inch koi weighs 10-15 gms) showed a moribund fish released between 10,000 and 100,000,000 bacteria per hour! In open sea cages where discharged pathogens would be washed away and diluted a study in Norway showed the incidence of a certain viral disease could be reduced by 66% by removing dead fish daily.
DISEASE SPECIFICITY
Some pathogens cause disease in only one species, for example KHV apparently affects only the common carp Cyprinus carpio. Others are found on many species globally and affect many species eg Icthyophthirius multifilis.
INFECTIVE DOSE OR VIRULENCE
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The numbers of a particular pathogen that are required to cause disease is known as the infective dose. The more virulent the smaller the infective dose of pathogen is. Different pathogens and strains of pathogens will have different virulence and different infective doses will be required to cause disease. The exact infective dose is variable and dependant on the pathogen, the species of fish, the environment and the fishs general state of health. Aeromonas species can cause disease when there are 10,000 bacteria per ml of water for a short time. That sounds a lot, but one medium sized moribund goldfish can potentially infect 150 litres (approximately 35 gallons) of water to that level between 5pm and 8am the next morning in a closed system with no UV or ozonization. Just 100 bacteria per ml is required to cause disease over an extended period of three weeks exposure. The moribund goldfish could infect a system of 1000 litres to that level in just one hour.
AVOIDING AN INFECTIVE DOSE

Exclude the pathogen from the chain of supply and entry to your site. Treat for the pathogen as a matter of routine to continually reduce numbers present (but avoid creating strains resistant to treatment). Make the water a hostile environment for the pathogen e.g. by use of ozone or UV. Ensure the fish are well cared for, thus enhancing their immune system. The more active the immune system, the bigger the pathogen dose required to cause an outbreak of disease. Remove dead or dying fish promptly.
PATHOGEN PERSISTENCE
Some pathogens cannot exist away from their hosts. In a system with no fish present Ich will die out in days as apparently will the KHV virus. In the latter case sterilisation may still be a better option for control. On the other hand other viruses e.g. IPN in salmonids will still be infective weeks or months after the fish are removed from a system.
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6.9 INFECTION ROUTES:

Direct Contact with skin
Infective stages of some pathogens can leave a host and travel through the water directly to their next host. Normally the higher the stocking density the more likely the direct contact is to occur. Though it can also occur during mating and when territorial disputes occur. Keeping a fishs skin and the layer of mucus overlying it intact by careful handling and netting etc helps prevent infections by this route.
Inhalation
Infective particles can lodge in the gills of a fish and the pathogens can enter the blood stream directly, infect the cells of the gill, or remain in the gills and live there (e.g. the parasite which causes Amoebic gill disease in Salmon in Australia)
Ingestion
Infective particles may be taken into the digestive tract and can either enter the blood stream directly, infect the cells of the gut lining, or lodge in gut lumen and live there (e.g. the parasite Ligula oceanica or some bacteria such as that causing Enteric red mouth disease). Infected food can introduce pathogens to the gut. Feeding wild caught food eg Daphnia, or animals that have died (of either disease or natural causes) should be avoided as this carries a high risk of introducing infective agents to the gut.
Vectors and Fomites (See also 6.10)

A vector is an organism that can physically carry disease organisms, often without being affected by them. A fomite is an object on which a pathogen can be spread.
Intermediate hosts
Some parasites cannot complete their life cycle unless a secondary host (a snail for instance) is present. The continuous prevention of the vector/disease/host life cycle will keep the stock disease free. For instance, as is the case in a number of diseases, if a snail is the intermediate host then removal of the snail breaks the infection cycle and prevents the outbreak of disease.
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6.0 FUNDAMENTALS OF DISEASE TRANSMISSION 6.10 DISEASE TRANSMISSION ROUTES
WATERBORNE
Water can transmit pathogens. The water can be flowing, it can be stagnant, it can be a thin film, on clothes or in splashes and aerosols. Transfer of water (in the aquarium or pond, on nets, plants, vehicles, hands, clothes, puddles on the floor, in fish body tissues or excretions - or even on the fur, feathers or skin of mammals, birds or amphibians) is a way that fish diseases are frequently transmitted.
PATHOGENS CAN BE TRANSMITTED IN FILMS OF WATER ON FLOORS, WORK SURFACES, NETS, HANDS, VEHICLES ETC.
Viruses can be stored as crystals but require a host and water to reproduce. Some bacteria can be dried and stored. Some cannot stand drying out (desiccation). All bacteria need the presence of water to reproduce. Many parasites are killed by drying them out. Others can form cysts that can resist desiccation for varying periods.
All parasites require the presence of water to grow and reproduce! Pathogens can be transmitted in water including films, droplets and fluids!
ALL THE FOLLOWING CAN ALSO SPREAD DISEASE IN WATER:

Individual pathogens or clumps of pathogens The infective stages of parasites or fungi Particles of debris which contain the resting stages of pathogens Waste products from infected animals e.g. mucus, faeces and urine Infected materials lost from wounds etc e.g. blood or infected cells sloughed off from the kidney or digestive tract
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EXAMPLES OF VECTORS AND FOMITES FISH

Direct contact between fish is, except at breeding and during territorial disputes, rare in nature. In captivity, dense stocking in systems or transport bags and catching fish in large nets full may mean direct contact is an important route of disease transmission. Obligate pathogens must have a host, that is the fish. In the absence of any fish they tend to die. Though the time periods over which this happens varies eg Ich in a system at 24 o C dies in 4 days. Viruses may persist in the aquatic environment for days, weeks, months or years but can only reproduce inside a living organism Fish can act as passive carriers. Fish can shed pathogens, even when they show no signs of diseases, but especially when they are either morbid or dead.
OTHER LIVING ORGANISMS

Leeches spread carp trypanosomiaisis between fish; the eradication of leeches prevents the spread of the disease. Without treatment infected fish will remain infectious for long periods and the disease can be spread again if leeches find their way back into that system. Full eradication can only be completed if the fish are treated and the leeches eradicated.
PARASITES
Parasites can cause disease in their own right. Trichodina, Ich, Dactylogyrus, and Gyrodactylus, Argulus and Lernea are a few of the parasites that might be found on fish. They all either puncture the body and draw off fluids, graze off layers of the skin, or feed on the mucus on the skin, gills or gut. They may ingest infected materials and carry them to their next host. The wounds they cause also open up a perfect entry point for some bacteria and viruses. Several scientific papers have suggested that parasites physically carry pathogens. SVC and ulcer disease are cited as examples of diseases carried by parasites while the anchor worm, Lernea is capable of transferring bacteria.
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FOOD
If fresh minced or chopped fish is fed it can be a continual source of infection unless it has been treated by pasteurisation, UV, irradiation or the like. Plankton samples collected from the wild can carry diseases or parasites. Artemia cysts can carry bacteria such as Vibrio. The capsule can be removed or disinfected. Heat treatment, irradiation and the effects of freezing can eliminate pathogens. Badly stored food can become a source of illness and disease. Food inappropriate to the species can promote disease. Diets consisting of material derived from the wild, such as fish fillets or wild caught Daphnia culex are often perceived as having great nutritional benefits as they are believed to contain many macro nutrients essential to the good health of the animal. However, there is an inherent risk in using wet diets as they may introduce pathogens to the stockfish. These many take the form of parasites that use an intermediate host to enter their final host (e.g. Diphylobothrium), or simply be concentrated by the feeding of the prey item to form an infectious particle which when ingested can establish the infection. Potentially the highest risk here is the use of wet fish in diets which can, under appropriate conditions, be a very high risk factor allowing large amount of pathogen to enter the diet if the source material was infected. This is believed to be how VHSV entered the Gigha turbot farm in Scotland in 1994 that led to the compulsory slaughter of the stock and fallowing of the farm. The requirement to use wet feed for Koi and other cold water species is limited as there are very good commercial diets available, and if it is deemed necessary for other species or for wild caught animals unused to a captive diet, then pasteurised or irradiated feeds should be used if possible. Good quality food will promote a healthy immune system and enable fish to resist pathogens.
FAECES - SURVIVAL CAPSULE FOR PATHOGENS!

Faeces can provide a survival capsule for some fish pathogens. Faeces is made of what remains of a fishs food after digestion, mucus, cells lining the gut that are shed continually, dissolved chemicals such as ammonia and harmless (and even helpful) bacteria naturally found in the gut and pathogens. Lipids (fats) can create a waterproof coating on the outside of the faecal material, thus the contents, including any pathogens present, are at least for a time insulated in a cocoon of material that is conducive to their survival.
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CLOTHES AND PERSONAL CONTACTS

DOCTORS TIES AND LECTURES ON DISEASE PREVENTION SPREAD DISEASES Disinfection of personnel and equipment is very important in preventing the spread of disease around a site and to new stock. Stock that has been infected with a pathogen or developed disease should be isolated, ideally in a separate area away from healthy animals, with different equipment and protective clothing used just in that area and for that job. Obviously we have all seen the Now wash your hands signs in the bathroom because pathogens can be spread on our skin or under our finger nails. Diseases can be spread by less obvious, not to say odd, ways. There is the case of an eminent medical professor who made a point of shaking the hand of everyone attending his lecture on preventing diseases spreading in hospitals. Unbeknown to anyone, he covered his hands with a fluorescent pigment, at the end of the lecture he would turn on a black light to show where his hands had been, and it was easy to see how pathogens are acquired. Concerns about disease transmission in hospitals still make the news. One of the latest concerns being that doctors neckties may spread infection from patient to patient by touching an infected area on one person and spreading it to the next as the doctor moves on. Even seemingly trivial issues can be the source of biosecurity risks and should be managed.
CHAPTER 7 SYSTEM OR FARM MANAGEMENT

7.1 BASIC WORK PRACTICES
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7.0 SYSTEM OR FARM MANAGEMENT
Isolate and acclimatise newly arrived fish whenever possible. If the fish you buy are carrying any pathogens then this will help, along with appropriate treatments, prevent them transferring them to your existing stock.
Work clean to dirty; work routines should start with the species and groups that are cleanest i.e. those groups likely to have the least pathogen load, such as newly treated fish, and the most vulnerable, young or stressed fish. Newly arrived fish may need to be treated with caution and care should be taken to ensure diseases are not transferred to or from them. Deal with fish requiring disease treatment last. Working in this way reduces the potential for pathogen transfer to clean fish.
Clean
Dirty!
Each aquarium, pond or system should as far as is possible be allocated separate equipment. This should be strictly adhered to especially in any isolation or acclimatisation area Sterilising and disinfecting equipment: nets and other equipment can carry pathogens between tanks, ponds or holding systems. Either equipment should be allocated and separated for use in a specific area, or else it should be disinfected as it is moved around a site. Hands are bits of equipment that can carry diseases, and so should be at least thoroughly washed between systems. Sterilising and disinfection filters ( see UV or ozone see sections D and E below) should be maintained and managed according to the manufacturers instruction to be as effective as possible barrier to the transfer of pathogens in centralised systems.
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7.2 A.
WATER MANAGEMENT FLOW THROUGH SYSTEMS
Single use: in these systems water is used just once and then discarded. Any pathogens it picks up are continually diluted and removed from the system. To that extent such a system resembles ventilation. In Channel Catfish culture systems in the US increasing the flow of water through such systems eliminated or greatly reduced the presence of Ich on the fish. This was because the infective stages were washed out of the system before they could find a new host. Multiple use flow through: in these systems the water flows from one pond to one or more others before being discarded. Any problems in a pond are passed on with the water flow to every subsequent pond. On an individual site it is best practise to allow the youngest fish first use of the clean water, as they tend to be carrying fewest, if any, pathogens. They also tend to be most easily affected by pathogens and become diseased by a smaller infective dose. If you are purchasing the older fish from such a site you may buy the accumulated problems from all the ponds in which the water has been previously used.
B.
RECIRCULATION SYSTEMS:
Without treatment: such systems just recirculate the pathogens released by the fish. Each time the water is recirculated, it picks up more released pathogens. Thus the pathogen load builds up with each passage. In this sort of system the likelihood of transmission is greater as is the likelihood that an infective dose of a pathogen will be reached. Using sterilising filters (see sections D and E below) UV or ozone: carefully managed sterilising filters can reduce or eliminate the build up of pathogens shed by fish in a holding system.
WATER MANAGEMENT
Spring Borehole or treated water
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Water recycled
Water recycled
No treatment
Holding system
Effective UV and/or Effective UV and/or Ozone Ozone or other effective treatment
Pathogen load multiplies with each pass No bio-security
Single pass flow through
Preventative treatments e.g. Preventative low level copper treatments e.g. low anti paraciticides in level copper anti marine systems may paraciticides in marine reduce infective load of systems may reduce narrower range ineffective load of of potential pathogens narrower range of
potential pathogens. Bio-secure dependant on careful maintenance of UV lamp, ozone doser etc.
Bio-secure
But if water is reused with no treatment it becomes increasingly bio insecure with number of times it is used
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C.
INFECTION OR RE-INFECTION? WHICH IS THE GREATER PROBLEM?
If a disease causing organism is present in a population of fish, that population can be described as infected. Many organisms present in fish populations, that can cause disease, do so only rarely. Fish can shed infectious materials even when a disease is not apparent. The fishs immune system has evolved to be able to deal with some organisms by either eliminating or controlling them. In the wild, or in farms where water is used only once and then discharged, those infective materials may rapidly become diluted and dispersed in the river, lake or sea. Thus no other fish is exposed to an infective dose nor is the fish discharging infective materials subject to re-infection so there is no outbreak of disease. In recirculation systems little such dilution or dispersion can occur. Thus not only is infection passed to clean fish, but the infected fish are subject to re-infection. While a fish may be able to deal with a small dose of a pathogen, if subjected to continual re-infection a disease may be caused. This is especially so as in an untreated re-circulation or under gravel system, the amount of the disease causing organism in the water rises and often the water quality falls, both of which make the fish more susceptible to infection and disease. Some organisms are killed when they pass through the UV filters. This action can only take place as the water passes through the filter and thus has no direct impact on biological material at any other point in the system. Ozone can be used to make the water in the whole system a hostile environment , in which many pathogens cannot survive.
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7.0 SYSTEM OR FARM MANAGEMENT D. ULTRA VIOLET-UV (including sunlight) See also appendix 5
SUNLIGHT
Strong sunlight has high levels of UV that can disinfect water. However the degree of disinfection achieved will vary considerably and be dependant on, among other things, water clarity and cloud conditions. As the outcome in ponds may be highly variable leaving water in sunlight to help disinfect it should be regarded with caution.
Conversion factors
You may come across a number of different units being used to describe the lethal dose of UV. Different forms are used. 1,000W/cm2 = 1mW/cm2 = 1mJ/cm2 = one millionth All of the values below are the same: 20,000 Watts/cm2, 20,000 W/cm2 or 20,000 microW/cm2 20 mWatts/cm2, 20 mJoules/cm2, 20 mW/cm2 20 mJ/cm2 or 20 milliWatts/cm2 or 20 milliJ/cm2
Effective dose of UV
UV kills by disrupting the genetic material in the nucleus of cells; generally the larger the organism the greater protection the genetic material has from the cellular material surrounding it (which absorbs UV) and hence the greater the dose of UV required to be effective. Different organisms have different tolerances to exposure to UV (See Appendix 5). The OIE recommends 10mJ/cm2 UV as the minimum lethal dose for bacteria. Generally it is acknowledged few bacteria can survive 25mJ/cm2. To kill some viruses requires 125 200mJ/cm2, while small parasites may require 1,000 mJ/cm2. UV is an effective killer of pathogens in clear water. It is ineffective if the water is heavily contaminated with suspended material or some dissolved minerals which prevent light penetration, or if the bulbs have exceeded their recommended working life. UV systems must be maintained according to manufacturers instructions to achieve best results. UV light is harmful to the eyes. One should never look at an illuminated UV source.
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E.
OZONE (See also appendix 6)
Ozone is a very effective oxidising agent rapidly degrading cell membranes, organelles, and the nucleus of pathogens. It is highly reactive with organic material and can penetrate particulate matter very rapidly. To achieve disinfection a residual ozone level of 0.4g/m3 per hour (equivalent to 0.4mg/l source http://www.unep.or.jp/ietc/Publications/TechPublications/TechPub-8f/B/Disinfection2.asp) with a contact time with the pathogen of around 4 minutes is required. This level of disinfection is very easy to achieve in a re-circulation system by injecting ozone into a foam fractionator column (protein skimmer or similar) and allowing the reaction and contact time to complete there. It is possible to automate this process by means of an ozone dosing computer which will measure the redox potential in the reaction vessel and add more or less ozone as required to ensure effective sterilisation. However it should not be assumed that ozone is the answer in all situations, to every disinfection problem and the advantages and disadvantages must be considered.
ADVANTAGES
The advantages of this technology are as follows: it has a high efficiency of disinfection that virtually guarantees the elimination of infectious microbes from the water; it is a clean technology that does not impart taste or other traits to the water; the disinfecting process is not affected by different water pH values.
DISADVANTAGES
The disadvantages of this technology are as follows: it is relatively expensive for instance the electric arc generation poles required to oxidize the air and convert it to ozone are expensive operation of the ozone-generating equipment requires skilled technicians, as escaping ozone is a hazardous to staff and other livestock; it requires a continuous and constant source of electricity; inspite of its complete disinfecting power, the residual presence of ozone in the water is limited, it has a negative impact on silicone glues.
Redox potential effective levels

Ozone can be produced and delivered by metered doses automatically. The amount of free ozone remaining in the water, after absorption by organic matter including pathogens, can be assessed by measuring the redox potential. Fish tolerate redox potentials up to 410-420V; above this reading the ozone present is likely to be toxic to the fish. Complete sterilisation can be achieved by maintaining a redox potential of 700V for 10 minutes.
F.
CALCULATION OF TURNOVER TIME
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There are at least two considerations in calculating turnover times. Most frequently the calculation used is simply: Volume of system in litres Rate of pumping litres per min = turnover time in minutes
Thus in a system with a volume of 1000 litres and a pump producing 100 litres/min 1000 100 = 10 minutes
That does not mean that in 10 minutes all the water has passed through the pump and any UV filter attached to it. It takes much longer to ensure that all the water to pass through the pump. The explanation is that in the first minute 100 litres (10% of the total volume) of water passes through the pump. At the end of that minute the system contains 100 litres that has been through the pump and 900 litres which has not. Assuming there is good mixing in the next minute 100 litres passes through the pump but 10% of this will already have passed through the pump and UV. Thus at the end of two minutes 190 litres not 200 has been treated. This explanation is illustrated in the table below. Time minutes Volume pumped lts Volume litres treated for first time if mixing of water perfect 100 90 81 74 67 60 54 47 43 38 Volume treated
1 2 3 4 5 6 7 8 9 10
100 200 300 400 500 600 700 800 900 1000
100 190 271 345 412 472 526 573 616 654
The formula to use to work out the time taken for all the water in a system to pass through the pump or UV is: Volume of system in litres Rate of pumping litres per min Thus in the example above 1000 100 x9 = 90 minutes x9 = time in minutes for all the water to pass through the pump
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7.3
STOCKING OF SYSTEMS
The stocking policy of systems, particularly fishponds, can have a significant impact on the pathogens carried by fish that are produced in them.
DRAINING, DRYING AND/OR TREATMENT BETWEEN STOCKINGS

Sterilisation may be achieved by use of the following techniques (for further details see ww.oie.int) Process(es) Desiccation and light Calcium oxide Calcium cyanamide Usage Earthen bottomed ponds Apply to dry earth base Spores on earthen bottoms Method Dry for 3 months at 180C 0.5kg/m2 for 4 weeks 3 tonnes/hectare leave in contact for 1 month Comment Period can be reduced if chemical disinfectant used Keep water at pH 8.5 or above
Sterile ponds are free of pathogens so cant transmit them to fish stocked in the pond or system subsequently.
FALLOWING BETWEEN STOCKINGS

Fallowing means the removal of all livestock from but not draining systems between stockings. If their host is not present, many pathogens will die. The infective stages of Ich will die after a short temperature dependant period if a suitable host is not present. Fungi or other pathogens which produce resistant egg or spore stages may not. You must assure yourself that: all the fish were removed from the ponds. To achieve this it is likely that a technique using electrofishing or poisons would be required, netting does not always catch 100% of fish the time the pond was left empty of fish was sufficient for the pathogen of concern to have finished its lifecycle and died because of the absence of a host.
CONTINUAL STOCKING
Continual stocking of ponds may mean the continual availability of hosts so pathogens dont die but continue to multiply. The use of an all in, all out policy may slightly reduce the risks of disease transfer from one batch of fish to the next. Co-mingling of batches in a continually stocked pond (especially if the fish stocked come from a number of sources) multiplies the risk. The fish you buy could come from source A, but may have pathogens originating at many farms. After all is said and done, if the pathogen you were concerned about was never present, no amount of management can reduce further the risk. However, it would be wise to instigate a system of checks to make sure the situation remains the same.
7.4
VENTILATION
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Aeration and splashing creates small water droplets than can become suspended in the air as an aerosol. Aerosols can contain small pathogens such as bacteria and viruses. Particularly in humid environments aerosols can be long lived and thus act as a transmission agent for diseases between holding systems. Some pathogens found on fish may cause diseases in humans (see appendix 12). In damp environments mould and fungal growth are encouraged. These may damage the fabric of a building. The spores from moulds and fungi may pose a risk to humans, either by direct inhalation or by sensitising the skin or lungs and potentially triggering dermatitis or asthma. Good ventilation of a fish room can help prevent the transmission of diseases between fish holding system in at least two ways: Dilution: for instance by replacing 50% of the air in a room, 50% of the aerosols formed and hence 50% of the pathogens carried by them may be removed. The chances of an infective dose being transmitted are, all other things being equal, reduced by 50%. Drying: by continually replacing humid air, any water present on surfaces will tend to evaporate. Many pathogens find it easier to survive in damp atmospheres or damp surfaces than dry ones. Puddles and damp surfaces may permit fish pathogens to survive for a long period thus increasing the pathogen population present in the room, increasing the places an infective dose can be found in the room thus increasing the chances and outbreak of disease. Infective spores of the marine protozoan parasite Amyloodinium ocellatum can be transported at least 4 metres in an aerosol droplet. Whitespot Ichthyophthirius multifilis and the bacteria Aeromonas salmonicida have also been shown to be transported by aerosols. Ventilation can help by drying water droplets present in the air as an aerosol. Aerosol droplets persist longer in damp air. Pathogens by Air Aemononas sps bacteria have been known to spread at least 7m in aerosols. That was the size of the room in which the experiment took place and may not represent the distance could travel in ideal conditions. Remember viruses are smaller and maybe carried further still. At its simplest, ventilation may be just allowing air to blow through the fish area. Normal outside air usually carries very few organisms let alone fish pathogens. Heat lost (or more rarely gained) can make this simple solution expensive. Most sophisticated ventilation systems reclaim heat from the air discharged so reducing costs. It may be a wise precaution to ensure the inlet to your ventilation system is not placed close to the outlet from the system so that contaminated aerosols are not pumped straight back into your fish house.
DRY SURFACES PATHOGENS?
VS
WET SURFACES AS HARBOURS FOR
It is an unfortunate fact in our industry that most surfaces will come into contact with water at some time and become damp. This causes problems as damp surfaces provide a much better environment for the survival of pathogens than dry ones. Desiccation or drying is a very effective method for killing pathogens that need a small amount of water to survive. If a surface is perpetually wet or wetted regularly, then there is the potential for any pathogen to survive for long periods, as there is no opportunity to dry out and kill the pathogen. If the surface is very wet numerous bacteria, algae and protozoans may begin to grow on that surface and form a biofilm. This can trap undesirable pathogens and allow even longer survival times as they can provide an ideal habitat for bacterial growth and nutrition or provide an environment where natural
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oxidation, desiccation etc are limited. The build up of a bio-film may hinder disinfection of that surface by either preventing penetration of the active ingredient into the film, or exhausting the active ingredients due to its high organic content.
7.5
BIOFILTRATION AND DISEASE PREVENTION
It is not the aim of this report to explain how filters work to improve water quality. However, it is worth reiterating the point that the stress caused by poor water quality does create the conditions in which pathogens are likely to cause disease. Fish kept in good quality water are better able to resist pathogen invasion because it is more likely that their immune system is in better condition. That said, bio-filters can act as a means of reducing pathogen loads but care is required as they can also harbour pathogens.
HOW BIOFILTERS CAN REDUCE PATHOGEN NUMBERS IN A SYSTEM

A well established biofilter can reduce the population of pathogens in an aquarium or pond system. The reasons for this are very complex and poorly understood, however, one of the most important consideration is the interaction between the bacteria and protozoa that colonise a biofilter. The normal flora of a bio-filter consists of a well established population of bacteria and a varied population of protozoa either living in, on or in close association with the filter media. These organisms are very well adapted to their environments and: Produce enzymes that digest other bacteria and viruses from the water column Feed on bacteria or viruses directly (in the case of protozoa) Produce aggressins that mop up micro-nutrients from the surrounding environment, for example chemicals known as siderophores, take up iron directly from the environment. Aggressins are released to starve competing organisms. Produce natural antibiotics to prevent the new microbe becoming established.
They also have two other minor roles acting as a: Reservoir for bacteriophages (a type of virus) that can kill pathogenic strains of bacteria Mechanical filter trapping bacteria, viruses and parasites either in the media or on the biofilm.
The bio-film is a very hostile environment to new bacteria, each bacterial species that has already colonised the filter is competing for nutrients with each other; less aggressive species starve to death and, in turn their organic components are recycled. In addition to this the protozoa are consuming microbes continuously and again their waste and any dead protozoa will be recycled. All of these defence mechanisms used by the established bacteria can make it a slow process for a new species of microbe to become established in a filter. An obvious example of this is the long period of time it takes for a biological filter to mature from the ineffective filters colonised initially by Pseudomonas sp and Vibrio sp to a bacterial flora dominated by Nitromonas, Asetobacter & Nitrosomas species. In an established system a mature biological filter can significantly reduce the level of circulating pathogens.
Research has indicated that well established biological filters could reduce the levels of circulating Aeromonas salmonicida from 1 million CFU*/ml to 700,000 CFUs/ml (or a 30% reduction). If combined with an ozone tower prior to the bio-filter then they become more effective acting as a final sink for any pathogens that escape the ozonation process before the water is re-circulated around the system.
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OR INCREASE THEM!!!!!
As always there has to be a word of warning with this approach to reducing pathogen load. The pathogen may become established in the filter and ultimately act as a reservoir of pathogen continually shedding pathogen into the environment. This can happen when the load of pathogenic bacteria is so high that it out competes the established bacteria in the filter. The pathogen will have its own suite of aggressins that it uses to survive in the filter biofilm. If present in sufficient numbers it can out compete established bacteria populations. Should this happen then there is little option but to sterilise the bio-filter with hypochlorite or similar to remove the pathogen before restocking occurs. Some may argue that this should be done after any serious disease outbreak as part of the control regime. It is worth remembering that pathogenic viruses will need their host (the fish or invertebrate), to replicate in. They cannot replicate in the biofilm although they may become trapped in the filter. *CFU means colony forming unit. This gives an idea of how live pathogens are present.
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CHAPTER 8 DISINFECTION
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8.0 DISINFECTION
By the very nature of the trade (especially at retail level) many disinfection routines, such as footbaths at entrances, are just not possible. The information below may help to establish what is possible and practicable in different situations.
8.1
DEFINITIONS
Disinfection Killing 99.99% of bacteria present only 1 in 10,000 pathogens survive Sterilisation Killing 99.999999% of bacteria present only 1 in 10,000,000 pathogens survive
8.2
DISINFECTION OF WET SURFACES
Normally good quality disinfectants are very effective in treating wet or damp surfaces, in particular halogens (chlorine based bleaches or iodophores) are very reactive and have a short half life which means that used properly there is little risk to animals that may come into contact with the surfaces subsequently. If the area has substantial biofilm build up then pre-cleaning with detergents followed by disinfection should be considered. It is best to prevent wet surfaces becoming contaminated in the first place. This could be achieved by instigating a regular maintenance schedule, which could be as simple as drying after use and wiping down with disinfectant. In areas such as packing stations where many fish and lots of water from different aquariums may come into contact with surfaces, a formalised Standard Operating Procedure (SOP) should be developed that requires daily (or as frequently as the use of the area necessitates) stripping down of the area followed by a standardised disinfection.
DISINFECTANTS DONT DISINFECT DIRT Disinfectants, including UV and ozone, are inactivated by dirt Mud, biofilms, mucus and blood absorb and inactivate disinfectants Suspended particles, whether inorganic (soil and clay particles) or organic (faeces and algae) inactivate disinfectants Effective disinfection can only be achieved when surfaces have been thoroughly cleaned and rinsed or as much suspended materials and dissolved organic material as possible has been removed from the water.
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8.0 DISINFECTION
THE EFFECTIVENESS OF DIPPING, WASHING AND SCRUBBING WITH DISINFECTANTS

Experiments carried out on pig farms showed the importance of the techniques used to disinfect wellington boots. Though fish farms or retailers are different from pig farms, the overarching principle still holds true. The results were obtained by measuring the number of bacteria present on a small area of the boot (about 0.1 sq in) when cleaned it was cleaned in different ways. The results are expressed in comparative terms rather than in terms of the number of bacteria present to help clarity of understanding. In practice these results will differ according to the amount of organic matter present on the surface to be disinfected, the bacterial, viral or other species present and the disinfectant used. The clear message, which bears repetition, is that dirty surfaces CANNOT be disinfected. Description of disinfectant procedure Boot before any disinfection Step in and out of disinfectant bath Stand boots in footbath for 2 minutes with no cleaning Scrub boots in water for 30 seconds Scrub boots in water for 30 seconds then disinfect Boots scrubbed in disinfectant Relative number of bacteria present after treatment 139,000,000 88,000,000 1,295,000 5,200 6 1 % of bacteria removed 0 37 99.1 99.997 99.9999957 99.99999993
8.3
HUMAN SKIN
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8.0 DISINFECTION
Bacteria on the skin 10 trillion bacteria (10,000,000,000,000) live on the skin of an average human. 10 billion (10,000,000,000) will be left after 99.99% are removed by disinfection. However those bacteria remaining tend to be those that are deeply embedded in the skin Recently deposited bacteria such as those picked up from immersing hands in an aquaria or pond are the most likely to be killed by disinfection.
SKIN CLEANSING AND DISINFECTION

Good personal hygiene can prevent the spread of fish diseases and zoonoses (organisms found on fish which can cause disease in humans see Appendix 10). Method Washing hands with soap and hot water (around 60C*) Impact on bacteria Reduces the number of bacteria present on the skin by 30-50% Outcome The soap disrupts bacterial cell walls, causes the bacteria to become dislodged and washed off the skin and the hot water will thermally damage some bacteria causing them to die.
Use of bactericidal soap (such as a coal tar soap or one containing triclosan or tricloban) Washing hands in 40-60% alcohol after washing with soap and water Adding iodine or chlorhexadine gluconate
Can reduce bacterial numbers by 60-90%
Can reduce bacterial numbers by 99.5%
99.99%
* 60C is the hottest temperature that can be tolerated without blistering. Great care is required to avoid injury at these temperatures. (To achieve complete sterilisation 137 deg C for 1 hour is regarded as incompatible with life)
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8.0 DISINFECTION
BARRIERS FOR THE SKIN

As is explained earlier there are always bacteria on the skin regardless of how thorough the disinfection is. However, total exclusion of the skin becoming contaminated from the aquatic environment is easily achieved by the use of sterile disposable gloves, which, can be used to remove dead animals or disinfect contaminated equipment or aquariums and then disposed of. Care should be taken to prevent water getting in to the gloves and holding contaminated water next to the skin and being transferred between systems. The use of barrier creams (creams that leave an impervious membrane on the skin) are also very useful in preventing contamination of the skin. However, the use of barrier creams is more difficult than sterile gloves as they have to be removed properly to prevent contamination of the users skin with bacteria trapped on the surface of the barrier, as well as ensuring that the barrier is completely removed to prevent transmission of pathogens.
8.4
CLOTHES
Clothes, like skin, can be the source of cross infections so care must be exercised in preventing transmission of a pathogen from a disease outbreak or high biosecurity unit to the rest of the facility. The easiest way of achieving this is to have dedicated clothes for use in the disease or high biosecurity areas and make sure that all people entering high risk area change from their outside clothes into suitable personal protective clothing for that unit e.g. lab coats or other overalls. Often this is achieved by having a changing area or ideally changing room adjacent to the high risk area.
DISINFECTION OF CLOTHES
Normally a boil wash with a bio-cidal washing powder is sufficient to achieve good disinfection of clothing. It should be remembered that a minimum temperate of 60C must be reached for at least 20 minutes for this to be effective. Ideally there should be regular washing of work clothes scheduled in to the working regime and clothes should be washed centrally rather than relying on staff to take them home, risking the spread of disease to home aquarium and hence the wider environment. In cases of severe disease outbreak or where the operator wants to be extremely bio secure then it is prudent to arrange for the personal protective clothing (PPC) supplied by the company to be soaked in strong hypochlorite (minimum of 10ppt free chlorine) overnight prior to laundering. New PPC should be issued on request.
8.5
FOOTWEAR
Ideally all footwear should be scrubbed clean and dipped into a strong disinfectant (such as hypochlorite or iodophores) to prevent transmission of disease between unit and the external environment. The use of shallow foot baths or sponge mats is the easiest way to achieve this. However, some staff and visitors are concerned with disinfectants damaging their footwear (halogens are aggressive chemicals) so it may be worth considering providing Wellington boots, or similar, for staff and visitors to areas about which you are particularly concerned. While disinfection of clothes or footwear may be regarded as impractical in many situations the fact that if contaminated they can transmit disease should not be forgotten.
8.6
EQUIPMENT
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8.0 DISINFECTION
Like skin and clothes disease can be spread on the surface of contaminated equipment such as nets, vehicles etc.. Ideally all equipment that comes into contact with stock (diseased or healthy) should be immersed into a strong biocide regularly (such as hypochlorite or iodophores), ideally after each use, to achieve sterilisation (killing 99.999999% of all the bacteria present). As a minimum nets, buckets, hoses etc should be sterilised on a daily basis and never used on a different aquarium or moved around the site without sterilisation.
8.7
DISCHARGE WATER
Most wastewater from wholesalers and retailers is discharged to foul sewers. Foul sewers are hostile environments to many pathogens and massively dilute any pathogens that are present. UV or ozone can be used as required to sterilise water discharged from a site. Alternatively, chlorine based bleaches can be used but these usually have to be neutralised by the addition of chemicals e.g. thiosulphates. If required to undertake discharge sterilisation further advice and approval may be required from Government agencies.
8.8
DISINFECTING SYSTEMS
It is wise to regularly empty and disinfect systems to try to ensure that pathogens cannot build up in a system. You may choose to do this at the end of a season to ensure any pathogens are not lying in wait to infect new animals. When disinfecting a system remember to clean all parts including the pipework and dont restock it with fish held in it prior to disinfection they may carry all last seasons pathogens with them, this may put you back to where you started rather than give you a fresh start.
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8.0 DISINFECTION
CHAPTER 9 IDENTIFYING DISEASES

TECHNIQUES FOR DIAGNOSING, AND SCREENING FOR, FISH DISEASES
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9.0 IDENTIFYING DISEASES
The identification of disease is a very important, and skilled, procedure in fish keeping and aquaculture. It is the cornerstone of good disease management as it allows the correct intervention, use of the correct and most effective therapeutics and husbandry. There are legal implications as some diseases are notifiable, such as Spring Viraemia of Carp (SVC) in the UK, and failure to report the presence or suspicion of the presence of these diseases may leave the responsible person open to prosecution by the appropriate authorities. There are a bewildering array of identification techniques available ranging from simple observation to scientific techniques requiring expertise or equipment that is only available in a very few Government or university laboratories. The techniques available often have long names that are rarely used, instead acronyms are substituted, a guide to these is included in the descriptions below. Below are brief descriptions of some of the more common techniques you might already be aware of or hear discussed as time passes. Note of caution! A technique is only as effective as the skill and care of the person carrying it out. Purchasing tests from centres of excellence that are quality assured (ISO 9000, UKAS accredited etc) should give a guarantee of quality
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SIMPLE ON SITE INVESTIGATIONS

(Easily carried out by site staff)
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9.1 WATER QUALITY 9.0 IDENTIFYING DISEASES
Whether by high tech filter systems or simply regular water changes good water quality should be maintained. This will promote the immune system function and general health of the fish being kept. It has been said that the greatest step forwards in human health has been the introduction of municipal sewerage works. It is also estimated that 90% or more of fish disease is caused directly or indirectly by poor water quality. The OATA Water Quality criteria should be used as a minimum standard (Appendix 1).
9.2
PHYSICAL SIGNS
PATTERNS OF MORTALITY IS THE PROBLEM INFECTIOUS?

Occasional and/or sporadic deaths may be caused by tumours and the like. If the majority or all the fish in a setup die very quickly, then an acute episode (this might include a problem associated with a filter break down, poisoning, or overstocking catastrophic infectious disease cannot be discounted) should be considered. If mortalities start very low, rise to a peak and diminish again, especially if accompanied mortality is significant, then an infectious disease should be suspected. The prevalence of the symptoms of a disease in an affected population can be used to establish the likelihood of it being infectious or not. If symptoms occur in: more than 0.05% of the population per day then it may be infectious less than 0.05% of the population per day then the chance is it is non-infectious, although, it may be the very start of an infectious outbreak and the stock should be monitored closely. Careful interpretation of observations will be needed.
DISEASE CALLING CARDS AS AN IDENTIFICATION TOOL

The pattern of deaths during a disease outbreak may enable a tentative diagnosis of a problem. For instance KHV should be considered as a potential culprit (not definitely the culprit) if: Only carp (Cyprinus carpio) including koi, ghosts and common varieties are affected and killed. Other species such as goldfish and grass carp in the same system remain unaffected. Mortalities occur at a water temperature between 18-30C. If during an outbreak the temperature rises above or falls below this range mortalities diminish or stop. Mortalities are very rapid. Seemingly healthy fish become ill and die in 24 to 48 hours. Very severe mortalities. 80-100% mortalities occur within 10 days of disease outbreak.
CLINICAL SIGNS
The clinical signs, that is what can be directly observed by eye, associated with fish disease are generally quite indicative of the disease group. For example large sores often indicate a bacterial infection, spinning may indicate a neurological disorder or flicking off surface a parasite infestation.
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The gross pathology of a disease can often give a good indication as to the cause, for example, the presence of the open furuncles (abcesses) in goldfish is indicative of Aeromonas infection. However, it is not possible to tell the species of bacteria causing the infection, which may be A. salmonicida, A. sobria or A. hydrophila. In some instances gross signs are all one needs to accurately diagnose disease, for example if a fish with a distended abdomen is euthanased and found to contain a huge parasitic worm, then there may be no need to proceed further. Secondary invasions of bacteria and parasites might be the most obvious problem on an individual fish, but these signs may obscure the presence of the primary pathogen. For instance the clinical and physical signs of KHV include: Bleeding from the gills White patches (where excess mucus has been produced or tissue has died) which may be small to very extensive on the gills Pale patches and blistering on the skin Sunken eyes Occasionally fish have shown signs of nervous problems (that is periods of inactivity followed by hyperactive behaviour triggered by a very small stimulant)
But in the years following the disease becoming a problem it was often misdiagnosed because of the obvious presence of secondary infection with bacteria and parasites which were judged the culprits. The presence of the majority of fish diseases especially those caused by bacteria or viruses cannot be definitively determined purely by observing the fishes appearance or clinical signs alone. With few exceptions it is very difficult to identify parasites by observation by eye.
CLINICAL SIGNS CAN DECEIVE THE EYE

Fish diseases often have clinical signs that can be easily mistaken for a number of diseases. For example dropsy may be caused by acute liver failure, liver carcinoma, kidney failure or infection, gill, intestine or skin damage, a viral infection or bacterial septicaemia. Hence, the presence of the symptoms of dropsy may indicate a range of both infectious and non-infectious problems.
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MORE COMPLEX ON SITE INVESTIGATIONS

(Likely to require additional training experience or expertise for them to provide accurate results and for these to be interpreted).
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9.3
HAND LENS
Examining the skin, lesions or internal organs using a simple x10 hand lens may show up small parasites or damage to the tissues that are not seen with the naked eye.
9.4
AGGLUTINATION TEST
There is the potential to use this diagnostic test for the rapid identification of pathogens in the field. It relies on antibody tests where the antibody is bound to a latex bead. These are then mixed with a suitable sample of the fish tissue. If the pathogen is present the antibodies cross link with the pathogens, and the latex beads precipitate to form a solid, and this indicate the presence of the pathogen. If the solution stays milky (because the latex balls have remained in suspension) , then there is no evidence for the pathogens presence given. These agglutination tests are very useful for rapid diagnosis, but they are not as sensitive as laboratory based immunological (see 9.11) tests and a negative result is a good indication of the absence of the pathogen, but these tests may fail to detect a low level infection.
SAMPLING
When samples are taken for laboratory examination, great care must be taken to prevent cross contamination between: organs within an individual animal individual animals if several are sampled the animal and the investigator.
The most common errors associated with each procedure can be listed thus:
9.5
MICROBIAL SAMPLING (VIRUSES AND BACTERIA)
Samples are usually taken by sterile swab. o Swab contaminated by the user i.e. the sterile swab touched the users skin and contaminates it with human microbes and microbes from other samples if the operator dont clean their hands adequately. Swab contaminated by the faeces or gut contents of the animal i.e. the sterile swab is swamped with the bacteria from a naturally rich microbial flora masking pathogens Swab contaminated by the environment (eg dropped on the floor!) again the sterile swab is swamped with the bacterial from a naturally rich microbial flora masking pathogens
9.6
BLOOD SAMPLING
Samples are usually taken by needle and syringe. o Not using a clean syringe between fish, thus there is carry over from one sample to the other, making it impossible to tell which fish is infected or has antibodies.
9.7
TAKING SAMPLES FOR MICROSCOPY
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Samples are taken following dissection. The commonest error here is sampling the wrong organ! Remember the kidney of fish runs below the spine (many people think this is the main blood vessel, the aorta) while the spleen sits where you would expect the kidney to be if a fish was a mammal. It is not unknown for very experienced investigators used to working on mammals to sample the spleen rather than the kidney thus, missing the most important fish organ for diagnosing infectious disease.
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LABORATORY INVESTIGATIONS
(Individual farmers, wholesalers or retailers are unlikely to have the facilities to undertake these tests. They are likely to be undertaken on samples sent to specialised laboratories. The cost of tests may vary considerably both between diseases and the laboratory chose.)
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9.0 IDENTIFYING DISEASES 9.8 SHOULD YOU HAVE AN ON SITE LABORATORY?
There are points both in favour of and against having a laboratory (or an area in which sick fish are examined on a site). Some of the key points to consider are:
IN FAVOUR
Accurate identification of pathogens may permit selection of appropriate effective treatment Earlier detection allows earlier treatment, which is usually most effective. Sending samples off site may lead to significant delays in diagnosis.
AGAINST
Good quality equipment is required and that may be expensive. Accurate diagnosis requires experienced and/or trained staff. Inaccurate diagnosis can be costly. Potentially all the pathogens entering your site will enter the examination area. Unless hygiene is carefully managed diseases could also spread from it to other parts of the site.
9.9 MICROSCOPY
A microscope can be used to see and accurately identify many of the common fish parasites by using wet preparations or skin scrapes. Similarly damage to the gills can be seen, but it may not be possible to establish the cause of the damage using a microscope alone. You may also just be aware of large numbers of bacteria in the mucus though you cannot identify them. This is the limit of the investigations possible in even those retail outlets possessing and using a light microscope, a diagnosis cannot always be made on this information alone.
HISTOLOGICAL TECHNIQUES
In heavily infected fish, scientific laboratories are able to prepare and stain very thin sections of tissue (this is known as histology) in which using microscopes they may be able to see signs characteristic of various diseases. Under extremely powerful electron microscopes individual virus particles can be seen, however this does not identify the strain of virus, just the presence of virus like particles. Examination of the tissues by histology is often desirable as this can indicate which organs have been infected, and can detect the physical presence of pathogens, (not the species of bacteria or virus although fungi and parasites can be accurately identified based on their morphology). Often this technique can be
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used to eliminate a pathogen, e.g. by identifying a neoplasm (tumour or cancer) or toxin induced cellular changes. However, when combined with culture (9.10), immunological (see 9.11 ) or molecular techniques (see 9.12) identification of pathogens can be very accurate.
9.10 CULTURE OF PATHOGENS

Some pathogens e.g. bacteria, virus, fungi or parasites can be grown in the laboratory and directly identified based on their morphology, biochemical, genomic and immunological profiles. Often this is the gold standard of identification as it is direct evidence of the presence of the pathogen in the animals being investigated. It will provide information on the strain of the pathogen, be useful for identifying the potential source of infection and the most suitable treatment options e.g. antibiotics that a bacterial pathogen is susceptible or resistant to. However, such tests often require the euthanasia and dissection of the animal to obtain the samples, which may be problematic if it is desirable to keep the animal alive.
9.11 IMMUNOLOGICAL TECHNIQUES

The most common techniques for the detection of pathogens are ELISA, IHC and IFAT (see below). These tests rely on the specificity of antibodies (chemicals produced by an animals immune systems which recognise and destroy foreign material such as bacteria and viruses), which tend to bind only with proteins or other molecules present on specific pathogens. A pathogen provokes the fishes immune system to produce antibodies which are detected, it must be remembered that the level of antibodies diminishes with time after the fish has eliminated the disease (See also 10.1 The fishes immune system) These tests may not be absolutely specific. Thus, if the body reacts to a part of a pathogen that is also present on other pathogens, then the test may detect a number of organisms.
A.
ELISA (Enzyme Linked Immuno-Sorbant Assay)
If any of you have been unfortunate enough to be involved in an investigation of a possible SVC outbreak, you may well have seen samples for ELISA testing being taken. These tests very rapidly (often within 4 hours) give an indication of the presence or absence of a disease. The results are only regarded as, at the moment, presumptive and require confirmation by cell line isolation testing before confirmation of the presence of a disease. Samples from, preferably clearly diseased fish are returned to the laboratory for the cell isolation test. It is interesting to note that ELISA tests are used to confirm outbreaks of swine fever and foot and mouth disease, and it is possible, as testing techniques are developed that confirmed recognition of fish diseases by these tests will be accepted at some point in the future. ELISA tests rely on a level of knowledge of the antibodies produced by the fishes immune system to a particular pathogen which is not yet available for all fish diseases. Latent or dormant infections may not produce very large amounts of antibodies making them difficult to detect. There are two types of ELISA test: Capture ELISA: in this test antibodies are used to capture and detect the presence of the pathogen. In effect this test will tell you that the pathogen, or at least the parts attacked by the antibodies are present. The test is set up in such a way that a colour change occurs and is used as a measure of the presence of a particular pathogen.
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Screening ELISA: In this test the process is reversed in that the known pathogen or pathogens are used to detect the presence of antibodies. Antibodies are the chemicals fish naturally release to attack the pathogen. The test allows the recognition of the antibodies, if present, by a colour change. Any colour shows evidence of the animal having been exposed to the pathogen at some time in its life, and the intensity of the colour (which is proportional to the degree of binding of the fishs antibodies to the pathogen) can give an indication of how recent this was. The advantages of this method is that it allows many pathogens to be screened for at once and will give an indication of the diseases the fish or population of fish have been exposed to during their lives. Thus, this can be a very useful tool in assessing disease risk when importing fish from abroad as exposure to pathogens of concern can be assessed.
B.
IHC Immuno-Histo-Chemistry
Thin sections are cut from potentially diseased animals and instead of being stained with a series of dyes, antibodies are used to bind to specific pathogens. The slides are then stained so that any of the specific pathogens present can be seen under a microscope.
C.
IFAT-Indirect Fluorescent Antibody Test
A very similar technique to IHC except the detecting antibody has been tagged with a fluorescent dye such as fluorescein. The staining principle is identical for IHC but the slide is examined under a special microscope to enable the fluorescence to be seen. The technique is more versatile than IHC as different coloured dyes can be used to identify different antibodies and hence pathogens or strains of pathogens in a section. However, the high cost of the specialised microscope (around 40k) restricts this to dedicated diagnostic laboratories.
D.
ISH (In Situ Hybridisation) and FISH (Fluorescent In Situ Hybridisation)
In many ways ISH and FISH can be considered the molecular equivalents of IHC and IFAT. However, instead of relying on antibodies to detect proteins, DNA or RNA probes are used which are tagged with either a dye (ISH) or a fluorescent pigment. Treated slides are examined under the microscope for the presence of the dye or fluorescence, which demonstrates the location of the pathogens genome within the tissues. These techniques can be used to look at material preserved for long periods. It can be used to take a fresh look at materials from old disease episodes using new tests to try to determine what caused them. For instance these sorts of techniques are being used to re-examine disease outbreaks of disease in carp to see if they were caused by KHV.
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9.12 MOLECULAR TESTS

These are tests that require a scientific laboratory and/or training to use effectively.
A.
POLYMERASE CHAIN REACTION PCR
WHAT IS PCR?
PCR is a technique used to produce a large number of copies of a specific region or sequence of an organisms DNA or RNA, its genetic code. The DNA or RNA code or sequence is unique to the organism of interest, and by amplifying this specific sequence using the PCR, a very small quantity of material from the pathogen can be detected. In PCR the DNA or RNA from a sample in which it is suspected is the pathogen is extracted and placed into a PCR machine (sometimes called a thermocycler) with a genetic template (primers) from the pathogen of interest and bases - chemicals that are the building blocks of DNA and RNA. The mix is then repeatedly heated and cooled, which allows the primers to bind with the target sequence in the pathogen of interests gene fragment, and produces a replica set. Each time the thermocycler runs a heating/cooling sequence the number of copies of the template doubles until either the operator finishes the process after a known series of replication sets, or until the bases are exhausted. The initial copies of the genes are significantly amplified (each cycle doubles the number of copies of the gene fragment in the mix). Once the cycling has finished, the mix is taken out and the gene fragments are visualised on an agarose gel and the presence or absence of the known gene template indicates the presence or absence of the pathogen. If the pathogen is not present then nothing will be found on the agarose gel. PCR becomes even more sensitive as scientists are able to increase the number of the segments they can detect in a sample making detection easier. If not one, but two sequences of unique genetic code are used for detection. The results may be more accurate and are known as nested PCRs. It has been said that this technique can potentially detect a single viral particle in a sample.
The difference between single and nested PCRs

A single PCR would be like trying to identify a street from the picture of a single house. There are few houses - Buckingham Palace? - that are so completely different and unique that you could accurately identify their location immeadiately. Many houses, on many estates, in many towns, look similar or indeed are identical. The opportunities for misidentification are clear. The genetic material from many organisms is also very similar. However if you have pictures of say two or three houses from the street, then misidentification becomes less likely while a picture of the whole street makes mistakes less likely still. Clearly technical aspects of the photos such as their size and resolution and even your own eyesight can affect the accuracy of the results, in the same way technical issues determine the accuracy of PCRs themselves.
INTERPRETING RESULTS FROM PCR TESTS
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It must be remembered that molecular tests are limited by technical constraints and they are not usually considered as definitive tests for the detection of a pathogen, instead they should be considered along with other more traditional tests such as culture (9.10) and cell line isolations (9.13) The method doesnt necessarily detect the whole disease organism. PCRs may be described as proxy tests, that is they detect the presence of small chains of genetic material not necessarily a whole organism. Thus a PCR can detect the debris of organisms long since gone, or certainly no longer capable of producing disease. Because PCR amplifies the signal from a single fragment of a gene, there is the potential to detect levels of the pathogen that are not sufficient to give an infective dose to the host. However, this sensitivity is very useful for detecting the presence of an organism on a site and thus is very useful in ascertaining risk due to the presence of a pathogen. If only a small genetic probe, that is one that only looks for a small sequence of DNA or RNA has been developed, there is the potential for it to cross-react with related genes in non-target organisms. This is equally true of antibody based tests too, although their sensitivity is less as the signal is not amplified in the same way as a PCR test. Thus a full understanding of the genome of the organism is required to be fully confident of the test (again this is also true of antibody based tests too). Certain PCR test require RNA rather than DNA to be collected. Unfortunately RNA is less stable than DNA, so unless appropriate storage has been carried out (e.g. during in transport) then it is possible for the sample to degrade and any signal to be lost.
FALSE RESULTS
The standard method can show false positives and negatives if the methods are not very carefully controlled in other diseases. Vaccination, with live attenuated vaccines might cause false positives due to the introduction of the pathogens.
THE CONSEQUENCES OF FALSE NEGATIVES

Missed opportunities to manage a disease outbreak or spread which could lead to economic harm.
THE CONSEQUENCES OF FALSE POSITIVES

Stock slaughtered unnecessarily. Movement restrictions (no sales). Missed opportunities to import or export.
1009.13 CELL LINE ISOLATION

Cells can be grown in thin layers in shallow dishes. A whole range of types of cell are now grown in labs routinely, for instance Fat Head Minnow (FHM) and Koi Fin (KF) cells. Some viruses such as the SVC virus grow in a wide range of cell lines, but others can be very specific and only grow in cells derived from the original host. Currently KHV can only be cultured in koi fin cells. Viruses, or samples thought to contain viruses, can be inoculated on to these cells. Cells that become infected will show definite signs of infections, these signs may be specific to a particular virus. A PCR can be used as a tool, to confirm the presence of a virus, this does require the PCR to be sensitive enough to detect viruses at the level at which it can cause signs of infection in the cells.
9.14 SUMMARY TABLE OF USES OF ON-SITE AND LABORATORY TESTS
Test Pathogen group Bacteria Viruses Parasites Fungi Poisons (weedkillers etc) ELISA ++++ ++++ ++++ ++++ ++++ PCR +++ ++++ ++ + IFAT +++ +++ ++ + ISH (FISH) +++ +++ ++ + Histology ++ ++ +++++ ++++ +++ Culture +++++ +++++ + +++++ Gross pathology ++ ++ ++++ ++ ++ Anatomical features +++++ ++++ Toxicology screening +++++
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Algae toxins Environmental problems (anoxia etc) Predators
++++ -
+++ ++++
+++
+++++ -
++++
++++
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10.0 TREATMENTS
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10.0 TREATMENTS CHAPTER 10 TREATMENTS
Some treatments can be used as prophylactics, to deal with a pathogen before it causes a disease, or when a disease breaks out, to hopefully cure it. Treatments are to a greater or lesser extent regarded as veterinary medicines by Governments around the world. The use of veterinary medicines are governed by a variety of laws, it would be impossible to summarise these rules as they differ from one country to the next. In Appendix 7 is a dated note of some of the points, concerning the situation in the UK. It is axiomatic that intensive farming of animals goes hand in hand with culture of their pathogens. The culture of fish has had severe problems from time to time as a consequence of infectious diseases. In Atlantic salmon farming, Norway was initially plagued with vibriosis disease and Scotland and Norway suffered badly from furunculosis in the late 1980s. There is little doubt that these bacterial diseases have been very successfully brought under control by vaccines. However, there are still many diseases for which vaccines are not available and this includes the vast majority of diseases that affect ornamental fish. Think how useful a white spot, SVC or KHV vaccine might be. However, the costs are often considered prohibitive as the licensing of a single vaccine costs in the region of 10,000 and this does not include the cost of the research programme, which may run into hundreds or thousands of pounds, that is required to ensure safety and efficacy as part of the licence application. All treatments should be used in accordance with the manufacturers instructions and according to the law in any given country. Great care should be taken to avoid misuse of treatments as they might cause harm to the staff using them, the fish being treated or the environment.
10.1 THE FISHS IMMUNE SYSTEM A NATURAL REMEDY FOR DISEASES?

Healthy fish normally rely on their own immune system to prevent or fight disease. The immune system has arisen from millions of years of evolution. When functioning efficiently the immune system either prevents diseases by prohibiting the entry of pathogens into the body and/or by killing them facilitates treatment eg with antibiotics or is deliberately stimulated as a part of the treatment eg immunostimulants and vaccines
The immune systems response to infection is particularly reduced by:
Temperatures outside the optimum range for the particular fish species Stress
TERMINOLOGY
In any discussion of the fishs immune system it is useful to be aware of the meanings of some some key words ANTIGENS ANTIBODIES c hemicals, pathogens or foreign bodies entering a fish c hemicals produced by the body in response to the presence of an antigen.
Continued on page 98
104Fishs Basic Response to Pathogens

10.0 TREATMENTS
T Lymphocytes kill or engulf infected cells and digest the pathogen
Antigens Pathogen
B Lymphocytes produce antibodies
Non-specific immune response
Lymphocytes = white blood cells
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STRESS AND THE FISHS IMMUNE SYSTEM
The immune systems of fish have evolved over millions of years to protect them against pathogens. This system should be regarded as an integral part of any biosecurity management scheme. Mucus Mainly physical barriers to pathogens Skin (including gut lining)
10.0 TREATMENTS
Avoid physical damage to either or both. Damage permits pathogens access to the body as well as causing osmoregulation problems which will stress the fish
The INNATE (non specific) immune system Consists of chemical released into body fluids by various and system defends cells. This against any antigen entering the body. Various body cells
Promoted by immuno stimulants , good husbandry and nutrition
ACTIVITY OF BOTH IMMUNE SYSTEMS SEVERELY DEPRESSED BY STRESS AND TEMPERATURES OUTSIDE (PARTICULARLY LOWER THAN) THE FISHS OPTIMAL RANGE AND PROMOTED BY GOOD HUSBANDRY AND NUTRITION
The specific immune systems Reacts to individual antigens. This system relies on chemicals know as antibodies and cells which engulf and digest invading pathogens. White blood cells
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10.0 TREATMENTS
INNATE IMMUNITY
consists of chemicals released into body fluids by various cells. This system defends against any antigen entering the body. It does not react specifically to any one antigen. Reacts to individual antigens. This system relies on chemicals known as antibodies and cells which engulf and digest invading pathogens. This system has a memory and can react more powerfully to an antigen if the fish is subsequently exposed again to the same antigen (see 10.7). The mechanism of protection is dependent upon a population of white blood cells called the lymphocytes. These cells carry receptors on their surface which can recognise chemicals (known as antigens) present on any material foreign to the fishs body (including pathogens). In a fish not previously exposed to a particular disease and its characteristic antigens only a small subpopulation of lymphocytes can recognise the antigen and react to it immediately. These lymphocytes react by dividing rapidly to produce identical copies of themselves. The number of cells that can recognise the antigen on the invading pathogen therefore increases rapidly.
SPECIFIC IMMUNITY
Antibody level
Exposure to antigen (pathogen) or vaccine
Time
Two types of lymphocytes produce different reactions to the presence of antigens: B lymphocytes are responsible for producing antibodies. These bind the antigen on the pathogen leading to the pathogen being killed by the body. T lymphocytes can kill cells that are infected by a pathogen. They can also produce chemicals known as cytokines which are important in activating special blood cells known as phagocytes which engulf and kill the pathogen.
KEY FACTORS AFFECTING THE IMMUNE SYSTEM
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10.0 TREATMENTS
The immune response depends upon a great deal of cell division and the production of proteins. The two main factors which decrease the rate of cell division and protein production are: STRESS Stress shuts down those aspects of metabolism which facilitate cell division and the production of protein in favour of providing carbohydrate energy sources, which might be required for fight or flight reactions.
STRESS
FISHS IMMUNE SYSTEMS ABILITY TO DEAL WITH PATHOGENS
LOW TEMPERATURE Reduces the rate of chemical reactions and thus slows cell metabolism. The optimal temperature for vaccination of a given species of fish is related to its preferred or normal temperature range. Thus for Atlantic salmon the optimal temperature for protein production and cellular activity is about 12C and is extremely slow at 4C, while for sea bass the optimum temperature is about 22C and at temperatures of about 10C the immune system of such warm water species is suppressed. The rate of induction of an immune response is faster in warm water species. At their optimum temperature, cold water Atlantic salmon will take about 6 weeks to start producing antibodies and the response will reach a peak at about 10 weeks post-vaccination, while warm water sea bass start producing antibodies within 1 week, reaching a peak in about 2 weeks. Thus, if fish are to be moved to a location where the risk of a particular disease is high, they need to be vaccinated at a certain minimum period of time prior to movement in order to ensure that they are protected. This minimum period depends upon the species and the temperature.
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10.2 CHEMICALS
A variety of chemicals are routinely used as treatments for fish. It is not appropriate to review the range of chemicals used either currently or historically. Many reference books are available that fulfil this purpose. In fish farming concerns over the safety and potential environmental impact of some chemicals has led to their withdrawal from the market or for bans on usage to be put in place.
10.0 TREATMENTS
10.3 ANTIBIOTICS
Since they were first discovered antibiotics have revolutionised the treatment of a whole range of previously deadly diseases in man and animals. That said the greatest step forward in reducing human disease was the introduction of municipal sewage systems and a ready supply of clean drinking water. Equally it is true that the greatest gains in fish health are to be made by careful husbandry; after all fish have spent hundreds of thousands, if not millions, of years evolving mechanisms to deal with the disease causing organisms in their environment. Despite this antibiotics have their place in treating fish diseases and fish farming. Antibiotics have received massive adverse publicity over the past couple of years. The reason, in the first instance, was the growth in prevalence of antibiotic resistant bacteria in hospitals. This was generally blamed on doctors for over-prescribing antibiotics. Blame was also put on the general public for insisting that they receive antibiotics for illnesses, like the common cold, which being viral in origin are not killed by them. Over the last couple of years the veterinary use of antibiotics has been the subject of media attention. In farming antibiotics can be used as growth promoters, to treat specific diseases or as prophylactics. In particular the use in the veterinary sphere of antibiotics either used in human medicine or closely related to those used in human medicine has given rise to public debate. The concern is that their use could: Or Remain in the meat of the treated animals and thus enter the human food chain. Promote the emergence of resistant strains of bacteria that can cause disease in humans.
An additional concern has been that bacteria that do not cause problems to humans could transmit their antibiotic resistance to other species of bacteria which might cause illness to humans. Already a report has appeared in the press implicating misuse of antibiotics on a fish farm (not in ornamental fish or on an ornamental site) for the resistance to treatment of an infection in a woman who died. The problem was apparently cause by a Vibrio spp. bacteria which infected a puncture wound cause by the spine in the dorsal fin of a gilthead bream. Twenty other people have been reported as being infected by similar bacteria following puncture wounds in the hands caused by handling Tilapia spp. This resulted in swelling and uncontrollable bleeding from the wound. In half the cases the hands were amputated to save the patient. Vibrio spp. bacteria are very common in water and the species involved in this in this incident usually cause no problems.
RESISTANCE IS NOT NEW
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10.0 TREATMENTS
Resistance to antibiotics is not a new phenomenon. Bacteria resistant to ampicillin, timethoprim, nalidixic acid, tetracycline, cefazimide, carbenicillin, cefazolin and cephamandole were found in 2000 year old ice from the Arctic. These same bacteria were not resistant to streptomycin, gentamycin, chloramphenicol or ciprofloxacin. That saidp resistance to most antibiotics is absent from bacteria collections from the early part of this century.
WHY IS THERE ANY NATURAL RESISTANCE TO ANTIBIOTICS?

The simple answer is because antibiotics are largely natural in origin. In places like the soil many species of bacteria and fungi grow together and compete for space. To help them compete they release chemicals to inhibit the growth of their competitors so that they are not overwhelmed. It is these chemicals which were first isolated and used as antibiotics. More recently these chemicals, or man made versions of them, have been produced in large quantities in factories.
HOW DO RESISTANT POPULATIONS ARISE?

Bacteria are usually present in vast numbers. There are genetic differences between individual bacteria. Within each population will be a small number that are, by chance, resistant to a therapeutic dose of antibiotic. Thus a small number of resistant bacteria will survive treatment. In the ideal world an animals own immune system will kill this very small residual population. If, for any reason, the immune system is not functioning optimally or the antibiotic dose leaves a large number of bacteria alive (for instance if too small a dose is given, or the treatment is not completed) then these will multiply to give a population, the vast majority of which, are resistant to further attempts to treat them. Increasing the dose of antibiotic used may remedy the situation; however it must be remembered that all treatments are biocides. These work because they kill biological systems. It is usually only a matter of the dose used which determines that bacteria are killed by a treatment but the fish are not. If the dose of a treatment is increased too much then both the bacteria and the fish may be killed.
DO RESISTANT POPULATIONS LAST FOREVER?

Generally it is thought that once the antibiotic is removed, the population may gradually revert to the normal antibiotic susceptible form. Thus if resistant strains have built up in a farm, continued use of different antibiotics, or higher doses of the same one, are not the only ways forward. However a paper has appeared in the scientific press challenging this view and proposing that in some instances antibiotic resistant bacterial populations were persistent.
ARE ALL BACTERIA BAD?

The answer is no. Humans rely on some bacteria to produce vital nutrients in our intestines, such as vitamin K, without which our blood would not clot. Many other bacteria species grow harmlessly in and on the bodies of animals and plants. Their removal may leave gaps into which the pathogens (disease causing bacteria) may grow and become established. Remember when you dig a new flower bed the weeds always get there first!!
MAGIC BULLETS? 110ANTIBIOTICS Some categories of antibiotics kill a wider spectrum of bacteria than others. 10.0 TREATMENTS
That said antibiotics do not specifically kill a single species or strain of bacteria. Indeed one of the problems associated with antibiotic use is that they kill both pathogenic and useful or harmless bacteria. Antibiotics are not well aimed magic bullets that hit a very specific target but more closely resemble shotgun pellets whose effects spread outside the intended target to a greater or lesser extent.
WHAT RANGE OF ANTIBIOTICS ARE AVAILABLE TO THE INDUSTRY?

The conditions under which antibiotics are supplied varies around the world. In some areas they are available over the counter from retailers. In other regions they are available only by prescription from veterinary surgeons. The range available is also quite variable. In 1995 the use of over 25 antibiotics were permitted for use in fish in Japan while only four were licensed in the UK.
HOW CAN ANTIBIOTIC RESISTANCE SPREAD?

Once a genetic mutation, giving rise to a bacteria resistant to antibiotics, has occurred its population develop as quickly as the bacteria can replicate itself (this can be either by sexual or asexual means). Bacteria can divide at an enormous rate given the correct conditions. However there is another way that resistance can be spread very rapidly both within and BETWEEN bacterial species. The technical term for the process is plasmid transfer. Plasmids are pieces of genetic materials that bacteria can release and absorb. Thus a bacteria of species A could release plasmids which in turn could be absorbed by other individuals of species A or indeed species B, C and D. It is the spread of plasmids among species that gives rise to the fear that antibiotic resistance in bacteria usually associated with animals could spread to bacteria causing disease in human. There are those who say this could happen and those who argue equally strongly that it could not: the jury is out on this issue! Monitoring the antibiotic resistance profiles of bacteria on imported food and animals including ornamental fish has been started by some Governments. Official reports of these findings have already been published mentioning the presence of antibiotic resistant bacteria on ornamental fish.
10.4 PROBIOTICS
Imagine adding bacteria to your fish holding system or transport. It goes against normal experience, but this may be one way forward in disease prevention particularly in young fish. Bacteria will take advantage of any opportunity to colonise an area where food is available for them. This could be the skin, gills or gastro-intestinal tract. The theory behind the use of probiotics is that if you can ensure every site where bacteria can grow is full of harmless bacteria, then pathogens will not get a chance to become established. The benign bacteria form what is in effect a protective sheath over the living fish tissue. There have also been a number of scientific papers published over the last twenty years that indicate bacteria isolated from the gut mucus produce substances which inhibit pathogens. Development of a pattern of use of probiotics in fish is some way off. That said, interest is being shown in the technology. The use of probiotics such as natural yoghurt have been used to help recolonise the guts of animals and humans following antibiotic treatment which killed the bacteria population normally present.
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10.5 PREBIOTICS
The bacteria in our guts is altered by the food we eat. Thus the gut bacteria in a person eating a diet high in fibre will be different to those in the gut of a person on a high fat diet. The same is true of the bacteria in fish intestines. Prebiotics are foods used to promote the growth of helpful bacteria. This technology is in the earliest stages of development for use with fish.
10.0 TREATMENTS
COMPARISON OF THE USE AND EFFICACY OF SOME DIFFERENT TREATMENT TYPES

Chemicals When are they normally used At the time of the disease Antibiotics At the time of a disease outbreak or exposure to a pathogen Excellent (resistance can be a problem) Limited Vaccines (see chapter 11) Prior to disease- little use when disease present Excellent Immunostimulants Prior to disease outbreak
Effectiveness*
Excellent
Good
Range of activity
From wide to narrow
Limited
Wide Short
Duration of Short Short Long protection *assumes accurate diagnosis and appropriate treatment used
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10.6 IMMUNOSTIMULANTS
Immunostimulants can be defined as substances that improve a fishs protection from diseases by stimulating its non-specific immune responses. The non-specific immune system is a series of reactions to foreign material, such as bacteria, that do not involve the specific immune response e.g. antibody production. They react on first contact with a pathogen to slow or stop the establishment of an infection. These responses are short lived and can be overcome by aggressive pathogens. The level of protection induced by an immunostimulant can be directly measured by looking at the non-specific defence proteins such as the opsonising protein ORP, before and after treatment. The level of this and other proteins are elevated for a period after treatment with an immunostimulant and by inference the resistance of the host to infection is also enhanced during this period.
10.0 TREATMENTS
WHAT MAKES A GOOD IMMUNO-STIMULANT?

Immunostimulants are derived from many sources yeast (glucans), other fungi (glucans & chitin), invertbrates (chitin and chitosan), bacteria (LPS and MPB) mammals (lactoferrin and transferrin) or they may be artificial (levamisole). They are effective because vertebrates (including fish) see these materials as foreign and react to them by activating the non-specific immune system. Although many immunostimulants are obtained from non-host structural materials, others have physiological roles such as lactoferrin and transferrin, which are found in fish. Many immunostimulants can be considered to be dietary supplements rather than true therapeutic drugs so their incorporation in the diets of larval and adult fish is not licensed to the same degree as say an antibiotic. (However the legal situation which can change must always be reviewed in each instance). Thus they have the potential to be widely adopted in animal diets. However, it must be understood that their effect may vary between species and, as will be discussed later, not all of the effects may be beneficial.
THE EFFECT OF IMMUNOSTIMULANTS ON THE IMMUNE SYSTEM OF FISH

The vast majority of immunostimulants studies with fish have shown major benefits, either by elevating the non-specific defence mechanisms of fish prior to exposure to a pathogen, or improved survival following exposure to a specific pathogen when treated with them. The following groups of immunostimulants have been found to be effective against various diseases: Viral infections; Fungal infections; Bacterial infections; Parasitic infections;
-glucans, levamisole, MDP CpG, ssRNA, LPS -glucans -glucans, MDP, FK-565, peptidoglycan -glucans, levamisole, MDP
BENEFITS OF IMMUNOSTIMULANTS
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10.0 TREATMENTS
The perceived benefits of immunostimulants are many and varied. As most immunostimulants occur naturally the molecules that can be obtained from a natural source in large amounts., e.g. glucans from yeast or chitin from arthropods (such as shrimps shell meal) can make cost effective dietary supplements. The addition of immunostimulants as dietary supplements, can improve the non-specific defences of the fish providing resistance to pathogens during periods of high stress, such as grading or transport. Their use in vaccine formulations, experimentally at least, has given very good antibody responses without the adverse side effects that have been reported for some other types of chemicals (known as adjuvants) historically used to boost the bodys response to vaccination. Theoretically, larval fish can be bathed in a solution of an immunostimulant or it can be incorporated into their diet (e.g. in enriched rotifers or Artemia) to provide improved protection through crisis periods such as end of yolk sac/first feeding or metamorphosis. It should be stressed that the use of immunostimulants in larval fish must be approached with caution as there is evidence that feeding an immunostimulant to larval fish may be detrimental to the optimal development of the full immune system. However there is great potential to improve larval survival against bacterial and viral pathogens by the judicious use of immunostimulants.
STRATEGIES FOR THE USE OF IMMUNOSTIMULANTS IN LARVAL FISH

Feeding immunostimulants continually during larval development may induce tolerance. Worse, it may damage the developing immune system causing the immune system to stop responding to that type of stimuli, or reducing non-specific responses. Further research is required to establish what adverse effects immunostimulants might have on larval fish if they are given inappropriately. What is clear from the limited number of studies that have been carried out in this field is that immunostimulants should be used cautiously in larval fish and only given for short periods during periods of high risk. Certainly the routine incorporation of immunostimulants in diets should be approached with caution, as their effects may not be completely beneficial.
STRATEGIES FOR THE USE OF IMMUNOSTIMULANTS IN IMMUNOLOGICALLY MATURE FISH

In adult fish i.e. fish that have undergone metamorphosis and have a fully developed immune system, there are only two effective strategies for immunostimulisation. These are continual feeding of the immunostimulant, or, pulse feeding of the immunostimulant. Bathing the fish in a solution of immunostimulant is a third strategy but does not seem to have been effective in studies on adult fish, although it may be beneficial. Continual feeding of immunostimulants has generally been abandoned, in adult fish, in favour of pulse feeding. There are two possible outcomes of continually feeding of an immunostimulant. The immunostimulant may up regulate the immune system to heightened level and this is maintained indefinitely. However, this is a very rare occurrence. Continual exposure to an immunostimulant can induce tolerance. This is caused by the immune system of the host becoming de-sensitised to the immunostimulant and the response is lost. In extreme circumstance the continued exposure to the immunostimulant causes the immune system to become suppressed, giving a lower level of non-specific protection than that of untreated fish.
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To overcome this, most immunostimulation use involves pulse feeding the immunostimulant for a short period, usually 4-6 weeks. The level of immunostimulation then falls back towards the resting level before another dose of immunostimulants is given. This tends to cause the host immune system to return to the stimulated level then to fall when the immunostimulant is withdrawn. The process can then be repeated. Such a strategy offers immense flexibility in disease management and risk reduction as the immunostimulant can be fed during periods of increased disease risk. Such periods of risk include: spring and autumn temperature changes; just prior to the breeding season; or prior to shipping.
10.0 TREATMENTS
SUMMARY
Immunostimulants offer the ability to protect valuable stock against short term threats from pathogens and can be administered a few days prior to stressful husbandry procedures. They have the potential to enable larval fish to protect themselves from infection providing the immune system is sufficiently developed to benefit from their administration. Since most immunostimulants are natural products, which have short, non-hazardous, breakdown routes, they may be easier to license for use. This may give the industry an effective method of providing animals with short-term protection at particular points in the production cycle. However, it is unknown how they may adversely affect the developing immune system of larval fish and inappropriate use may be detrimental to their survival in the long term.
10.7 IMMUNISATION AND VACCINATION (see also 10.1)

HISTORY
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10.0 TREATMENTS
Vaccines are relatively recent additions to the medicines available to treat fish. The table below summarises the impact that the introduction of vaccines had on the use of antibiotics in the salmon farming industry in Norway. It may be that the whole of this change cannot be explained just by the introduction of vaccines, but may be partially explained by the development of generally better husbandry techniques. Production of salmon tonnes 60,000 342,000 % reduction in use of antibiotics Total weight of antibiotics used kg 50,000 679 Antibiotics used per tonne of production grams 830 1.2 99.85
Year 1987 1998
The reasons for the salmon industry to move away from antibiotics included the: short period of protection they offer need for repeated doses to be administered particularly for recurrent diseases difficulties caused by resistant strains of bacteria increased concern about and control of residues present in the human food chain. increased concern about the impacts that antibiotics and other chemicals used for disease treatment could have on the environment
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10.0 TREATMENTS
PROTECTION BY VACCINES SPECIFIC IMMUNITY
A vaccine is best regarded as inducing protection against a particular disease that is specific for that particular disease and does not protect against other diseases. Vaccines induce long-term protection and in aquaculture (fish are usually kept and grown for 2 or 3 years prior to sale) a single administration is usually sufficient to induce protection for the lifetime of the fish. This long term protection is because the body retains a memory for that specific disease and can react quickly and effectively when the causative pathogen arrives.
NON-SPECIFIC IMMUNITY
Vaccines may have more general immunostimulatory properties that can also activate the non-specific defence mechanisms. These can increase disease resistance levels but only for a short period of time. Due to their capacity to induce such responses they are also used in shellfish culture, especially of shrimps, but strictly speaking this function should be considered as that of an immunostimulant.
Strictly vaccines are used only as a prophylactic measure in vertebrates, including fish. They do not work generally very well, or at all, if administered while fish are affected by the pathogen the vaccine is intended to control.
TYPES OF IMMUNITY AND IMMUNIZATION NATURAL OR INHERITED RESISTANCE

Pathogens cannot infect all species. Even among species that pathogens can infect there are often populations or strains which are either completely or partially resistant to infection. This resistance is not induced by any means and is often passed on from generation to generation.
NATURAL EXPOSURE
Animals and their pathogens regularly come into contact with one another and diseases occur naturally. Equally naturally some of the animals affected recover and become resistant to the disease. This may over time lead to naturally immune populations. However, the pathogen might keep changing so that it can continually infect its hosts and stay one step ahead of their immune system.
KILLED VACCINES
Killed pathogen are injected or introduced to an animal. Its immune response will react to the killed pathogen and remember it. When confronted with a live pathogen of the same species the immune system can react actively and attack and eliminate it. Animals tend to react less strongly to killed vaccines than live pathogens. Animals may need booster doses on a regular basis to ensure the immune system is primed for the disease the vaccine is designed to protect them from.
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LIVE ATTENUATED VACCINES
These vaccines use a live rather than killed pathogen to promote immunity. However rather than using the normal strains of pathogen they use attenuated or weakened pathogens. Because the vaccine is live a good response is promoted by the animal but because the pathogen is weakened it should not cause disease. A potential problem with this sort of vaccine is if the pathogen reverts to its original disease causing type. However, before vaccines are permitted to enter most markets in the world extensive tests have to carried out to prove that reversion does not happen.
10.0 TREATMENTS
METHODS OF VACCINATION
There are two main methods of administering vaccines to fish, immersion in a dilute suspension of the vaccine or by injection into the body cavity. For practical reasons the latter method require the fish to be over about 15 grams in weight and so it is not usually feasible for ornamental species due to their small size.
IMMERSION VACCINATION
This is effective for some, but not all vaccines. The vaccine against the bacterial disease vibriosis is highly effective when administered by immersion. It is used widely in marine fish aquaculture. However, with the exception of the vaccine against Enteric Red Mouth (a disease of trout), which is delivered by immersion to fish in freshwater hatcheries other vaccines are delivered by injection in order to achieve effective protection. Immersion vaccines are not formulated with any adjuvant and comprise a whole bacterial culture inactivated by addition of formalin. Small batches of fish held in a net are immersed for about 30 seconds in vaccine which is usually diluted about 1:10 with water. Each litre of undiluted vaccine is sufficient to immunise about 100kg of fish. The method is ideal for vaccinating large numbers of small fish.
INJECTION VACCINATION
This is the most effective route of vaccination as it allows the incorporation of adjuvants that induce longterm protection. However, it is obviously very labour intensive. Adjuvants are oils which prolong the duration of protection given by a vaccine and also act as immunostimulants. However the use of adjuvant has been associated with side effects which can result in the viscera adhering to the body wall of the fish.
ORAL DELIVERY OF VACCINES

While this is an ideal method of vaccinating fish logistically, unfortunately at the time of writing it is not used on a widespread basis and it can be of varying effectiveness. There is still much research being conducted on the development of effective oral delivery of vaccines. Oral vaccines would need to pass through the stomach and foregut to the hindgut to be effective. However, it appears that antigens administered orally are digested in the foregut. Methods of protecting them as they pass through this area and releasing them in the hindgut are still being researched.
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10.0 TREATMENTS
BOOSTER VACCINATION
Antibody levels may fall after vaccination. Eventually they will fall to a level where they do not offer adequate protection. At that point the fish may be vaccinated again with a booster. This vaccination, which may have been repeated at regular intervals returns antibodies to protective levels.
Antibody levels
Fish protected
Antibody levels not sufficient to offer disease protection.
Vaccination
Booster Time
Ideally the booster would be given before the antibody level fell below levels that protected against disease.
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QUESTIONNAIRES
QUESTIONNAIRES
1. Your business attitude to risk
2.
Identifying pathogens of concern to you
3.
Assessment of pathways by which pathogens may enter a site
4.
Site biosecurity appraisal
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YOUR BUSINESS ATTITUDE TO THE RISK OF PATHOGENS ENTERING (AND LEAVING) YOUR SITE.
High biosecurity 9 8 7 6 5 4 3 2 1 Prepared to accept and fund the costs of any outbreak of disease Not prepared to fund any measures to prevent pathogens entering your site or the disease they may cause Prepared to invest in biosecurity measures to prevent the entry of specific pathogens Prepared to invest in comprehensive, effective biosecurity measures Prepared to fund prevention of pathogen entry and treatment of disease including the regular additional use of external professional advice, e.g. vets Prepared to invest in training staff, cost effective disease prevention and treatment
QUESTIONNAIRE 1
Low biosecurity
Try to score your attitude on a scale of 1 to 9.

The comments alongside the scale are indicative of what would constitute attitudes that could lead to high (9), medium (5) or low (1) biosecurity. You may you feel that your attitude falls between two of the indicative scores.
IDENTIFYING PATHOGENS OF CONCERN TO YOUR BUSINESS (Chapter 3)

List the pathogens (including zoonoses see Apendix 12) you want : a. b. c. d. e. To completely exclude from your site Are prepared to manage after arrival on your site To prevent leaving your site entirely Reduce the numbers leaving your site Are unconcerned about. (3.3)
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QUESTIONNAIRE 2
A risk evaluation table may be used to help make decisions. (3.2) Risk evaluation table
Probability
Before a pathogen or potential pathogen can be allocated to these categories a range of information about each of them should be gathered. Answering the questions below may help you gather the required information for each pathogen or potential pathogen and place it in a category with more confidence. 1. Will the pathogen have an impact on your business: cause fish losses on your site? cause reduction of saleability of the fish while on your site? be passed to your customers, or subsequent customers sites and lead to loss of confidence in you as a supplier?
1223. Are the pathogens, or potential pathogens, of concern found:

QUESTIONNAIRE 2
everywhere (and thus may not be excluded)? (6.3) present on one of more of your suppliers sites? on your site? on both your site and that of at least one of your suppliers?
2. What will be the cost to your business of those impacts?
4. Is the pathogen (6.3) always going to cause disease (obligate pathogen)? only going to cause disease under certain circumstances (facultative pathogen)?
5. Are the pathogens of concern transmitted (6.9) horizontally? Vertically? Both? is a vector, intermediate host or fomite required?
6. Under what conditions will the presence of the pathogen cause disease? (4.4) 7. Could the pathogen of concern become established on your site (does the stocking density, temperature, host or vector availability, pathogen loading, dispersal or transport technique permit transfer)? (4.4, 6.0, 7.0, 8.0) Yes Uncertain No-if there is no risk of the disease becoming established on your site or being carried passively through your site then the assessment can be stopped at this point
8. Which techniques must be used to accurately identify the pathogens concerned? (9) Are these techniques available to your supplier either by employing an off-site consultant (including Government laboratory) or on-site? Are these techniques available to you either by off-site consultant (including Government laboratory) or on-site? The pathogen cant be accurately and reliably detected and hence risk is uncertain.
9. Are there treatments available for the pathogen? (10.0) What are those treatments? How effective are they? Are there legal constraints on their use eg requirement for veterinary prescription and supervision? What are the costs associated with treatment?
ASSESSMENT OF PATHWAYS BY WHICH PATHOGENS MAY ENTER A SITE

This assessment can be used to assess the likelihood of a pathogen entering a site. It can be applied to any site at any point of the supply chain. Thus it could be applied to either the producer at the start of the chain or the retailer at its end.
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QUESTIONNAIRE 3
Examples of routes of entry Fish Plants Water Packaging material Wildlife Equipment Staff Customers
Indicative list of sources Wild fish swimming onto site, commercial supplies, customer returns Commercial supplies, customer returns River, tapwater, borehole, water used for transport Fish and plant transport boxes and bags. Any animals , reptiles, amphibians, rats , invertebrates etc. Nets, bags, vehicles Hands, clothes, protective gear Hands, clothes
Likelihood of * pathogen or disease entering
List preventative measures in place*
Effectiveness of * preventative measures
Rate on a scale of
1 9
Highly likely Highly unlikely
Ineffective Highly effective
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QUESTIONNAIRE 4
SITE BIOSECURITY APPRAISAL

This questionnaire can be used by any business which holds or keeps livestock on their site. Thus it is relevant to importers, breeders, wholesalers and retailers. INTERPRETATION: As the saying goes a chain is only as strong as its weakest link. Thus just one ineffective preventative measure renders a site open to biosecurity problems. *It is suggested that you use the following scoring system. The higher the score the more effective the biosecurity is.
Likelihood of occurrence
Very likely Likely Possible Unlikely Very unikely Not known
Biosecurity score
1 3 5
Effectiveness of measures in place

Absent or completely ineffective Ineffective
Effective 9 If an estimate cannot be made then it may be best to assign a low score ie assume low biosecurity Highly effective Not known
As appropriate this assessment can be applied to your business, the businesses supplying you directly and in turn down the supply chain to the point of production or collection. The indicative score is based on the scoring system described above ie the higher the value given (between 1 and 9) the higher the biosecurity.
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QUESTIONNAIRE 4
The indicative score is given only as a guide. Individual circumstances might greatly vary the score given. For instance for question 1 the scores given indicate that if a member of your staff has visited a site then that is likely to promote biosecurity. However this will depend on several factors including: the knowledge and experience of the person making the visit, the quality, accuracy and thoroughness of the report they provide effective management action being taken on the basis of that report.
In the absence of any one of these conditions the value of the measure is reduced and thus reduces its value as a biosecurity safeguard. Many of the scores will in reality depend on the quality of management. A good site poorly managed may present a significant risk. No absolute reliance should be given to the indicative scores included in the table. The scores you give can be used to calculate an average biosecurity score for a site, either yours or those of your suppliers, this may prove useful in deciding whether to continue trading with a particular supplier or site, or to ask for improvements to be made or to change the procedures eg treatment on arrival, you use when accepting fish from that site so as to limit the biosecurity risk to your site.
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QUESTIONNAIRE 4
1.
Question
Indicative biosecurity score if answer is yes
Text reference(s)
Comments
Your score
Supply chain
Do you know how many facilities the fish you buy enter en-route to your site?
4.4
Knowledge of livestock sources

Have you or your staff visited the site (farm, wholesaler etc) supplying the fish and other aquatic organisms you purchase? In turn has your supplier visited the original supplying site and reported accurately to you on its condition? Are you buying site unseen? Up to 9
4.1
Up to 9 Down to 1
3.
Livestock
Closed cycle production on site (applies to farms) Under what conditions and when were broodstock introduced to the site? Stock is bought in from how many other farms or sites? (Generally the higher the number the greater the biosecurity risks). Livestock from individual farms isolated Detailed information available on suppliers Up to 9 Up to 9 8-9 1-9 1-9 4.4
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QUESTIONNAIRE 4
4.
Preventative acclimatization
Where appropriate are preventative acclimatisation methods used to prevent entry of pathogens? Are fish acclimatised on arrival? Are fish treated prophylactically for pathogens of concern on or after arrival? Is the acclimatisation area separate from fish already held ? Are newly arrived fish separated on a centralised system by use of either UV or ozone? Or Are newly arrived fish put in separate holding systems, such as single aquaria on UG filtration? Are newly arrived fish mixed with old stock? All 1-9 depending on the measures applied and how rigorously
5.2
5.2 6.8, 6.9 7.2 7.2
6.8,6.9
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QUESTIONNAIRE 4
Water source on site

Borehole, spring or well? Is the water under the control of the site manager from source to farm? Is the incoming water UV or ozone treated? Is it treated tapwater? Potable drinking water? River or other water body containing fish? Are fish excluded from the water source? Does the site flood? Can fish swim onto the site? 9 7 5 to 9 1 to 9 8-9 1-2 2-4 1 1
6.0
Water flow management

Water is used once and discarded Water flows from one pond or aquarium system to the next without treatment Water is recycled; Untreated Via a properly managed UV filter Via a properly management ozone treatment 1 4-5 7 9 1 to 3
7.2
7a
Aquarium system management

Dried and disinfected between stockings? Dried between stockings? Fallowed between stockings? (Prior to fallowing were all fish removed? ) Stocked continuously using: All in all out policy for stocking system Continual stocking and mixing of batches
9 7 1-4
7.3
129
QUESTIONNAIRE 4
Down to 1
Pond management
Dried and limed between stocking? Dried between stockings? Treated with lime but not drained between stocking? Fallowed between stocking? How long for? Prior to fallowing were all fish removed? By what technique: Netting Poisoning Electro-fishing Other 2 9 1-4 ? 2 1 9 7-8 4-5 3-4 7.3
Stocked continuously using: All in all out policy for stocking ponds? Continual stocking and mixing of batches?
130
QUESTIONNAIRE 4
Water quality monitoring

Is water quality monitored? Parameters measured Free ammonia Nitrite Nitrate pH Dissolved oxygen Other - list: Are records kept? Is effective action taken if adverse results found? Yes No 4-9 Up to 9 Down to 1 4- 9 Appendix II
Health monitoring
No formal system On-site visual On-site microscope Vet visits as needed Vet visits at regular intervals Regular sampling programme (wet preparations, histology, bacteriology) 1 3 5 6 7-8 7-8 10.0
Official services visit to assess health of stock as needed Official services visit to assess health of stock regularly Official services undertake regular sampling programme (wet preparations, histology, bacteriology, virology) Other techniques 10
6-9 6-9 9
131
QUESTIONNAIRE 4
Fish treatments
Are the fish stocks treated prophylactically for the pathogens of greatest concern to you? Are fish only treated for pathogens when they are visible to the naked eye or cause disease? Are the fish vaccinated against important pathogens? Nothing is known of the fish treatment regime at the supply site No treatments are undertaken Up to 9 4-7 Up to 9 1 1 9.0
132
QUESTIONNAIRE 4
133
Below, as an aide memoire, are lists of some risk reduction strategies that might be applied as and where appropriate. Some such as those applying to moving fish or equipment around a site might apply to any business. Others such as those concerning hand disinfection may for practical reasons apply less widely. They can be used to inform management of a site or as a guide of what to look out for when evaluating a site (for instance that of a supplier). These are not a substitute to answering the questionnaires, nor are they comprehensive, but may add to the value of the evaluation process that should be undertaken preferably after the questionnaires have been completed. * A 5.2 C Q Appendix Chapter section Chapter (whole) Questionnaire Section* Q1 C2 Whole report, A1
General policy & management

Decide your businesses attitude to the risk of bringing pathogens and disease on to, around and off your site Identify hazards you want to manage or eliminate and critical control points associated with them Establish biosecurity policy (ensuring you identify any laws which govern fish disease or fish movements either locally or in the areas or countries to which you send livestock) Communicate your biosecurity policy to suppliers, staff and customers as appropriate and necessary Provide training, technical back up or equipment to staff as required to enable them to implement the policy Establish working relationship with a suitably interested, experienced or qualified vet or fish biologist Review the biosecurity policy and any requirements for change or additional training etc. regularly
Whole report
134
General husbandry
Ensure the appropriate staff know the biology of the fish species in which you trade and can, as appropriate, pass that knowledge on to other staff members and customers Ensure the appropriate staff members know enough of the biology and characteristics of fish diseases, especially those caused by the pathogens of greatest concern, to recognise, treat or call for additional assistance as appropriate Monitor stock health regularly (using appropriate techniques for detection of pathogen of concern) Be aware of the potential for new diseases to emerge or old diseases to re-emerge Know how and when the available disease treatments may be used. Be aware of the manufacturers recommendations concerning use, health and safety and disposal Maintain water quality appropriate for the species (remembering to maintain an appropriate temperature for the species) Maintain records of water quality, fish health, imports and the like Maintain the health of the fishs own immune system by appropriate husbandry including resting/acclimatization periods Avoid unnecessary stressing of fish eg over or under stocking depending on species, using correct transport and handling methods Be aware of how disinfectants work and how they may be used. Clean and disinfect surfaces, nets and equipment as required. Regularly sterilize systems (this could be between stockings at regular intervals or end of years) as appropriate. Always remember the pipework is part of the system and must also be sterilised. Keep holding areas well ventilated and surfaces as dry as possible. 10.1 10.1 C9, A9 8.0 C4, C5, C6, C7, A7 C9 6.4,6.5 C10 A2
Section
7.4
135
Avoid bringing disease on to a site
Identify the pathways by which pathogens could arrive on your site Establish a plan to minimize or eliminate where possible the risks identified with each pathway Wherever possible purchase from a source that matches the EU Approved Farm model and/or sites that are not infected, and not supplied by sites which are infected, with the pathogen of concern When possible visit the sites you buy fish from. Know your supplier! As far as possible know your supply chain. The more participants in a supply chain (especially if fish from different sources are mixed) the greater the potential for problems, especially if adequate management is not in place at each site When available buy fish which are free of the pathogens of concern to you (or, if appropriate or possible, which have been vaccinated against them but which do not act as carriers) Isolate newly purchased fish, especially those from different sources, acclimatize them and treat them either promptly upon the discovery of a pathogen as required or prophylactically as experience dictates Remove or prevent the entry of vectors and fomites (including equipment and organisms that carry diseases other than the livestock you intend to keep such as unwanted fish, snails, birds and amphibians) that could bring disease on to, or move disease around your site 5.2 4.4
Section
C4, 6.10 Whole report Q3 4.1
6.10
Avoid moving disease around a site

Remove dead fish as soon as they are found and try to prevent them being consumed by their tank or pond mates (and dispose of safely) Reduce the pathogen loading in the water used by using a one use flow through system or in re-circulation systems by using sterilizing filters maintained according to manufacturers instructions (UV and ozone) Wherever possible daily routines such as cleaning should work from systems with the least pathogens present to those with the most (clean to dirty) Ensure that routine washing and sterilization of equipment eg nets, buckets etc. is carried out according to manufacturers instructions after each use (making sure they are rinsed free of disinfectant prior to reuse). Preferably would also be dried in each time it was used equipment. After a serious disease outbreak use effective disinfection methods, remembering biofilms such as those in biofilters can protect pathogens so must be thoroughly cleansed. Rinse disinfectant from system as per manufacturers instructions
Section
6.8, A4 6.8, 7.2, A4, A5 7.1 8.0
C8, 7.5
136
Avoid moving disease off a site

Treat fish as required prior to removal from a site Treat water if required prior to discharge Dispose of mortalities safely or as required by local law
Section
C7, 8.7
High risk areas such as hatcheries, nurseries, disease investigation areas

Install a hand-washing regime with sinks and skin disinfection available at all entrances and exits to the facility. Train all staff to wash their hands prior to coming into contact with and after manipulating animals. Consider the use of disposable sterile gloves in high risk situations such as mortality removal. Provide Personal Protective Clothing (overalls and Wellingtons) and the facility to disinfect work clothing on site. 8.3
8.4
Install footbaths at the entrance and exit
8.2
Ensure that routine sterilisation of nets, transport buckets etc is carried out, preferably after each use (making sure they are used free of disinfectant prior to reuse).
8.6
137
Appendices
138
139
APPENDIX 1 WATER QUALITY CRITERIA
ORNAMENTAL AQUATIC TRADE ASSOCIATION (OATA)
WATER QUALITY CRITERIA
140
CONTENTS
1. AMMONIA
SOURCES MEASUREMENT OF AMMONIA SAFE LEVELS OF FREE AMMONIA
2.
NITRITE
SOURCES MEASUREMENT OF NITRITE SAFE LEVELS OF NITRITE
3.
NITRATE
SOURCES MEASUREMENT OF NITRATE SAFE LEVELS OF NITRATE
4. 5. 6.
PH
THE PH SCALE
BUFFERING
RISING PH
DISSOLVED OXYGEN
SATURATION SOLUBILITY OF OXYGEN WEATHER MEASUREMENT OF DISSOLVED OXYGEN OXYGEN CONSUMPTION
7. 8.
BIOLOGICAL FILTRATION APPENDIX

STOCKING DENSITIES- ORNAMENTAL FISH WATER QUALITY CRITERIA GUIDE STOCKING DENSITIES
141
1. AMMONIA
SOURCES
Table 1: Levels of TOTAL AMMONIA (mg.l or ppm) that maintain FREE AMMONIA at or below 0.02mg/l (ppm) at a range of pH and emperatures
PH/Temp C 0
5
10
15
20
25
30
6 250
154
105
74
50
35
25
6..5 77
50
34
23
16
11
8
7 24
16
11
7.5
5
3.5
2.5
7.5 7.7
5
3.4
2.3
1.6
1.1
0.8
8 2.4
1.6
1.1
0.75
0.52
0.37
0.27
8.5 0.78
0.52
0.36
0.25
0.18
0.13
0.1
9 0.1
0.07
0.05
0.04
0.04
0.03
0.03
These figures apply to freshwater. To meet criteria for marine fish these figures should be halved.
142
Levels can be reduced by: Improvement of stocking, feeding and general husbandry procedures Improvement of biological filtration Use of ion exchange materials Dilution by water changes
In aquaria and ponds the principal sources of ammonia are: Excretion by fish and other livestock as a normal part of their metabolism The breakdown of protein in uneaten food or dead livestock which remains undetected. It is therefore of great importance that careful cleaning is undertaken at suitable intervals.
As ammonia is released into the water by either of these processes it may take one of two forms: Free Ammonia (unionised ammonia, chemical symbol NH3 ). This form of ammonia is highly toxic to fish Ammonium (ionised ammonia, chemical symbol NH4). This form of ammonia is virtually non-toxic to fish
NH3 Total Ammonia

Dissolves
NH4+ Free Ammonia

+
NH3 Ammonium
The balance between Free Ammonia and Ammonium is determined by the pH and temperature of the water and may be summarised :
High temperature High pH
High free ammonia (low ammonium) Dangerous to fish
Low temperature Low pH
Low free ammonia (high ammonium)
Relatively safe for fish
143
MEASUREMENT OF AMMONIA
Test kits and electronic meters usually measure all ammonia present, this is TOTAL AMMONIA. Some test kits measure Ammonium-Nitrogen and apply a conversion factor to determine the ammonia. This will usually be explained in the instructions provided with the test kit.
TOTAL AMMONIA = FREE AMMONIA + AMMONIUM SAFE LEVELS OF FREE AMMONIA

OATA recommends that FREE AMMONIA should not exceed 0.02ppm (mg/l) in freshwater and 0.01ppm in seawater. Above this level free ammonia causes the fish stress and at higher levels it may cause damage to gills and many internal organs eventually resulting in fish deaths.
144
2.
NITRITE
SOURCES
Levels can be reduced by: Improvement of stocking, feeding and general husbandry procedures; Improvement of biological filtration Dilution by water change;
In aquaria and ponds nitrites are produced by Nitrosomonas bacteria when ammonia is broken down.
MEASUREMENT OF NITRITE
Test kits are available. Some of these measure Nitrite-Nitrogen a conversion factor being applied to the result to obtain a true Nitrite reading; full instructions should be available with the test kit used.
SAFE LEVELS OF NITRITE

OATA recommends that Nitrite should not exceed 0.2mg/l in freshwater and 0.125mg/l in seawater. Nitrite poisons the fish by binding the haemoglobin in the blood preventing it carrying oxygen, in effect suffocating the fish. The gills of fish dying as a result of nitrite poisoning are a characteristic brown colour. In freshwater the toxicity of nitrite may be reduced by the addition of small amounts of certain salts.
3.
NITRATE
145
SOURCES
Levels can be reduced by: Dilution by water change, ensure water used for change has a lower nitrate level; Use of ion exchange materials; Increase plant density; Use of denitrifying biological filtration
Nitrates are: Produced as Nitrobacter breaks down nitrites; Introduced in tap water. In some areas of the country in tap water nitrate levels exceed 130mg/l
MEASUREMENT OF NITRATE
Test kits are available. Some measure Nitrate-Nitrogen a conversion factor then being applied to obtain a true Nitrate reading; full instructions should be available with the kits used.
146
SAFE LEVELS OF NITRATE

Nitrate is generally of low toxicity though some species, especially marines, are sensitive to its presence. When nitrate levels are high, as a result of biological filtration, other toxic chemicals produced in this process may be present at levels that adversely affect fish health. In the Water Quality Criteria, OATA recommends that nitrate levels in freshwater ponds do not exceed those in the tap water supply be 50mg/l. As the livestock is more sensitive in marine systems Nitrate should not exceed 40mg/l.pH.
4.
PH
147
THE PH SCALE..
10 11
12
13 14
TAPWATER
...is logarithmic
This means that the ph of a pond or aquaria alters by one units its acidity or alkalinity alters by x 10; Thus ph6 is x 10 more acid than ph7, And ph5 is x 100 more acid than ph7
148
5. BUFFERING
Hard water and seawater contain dissolved materials that prevent rapid change in pH they are BUFFERED. The buffering system in seawater is overcome at about pH 8.1. Once the buffering system runs out a very rapid fall in the pH may occur jeopardising livestock.
RISING PH
At low pHs the toxicity of ammonia is low. Low pHs may be bought about by carbon dioxide, produced by animals all the time and by plants at night, dissolving in water and forming carbonic acid. If water of a higher pH is added, particularly if buffered, then there may be a sudden increase in pH. Associated with this rise will be a rapid increase in the toxicity of any ammonia present. This situation may arise in the transport of livestock in either hard or seawater.
6.
DISSOLVED OXYGEN
1 litre of oxygen weighs 1,428mg 1 litre of air contains 285mg oxygen I litre of freshwater contains 14.6mg oxygen at 0C
149
Water is therefore an oxygen poor environment. It contains only 5% of the oxygen that the same volume of air does.
Oxygen depleted by:
Plants at night
Biological filtration Livestock
Oxygen replenished by:
Plants during the day
By diffusion at airwater interfaces
150
SATURATION
If a beaker of sterile freshwater is left to stand at 25C then the normal maximum amount of oxygen that it can dissolve is 8.2mg/l. At this point the water sample is said to SATURATED. If it contained only 4.1mg/l then it would be 50% saturated. A level of dissolved oxygen of 6mg;l as recommended in the OATA Water Quality Criteria is equivalent to 73% saturation.
SOLUBILITY OF OXYGEN
As the table below demonstrates, that as the water temperature rises the amount of oxygen it may dissolve before becoming saturated diminishes. Seawater dissolves less oxygen than freshwater before it becomes saturated. Table 2: Solubility of oxygen Temp C Mg/l oxygen freshwater 14. 612.8 11.3 10.1 9.1 8.2 7.5 OATA criteria in terms of % saturation 41 47 53 59 66 73 80 Mg/l oxygen saltwater 11.7 10.4 9.3 8.5 7.8 7.1 6.5 OATA criteria in terms of % saturation 47 52 58 65 71 77 85
0 5 10 15 20 25 30
Altitude and atmospheric pressure play a small part in determining oxygen solubility. For practical purposes both may be ignored.
WEATHER
The weather may combine all of the factors to create problems (some of them may arise also in fish houses). Sunlight increases water temperature and hence decreases oxygen solubility. Freezing ice seals the surface preventing the entry of oxygen and the escape of toxic gases. Still periods when there is little wind such as before thunder storms, the rate of diffusion of oxygen is diminished by the reduction of the pond surface area (ripple in a light wind may increase the surface area of a pond by two or three times).
MEASUREMENT OF DISSOLVED OXYGEN

Chemical test kits are available.
OXYGEN CONSUMPTION

151
Size of livestock small fish use relatively more oxygen than large fish. Total weight of livestock Oxygen consumption of livestock doubles for each 10C rise in temperature, but oxygen availability is reduced as its solubility is also reduced by the temperature rise. Biofilter activity ammonia production mirrors oxygen consumption.
152
7.
BIOLOGICAL FILTRATION
Biological filtration is the process by which waste, principally ammonia, products in ponds and aquaria are broken down by bacteria.
BIOLOGICAL FILTRATION
AMMONIA
NH3 BACTERIA
NITRITE
NO2
} }
NITROSOMONAS
(BOTH REQUIRE PRESENCE OF OXYGEN TO PROCEED ie THEY ARE AEROBIC)
NITROBACTER
NITRATE
NO3
OR
NITROGEN GAS
N2
AMMONIA
NH3
} }
(REQUIRES THE ABSENCE OF OXYGEN TO PROCEED ie ANAEROBIC)
VARIOUS
DENITRIFICATION
The bacteria which are responsible for nitrification require:
153
A surface on which to grow (the larger the surface area, the greater the population which may grow) A good supply of dissolved oxygen and an active filter may use more oxygen that the livestock in the water it processes A supply of nutrients ammonia and nitrates
The bacteria responsible for denitrification require: A surface on which to grow; A supply of nutrients o Principally nitrates; o Secondarily, if methanol or similar chemical is present, nitrogen gas will be formed; o If other organic materials are present, ammonia may be formed.
Oxygen kills the bacteria responsible for this process. A filter may be said to mature when any ammonia entering a tank is instantaneously converted to nitrite and then in turn to nitrate. Biological filters are only mature for specific conditions; if the stocking density or feeding increases then the filter needs a further period of maturation.
154
8.
APPENDIX A
STOCKING DENSITIES ORNAMENTAL FISH

It is virtually impossible to determine the quality of fish to be kept in a system purely on weight or number of fish per unit volume or area of water surface area. The variation in holding system used, the quality of husbandry and types of fish stocked vary so greatly that it would render any such system too complicated to be practical; or too simple to be useful. The maintenance of water quality standards can be used to determine working stocking densities.
WATER QUALITY CRITERIA COLD WATER SPECIES

*Dissolved Oxygen *Free Ammonia Nitrite Nitrate - min 6mg/litre - max 0.02mg/litre - max 0.2mg/litre - max 50mg/litre above ambient tap water
TROPICAL SPECIES
*Dissolved Oxygen *Free Ammonia Nitrite Nitrate - min 6mg/litre - max 0.02mg/litre - max 0.2mg/litre - max 50mg/litre above ambient tap water
TROPICAL MARINE SPECIES

*Dissolved Oxygen *Free Ammonia Nitrite Nitrate *pH - min 5.5mg/litre - max 0.01mg/litre - max 0.125mg/litre - max 40mg/litre absolute - min 8.1
Factors marked * should be measured in the first instance, if they prove satisfactory, and the fish appear healthy, then further investigation may not be necessary.
FISH UNDER TREATMENT

It may not be possible to maintain levels given when effective disease treatments are in use.
GUIDE STOCKING DENSITIES

The water quality standards should not be met at the expense of a correct feeding reginme.
155
COLD FRESHWATER
8kg/1000 l
TROPICAL FRESHWATER
Fish up to 5cm (2) 1.5kg/1000 l Fish over 5cm (2) 2.5kg/1000 l
TROPICAL MARINE
Fish up to 5cm (5) 1kg/1000 l Fish over 5cm (2) 2kg/1000 l Guide stockings are ADVISORY only. They may be exceeded if the water quality standards are satisfied. When the water quality standards are exceeded at a lower stocking, this must be considered as the maximum stocking density permissible. The TOTAL volume of the system must be measured and taken into account in determining actual stocking densities.
TECHNICAL NOTE
The above figures should be read in the following manner: Free Ammonia as NH3 Nitrite as NO2 Nitrate as NO3
156
APPENDIX 2 FISH EUTHANASIA
FISH EUTHANASIA
It now is widely accepted that fish feel pain or something similar to it. Euthanasia must be carried out using a technique that brings about death quickly with as little pain as possible. A well-aimed blow to the head which kills fish rapidly by rendering the animal unconscious at the same time as destroying the brain is considered one of the most humane ways of euthanizing a fish. If faced with a large number of fish to destroy, a member may be best advised to seek help from his or her vet. A fish anaesthetic such as benzocaine which is highly effective may be considered. Fish are netted into a tub of benzocaine where they are allowed to become overdosed. Benzocaine is relatively insoluble in water. It must first be dissolved in alcohol at a rate of 40g to the litre. This is then dissolved in water at a rate of 20ml per 2 gallons (9 litres) which gives a benzocaine level of 100 ppm. This will rapidly anaesthetise the fish. Fish should be left in the solution for 2 hours to ensure that death has taken place. Dead fish should be disposed of with due consideration to health and safety, and in accordance with disposal of waste legislation that may be applicable. They should not be thrown in the bin or flushed down the loo!
DISPOSAL OF CLINICAL WASTE
157
APPENDIX 3 DISPOSAL OF CLINICAL WASTE
Two years ago in a BBC Watchdog program about the company Petsmart concerning their care of mammals, the issue of the disposal of dead animals was raised. The program alleged that dead animals that should have been treated as clinical waste were thrown in the rubbish skip rather than in yellow bags.
WHAT IS CLINICAL WASTE?

Clinical waste includes any waste which consists wholly or partly of human or animal tissue, blood, other body fluids, excretions, dressings, syringes or needles which unless rendered safe may prove hazardous to any person coming into contact with it. Dead fish would appear to fit into this definition. Certainly the bodies of dead animals from veterinary surgeries are defined as clinical waste. A fuller definition is given in the Controlled Waste Regulations 1992.
RISK ASSESSMENT
As with any operation in the work place, a risk assessment may be required for operations handling dead animals under the Health and Safety or COSHH legislation. This should include any risks to health from handling dead animals, including any precautions thought necessary (in the case of fish good personal hygiene and the provision of gloves may reduce any risks from zoonoses).
HOW SHOULD CLINICAL WASTE BE DISPOSED OF?

In yellow plastic sacks provided by a specialist collection company. They can then be picked up at appropriate intervals.
DO I HAVE TO TREAT DEAD FISH AS CLINICAL WASTE?

Dead fish may come within the definitions in the relevant act. However if you wish to treat dead fish as ordinary waste, you would have to prove to the relevant authority that it constituted no greater risk than ordinary waste. Advice on appropriate treatments may be available from the EA, SEPA or HSWE. Already one member has reported discussing this issue as part of his pet shop licence renewal. The conclusion reached was that so little waste was generated, that it could be treated as ordinary waste material. As you may be aware, each district authority can interpret the law differently. Thus before incurring any costs it would be wise to seek their advice on what they deem appropriate.
158 UV
APPENDIX 4 LETHAL DOSE OF UV FOR VARIETY OF ORGANISMS
The following list is not comprehensive but shows the great variety of dose required to kill different groups of potential pathogens BACTERIA Bacillus anthracis (Anthrax bacillus) B. megatherium species (veg) B. megatherium species (spores) B. pratyphosus B. subtilis (mixed) B. subtilis (spores) Clostridium tetani Corynebacterium, C. deptheriae Dysentery bacilli (Shigella sonni) Eberthela typhosa (Typhus bacillus) Micrococcus candidus M. piltonensis M. spaeroides Myxobacterium tuberculosis Neisseria catarrhalis Phytomonas tumefaciens Proteus vulgaris Pseudomonas aerugenosa P. fluorescens Salmonella species Salmonella enteritidis S. typhimurium (ave) Sarcina lutea Serratia marcescens Shigillia paradysenderiae Spirillum rubsum Staphylococcus albus S. aureus S. hemolyticus Streptococcus lactis S. viridans FUNGI Saccharomyces ellipsoideus Saccharomyces. species S. cerevisiae Brewers Yeast Killing dose ( W s/cm) 8,700 2,500 5,200 6,100 11,000 22,000 22,000 6,500 4,200 4,100 12,300 15,000 15,400 10,000 8,500 8,500 6,600 10,500 6,600 10,000 7,600 15,200 26,400 6,160 3,400 6,160 5,700 6,600 5,500 8,800 3,800 13,200 17,600 13,200 6,600
Bakers Yeast Common Yeast Cake Penicillium roqueforti P. expansum P. digitatum Aspergillus glaucus A. flavus A. niger Rhisopus nigricans Mucor racemosus A M. racemosus B Oospora lactis VIRUSES Bacteriophage (escherichia coli) Tobacco mosaic Influenza PROTOZOA Paramecium PARASITES Nematode Eggs Algae Chlorella vulgaris
8,800 13,200 26,400 22,000 88,000 88,000 99,000 330,000 220,000 35,200 35,200 11,000 6,600 440,000 3,400 200,000
159
APPENDIX 4 LETHAL DOSE OF UV FOR VARIETY OF ORGANISMS
92,000 22,000
160 EFFICACY OF OZONE - TYPICAL DOSAGE AND REACTION TIMES

The OIE recommendation (AAHC 2001) for the sterilisation of water of fish pathogens is 0.2-1 mg/litre for 3 minutes. However it is a very effective disinfectant, and the following pathogen groups are readily killed by ozone (source http://www.trio3.com/moldsFungus.htm) Aspergillus Niger (black Mount): destroyed by 1.5 to 2 mg/1. Bacillus Bacteria: destroyed by 0.2 mg/1 within 30 seconds Bacillus Anthracis: causes anthrax in sheep, cattle and pigs. A human pathogen. Ozone susceptible. Clostridium Bacteria: ozone-Susceptible. Clostridium Botulinum Spores: the toxin produced by this bacterium paralyzes the central nervous system, it multiplies in food. 0.4 to 0.5 mg/1. Diphtheria Pathogen: destroyed by 1.5 to 2 mg/1. Eberth Bacillus (Typhus abdominalis): destroyed by 1.5 to 2 mg/1. Echo Virus 29: this virus most sensitive to ozone. After a contact time of 1 Minute at 1 mg/1 of ozone, 99.999% is killed. Escherichia Coli Bacteria (from faeces): destroyed by 0.2 mg/1 within 30 seconds. Encephalomyocarditis Virus: destroyed to zero level in less than 30 seconds with 0.1 to 0.8 mg/1. Enterovirus Virus: destroyed to zero level in less than 30 seconds with 0.1 to 0.8 mg/1. GDVII Virus: destroyed to zero level in less than 30 seconds with 0.1 to 0.8 mg/1. Herpes Virus: destroyed to zero level in less than 30 seconds with 0.1 to 0.8 mg/1. Influenza Virus: 0.4 to 0.5 mg/1. Klebs-Loffler Virus: destroyed by 1.5 to 2 mg/1. Poliomyelitis Virus: kill of 99.999% with 0.3 to 0.4 mg/1 in 3 to 4 minutes. Proteus Bacteria: very Susceptible. Pseudomonal Bacteria: very Susceptible. Rhabdovirus Virus: destroyed to zero level in less than 30 seconds. Salmonella Bacteria: very Susceptible. Staphylococci: Destroyed by 1.5 to 2 mg/1. Stomatitis Virus: destroyed to zero level in less than 30 seconds with 0.1 to 0.8 mg/1. Streptococcus Bacteria: destroyed by 0.2 mg/1 within 30 seconds.
APPENDIX 5 EFFICACY OF OZONE
As can be seen from this data, the major groups of pathogens that will affect cultures of fish (psuedomonads, representing the Aeromonas and Vibrio group) Rhabdoviruses (to which SVC belongs) and the Herpes Viruses (to which KHV belongs) are rapidly destroyed by ozone, clearly demonstrating how effective this disinfectant can be.
NOVEMBER 2005 VETERINARY MEDICINES
161
APPENDIX 6 - VETERINARY MEDICINE
Veterinary medicines legislation in the UK has been thoroughly overhauled and will now be covered by one piece of legislation, The Veterinary Medicines Regulations 2005, SI No 2745. Any amendments to the Regulations will be made annually. For the last decade mainstream fish treatments have been in a grey area. They technically required full authorisation which few or none had, but were well known to the authorities. This new regulatory framework will establish the Exemption Scheme for small pet animals which will resolve this problem. In accordance with the Scheme products will be clearly labelled and easy to recognise. This briefing note is just to give an outline of the changes coming over the next two years. Members are referred to more detailed sources of official information at the foot of this document. You may need to review the product ranges you stock and also ensure all stock not meeting the labelling requirements are sold by November 2007.
WHEN WILL THE RULES APPLY?

In general from 30 October 2005 but as there is a transition period of two years it will be fully applied for many of the fish treatments on the market from 1 November 2007. For further details see Exemption Scheme for small pet animal medicines. However importers, manufacturers and wholesalers should start taking action now.
WHAT IS A VETERINARY MEDICINE?

A veterinary medicinal product is defined as: any substance or combination of substances presented as having properties for treating or preventing disease in animals, or any substance or combination of substances which may be used in, or administered to, animals with a view to either restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or to making a medical diagnosis.
If a product is either presented as a medicine or has a medicinal function then it will be covered by this definition. Treatments for ectoparasites are normally regulated by veterinary medicines law rather than that covering pesticides. A few small exceptions exist for vaccines made and used on a single site and for veterinary medicines based on radioactive isotopes.
162 HOW MIGHT I BREAK THE LAW?

The main ways to break the law are by: Importing Possessing Supplying Administering (this applies even if the animals treated are owned by you!)
APPENDIX 6 VETERINARY MEDICINE
illegal veterinary medicinal products, including herbal and homeopathic remedies that are covered by the definition above. Authorised products must be used for the species and purposes for which they were authorised. Vets may use treatments outside of this requirement subject to prescribing cascade rules. Vets are fully aware of these rules. Any use off label i.e. not for the purpose the medicine was authorised, automatically means that use is subject to a veterinary prescription. It is quite clear that importing products labelled only in Chinese, for example, is illegal.
HOW DO I KEEP WITHIN THE LAW?

By selling or using authorised products for the specified species or selling or using treatments included in the Exemption Scheme for small pet animals.
WILL I NEED AN AUTHORISATION OR A LICENCE?

Manufacturers Importers Wholesalers Retailers Yes Yes Yes No
More information is available from the web sources referenced at on page 164.
WHAT IS AN AUTHORISED PRODUCT?

Veterinary medicines in general require a marketing authorisation before they may be legally sold in the UK. Only vaccines produced from pathogens or antigens on a particular site and used on that site, medicines based on radioisotopes and treatments subject to the small pet animal scheme (see below) are exempt from the requirement for authorisation. To become fully authorised a product needs to satisfy safety, quality and efficacy criteria.
WHAT IS THE EXEMPTION SCHEME FOR SMALL PET ANIMALS?
The costs of completing the full authorisation process are considerable. These costs would mean that products for some species would never be financially viable and so would never be available. This could cause significant welfare problems.
163
APPENDIX 6 - VETERINARY MEDICINE
The Exemption Scheme relieves manufacturers of the necessity to undertake expensive testing. However it does not relieve manufacturers of the need to use Good Manufacturing Practice , which will include on site inspection of their facilities, and as appropriate other controls applied to fully authorised medicines.
WHAT PRODUCTS WILL BE COVERED BY THE SCHEME?

The exemption only applies to animals that are not intended to be used for food. It is in the best interests of all concerned that all in the industries using exempt products work to prevent misuse of them in food producing species. It covers only products that are not controlled by other controls. Thus antibiotics which require veterinary prescriptions will not be eligible for inclusion in this Scheme. Products that are intended for other species if used on exempted species will require a veterinary prescription. Organophosphates (pesticides used in sheep dips) are controlled chemicals and will not be permitted in exempt products. Treatments for ornamental fish kept in closed ponds and aquaria are exempt. The pack size of remedies is limited to 25,000 litres. (Cage birds, homing pigeons, terrarium animals, small rodents, ferrets and rabbits are the other exempt groups). From November 1 2007 the active ingredients of exempt treatments will have to be included on a list kept by the Veterinary Medicines Directorate.
HOW WILL I TELL IF A PRODUCT IS LEGAL?

By November 2007 only properly labelled products should be placed on the market. These will carry the statement This veterinary medicine is marketed in accordance with the small pet animals exemption scheme. There are detailed labelling requirements. Further details are available from VMD or from their website.
WILL ANYTHING CHANGE?

The simple answer is yes! Products that are being sold legally will be easy to spot. As the situation becomes established illegal treatments will be equally easy to spot. VMD and Trading Standards Officers will take rigorous action when illegal products are found and significant penalties may result!!!
164 INFORMATION SOURCES ON THE WEB

The Veterinary Medicines directorate website can be found at: http://www.vmd.gov.uk/ Veterinary Guidance Notes of most relevance are; 1. An Introduction to Marketing Controls On Veterinary Medicines 2. Manufacturing and Wholesale Dealer's Authorisations for Veterinary Medicines 3. Marketing Authorisation Exemption Scheme for Pet Animal Medicines
APPENDIX 6 VETERINARY MEDICINE
NATIONAL AND INTERNATIONAL LAWS
165
APPENDIX 7 - NATIONAL AND INTERNATIONAL LAWS
This is a rather dry subject but is included so that the background to how and why laws to prevent the entry of certain fish diseases to countries are made. International trade is governed by the rules of the World Trade Organisation (WTO). In a nutshell the WTO rules state that member countries may not protect industries in their own countries by putting in place tariffs, duties or other barriers to the import of products from other countries without justification. 49 countries are members of the WTO. The WTO permits barriers to trade that protect countries human, animal or plant health within what is known as the Sanitary and Pytosanitary agreement most commonly referred to as the SPS (see box below).
WTO Sanitary and Phytosanitary Agreement:

1. Members have the right to take sanitary and phytosanitary measures necessary for the protection of human, animal or plant life or health, provided that such measures are not inconsistent with the provisions of this Agreement. 2. Members shall ensure that any sanitary or phytosanitary measure is applied only to the extent necessary to protect human, animal or plant life or health, is based on scientific principles and is not maintained without sufficient scientific evidence. 3. Members shall ensure that their sanitary and phytosanitary measures do not arbitrarily or unjustifiably discriminate between Members where identical or similar conditions prevail, including between their own territory and that of other Members. Sanitary and phytosanitary measures shall not be applied in a manner, which would constitute a disguised restriction on international trade. The whole text can be seen at: http://www.wto.org/english/tratop_e/sps_e/spsagr_e.htm The WTO uses standards setting organizations to help decide what is fair and appropriate standards for the implementation of the SPS agreement. For animal diseases the standards setting body is the OIE (Office Internationale Epizooites, otherwise known as the World Organization for Animal Health). Within the OIE there is an Aquatic Animal Health Commission, which advises on which diseases are serious enough to require countries to notify the OIE of their presence on their territory. When the OIE makes a disease notifiable in this way it does not require any action to be taken about the disease except that when it occurs the OIE must be notified. The information about the presence or absence of diseases from countries is then made available by the OIE. The OIE also publishes a code and manual on aquatic diseases setting out in detail which diseases are notifiable, how they may be detected, how a country may declare the whole or part of its territory free of a disease and how sites are to be cleared and disinfected after an outbreak of a disease if that is what a country wishes to do (this is not a measure made compulsory by the OIE). http://www.oie.int OIE Home page http://www.oie.int/eng/normes/fcode/en_sommaire.htm The OIE Aquatic Health Code contain import risk assessments and other technical information
The OIE Aquatic Manual which includes lists of OIE 166 http://www.oie.int/eng/normes/fmanual/A_summry.htm notifiable diseases and diagnosis procedures.
APPENDIX 7 NATIONAL AND INTERNATIONAL LAWS
Individual countries or trade blocs such as the EU can then decide what diseases they want to protect themselves against. The EU has in essence three different scenarios
Disease-free zone: where after exhaustive testing using procedures set out by the OIE a country or zone is found to be free of a pathogen then it may apply rigorous standards of fish health (including intensive testing) to any imports of species susceptible to the pathogen. Eradication zone; where the pathogen has occurred or occurs occasionally in a member state but the member state applies strict control measures when it is found. It may act as though it is disease-free as far as imports are concerned. Diseased or infected zone where the pathogen is present (or hasnt been proved by a variety of methods) in a member state and no action is being taken to control or eradicate the disease then no barriers are put in place to trade with other countries or zones within the EC in which the same disease occurs.
The EU also required the eradication of animals under some circumstances when the pathogen is found. Thus a notifiable disease in the EU has a different meaning to a disease notifiable to the OIE. Spring Viraemia of Carp can be used as an example*; Countries** Finland, Denmark, Sweden, Ireland and Northern Ireland United Kingdom Disease status Extensive testing allows these countries and areas to be deemed disease-free for SVC. Where imports are permitted from May only import susceptible species from countries or zones tested and found to be SVC free or which can prove that SVC has never occurred in their country Where they may export to Anywhere
Has had occasional outbreaks of SVC but eradication or control measure are enforced. Known as an eradication zone Either SVC is known to occur or they have not undergone the testing to prove absence of the disease
May only import susceptible species from countries or zones tested and found to be free of SVC or which can prove that SVC has never occurred in there country Anywhere. General health rules will apply but no requirement for SVC susceptibles to be tested for SVC.
As it is not regarded as disease-free can only export to areas of lesser health status, within the EU this means the other 20 member states. Cannot export SVC susceptible species to disease-free, eradication zones.
The other 20 member states
*Believed correct February 2006 Questionnaire 1 Questionnaire 3 Contents
**Individual zones or farms may have a different status from the country as a whole
ADVICE TO DEALERS, TRADERS AND FARMERS ON STEPS TO PREVENT THE SPREAD OF SVC
167
APPENDIX 8 DEFRAS ADVICE ON AVOIDING SVC
Taken from: Spring Viraemia of Carp, produced by Ministry of Agriculture, Fisheries and Food Welsh Office Agriculture Department (now DEFRA) several years ago. Copies can be obtained from MAFF Fish Diseases Laboratory T: 01305 206600. Dealers, traders and farmers should report any knowledge or suspicions of SVC to the Fish Diseases Laboratory, Weymouth or to their local Water Authority. Carp, just like any other fish, have increased susceptibility to disease when they are stressed. Stress can be caused by a number of factors such as rapid changes in water temperature, movement from one site to another, poor handling or overstocking. The risk of disease and mortalities can be significantly reduced by keeping stress to a minimum wherever possible. Dealers should avoid collecting and mixing fish from different sites, especially when the fish are intended for immediate sale. If feasible, dealers should also consider a period of quarantine (preferably for two weeks) for recently acquired stocks. This may prevent the introduction of SVC to their own stocks and to those of customers. When netting stocks, dealers should ensure that nets and equipment are disinfected before use at another site. It will obviously benefit dealers, traders and farmers to ask appropriate questions of importing agents or wholesale suppliers as to the health of fish before accepting consignments. Importers of ornamental varieties should take all reasonable steps to determine whether overseas suppliers can give assurances that their fish stocks are free from infection with SVC virus, or other serious diseases. Importers should consider seeking satisfactory responses from suppliers on points such as: Are regular examinations for disease carried out on fish stocks held on the suppliers site/ premises? Are all new consignments of fish brought into the suppliers site/premises examined for disease? Who undertakes these examinations (e.g. own employee, private veterinarians, state veterinary service)? Are only sick fish examined, or are random samples of apparently healthy fish also examined routinely? Are samples taken for laboratory tests for bacteria, viruses, parasites? If yes, which laboratory carries out the test? Can the supplier provide a list of tests carried out on his site/premises and the results? What steps does the supplier take to ensure that the health status of his own suppliers fish stocks is acceptable?
168 DISINFECTION
Ideally, disinfection should involve the treatment of nets, sacks, footwear and other equipment such as boats, prior to moving to other waters. Equipment used in fishery remedial work, such as bank repairs and stock netting, might similarly be treated. Iodine-based preparations (iodophors) are recommended for disinfecting equipment: such preparations, by way of example, include Wescodyne (Ciba Geigy Ltd) and FAM-30 (Vanodine International). Advice on local sources of supply or suitable alternatives may be obtained from local veterinary surgeons, Water Authorities, or the Ministrys Fish Disease Laboratory at Weymouth (T: 01305 206600). As a broad guide Wescodyne should be diluted: 1 parts in 100 with water and FAM-30: 1 part in 100 with water. Other iodophores should be diluted to provide a final concentration of 250 parts per million active iodine. Suppliers, or scientists at the Fish Disease Laboratory, Weymouth can advise in cases of doubt. Disinfection is best achieved by first cleaning off all mud etc followed by immersion for 15 to 30 minutes; or by application to surfaces using a pad soaked with disinfectant. (Reference to specific disinfectant products should not be construed as any criticism of similar products). For regular disinfection of heavily soiled footwear, a bath of 1% caustic soda (sodium hydroxide) solution is more appropriate. The strength of the caustic soda should be checked daily and the disinfectant replaced if the pH is 11 or below, as shown by indicator papers. Protective clothing should be used when handling caustic soda to protect the skin and eyes. This disinfectant can corrode metals. Iodophors and many other disinfectants are extremely poisonous to fish. Footwear and all equipment, especially nets, should be thoroughly rinsed with tap water after disinfecting. Disinfectant and washings must be disposed of in a way, which does not harm the environment. They should never be tipped into water containing fish or other aquatic life.
APPENDIX 8 - DEFRAS ADVICE ON AVOIDING SVC
EMERGENCE OF INFECTION
For an epidemic to occur a pathogen must spread to new hosts faster than its current hosts recover or die. Formally: if the coefficient R>1 then an epidemic can occur if the pathogen is introduced (e.g. with new stock). Rs value depends on transmission coefficient, , initial population density of nave hosts, S, and the rate of removal of infection, . R = S > 1
169
APPENDIX 9 EMERGENCE OF INFECTIONS
This formula illustrates how the risk of epidemics can be reduced by preventing over-crowding or stress (to reduce S and ) and by removing sick fish (to increase ).
Pathogens that are normally rare may emerge as a serious disease problem under systems that produce and move large numbers of live fish at high density (increasing and S). Pathogens may also evolve increased virulence under such conditions, as high rates of missing of hosts select for rapidly spreading pathogens that can take advantage of the opportunities, even if damage to the existing host is increased. One potential source of new pathogens is the mixing of stocks from different sources. Some stocks may be resistant carriers that spread a pathogen to nave stocks of the same or similar species, resulting in disease. A more complex emergence can occur when pathogens jump to different species. Cross-species transmission may be inherently unlikely ( is small ) but if species are mixed in large numbers and in close proximity (S is large) the unlikely step has many chances to occur. Once the pathogen has jumped species it may cause a dead end infection. Perhaps killing its new host, but not spreading. Far more seriously it may be able to adapt and transmit to others of the new species; this adaptation may be a slow process with the pathogen improving its ability to transmit ( increasing) with each successful transmission. If the new hosts are held at high densities, or are stressed, then the pathogen is more likely to survive this period of inefficient transmission, allowing a new disease to emerge. The best way to reduce the risk of disease emergence is to adopt good husbandry: do not overcrowd or otherwise stress fish, avoid mixing stocks or species from different sources and remove sick fish as quickly as possible.
170 ZOONOSES: THE RISKS

APPENDIX 10 - ZOONOSES
Zoonosis (zoonoses, plural): a disease of animals that may be transmitted to Man under natural conditions. Although many aquatic organisms can potentially infect Man, very few fish diseases have been proven to be zoonotic. Humans become infected by handling diseased fish or contaminated equipment, or by ingesting water from facilities that contain diseased fish. Therefore the use of sterilizing equipment, wearing disposable gloves and thorough hand washing should be routine precautions to minimize the risk. It should be assumed that zoonotic organisms are always present and therefore the following safety measures should be used to limit the spread of these diseases: Use disposable gloves for food preparation, post-mortem examinations If open wounds are present then cover with a waterproof bandage and wear rubber or plastic gloves of a suitable length Suitable hand-washing facilities and warning signs should be provided for staff. Areas of exposed skin that have been in contact with water from tanks or ponds should be washed, and rinsed thoroughly, as soon as practicable and certainly before eating drinking or smoking. If used remember antiseptic cleansers may provoke allergic reactions in some people and residues on hands may prove harmful to fish Avoid sharing equipment between systems and where necessary sterilize with suitable disinfectants at the correct concentration for the recommended length of time Identify and mark infected systems, and where possible, avoid further stocking until the system is disinfected Wash and disinfect contaminated work surfaces regularly As far as is possible feed live fish (anyway a practice that is likely to be made illegal shortly) or raw fish offal to large carnivorous fish Regard all dead fish as clinical waste and dispose of them carefully in accordance to current legislation Do not prime water siphons by mouth Do not eat, drink or smoke outside designated areas Do not wash nets and equipment in sinks intended for human use Immunosuppressed persons (infected with HIV or receiving chemotherapy) should not handle potentially infected materials
MYCOBACTERIUM
Cause: Mycobacterium marinum, M fortuitum and other Mycobacterium species; common bacteria in all aquatic environments Comment: the most common chronic disease that affects aquarium fish, commonly called mycobacteriosis or fish tuberculosis (TB). It may take two or more years for the number of organisms to grow to detectable levels. Transmission: bacteria are shed from infected skin ulcers and the intestine into the water. Fish are infected by contact with organisms in water, cannibalism and eating uncooked infected fish. Signs in fish: most show few or no external signs of disease but advanced cases may exhibit poor growth, weight loss, colour change, lethargy, chronic non-healing ulcers, skeletal deformity, abdominal swelling, incoordination, sudden death.
Signs in humans: single or multiple (sporothricoid) non-healing ulcers on hands or arms, commonly called fish tank or swimming pool granuloma, aquarists nodule, aquarists finger, aquarists arm. Small lesions may heal spontaneously but others require courses of multiple antibiotics over several months. Control: there are no effective non-lethal tests to identify infected fish and no vaccine
171
VIBRIO
Cause: various Vibrio species; common bacteria in marine and brackish environments Transmission: infection by contact with bacteria in water Signs in fish: ulcers, lethargy, inflamed areas on skin, deaths Signs in humans: ingestion may cause vomiting and diarrhoea; wound infection produces local inflammation and severe tissue reaction
AEROMONAS
Cause: Aeromonas hydrophila and other Aeromonas species; common bacteria in freshwater environments Transmission: infection by contact with bacteria in water Signs in fish: ulcers, lethargy, inflamed areas on skin, deaths Signs in humans: ingestion may cause vomiting and diarrhoea; puncture wound infection produces local inflammation and severe tissue reaction.
OTHERS
Several other organisms of aquatic origin have been linked to human disease but in most cases these have been due to contamination of the environment rather than direct infection from fish. These include Salmonella, Leptospirosis (Weils disease, usually associated with surfaces or water infected by the waste products of rats), Streptococcus, Erysipelothrix (fish rose, erysipeloid), Cryptosporidium. Details of these and other rare cases can be found in the reference below.
REFERENCES
Nemetz, TG & Shotts, EB Jr (1993) Zoonotic diseases. In: Fish Medicine (ed. MK Stoskopf) WB Saunders Co., Philadelphia
FISH TUBERCULOSIS
Fish tuberculosis or mycobacteriosis is most commonly caused by Mycobacterium marinum or Mycobactenum fortuitum. The disease is spread by various methods; skin granulomas may release bacteria directly into the water, bacteria may be shed via the faeces from tuberculous granulomas in the liver or gut lining, or bacteria may be shed in the urine from the kidney. Another more direct route involves dead fish in the tank or pond being cannibalised. This is very common and usually the heavily infected abdominal organs are eaten first. Disease can also be spread by the aquarist who feeds live infected fish to his large specimen fish such as oscars (Astronotus ocellatus) or piranha (Serrasalmus spp). The disease is zoonotic and in man is often called aquarists arm; it is occasionally seen in people working in aquarium shops, the commonest site of infection are the hands and arms due to minor scratches becoming infected. As an infection of man it is difficult to treat, often taking months to resolve. In fish TB is in my experience impossible to eliminate once it has occurred in a tank or pond, affected collections offish
not have any new additions and when the collection becomes depleted it is better perhaps to 172 should sacrifice the remaining fish and disinfect the system before starting again.
Because of the possibility of human infection, hygiene is very important when dealing with fish tanks. Wash hands and arms well after doing any tank servicing and pay particular attention to dressing any cuts and scratches. Do not use siphons that involve you getting a mouthful of water! Do not wash out tank filters in the kitchen sink or pour wastewater down it, if you have to then make sure it is well washed afterwards. Simple, sensible hygiene practices should be more than enough to reduce greatly the already very low risk of infection.
OTHER BACTERIAL DISEASES

SALMONELLA
Fish are not known as natural hosts of Salmonella, although they do have a number of related organisms causing disease. This is a major benefit offish over most other pets. In the last couple of years there has been one outbreak of related cases where it appears that Salmonella Java was transmitted by fish to a small number ofaquarists. This organism was not causing any problem in the fish that appear simply to have mechanically transferred it from a more conventional source to the aquarists who were affected. It is possible that other infections such as campylobacter might also spread in this fashion. Weils Disease (Leptospirosis) This disease is not a fish transmitted zoonosis, it is associated with water and fish because it is transmitted by rats which are often associated with water. Rats are certainly associated with waste food left around ponds so this is to be avoided. Weils Disease is caused by Leptospira icterohaemorrhagiae and Canicola fever by Leptospira canicola (henceforth referred to as Li and L.c). Both infections affect both man and dog, in both species Li is by far the worst causing a serious hepatitis (liver disease) and jaundice, Lc can cause jaundice but is usually milder, exposure to Lc can cause chronic nephritis (kidney disease). One should also stress that leptospiral meningitis is possibly the commonest manifestation of leptospirosis associated with either infectious organism, signs probably being ascribed to flu due to headaches and weakness. Treatment is possible but it is urgent, the disease can be fatal. Li is more common than Lc, because Lc is only slightly pathogenic to rats, and is usually eliminated by them, whereas infection of rats with Li causes chronic kidney disease in rats which are then long term carriers. An important aspect of controlling Leptospirosis is to ensure that dogs are regularly (annually) vaccinated, both forms of Leptospirosis are present in the routine dog vaccination and it is very simply carried out. Dogs live in much closer proximity to people than do rats and are often living in the house with children, if unprotected they can become infected just as easily as humans and can infect the whole family. Although acidic dog urine doesnt harbour Leptospires for long, dilution in water as may occur in and around the pond permits them to survive longer and makes it important to prevent them becoming infected. Aeromonads and Psedomonads common in fish may infect wounds; they have also been associated with enteritis. Various other Enterbnacteria have also been linked between fish/water and man. The major problem in this regard is by ingestion i.e. contaminated food or drink, licking fingers or biting fingernails. Various gram-positive organisms. Streptococcus, Staphylococcus and Clostridia are associated both with fish and the aquatic habitat. The latter two with relation to man are associated with food poisoning. Streptoccus has caused disease in both fish and man, although with no demonstrated link between the two. Erysipefothrix rhusiopathiae is found worldwide and has been associate with fish skin and mucus (and causes disease in a range of animals including man). Localised skin infections similar to tubercular granulomas may be seen - called Fish Rose or erysipeloid. Septicaemia is rare but chronic effects on the heart are also thought to be involved. Nocardia asteroides infection also produces a rather similar chronic skin granuloma. This can be difficult to distinguish from Mycobacterioisis (TB).
OTHER ORGANISMS
Cryptosporidiosis Cryptosporidium is a strange protozoan organism causing enteric problems in man, again although it has been found in fish it is actually frequently transmitted by water. The organism has been found to be present in tap water. Again this is the link with fish, there is no evidence of transmission from fish to man.
173
PREVENTION
Basic commonsense hygiene will prevent transmission of infections being transmitted to aquarists and pondkeepers. Hand sterilising preparations are useful in the shop situation, good thorough cleaning and disinfection of tanks, nets etc are important.
174 FURTHER INFORMATION

Information sources should be subject to a risk analysis. Not everything in print is factual or correct. For a variety of reasons information may be misguided, misinterpreted or misrepresented. Information may become dated or what was thought to be fact is proved incorrect. Of course it may be that there are two or more entirely reasonable interpretations of the facts and you must choose which to use. Remember if we go back just a century or so scientists believed disease was caused by evil spirits the configuration of the stars and the wrath of the gods. The following comments might be useful in evaluating information sources: Scientific Journals-these may or may not be peer reviewed. Information in peer reviewed journals has greater creditability because the information has been read and edited by several other scientists before being published. Textbooks: these may also be peer reviewed. They may also just reflect the authors own ideas or their interpretation of others ideas and findings. Magazines: care should be taken when evaluating information from this source. There may for instance be links between the article and the products sold by the author. This does not necessarily mean there is anything wrong just that a cautious approach may be best. The Web: anyone can publish anything on the web. It could be the latest brilliant piece of science, effective practical tips or garbage; you must decide.
APPENDIX 11 FURTHER HELP

Biosecurity Oata

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BIOSECURITY

ORNAMENTAL FISH INDUSTRY

ORNAMENTAL AQUATIC TRADE ASSOCIATION (OATA)

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

FLOW THROUGH SYSTEMS RECIRCULATION

Questionnaire 2 Questionnaire 4 Risk reduction strategies

C. INFECTION OR REINFECTION? WHICH IS THE GREATER PROBLEM?

ULTRA VIOLET SUNLIGHT CONVERSION FACTORS EFFECTIVE DOSE OF UV

OZONE ADVANTAGES DISADVANTAGES REDOX POTENTIAL EFFECTIVE LEVELS

8.3 HUMAN SKIN

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

IHC (IMMUNO-HISTOCHEMISTRY) IFAT - (INDIRECT FLUORESCENT ANTIBODY TEST)

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

10.2 CHEMICALS 10.3 ANTIBIOTICS 10.4 PROBIOTICS 10.5 PREBIOTICS COMPARISON

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

SUMMARY OF RISK REDUCTION STRATEGIES

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

STRUCTURE OF THE REPORT

Risk Analysis and HACCP

Deciding which are the bad bugs

Keeping out the bad bugs

Stopping the bad bugs spreading

Disease treatments, Identifying Disease

Site biosecurity and risk reduction strategies

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

AIM OF THE REPORT

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

HOW TO USE THIS REPORT

Decide which pat ho gens or diseases yo u are co ncerned abo ut

Review management of bio securit y and implement changes

Acce pta ble

Assess or re-assess t he biosecurity of yo ur suppliers

Negot iat e changes

Assess or re-assess t he bio securit y o n yo ur o wn site

Review management o f biosecurity on yo ur o wn site and implement changes

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

WHICH QUESTIONNAIRE TO USE

R eview annually o r as required

Have you told everybody about your biosecurity policy?

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

WHAT THIS REPORT DOES NOT AIM TO DO

Questionnaire 1 Questionnaire 3 Contents

Questionnaire 2 Questionnaire 4 Risk reduction strategies

CHAPTER 1 WHAT IS BIOSECURITY?

Questionnaire 1 Questionnaire 3 Contents