6.4 Diagnostics

Diagnostics play two essential roles in aquatic animal health management and disease control, i.e. (1) to screen healthy animals to ensure that they are not carrying infection at subclinical levels by specific pathogens; and (2) to determine the cause of unfavourable health or other abnormality (such as spawning failure, slow growth, behaviour, etc.) in order to recommend mitigating measures applicable to that particular condition.

The former, commonly done on stocks or populations of aquatic animals destined for live transfer from one area or country to another, reduces the risks on two fronts: (1) risk of animals carrying opportunistic pathogens which may proliferate during shipping, handling or change in environment; and (2) risk of resistant or tolerant animals transferring a significant pathogen to a population which might be susceptible to infection. The latter is the most immediate and clearly recognized role of diagnostics in aquatic animal health biosecurity (Bondad-Reantaso et al., 2001).
Diagnosis of aquatic animal diseases is a specialized field and laboratories that traditionally have dealt with terrestrial animal diseases may need to develop specialized expertise and materials to deal with aquatic animal diseases. Many of the components of the terrestrial animal disease diagnostic laboratory can, however, be applied to aquatic animal diseases. The pathology, histopathology, bacteriology and virology components of the terrestrial diagnostic laboratory are relevant to many, but not all aquatic animal diseases. Few aquatic animal diseases show pathognomonic macroscopic signs of infection by which a definitive diagnosis can be made. Morphological pathology, including direct light microscopy, histopathology and electron microscopy, is therefore an important and essential component of the diagnostic investigation of aquatic animal diseases.

Standard laboratory culture methods are applied to viral, bacterial and mycotic pathogens, particularly those of finfish. Increasingly, molecular techniques are applied for detailed identification of pathogenic organisms and provide the possibility of a more rapid diagnosis. Virus isolation is still the gold standard for the diagnosis of many aquatic viral infections of finfish. These methods are often disease specific, and laboratories need to develop the tests appropriate to the aquatic animal health needs of their country.
The laboratory isolation of finfish viruses is done on pathogen-specific tissue cell lines. An appropriate variety of tissue cell lines need to be sourced by laboratories intending to offer virus isolation tests. Relevant viruses for positive controls need to be sourced and maintained as well as the respective antisera.
The type of sample material depends on the species, life stage and size of the target animal, and the epidemiological situation. It is influenced by the objective of the diagnostic testing, whether for the detection of overt disease or for targeted sampling (OIE, 2015).
A large amount of information can be gleaned from histological examination, and this technique is often recommended as an initial screening method and where abnormal disease or mortality occurs (OIE, 2015). Electron microscopy and molecular techniques may be needed to confirm pathogen identity.
Details of relevant diagnostic methods for the OIE-listed diseases of aquatic animals are regularly updated in the OIE Manual of Diagnostic Tests for Aquatic Animals (OIE, 2015), and laboratories should consult the recommended diagnostic techniques for specific pathogen confirmation and disease surveillance. The manual also provides important information on the quality management in veterinary laboratories and the principles and methods of validation of diagnostic assays for infectious diseases (OIE, 2015). Diagnostic techniques employed for the examination of material from the three major groups of OIE-defined aquatic animals, finfish, crustaceans and bivalves, differ somewhat.
Crustacean viruses are not routinely isolated, limiting the use of virus-culture-based assays in the diagnosis of viral diseases of crustaceans. Antibody-based tests are precluded from both crustacean and molluscan diagnostic techniques due to the inability of crustaceans and molluscs to produce antibodies (OIE, 2015).
Diagnostic techniques appropriate to OIE-listed finfish diseases include (OIE, 2015):
• virus isolation on tissue cell cultures;
• serology;
• direct microscopy;
• histological techniques;
• electron microscopy; and
• molecular techniques for confirmatory testing and diagnosis.
Diagnostic techniques appropriate to OIE-listed crustacean diseases include (OIE, 2015):
• 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 or monoclonal antibodies; and
• molecular methods (including sequencing where appropriate for strain determination).
Diagnostic techniques appropriate to OIE-listed bivalve diseases include (OIE, 2015):
• macroscopic examination;
• histological techniques;
• transmission electron microscopy; and
• molecular methods.