D3525.PDF

Original: English
July 2006
REPORT OF THE MEETING OF THE OIE AD HOC GROUP ON
AQUATIC ANIMAL HEALTH SURVEILLANCE
Paris, France, 24–26 July 2006
_______
The meeting of the OIE ad hoc Group on Aquatic Animal Health Surveillance was held at the OIE Headquarters
in Paris from 24 to 26 July 2006.
Dr Gideon Bruckner, Head of the OIE Scientific and Technical Department, welcomed the members on behalf of
the OIE Director General, Dr Bernard Vallat.
The meeting was chaired by Prof. Barry Hill. The Agenda, the list of participants and the Terms of Reference for
the ad hoc Group are presented as Appendices I, II and III, respectively.
1.
Terrestrial Animal Health Code Appendix 3.8.1 General guidelines for animal health
surveillance
The ad hoc Group reviewed Appendix 3.8.1 of the OIE Terrestrial Animal Health Code on General
guidelines for animal health surveillance and adapted them to aquatic animal health surveillance. The
Group noticed areas in the Terrestrial Appendix where improvements could be made, but could not fully
address them due to time constraints. The Group proposed to address these issues at a future meeting.
Furthermore, the Group identified a number of definitions in the Terrestrial Code that need to be revised,
such as targeted surveillance and outbreak. In addition, there is a need to include some new definitions such
as active and passive surveillance
The adapted guidelines are presented at Appendix IV and are proposed for inclusion in the Aquatic Animal
Health Code.
2.
Manual of Diagnostic Tests for Aquatic Animals Chapter 1.1.4 Requirements for
surveillance for international recognition of freedom from infection
The Group reviewed and updated Chapter 1.1.4 of the Manual of Diagnostic Tests for Aquatic Animals on
Requirements for surveillance for international recognition of freedom from infection.
One major revision identified by the Group was to expand the chapter to include surveillance to describe
occurrence and distribution of selected endemic diseases. For this reason, the Group began the process of
incorporating issues relevant to surveillance of endemic diseases. This task proved to be more extensive
than originally envisioned and will require further work by the Group at future meetings.
To reflect the change in scope of the chapter, the Group changed the title of the chapter to Guidelines for
Aquatic Animal Health Surveillance.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
1
The Group discussed the effect of sensitivity and specificity on sample size required for surveys to
substantiate disease freedom and decided to include a table reporting sample sizes and acceptable number
of false positives under varying parameter values.
In the original version of chapter 1.1.4, both the terms disease and infection were used. However because
disease is an expression of infection under specific conditions, the Group decided to focus its efforts on
developing guidelines for surveillance to substantiate freedom from infection.
The chapter is presented at Appendix V.
3.
Aquatic Manual chapters I.1, I.2 and I.3 on General information (on diseases of fish,
molluscs and crustaceans)
The Group noticed that considerable efforts were required to harmonise the three introductory chapters on
general information for diseases of fish, molluscs and crustaceans. Although work on harmonising the
chapters has progressed, a number of discrepancies remain among the three documents. A structure is now
provided for the authors of the chapters to fill in the gaps in the information. Some information that was
common to the three chapters was incorporated into chapter 1.1.4.
The chapters are presented at Appendices VI, VII and VIII.
4.
Revise the specific disease chapter template of the Aquatic Manual to ensure that
scientific information necessary to develop appropriate surveillance programmes for
diseases can be formulated
A number of topic headings were added to the disease chapter template (see Appendix IX). These will be
further developed at the next meeting.
The Group identified the need to include in the disease chapters considerable information on the
development of surveillance systems for the specific disease. The Group realised that generating such
information might require a multidisciplinary approach and suggested that the revisions to the chapters may
also include input from epidemiologists.
5.
Develop guidelines for Aquatic Manual chapter authors to follow in specifying the
surveillance requirements for individual diseases
Due to time constraints, the Group made limited progress on this item and will develop these guidelines at a
future meeting.
6.
Steps in the design of a surveillance system
The Group initiated a description of steps involved in the design of a surveillance system with the intention
of including it in the guidelines. This will be further developed at the next meeting and harmonized within
the structure of Chapter 1.1.4. The steps are presented at Appendix X.
_______________
…/Appendices
2
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix I
REPORT OF THE MEETING OF THE OIE AD HOC GROUP ON
AQUATIC ANIMAL HEALTH SURVEILLANCE
Paris, France, 24–26 July 2006
_____
Agenda
1.
Terrestrial Animal Health Code Appendix 3.8.1 General guidelines for animal health surveillance
2.
Manual of Diagnostic Tests for Aquatic Animals Chapter 1.1.4 Requirements for surveillance for
international recognition of freedom from infection
3.
Aquatic Manual chapters I.1, I.2 and I.3 on General information (on diseases of fish, molluscs and
crustaceans)
4.
Revise the specific disease chapter template of the Aquatic Manual to ensure that scientific information
necessary to develop appropriate surveillance programmes for diseases can be formulated
5.
Develop guidelines for Aquatic Manual chapter authors to follow in specifying the surveillance
requirements for individual diseases
6.
Steps in the design of a surveillance system
_______________
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
3
Appendix II
REPORT OF THE MEETING OF THE OIE AD HOC GROUP ON
AQUATIC ANIMAL HEALTH SURVEILLANCE
Paris, France, 24–26 July 2006
_____
List of Participants
MEMBERS
Prof. B.J. Hill (Chairman)
The Centre for Environment,
Fisheries & Aquaculture Science
(CEFAS), Barrack Road, The Nothe,
Weymouth, Dorset DT4 8UB,
UNITED KINGDOM
Tel.: (44-1305) 20.66.26,
Fax: (44-1305) 20.66.27
E-mail: [email protected]
Dr Marios Georgiadis
Lecturer of Epidemiology,
Laboratory of Animal Production
Economics, Department of Animal
Production, Ichthyology, Ecology
and Protection of Environment,
Faculty of Veterinary Medicine,
Aristotle University of Thessaloniki,
BOX 410, 54124 Thessaloniki,
GREECE
Tel.: (30-2310) 99.99.30
FAX: (30-2310) 99.99.19
E-mail: [email protected]
Dr Flavio Corsin
Network of Aquaculture Centres in
Asia-Pacific (NACA), NAFIQAVED
Building, 10 Nguyen Cong Hoan, Ba
Dinh, Hanoi,
VIETNAM
Tel.: (84-91) 277.69.93
FAX: (84-4) 8317221
E-mail: [email protected]
Dr Gideon Bruckner
Head, Scientific and Technical Dept
E-mail: [email protected]
Ms Sara Linnane
Scientific Editor, Scientific and
Technical Dept
E-mail: [email protected]
Dr Larry Hammell
Professor, Department of Health
Management, and Director, AVC –
Centre for Aquatic Health Sciences,
Atlantic Veterinary College,
University of Prince Edward Island,
550 University Avenue,
Charlottetown, PE C1A 4P3
CANADA
Tel.: (1-902) 566.07.28
Fax: (1-902) 566.08.23
E-mail: [email protected]
CENTRAL BUREAU
Dr Bernard Vallat
Director General
OIE 12 rue de Prony
75017 Paris, FRANCE
Tel.: (33-1) 44.15.18.88
Fax: (33-1) 42.67.09.87
E-mail: [email protected]
_______________
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
5
Appendix III
Terms of Reference for the OIE ad hoc Group on Aquatic Animal Health Surveillance
1.
Review, revise and update Chapter 1.1.4 (Surveillance) of the Aquatic Manual, taking into account the
general guidelines for surveillance in the Terrestrial Code;
2.
Draft an appendix on general guidelines for aquatic animal health surveillance for inclusion in the Aquatic
Code;
3.
Review, revise and update chapters I.1, I.2 and I.3 of the Aquatic Manual and ensure consistency and
continuity with Chapter 1.1.4 (Surveillance) while including specific provisions related to aquatic animal
health surveillance for fish, molluscs and crustaceans;
4.
Revise the specific diseases chapter template of the Aquatic Manual to ensure that scientific information
necessary to develop appropriate surveillance programmes for diseases can be formulated;
5.
Develop guidelines for Aquatic Manual chapter authors to follow in specifying the surveillance
requirements for individual diseases;
6.
To submit a report to the OIE Aquatic Animal Health Standards Commission preferably by October 2006.
_______________
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
7
Appendix IV
APPENDIX
X.X.X.
GENERAL GUIDELINES FOR AQUATIC ANIMAL
HEALTH SURVEILLANCE
Article 3.8.1.1.
Introduction and objectives
1.
In general, surveillance is aimed at:
-
demonstrating the absence of disease or infection,
-
determining the occurrence or distribution of disease or infection, including changes to their
incidence or prevalence (or its contributing factors),
-
detecting as early as possible exotic or emerging diseases.
The type of surveillance applied depends on the desired outputs needed to support decision-making.
The following guidelines may be applied to all diseases, their agents and susceptible species as listed in
the Aquatic Code, and are designed to assist with the development of surveillance methodologies.
Except where a specific surveillance method for a certain disease or infection is already described in the
Aquatic Code, the guidelines in this Appendix may be used to further refine the general approaches
described for a specific disease or infection. Where detailed disease/infection-specific information is not
available, suitable approaches should be based on these guidelines.
Surveillance data determine the quality of disease status reports and should satisfy information
requirements for accurate risk analysis both for international trade as well as for national decisionmaking.
2.
3.
Animal health surveillance is an essential component of programmes aimed to [necessary
diseases]:
to detect
-
monitor disease trends,
-
control endemic [and exotic] diseases,
-
prevent introduction of exotic diseases,
_
support claims for freedom from disease or infection,
-
provide data to support the risk analysis process, for both animal health and/or public health
purposes,
-
substantiate the rationale for sanitary measures.
Essential prerequisites to enable a Member Country to provide information for the evaluation of its
animal health status are:
a.
that the particular Member Country complies with the provisions of Chapter 1.4[3].3. of the
Aquatic Code on the quality and evaluation of the Competent Authorities;
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
9
Appendix IV cont.
4.
b.
that, where possible, surveillance data be complemented by other sources of information (e.g.
scientific publications, research data, documented field observations and other non-survey data);
c.
that transparency in the planning and execution of surveillance activities and the analysis and
availability of data and information, be maintained at all times, in accordance with
Chapter 1.2.1[1.2]. of the Aquatic Code.
The objectives of this Appendix are to:
a. provide guidance to the type of outputs that a surveillance system should generate;
b. provide guidelines to assess the quality of disease surveillance systems.
Article 3.8.1.2.
Definitions
The following definitions apply for the purposes of this Appendix:
Bias: A tendency of an estimate to deviate in one direction from the [a] true value of a population
parameter.
Case definition: A case definition is a set of criteria used to distinguish [classify] a case animal or
epidemiological unit from a non-case [as a case].
Confidence: In the context of demonstrating freedom from infection, confidence is the probability that the
type of surveillance applied would detect the presence of infection if the population were infected. The
confidence depends on, among other parameters, the assumed level of infection in an infected population.
The term refers to confidence in the ability of the surveillance applied to detect disease, and is equivalent to
the sensitivity of the surveillance system.
Early detection system: an efficient system for ensuring the rapid recognition of signs that are
suspicious of a listed disease, or an emerging disease situation, or unexplained mortality, in aquatic animals in an
aquaculture establishment or in the wild, and the rapid communication of the event to the Competent Authority,
with the aim of activating diagnostic investigation with minimal delay. Such a system will include the
following characteristics:
a. broad awareness, e.g. among the personnel employed at aquaculture establishments or involved in processing,
of the characteristic signs of the listed diseases and emerging diseases;
b.
veterinarians or aquatic animal health specialists trained in recognising and reporting suspicious disease
occurrence;
c.
ability of the Competent Authority to undertake rapid and effective disease investigation;
d.
access by the Competent Authority to laboratories with the facilities for diagnosing and differentiating
listed and emerging diseases.
[Early detection system: A system for the timely detection and identification of an incursion or emergence of disease/infection
in a country, zone or compartment. An early detection system should be under the control of the Veterinary Services and should
include the following characteristics:
a.
representative coverage of target animal populations by field services;
b.
ability to undertake effective disease investigation and reporting;
c.
access to laboratories capable of diagnosing and differentiating relevant diseases;
10
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IV cont.
d.
a training programme for veterinarians, veterinary para-professionals and others involved in handling animals for detecting
and reporting unusual animal health incidents;
e.
the legal obligation of private veterinarians in relation to the Veterinary Administration;
f.
timely reporting system of the event to the Veterinary Services;
g.
a national chain of command.]
Outbreak definition: An outbreak definition is a set of criteria used to classify the occurrence of one or
more cases in a group of animals or units as an outbreak.
Probability sampling: A sampling strategy in which every unit has a known non-zero probability of
inclusion in the sample.
Sample: The group of elements (sampling units) drawn from a population, on which tests are performed
or parameters measured to provide surveillance information.
Sampling units: The unit that is sampled, either in a random survey or in non-random surveillance. This
may be an individual animal or a group of animals (e.g. an epidemiological unit). Together, they comprise the
sampling frame.
Sensitivity: The proportion of truly positive units that are correctly identified as positive by a test.
Specificity: The proportion of truly negative units that are correctly identified as negative by a test.
Study population: The population from which surveillance data are derived. This may be the same as the
target population or a subset of it.
Surveillance: The systematic ongoing collection, collation, and analysis of data, and the timely
dissemination of information to those who need to know so that action can be taken.
Surveillance system: A method of surveillance that may involve one or more component activities that
generates information on the health, disease or zoonosis status of animal populations.
Survey: An investigation in which information is systematically collected, usually carried out on a sample
of a defined population group, within a defined time period.
Target population: The population about which conclusions are to be inferred.
Test: A procedure used to classify a unit as either positive, negative or suspect with respect to an infection
or disease.
Test system: A combination of multiple tests and rules of interpretation that are used for the same
purpose as a test.
Article 3.8.1.3.
Principles of surveillance
1.
Types of surveillance
a. Surveillance may be based on many different data sources and can be classified in a number of
ways, including:
i.
the means by which data are collected (active versus passive surveillance);
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
11
Appendix IV cont.
ii.
the disease focus (pathogen-specific versus general surveillance); and
iii.
the way in which units for observation are selected (structured surveys versus non-random
data sources).
b. In this Appendix, surveillance activities are classified as being based on:
EITHER
i.
structured population-based surveys, such as:
ƒ systematic sampling at slaughter;
ƒ random surveys;
OR
ii.
structured non-random surveillance activities, such as:
ƒ disease reporting or notifications;
ƒ control programmes/health schemes;
ƒ targeted testing/screening;
ƒ ante-mortem and post-mortem inspections;
ƒ laboratory investigation records;
ƒ biological specimen banks;
ƒ sentinel units;
ƒ field observations;
ƒ farm production records.
c. In addition, surveillance data should be supported by related information, such as:
i.
data on the epidemiology of the infection, including environmental, host population
distribution, and climatic information;
ii.
data on farmed and wild animal movements and trading patterns for aquatic animals and
aquatic animal products;
iii.
national animal health regulations, including information on compliance with them and their
effectiveness;
iv.
history of imports of potentially infected material; and
v.
biosecurity measures in place.
d. The sources of evidence should be fully described. In the case of a structured survey, this should
include a description of the sampling strategy used for the selection of units for testing. For
structured non-random data sources, a full description of the system is required including the
source(s) of the data, when the data were collected, and a consideration of any biases that may
be inherent in the system.
12
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IV cont.
2.
Critical elements
In assessing the quality of a surveillance system, the following critical elements need to be addressed
over and above quality of Competent Authority [Veterinary Services] (Chapter 1.4[3].3.).
a. Populations
Ideally, surveillance should be carried out in such a way as to take into account all animal species
susceptible to the infection in a country, zone or compartment. The surveillance activity may cover
all individuals in the population or part of them. When surveillance is conducted only on a
subpopulation, care should be taken regarding the inferences made from the results.
Definitions of appropriate populations should be based on the specific recommendations of the
disease chapters of the Aquatic Manual [Terrestrial Code].
b. Epidemiological unit
The relevant epidemiological unit for the surveillance system should be defined and documented to
ensure that it is representative of the population. Therefore, it should be chosen taking into
account factors such as carriers, reservoirs, vectors, immune status, genetic resistance and age,
sex, and other host criteria.
c. Clustering
Infection in a country, zone or compartment usually clusters rather than being uniformly or
randomly distributed through a population. Clustering may occur at a number of different levels
(e.g. tank, pond, farm, [a cluster of infected animals within a herd, a cluster of pens in a building], or
compartment). Clustering should be taken into account in the design of surveillance activities and
the statistical analysis of surveillance data, at least at what is judged to be the most significant
level of clustering for the particular animal population and infection.
d. Case and outbreak definitions
Clear and unambiguous case and outbreak definitions should be developed and documented for
each pathogen under surveillance, using, where they exist, the standards in the Aquatic Code
[Terrestrial Code].
e. Analytical methodologies
Surveillance data should be analysed using appropriate methodologies, and at the appropriate
organisational levels to facilitate effective decision making, whether it be planning interventions
or demonstrating status.
Methodologies for the analysis of surveillance data should be flexible to deal with the
complexity of real life situations. No single method is applicable in all cases. Different
methodologies may be needed to accommodate the relevant pathogens, varying production and
surveillance systems, and types and amounts of data and information available.
The methodology used should be based on the best available information that is in accord with
current scientific thinking. The methodology should be in accordance with this Appendix and
fully documented, and supported by reference to the scientific literature and other sources,
including expert opinion. Sophisticated mathematical or statistical analyses should only be
carried out when justified by the proper amount and quality of field data.
Consistency in the application of different methodologies should be encouraged and
transparency is essential in order to ensure fairness and rationality, consistency in decision
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
13
Appendix IV cont.
making and ease of understanding. The uncertainties, assumptions made, and the effect of these
on the final conclusions should be documented.
f. Testing
Surveillance involves the detection of disease or infection by the use of appropriate case definitions
based on the results of one or more tests for evidence of infection [or immune] status. In this
context, a test may range from detailed laboratory examinations to field observations and the
analysis of production records. The performance of a test at the population level (including field
observations) may be described in terms of its sensitivity and specificity and predictive values.
Imperfect sensitivity and/or specificity will have an impact on the conclusions from
surveillance. Therefore, these parameters should be taken into account in the design of
surveillance systems and analysis of surveillance data.
Although not determined for many aquatic diseases, sensitivity and specificity should be
estimated as best as possible. The values of sensitivity and specificity for the tests used should
be specified, and the method used to determine or estimate these values should be documented.
Alternatively, where values for sensitivity and/or specificity for a particular test are specified in
the Aquatic Manual [Terrestrial Code], these values may be used as a guide.
Samples from a number of animals or units may be pooled and subjected to a testing protocol.
The results should be interpreted using sensitivity and specificity values that have been
determined or estimated for that particular pool size and testing procedure.
g. Quality assurance
Surveillance systems should incorporate the principles of quality assurance and be subjected to
periodic auditing to ensure that all components of the system function and provide verifiable
documentation of procedures and basic checks to detect significant deviations of procedures
from those documented in the design.
h. Validation
Results from animal health surveillance systems are subject to one or more potential biases.
When assessing the results, care should be taken to identify potential biases that can
inadvertently lead to an over-estimate or an under-estimate of the parameters of interest.
i. Data collection and management
The success of a surveillance system is dependent on a reliable process for data collection and
management. The process may be based on paper records or computerised. Even where data
are collected for non-survey purposes (e.g. during disease control interventions, inspections for
movement control or during disease eradication schemes), the consistency and quality of data
collection and event reporting in a format that facilitates analysis, is critical. Factors influencing
the quality of collected data include:
14
ƒ
the distribution of, and communication between, those involved in generating and
transferring data from the field to a centralised location;
ƒ
the ability of the data processing system to detect missing, inconsistent or inaccurate data,
and to address these problems;
ƒ
maintenance of disaggregated data rather than the compilation of summary data;
ƒ
minimisation of transcription errors during data processing and communication.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IV cont.
Article 3.8.1.4.
Structured population-based surveys
In addition to the principles for surveillance discussed above, the following guidelines should be used
when planning, implementing and analysing surveys.
1.
Types of surveys
Surveys may be conducted on the entire target population (i.e. a census) or on a sample. A sample
may be selected in either of the two following ways:
a. non-probability based sampling methods, such as:
i.
convenience;
ii.
expert choice;
iii.
quota;
b. probability based sampling methods, such as:
i.
simple random selection;
ii.
cluster sampling;
iii.
stratified sampling;
iv.
systematic sampling.
[Non-probability based sampling methods will not be discussed further.]
Periodic or repeated surveys conducted in order to document disease freedom should be done using
probability based sampling methods so that data from the study population can be extrapolated to
the target population in a statistically valid manner. Recognising the inherent impracticalities in
sampling from some aquatic populations, non-probability based sampling could be used when biases
have been estimated.
The sources of information should be fully described and should include a detailed description of the
sampling strategy used for the selection of units for testing. Also, consideration should be made of
any biases that may be inherent in the survey design.
2.
Survey design
The population of epidemiological units should first be clearly defined; hereafter sampling units
appropriate for each stage, depending on the design of the survey, should be defined.
The design of the survey will depend on the size and structure of the population being studied, the
epidemiology of the infection and the resources available.
3.
Sampling
The objective of sampling from a population is to select a subset of units from the population that is
representative of the population with respect to the object of the study such as the presence or
absence of infection. Sampling should be carried out in such a way as to provide the best likelihood
that the sample will be representative of the population, within the practical constraints imposed by
different environments and production systems. In order to detect the presence of an infection in a
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
15
Appendix IV cont.
population of unknown disease status, targeted sampling methods that optimise the detection of
infection can be used. In such cases, care should be taken regarding the inferences made from the
results.
4.
Sampling methods
When selecting epidemiological units from within a population, probability sampling (e.g. simple random
selection) should be used. When this is not possible, sampling should provide the best practical
chance of generating a sample that is representative of the target population.
In any case, the sampling method used at all stages should be fully documented and justified.
5.
Sample size
In general, surveys are conducted either to demonstrate the presence or absence of a factor (e.g.
infection) or to estimate a parameter (e.g. the prevalence of infection). The method used to calculate
sample size for surveys depends on the purpose of the survey, the expected prevalence, the level of
confidence desired of the survey results and the performance of the tests used.
Article 3.8.1.5.
Structured non-random surveillance
Surveillance systems routinely use structured non-random data, either alone or in combination with
surveys.
1.
Common non-random surveillance sources
A wide variety of non-random surveillance sources may be available. These vary in their primary
purpose and the type of surveillance information they are able to provide. Some surveillance systems
are primarily established as early detection systems, but may also provide valuable information to
demonstrate freedom from infection. Other systems provide cross-sectional information suitable for
prevalence estimation, either once or repeatedly, while yet others provide continuous information,
suitable for the estimate of incidence data (e.g. disease reporting systems, sentinel sites, testing
schemes). Surveillance systems routinely use structured non-random data, either alone or in
combination with surveys.
a. Disease reporting or notification systems
Data derived from disease reporting systems can be used in combination with other data
sources to substantiate claims of animal health status, to generate data for risk analysis, or for
early detection. Effective laboratory support is an important component of any reporting
system. Reporting systems relying on laboratory confirmation of suspect clinical cases should
use tests that have a high specificity. Reports should be released by the laboratory in a timely
manner, with the amount of time from disease detection to report generation minimized (to
hours in the case of introduction of a foreign animal disease).
b. Control programmes / health schemes
Animal disease control programmes or health schemes, while focusing on the control or
eradication of specific diseases, should be planned and structured in such a manner as to
generate data that are scientifically verifiable and contribute to structured surveillance.
c. Targeted testing / screening
This may involve testing targeted to selected sections of the population (subpopulations), in
which disease is more likely to be introduced or found. Examples include testing culled and
16
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IV cont.
dead animals, swill fed animals, those exhibiting clinical signs, animals located in a defined
geographic area and specific age or commodity group.
d. Ante-mortem and post-mortem inspections
Inspections of aquatic animal slaughter premises or processing plants [animals at abattoirs] may
provide valuable surveillance data. The sensitivity and specificity of the particular [slaughterhouse]
inspection system for detecting the presence of infectious agents of surveillance interest under
the particular inspection arrangements applying in a country should be pre-determined by the
Competent Authority if the data is to be fully utilised. The accuracy of the inspection system will be
influenced by:
i.
the level of training and experience of the staff doing the inspections, and the ratio of staff
of different levels of training;
ii.
the involvement of the Competent Authorities in the supervision of ante-mortem and postmortem inspections;
[iii.
the quality of construction of the abattoir, speed of the slaughter chain, lighting quality, etc; and]
iii[iv]. staff morale/motivation for accurate and efficient performance.
Post-harvest [Abattoir] inspections are likely to provide good coverage only for particular age
groups and geographical areas. Post-harvest [Abattoir] surveillance data are subject to obvious
biases in relation to target and study populations (e.g. only animals of a particular class and age
may be slaughtered for human consumption in significant numbers). Such biases need to be
recognised when analysing surveillance data.
Both for traceback in the event of detection of disease and for analysis of spatial and population
[herd]-level coverage, there should be, if possible, an effective identification system that relates
each animal in the slaughter premises/processing plant [abattoir] to its locality of origin.
e. Laboratory investigation records
Analysis of laboratory investigation records may provide useful surveillance information. The
coverage of the system will be increased if analysis is able to incorporate records from national,
accredited, university and private sector laboratories. Valid analysis of data from different
laboratories depends on the existence of standardised diagnostic procedures and standardised
methods for interpretation and data recording. As with post-harvest [abattoir] inspections, there
needs to be a mechanism to relate specimens to the farm of origin.
f. Biological specimen banks
Specimen banks consist of stored specimens, gathered either through representative sampling or
opportunistic collection or both. Specimen banks may contribute to retrospective studies,
including providing support for claims of historical freedom from infection, and may allow
certain studies to be conducted more quickly and at lower cost than alternative approaches.
g. Sentinel units
Sentinel units/sites involve the identification and regular testing of one or more of animals of
known health/exposure [immune] status in a specified geographical location to detect the
occurrence of disease [(usually serologically)]. They are particularly useful for surveillance of
diseases with a strong spatial component, such as vector-borne diseases. Sentinel units provide
the opportunity to target surveillance depending on the likelihood of infection (related to vector
habitats and host population distribution), cost and other practical constraints. Sentinel units
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
17
Appendix IV cont.
may provide evidence of freedom from infection, or provide data on prevalence and incidence
as well as the distribution of disease.
h. Field observations
Clinical observations of epidemiological units [animals] in the field are an important source of
surveillance data. The sensitivity and/or specificity of field observations may be relatively low,
but these can be more easily determined and controlled if a clear, unambiguous and easy to
apply standardised case definition is applied. Education of potential field observers in
application of the case definition and reporting is an important component. Ideally, both the
number of positive observations and the total number of observations should be recorded.
i. Farm production records
Systematic analysis of farm production records may be used as an indicator of the presence or
absence of disease at the population [herd or flock] level. In general, the sensitivity of this
approach may be quite high (depending on the disease), but the specificity is often quite low.
2.
Critical elements for structured non-random surveillance
There is a number of critical factors that should be taken into account when using structured nonrandom surveillance data such as coverage of the population, duplication of data, and sensitivity and
specificity of tests that may give rise to difficulties in the interpretation of data. Surveillance data
from non-random data sources may increase the level of confidence or be able to detect a lower level
of prevalence with the same level of confidence compared to structured surveys.
3.
Analytical methodologies
Different methodologies may be used for the analysis of non-random surveillance data.
Different scientifically valid methodologies may be used for the analysis of non-random surveillance
data. Where no data are available, estimates based on expert opinions, gathered and combined using a
formal, documented and scientifically valid methodology may be used.
4.
Combination of multiple sources of data
The methodology used to combine the evidence from multiple data sources should be scientifically
valid, and fully documented including references to published material.
Surveillance information gathered from the same country, zone or compartment at different times may
provide cumulative evidence of animal health status. Such evidence gathered over time may be
combined to provide an overall level of confidence. For instance, repeated annual surveys may be
analysed to provide a cumulative level of confidence. However, a single larger survey, or the
combination of data collected during the same time period from multiple random or non-random
sources, may be able to achieve the same level of confidence in just one year.
Analysis of surveillance information gathered intermittently or continuously over time should, where
possible, incorporate the time of collection of the information to take the decreased value of older
information into account. The sensitivity, specificity and completeness of data from each source
should also be taken into account for the final overall confidence level estimation.
Article 3.8.1.6.
Surveillance to demonstrate freedom from disease/infection
1.
18
Requirements to declare a country, zone or compartment free from disease/infection without
pathogen specific surveillance
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IV cont.
This Article provides general principles for declaring a country, zone or compartment free from
disease/infection in relation to the time of last occurrence and in particular for the recognition of
historical freedom.
The provisions of this Article are based on the principles described in Article 3.8.1.3. of this
Appendix and the following premises:
o in the absence of disease and vaccination, the farmed and wild animal populations would become
susceptible over a period of time;
o the disease agents to which these provisions apply are likely to produce identifiable clinical signs
in observable susceptible animals;
o competent and effective Competent Authority [Veterinary Services] will be able to investigate, diagnose
and report disease, if present;
o the absence of disease/infection over a long period of time in a susceptible population can be
substantiated by effective disease investigation and reporting by a Member Country.
a. Historically free
Unless otherwise specified in the relevant disease chapter, a country, zone or compartment may be
recognised free from infection without formally applying a pathogen-specific surveillance
programme when:
i.
there has never been occurrence of disease, or
ii.
eradication has been achieved or the disease/infection has ceased to occur for at least 25 years,
provided that for at least the past 10 years:
iii.
it has been a notifiable disease;
iv.
an early detection system has been in place;
v.
measures to prevent disease/infection introduction have been in place; no vaccination against
the disease has been carried out unless otherwise provided in the Aquatic Code [Terrestrial
Code];
vi.
infection is not known to be established in wildlife within the country or zone intended to
be declared free. (A country or zone cannot apply for historical freedom if there is any
evidence of infection in wildlife. However, specific surveillance in wildlife is not necessary.)
b. Last occurrence within the previous 25 years
Countries, zones or compartments that have achieved eradication (or in which the disease/infection has
ceased to occur) within the previous 25 years, should follow the pathogen-specific surveillance
requirements in the Aquatic Code [Terrestrial Code] if they exist. In the absence of specific
requirements for surveillance in the Aquatic Code [Terrestrial Code], countries should follow the
general guidelines for surveillance to demonstrate animal health status outlined in this Appendix
provided that for at least the past 10 years:
i.
it has been a notifiable disease;
ii.
an early detection system has been in place;
iii.
measures to prevent disease/infection introduction have been in place;
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
19
Appendix IV cont.
2.
iv.
no vaccination against the disease has been carried out unless otherwise provided in the
Aquatic Code [Terrestrial Code];
v.
infection is not known to be established in wildlife within the country or zone intended to be
declared free. (A country or zone cannot apply for freedom if there is any evidence of
infection in wildlife. However, specific surveillance in wildlife is not necessary.)
Guidelines for the discontinuation of pathogen-specific screening after recognition of freedom from
infection
A country, zone or compartment that has been recognised as free from infection following the
provisions of the Aquatic Code [Terrestrial Code] may discontinue pathogen-specific screening while
maintaining the infection-free status provided that:
3.
a.
it is a notifiable disease;
b.
an early detection system is in place;
c.
measures to prevent disease/infection introduction are in place;
d.
vaccination against the disease is not applied;
e.
infection is known not to be established in wildlife. (Specific surveillance in wildlife has
demonstrated the absence of infection.)
International recognition of disease/infection free status
For diseases for which procedures exist whereby the OIE can officially recognise the existence of a
disease/infection free country, zone or compartment, a Member Country wishing to apply for recognition
of this status shall, via its Permanent Delegate, send to the OIE all the relevant documentation
relating to the country, zone or compartment concerned. Such documentation should be presented
according to guidelines prescribed by the OIE for the appropriate animal diseases.
4.
Demonstration of freedom from infection
A surveillance system to demonstrate freedom from infection should meet the following
requirements in addition to the general requirements for surveillance outlined in Article 3.8.1.3. of
this Appendix.
Freedom from infection implies the absence of the pathogenic agent in the country, zone or
compartment. Scientific methods cannot provide absolute certainty of the absence of infection.
Demonstrating freedom from infection involves providing sufficient evidence to demonstrate (to a
level of confidence acceptable to Member Countries) that infection with a specified pathogen is not
present in a population. In practice, it is not possible to prove (i.e., be 100% confident) that a
population is free from infection (unless every member of the population is examined simultaneously
with a perfect test with both sensitivity and specificity equal to 100%). Instead, the aim is to provide
adequate evidence (to an acceptable level of confidence), that infection, if present, is present in less
than a specified proportion of the population.
However, finding evidence of infection at any level in the target population automatically invalidates
any freedom from infection claim unless otherwise stated in the relevant disease Chapter.
Evidence from targeted, random or non-random data sources, as stated before, may increase the level
of confidence or be able to detect a lower level of prevalence with the same level of confidence
compared to structured surveys.
20
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IV cont.
Article 3.8.1.7.
Surveillance for distribution and occurrence of infection
Surveillance to determine distribution and occurrence of infection or of other relevant health related
events is widely used to assess the prevalence and incidence of selected disease/infection [progress in the control
or eradication of selected diseases and pathogens and] as an aid to decision making, for example implementation
of control and eradication programmes. It has, however, relevance for the international movement of
animals and products when movement occurs among infected countries.
In contrast to surveillance to demonstrate freedom from infection, surveillance for the distribution and
occurrence of infection [used to assess progress in control or eradication of selected diseases and pathogens] is usually
designed to collect data about a number of variables of animal health relevance, for example:
1.
prevalence or incidence of infection in wild and cultured animals;
2.
morbidity and mortality rates;
3.
frequency of disease/infection risk factors and their quantification;
4.
frequency distribution [herd sizes or the sizes of other] of epidemiological units;
[5.
frequency distribution of antibody titres]
[6.
proportion of immunised animals after a vaccination campaign]
5.
frequency distribution of the number of days elapsing between suspicion of infection and laboratory
confirmation of the diagnosis and/or to the adoption of control measures;
6.
farm production records, etc.
_______________
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
21
Appendix V
CHAPTER
1.1.4.
GUIDELINES FOR AQUATIC ANIMAL
HEALTH SURVEILLANCE [REQUIREMENTS
FOR
SURVEILLANCE FOR INTERNATIONAL RECOGNITION OF
FREEDOM FROM INFECTION]
PART 1
A. GUIDELINES FOR SELF DECLARATION [INTERNATIONAL RECOGNITION] OF
FREEDOM FROM INFECTION
[1. GENERAL PRINCIPLES
General principles are provided below for declaring a country, zone or aquaculture establishment free from infection in
relation to the time of last occurrence, and in particular for the recognition of historical freedom.
An essential prerequisite to provide the guarantees required for the recognition of freedom from infection is that the
particular Member Country complies with the requirements of Chapter 1.4.3 of the Aquatic Code for the evaluation of the
Competent Authorities.
The general principles are:]
The general assumptions regarding self declarations of freedom from infection are:
•
in the absence of infection or vaccination, the animal population would be susceptible to [to clinical
disease, or] infection, over a period of time;
•
the disease agents to which these provisions apply are likely to produce identifiable clinical or
pathological signs in susceptible animals under conducive situations;
•
an animal population may be free from some specified pathogens but not from others;
•
[there are competent and effective personnel of] the Competent Authority is able and has the means to
effectively investigate, detect and report disease or infection, if present;
•
the historical absence of infection [over a long period of time] in susceptible populations can be
substantiated by effective [disease investigation] surveillance and reporting by the Competent
Authority [of the Member Country].
Demonstrating freedom from infection involves providing sufficient evidence to demonstrate that
infection with a specified agent is not present in a specified population. In practice, it is not possible to
definitively prove that a population is free from infection (unless every member of the population is
examined simultaneously with a perfect test with both sensitivity and specificity equal to 100%). Instead,
the aim is to provide adequate evidence (to an acceptable level of confidence), that infection, if present, is
present in less than a specified proportion of the population.
1. CRITERIA
[REQUIREMENTS] TO DECLARE
] FREE FROM INFECTION WITH
ESTABLISHMENT
A
COUNTRY,
ZONE
OR
COMPARTMENT
[AQUACULTURE
A SPECIFIED PATHOGEN
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
23
Appendix V cont.
The criteria [requirements] to declare a country, zone or compartment [aquaculture establishment] free from
infection differ depending on the previous infection status of the country, zone or compartment
[aquaculture establishment], namely:
•
Absence of susceptible species;
•
Historically free;
•
Last known occurrence within the previous 25 years;
•
Previously unknown infection status.
1.1. Absence of susceptible species
Unless otherwise specified in the relevant disease chapter, a country, zone or compartment
[aquaculture establishment] may be recognised as being free from infection without applying targeted
surveillance if there are no susceptible species (as listed in the relevant chapter of the Aquatic Code,
or in the scientific literature) present in that country, zone or compartment [aquaculture
establishment], provided that the basic [prescribed] biosecurity conditions have been in place
continuously in the country, zone or compartment [aquaculture establishment] for at least the
previous 10 years.
Basic biosecurity means a set of conditions applying to a particular disease or infection, and
particular conditions zone or country, required to ensure adequate biosecurity, namely:
a)
the disease is compulsorily notifiable to the Competent Authority;
b)
an early detection system is in place within the zone or country;
c)
no vaccination against the disease is carried out;
d)
infection is not known to be established in wild populations;
e)
import requirements to prevent the introduction of disease or infection into the country or
zone, as outlined in the Aquatic Code, are in place.
1.2. Historically free
Unless otherwise specified in the relevant disease chapter, a country, zone or compartment
[aquaculture establishment] may be recognised as being free from infection without formally applying
targeted surveillance when:
•
there has never been any observed occurrence of disease;
•
eradication has been achieved or the disease has ceased to occur for at least 25 years,
or
provided that the basic [prescribed] biosecurity conditions have been in place continuously in the
country, zone or compartment [aquaculture establishment] for at least the previous 10 years.
1.3. Last known occurrence within the previous 25 years
For countries or zones that have achieved eradication (or in which the disease has ceased to
occur) within the previous 25 years, in addition to the basic [prescribed] biosecurity conditions,
appropriate targeted surveillance must have been applied to demonstrate the absence of the
infection, consistent with the provisions of Section B of this chapter.
1.4. Previously unknown infection status
24
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
For countries or zones with previously unknown infection status, or which have not previously
met the requirements of the Sections A.2.1, A.2.2 or A.2.3 above, the basic [prescribed] biosecurity
conditions must be introduced in addition to targeted surveillance consistent with the provisions of
Section B of this chapter.
2. GUIDELINES
FOR THE MAINTENANCE OF CONTINUED RECOGNITION OF FREEDOM FROM INFECTION
A country, zone or compartment [aquaculture establishment] that has been recognised free from infection
following the provisions of Sections A.2.1 or A.2.2, may maintain its official status as infection-free
provided that the basic [prescribed] biosecurity conditions are continuously maintained.
A country, zone or compartment [aquaculture establishment] that has been recognised free from infection
following the provisions of Sections A.2.3 or A.2.4, may discontinue targeted surveillance and maintain
its official status as infection-free provided that the basic [prescribed] biosecurity conditions are
continuously maintained.
The different paths to recognition of freedom from infection are summarised in the diagram below.
Absence of
susceptible species
Historically free
Last occurrence wtihin
the previous 25 years
Previously unknown
disease status
Meet basic
biosecurity conditions
Meet basic
biosecurity conditions
and
Implement targeted
surveillance
(Section B)
Freedom from Infection
Maintain basic
biosecurity conditions
Discontinue
targeted surveillance
B. GUIDELINES FOR AQUATIC ANIMAL HEALTH SURVEILLANCE
1. INTRODUCTION
[This
section provides standards to be applied when demonstrating country, zone or aquaculture establishment freedom
from infection, in accordance with the principles of Section A. Standards described in this section]
In general,
surveillance is aimed at:
-
demonstrating the absence of disease or infection,
-
determining the occurrence or distribution of disease or infection, including changes to their
incidence or prevalence (or its contributing factors),
-
detecting as early as possible exotic or emerging diseases.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
25
Appendix V cont.
The type of surveillance applied depends on the desired outputs needed to support decision-making.
The following guidelines may be applied to all diseases, their agents and susceptible species as listed in
the Aquatic Code, and are designed to assist with the development of surveillance methodologies. [More
detailed information in each disease chapter (] Except where [it exists) of this Aquatic Manual] a specific
surveillance method for a certain disease or infection is already described in the Aquatic Code, the
guidelines in this Appendix may be used to further refine the general approaches described for a
specific disease or infection. Where detailed disease/infection-specific information is not available, suitable
approaches [values] should be [chosen] based on these guidelines [in this chapter].
Surveillance data determine the quality of disease status reports and should satisfy information
requirements for accurate risk analysis both for international trade as well as for national decisionmaking.
Animal health surveillance is an essential component of programmes aimed to:
-
monitor disease trends
-
control endemic diseases,
-
prevent introduction of exotic diseases,
_
support claims for freedom from disease or infection,
provide data to support the risk analysis process, for both animal health and/or public health
purposes,
-
substantiate the rationale for sanitary measures.
There is sometimes a perception that surveillance can only be conducted using sophisticated
methodologies. However, an effective surveillance system can also be developed by making use of
gross observations and available resources.
Surveillance of endemic diseases provides valuable information for day-to-day health management and
can act as the foundation for detecting outbreaks of exotic disease and demonstrating specific disease
freedom.
Surveillance may address both infectious and non-infectious diseases of concern to the country.
Section B provides standards to be applied when: (a) demonstrating country, zone or compartment
[aquaculture establishment] freedom from infection, in accordance with the principles of Section A and
(b) assessing the occurrence and distribution of a specific infection/disease or syndrome
Standards described in this section may be applied to all diseases, their agents and susceptible species as
listed in the Aquatic Code, and are designed to assist with the development of surveillance
methodologies. Nevertheless surveillance may include also non listed diseases
It would be impractical to try to develop a surveillance system, for all the known aquatic animal
diseases in a country. Therefore prioritising the diseases to be included in a surveillance system should
be conducted considering:
26
-
the needs to provide assurance of disease status for trade purposes
-
the resources of the country
-
the financial impact or threat posed by the different diseases
-
the importance of an industry-wide disease control program within a country or region
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
The concept of risk encompasses both the probability of the disease occurring and the severity of its
consequences
More detailed information in each disease chapter (where it exists) of this Aquatic Manual may be used
to further refine the general approaches described in this chapter. Where detailed disease/infectionspecific information is not available, surveillance can also be conducted following the guidelines in this
chapter. Access to epidemiological expertise would be invaluable for the design, implementation of the
system and interpretation of results derived from a surveillance system.
2. GENERAL
PRINCIPLES
[Demonstrating freedom from infection involves providing sufficient evidence to demonstrate that infection with a specified
agent is not present in a specified population. In practice, it is not possible to definitively prove that a population is free
from infection (unless every member of the population is examined simultaneously with a perfect test with both sensitivity
and specificity equal to 100%). Instead, the aim is to provide adequate evidence (to an acceptable level of confidence),
that infection, if present, is present in less than a specified proportion of the population.
Methodologies to demonstrate freedom from infection should be] Surveillance methodologies should be flexible to
deal with the complexity of real life situations. No single method is applicable in all cases.
Methodologies must be able to accommodate the variety of aquatic animal species, the multiple
diseases of relevance, varying production [and surveillance] systems, and types and amounts of data and
information available.
The methodology used should be based on the best available information that is in accord with current
scientific thinking. The methodology should be well documented and supported with references to the
scientific literature and other sources, including expert opinion. Efforts should be made to address the
information gaps wherever possible.
[Consistency in methodologies should be encouraged and transparency is] Methodologies that are consistent and
transparent are essential to ensure fairness and rationality, consistency in decision making and ease of
understanding by all the interested parties. [Applications for] The presentation of the results generated
through surveillance (e.g. recognition of infection-free status) should document the uncertainties, the
assumptions made, and the potential effect of these on the final estimate.
3. SURVEILLANCE
INFECTION]
SYSTEM CONSIDERATIONS
[GENERAL
REQUIREMENTS FOR DEMONSTRATION OF FREEDOM FROM
3.1. Objectives [Population]
[The target
population to which the demonstration of freedom from infection applies is all individuals of all species
susceptible to the infection in a country, zone or aquaculture establishment.
The study population may be the same as the target population or a subset of it. The study population should be (in
order of preference):
•
The appropriate study population as defined in the relevant disease chapter of the Aquatic Code (if such a
definition exists),
•
A subset of the target population that defines a group of animals which, if infection were present, would be
most likely to have a higher prevalence of infection than the target population. This subset should be defined in
terms of:
•
species;
•
time (e.g. season or month of year);
•
stage of life-cycle or growth period;
•
production system and/or management characteristics;
•
location;
•
readily identifiable physical or behavioural characteristics.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
27
Appendix V cont.
•
The same as the target population,
•
A subset of the target population with the same or lower probability of infection. The nature and impact of any
biases on the results of the analysis must be considered, documented and taken into account in the analysis.]
The objectives of surveillance systems can be to describe disease occurrence and distribution, to
demonstrate freedom from disease in a particular compartment, or to detect emerging diseases.
The objectives determine the specific surveillance components and therefore require planning
prior to design and implementation. Resource limitations will dictate the scope and precision of
the programme.
3.2. Population
The target population consists of all individuals of all species susceptible to the disease or infection
in a country, zone or compartment to which the surveillance results apply. Sometimes
components of the target population are at higher risk of being the point of introduction for an
exotic disease. In these cases, it is advisable to focus surveillance efforts on this part of the
population, such as farms on a geographic border. Similarly, some local areas within a region may
be known to be absent of the disease of concern, allowing resources to be concentrated on
known positive areas for greater precision of prevalence estimates and only verification of
expected 0 prevalence areas.
3.3. Sources of evidence
Surveillance data may originate [Evidence of freedom from infection may be based on a] from a number of
different sources, including:
•
structured, population-based surveys using one or more tests to detect [for the presence of] the
agent;
•
other [surveillance, including] structured non-random [surveillance]sources, such as:
•
sentinel sites;
•
disease notifications and laboratory investigation records;
•
academic and other scientific studies;
•
a knowledge of the biology of the agent, including environmental, host population
distribution, known geographical distribution, vector distribution and climatic information;
•
history of imports of potentially infected material;
•
biosecurity measures in place;
[•
evaluation of the official services; or]
•
any other sources of information that provide contributory evidence regarding disease or
[that] infection [is not present] in the country, zone or compartment [aquaculture establishment].
The sources of evidence [used to demonstrate freedom from infection] must be fully described. In the
case of a structured survey, this must include a description of the sampling strategy used for the
selection of units for testing. For complex surveillance systems, a full description of the system is
required including consideration of any biases that may be inherent in the system. Evidence to
support claims of freedom of disease can use structured non-random sources of information
provided any potential error is to detect rather than miss positive cases. Evidence to support
changes in prevalence/incidence of endemic disease must be based on valid, reliable methods to
generate precise estimates with known error.
28
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
3.3. Statistical methodology
Analysis of data for evidence of freedom from infection involves estimating the probability (α)
that the evidence observed (the results of surveillance) could have been produced under the null
hypothesis that infection is present in the population at a specified prevalence(s) (the design
prevalence[s]). The confidence in (or, equivalently, the sensitivity of) the surveillance system that
produced the evidence is equal to 1–α. If the confidence level exceeds a pre-set threshold, the
evidence is deemed adequate to demonstrate freedom from infection.
The required level of confidence in the surveillance system (probability that the system would detect
infection if infection were present at the specified level) must be greater than or equal to 95%.
The power (probability that the system would report that no infection is present if infection is
truly not present) may be set to any value. By convention, this is often set to 80%, but may be
adjusted according to the country’s or zone’s requirements.
Different statistical methodologies for the calculation of the probability α, including both
quantitative and qualitative approaches, are acceptable as long as they are based on accepted
scientific principles.
The methodology used to calculate the confidence in the surveillance system must be scientifically
based and clearly documented, including references to published work describing the
methodology.
[3.4.
Clustering of infection
Infection in a country, zone or aquaculture establishment usually clusters rather than being uniformly distributed
through a population. Clustering may occur at a number of different levels (e.g. a cluster of moribund fish in a pond, a
cluster of ponds in a farm, or a cluster of farms in a zone). Except when dealing with demonstrably homogenous
populations, approaches to demonstrating freedom must take this clustering into account in the design and the
statistical analysis of the data, at least at what is judged to be the most significant level of clustering for the particular
animal population and infection.
3.5.
Design prevalence]
Statistical analysis of surveillance data often requires assumptions about population parameters or
test characteristics. These are usually based on expert opinion, previous studies on the same or
different populations, expected biology of the agent, and so on. The uncertainty around these
assumptions must be quantified and considered in the analysis (e.g. in the form of prior
probability distributions in a Bayesian setting).
For surveillance systems used to describe disease patterns, the purpose is to estimate prevalence
or incidence with confidence intervals or probability intervals. The magnitude of these intervals
expresses the precision of the estimates and is related to sample size. Narrow intervals are
desirable but will require larger sample sizes and more dedication of resources. The precision of
the estimates and the power to detect differences in prevalence between populations or between
time points depends not only on sample size, but also on the actual value of the prevalence in the
population or the actual difference. For this reason, when designing the surveillance system, a
prior estimate / assumption of expected prevalence or expected difference in prevalence must be
made.
For surveillance systems used to demonstrate freedom from specific diseases, calculation of the
confidence of a surveillance system is based on the null hypothesis that infection is present in the
population. The level of infection is specified by the design prevalence. In the simplest case, this is
the prevalence of infection in a homogenous population. More commonly, in the presence of
disease clustering, two design prevalence values are required, for instance, the animal-level
prevalence (proportion of fish infected in an infected farm) and the group-level prevalence
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
29
Appendix V cont.
(proportion of infected farms in the country, zone or compartment [aquaculture establishment]).
Further levels of clustering may be considered, requiring further design prevalence values.
The values for design prevalence used in calculations must be those specified in the relevant
disease chapter (if present) of this Aquatic Manual. If not specified for the particular disease,
justification for the selection of design prevalence values must be provided, and should be based
on the following guidelines:
•
At the individual animal level, the design prevalence is based on the biology of the infection
in the population. It is equal to the minimum expected prevalence of infection in the study
population, if the infection had become established in that population. It is dependent on the
dynamics of infection in the population and the definition of the study population (which may
be defined to maximise the expected prevalence in the presence of infection).
•
A suitable design prevalence value at the animal level (e.g. prevalence of infected animals in a
cage) may be:
•
•
between 1% and 5% for infections that are transmitted slowly; and
•
over 5% for more contagious infections.
At higher levels (e.g. cage, pond, farm, village, etc.) the design prevalence usually reflects the
prevalence of infection that is practically and reasonably able to be detected by a surveillance
system. Detection of infection at the lowest limit (a single infected unit in the population) is
rarely feasible in large populations. The expected behaviour of the infection may also play a
role. Infections that have the ability to spread rapidly between farms may have a higher farmlevel design prevalence than slow-moving infections.
A suitable design prevalence value for the first level of clustering, (e.g. proportion of infected
farms in a zone) may be up to 2%.
When surveillance objectives are to estimate prevalence/incidence or changes in disease patterns,
statistical analysis must account for sampling error. Analytic methods should be thoroughly
considered and consultation with biostatistician/quantitative epidemiologist consulted beginning
in the planning stages and continued throughout the programme.
Surveillance data are used to estimate incidence and prevalence measures for the purpose of
describing disease occurrence in terms of animal unit, time and place. These measures can be
calculated for an entire population and specific time period, or for subsets defined by host
characteristics (e.g. age-specific incidence). Incidence estimation requires on-going surveillance to
detect new cases while prevalence is the estimated proportion of infected individuals in a
population at a given time point. The estimation process must consider test sensitivity and
specificity. (consider whether this fits in this location)
3.4. Clustering of infection
Infection in a country, zone or compartment [aquaculture establishment] usually clusters rather than
being uniformly distributed through a population. Clustering may occur at a number of different
levels (e.g. a cluster of moribund fish in a pond, a cluster of ponds in a farm, or a cluster of farms
in a zone). Except when dealing with demonstrably homogenous populations, surveillance must
take this clustering into account in the design and the statistical analysis of the data, at least at
what is judged to be the most significant level of clustering for the particular animal population and
infection. For endemic diseases, it is important to identify characteristics of the population which
contribute to clustering and thus provide efficiency in disease investigation and control.
3.5. Expected prevalence (section already incorporated into statistical methods)
30
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
3.5. Test characteristics
All surveillance involves performing one or more tests for evidence of the presence of current or
past infection, ranging from detailed laboratory examinations to farmer observations. The
performance level of a test at the population level is described in terms of its sensitivity and specificity.
Imperfect sensitivity and/or specificity impact on the interpretation of surveillance results and
must be taken into account in the analysis of surveillance data. For example, in populations with
low prevalence of infection, a large proportion of positive tests may be false unless the tests used
have perfect specificity. To ensure detection in such instances, a highly sensitive test is frequently
used for initial screening and then confirmed with highly specific tests.
All calculations must take the performance level (sensitivity and specificity) of any tests used into
account. The values of sensitivity and specificity used for calculations must be specified, and the
method used to determine or estimate these values must be documented. [Where] Test sensitivity
and specificity can be different when applied to different populations and testing scenarios. For
example, test sensitivity may be lower when testing carrier animals with low level infections
compared to moribund animals with clinical disease. Alternatively, specificity depends on the
presence of cross-reacting agents, the distribution of which may be different under different
conditions or regions. Ideally, test performance should be assessed under the conditions of use
otherwise increased uncertainty exists regarding their performance. In the absence of local
assessment of tests, values for sensitivity and/or specificity for a particular test that are specified in
this Aquatic Manual may be used but the increased uncertainty associated with these estimates
should be incorporated into the analysis of results [these values may be used without justification].
Where more than one test is used in a surveillance system (sometimes called using tests in series or
parallel), the overall test system sensitivity and specificity must be calculated using a scientifically valid
method.
Pooled testing involves the pooling of specimens from multiple individuals and performing a
single test on the pool. Pooled testing is an acceptable approach in many situations. Where pooled
testing is used, the results of testing must be interpreted using sensitivity and specificity values
that have been determined or estimated for that particular pooled testing procedure and for the
applicable pool sizes being used. Analysis of the results of pooled testing must, where possible, be
performed using accepted, statistically based methodologies, which must be fully documented,
including published references.
Test results from surveillance for endemic disease will provide estimates of apparent prevalence.
Using sensitivity and specificity, true prevalence should be calculated with the following formula:
FORMULA TO BE ADDED
In addition, it should be remembered that laboratories may disagree for various test, host, or
procedure-related reasons. Therefore, sensitivity and specificity parameters should be validated
for the particular laboratory and process.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
31
Appendix V cont.
3.7. Multiple sources of evidence
Where multiple different data sources providing evidence of freedom from infection exist or are
generated for disease trends, each of these data sources may be analysed according to the
provisions of Sections B.3, B.4 (for structured surveys) and B.5 (for complex data sources). The
resulting estimates of the confidence in each data source may be combined to provide an overall
level of confidence for the combined data sources.
The methodology used to combine the estimates from multiple data sources:
•
must be scientifically valid, and fully documented, including references to published material;
and
•
should, where possible, take into account any lack of statistical independence between
different data sources.
Surveillance information gathered from the same country, zone or compartment [aquaculture
establishment] at different times may provide cumulative evidence of freedom from infection. Such
evidence gathered over time may be combined into an overall level of confidence. For instance,
repeated annual surveys may be analysed to provide a cumulative level of confidence. However, a
single (larger) survey may be able to achieve the same level of confidence in just 1 year.
Analysis of surveillance information gathered intermittently or continuously over time should,
where possible, incorporate the time of collection of the information to take the decreased value
of older information into account. Changes in disease occurrence of endemic diseases must
account for other factors influencing detection proficiency.
4.
SPECIFIC
REQUIREMENTS FOR STRUCTURED SURVEY DESIGN AND ANALYSIS
Since the focus of this section is still on [One method of generating evidence for] freedom from infection,
further consideration of the following section is needed to include recommendations for surveillance
for endemic diseases.
One component of surveillance is the use of structured, population-based, targeted surveys. In
addition to the requirements specified in Section B.3, the following guidelines should be used when
implementing and analysing surveys to demonstrate freedom from infection or for describing disease
patterns.
4.1. Survey design
The most important unit of diagnosis is the epidemiological unit. The population of epidemiological
units must be clearly defined.
The design of the survey will depend on the size and structure of the population being studied. If
the population is relatively small and can be considered to be homogenous with regards to risk of
infection, a single-stage survey can be used.
In larger populations where a sampling frame is not available, or when there is a likelihood of
clustering of disease, multi-stage sampling is required. In two-stage sampling, at the first stage of
sampling, groups of animals (e.g. ponds, farms or villages) are selected. At the second stage,
animals are selected for testing from each of the selected groups.
Stratification may be used in survey design.
4.2. Sampling
The objective of sampling from a population is to select a subset of units from the population that is
representative of the population with respect to the characteristic of interest (in this case, the
32
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
presence or absence of infection). Sampling should be carried out in such a way as to provide the
best likelihood that the sample will be representative of the population, within the practical
constraints imposed by different environments and production systems.
Biased or targeted sampling in this context involves sampling from a defined study population that
has a different probability of infection than the target population of which it is a subpopulation.
Once the study population has been identified, the objective is still to select a representative sample
from this subpopulation.
4.3. Sampling methods
The survey design may involve sampling at several levels.
For sampling at the level of the epidemiological units or higher units, a formal probability sampling
(e.g. simple random sampling) method must be used.
When sampling below the level of the epidemiological unit (e.g. individual animal), the sampling
method used should provide the best practical chance of generating a sample that is representative
of the population of the chosen epidemiological unit. Collecting a truly representative sample of
individual animals (whether from a pond, cage or fishery) is often very difficult. To maximise the
chance of finding infection, the aim should be to bias the sampling towards infected animals, e.g.
selecting moribund animals, life stages with a greater chance of active infection, etc.
The sampling method used at all levels must be fully documented and justified.
4.4. Sample size
The number of units to be sampled from a population should be calculated using a statistically valid
technique that takes at least the following factors into account:
•
The sensitivity and specificity of the diagnostic test, or test system;
•
The design prevalence (or prevalences where a multi-stage design is used);
•
The level of confidence that is desired of the survey results.
Additionally, other factors may be considered in sample size calculations, including (but not
limited to):
•
The size of the population (but it is acceptable to assume that the population is infinitely large);
•
The desired power of the survey;
•
Uncertainty or variability in estimates of sensitivity and specificity.
The specific sampling requirements will need to be tailor-made for each individual disease, taking
into account its characteristics and the specificity and sensitivity of the accepted testing methods
for detecting the disease agent in host populations.
FreeCalc (add link) is a suitable software for the calculation of sample sizes at varying parameter
values. The table below provides examples of sample sizes generated by the software.
ADD TABLE + EXPLANATION (Flavio)
[Detailed guidelines are to be provided in the next (fifth) edition of the Aquatic Manual. In the meantime, the sampling
procedures given in Chapters I.1, I.2 and I.3 may be applied.]
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
33
Appendix V cont.
4.5. Data analysis
Analysis of test results from a survey shall be in accordance with the provisions of Section B.3 and
take at least the following considerations into account:
•
The survey design;
•
The sensitivity and specificity of the test, or test system;
•
The design prevalence (or prevalences where a multi-stage design is used);
•
The results of the survey.
4.6. Quality assurance
Surveys should include a documented quality assurance system, to ensure that field and other
procedures conform to the specified survey design. Acceptable systems may be quite simple, as
long as they provide verifiable documentation of procedures and basic checks to detect significant
deviations of procedures from those documented in the survey design.
5.
SPECIFIC
REQUIREMENTS FOR COMPLEX NON-SURVEY DATA SOURCES
Data sources that provide evidence of freedom from infection, but are not based on structured
population-based surveys may also be used to demonstrate freedom, either alone or in combination
with other data sources. Different methodologies may be used for the analysis of such data sources,
but the methodology must comply with the provisions of Section B.3. The approach used should,
where possible, also take into account any lack of statistical independence between observations.
Analytical methodologies based on the use of step-wise probability estimates to describe the surveillance
system may determine the probability of each step either by:
•
the analysis of available data, using a scientifically valid methodology; or where no data are
available,
•
the use of estimates based on expert opinion, gathered and combined using a formal, documented
and scientifically valid methodology.
Where there is significant uncertainty and/or variability in estimates used in the analysis, stochastic
modelling or other equivalent techniques should be used to assess the impact of this uncertainty
and/or variability on the final estimate of confidence.
PART 2
EXAMPLE SURVEILLANCE SYSTEMS
Three additional examples are required. These should be based on the current three examples but refer to
a situation in which the disease used as example is already present in the population
The following examples describe surveillance systems and approaches to the analysis of evidence that are
able to meet the requirements of Part 1 of this chapter. The purpose of these examples is:
•
to illustrate the range of approaches that may be acceptable;
•
to provide practical guidance and models that may be used for the design of specific surveillance
systems; and
34
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
•
to provide references to available resources that are useful in the development and analysis of
surveillance systems.
While these examples demonstrate ways in which freedom from infection may be successfully
demonstrated, they are not intended to be prescriptive. Countries are free to use different approaches, as
long as they meet the requirements of Part 1 of this chapter.
The examples deal with the use of structured surveys and are designed to illustrate different survey
designs, sampling schemes, the calculation of sample size, and analysis of results. It is important to note
that alternative approaches to demonstrating freedom using complex non-survey-based data sources are
also currently being developed and may soon be published1.
EXAMPLE 1 –
ONE-STAGE STRUCTURED SURVEY
(FARM
ACCREDITATION)
Context
A freshwater aquaculture industry raising fish in tanks has established a farm accreditation scheme. This
involves demonstrating farm-level freedom from a particular (hypothetical) disease (Disease X). The
disease does not spread very quickly, and is most common during the winter months, with adult fish at the
end of the production cycle being most severely affected. Farms consist of a number of grow-out tanks,
ranging from 2 to 20, and each tank holds between 1000 and 5000 fish.
Objective
The objective is to implement surveillance that is capable of providing evidence that an individual farm is
free from Disease X. (The issue of national or zone freedom, as opposed to farm freedom, is considered
in the next example.)
Approach
The accreditation scheme establishes a set of standard operating procedures and requirements for
recognition of freedom, based on the guidelines given in this chapter. These require farms to undertake a
structured survey capable of producing 95% confidence that the disease would be detected if it were
present. Once farms have been surveyed without detecting disease, they are recognised as free, as long as
they maintain a set of minimum biosecurity standards. These standards are designed to prevent the
introduction of Disease X into the farm (through the implementation of controls specific to the method
of spread of that disease) and to ensure that the disease would be detected rapidly if it were to enter the
farm (based on evidence of adequate health record keeping and the prompt investigation of unusual
disease events). The effective implementation of these biosecurity measures is evaluated with annual onfarm audits conducted by independent auditors.
Survey standards
Based on the guidelines given in this chapter, a set of standards are established for the conduct of surveys
to demonstrate freedom from infection with causative agent of Disease X. These standards include:
•
The level of confidence required of the survey is 95% (i.e. Type I error = 5%).
•
The power of the survey is arbitrarily set at 95% (i.e. Type II error = 5%, which means that there is a
5% chance of concluding that a non-diseased farm is infected).
•
The target population is all the fish on the farm. Due to the patterns of disease in this production
system, in which only fish in the final stages of grow-out, and only in winter are affected, the study
population is defined as grow-out fish during the winter months.
1
International EpiLab, Denmark, Research Theme 1: Freedom from disease.
http://www.vetinst.dk/high_uk.asp?page_id=196
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
35
Appendix V cont.
•
The issue of clustering is considered. As fish are grouped into tanks, this is the logical level at which
to consider clustering. However, when a farm is infected, the disease often occurs in multiple tanks,
so there is little evidence of strong clustering. Also, the small number of tanks on a single farm means
that it is difficult to define a design prevalence at the tank level (i.e. the proportion of infected tanks
that the survey should be able to detect on the farm). For these reasons, it is decided to treat the
entire grow-out population of each farm as a single homogenous population.
•
Stratification is also considered. In order to ensure full representation, it is decided to stratify the
sample size by tank, proportional to the population of each tank.
•
The design prevalence at the animal level is determined based on the epidemiology of the disease.
The disease does not spread quickly, however, in the defined target population, it has been reported
to affect at least 10% of fish, if the population is infected. In order to take the most conservative
approach, an arbitrarily low design prevalence of 2% is used. A prevalence of 10% may have been
used (and would result in a much smaller sample size), but the authorities were not convinced by the
thought that the population could still be infected at a level of say 5%, and disease still not be
detected.
•
The test used involves destructive sampling of the fish, and is based on an antigen-detection enzymelinked immunosorbent assay (ELISA). Disease X is present in some parts of the country (hence the
need for a farm-level accreditation programme). This has provided the opportunity for the sensitivity
and the specificity of the ELISA to be evaluated in similar populations to those on farms. A recent
study (using a combination of histology and culture as a gold standard) estimated the sensitivity of
the ELISA to be 98% (95% confidence interval 96.7–99.2%), and the specificity to be 99.4% (99.2–
99.6%). Due to the relatively narrow confidence intervals, it was decided to use the point estimates of
the sensitivity and specificity rather than complicate calculations by taking the uncertainty in those
estimates into account.
Sample size
The sample size required to meet the objectives of the survey is calculated to take the population size, the
test performance, the confidence required and the design prevalence into account. As the population of
each farm is relatively large, differences in the total population of each farm have little effect on the
calculated sample size. The other parameters for sample size calculation are fixed across all farms.
Therefore, a standard sample size (based on the use of this particular ELISA, in this population) is
calculated. The sample size calculations are performed using the FreeCalc software2. Based on the
parameters listed above, the sample size required is calculated to be 410 fish per farm. In addition, the
program calculates that, given the imperfect specificity, it is still possible for the test to produce up to five
false-positive reactors from an uninfected population using this sample size. The authorities are not
comfortable with dealing with false-positive reactors, so it is decided to change the test system to include a
confirmatory test for any positive reactors. Culture is selected as the most appropriate test, as it has a
specificity that is considered to be 100%. However, its sensitivity is only 90% due to the difficulty of
growing the organism.
As two tests are now being used, the performance of the test system must be calculated, and the sample
size recalculated based on the test system performance.
Using this combination of tests (in which a sample is considered positive only if it tests positive to both
tests), the specificity of the combined two tests can be calculated by the formula:
Sp Combined = Sp1 + Sp 2 − ( Sp1 × Sp 2 )
which produces a combined specificity of 1 + 0.994 – (1 × 0.994) = 100%
2
36
FreeCalc – Cameron, AR. Software for the calculation of sample size and analysis of surveys to demonstrate freedom from
disease. Available for free download from http://www.ausvet.com.au.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
The sensitivity may be calculated by the formula:
SeCombined = Se1 × Se
which produces a combined sensitivity of 0.9 × 0.98 = 88.2%
These new values are used to calculate the survey sample size yielding a result of 169 fish. It is worth
noting that attempts to improve the performance of a test (in this case increase specificity) generally result
in a decrease in the performance of the other aspect of the test performance (sensitivity in this example).
However, in this case, the loss of sensitivity is more than compensated for by the decreased sample size
due to the improved specificity.
It is also worth noting that, when using a test system with 100% specificity, the effective power of the
survey will always be 100%, regardless of the figure used in the design. This is because it is not possible to
make a Type II error, and conclude that the farm is infected when it is not.
A check of the impact of population size on the calculated sample size is worthwhile. The calculated
sample size is based on an infinitely large population. If the population size is smaller, the impact on
sample size is shown in the following table:
Population size
Sample size
1000
157
2000
163
5000
166
10,000
169
Based on these calculations, it is clear that, for the population sizes under consideration, there is little
effect on the sample size. For the sake of simplicity, a standard sample size of 169 is used, regardless of
the number of grow-out fish on the farm.
Sampling
The selection of individual fish to include in the sample should be done in such a manner as to give the
best chance of the sample being representative of the study population. A fuller description of how this
may be achieved under different circumstances is provided in Survey Toolbox3. An example of a single farm
will be used to illustrate some of the issues.
One farm has a total of eight tanks, four of which are used for grow-out. At the time of the survey (during
winter), the four grow-out tanks have 1850, 4250, 4270 and 4880 fish, respectively, giving a total
population of 15,250 grow-out fish.
Simple random sampling from this entire population is likely to produce sample sizes from each tank
roughly in proportion to the number of fish in each tank. However, proportional stratified sampling will
guarantee that each tank is represented in proportion. This simply involves dividing the sample size
between tanks in proportion to their population. The first tank has 1850 fish out of a total of 15,250,
representing 12.13%. Therefore 12.13% of the sample (21 fish) should be taken from the first tank. Using
a similar approach the sample size for the other three tanks is 47, 47 and 54 fish, respectively.
3
Survey Toolbox for Aquatic Animal Diseases – A Practical Manual and Software Package. Cameron A.R. (2002). Australian
Centre for International Agricultural Research (ACIAR), Monograph No. 94, 375 pp. ISBN 1 86320 350 8. Printed version
available from ACIAR (http://www.aciar.gov.au) Electronic version available for free download from
http://www.ausvet.com.au.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
37
Appendix V cont.
Once the sample for each tank is determined, the problem remains as to how to select 21 fish from a tank
of 1850 so that they are representative of the population. Several options exist.
•
If the fish can be handled individually, random systematic sampling may be used. This is likely to be
the case if, for example:
•
fish are harvested during winter and samples can be collected at harvest; or
•
routine management activities involving handling the fish (such as grading or vaccination) are
conducted during the winter.
If fish are handled, systematic sampling simply involves selecting a fish at regular intervals. For
instance, to select 21 from 1850, the sampling interval should be 1850/21 = 88. This means that
every 88th fish from the tank should be sampled. To ensure randomness, it is good practice to use a
random number between 1 and 88 (in this case) to select the first fish (e.g. using a random number
table), and then select every 88th fish after that.
•
If fish cannot be handled individually (by far the most common, and more difficult, circumstance)
then the fish to be sampled must be captured from the tanks. Fish should be captured in the most
efficient and practical way possible, however every effort should be made to try to ensure that the
sample is representative. In this example, a dip net is the normal method used for capturing fish.
Using a dip net, convenience sampling would involve capturing 21 fish by repeatedly dipping at one
spot and capturing the easiest fish (perhaps the smaller ones). This approach is strongly discouraged.
One method of increasing the representativeness is to sample at different locations in the tank –
some at one end, some at either side, some at the other end, some in the middle, some close to the
edge. Additionally, if there are differences among the fish, an attempt should be made to capture fish
in such a way as to give different groups of fish a chance of being caught (i.e. do not just try to catch
the small ones, but include big ones as well).
This method of collecting a sample is far from the ideal of random sampling, but due to the practical
difficulties of implementing random sampling of individual fish, this approach is acceptable, as long
as the efforts made to increase the representativeness of the sample are both genuine and fully
documented.
Testing
Specimens are collected, processed and tested according to standardised procedures developed under the
accreditation programme and designed to meet the requirements of this Aquatic Manual. The testing
protocol dictates that any specimens that test positive to ELISA be submitted for culture, and that any
positive culture results indicate a true positive specimen (i.e. that the farm is not free from disease). It is
important that this protocol be adhered to exactly. If a positive culture is found, then it is not acceptable
to retest it, unless further testing is specified in the original testing protocol, and the impact of such testing
accounted for in the test system sensitivity and specificity estimates (and therefore the sample size).
Analysis
If the calculated sample size of 169 is used, and no positive reactors are found, then the survey will have a
confidence of 95%. This can be confirmed by analysing the results using the FreeCalc software mentioned
above (which reports a confidence level of 95.06%).
It may happen in some cases that the survey is not conducted exactly as planned, and the actual sample
size is less than the target sample size. However, the size of the farm may also be smaller. In these cases, it
is advisable to analyse the farm data on a farm-by-farm basis. For example, if only 165 specimens were
collected from a farm with only 2520 fish, the resulting confidence would still be 95%. If only 160 fish
38
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
were collected, the confidence is only 94.5%. If a rigid target of 95% confidence is used, then this survey
would fail to meet that target and more evidence would be required.
EXAMPLE 2 –
TWO-STAGE STRUCTURED SURVEY
(NATIONAL
FREEDOM)
Context
A country aims to declare freedom from Disease Y of crustaceans. The industry in this country is based
largely on small-holder ponds, grouped closely together in and around villages. The disease is reasonably
highly contagious, and causes mass mortality mid to late in the production cycle, with affected animals
becoming moribund and dying in a matter of days. Affected animals show few characteristic signs, but an
infected pond will almost invariably break down with mass mortality unless harvested beforehand. It is
more common in late summer, but can occur at any time of year. It also occurs occasionally early in the
production cycle. In this country, there are some limitations to the availability of laboratory facilities and
the transport infrastructure. However, there is a relatively large government structure, and a
comprehensive network of fisheries officers.
Objective
The objective is to establish national freedom from Disease Y. The surveillance system must meet the
requirements of Part 1 of this chapter, but must also be able to be practically implemented in this smallholder production system.
Approach
The aquaculture authorities decide to use a survey to gather evidence of freedom, using a two-stage survey
design (sampling villages at the first level, and ponds at the second). Laboratory testing of specimens from
a large number of farms is not considered feasible, so a combined test system is developed to minimise
the need for expensive laboratory tests.
The unit of observation and analysis is, in this case, the pond, rather than the individual animal. This
means that the diagnosis is being made at the pond level (an infected pond or a non-infected pond) rather
than at the animal level.
The survey is therefore a survey to demonstrate that no villages are infected (using a random sample of
villages and making a village-level diagnosis). The test used to make a village-level diagnosis is, in fact,
another survey, this time to demonstrate that no ponds in the village are affected. A test is then performed
at the pond level (farmer observation followed, if necessary, by further laboratory testing).
Survey standards
•
The confidence to be achieved by the survey is 95%. The power is set at 95% (but is likely to be
virtually 100% if the test system used achieves nearly 100% specificity, as demonstrated in the
previous example).
•
The target population is all ponds stocked with shrimp in the country during the study period. The
study population is the same, except that those remote areas to which access is not possible are
excluded. As outbreaks can occur at any time of year, and at any stage of the production cycle, it is
decided not to further refine the definition of the population to target a particular time or age.
•
Three tests are used. The first is farmer observation, to determine if mass mortality is occurring in a
particular pond. If a pond is positive to the first test (i.e. mass mortality is detected), a second test is
applied. The second test used is polymerase chain reaction (PCR). Cases positive to PCR are further
tested using transmission experiments.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
39
Appendix V cont.
•
Farmer observation can be treated as a test just like any other. In this case, the observation of
mass mortality is being used as a test for the presence of Disease Y. As there are a variety of
other diseases that are capable of causing mass mortality, the test is not very specific. On the
other hand, it is quite unusual for Disease Y to be present, and not result in mass mortality, so
the test is quite sensitive. A standard case definition is established for ‘mass mortality’ (for
instance, greater than 20% of the pond’s population of shrimp observed dead in the space of
less than 1 week). Based on this definition, farmers are able to ‘diagnose’ each pond as having
mass mortality. Some farmers may be over-sensitive and decide that mass mortality is occurring
when only a small proportion of shrimp are found dead (false positives, leading to a decrease in
specificity) while a small number of others fail to recognise the mortalities, decreasing sensitivity.
In order to quantify the sensitivity and specificity of farmer observation of mass mortalities, as a
test for Disease Y, a separate study is carried out. This involves both a retrospective study of the
number of mass mortality events in a population that is thought to be free from disease, as well
as a study of farmers presented with a series of mortality scenarios, to assess their ability to
accurately identify a pond with mass mortality. By combining these results, it is estimated that
the sensitivity of farmer-reported mass mortalities as a test for Disease Y is 87% while the
specificity is 68%.
•
When a farmer detects a pond with mass mortality, specimens are collected from moribund
shrimp following a prescribed protocol. Tissue samples from 20 shrimp are collected, and
pooled for PCR testing. In the laboratory, the ability of pooled PCR to identify a single infected
animal in a pool of 20 has been studied, and the sensitivity of the procedure is 98.6%. A similar
study of negative specimens has shown that positive results have occasionally occurred,
probably due to laboratory contamination, but maybe also because of the presence of non-viable
genetic material from another source (shrimp-based feed stuffs are suspected). The specificity is
therefore estimated at 99%.
•
Published studies in other countries have shown that the sensitivity of transmission tests, the
third type of test to be used, is 95%, partly due to variability in the load of the agent in
inoculated material. The specificity is agreed to be 100%.
•
Based on these figures, the combined test system sensitivity and specificity are calculated using
the formulae presented in Example 1, first with the first two tests, and then with the combined
effect of the first two tests and the third test. The result is a sensitivity of 81.5% and a specificity
of 100%.
•
The design prevalence must be calculated at two levels. First, the pond-level design prevalence (the
proportion of ponds in a village that would be infected if disease were present) is determined. In
neighbouring infected countries, experience has shown that ponds in close contact with each other
are quickly infected. It is unusual to observe an infected village with fewer than 20% of ponds
infected. Conservatively, a design prevalence of 5% is used. The second value for design prevalence
applies at the village level, or the proportion of infected villages that could be identified by the
survey. As it is conceivable that the infection may persist in a local area without rapid spread to other
parts of the country, a value of 1% is used. This is considered to be the lowest design prevalence
value for which a survey can be practically designed.
•
The population of villages in the country is 65,302, according to official government records. Those
with shrimp ponds number 12,890, based on records maintained by the aquaculture authorities.
These are generated through a five-yearly agricultural census, and updated annually based on reports
of fisheries officers. There are no records available of the number of ponds in each of these villages.
Sample size
Sample size is calculated for the two levels of sampling, first the number of villages to be sampled and
then the number of ponds to be sampled. The number of villages to be sampled depends on the
40
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
sensitivity and the specificity of the test used to classify villages as infected or not infected. As the ‘test’
used in each village is really just another survey, the sensitivity is equal to the confidence and the
specificity is equal to the power of the village-level survey. It is possible to adjust both confidence and
power by changing the sample size in the village survey (number of ponds examined), which means that
we can determine, within certain limits, what sensitivity and specificity we achieve.
This allows a flexible approach to sample size calculation. If a smaller first-stage sample size is desired (a
small number of villages), a high sensitivity and specificity are needed, which means that the number of
ponds in each village that need to be examined is larger. A smaller number of ponds will result in lower
sensitivity and specificity, requiring a larger number of villages. The approach to determining the optimal
(least cost) combination of first- and second-stage sample sizes is described in Survey Toolbox.
A further complication is presented by the fact that each village has a different number of ponds. In order
to achieve the same (or similar) confidence and power (sensitivity and specificity) for each village, a
different sample size may be required. The authorities choose to produce a table of sample sizes for the
number of ponds to sample in each village, based on the total ponds in each village.
An example of one possible approach to determining the sample size follows:
The target sensitivity (confidence) achieved by each village-level survey is 95%. The target specificity is
100%. Using the FreeCalc software, with a design prevalence of 1% (the survey is able to detect disease if
1% or more villages are infected), the first-stage sample size is calculated as 314 villages. Within each
village, the test used is the combined test system described above with a sensitivity of 81.5% and a
specificity of 100%. Based on these figures the following table is developed, listing the number of ponds
that need to be sampled in order to achieve 95% sensitivity.
Population
Sample size
30
29
40
39
60
47
80
52
100
55
120
57
140
59
160
61
180
62
200
63
220
64
240
64
260
65
280
65
300
66
320
66
340
67
360
67
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
41
Appendix V cont.
380
67
400
67
420
68
440
68
460
68
480
68
500
68
1000
70
Sampling
First-stage sampling (selection of villages) is done using random numbers and a sampling frame based on
the fisheries authorities list of villages with shrimp ponds. The villages are listed on a spreadsheet with
each village numbered from 1 to 12,890. A random number table (such as that included in Survey Toolbox)
or software designed for the generation of random numbers (such as EpiCalc4) is used.
The second stage of sampling involves random selection of ponds within each village. This requires a
sampling frame, or list of each pond in the village. The fisheries authorities use trained local fisheries
officers to coordinate the survey. For each selected village, the officer visits the village and convenes a
meeting of all shrimp farmers. At the meeting, they are asked how many ponds they have and a list of
farmers’ names and the number of ponds is compiled. A simple random sample of the appropriate
number of ponds (between 29 and 70, from the table above, depending on the number of ponds in the
village) is selected from this list. This is done either using software (such as Survey Toolbox’s
RandomAnimal program), or manually with a random number table or decimal dice for random number
selection. Details of this process are described in Survey Toolbox. This selection process identifies a
particular pond in terms of the name of the owner, and the sequence number amongst the ponds owned
(e.g. Mr Smith’s 3rd pond). Identification of the actual pond is based on the owners own numbering
system for the ponds.
Testing
Once ponds have been identified, the actual survey consists of ‘testing those ponds’. In practice, this
involves the farmers observing the ponds during one complete production cycle. The local fisheries
officer makes weekly visits to each farmer to check if any of the selected ponds have suffered mass
mortality. If any are observed (i.e. the first test is positive), 20 moribund shrimp are collected for
laboratory examination (first PCR, and then, if positive, transmission experiments).
Analysis
Analysis is performed in two stages. First, the results from each village are analysed to ensure that they
meet the required level of confidence. If the target sample size is achieved (and only negative results
obtained), the confidence should be 95% or greater in each village. At the second stage, the results from
each village are analysed to provide a country level of confidence. Again, if the target sample size (number
of villages) is achieved, this should exceed 95%.
4
42
http://www.myatt.demon.co.uk/epicalc.htm
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
EXAMPLE 3 –
SPATIAL SAMPLING AND THE USE OF TESTS WITH IMPERFECT SPECIFICITY
Context
A country has an oyster culture industry, based primarily on rack culture of oysters in 23 estuaries
distributed along the coastline. In similar regions in other countries, Disease Z causes mortalities in late
summer/early autumn. During an outbreak a high proportion of oysters are affected, however, it is
suspected that the agent may be present at relatively low prevalence in the absence of disease outbreaks.
Objective
The national authorities wish to demonstrate national freedom from Disease Z. If the disease should be
detected, a secondary objective of the survey is to collect adequate evidence to support zoning at the
estuary level.
Approach
The authorities conclude that clinical surveillance for disease outbreaks is inadequate because of the
possibility of low level subclinical infections. It is therefore decided to base surveillance on a structured
two-stage survey, in which sampled oysters are subjected to laboratory testing. The first stage of the
survey is the selection of estuaries. However, due to the objective of providing evidence for zoning
(should disease be found in any of the estuaries), it is decided to use a census approach and sample every
estuary. In essence this means that there will be 23 separate surveys, one for each estuary. A range of
options for sampling oysters are considered, including sampling at harvest or marketing, or using farms
(oyster leases) as a level of sampling or stratification. However the peak time of activity of the agent does
not correspond to the harvest period, and the use of farms would exclude the significant numbers of wild
oysters present in the estuaries. It is therefore decided to attempt to simulate simple random sampling
from the entire oyster population in the estuary, using a spatial sampling approach.
Survey standards
•
The target population is all of the oysters in each of the estuaries. The study population is the oysters
present during the peak disease-risk period in late summer early autumn. Wild and cultured oysters
are both susceptible to disease, and may have associated with them different (but unknown) risks of
infection. They are therefore both included in the study population. As will be described below,
sampling is based on mapping. Therefore the study population can more accurately be described as
that population falling within those mapped areas identified as oyster habitats.
•
A design prevalence value is only required at the oyster level (as a census is being used at the estuary
level). While the disease is often recognised with very high prevalence during outbreaks, a low value
is used to account for the possibility of persistence of the agent in the absence of clinical signs. A
value of 2% is selected.
•
The test used is histopathology with immuno-staining techniques. This test is known to produce
occasional false-positive results due to nonspecific staining, but is very sensitive. Published studies
indicate values of 99.1% for sensitivity and 98.2% for specificity. No other practical tests are
available. This means that it is not possible to definitively differentiate false positives from true
positives, and that in a survey of any size, a few false positives are expected (i.e. 1.8%).
•
The confidence is set at 95% and the power at 80%. In the previous examples, due to the assumed
100% specificity achieved by use of multiple tests, the effective power was 100%. In this case, with
imperfect specificity, there will be a risk of falsely concluding that a healthy estuary is infected, so the
power is not 100%. The choice of a relatively low figure (80%) means that there is a 1 in 5 chance of
falsely calling an estuary infected when it is not infected, but it also dramatically decreases the survey
costs, through a lower sample size.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
43
Appendix V cont.
Sample size
Based on the assumption that the sampling procedure will mimic simple random sampling, the sample size
(number of oysters to sample per estuary) can be calculated with FreeCalc. The population size (number of
oysters per estuary) is assumed to be very large. The calculated sample size, using the sensitivity, specificity
and design prevalence figures given above, is 450. FreeCalc also reports that, based on this sample size and
the specificity of the test, it is possible to get 10 or fewer false-positive test results, and still conclude that
the population is free from disease. This is because, if the population were infected at 2% or greater, the
anticipated number of positive reactors from a sample of 450 would be greater than 10. In fact, we would
expect 9 true positives (450 × 2% × 99.1%) and 8 false positives (450 × 98% × 1.8%) or a total of
17 positives if the population were infected at a prevalence of 2%.
This illustrates how probability theory and adequate sample size can help differentiate between true- and
false-positive results when there is no alternative but to use a test with imperfect specificity.
Sampling
The aim is to collect a sample of 450 oysters that represent an entire estuary. Simple random sampling
depends on creating a sampling frame listing every oyster (not possible) and systematic sampling depends
on being able to (at least conceptually) line up all the oysters (again, not possible). The authorities decide
to use spatial sampling to approximate simple random sampling. Spatial sampling involves selecting
random points (defined by coordinates), and then selecting oysters near the selected points. In order to
avoid selecting many points with no oysters nearby, the estuary is first mapped (the fisheries authorities
already have digital maps defining oyster leases available). To these maps areas with significant
concentrations of wild oysters are also added, based on local expertise. Pairs of random numbers are
generated such that the defined point falls within the defined oyster areas. Other schemes are considered
(including using a rope marked at regular intervals, laid out on a lease to define a transect, and collecting
an oyster adjacent to each mark on the rope) but the random coordinate approach is adopted.
Survey teams then visit each point by boat (using a GPS [Global Positioning System] unit to pinpoint the
location). A range of approaches is available for selecting which oyster to select from a densely populated
area, but it should involve some effort at randomness. Survey staff opt for a simple approach: when the
GPS receiver indicates that the site has been reached, a pebble is tossed in the air and the oyster closest to
the point where it lands is selected. Where oysters are arranged vertically (e.g. wild oysters growing up a
post), a systematic approach is used to determine the depth of the oyster to select. First, an oyster at the
surface, next, an oyster halfway down, and thirdly, an oyster as deep as can be reached from the boat.
This approach runs the risk of bias towards lightly populated areas, so an estimate of the relative density
of oysters at each sampling point is used to weight the results (see Survey Toolbox for more details).
Testing
Specimens are collected, processed, and analysed following a standardised procedure. The results are
classified as definitively positive (showing strong staining in a highly characteristic pattern, possibly with
associated signs of tissue damage), probably positive (on the balance of probabilities, but less characteristic
staining), and negative.
Analysis
The interpretation of the results when using a test with imperfect specificity is based on the assumption
that, in order to conclude that the population is free from infection, any positive result identified is really a
false positive. With a sample size of 450, up to 10 false positives may be expected while still concluding
that the population is free from disease. However, if there is reasonable evidence that there is even a single
true positive, then the population cannot be considered free. This is the reason for the classification of
positive results into definitive and probable positives. If there are any definitive positives at all, the
population in that estuary must be considered infected. The probable positives are consistent with false
44
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix V cont.
positives, and therefore up to 10 may be accepted. Using FreeCalc the actual confidence achieved based on
the number of (presumed) false positives detected can be calculated. For instance, if 8 ‘probably positive’
results were detected from an estuary, the confidence level for the survey would be 98.76%. On the other
hand, if 15 ‘probably positive’ results were detected, the confidence is only 61.9%, indicating that the
estuary is likely to be infected.
Discussion
Normally, it may be safely assumed that a surveillance system aimed at demonstrating freedom from
disease is 100% specific. This is because any suspected occurrence of disease is investigated until a
definitive decision can be made. If the conclusion is that the case is truly a case of disease, then there is no
issue of declaring freedom – the disease is known to be present. This example presents a different
situation where, due to lack of suitable tests, it is not possible for the surveillance system to be 100%
specific. This may represent an unusual situation in practice, but illustrates that methods exist for dealing
with this sort of problem. In practice, a conclusion that a country (or estuary) is free from infection, in the
face of a small (but statistically acceptable) number of positive results, will usually be backed up by further
evidence (such as the absence of clinical disease).
*
* *
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
45
Appendix VI
SECTION
2.1.
DISEASES OF FISH
CHAPTER
I.1.
GENERAL INFORMATION
[A. GENERAL BASIS FOR FISH HEALTH SURVEILLANCE/CONTROL PROGRAMMES]
A. OVERALL APPROACH TO FISH HEALTH MANAGEMENT
[1. TARGET PATHOGENS AND DISEASES
Target pathogens and fish diseases are included in the Aquatic Animal Health Code (the Aquatic Code) according to the
following basic considerations: they resist or respond poorly to therapy, have a restricted geographical range, are of high
socio-economic importance, and occur in species involved in international trade. For the current OIE list of diseases of fish,
please consult the current edition of the Aquatic Code. This Aquatic Manual includes chapters on diseases listed in the
Aquatic Code and chapters on certain other diseases of importance to trade.
2. OVERALL APPROACH FOR ANIMAL HEALTH CONTROL IN FISH CULTURE]
A comprehensive approach for animal health control in fish culture requires:
•
Assessment of the health status of fish using methods based on the provision in chapter 1.1.4.
•
The constraint of restocking open waters and farming facilities only with fish having a health status
higher than or equal to that of fish already living in the considered areas.
•
Eradication of disease when possible, by slaughtering infected stocks, disinfecting facilities and
restocking with fish from approved disease-free sources.
•
Notification by every Member Country of its particular requirements, besides those provided by the
Aquatic Code, for importation of aquatic animals and aquatic animal products.
If the above procedures are followed, it becomes possible to give adequate assurance of the health status
of aquaculture products for specified diseases, according to their country, zone or compartment.
The issue of a health certificate by the appropriate official, based on a health status report and
examinations of aquatic animals, provides assurance that the aquaculture products in a defined
consignment originate from a whole country, a zone or a farm/harvesting site free of one or more of the
specified diseases listed in the Aquatic Code and possibly of other specified diseases (see model of
international certificate in the Aquatic Code).
The assessment of the health status of fish stocks is based on inspection of fish production sites and
further laboratory examination of samples originating from fish specimens taken among the stock of a
defined fish population. This endeavour requires the fish sample to be collected according to defined
sampling size charts and the samples to be processed according to accepted methods.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
47
Appendix VI cont.
Several techniques are applicable for fish pathogens. For screening and diagnostic purposes, the Aquatic
Manual has established two types of examination procedures that will be acceptable for such work;
1) Screening methods, and 2) Diagnostic methods. The accepted methods are listed under each disease
chapter.
B. TARGET PATHOGENS AND DISEASES
Target pathogens and fish diseases are included in the Aquatic Animal Health Code (the Aquatic Code)
according to the following basic considerations: they resist or respond poorly to therapy, have a restricted
geographical range, are of high socio-economic importance, and occur in species involved in international
trade. For the current OIE list of diseases of fish, please consult the current edition of the Aquatic Code.
This Aquatic Manual includes chapters on diseases listed in the Aquatic Code and chapters on certain other
diseases of importance to trade.
Surveillance can also be developed for non-listed diseases especially for emerging diseases.
It would be impractical to try to develop a surveillance system, for all the known aquatic animal diseases in
a country. Therefore prioritising the diseases to be included in a surveillance system should be conducted
considering:
•
the needs to provide assurance of disease status for trade purposes,
•
the resources of the country,
•
the risk posed by the different diseases,
The concept of risk encompasses both the probability of the disease occurring and the severity of its
consequences.
C. SAMPLING [PROCEDURES]
1.
SAMPLING
OBJECTIVES
There are at least three purposes for which fish stocks may be sampled. These are: 1) surveillance;
2) issuing health certificates and 3) disease outbreak investigation. The number and type of samples to
be taken for analysis and the sampling procedure varies greatly according to which of these purposes
applies.
2.
NUMBER
OF EPIDEMIOLOGICAL UNITS TO BE SAMPLED
Sample sizes for surveillance or for issuing health certificates should be calculated using the provisions
in chapter 1.1.4 of this Aquatic Manual.
When conducting disease outbreak investigations, the number of cases and non-cases will depend on
the case definition to be found in the appropriate chapter of the Aquatic Manual. If a case definition is
not available, a case definition describing the disease of interest as accurately as possible should be
developed and the number and status of units to be sampled should be determined in accordance with
that case definition.
Fish collection should encompass a statistically significant number of specimens, but it is obvious that
failure to detect certain pathogens from the sample does not guarantee the absence of these agents in
the specimen examined or in the stock. This is particularly true of free-ranging or feral stocks from
which it is difficult to collect a representative and random sample. However, the risk of a pathogen
escaping the surveillance system is reduced in fish farms whose fish stocks have been inspected and
checked for pathogens for several years (at least 2), insofar as they are not exposed to possible
recontamination by feral fish.
48
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VI cont.
3.
SAMPLING
PROTOCOL
3.1. Selection of epidemiological units and sampling methods
Fish sampled should be alive when collected.
During disease outbreak investigations, if any moribund fish are present in the fish population to
be sampled, they should be selected first for sample collection and the remainder of the sample
should comprise randomly selected live fish from all rearing units that represent the lot being
examined.
Add information on different methods to be used for sampling and discuss potential bias of each
method
When a given fish production site harbours a salmonid broodstock, for testing for Renibacterium
salmoninarum and some of the viral disease agents, it is preferable for one of the sample collections
made each to be focused on the sexual products (sperm and ovarian fluid) released by broodfish
at the time of spawning. If an adult broodstock includes fish of different ages, the older fish
should be selected for sampling.
3.2. Post-sampling procedures
As in the case of clinically infected fish, organ and fluid samples should be taken and processed as
soon as possible after fish specimen collection. Sample freezing should be avoided.
Fish samples should be sent to the laboratory alive or killed and packed separately in sealed
aseptic refrigerated containers or on ice. The freezing of collected fish should be strictly avoided.
However, it is highly preferable and recommended to collect organ samples from the fish
immediately after they have been selected at the fish production site and to store and process the
samples as described in Sections 2 and 3. An identification label that includes information on the
place, time, date, species, number of samples collected, dead/moribund state on collection, and
the name and contact information of the individual collecting the sample(s) should be attached to
the sample(s).
[1. COLLECTION OF FISH SPECIMENS
1.1.
Diagnosis in a disease situation
A minimum number of ten moribund fish or ten fish exhibiting clinical signs of the diseases in question should be
collected: fish should be alive when collected, and should be sent to the laboratory alive or killed and packed
separately in sealed aseptic refrigerated containers or on ice. The freezing of collected fish should be strictly avoided.
However, it is highly preferable and recommended to collect organ samples from the fish immediately after they have
been selected at the fish production site and to store and process the samples as described in Sections 2 and 3. An
identification label that includes information on the place, time, date, species, number of samples collected,
dead/moribund state on collection, and the name and contact information of the individual collecting the sample(s)
should be attached to the sample(s).
1.2.
Fish appear to be clinically normal
Fish collection should encompass a statistically significant number of specimens, but it is obvious that failure to detect
certain pathogens from the sample does not guarantee the absence of these agents in the specimen examined or in
the stock. This is particularly true of free-ranging or feral stocks from which it is difficult to collect a representative and
random sample. However, the risk of a pathogen escaping the surveillance system is reduced in fish farms whose fish
stocks have been inspected and checked for pathogens for several years (at least 2), insofar as they are not exposed
to possible recontamination by feral fish.
When a given fish production site harbours a salmonid broodstock, for testing for Renibacterium salmoninarum and
some of the viral disease agents, it is preferable for one of the sample collections made each year to be focused on
the sexual products (sperm and ovarian fluid) released by broodfish at the time of spawning (see below). If an adult
broodstock includes fish of different ages, the older fish should be selected for sampling:
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
49
Appendix VI cont.
•
Samples should comprise all susceptible species on the site (see relevant chapters of this Aquatic Manual for
the list of species susceptible to each disease), with each lot of a species being represented in the sample
group. A lot is defined as a group of the same fish species that shares a common water supply and that
originates from the same broodfish or spawning population.
The geographical origin of samples should be defined by the name of the sampling site associated with either its
geographical co-ordinates or its location along a river course or body of water.
1.3.
•
If any moribund fish are present in the fish population to be sampled, they should be selected first for sample
collection and the remainder of the sample should comprise randomly selected live fish from all rearing units
that represent the lot being examined.
•
A general approach to surveillance and sampling is given in chapter 1.1.4 of this Aquatic Manual. The sampling
should be designed in order to enable detection, at a 95% confidence level, of infected animals. The following
section gives information relevant to sampling finfish. Until disease-specific details are included in the individual
disease chapters in this Aquatic Manual, Table 1 can be used to calculate sample size.
•
As in the case of clinically infected fish, organ and fluid samples should be taken and processed as soon as
possible after fish specimen collection. Sample freezing should be avoided.
Sampling specifications according to the objectives of a given fish surveillance programme
A general approach to surveillance and sampling is given in chapter 1.1.4 of this Aquatic Manual. The sampling
should be designed in order to enable detection, at a 95% confidence level, of infected animals. The following section
gives information relevant to sampling finfish. For those diagnostic tests where the sensitivity and specificity have
been established, sample size may be determined using methods such as FreeCalc (www.ausvet.com.au) or similar
programs as outlined in Chapter 1.1.4 Requirements for surveillance for international recognition of freedom from
infection. However, for those diagnostic tests where the sensitivity and specificity have not been established, the
default sample size should be determined from Table 1 in the present chapter.
Table 1. Random sample size based on assumed pathogen prevalence in lot
and assuming 100% sensitivity and specificity of the technique
Lot size
At 2% prevalence,
size of sample
At 5% prevalence,
size of sample
At 10% prevalence,
size of sample
50
50
35
20
100
75
45
23
250
110
50
25
500
130
55
26
1000
140
55
27
1500
140
55
27
2000
145
60
27
4000
145
60
27
10,000
145
60
27
100,000 or more
150
60
30
After Ossiander & Wedemeyer, 1973.
a) Achievement of the health status of a fish stock/population at a given inspection site
•
A fish culture unit should be inspected twice a year for 2 years at the appropriate life stage of the fish and at
times of the year when temperature and season offer the best opportunity for observing clinical signs and
isolating pathogens. On each occasion, fish of the susceptible species listed in the Aquatic Code for the
disease in question should be collected in order to detect a prevalence of infection equal to or higher than
2% at 95% confidence level. Most often, 150 fish will thus be collected on each occasion. If broodfish are
present during one of the two inspections, up to 150 ovarian fluid samples will be taken from broodfish in the
given fish culture unit.
•
If fish health surveillance is focused on wild fish populations at a given site of inspection or on rearing ponds
without holding facilities in which different fish crops may be pooled, 150 fish specimens should be collected
once a year for 2 years. Insofar as it is possible, specimens of the oldest fish and/or salmonid ovarian fluid
should be collected as a priority.
•
During this 2-year period, the fish production unit may only receive fish from a unit whose health status has
already been approved and is equal to or higher than the health status sought for the facility being
inspected.
b) Maintenance of the health status
50
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VI cont.
•
Once a fish production unit, including pond fish production units equipped with holding facilities, has been
recognised to be free from all or certain diseases listed in the Aquatic Code after 2 years of surveillance
with laboratory tests and in the absence of any suspect clinical signs, twice-yearly inspections should
continue. An aquaculture establishment that has been recognised free from infection may maintain its
official status as infection-free provided that the basic biosecurity conditions are continuously maintained.
An aquaculture establishment that has been recognised free from infection may discontinue targeted
surveillance and maintain its official status as infection-free provided that the basic biosecurity conditions
are continuously maintained.
•
The fish production unit may only receive fish having a health status higher than or equal to that of those
already present.
The above sampling specifications for the achievement and maintenance of the health status of fish at given
fish production sites imply that all provisions given in Section A.2 (overall approach for animal health control in
fish culture) are in force.]
4.
ASSESSING
THE HEALTH STATUS OF THE EPIDEMIOLOGICAL UNIT
4.1. Sample material to be used in viral and bacteriological tests
Sample material depends both on the size of animals and the objective of testing, i.e. diagnosis of
overt disease or detection of fish that are subclinical pathogen carriers.
4.2. Specifications according to fish size
Alevin and yolk sac fry: sample the entire fish but remove the yolk sac if present.
Fish 4 to 6 cm: take the entire viscera including the kidney. A piece of encephalon can be
obtained after severing the head at the level of the rear edge of the operculum and pressing it
laterally.
Fish over 6 cm: take the kidney, spleen, and heart or encephalon.
Broodfish: take the ovarian fluid and/or tissues.
4.2. Specifications according to clinical status
In the case of clinical infection, besides whole fry or entire viscera, organs to be sampled are
anterior kidney, spleen and heart or encephalon for virus tests, kidney and spleen for bacterial
tests, and skin or muscle when sampling for tests for epizootic ulcerative syndrome. Samples from
ten diseased fish will thus be taken and combined to form pools of a maximum of five fish each.
For detecting subclinical carriers, samples may be combined as pools of no more than five
fish/pool. Pools of ovarian fluid from five broodfish should not exceed a total volume of 5 ml,
i.e. 1 ml/broodfish. These ovarian fluid samples are to be taken individually from every sampled
female and not collected following the pooling of ova.
Once aseptically removed from fish, the organs and/or ovarian fluid sampled are each split into
two parts if both bacteriological and virological examinations are to be done.
5.
GENERAL PROCESSING
OF
ORGANS/FLUID SAMPLES
FOR
VIROLOGICAL EXAMINATION
5.1. Transportation and antibiotic treatment of samples
Pools of organs or of ovarian fluids are placed in sterile vials and stored at 4°C until virus
extraction is performed at the laboratory. Virus extraction should optimally be carried out within
24 hours after fish sampling, but is still acceptable for up to 48 hours.
Organ samples may also be transported to the laboratory by placing them in vials containing cell
culture medium or Hanks’ balanced salt solution (HBSS) with added antibiotics to suppress the
growth of bacterial contaminants (one volume of organ in at least five volumes of transportation
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
51
Appendix VI cont.
fluid). Suitable antibiotic concentrations are: gentamycin (1000 µg/ml) or penicillin
(800 International Units [IU]/ml) and streptomycin (800 µg/ml). The antifungal compounds
Mycostatin® or Fungizone® may also be incorporated into the transport medium at a final
concentration of 400 IU/ml. Serum or albumen (5–10%) may be added to stabilise the virus if the
transport time will exceed 12 hours.
5.2. Virus extraction
•
This procedure is conducted below 15°C and preferably at between 0 and 5°C.
•
Decant antibiotic-supplemented medium from organ sample.
•
Homogenise organ pools in transport medium at a final dilution of 1/10 using a mortar and
pestle or electric homogeniser until a paste is obtained.
•
Centrifuge the homogenate in a refrigerated centrifuge at 2–5°C at 2000 to 4000 g for
15 minutes, collect the supernatant and treat for either four hours at 15°C or overnight at
4°C with antibiotics, e.g. gentamicin 1 mg/ml. If shipment of the sample has been made in a
transport medium (i.e. with exposure to antibiotics) the treatment of the supernatant with
antibiotics may be omitted. The antibiotic treatment makes filtration through membrane
filters unnecessary.
•
Likewise, ovarian fluid samples may be treated with antibiotics to control microbial
contamination. In neither case can homogenates or ovarian fluid samples be diluted more
than twofold.
•
Ovarian fluid samples should be centrifuged in the same way as organ homogenates, and
their supernatants used directly in subsequent steps.
5.3. Treatment to neutralise enzootic viruses
In some countries, fish are often subclinical carriers of enzootic viruses, such as infectious
pancreatic necrosis virus (IPNV), which induce a CPE in susceptible cell cultures and thus
complicate isolation and identification of target pathogens. In such situations, the infectivity of
the enzootic viruses should be neutralised before testing for the viruses listed in the Aquatic Code.
However, when it is important to determine whether one of the enzootic viruses is present,
samples should be tested with and without the presence of neutralising antibodies (NAbs).
To neutralise aquatic birnaviruses, mix equal volumes (200 µl) of a solution of NAbs against the
indigenous birnavirus serotypes with the supernatant to be tested. Allow the mixture to react for
1 hour at 15°C or overnight at 4°C prior to inoculation on to susceptible cell monolayers. The
titre of the NAb solution used (it may be a multivalent serum) should be at least 2000 in a 50%
plaque reduction test versus the viral serotypes present in the given geographical area.
When samples are from a country, region, fish population or production unit considered to be
free from enzootic viral infections, this treatment of the organ homogenate should be omitted.
This approach can also be used to neutralise other viruses enzootic to the area being tested.
6.
GENERAL PROCESSING
OF
SAMPLES INTENDED
FOR
BACTERIOLOGICAL EXAMINATION
As in viral infections, internal organs may be used as a source of isolation whenever systemic infection
is suspected. However, active proliferation of saprophytic microorganisms is such a disadvantage that
freshly taken tissue is used for bacteriological examination. The fact that no antibiotic substances may
be added to the transport medium in which the samples are collected reinforces this preference.
52
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VI cont.
D. MATERIALS AND BIOLOGICAL PRODUCTS REQUIRED FOR THE
ISOLATION AND IDENTIFICATION OF FISH PATHOGENS
1.
FISH VIRUSES
1.1. Fish cell lines
The following four fish cell lines will be required to test for the fish pathogens mentioned in the
Aquatic Code:
Bluegill fry (BF-2)
Chinook salmon embryo (CHSE-214)
Epithelioma papulosum cyprini (EPC)
Rainbow trout gonad (RTG-2)
Fathead minnow (FHM)
Grunt fin (GF)
Salmon head kidney (SHK1)
Atlantic salmon kidney (ASK)
1.2. Culture media
Traditional Eagle’s minimal essential medium (MEM) with Earle’s salt supplemented with 10%
fetal bovine serum (FBS), antibiotics and 2 mM L-glutamine is the most widely used medium for
fish cell culture.
Stoker’s medium, however, which is a modified form of the above medium comprising a doublestrength concentration of certain amino acids and vitamins, is particularly recommended to
enhance cell growth, using the same supplementations as above + 10% tryptose phosphate.
These media are buffered with either sodium bicarbonate, 0.16 M tris-hydroxymethyl
aminomethane (Tris) HCl, or, preferably, 0.02 M N-2-hydroxyethyl-piperazine-N-2ethanesulfonic acid (HEPES). The use of sodium bicarbonate alone is restricted to those cell
cultures made in tightly closed cell culture vessels.
Alternatively, Leibovitz medium (L15) supplemented with FBS (5% or 10%), L-glutamine (4 mM)
and gentamicin (50 µg/ml) can be used for some cell lines e.g. SHK1.
For cell growth, the FBS content of the medium is usually 10%, whereas for virus isolation or
virus production it may be reduced to 2%. Similarly, the pH of the culture medium for cell growth
is 7.3–7.4 and is adjusted to 7.6 for virus production or virus assay.
The composition of the most frequently used antibiotic mixture is penicillin (100 IU/ml) and
dihydrostreptomycin (100 µg/ml). Mycostatin (50 IU/ml) may be used if fungal contamination is
expected. Other antibiotics or antibiotic concentrations may be used as convenient for the
operator depending on the antibiotic sensitivity of the bacterial strains encountered.
1.3. Virus positive controls and antigen preparation
a) Virus nomenclature
•
Epizootic haematopoietic necrosis virus (EHNV)
Infectious haematopoietic necrosis virus (IHNV)
•
Spring viraemia of carp virus (SVCV)
•
Viral haemorrhagic septicaemia virus (VHSV) (synonym Egtved virus)
•
Infectious salmon anaemia virus (ISAV)
•
Red sea bream iridovirus (RSIV)
•
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
53
Appendix VI cont.
•
Infectious pancreatic necrosis virus (IPNV)
b) Virus production
For the production of most of these viruses, the susceptible cell cultures (see relevant sections
in this Aquatic Manual) should be inoculated with fairly low multiplicities of infection (m.o.i.),
i.e. 10–2 to 10–3 plaque-forming units (PFU) per cell.
The optimal temperatures for virus propagation are:
•
15°C for IHNV, VHSV, ISAV and IPNV
•
20°C for SVCV
•
22°C for EHNV
•
25°C for RSIV
c) Virus preservation and storage
•
Centrifuge infected cell cultures at 2–5°C and 2000–4000 g for 15 minutes then dilute the
virus-containing supernatants in order to obtain virus titres averaging 1–2 × 106 PFU/ml.
•
Dispense the resulting viral suspensions into sterile vials at volumes of 0.3–0.5 ml each.
•
Freeze and store each series of standard virus stocks at –80°C or liquid nitrogen, and
check the titre of each virus stock at regular intervals if it has not been used during that
time period.
Lyophilisation: long-term storage (decades) of the seeds of standard virus strains is achievable
by lyophilisation. For this purpose, viral suspensions in cell culture medium supplemented
with 10% FCS are mixed (v/v) with an equal volume of cryopreservative medium (such as
20% lactalbumine hydrolysate in distilled water) before processing. Seal or plug under vacuum
and store at 4°C, in the dark.
2.
FISH BACTERIA
2.1. Culture media
Few species of fish pathogenic bacteria require special media for cultivation, and most of the
commonly used isolation peptones (trypsin-soya, brain–heart, etc.) can conveniently be employed.
However, the low optimal temperatures of some species result in slow growth, and small colonies
are frequently obtained during isolation. In these cases, it is usual to add enrichment factors, such
as serum or blood at 5–10%, in order to improve cultivation. Conversely Renibacterium
salmoninarum is a very fastidious organism and requires special media enriched with cysteine
(No. A11B).
Bacteriology of fish is generally conducted at temperatures between 20 and 26°C. It is sometimes
necessary or useful to have access to several incubators. Renibacterium salmoninarum, Flavobacterium
psychrophilus and others need 15°C for optimal growth and many of the bacteria isolated from
warm water fish may be incubated at 30°C to accelerate the diagnostic steps.
54
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VI cont.
2.2. Storage of cultures
Bacterial strains can be stored for a short term on ordinary media, placing the slants or broths at
4°C. For most strains, the use of commercial media or agar slants with mineral oil overlay will
extend viability to 1–2 years under the same conditions without further special requirements.
Freezing is probably the best way to preserve bacterial suspensions of high titre. However, it does
not always prevent some phenotype characteristics from changing. When stability of
characteristics such as virulence is important, it may be better to use lyophilisation, although the
number of viable bacteria may be decreased dramatically.
Different kinds of supports have been proposed to improve the efficacy of freezing and
lyophilisation, namely glycerol (5–15%) in the former case, and skim milk, lactose, and dextran
(5–10%) in the latter. There is no general rule, and convenient conditions have to be determined
in prior trials for all species. The addition to fresh cultures of one volume of support containing
Bactopeptone 11% + Dextran 4% has provided excellent results for lyophilisation of certain fish
bacteria, but other formulae would be worth testing in many cases.
3.
SEROLOGY
3.1. Production of rabbit antisera and polyclonal antibodies to fish viruses
There are various ways in which antibodies against fish viruses can be raised in rabbits. Titre and
specificity are influenced, however, by the inoculation programme used. The following
immunisation protocols may be used to produce antisera for use in the virus isolation and/or
identification procedures described later.
a) Antisera to infectious pancreatic necrosis virus
Intravenous injection with 50–100 µg of purified virus on day 0, followed by an identical
booster on day 21, and bleeding 5–7 days later. Rabbits may be reused if not bled completely.
b) Antisera to other viruses
The immunisation protocols alternate an intramuscular or intra-dermal injection with further
intravenous boosters:
Day 0: primary injection, 500–1000 µg of purified virus is mixed (v/v) with adjuvant (Freund’s
incomplete or other5) giving a total volume of 1.2 ml. This antigen is delivered to the rabbit as
multipoint intradermal injections (20 points on each side) after the animal has been shaved.
Day 21: collect about 20 ml of blood and check for reactivity (neutralisation, fluorescence);
boost intravenously with the same amount of purified virus as in the primary injection, but
without adjuvant.
Prior to the intravenous booster injection, the rabbit should be treated with promothazine
(12 mg intramuscularly) to prevent possible anaphylactic response.
Day 28: sample the blood, check the serum reactivity and bleed or boost according to the
results.
For rhabdoviruses, this immunisation procedure is well suited to production of antisera to be
used in immunofluorescence and enzyme-linked immunosorbent assay. However, a more
5
Use of Freund’s complete adjuvants may be restricted on animal welfare grounds. Alternative synthetic
adjuvants include trehalose dimycolate and monophosphate lipid A.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
55
Appendix VI cont.
efficient method for production of neutralising antisera is regular intravenous injection
without adjuvant (0.2 ml) every 3-4 days (twice a week). As many as 15 injections may be
necessary; 1 week after the last injection, a serum sample should be collected and tested.
3.2. Antisera to fish bacteria
It is still difficult to obtain antimicrobial sera in large amounts from commercial sources, and it
will often be necessary to prepare such antisera. The general methods are the same as for viruses.
Bacterial antigens are often used as crude preparations killed by heating or by formalin (0.35%
formalin). Rabbit injection at increasing doses can be either intramuscular, each week, with
adjuvant the first time, or intravenous at 3–4-day intervals without adjuvant. A booster injection is
often required after 15 days.
A special multipoint intradermal schedule has proven very efficient for anti-Renibacterium sera
production, and is also valuable for other bacteria of low antigenicity in rabbits.
The antigen is heat killed (60°C, 45 minutes) and adjusted to 2 mg/ml. The flanks of the animals
are thoroughly shaved, and multipoint intradermal injections are performed using total amounts
of 1 mg bacteria/animal, according to the following schedule:
Day 1
1 mg + complete Freund’s adjuvant (CFA) (v/v)
Day 21 1 mg + incomplete Freund’s adjuvant (IFA) (v/v)
Day 42 1 mg + IFA
Day 63 1 mg antigen
Day 68 Collecting of blood samples (about 30 ml)
Withdrawal injections and bleeding for serum collection may be repeated at 1-month intervals.
3.3. Processing and storage of immune sera
After blood clotting, collect and centrifuge the serum at 20°C and heat it for 30 minutes at 56°C.
Filter the resulting heat-inactivated serum through a membrane filter (450 nm pore size) and
temporarily store it at 4°C for the time necessary for the screening of its reactivity and specificity
and for checking that these properties are not affected by preservation conditions (e.g. freezing or
lyophilisation). Sterile rabbit sera can be kept for at least 2 months at 4°C without any change in
their properties. Dispense (usually as small volumes) and freeze at –20°C or lyophilise.
Immunoglobulins (Ig) may be extracted from antisera using conventional methods suitable for Ig
purification. Selective attachment to protein A constitutes a reliable and effective method. The
concentration of Ig solutions is adjusted to the values required for further conjugate preparation
or storage.
Preservation of Ig: Mix a solution of Ig of concentration 2 mg/litre with sterile pure glycerol (v/v)
and keep at –20°C. Solutions of Ig with a higher concentration may also be prepared in glycerol.
3.4. Mouse monoclonal antibodies to fish viruses and bacteria
Monoclonal antibodies (MAbs) to most of the fish viruses have been raised over the past years.
Some of them, singly or as two or three associated MAbs, have given rise to biological reagents
suitable for the identification of virus groups (IPN, VHS, IHN). Other MAbs, taken individually
or as components of Ab panels, allow accurate typing of VHSV and IHNV. These MAbs can be
obtained from the Reference Laboratories listed at the end of this Aquatic Manual.
56
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VI cont.
The production of MAbs to bacteria has also been described. It has resulted in the development
of commercial diagnostic kits for Renibacterium salmoninarum, but in most cases remains limited to
specialised laboratories.
In theory, mouse monoclonal IgGs can be processed and stored as for polyclonal IgGs. However,
the reactivity of certain MAbs may be impaired by processes such as enzymatic- or radio-labelling
or lyophilisation. It is thus necessary to test various MAbs for the conditions under which they
will be used.
3.5. Use of molecular techniques for confirmatory testing and diagnosis
Molecular techniques including nucleic acid probes and the polymerase chain reaction (PCR) have
been developed for the identification of many pathogens of aquatic animals. However, as is the
case with several other diagnostic techniques, an advantage in sensitivity is frequently offset by
problems in interpretation or susceptibility to technical problems. Whereas methods based on
direct culture or serology are relatively robust, PCR can be quite dependent on the conditions
under which it is run and can be highly subject to laboratory contamination by previous PCR
products, yielding false-positive results. Thus, while several nucleic acid probe and PCR protocols
are included in this version of this Aquatic Manual as diagnostic or confirmatory methods, where
possible, well-established techniques (e.g. virus isolation) are specified as standard screening
methods. Whenever these newer molecular techniques are used, they should be performed with
caution and with special attention to the inclusion of adequate positive and negative controls.
· 3.5.1. Sample preparation
For these techniques, samples should be prepared to preserve the DNA of the pathogen.
Likewise, samples intended for testing with antibody-based methods should be preserved to
retain the reactive antigenic sites for the antibodies used.
Samples selected for DNA-based or antibody-based diagnostic tests should be handled and
packaged with the greatest care to minimise the potential for cross contamination among the
samples or target degradation before the assay can be performed. To prevent contamination,
new containers (plastic sample bags or bottles) should be used. A water-resistant label, with
the appropriate data filled out, should be placed within each package or container for each
sample set.
Some suitable methods for preservation and transport of samples taken for molecular or
antibody-based tests are:
·
Live iced specimens or chilled specimens: for specimens that can be rapidly transported to the
laboratory for testing within 24 hours, pack samples in sample bags surrounded by an
adequate quantity of wet ice around the bagged samples in an insulated box and ship to
the laboratory.
·
Frozen whole specimens: select live specimens according to the purpose of sampling, quick
freeze in the field using crushed dry-ice, or freeze in a field laboratory using a mechanical
freezer at –20°C or lower temperature. Prepare and insert the label into the container
with the samples, pack samples with an adequate quantity of dry-ice in an insulated box,
and ship to the laboratory.
·
Alcohol-preserved samples: in regions where the storage and shipment of frozen samples is
problematic, 90–95% ethanol may be used to preserve, store, and transport certain types
of samples. Pack for shipment according to the methods described above.
·
Fixed tissues for in-situ hybridisation and immuno-histochemistry: for this purpose, classic
methods for preservation of the tissues are adequate. Davidson’s solution is usually a
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
57
Appendix VI cont.
good choice for later use of molecular probes. For DNA, specifically, over-fixation (over
24–48 hours) should be avoided.
· 3.5.2 Preservation of RNA and DNA in tissues using RNAlater
Tissue is cut to be less than 0.5 cm in one dimension and submerged in 10 volumes of
RNAlater (e.g. a 0.5 g sample requires about 5 ml of RNAlater). Small organs such as kidney,
liver and spleen can be stored whole in RNAlater. These samples can be stored at 4°C for one
month, at 25°C for 1 week or at –20°C indefinitely. Archive RNAlater-treated tissues at
–20°C.
· 3.5.3. DNA extraction
For DNA extraction, ground the preserved tissues to powder. Around 10 volumes of
extraction buffer (NaCl [100 mM], ethylene diamine tetra-acetic acid [EDTA, 25 mM], pH 8,
sodium dodecyl sulfate [SDS, 0.5%]) are added with proteinase K (100 µg/ml). Following
overnight incubation at 50°C, DNA is extracted using a standard phenol/chloroform
protocol, and precipitated with ethanol.
Considering time constraints and risks for laboratory staff, commercially available kits may
provide satisfactory technical alternative. Use of commercial kits should be validated by
comparison with standard phenol/chloroform protocol prior to its routine use in diagnostic
laboratories.
· 3.5.4. RNA extraction
To isolate RNA from tissues preserved in RNAlater, simply remove the tissue from RNAlater
and treat it as though it was just harvested. Most tissues can be homogenised directly in lysis or
extraction buffer.
· 3.5.5. Preparation of slides for in-situ hybridisation
For in-situ hybridisation (ISH), tissues are fixed in Davidson’s fixative for approximately
24 hours and then embedded in paraffin according to standard histology methods. Sections are
cut at 5 µm thick and placed on aminoalkylsilane-coated slides, which are then baked overnight
in an oven at 40°C. The sections are de-waxed by immersing in xylene for 10 minutes. This
step is repeated once and then the solvent is eliminated by immersion in two successive
absolute ethanol baths for 10 minutes each. The sections are then rehydrated by immersion in
an ethanol series. The protocol may require a step of membrane permeabilisation enabling
access to the target DNA. For this purpose, sections are treated with proteinase K (100 µg/ml)
in TE buffer (Tris [50 mM], EDTA [10 mM]), at 37°C for 30 minutes. For ISH tests, it is
essential that both a known positive and a known negative slide be stained to eliminate false
positive results due to nonspecific staining/stain dropout, and false negative results due to
errors in the staining protocol.
4.
ADDITIONAL
INFORMATION TO BE COLLECTED
The geographical origin of samples should be defined by the name of the sampling site associated with
either its geographical co-ordinates or its location along a river course or body of water.
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62. WOLF K. & DARLINGTON R.W. (1971). Channel catfish virus: a new herpesvirus of Ictalurid fish. J.
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65. WOLF K. & QUIMBY M.C. (1973). Fish viruses: Buffers and methods for plaquing eight agents under
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66. WOLF K., SNIESZKO S., DUNBAR C. & PYLE E. (1960). Virus nature of IPN in trout. Proc. Soc. Exp.
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67. WUNNER W.H. & PETERS D. (1991). Family Rhabdoviridae. In: Classification and Nomenclature of
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New York, USA and Vienna, Austria, 250–262.
*
* *
62
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII
SECTION
2.2.
DISEASES OF MOLLUSCS
CHAPTER
I.2.
GENERAL INFORMATION
A. OVERALL APPROACH TO MOLLUSC HEALTH MANAGEMENT
General information on how to approach mollusc health should be added.
Several techniques are applicable for mollusc pathogens. For screening and diagnostic purposes, the
Aquatic Manual has established two types of examination procedures that will be acceptable for such
work; 1) Screening methods, and 2) Diagnostic methods. The accepted methods are listed under each
disease chapter.
B. TARGET PATHOGENS AND DISEASES
Target pathogens and mollusc diseases are included in the Aquatic Animal Health Code (the Aquatic Code)
according to the following basic considerations: they resist or respond poorly to therapy, have a restricted
geographical range, are of high socio-economic importance, and occur in species involved in international
trade. For the current OIE list of diseases of molluscs, please consult the current edition of the Aquatic
Code. This Aquatic Manual includes chapters on diseases listed in the Aquatic Code and chapters on certain
other diseases of importance to trade.
Surveillance can also be developed for non-listed diseases especially for emerging diseases.
It would be impractical to try to develop a surveillance system, for all the known aquatic animal diseases in
a country. Therefore prioritising the diseases to be included in a surveillance system should be conducted
considering:
•
the needs to provide assurance of disease status for trade purposes
•
the resources of the country
•
the risk posed by the different diseases
The concept of risk encompasses both the probability of the disease occurring and the severity of its
consequences.
[1. DISEASES OF MOLLUSCS IN THE OIE AQUATIC MANUAL
Over the past few decades, world mollusc production has been adversely affected by a number of diseases and, given their
severe impact on economic and socio-economic development in many countries; some of these diseases have become a
primary constraint to the development and sustainability of mollusc aquaculture. Disease agent transfer via transfers of live
molluscs has been a major cause of disease outbreaks and epizootics.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
63
Appendix VII cont.
For the OIE list of diseases of molluscs, please consult the current edition of the Aquatic Code. For all of these diseases,
the OIE Reference Laboratory may provide technical assistance on the practical implementation of the standards included
in the Aquatic Code and Aquatic Manual.]
C. SAMPLING
1. SAMPLING
OBJECTIVES
There are at least three purposes for which mollusc stocks may be sampled. These are: 1) surveillance;
2) issuing health certificates and 3) disease outbreak investigation. The number and type of samples to
be taken for analysis and the sampling procedure varies greatly according to which of these purposes
applies.
[2.
SAMPLING PROCEDURES
There are at least three purposes for which mollusc stocks may be sampled. These are: 1) surveillance; 2) disease outbreak
or suspicion; and 3) confirmatory diagnosis. The number and type of samples to be taken for analysis varies greatly
according to which of these purposes applies.]
2. NUMBER
OF EPIDEMIOLOGICAL UNITS TO BE SAMPLED
Sample sizes for surveillance or for issuing health certificates should be calculated using the provisions
in chapter 1.1.4 of this Aquatic Manual.
When conducting disease outbreak investigations, the number of cases and non-cases will depend on
the case definition to be found in the appropriate chapter of the Aquatic Manual. If a case definition is
not available, a case definition describing the disease of interest as accurately as possible should be
developed and the number and status of units to be sampled should be determined in accordance with
that case definition.
3. SAMPLING
PROTOCOL
3.1. Selection of epidemiological units and sampling methods
[2.1.
Collection of specimens
A general approach to surveillance and sampling is given in Chapter 1.1.4 of this Aquatic Manual. The sampling
should be designed to enable detection, at a 95% confidence level, of infected animals. The following section gives
information relevant to sampling molluscs. For those diagnostic tests where the sensitivity and specificity have been
established, sample size may be determined using methods such as FreeCalc (www.ausvet.com.au) or similar
programs as outlined in Chapter 1.1.4 Requirements for surveillance for international recognition of freedom from
infection. However, for those diagnostic tests where the sensitivity and specificity have not been established, the
default sample size should be determined from Table 1 in the present chapter.
Table 1. Random sample size based on assumed pathogen prevalence in lot
and assuming 100% sensitivity and specificity of the technique
64
Lot size
At 2% prevalence,
size of sample
At 5% prevalence,
size of sample
At 10% prevalence,
size of sample
50
50
35
20
100
75
45
23
250
110
50
25
500
130
55
26
1000
140
55
27
1500
140
55
27
2000
145
60
27
4000
145
60
27
10,000
145
60
27
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII cont.
100,000 or more
150
60
30
After Ossiander and Wedemeyer, 1973.
2.2.
Specific recommendations for sampling molluscs]
The timing and frequency of sampling should be determined by the cycle of infection by the
disease agent and the pre-patent period. An adequate time period should be allocated when
sampling for seasonal diseases, for example infection with Marteilia refringens or Haplosporidium
nelsoni, to ensure optimal detection. As disease agents may increase in intensity of infection
associated with loss of host condition following spawning, post-spawning sampling is also
recommended. Sampling periods should also take account of the transfer of juveniles and spats
into outgrowing areas, and the transfer of adults for further fattening or relaying.
Samples should also cover a range of size groups, or target the most susceptible age group when
this is known. During sampling, any molluscs showing abnormalities (abnormal growth, gaping
valves, elevated or high mortality rates) should be selected. Diseases have sub-clinical stages of
development that can escape detection using routine screening methodology. However, the
probability of detection of infection may be increased by holding the bivalves in quarantine for a
long period and subjecting them to stress (crowding, handling, temperature and salinity changes,
etc.). To detect infection, species of molluscs that are more susceptible to infection should be
examined histologically. For example, members of the Arcidae (Arca, Barbatia), Malleidae
(Malleus), Isognomonidae (Isognomon), Chamidae (Chama), Tridacnidae (Tridacna) and Veneridae
tolerate high prevalence of Perkinsus olseni infection and are good indicators of the presence of
P. olseni.
Prevalence of infection is an important factor affecting the chance of detection. When molluscs
are to be moved from natural beds into a farm site or between natural beds in different zones,
large numbers of bivalves may be required to detect low prevalence of infection. For example, in
Western Australia, Marteilia sydneyi and Perkinsus sp. occur at 0.1% prevalence in isolated beds
untouched by humans. This level of sampling should be commensurate with the level of risk to
the receiving waters and the aquatic resources they support.
For each zone, a number of sampling sites should be selected in the most practical way so as to
maximise the chances of detecting disease agents. The number of sites should be increased for
large zones that contain several discrete areas of cultivation of the susceptible species. Account
should be taken of parameters that have an effect on the development of the disease agents, such
as stocking density, water flow, and the developmental cycle of the molluscs.
[It is an important requirement that the screening techniques used be the optimum methods available for detection of
the disease agent in question, and that when the infection is present, it can be detected. For screening methods,
sensitivity and specificity should have been assessed. This information has a strong influence on sample size (see
Section 2.1).
2.3. Shipment of samples]
3.2. Post-sampling procedures
All sampled molluscs should be delivered to the approved diagnostic laboratory and should arrive
live. The laboratory should be informed of the estimated time of arrival of the sample so the
required materials to process the molluscs can be prepared before reception of samples.
Mollusc samples should be packed in accordance with current standards in order to keep them
alive. If the sampling site is a long distance from the laboratory, moribund animals or those with
foul-smelling tissues may be of little use for subsequent examination. Required samples should be
shipped as soon as possible after collection from the water, in order to reduce air storage and
possible mortality during transportation, especially for moribund diseased molluscs.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
65
Appendix VII cont.
For samples that cannot be delivered live to the diagnostic laboratory, due to advanced stages of
disease, long distance or slow transportation connections, etc., specimens should be fixed on site
as recommended in the following sections of this chapter or the individual disease chapters of this
Aquatic Manual. While this is suitable for, for example, subsequent histology or transmission
electron microscopy examination, other techniques, such as fresh smears, tissue imprints, routine
bacteriology, mycology or Ray’s fluid thioglycollate culture of Perkinsus spp., cannot be performed.
Diagnostic needs and sample requirements should be discussed with the diagnostic laboratory
prior to collection of the sample.
Samples should be accompanied with background information, including the reason for
submitting the sample (surveillance, abnormal mortality, abnormal growth, etc.), gross
observations and associated environmental parameters, approximate prevalence and patterns of
mortality, origin and nature of the molluscs (species, age, whether or not the samples are from
local mollusc populations or stocks transferred from another site, date of transfer and source
location, etc.). This information should identify possible changes in handling or environmental
conditions that could be a factor in mortality in association, or not, with the presence of
infectious agents.
4. ASSESSING
THE HEALTH STATUS OF THE EPIDEMIOLOGICAL UNIT
4.1. Macroscopic examination
The gross observation of molluscs should target, as far as possible, animal behaviour, shell
surface, inner shell and soft tissues.
It is often difficult to observe the behaviour of molluscs in open waters. However, observation of
molluscs in certain rearing facilities such as brood-stock in tanks and larvae in hatcheries can
provide useful indications of disease-related behavioural changes. If signs are noted (e.g. presettlement of larvae on the bottom, food accumulation in tanks, signs of weakening, etc.), samples
may be examined for gross signs, including observation under a dissecting microscope for
abnormalities and deformities, fouling organisms, and fixed for further processing as
recommended below. For adults and juveniles, signs of weakening may include gaping,
accumulation of sand, mud and debris in the mantle and on the gills, mantle retraction away from
the edge of the shell, decreased activity (scallop’ swimming, clam’ burrowing, abalone’ grazing),
etc. The righting reflex of abalone after being inverted is not possible in weakened animals, and it
is a good indicator of weakness. Open-water mortality should be monitored for patterns of losses
and samples collected for further analysis. Environmental factors pre- and post-mortality should
be recorded.
Even under culture conditions, the shells of molluscs may not be clean and fouling organisms are
normal colonists of mollusc shell surfaces. Organisms such as barnacles, limpets, sponges,
polychaete worms, bivalve larvae, tunicates, bryozoans, etc., do not normally threaten health of
molluscs. Culture systems, such as suspension and shallow water culture, can even increase
exposure to fouling organisms and shells may become covered by other animals and plants. This
can affect the health directly by impeding shell opening and closing or indirectly through
competition for food resources. Signs of weakening associated with heavy fouling should be a
cause for concern rather than fouling itself. Shell damages by boring organisms such as sponges
and polychaete worms are usually benign, but under certain conditions may reach proportions
that make the shell brittle or pierce through to the soft-tissues. This degree of shell damage can
weaken the mollusc and render it susceptible to inter-current pathogen infections. Shell
deformities (shape, holes in the surface), fragility, breakage or repair should be noted, but are not
usually indicative of a disease concern. Abnormal coloration and smell, however, may indicate a
possible soft-tissue infection that may need to be examined at a laboratory.
The molluscs should be opened carefully so as not to damage the soft tissues, in particular the
mantle, gills, heart and digestive gland. The presence of fouling organisms on the inner shell
66
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII cont.
surface is a clear indication of weakness. The inner surface of the shell is usually smooth and clean
due to mantle and gill action. Perforation of the inner surface may occur, but can be sealed off by
the deposition of additional conchiolin and nacre. This may result in formation of mud- or waterfilled blisters. Blisters may also form over superficial irritants such as foreign bodies. The degree
of shell perforation can be determined by holding the shell up to a strong light. Where
abnormalities occurring within the matrix of the shell warrant further investigation, freshly
collected specimens can be brought intact to the laboratory or fixed for subsequent
decalcification, as required. The appearance of the soft-tissues is frequently indicative of the
physiological condition of the animal. Soft tissues should be examined for the presence of abscess
lesions, pustules, tissue discoloration, pearls, oedema, overall transparency or wateriness, gill
deformities, etc., and, when found in association with weak or dying animals, these abnormalities
should be a cause for concern.
Abnormalities and lesions of the tissues should be noted and recorded, as well as any shell
deformities, shell-boring organisms and conspicuous mantle inhabitants. Levels of tissue damage
should be recorded and samples of affected and unaffected animals collected for laboratory
examination as soon as possible.
4.2. Examination of stocks where abnormal mortality occurs
Abnormal mortality of molluscs is usually recognised as a sudden sizeable mortality that occurs in
a short time between two observations or inspections of the stocks (for example, about 15 days in
the case of facilities located in inter-tidal zone). In a hatchery, abnormal mortality is the failure of
successive production of larvae coming from different brood-stock. Given the broad spectrum of
species, environments and culture conditions, these definitions should be adapted when and
where necessary.
Whenever abnormal mortality occurs in stocks of molluscs, an urgent investigation should be
carried out to determine the disease determinants [aetiology].
The samples taken should be collected, preserved or fixed and stored in accordance with the
procedures described in this Aquatic Manual.
Where and when available, unaffected or control molluscs should also be fixed for histological
comparison with abnormal tissues. Whatever the fixative, it is essential that the shell be removed
to allow easy ingress of the fixative. Bivalves and operculated gastropods can keep the shell shut
against fixative until autolysis begins.
4.3. Diagnostic methods
Techniques applicable to molluscan disease agents are limited to direct detection of the causative
agent. Classic serological methods cannot be used for diagnostic purposes because molluscs do
not produce antibodies. In addition to histology and cytology, immunoassays using monoclonal
antibodies or nucleic acid probes can be used for the detection of listed disease agents. From this
point of view, the development of DNA-based diagnostic techniques for mollusc disease agents
has certainly been the most significant advancement in recent years. Given the development and
potential for widespread application of these diagnostic techniques and the inherent problems
currently associated with their use, the issue of validation is of the utmost importance.
Three levels of examination procedures are proposed in the following sections. Histology is
recommended as a standard screening method because it provides a large amount of information.
It is particularly important because macroscopic examination usually gives no pathognomonic
signs or solid indicative information. Also, mortality may be due to several disease agents or
physiological problems, such as loss of condition following spawning, and this can only be
determined using histology. Screening (surveillance) is routinely performed by histology.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
67
Appendix VII cont.
According to each epidemiological situation, and when it is justified, targeted surveillance may rely
on other techniques.
When abnormal mortality outbreaks occur, histology is also recommended. It is particularly
important because macroscopic examination usually gives no pathognomonic signs or solid
indicative information. Also, mortality may be due to several disease agents or physiological
problems, such as loss of condition following spawning, and this can only be determined using
histology. Various presumptive diagnostic methods can be used in addition to histology, among
which, tissue imprints, Ray’s fluid thioglycollate medium (RFTM) culture or polymerase chain
reaction (PCR) may be used, as recommended in the individual disease chapters. Such methods
may provide advantages of quick and/or cheap procedures as an answer to suspicion of infection
with a given disease agent.
When a disease agent is encountered during screening or mortality outbreaks, molecular methods
are increasingly being used, in addition to electron microscopy, for specific identification. Some of
the OIE listed diseases for molluscs are caused by disease agents belonging to genera
encompassing closely related species. Specific protocols designed to detect certain listed agents
are recommended in the following chapters, to be used to confirm histological examination
results and/or to give a species-specific diagnosis.
4.4. Histological techniques
Because of the generic use of histology in diagnostic procedures for diseases of molluscs, a
detailed technical guideline is provided in this chapter.
Histology is a technique that is used to study the structure of cells and tissues under light
microscopy. Tissue preparation involves different steps, including tissue fixation, dehydration,
impregnation and embedding of samples, preparation of sections, staining and mounting of slides.
Live moribund animals or freshly dead (within minutes) animals provide the optimum conditions
under which to collect tissues. A standard section should be taken through the digestive gland, to
include the gills, mantle and palps, where possible. Alternatively for large specimens, several
sections should be taken to include all the important tissues.
· 4.4.1. Tissue fixation
The role of the fixative is to maintain the morphology of the tissues as close to in-vivo
morphology as possible and to prevent post-sampling necrosis. Recommended fixatives used
for the study of marine molluscs are Davidson’s solution and Carson’s solution for large
specimens. For smaller specimens, glutaraldehyde fixatives may be used and are compatible for
electron microscopy use. The ratio of fixative to tissue volume should be at least 10:1 to
ensure good fixation.
6
68
Davidson’s solution:
Sea water
95% Alcohol
35–40% Formaldehyde6
Glycerol
Glacial acetic acid
1200 ml
1200 ml
800 ml
400 ml
10% (add extemporaneously)
Carson’s solution:
NaH2PO4.2H2O
NaOH
23.8 g
5.2 g
A saturated 37–39% aqueous solution of formaldehyde gas.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII cont.
Distilled water
40% Formaldehyde1
Adjust the pH to 7.2–7.4
900 ml
100 ml
There is no universal fixative and choice should be made taking into account later use of fixed
material as well as practical aspects of fixative use (price, component availability, etc.).
Davidson’s solution is an excellent choice for preserving the structure of the tissues. In
addition, tissue sections fixed in Davidson’s solution can be stained later by different
histochemical methods, as well as in-situ hybridisation with DNA probes. For this purpose,
over-fixation (over 24–48 hours) should be avoided. Carson’s solution may not be as good as
Davidson’s solution for histological analysis. Nevertheless it does allow good preservation of
the ultrastructure and may be used to preserve samples for later study by electron microscopy.
Because electron microscopy may be a valuable adjunct in diagnosing or confirming infections
in molluscs, fixation of some samples (especially smaller samples) using glutaraldehyde, as
described in Section 6.2 of this chapter, may be considered. Otherwise, material fixed in
Carson’s solution, and shown to contain adequate levels of targeted disease agents or
abnormalities, can be refixed in glutaraldehyde. It is recommended that part of the mollusc be
fixed in Davidson’s solution while the other part be fixed in Carson’s solution for further
investigation. This should be done in order to ensure fixation of all tissues/organs in the two
fixatives. If neither are available, 10% formalin buffered with filtered seawater is adequate.
Within each country, the molluscan aquaculture industry should agree on the most effective
way of ensuring adequate fixation.
· 4.4.2. Dehydration, impregnation and embedding of the samples
The embedding of the samples in paraffin requires several steps during which the water
contained in the tissues is progressively replaced, first by alcohol, then by xylene or equivalent
less toxic clearing solution, and lastly by paraffin.
After having fixed the samples in Carson’s or Davidson’s solution, they are transferred
through graded alcohols (70–95 [v/v]) before final dehydration in absolute ethanol. The
alcohol contained in the tissues is next eliminated by immersing them in xylene. The tissues are
then impregnated with paraffin, which is soluble in xylene, at 60°C. These steps may be all
carried out automatically using a machine.
Blocks are produced by letting the tissues cool in moulds filled with paraffin on a cooling
table; cooling and moisturising are essential to section cutting.
· 4.4.3. Preparation of the sections
After the blocks have
histological sections of
on histological slides,
temperature allows the
slides.
been cooled on a cold plate, which allows the paraffin to solidify,
about 2–3 µm are cut using a microtome. The sections are recovered
drained and dried overnight at 60°C. Drying the samples at this
excess moisture to be eliminated and thus the sections adhere to the
· 4.4.4. Staining and mounting the slides
Before staining, the paraffin is removed from the sections by immersing them in xylene or
equivalent less toxic clearing solution for 10–20 minutes. This is repeated once and then the
solvent is eliminated by immersion in two successive absolute ethanol baths for 10-minute
periods each and rehydrated by immersion in a bath of tap water for 10 minutes. Different
topographical or histochemical staining techniques can then be performed.
When staining with haematoxylin–eosin (H&E) is used, (haematoxylin or equivalent) nuclear
and basophilic structures stain a blue to dark purple colour, the endoplasmic reticulum stains
blue, while the cytoplasm takes on a grey colour. The acid dye eosin stains the other structures
pink. This staining technique is simple and reproducible and, although it only allows a limited
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
69
Appendix VII cont.
differentiation of cell structures, it is possible to detect any abnormalities in tissue and cellular
structure. Other techniques may be applied to demonstrate particular structures or features as
required (e.g. trichrome for connective tissue and cytoplasmic granules).
4.5. Transmission electron microscopy methods
Because of the very frequent use of transmission electron microscopy in confirmatory
identification of disease agents in diagnostic procedures for diseases of molluscs, detailed
technical guidelines are provided in this chapter for indication.
Fixation for electron microscopy should be done immediately after the animal has been killed,
before fixation for histology. Only samples taken rapidly from live animals will be of any use. The
preparation of samples for electron microscopy involves the following steps: tissue fixation,
decalcification of the samples (when necessary), dehydration, impregnation and embedding of the
samples, preparation and counterstaining of the sections.
· 4.5.1. Tissue fixation
For tissues that are to be examined by electron microscopy, it is important that the fixation be
performed correctly in order to cause as little damage as possible to the ultrastructure. The
specimens are cut such that their dimensions do not exceed 1–2 mm. This small size allows
the various solutions to penetrate rapidly into the sample.
Fixation of the samples is carried out directly in 3% glutaraldehyde for 1 hour. Fixation for
longer periods leads to membranous artefacts. The samples are washed in buffer three times,
then fixed in 1% osmic acid and washed twice again in buffer. Various formulations of
glutaraldehyde fixative and buffers work equally well.
In order to cause as little damage as possible to the ultrastructure, the samples are treated with
solutions that have an osmolarity close to that of the tissues. Thus, mollusc tissues are treated
with solutions with an osmolarity of around 1000 mOsm. The osmolarity of the solutions is
adjusted with NaCl. As mollusc tissues are nearly iso-osmotic with seawater, it is possible to
make the glutaraldehyde up with 0.22 µm filtered seawater, and use the filtered seawater for
subsequent washes.
Sodium cacodylate
0.4 M: 8.6 g in 100 ml of distilled water
Sodium chloride
10% in distilled water
Cacodylate buffer, pH 7.4:
1000 mOsm
Sodium cacodylate
NaCl
Distilled water
Adjust the pH to 7.4
70
50 ml from 0.4 M stock solution
20 ml from 10% stock solution
30 ml
3% Gluteraldehyde:
1000 mOsm
25% gluteraldehyde
0.4 M sodium cacodylate
10% NaCl
Distilled water
2.5 ml
5 ml
3.5 ml
9 ml
1% Osmic acid:
1000 mOsm
4% Osmic acid
0.4 M sodium cacodylate
1 volume
1 volume
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII cont.
NaCl
Distilled water
1 volume from 10% stock solution
1 volume
5% EDTA:
Disodium EDTA
Cacodylate buffer
5g
100 ml
EDTA dissolves when the pH is above 8. When the solution becomes clear adjust the pH to
7.4 by adding concentrated HCl.
If the samples have been previously fixed and stored in Carson’s solution, they should be
washed several times in a bath of buffer before fixation with 3% glutaraldehyde.
· 4.5.2. Dehydration, impregnation and embedding of the samples
The samples are dehydrated in successive baths of ethanol: 70% ethanol once, 95% ethanol
twice, absolute ethanol three times. The dehydration is completed by two baths of propylene
oxide, which allows the subsequent impregnation with Epon or other resin.
The samples are impregnated progressively. After a first bath in a mixture of polypropylene
oxide–Epon (50/50), the samples are placed in a bath of Epon. The longer the incubation, the
better is the impregnation of the tissues.
Embedding is carried out by placing the samples in moulds filled with Epon resin. A label
identifying the sample is included in each block and the blocks are then placed at 60°C (the
temperature at which Epon resin polymerises) for 48 hours.
· 4.5.3. Preparation of the sections and the counterstaining
The blocks are cut to appropriate sizes with a razor blade and the sections are then cut using
an ultra microtome. Semi-thin sections (0.5–1 µm) are cut and placed on glass slides. These
will be used to control the quality of the samples by light microscopy and to find the areas of
interest on the section.
The semi-thin sections are stained at 90–100°C with 1% toluidine blue solution. After drying,
the slides are mounted under cover-slips with a drop of synthetic resin and observed under the
light microscope.
Ultra-thin sections 80–100 nm thick are placed on mesh copper grids for electron microscopy
analysis. Uranyl acetate and lead citrate are used to counterstain the ultra-thin sections.
4.6. Molecular methods
Molecular techniques usually offer an advantage in sensitivity that is frequently offset by technical
problems. PCR is particularly dependent on the conditions under which it is run, yielding falsepositive or false-negative results. Whenever molecular techniques are used, they should be
performed with caution and with special attention to the inclusion of adequate positive and
negative controls in order to overcome the possible lack of robustness and to maintain adequate
accuracy.
PCR, PCR-RFLP (restriction fragment length polymorphism), sequencing, in-situ hybridisation
and immuno-histochemistry are increasingly used in confirmatory identification of disease agents.
For these techniques, samples should be prepared to preserve the DNA of the pathogen.
Likewise, samples intended for testing with antibody-based methods should be preserved to retain
the reactive antigenic sites for the antibodies used.
· 4.6.1. Sample preparation
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
71
Appendix VII cont.
Samples selected for DNA-based or antibody-based diagnostic tests should be handled and
packaged with the greatest care to minimise the potential for cross contamination among the
samples or target degradation before the assay can be performed. To prevent contamination,
new containers (plastic sample bags or bottles) should be used. A water-resistant label, with
the appropriate data filled out, should be placed within each package or container for each
sample set.
Some suitable methods for preservation and transport of samples taken for molecular or
antibody-based tests are:
·
Live iced specimens or chilled specimens: for specimens that can be rapidly transported to the
laboratory for testing within 24 hours, pack samples in sample bags surrounded by an
adequate quantity of wet ice around the bagged samples in an insulated box and ship to
the laboratory.
·
Frozen whole specimens: select live specimens according to the purpose of sampling, quick
freeze in the field using crushed dry-ice, or freeze in a field laboratory using a mechanical
freezer at –20°C or lower temperature. Prepare and insert the label into the container
with the samples, pack samples with an adequate quantity of dry-ice in an insulated box,
and ship to the laboratory.
·
Alcohol-preserved samples: in regions where the storage and shipment of frozen samples is
problematic, 90–95% ethanol may be used to preserve, store, and transport certain types
of samples. Whole molluscs (when the specimen is small), excised tissues from larger
molluscs. Pack for shipment according to the methods described above.
·
Fixed tissues for in-situ hybridisation and immuno-histochemistry: for this purpose, classic
methods for preservation of the tissues are adequate. Davidson’s solution is usually a
good choice for later use of molecular probes. For DNA, specifically, over-fixation (over
24–48 hours) should be avoided.
· 4.6.2. DNA extraction
For DNA extraction, grind the preserved tissues to powder. Around 10 volumes of extraction
buffer (NaCl [100 mM], ethylene diamine tetra-acetic acid [EDTA, 25 mM], pH 8, sodium
dodecyl sulfate [SDS, 0.5%]) are added with proteinase K (100 µg/ml). Following overnight
incubation at 50°C, DNA is extracted using a standard phenol/chloroform protocol, and
precipitated with ethanol.
Considering time constraints and risks for laboratory staff, commercially available kits may
provide satisfactory technical alternative. Use of commercial kits should be validated by
comparison with standard phenol/chloroform protocol prior to its routine use in diagnostic
laboratories.
· 3.3.3. Preparation of slides for in-situ hybridisation
For in-situ hybridisation, molluscs are fixed in Davidson’s fixative for approximately 24 hours
and then embedded in paraffin according to methods described above for histology. Sections
are cut at 5 µm thick and placed on aminoalkylsilane-coated slides, which are then baked
overnight in an oven at 40°C. The sections are dewaxed by immersing in xylene for
10 minutes. This step is repeated once and then the solvent is eliminated by immersion in two
successive absolute ethanol baths for 10 minutes each. The sections are then rehydrated by
immersion in an ethanol series. The protocol may require a step of membrane
permeabilisation enabling access to the target DNA. For this purpose, sections are treated with
proteinase K (100 µg/ml) in TE buffer (Tris [50 mM], EDTA [10 mM]), at 37°C for
30 minutes.
72
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII cont.
5. ADDITIONAL
INFORMATION TO BE COLLECTED
The geographical origin of samples should be defined by the name of the sampling site associated with
either its geographical co-ordinates or its location along a river course or body of water.
KEY REFERENCES
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ALMEIDA M., BERTHE F., THEBAULT A. & DINIS M.T. (1999). Whole clam culture as a quantitative
diagnostic procedure of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams Ruditapes decussatus.
Aquaculture, 177, 325–332.
2.
ANDERSON T.J., MCCAUL T.F., BOULO V., ROBLEDO J.A.F. & LESTER R.J.G. (1994). Light and electron
immunohistochemical assays on paramyxean parasites. Aquatic Living Resources, 7, 47–52.
3.
AZEVEDO C., CORRAL L. & CACHOLA R. (1990). Fine structure of zoosporulation in Perkinsus atlanticus
(Apicomplexa: Perkinsea). Parasitology, 100, 351–358.
4.
BERTHE F.C.J., BURRESON E. & HINE M. (1999). Use of molecular tools for mollusc disease
diagnosis. Bull. Eur. Ass. Fish Pathol., 19(6), 277–278.
5.
BERTHE F.C.J., LE ROUX F., ADLARD R.D. & FIGUERAS A.J. (2004). Marteiliosis of molluscs: a
review. Aquatic Living Resources, 17(4), 433–448.
6.
BONDAD-REANTASO M.G., MCGLADDERY S.E., EAST I. & SUBASINGHE R.P. (2001). Asian
Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper, No. 402, supplement 2.
Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 240 pp.
7.
BOULO V., MIALHE E., ROGIER H., PAOLUCCI F. & GRIZEL H. (1989). Immunodiagnosis of Bonamia
ostreae (Ascetospora) infection of Ostrea edulis L. and subcellular identification of epitopes by monoclonal
antibodies. J. Fish Dis., 12, 257–262.
8.
BOWER S.M., MCGLADDERY S.E. & PRICE I.M. (1994). Synopsis of infectious diseases and parasites
of commercially exploited shellfish. Ann. Rev. Fish Dis., 4, 1–199.
9.
BURRESON E.M. & FORD S.E. (2004). A review of recent information on the Haplosporidia, with
special reference to Haplosporidium nelsoni (MSX disease). Aquatic Living Resources, 17(4), 499–517.
10. BUSHEK D., FORD S.E. & ALLEN SK (1994). Evaluation of methods using Ray’s fluid thioglycollate
medium for diagnosis of Perkinsus marinus infections in the eastern oyster, Crassostrea virginica. Ann. Rev.
Fish. Dis., 4, 201–217.
11. CARNEGIE R.B., BARBER B.J., CULLOTY S.C., FIGUERAS A.J. & DISTEL D.L. (2000). Development of a
PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the
Haplosporidia. Dis. Aquat. Org., 42, 199–206.
12. CARNEGIE R.B. & COCHENNEC-LAUREAU N. (2004). Microcell parasites of oysters: recent insights
and future trends. Aquatic Living Resources, 17(4), 519–528.
13. COCHENNEC N., LE ROUX F., BERTHE F. & GERARD A. (2000). Detection of Bonamia ostreae based on
small subunit ribosomal probe. J. Invertebr. Pathol., 76, 26–32.
14. CRANFIELD H.J., DUNN A., DOONAN I.J. & MICHAEL K.P. (2005). Bonamia exitiosa epizootic in Ostrea
chilensis from Foveaux Strait, southern New Zealand between 1986 and 1992. ICES J. Mar. Sci., 62(1), 3–
13.
15. DIGGLES B.K., COCHENNEC-LAUREAU N. & HINE P.M. (2003). Comparison of diagnostic
techniques for Bonamia exitiosus from flat oysters Ostrea chilensis in New Zealand. Aquaculture, 220,
145–156.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
73
Appendix VII cont.
16. DINAMANI P., HINE P.M. & JONES J.B. (1987). Occurrence and characteristics of the haemocyte parasite
Bonamia sp. in the New Zealand dredge oyster Tiostrea lutaria. Dis. Aquat. Org., 3, 37–44.
17. DUNGAN C.F. & ROBERSON B.S. (1993). Binding specificities of mono- and polyclonal antibodies to the
protozoan oyster pathogen Perkinsus marinus. Dis. Aquat. Org., 15, 9–22.
18. ELSTON R.A. (1999). Health Management, Development and Histology of Seed Oysters. World
Aquaculture Society. Baton Rouge, Louisiana, USA, 110 pp.
19. FARLEY C.A., WOLF P.H. & ELSTON R.A. (1988). A long-term study of ‘microcell’ disease in oysters
with a description of new genus Mikrocytos (g.n.), and two new species, Mikrocytos mackini (sp.n.) and
Mikrocytos roughleyi (sp. n.). Fish. Bull,. 86, 581–593.
20. FONG D., RODRIGUEZ R., KOO K., SUN J., SOGIN M.L., BUSHEK D., LITTLEWOOD D.T.J. & FORD S.E.
(1993). Small subunit ribosomal RNA gene sequence of the oyster parasite Perkinsus marinus. Mol. Mar.
Biol. Biotechnol., 2, 346–350.
21. FORD S.E. (1992). Avoiding the transmission of disease in commercial culture of molluscs, with special
reference to Perkinsus marinus (Dermo) and Haplosporidium nelsoni (MSX). J. Shellfish Res., 11, 539–546.
22. GALTSOFF P.S. (1964). The American oyster, Crassostrea virginica Gmelin. Fishery Bull., 64, 480 pp.
23. GRIZEL H., COMPS M., BONAMI J-R, COUSSERANS F., DUTHOIT J.L. & LE PENNEC M.A. (1974).
Recherche sur l’agent de la maladie de la glande digestive de Ostrea edulis Linné. Science et Pêche. Bull
Inst. Pêches marit., 240, 7–30.
24. GRIZEL H., MIALHE E., CHAGOT D., BOULO V. & BACHÈRE E. (1988). Bonamiasis: a model study of
diseases in marine molluscs. In: Disease Processes in Marine Bivalve Molluscs, Fisher W.S., ed. American
Fisheries Society Special Publication 18, 1–4.
25. HERVIO D., BOWER S.M. & MEYER G.R. (1996). Detection, isolation, and experimental transmission of
Mikrocytos mackini, a microcell parasite of Pacific oysters Crassostrea gigas (Thunberg). J. Invertebr. Pathol., 67,
72–79.
26. HINE P.M., BOWER S.M., MEYER G.R., COCHENNEC-LAUREAU N. & BERTHE F.C.J. (2001). The
ultrastructure of Mikrocytos mackini, the cause of Denman Island disease in oysters Crassostrea spp. and
Ostrea spp. in British Columbia, Canada. Dis. Aquat. Org., 45, 215–227.
27. HINE P.M. & THORNE T. (2000). A survey of some parasites and diseases of several species of bivalve
mollusc in northern Western Australia. Dis. Aquat. Org., 40, 67–78.
28. HOWARD D.W. LEWIS E.J., KELLER J. & SMITH C.S. (2004). Histological techniques for marine
bivalve molluscs and crustaceans. NOAA Technical Memorandum NOS NCCOS 5, 218 pp.
29. KLEEMAN S.N., LE ROUX F., BERTHE F. & ADLARD R.D. (2002). Specificity of PCR and in situ
hybridization assays designed for detection of Marteilia sydneyi and M. refringens. Parasitology, 125, 131–
141.
30. KOTOB S., MCLAUGHLIN S.M., VAN BERKUM P. & FAISAL M. (1999). Discrimination between two
Perkinsus spp. isolated from the softshell clam, Mya arenaria, by sequence analysis of two internal
transcribed spacer regions and the 5.8S ribosomal RNA gene. Parasitology, 119, 363–368.
31. LE ROUX F., AUDEMARD C., BARNAUD A. & BERTHE F. (1999). DNA probes as potential tools for the
detection of Marteilia refringens. Mar. Biotechnol., 1, 588–597.
32. LE ROUX F., LORENZO G., PEYRET P., AUDEMARD C., FIGUERAS A., VIVARES C., GOUY M. &
BERTHE F. (2001). Molecular evidence for the existence of two species of Marteilia in Europe. J.
Eukaryot. Microbiol., 48, 449–454.
74
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VII cont.
33. LESTER R.J.G. & DAVIS G.H.G. (1981). A new Perkinsus species (Apicomplexa, Perkinsea) from the
abalone Haliotis ruber. J. Invertebr. Pathol., 37, 181–187.
34. LONGSHAW M., FEIST S.W., MATTHEWS R.A. & FIGUERAS A. (2001). Ultrastructural characterization of
Marteilia species (Paramyxea) from Ostrea edulis, Mytilus edulis and Mytilus galloprovincialis in Europe. Dis.
Aquat. Org., 44, 137–142.
35. MIALHE E., BACHERE E., BOULO V., CADORET J.P., ROUSSEAU C., CEDENO V., SARAIVA E.,
CARRERA L., CALDERON J. & COLWELL R.R. (1995). Future of biotechnology-based control of
disease in marine invertebrates. Molec. Mar. Biol. Biotechnol., 4, 275–283.
36. MURRELL A., KLEEMAN S.N., BARKER S.C. & LESTER R.J.G. (2002). Synonymy of Perkinsus olseni Lester
& Davis, 1981 and P. atlanticus Azevedo, 1989 and an update on the phylogenetic position of the genus
Perkinsus. Bull. Eur. Ass. Fish Pathol., 22, 258–265.
37. OSSIANDER F.J. & WEDEMEYER G. (1973). Computer program for sample size required to
determine disease incidence in fish populations. J. Fish. Res. Board Can., 30, 1383–1384.
38. RAY S.M. (1966). A review of the culture method for detecting Dermocystidium marinum with suggested
modifications. Proc. Natl Shellfish Assoc., 54, 55–69.
39. REECE K., SIDDALL M.E., BURRESON E.M. & GRAVES J.E. (1997). Phylogenetic analysis of Perkinsus
based on actin gene sequences. J. Parasitol., 83, 417–423.
40. RODRIGUEZ F. & NAVAS J.L. (1995). A comparison of gill and haemolymph assays for the thioglycollate
diagnosis of Perkinsus atlanticus (Apicomplexa, Perkinsea) in clams, Ruditapes decussatus (L.) and Ruditapes
philippinarum (Adams et Reeve). Aquaculture, 132, 145–152.
41. STOKES N.A. & BURRESON EM (1995). A sensitive and specific DNA probe for the oyster pathogen
Haplosporidium nelsoni. J. Eukaryot. Microbiol., 42, 350–357.
42. THOESON J.C. (1994). Suggested Procedures for the Detection and Identification of Certain Finfish
and Shellfish Pathogens, Fifth Edition. Bluebook, American Fisheries Society, Bethsada, USA.
43. VILLALBA A., REECE K.S., ORDÁS M.C., CASAS S.M. & FIGUERAS A. J. (2004). Perkinsosis in
molluscs. Aquatic Living Resources, 17(4), 411–432.
44. WALKER P. & SUBASINGHE R.P. (2000). DNA-based Molecular Diagnostic Techniques. Research
needs for standardization and validation of the detection of aquatic animal pathogens and diseases.
FAO Fisheries Technical Paper, n°395, 93 pp.
45. YARNALL H.A., REECE K.S., STOKES N.A. & BURRESON E.M. (2000). A quantitative competitive
polymerase chain reaction assay for the oyster pathogen Perkinsus marinus. J. Parasitol., 86, 827–837.
*
* *
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
75
Appendix VIII
SECTION
2.3.
DISEASES OF CRUSTACEANS
CHAPTER
I.3.
GENERAL INFORMATION
A. OVERALL APPROACH TO CRUSTACEAN HEALTH MANAGEMENT
General information on how to approach crustacean health should be added (e.g. adoption of better
management practices owing to the difficulties in preventing the entry of pathogens into a farming system,
etc.).
Several techniques are applicable for crustacean pathogens. For screening and diagnostic purposes, the
Aquatic Manual has established two types of examination procedures that will be acceptable for such
work; 1) Screening methods, and 2) Diagnostic methods. The accepted methods are listed under each
disease chapter.
B. TARGET PATHOGENS AND DISEASES
Target pathogens and crustaceans diseases are included in the Aquatic Animal Health Code (the Aquatic
Code) according to the following basic considerations: they resist or respond poorly to therapy, have a
restricted geographical range, are of high socio-economic importance, and occur in species involved in
international trade. For the current OIE list of diseases of crustacean, please consult the current edition of
the Aquatic Code. This Aquatic Manual includes chapters on diseases listed in the Aquatic Code and
chapters on certain other diseases of importance to trade.
Surveillance can also be developed for non-listed diseases especially for emerging diseases.
It would be impractical to try to develop a surveillance system, for all the known aquatic animal diseases in
a country. Therefore prioritising the diseases to be included in a surveillance system should be conducted
considering:
•
the needs to provide assurance of disease status for trade purposes,
•
the resources of the country ,
•
the risk posed by the different diseases.
The concept of risk encompasses both the probability of the disease occurring and the severity of its
consequences.
[1.
DISEASES OF CRUSTACEANS LISTED BY THE OIE
Crustaceans are adversely affected by a number of diseases. This is especially evident in penaeid shrimp from aquaculture.
All of the crustacean diseases that have significant social or economic impact are infectious diseases. The crustacean
diseases and their aetiological agents that are included in the Aquatic Animal Health Code (the Aquatic Code) have a
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
77
Appendix VIII cont.
restricted geographical range, have no therapeutic remedies or treatments, are potentially excludable, and are of significant
social and economic importance. For the current OIE list of diseases of crustaceans, please consult the current edition of
the Aquatic Code.]
Because of the size and importance of the penaeid shrimp aquaculture industry, the principles and
methods discussed in this chapter will, of necessity, emphasise penaeid shrimp. The taxonomy of the
penaeid shrimp was revised in 1997 (14). Penaeid subgenera were raised to being full genera. Because
this change in penaeid taxonomy has not been universally accepted, for the purposes of this Aquatic
Manual, the taxonomy of the penaeids as outlined by Holthuis (8) will be used in this Aquatic Manual.
[Section 2 below has been moved to section C.5
2. DIAGNOSTIC METHODS
The methods available for diagnosis of the above-listed diseases include the traditional methods of morphological pathology
(direct light microscopy, histopathology, and electron microscopy), bioassay methods with susceptible indicator hosts, and
molecular methods (gene probes and polymerase chain reaction [PCR]). While tissue culture is considered to be a standard
tool in medical, veterinary, and fish diagnostic laboratories, it has yet to be developed as a usable, routine diagnostic tool for
crustacean pathogens. Clinical chemistry has not become a routinely used diagnostic tool by crustacean pathologists.
2.1.
Diagnostic methods for diseases of crustaceans
At the time of writing this section of the Aquatic Manual, the available diagnostic methods that may be selected for
diagnosis of the OIE listed crustacean diseases or detection of their aetiological agents are based on:
·
Gross and clinical signs
·
Direct bright-field, phase-contrast or dark-field microscopy with whole stained or unstained tissue wet-mounts,
tissue squashes, and impression smears; and wet-mounts of faecal strands
·
Histology of fixed specimens
·
Bioassays of suspect or subclinical carriers using a highly susceptible host (life stage or species) as the
indicator for the presence of the pathogen
·
Transmission or scanning electron microscopy
·
Antibody-based tests for pathogen detection using immune sera polyclonal antibodies (PAbs) or monoclonal
antibodies (MAbs)
·
Molecular methods
DNA probes in dot-blot hybridisation assays directly with fresh tissue samples or with extracted DNA
DNA probes or RNA probes for in situ hybridisation assays with histological sections of fixed tissues
Standard and real-time PCR and reverse-transcription (RT)-PCR for direct assay with fresh tissue samples or
with extracted DNA or RNA.
The detailed procedures for each of the available methods (screening, presumptive, and confirmatory) for diagnosis of
each of the OIE listed crustacean diseases are outlined in the individual disease chapters of this Aquatic Manual.
There is a paucity of antibody-based diagnostic tests available for the pathogens that cause crustacean diseases. As
crustaceans do not produce antibodies, antibody-based diagnostic tests are limited in their application to pathogen
detection. While a number of antibody-based diagnostic methods have been developed and are described in the
literature, these were developed with mouse or rabbit antibodies generated to viruses purified from infected hosts.
Because crustacean viruses (and the necrotising hepatopancreatitis [NHP] bacterium) cannot be routinely cultured in
vitro (i.e. produced in tissue culture), purified virus from infected hosts must be used to produce antibody. This has
severely limited the development and availability of this diagnostic tool. The recent application of MAb technologies to
this problem has begun to provide a few antibody-based tests. MAbs are available for the agents of several of the OIE
listed crustacean diseases (White spot syndrome virus [WSSV], Taura syndrome virus [TSV], yellowhead virus [YHV],
infectious hypodermal and haematopoietic necrosis virus [IHHNV], and the necrotising hepatopancreatitis bacterium
[NHP-B]). Antibody-based diagnostic kits/reagents for TSV, WSSV, YHV, NHP-B infections are currently available
from commercial sources.
Molecular methods have been developed and some methods are in widespread use for the detection of many of the
viral, bacterial, and protozoan pathogens of the penaeid shrimp. Nucleic acid-based detection methods are readily
available from the literature and some are available in kit form from commercial sources for the OIE listed pathogens
TSV, WSSV, and yellowhead disease virus (YHV/GAV), IHHNV, Penaeus monodon-type baculovirus (MBV),
78
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VIII cont.
Baculovirus penaei (BP), and infectious myonecrosis virus (IMNV). PCR or RT-PCR methods are available for all of
these viruses and are in routine use by certain sectors of the crustacean aquaculture industry. For the agents of other
OIE listed diseases, specific DNA probes tagged with nonradioactive labels are either reported in the literature or
available commercially for application in dot-blot formats with haemolymph or tissue extracts, or for use with routine
histological sections using in-situ hybridisation.]
C. SAMPLING
1. SAMPLING
OBJECTIVES
There are at least three purposes for which crustaceans stocks may be sampled. These are:
1) surveillance; 2) issuing health certificates and 3) disease outbreak investigation. The number and type
of samples to be taken for analysis and the sampling procedure varies greatly according to which of
these purposes applies.
[There are at least three purposes for which crustacean stocks may be sampled with regard to the OIE listed crustacean
diseases. These are: 1) surveillance; 2) stock or facility ‘certification’; and 3) disease diagnosis. The number and type of
samples to be taken for analysis varies greatly according to which of these purposes applies.
A general approach to surveillance and sampling is given in chapter 1.1.4 of this Aquatic Manual. The sampling should be
designed to enable detection, at a 95% confidence level, of infected animals. The following section gives information
relevant to sampling crustaceans. For those diagnostic tests where the sensitivity and specificity have been established,
sample size may be determined using methods such as FreeCalc (www.ausvet.com.au) or similar programs as outlined in
Chapter 1.1.4 Requirements for surveillance for international recognition of freedom from infection. However, for those
diagnostic tests where the values for sensitivity and specificity have not been established, the default sample size should be
determined from Table 1 in the present chapter.]
2. NUMBER
OF EPIDEMIOLOGICAL UNITS TO BE SAMPLED
Sample sizes for surveillance or for issuing health certificates should be calculated using the provisions
in chapter 1.1.4 of this Aquatic Manual.
When conducting disease outbreak investigations, the number of cases and non-cases will depend on
the case definition to be found in the appropriate chapter of the Aquatic Manual. If a case definition is
not available, a case definition describing the disease of interest as accurately as possible should be
developed and the number and status of units to be sampled should be determined in accordance with
that case definition.
3. SAMPLING
PROTOCOL
3.1. Selection of epidemiological units and sampling methods
Add some more information on different methods (castnet, feedtray, harvest) to be used for
sampling and discuss potential bias of each method.
3.2. Diagnosis in disease situations
In clinical disease episodes, carefully selected quality specimens with representative lesions should
be obtained from live or moribund crustaceans. Every effort should be made to sample those
specimens for diagnosis that are representative of the disease(s) that is (are) affecting the
crustacean stock of interest, and that are moribund or clinically diseased. Collection of dead
specimens should be avoided. When cultured or wild crustacean stocks are presenting clinical
signs of an active disease that are consistent with, or suggestive of, any one of the OIE listed
crustacean diseases, care should be taken to ensure that the samples collected are preserved
appropriately for the anticipated diagnostic tests (see sample preservation section for
recommended methods).
The recommended minimum numbers of specimens to collect for diagnostic testing are 100 for
the larval stages of most crustaceans; 50 for the postlarval stages; and 10 for juveniles and adults.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
79
Appendix VIII cont.
Sample numbers may be greater if clinically diseased specimens are readily apparent and collected.
Nonetheless, these recommended ‘minimum’ sample numbers are provided as guidelines, and it
must be emphasised that carefully selected, quality specimens are far more valuable (and costeffective) diagnostic specimens than dozens or hundreds of specimens taken at random to ‘fill
out’ the sample.
3.3. Diagnosis in subclinical crustacean carriers
When samples are to be taken for surveillance, for testing of subclinical carriers of previous
disease epizootics, for ‘certification’ of specific pathogen free (SPF) status, or for freedom of
particular disease within a country, zone, or facility the sample size to be taken should be
determined using the provisions reported in Chapter 114 [statistical table or a program such as FreeCalc.
The minimum sample size for each lot tested should provide a 95% level of confidence that infected specimens, if
present, will be in the sample, assuming a defined minimum prevalence of infection equal or greater than 2%, 5% or
10%. For surveillance and certification purposes for OIE listed diseases, the samples taken for diagnostic tests at any
given aquaculture site or from wild stocks, should include the appropriate number of specimens from each lot to be
tested according to Table 1 (or to the number calculated by FreeCalc, if applicable)].
For the OIE listed diseases it is highly recommended that the scheduling of sampling be planned
(i.e. by farm schedule, season, etc.) so that the particular life-stage(s) are sampled at a time when
the pathogen of concern is most likely to be detected. This is especially important when the
available diagnostic methods are dependent on simple microscopy or histological methods and do
not include molecular methods. For the baculoviruses BP and MBV larval and early postlarval are
the most appropriate samples; for TSV, IHHNV, WSSV, IMNV, NHP-B and YHV/GAV,
juveniles and subadults provide the best samples; and for crayfish plague, juveniles and adults are
suitable samples.
Samples taken for molecular or antibody-based tests for OIE-listed crustacean diseases may be
combined as pooled samples of no more than five specimens per pooled sample.
[3.3.
Testing for verification or maintenance of freedom from specific diseases
Once a crustacean production facility has been recognised to be free of all or certain diseases listed in the Aquatic
Code after 2 years of surveillance with laboratory tests and in the absence of any suspect clinical signs, twice-yearly
inspections should continue. However, collection of specimens for testing may be reduced to 30 crustaceans
(shrimp), including especially broodstock. Moribund shrimp observed during inspection visits must, however, be
collected for further laboratory examination
Table 1. Random sample size based on assumed pathogen prevalence in lot
and assuming 100% sensitivity and specificity of the technique
Lot size
At 2% prevalence,
size of sample
At 5% prevalence,
size of sample
At 10% prevalence,
size of sample
50
50
35
20
100
75
45
23
250
110
50
25
500
130
55
26
1000
140
55
27
1500
140
55
27
2000
145
60
27
4000
145
60
27
10,000
145
60
27
100,000 or more
150
60
30
After Ossiander & Wedemeyer, 1973.]
80
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VIII cont.
4. POST-SAMPLING
PROCEDURES
[SAMPLE TYPE AND PRESERVATION]
4.1. Samples for direct microscopy
Samples for direct microscopic examination should be examined as soon as possible after
collection. Use live specimens whenever possible, or use fresh, chilled, or 10% buffered formalinfixed specimens when live specimens are not practical. If an adequate field laboratory is available,
it should be used to process and examine samples near the site of collection.
4.2. Samples for histology
Collect shrimp by whatever means are available with a minimum of handling stress. Transport the
shrimp to the laboratory via a well oxygenated water-filled utensil. Supply adequate aeration to the
container if the shrimp are to be left for a short period of time before actual fixation. For the
study of presumably diseased shrimp, select those shrimp that are moribund, discoloured,
displaying abnormal behaviour, or otherwise abnormal, except in the case of intentional random
sampling for estimation of disease prevalence.
i)
Have ready an adequate supply of fixative. A general rule is that a minimum of ten volumes
of fixative should be used for one volume of tissue sample (i.e. a 10 g sample of shrimp
would require 100 ml of fixative).
ii)
Davidson’s AFA (alcohol, formalin, acetic acid) fixative
Davidson’s AFA fixative is recommended for most histological applications. The fixative is
rapid, reduces autolytic changes in tropical crustaceans (i.e. the penaeid shrimp), and its
acidic content decalcifies the cuticle. The formulation for Davidson’s AFA is (for 1 litre):
330 ml 95% ethyl alcohol
220 ml 100% formalin* (a saturated 37–39% aqueous solution of formaldehyde gas)
115 ml glacial acetic acid**
335 ml tap water (for marine crustaceans, sea water may be substituted)
Store the fixative in glass or plastic bottles with secure caps at room temperature.
*
Do not use previously made 10% formalin to prepare Davidson’s AFA because the
formalin content of the Davidson’s AFA will be inadequate to provide satisfactory
fixation.
**
Do not substitute other acids, such as HCl, for acetic acid. Histological sections
prepared from HCl–Davidson’s solution are not suitable for routine haematoxylin and
eosin histological staining.
iii) Fixation procedures with Davidson’s AFA
·
For larvae and postlarvae that are too small to be easily injected with fixative using a tuberculin
syringe: Using a fine mesh screen or a Pasteur pipette, select and collect specimens.
Immerse shrimp selected for sampling directly in the fixative. Fix for 12–24 hours in
fixative, then transfer to 50–70% ethyl alcohol for storage.
·
For larger postlarvae and very small juveniles that are too small to be injected: Select and collect
specimens as described in Section 3. Use a needle or fine-pointed forceps to incise the
cuticle. Immerse shrimp selected for sampling directly in the fixative. Fix for 12–24
hours in fixative, then transfer to 50–70% ethyl alcohol for storage.
·
For larger postlarvae, juveniles, and adults: Inject fixative (use 5–10% volume: weight) via
needle and syringe (needle gauge dependent on shrimp size, i.e. 27 gauge needle for
postlarvae and small juveniles) into the living shrimp.
The hepatopancreas (HP) should be injected first and at two or more sites, with a
volume sufficient to change the HP to a white to orange colour; then inject fixative into
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
81
Appendix VIII cont.
adjacent regions of the cephalothorax, into the anterior abdominal region, and into the
posterior abdominal region.
The fixative should be divided between the different regions, with the cephalothorasic
region, specifically the HP, receiving a larger share than the abdominal region.
A good guide to insure adequate fixation is to inject an equivalent of 5–10% of the
shrimp’s (or other crustacean’s) body weight; all signs of life should rapidly cease, and
visible colour change should occur in the injected areas.
Immediately following injection, slit the cuticle, with dissecting scissors, from the sixth
abdominal segment to the base of the rostrum, being particularly careful not to cut
deeply into the underlying tissue. The incision in the cephalothorasic region should be
just lateral to the dorsal midline, while that in the abdominal region should be
approximately mid-lateral.
·
For shrimp (and most other crustaceans) larger than ~12 g: After injection of fixative, the
body should then be transversely bisected, at least once, just posterior to the
abdomen/cephalothorax junction, and (optional) again mid-abdominally.
·
For very large crustaceans and crabs: The organs of interest may be excised after injection
of fixative. Completion of fixation of these tissue samples is then handled as outlined
previously.
Following injection, incisions and bisection/trisection, or excision of key organs,
immerse the specimen in the fixative (use 10:1 fixative:tissue ratio).
Allow fixation to proceed at room temperature for 24–72 hours depending on the size
of shrimp (or crustacean) being preserved. Longer fixation times in Davidson’s AFA
may be used to thoroughly decalcify the shell of crabs, lobsters, crayfish, etc.
Following fixation, the specimens should be transferred to 70% ethyl alcohol, where
they can be stored for an indefinite period.
Record a complete history of the specimens at the time of collection: gross observations
on the condition of the shrimp (or other crustacean), species, age, weight, source (wild,
or if culture pond or tank number, stock number, etc.), and any other pertinent
information that may be needed at a later time.
The label should stay with the specimens in the same container during fixation, storage
and transport to the laboratory. Always use No. 2 soft-lead pencil on water-resistant
paper (plastic paper is recommended; never use ink or marking pens as the ink is
dissolved by alcohol).
iv) Transport and shipment of preserved samples
Because large volumes of alcohol should not be posted or shipped, the following methods
are recommended: Remove the specimens from the 70% ethyl alcohol. For larvae,
postlarvae, or small juveniles, use leak-proof, screw-cap plastic vials if available; if glass vials
must be used, pack to prevent breakage. For larger specimens, wrap samples with white
paper towels to completely cover (do not use raw cotton). Place towel-wrapped specimens in
a sealable plastic bag and saturate with 70% ethyl alcohol. Insert the label and seal the bag.
Place the bag within a second sealable bag. Multiple small sealable bags can again be placed
within a sturdy, crush-proof appropriately labelled container for shipment (see Chapter 1.5.6
of the Aquatic Code for details).
4.3. Preservation of samples for antibody, DNA probe dot-blot tests, or polymerase chain
reaction
For routine diagnostic testing by PCR, RT-PCR or for dot-blot tests with DNA probes, samples
must be prepared to preserve the pathogen’s nucleic acid. Likewise, samples intended for testing
with antibody-based methods must be preserved to retain reactive antigenic sites for the
antibodies used.
82
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VIII cont.
· 4.3.1. Sample types
Samples selected for nucleic acid-based or antibody-based diagnostic tests should be handled
and packaged (in new plastic sample bags or bottles) with great care to minimise the potential
for cross contamination among the sample set taken from different (wild or farmed) stocks,
from tanks, ponds, farms, etc. New plastic sample bags or bottles must be used. A waterresistant label, with the appropriate data filled out in No. 2 pencil, should be placed within
each package or container for each sample set.
Some suitable methods for preservation and transport of samples taken for molecular or
antibody-based tests are:
·
Live specimens: These may be processed in the field or shipped to the diagnostic laboratory
for testing.
·
Haemolymph: This tissue is the preferred sample for certain molecular and antibody-based
diagnostic tests. Samples may be collected by needle and syringe by cardiac puncture,
from the haemocoel (i.e. the ventral sinus in penaeids), or from a severed appendage.
·
Iced or chilled specimens: This is for specimens that can be transported to the laboratory for
testing within 24 hours. Pack samples in sample bags surrounded by an adequate quantity
of wet ice around the bagged samples in a Styrofoam™-insulated box and ship to the
laboratory.
·
Frozen whole specimens: Select live specimens according to the criteria listed in Section 3,
quick freeze in the field using crushed dry-ice, or freeze in the field laboratories using a
mechanical freezer at –20°C or lower temperature. Prepare and insert the label into the
container with the samples, pack samples with an adequate quantity of dry-ice in a
Styrofoam™-insulated box, and ship to the laboratory.
·
Alcohol-preserved samples: In regions where the storage and shipment of frozen samples is
problematic, 90–95% ethanol may be used to preserve, store, and transport certain types
of samples. Whole crustaceans (any life stage provided the specimen is no larger than 2–
3 g), excised tissues (i.e. pleopods) from large crustaceans, or haemolymph may be
preserved in 90–95% ethanol, and then packed for shipment according to the methods
described in Section 4.2.v (see Chapter 1.5.6 of the Aquatic Code for details).
·
Preservation of RNA and DNA in tissues using RNAlater: Tissue is cut to be less than
0.5 cm in one dimension and submerged in 5 volumes of RNAlater (e.g. a 0.5 g sample
requires about 2.5 ml of RNAlater). Small organs such as kidney, liver and spleen can be
stored whole in RNAlater. These samples can be stored at 4°C for one month, at 25°C
for 1 week or at –20°C indefinitely. Archive RNAlater-treated tissues at –20°C.
5. ASSESSING
THE HEALTH STATUS OF THE EPIDEMIOLOGICAL UNIT
The methods available for diagnosis of the above-listed diseases include the traditional methods of
morphological pathology (direct light microscopy, histopathology, and electron microscopy), bioassay
methods with susceptible indicator hosts, and molecular methods (gene probes and polymerase chain
reaction [PCR]). While tissue culture is considered to be a standard tool in medical, veterinary, and fish
diagnostic laboratories, it has yet to be developed as a usable, routine diagnostic tool for crustacean
pathogens. Clinical chemistry has not become a routinely used diagnostic tool by crustacean
pathologists.
5.1. Diagnostic methods for diseases of crustaceans
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
83
Appendix VIII cont.
At the time of writing this section of the Aquatic Manual, the available diagnostic methods that
may be selected for diagnosis of the OIE listed crustacean diseases or detection of their
aetiological agents are based on:
·
Gross and clinical signs
·
Direct bright-field, phase-contrast or dark-field microscopy with whole stained or unstained
tissue wet-mounts, tissue squashes, and impression smears; and wet-mounts of faecal strands
·
Histology of fixed specimens
·
Bioassays of suspect or subclinical carriers using a highly susceptible host (life stage or
species) as the indicator for the presence of the pathogen
·
Transmission or scanning electron microscopy
·
Antibody-based tests for pathogen detection using immune sera polyclonal antibodies
(PAbs) or monoclonal antibodies (MAbs)
·
Molecular methods
DNA probes in dot-blot hybridisation assays directly with fresh tissue samples or with
extracted DNA
DNA probes or RNA probes for in situ hybridisation assays with histological sections of
fixed tissues
Standard and real-time PCR and reverse-transcription (RT)-PCR for direct assay with fresh
tissue samples or with extracted DNA or RNA.
The detailed procedures for each of the available methods (screening, presumptive, and
confirmatory) for diagnosis of each of the OIE listed crustacean diseases are outlined in the
individual disease chapters of this Aquatic Manual.
There is a paucity of antibody-based diagnostic tests available for the pathogens that cause
crustacean diseases. As crustaceans do not produce antibodies, antibody-based diagnostic tests are
limited in their application to pathogen detection. While a number of antibody-based diagnostic
methods have been developed and are described in the literature, these were developed with
mouse or rabbit antibodies generated to viruses purified from infected hosts. Because crustacean
viruses (and the necrotising hepatopancreatitis [NHP] bacterium) cannot be routinely cultured in
vitro (i.e. produced in tissue culture), purified virus from infected hosts must be used to produce
antibody. This has severely limited the development and availability of this diagnostic tool. The
recent application of MAb technologies to this problem has begun to provide a few antibodybased tests. MAbs are available for the agents of several of the OIE listed crustacean diseases
(White spot syndrome virus [WSSV], Taura syndrome virus [TSV], yellowhead virus [YHV],
infectious hypodermal and haematopoietic necrosis virus [IHHNV], and the necrotising
hepatopancreatitis bacterium [NHP-B]). Antibody-based diagnostic kits/reagents for TSV, WSSV,
YHV, NHP-B infections are currently available from commercial sources.
Molecular methods have been developed and some methods are in widespread use for the
detection of many of the viral, bacterial, and protozoan pathogens of the penaeid shrimp. Nucleic
acid-based detection methods are readily available from the literature and some are available in kit
form from commercial sources for the OIE listed pathogens TSV, WSSV, and yellowhead disease
virus (YHV/GAV), IHHNV, Penaeus monodon-type baculovirus (MBV), Baculovirus penaei (BP),
and infectious myonecrosis virus (IMNV). PCR or RT-PCR methods are available for all of these
viruses and are in routine use by certain sectors of the crustacean aquaculture industry. For the
agents of other OIE listed diseases, specific DNA probes tagged with nonradioactive labels are
either reported in the literature or available commercially for application in dot-blot formats with
84
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix VIII cont.
haemolymph or tissue extracts, or for use with routine histological sections using in-situ
hybridisation.
6. SAMPLE TYPE
7. ADDITIONAL
AND
PRESERVATION
INFORMATION TO BE COLLECTED
The geographical origin of samples should be defined by the name of the sampling site associated with
either its geographical co-ordinates or its location along a river course or body of water.
KEY REFERENCES
1.
ALDAY DE GRAINDORGE V. & FLEGEL T.W. (1999). Diagnosis of Shrimp Diseases. Food and
Agriculture Organization of the United Nations and Mulitmedia Asia, Bangkok, Thailand.
2.
BELL T.A. & LIGHTNER D.V. (1988). A Handbook of Normal Shrimp Histology. Special Publication
No. 1, World Aquaculture Society, Baton Rouge, Louisiana, USA.
3.
BONDAD-REANTASO M.G., MCGLADDERY S.E., EAST I. & SUBASINGHE R.P. (2001). Asian
Diagnostic Guide to Aquatic Animal Diseases. FAO Fisheries Technical Paper, No. 402, supplement 2.
Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 240 pp.
4.
BROCK J.A. & MAIN K. (1994). A Guide to the Common Problems and Diseases of Cultured Penaeus
vannamei. Published by the Oceanic Institute, Makapuu Point, P.O. Box 25280, Honolulu, Hawaii,
USA.
5.
CHANRATCHKOOL P., TURNBULL J.F., FUNGE-SMITH S.J., MACRAE I.H. & LIMSUWAN C. (1998).
Health Management in Shrimp Ponds. Aquatic Animal Health Research Institute, Department of
Fisheries, Kasetsart University Campus, Jatujak, Bangkok, Thailand, 152 pp.
6.
JOHNSON P.T. (1980). Histology of the Blue Crab, Callinectes sapidus. A Model for the Decapoda.
Prager, New York, USA, 440 pp.
7.
JOHNSON S.K. (1995). Handbook of Shrimp Diseases. TAMU-SG-90-601(r). Texas A&M Sea Grant
College Program, Texas A&M University, College Station, Texas, USA, 26 pp.
8.
HOLTHUIS L.B. (1980). FAO Species Catalogue. Vol. 1 - Shrimp and Prawns of the World. FAO
Fisheries Synopsis No. 125, FAO, Rome, Italy, 271 p.
9.
LIGHTNER D.V. (1996). A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases
of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, Louisiana, USA. 304 p.
10. LIGHTNER D.V. (1996). The penaeid shrimp viruses IHHNV and TSV: epizootiology, production
impacts and role of international trade in their distribution in the Americas. Rev. sci. tech. Off. int.
Epiz., 15, 579–601.
11. LIGHTNER D.V. & REDMAN R.M. (1998). Shrimp diseases and current diagnostic methods.
Aquaculture, 164, 201–220.
12. LIGHTNER D.V. & REDMAN R.M. (1998). Strategies for the control of viral diseases of shrimp in the
Americas. Fish Pathol., 33, 165–180.
13. LOTZ J.M. (1997). Special topic review: Viruses, biosecurity and specific pathogen-free stocks in
shrimp aquaculture. World J. Microbiol. Biotechnol., 13, 405–413.
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
85
Appendix VIII cont.
14. PEREZ FARFANTE I. & KENSLEY B.F. (1997). The Penaeoid and Sergestoid Shrimps and Prawns of
the World: Keys and Diagnosis for the Families and Genera. Editions du Museum national d’Histoire
Naturelle, Paris, France, 175, 1–233.
5
REDDINGTON J. & LIGHTNER D.V. (1994). Diagnostics and their application to aquaculture. World
Aquaculture, 25, 41–48.
*
* *
86
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IX
Aquatic Manual disease chapter template July 2006
CHAPTER
XXX.
DISEASE NAME
1.
CASE
DEFINITION
(Please start this chapter with a simple definition of the disease)
“For the purpose of this chapter, DISEASE NAME is considered to be INFECTION WITH
PATHOGEN NAME.”
Case definition for infection, for diseased animal and for disease holding unit
Case definition in endemic situations and in zero prevalence situations
2.
INFORMATION
FOR THE DESIGN OF SURVEILLANCE PROGRAMMES
(Information on some of the following items is required for the design of surveillance programmes as
described in Chapter 1.1.4 of the Aquatic Manual. Information on other items is needed to provide
details on possible risk management and disease control measures as described in the Aquatic Code.
There is no need to split the sections a) to d) into subsections to address each item. Also, it is
acknowledged that it will not be possible to provide – and reference – scientific data on each of these
items for each disease; some aspects will be well covered, while there will be a dearth of information
on others. Authors may wish to draw attention to areas where there is a significant lack of
information.)
For the following factors, what are the high-risk groups? Age, season, water temperature
How do these factors affect sensitivity and specificity? Age, season, water temperature
What is the preferred test for each of those categories?
Sensitivity and specificity estimates from previous studies – how were the evaluations made and what
situations do they concern?
Are there any tests that have proven to be useful in environments rather than hosts? Equipment, water
or soil samples and intermediate hosts/carrier
Can samples be preserved and how?
Incubation time
Opportunities for evidence collection and/or sampling at different points in the production cycle
(alternative sources of evidence)
a)
Agent factors
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
87
Appendix IX cont.
• Aetiological agent, agent strains
• Survival outside the host (i.e. in the natural environment)
• Stability of the agent (describe effective inactivation methods)
b)
• Life cycle
Host factors
• Susceptible host species (common and Latin names)
• Susceptible stages of the host
• Species or sub-population predilection (probability of detection)
• Target organs and infected tissue
• Persistent infection with lifelong carriers
• Vectors
c)
• Known or suspected wild fish carriers
Disease pattern
• Transmission mechanisms, coefficient of transmission, animal to animal, pond to pond, farm
to farm, etc., under different conditions and through different pathways. When introduced
into a naïve farm or area, does it have an obvious disease pattern (temporal/spatial
distribution)?
• If detailed information on transmission is not available, provide qualitative information on the
likelihood of transmission to occur under different situations (e.g. if one infected fish is
introduced into a pond, what proportion of the population would be expected to be infected
at different points in time)
• Within fish spread of the pathogen depending on different points of entry and under different
conditions
• If available, can a causal web or quantified risk factors be provided? Risk factors means factors
that either increase or decrease the risk
• What management practices increase or decrease the risk?
• Sources of stock – are they bought from outside, are they tested
• Prevalence (describe commonly observed prevalence in wild and farmed populations for the
detection method used, under different conditions)
• Geographical distribution
• Mortality and morbidity
d)
• Economic and/or production impact of the disease
Control and prevention
• Vaccination
• Chemotherapy
• Immunostimulation
• Resistance breeding
• Restocking with resistant species
• Blocking agents
• General husbandry practices
3.
88
SAMPLING
FOR SURVEILLANCE PURPOSES
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IX cont.
Surveillance to demonstrate the absence of disease or infection
- Sampling frequency and duration (to establish freedom and to maintain status)
- Risk factors that promote introduction of agents (e.g. isolation and avoiding exposure might lead
lower requirement for continuous sampling)
- Optimum water temperature
- Optimum age range or development stage of fish
- Selection of individual specimens
- Preservation of samples for submission
- Number of fish to be sampled
- Best organs or tissues
Surveillance to determine the occurrence or distribution of disease or infection
- Comment on fish / tissues that are not appropriate (i.e. when it is never possible to detect
- Priority areas for testing (fish; tissues, etc to be sampled for prevalence comparisons)
- Optimal diagnostic test combinations to determine prevalence (include ranking of costs)
4.
DIAGNOSTIC
METHODS
(Please provide a description of diagnostic methods.
The diagnostic methods should include the entire gambit of a disease investigation in a population or
an individual animal, i.e. provide descriptions of the clinical, histological etc picture, and not simply the
agent detection methods. In previous editions of the Aquatic Manual, this information was often
captured in the introduction.
It is acknowledged that not all methods listed will be applicable to all diseases. Only the ones that are
appropriate should be listed and described)
a)
Field diagnostic methods
(This includes observation of the animal and its environment, and gross clinical examinations)
b)
• Clinical signs
• Behavioural changes: specify which animals are likely to be affected or not affected by the
disease or infection within a tank? If different for acute or chronic conditions, provide details.
Clinical methods
(This includes methods that focus on the effects of the pathological agent on the host, rather than
on agent detection)
• Gross pathology
• Clinical chemistry
• Microscopic pathology
• Wet mounts
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
89
Appendix IX cont.
• Smears
• Fixed sections
c)
• Electron microscopy/cytopathology
Agent detection and identification methods
(This includes methods that detect, possibly isolate and amplify, and identify the agent. For each
method, information on the items in the text box on the right hand side should be provided. This
information is required to allow the reader to follow the technique, but also to provide the
necessary data – e.g. specificity and sensitivity – that are required for the development of a
sampling and surveillance programme.)
• Direct detection methods
i) Microscopic methods
• Wet mounts
• Smears
• Fixed sections
ii) Agent isolation and identification
• Cell culture/artificial media
• Antibody-based antigen detection methods (IFAT, ELISA, ….)
• Molecular techniques (PCR, ISH, sequencing….)
• Agent purification
• Indirect detection methods
• Serological methods
⇒ Samples to be taken
⇒ Technical procedure
ƒ
How to
controls
use
positive/negative
⇒ Levels of validation
ƒ
Specificity and sensitivity
ƒ
‘Gold’ standard
⇒ Interpretation of results
⇒ Availability
of test (from Reference
Laboratories, commercial sources or easily
synthesised)
4.
90
RATING
OF TESTS AGAINST PURPOSE OF USE
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix IX cont.
(This information is needed to determine which test is appropriate for various purposes. For example,
a particular method may be highly suitable to diagnose clinical cases of disease in individual animals of
a certain age group, but the same method may be rather unsuitable for assessing the infection status of
large numbers of clinically healthy animals. It is an assessment of the test’s ‘fitness for purpose’.)
The methods currently available for surveillance, detection, and diagnosis of WSSV are listed in Table
1. The designations used in the Table indicate: A = the method is the recommended method for
reasons of availability, utility, and diagnostic specificity and sensitivity; B = the method is a standard
method with good diagnostic sensitivity and specificity; C = the method has application in some
situations, but cost, accuracy, or other factors severely limits its application; and D = the method is
presently not recommended for this purpose. These are somewhat subjective as suitability involves
issues of reliability, sensitivity, specificity and utility. Although not all of the tests listed as category A or
B have undergone formal standardisation and validation (at least stages 1 and 2 of Figure 1 of chapter
1.1.2), their routine nature and the fact that they have been used widely without dubious results, makes
them acceptable.
Table 1. WSV surveillance, detection and diagnostic methods
Surveillance
Method
Presumptiv
e
Confirmator
y
Larvae
PLs
Juveniles
Adults
Gross signs
D
D
C
C
C
D
Bioassay
D
D
D
D
C
B
Direct LM
D
D
C
C
C
C
Histopathology
D
C
C
C
A
A
Transmission EM
D
D
D
D
D
A
Antibody-based assays
D
D
C
C
A
B
DNA Probes - in situ
D
D
C
C
A
A
PCR
D
B
A
A
A
A
Sequence
D
D
D
D
D
A
PLs = postlarvae; LM = light microscopy; EM = electron microscopy; PCR = polymerase chain reaction.
5.
CORROBORATIVE
DIAGNOSTIC CRITERIA
(Please define, based on the information provided in 1.-4., what constitutes a suspect case of disease,
and a confirmed case of disease. This information is required, for example for the purpose of disease
investigations, especially in cases where ‘free’ status is threatened. It would also be required when
surveillance of healthy populations yields controversial results, for example, positive PCR signals in the
absence of any other evidence of infection.
The definitions of ‘suspect’ and ‘confirmed’ will most likely be a combination of positive results from a
range of different methods as described under 3.
For example, a certain level of mortality at the right time of the year (see under 2b), in susceptible
animals (see under 2a.), together with matching clinical signs (see under 3a), liver lesions and
histopathology (see under 3b) could be sufficient for suspicion of DISEASE X. Several combinations
may be possible. A confirmed case could be defined where in addition to the above, the agent has been
detected. However, detection of viable agents without any disease signs could also constitute a
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
91
Appendix IX cont.
confirmed case. The definitions may differ between different species and may depend on whether case
is outside the known geographical/host range.
In accordance with Article VVVV of the Aquatic Code, all cases in other species should be referred
immediately to the appropriate OIE Reference Laboratory for confirmation, whether on not clinical
signs are associated with the case.)
a)
Definition of suspect case
b)
6.
Definition of confirmed case
PRESCRIBED
DIAGNOSTIC/DETECTION METHODS TO DECLARE FREEDOM
(Please prescribe the methods, based on the information provided in 1.-3., and assessed in 4., for
targeted surveillance to declare freedom from infection as outlined in the Aquatic Code)
REFERENCES
(Please provide a list key references that confirm the information in the chapter and references that
provide useful additional information. References should be to documents that are readily accessible.)
*
* *
92
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
Appendix X
Steps in a surveillance system
1.
Clearly define the objective and measurable outcomes
Prioritise the diseases to be included in the surveillance system (e.g. using a risk based approach)
2.
Define the populations (reference, target, study, etc.)
3.
Develop a case definition for each outcome
4.
Define the sources of data
-
-
Active
-
sampling strategy (frequency, population(s), what kinds of observations, collection of samples
and diagnostic tests),
-
training of people who will be gathering the data,
-
strictly defined procedures for data collection (standardisation of data collection, instruments of
measurement, exposure definition),
-
decide whether you want to collect data on risk factors.
Passive
-
Who are the eyes of the system (vets, farmers, laboratories)? What should they do if they see
something?
-
Education.
5.
Allocation of responsibilities
6.
Allocation of resources
7.
Flow of information
8.
Compilation of data at each level and presentation at the next level
9.
Data quality (cross check with other sources)
10. Actions at each level
11. Overall evaluation of results
12. Feedback
13. Link outcome of surveillance system to contingency plans
Ad hoc Group on Aquatic Animal Health Surveillance/July 2006
93
© World Organisation for Animal Health (OIE), 2006
This document has been prepared by specialists convened by the OIE. Pending adoption by the International Committee of
the OIE, the views expressed herein can only be construed as those of these specialists.
All OIE (World Organisation for Animal Health) publications are protected by international copyright law. Extracts may be
copied, reproduced, translated, adapted or published in journals, documents, books, electronic media and any other medium
destined for the public, for information, educational or commercial purposes, provided prior written permission has been
granted by the OIE.
The designations and denominations employed and the presentation of the material in this publication do not imply the
expression of any opinion whatsoever on the part of the OIE concerning the legal status of any country, territory, city or area
or of its authorities, or concerning the delimitation of its frontiers and boundaries.
The views expressed in signed articles are solely the responsibility of the authors. The mention of specific companies or
products of manufacturers, whether or not these have been patented, does not imply that these have been endorsed or
recommended by the OIE in preference to others of a similar nature that are not mentioned.