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Diagnosing disease

Before developing surveillance and zoning programmes, it is important to understand the principles behind surveillance of animal populations (Cameron 2002). Understanding different types of diagnosis and establishment of clear case definitions (criteria for an infection constituting a disease concern) are essential. Each of these is influenced by the nature of the disease under investigation, notably infection characteristics, local environmental factors influencing virulence, related human activities, and reliability (specificity/sensitivity) of available diagnostic tools.

Disease, in the classic medical sense, includes non-infectious diseases, but for most disease control programmes (national and international), infectious diseases are the focus of attention. Non-infectious disease management is generally the responsibility of farmers or local extension/field officers.

In the context of infectious disease control, animals may be considered as being “diseased” as soon as they become infected, even before clinical signs or pathological changes become evident. This is also true for carrier or reservoir hosts, that may carry and transmit viable infectious agents, but themselves exhibit no detectable pathology or signs of the disease in question. This means that case definitions are very important in the context of design of effective surveillance programs. For example, surveillance for clinical disease is significantly less complicated than screening healthy (non-clinical) infected or carrier hosts. However, in order to prevent disease or define the health status of aquatic animal populations as accurately as possible within defined zones, both clinical and non-clinical hosts need to be included.

The logistics of surveillance of healthy populations deemed susceptible to significant pathogens has recently come into question. The European Union (EU) and the OIE AAHSC are revisiting the cost-benefit of this all-inclusive effort under the assumption that, if the host population is indeed susceptible, and the disease agent is indeed highly virulent, then the first sign of the presence of the infection should be clinical disease. The debate over this assumption hinges on the fact that host-pathogen-environment interactions are complex, and some degree of risk of genetic or environmental suppression, is inherent in this assumption. Furthermore, clinical expression of disease may not be readily apparent, as is the case for many molluscan diseases. As epidemiological knowledge of significant diseases increases, however, the risk of missing or overlooking infected animals should be reduced.

Clinicians and pathologists devote substantial time and effort in arriving at the “correct” conclusive diagnosis when investigating disease. Competent investigators use judgment based on thorough knowledge of the literature, experience, diagnostic test results and, where appropriate, cross checks, as well as case-history observations (often provided by field personnel) to interpret their results and observations and reach an accurate diagnostic conclusion or result.

Table 2 lists a number of diagnostic methods that are applied to aquatic animal diseases. These may be used alone, or in combination, to arrive at a conclusive (confirmatory) diagnosis. All diagnostic methods, however, are subject to limitations. Random errors, due to lack of precision and non-random errors, due to false negative and false positive results, must always be taken into account. Sample sizes and collection strategies are the most commonly used methods to minimize both random and non-random diagnostic errors. Accurate quantitative assessments of the level of infection within a population (prevalence, intensity, and incidence) require further statistical analysis and correction factors to deal with sensitivity/specificity errors in diagnostic methodologies. Where infection levels become significant, general monitoring of endemic diseases (within “infected zones”) for levels exceeding a pre-defined tolerance threshold should trigger disease control action.

Table 2. Diagnostic methods routinely applied to aquatic animal diseases.

Field information

· history (recurrent losses, abnormal losses, stocking and related husbandry activities)

· economics (measure of losses due to sub-optimal production performance, or direct mortality)

· behaviour

· clinical signs

· physical examination (autopsy or gross observations)

· epidemiology (population disease dynamics)

· response to therapy (mainly finfish, some bacterial crustacean diseases)

Laboratory techniques

· microbiology

· tissue smears

· histopathology

· serology (immunological assays) (finfish)

· ultrastructural examinations (TEM, SEM, negative stain)

· tissue culture (cell-lines for certain finfish diseases only due to the current lack of cell lines for other groups)

· biomolecular analyses (PCR, ISH, etc.)

Experimental techniques

· physiology challenges (stress testing)

· transmission tests (bioassay infection challenges to assess host susceptibility)

Each investigation will yield information that can be applied to overall surveillance data gathering, and diagnosis with varying levels of certainty, depending on the complexity of the disease(s) of concern. In some instances, the investigation may not result in a conclusive diagnosis, but be limited to describing a “disease incident” (e.g. in terms of morbidity, mortality, duration of the problem, clinical signs, appearance of gross lesions). This is particularly common with aquatic animal diseases, many of which (especially microbial) are still “new” to science.

The level of diagnostic certainty will be largely determined by the investigator’s ability to recognize the characteristics of specific diseases, as well as whether or not the report needs to be followed up with a more detailed investigation by specialist expertise. In most instances, the highest level of diagnostic certainty for internationally recognized diseases is achieved when positive results are confirmed by an internationally accredited reference laboratory (usually a laboratory recognized with a high diagnostic capability in terms of facilities, staff expertise, experience and peer-reviewed scientific publications concerning a particular disease). For local or regional diseases, the same applies to confirmation by laboratories with adequate diagnostic facilities and recognized expertise on that disease.

Most first-time diagnoses of “new” or “emerging” diseases require confirmation by an independent reference laboratory with established expertise in the suspect pathogen or group of pathogens. Back up confirmation is an essential pre-requisite for any diagnosis that has significant zonation, disease control or trade implications. Even internationally recognized laboratories rely on cross-checks for diagnoses falling outside their area of specialization (e.g. exotic diseases). Thus, it is necessary to include an assessment of the diagnostic certainty (suspect, presumptive or confirmatory) with each record of a disease investigation.

Three levels of diagnosis have been defined to assist in the safe trans-boundary movement of aquatic animals and surveillance and control of their disease in the Asia-Pacific Region, however, these apply equally well to other areas of the world involved in aquatic animal disease diagnosis, since all laboratory diagnostics (whether Level II or III in technical complexity) benefit from Level I (field) information. The three levels of diagnosis are as described by Bondad-Reantaso et al. 2001 are summarized in Table 3. Level I diagnosis can be made for certain diseases at the field site without any laboratory confirmation. Most Level I information, however, is used to reinforce Level II diagnosis that requires some laboratory support. Level III diagnostic techniques require advanced laboratory infrastructure and training and are usually reserved for confirmation of diagnoses that remain presumptive at Levels I and II.

Establishing a case definition

It is important when investigating disease at a population level, that consistency of diagnosis is maintained, regardless of the diagnostic method(s) used. This involves developing a case definition, as well as undergoing field validation of the diagnostic techniques and developing a quality assurance/quality control system to ensure diagnostic consistency between diagnostic and field facilities. Failure to do so can lead to bias (non-random error) and inaccuracies in the surveillance programme. Such inaccuracies can cause significant errors in zonation and disease control decision-making, with significant disease or economic impacts.

A case definition is a set of standard criteria for deciding whether an individual study unit of interest has a particular disease or other outcome of interest. The study unit may be an individual animal or a group of animals such as a pond of shrimp, a cage of fish, a shellfish bed or an entire estuary.

For example, the investigator may be interested in comparing the occurrence of a particular disease in farmed fish in two different countries. Care is needed in such a comparison if one country uses microbiological screening techniques while the other country used observation of gross pathology alone to diagnose the same disease.

An optimal case definition depends on criteria that can be applied to any potential case in the source population. In many instances, it is difficult to define a set of criteria that includes all true cases of the disease of concern and exclude all similar, but unrelated, conditions. Few cases show the complete range of criteria attributed to a disease and there are always some “non-cases” which show clinical signs similar to those of the particular disease being investigated. This is particularly true for aquatic animal diseases, where clinical signs are rarely pathognomonic (i.e. specific to a single disease). A useful approach to development of a case definition is given by Stephen and Ribble (1996).

Table 3. Three levels of diagnostic information, associated requirements and responsibilities (Bondad-Reantaso et al. 2001).

Level - Activities

Skills and equipment



Level I - Activities
Observation of animal and the environment;
Clinical examination;
Gross pathology

Knowledge of normal (feeding, behaviour, growth of stock, etc); Frequent/regular observation of stock; Regular, consistent record-keeping and maintenance of records - including fundamental environmental information;
Knowledge contacts for health diagnostic assistance; Ability to submit and/or preserve representative specimens.

Fisheries Extension Officers;
Field Veterinarians;
Local Fisheries Biologists.

Field keys;
Farm record keeping formats;
Equipment list;
Model clinical data sheets;
Pond-side check list;
Protocols for preservation and transport of samples.

Level II - Activities

Laboratories with basic equipment and personnel trained/experienced in aquatic animal pathology; keep and maintain accurate diagnostic records; Preserve and store specimens; knowledge of/contact with different areas of specialization within Level II Knowledge of who to contact for Level III diagnostic assistance.

Fish biologists;
Aquatic Animal Veterinarians;

Model laboratory record-keeping system;
Protocols for preservation/transport of samples to Level III; Model laboratory requirements an equipment and consumable lists;
Contact information for accessing Level II and Level III specialist expertise;
Asia Diagnostic Guide to Aquatic Animal Diseases;
OIE Manual of Diagnostic Tests for Aquatic Animals;
Regional General Diagnostic Manuals.

Level III - Activities
Electron Microscopy
Molecular Biology

Highly equipped laboratory with highly specialized and trained personnel;
Keep and maintain accurate diagnostic records;
Preserve and store specimens;
Maintenance of contact with people responsible for sample submission.

Ultrastructural histopathologists;
Molecular biologists;

Model Laboratory requirements, equipment, consumable lists;
Model job descriptions skills for requirements;
Contact information for reference laboratories;
Protocols for preservation of samples for consultation and validation;
OIE Manual of Diagnostic Tests for Aquatic Animals;
General molecular and microbiology diagnostic references;
Asia Diagnostic Guide to Aquatic Animal Diseases.

Some examples of case definitions of use for investigating White Spot Disease (WSD) in shrimp are given in Table 4. The choice of a particular case definition depends on the objectives for the investigation and, no matter which case definition is used, it will not be perfect. For example, shrimp in some outbreaks of WSD can show no evidence of white spots in their carapaces, so use of the first case definition in Table 4 would produce false negative results for individual animals or stocks. False negative results are due to inadequate detection sensitivity, while false positive results are due to inadequate specific identification.

It is often necessary to define “suspect” cases, as well as confirmed cases. This is especially useful where it may take some time (e.g. weeks) to achieve diagnostic confirmation. Where a previously unrecognised and potentially serious disease is found, it is advisable to use a broad scope case definition to capture all possible cases. The definition, and associated surveillance and diagnostic protocols can be revised as more information is obtained.

Table 4. Examples of case definitions for white spot disease (WSD)[8] in shrimp.

Study Unit

Case definition


A shrimp with one or more visible, discrete white patches on the inside of the carapace.


A shrimp which yields positive PCR result for white spot syndrome virus.


A pond where one or more shrimp have one or more visible, discrete white patches on the inside of the carapace.


A pond where one or more shrimp yield a positive PCR result for white spot syndrome virus.


A pond subject to emergency harvest because, in the opinion of the manager, there is a risk of mass mortality from white spot syndrome.

Population (possible source of wild post-larvae)

A wild population where one or more shrimp have one or more visible, discrete white patches on the inside of the carapace.


A wild population where one or more shrimp yield return a positive PCR result for white spot syndrome virus.


A wild population subject to mass mortality from white spot syndrome.

Examples of presumptive (suspect) and confirmatory case definitions are given for epizootic ulcerative syndrome (EUS) in Table 5. All of the case definitions in Table 5 are legitimate for EUS, spanning the most pathogen specific, but least sensitive (first definition) to the most sensitive, but least specific (fourth definition) for individual animals.

Although still subject to debate, the consensus among experts is that EUS is a specific condition involving tissue damage due to the fungal agent, Aphanomyces piscicida/invadans, regardless of the pre-disposing factors.

Although the disease could be called aphanamycosis, implying Aphanomyces piscicida/invadans infection as the cause, other Aphanomyces spp. fungi can also cause fish lesions and disease with symptoms similar to EUS. Thus, caution is required in using genus-based “-osis” nomenclature. A classic human example of this is use of Herpes, which is now recognized to span a wide range of diseases and pathologies.

Table 5. Possible case definitions for epizootic ulcerative syndrome

Study Unit

Case definition


A fish with degeneration and hardening of the tissues of the eye-ball (necrotizing, glaucomatous dermatitis) and/or muscle tissue inflammation (myosistis) and/or localised blood cell aggregations (granulomas) in internal organs associated with the presence of Aphanomyces piscicida/invadans.


A fish with one or more granulomas with Aphanomyces piscicida/invadans in the lesion.


A fish with any lesions containing Aphanomyces piscicida/invadans.


A fish with one or more surface lesions which could be described as a “red spot”.


A pond with one or more fish meeting the descriptions above.


A river with one or more fish meeting the descriptions for individual animals.

The surveillance objective dictates the specificity or sensitivity of the case definition selected:

(i) Early detection is required because the disease has never been reported in an area and it presents a significant disease threat. Thus, any fish that may represent a case is important, and the most sensitive case definition is required - in this case “red spot”. Laboratory confirmation of the presence of Aphanomyces piscicida/invadans would be required to confirm (or refute) the presumptive diagnosis.

(ii) If an area is endemic for EUS, determining the prevalence of the condition would be important for monitoring for potential EUS outbreaks, and a more specific case definition required, particularly if there were other diseases present which produced similar “red spot” lesions.

Investigating disease outbreaks

The basis of all good surveillance is the ability of Competent Authorities and aquatic animal disease diagnostic services to investigate outbreaks of unusual disease events efficiently.

An outbreak investigation should be aimed at systematic identification of the cause(s) and source(s) of the infection, in order to:

In most situations, the primary objective of a disease outbreak investigation is to determine the cause and to identify ways of preventing further transmission (spread) of the disease agent. An infection from an exotic introduction usually shows a point-source “focus” of infection. Emergence of pathogenic levels of endemic diseases may centre on the most vulnerable groups within a susceptible population, or show more sporadic infections (chronic or random acute) increasing in frequency. It is the role of the investigating team to record and analyse these patterns to help meet the primary objective of preventing spread to unaffected susceptible populations. Disease outbreak investigations include the following activities.

Disease outbreak investigation

Outbreak Step 1 - The Diagnosis. The initial “presumptive” diagnosis of an outbreak is usually made on: clinical signs; crude patterns of infection, environmental and human activities associated with morbidity or mortality; and gross pathology. Whenever possible, laboratory tests should be undertaken as quickly as possible to verify the presumptive diagnosis. Since some laboratory procedures may require weeks, the implementation of control measures should be based on the presumptive diagnosis of significant diseases of concern.

Because any group of aquatic animals is likely to contain a range of pathogens and, even where there is a specific primary pathogen, there may be secondary infections, it is vital that a full range of specimens be taken from a number of animals at different stages of disease development - especially from any healthy animals in the vicinity of the outbreak, so comparative diagnostic observations can be made. When selecting healthy animals for examination, it is important to obtain them from at least two sources; (i) site(s) which appear(s) to be experiencing the particular problem, and (b) one or more sites in the same area, which have stocks showing no evidence of the disease problem. The geographic range of the latter will depend on the severity of the presumptive diagnosis and distribution of susceptible populations exposed directly, or indirectly, to the site of initial disease detection.

Outbreak Step 2 - Define a Case. Where large numbers of animals are dying rapidly, a case can simply be a recently dead (preferably moribund for microbial infections) animal.

As described above under “establishing a case definition”, where the disease aetiology is initially obscure, it is better to have a fairly broad case definition to ensure that all possible causes are included in the investigation. The case definition can be refined as more information becomes available and the data re-analysed accordingly.

Outbreak Step 3 - Confirm the Outbreak. This step may seem unnecessary but, in situations where a related disease is endemic, or where environmental extremes may cause physiological stress-based mortalities, such a confirmation is essential. For monitoring purposes (rather than surveillance-based zonation), a certain level of infection may be normal, however, any increase could lead to severe production losses if not identified quickly and accurately. Distinguishing a disease outbreak due to an increase in endemic infection levels, rather than a new/exotic disease outbreak is, therefore, critical.

Outbreak Step 4 - Characterize Outbreak. It is important to try and pin-point the time, population/stock and place associated with a disease outbreak where the cause is initially obscure. This is necessary for identifying possible source(s), mode(s) of transmission and chances of establishment of the infection:

(a) Time:

(b) Animal:

(c) Place:

(a) Time. For infectious diseases, identifying the index case is valuable for identifying the source of the outbreak (assuming it is a point source). The index case may be an individual animal, pond, farm, or stock (wild or farmed). One method of tracking an index-borne disease outbreak is to map an epidemic curve. This may have four or five segments; (1) the endemic level (where an infection is established), (2) an ascending branch, (3) a peak or plateau, (4) a descending branch, and (5) a secondary peak (Figure 5).

The duration of any epidemic is influenced by:

The slope of the ascending branch (2) can indicate the type of exposure. If transmission is rapid and the incubation period short, as with a significant infectious disease, then the ascending branch will be steeper than if transmission is slow or if the incubation period is long.

Figure 5. Segments of an epidemic curve.

The length of the plateau (3) and slope of the descending branch (4) indicate depends on the factors described for the duration of the epidemic, i.e. stocking densities, transmission mechanisms, and levels of susceptible stocks/reservoirs. Secondary peaks (5) are usually due to the introduction of new susceptible animals, a change in the mode of transmission, or temporary seasonal suppression of pathogenic proliferation (e.g. overwinter).

The choice of sampling frequency required to follow an epidemic curve is important. Appropriate time intervals may vary from several hours (e.g. some acute microbial infections) to months or years for slower progressing diseases, or diseases relying on seasonal intermediate host availability. Subtle differences in temporal patterns are missed, if sampling frequency is too far apart, e.g. secondary peaks (5) from animal-to-animal transmission, seasonal suppression of intermediate/carrier hosts or free-living infective developmental stages. Since the incubation period of most aquatic animal pathogens is highly variable and subject to the vagaries of hydrographic and climate conditions, at least one or two seasonal cycles (whether tropical or temperate) should be included. This is consistent with the OIE (OIE 2003b) standard of a minimum of two years before any facility/zone/country can be declared free of a listed disease.

In general, the OIE recommends an approach that is more flexible and disease-specific. The OIE Manual of Diagnostic Tests for Aquatic Animals (OIE 2003c) stipulates that 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; and the level of confidence that is desired of the survey results. 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[9].

In the case of exotic pathogens, exposure of a naïve host population will usually produce an epidemic curve similar to Figure 5. The initial exposure indicates rapid transmission within a defined water body or dense population, with large numbers affected over a short period of time. Survivors of the initial epidemic, may undergo subsequent outbreaks, as the disease establishes a cyclical infection pattern and environmental and host tolerance factors come into play.

In the short-term, rapid and accurate diagnosis have greatest priority. However, for effective, long-term, controls for diseases that cannot be eradicated, require understanding of the factors influencing the epidemic curve.

(b) Animal. The term "animal" is used for stocks, populations, sites, etc. Age, sex, geographical origin and genotype are frequently associated with varying susceptibility to disease impacts. However, infection patterns are also linked to the physiological and ecological characteristics of the infectious disease agent.

One method to analyse disease infection patterns within an outbreak, is measurement of attack rate (AR). Attack rate is the number of cases of a specific disease divided by the number of animals at risk at the beginning of the outbreak, e.g., EUS appears to affect small fish to a greater extent than large fish within in a given pond. In this case, the following calculations would be necessary:

If there were 1000 small fish in the pond and 300 had EUS, and there were 1000 large fish of which 100 had EUS during an outbreak, the AR’s would be 30 percent (AR1) and 10 percent (AR2), respectively. This indicates that small fish are three times more susceptible to EUS than large fish. Likewise this evaluation process could also be used to test the hypothesis that nutritionally stressed fish are more susceptible to infection than well-fed fish (Table 6).

Table 6. Attack Rate (AR) for EUS-infected fish with and without nutritional stress.

With nutritional stress

Without nutritional stress




AR (%)



AR (%)

AR Diff (%)





























In Table 6, ARs are expressed as percentages. The second last column (AR Diff) is the difference in attack rates between groups. The column “relative risk” (RR), gives the ratio of EUS found in fish with, and without nutritional stress. The higher the values for AR Diff and RR, the more significant the factor being analysed is in increasing the risk of disease. In this example, small fish are three times more likely to develop EUS than medium-sized fish, and six times more likely than large fish. Medium-sized fish are twice as susceptible as large fish.

This example also supports the hypothesis that nutritional stress is a factor in EUS size-related susceptibility.

In the context of surveillance and zonation, it is important to determine the relative importance of as many of the possible contributory factors (e.g. sudden acidification of the water for EUS) as possible. This will help focus surveillance efforts, whether aimed at early detection of endemic outbreaks, or surveillance to prove freedom from the disease. All surveillance programmes must focus on the most vulnerable sectors of the susceptible population as possible.

(c) Place. Defining the exact source of an outbreak can be helped by mapping an affected site/area or facility, recording the dates when cases were detected, and the stage of development of the infection. Such a map can indicate whether or not an outbreak is due to an infectious point-source, or other, source. Surveillance aimed mapping the extent of an outbreak should work inwards towards an apparent point-source. Such an approach reduces the risk of spread by surveillance activities radiating outward from a known infected farm/area/site.

Surveillance should also focus on neighbouring sites with documented (or undocumented) disease losses, and links to point-source waters or stocks though seed/broodstock or market-relay transfers. This will help develop accurate preliminary maps of the disease distribution. Negative results are as useful as positive results in tracking suspect highly virulent disease outbreaks, although more difficult to map as conclusively as positive results. Such maps are particularly important for aquatic animal diseases, since direct observation of infected animals can be difficult, requiring fishing, diving, specialized boat equipment, etc.

Outbreak Step 6 - Formulate hypothesis(es). Based on the analysis of time, place and animal data, options for controls and priorities for further investigation are developed. Any hypothesis must be compatible with the confirmed data and related epidemiological information. Control options can be developed based on such hypotheses, e.g. investigation of a WSD outbreak in two ponds at a research station led to the hypothesis of the sequence of events that contribute (Figure 6) to an outbreak of WSD in 70 day old post-larval shrimp.

Figure 6. Sequence of events leading to a WSD outbreak

Outbreak Step 7 - Intensive follow-up. Follow-up studies require analysis of available data, checking for cases that may be present at other locations (downstream or linked by stock transfers), examination of movement of stocks, feedstuff, or any other human activities associated with the affected stocks. Feeding or other challenge trials could be required where non-infectious agents are suspected. Transmission experiments, to establish infectious agent aetiology, where this is in doubt (Koch-Henle’s Postulate), may also be required.

Outbreak Step 8 - Implement control and prevention measures. An effective investigation will help define effective control options that reduce the risk of recurrence of similar outbreaks. Investigations may, however, indicate that the possibility of re-infection is inevitable, e.g., in open-water circumstances where, once established in susceptible or carrier/reservoir populations, eradication of the pathogen is not possible or economically feasible. In such instances, control measures are aimed at minimizing exposure to the established pathogen in affected waters and preventing spread to unaffected, susceptible, populations.

Outbreak Step 9 - Report findings and recommendations for dealing with future outbreaks. For isolated farms experiencing disease outbreaks, recommendations may take the form of a brief discussion with the farm manager, outlining the actions required for surveillance in order to prevent future outbreaks. A written report of the information, data and recommendations developed from the outbreak, provides a useful reference. For broader outbreaks, findings should be published in peer-reviewed scientific literature and, depending on the disease, reported to the OIE to ensure transparent reporting to trade partners. Reports of investigations of serious outbreaks should include: case-history background; methods applied to diagnostic and epidemiological investigations; results; hypotheses; financial and ecological impacts (as appropriate) and recommendations for control.

[8] In the OIE Aquatic Animal Health Standards Commission’s view, a PCR signal alone does not provide confirmation of the presence of viable and transmissible agents.
[9] See Chapter 1.1.4 “Requirements for surveillance for international recognition of freedom from infection”; OIE Manual of Diagnostic Tests for Aquatic Animals, 4th ed, 2003 -

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