Fish in culture are susceptible to a wide range of infectious pathogens in the form of bacterial, fungal, viral and parasitic agents. The diseases they cause pose a major threat to a thriving aquaculture industry, and result in considerable economic losses world-wide (Adams, Aoki, Berthe, Grisez & Karunasagar 2005). Transmission of these agents occurs between individual fish either through vertical or through horizontal transmission. Vertical transmission involves the transmission of pathogens from one or both parents to their offspring, while horizontal transmission primarily involves the spread of the pathogen through the water body. The environmental conditions under which the fish are maintained can exacerbate disease situations, especially because high numbers of fish maintained within ponds or cages can increase the levels of stress experienced by the fish, making them more susceptible to infection through immune suppression (Pottinger 2007). These crowded conditions also facilitate the spread of pathogens between fish horizontally through the water. In addition, farmed fish can be susceptible to infectious agents from wild fish populations within the vicinity of the farm and vice versa (Nicholson 2000).
Effective disease control is paramount within aquatic farming systems to stop the spread of infectious pathogens. Implementation of an effective health management programme consisting of well-organized management and husbandry practices, efficient biosecurity and hygiene measures, and improved resistance to disease through vaccination can all help to reduce and control disease at farm sites (Gudding, Lillehaug & Evensen 1999; Thompson & Adams 2004; RUMA Guidelines 2006; Adams 2009). However, disease monitoring in the form of surveillance and rapid diagnosis is also an important part of this process to allow appropriate action to be readily taken when pathogens are first detected and diseases are diagnosed, before they become a significant problem for the farmer. In addition to screening diseased animals, monitoring should include the screening of apparently healthy fish so that subclinical infections can be detected and used as an early warning signal of an imminent disease problem (World Organisation for Animal Health 2006). The detection and identification of pathogens in the environment, and between harvesting and re-stocking are also useful for reducing disease outbreaks.
Existing diagnostic methods
A variety of methods are available to detect pathogens both in fish and in the aquatic environment. These include traditional diagnostic methods and a diverse range of immunological and molecular methods (Cunningham 2004; Adams, Aoki, Berthe, Grisez & Karunasagar 2005). However, a combination of methods is often required for a definitive diagnosis of disease. A variety of methods used by Herath, Costa, Thompson, Adams and Richards (2009) for the detection of salmonid alpha virus (SAV-1) are shown in Fig. 1.
Traditional methods involve first culturing and isolating the pathogen from infected tissue and then identifying the organism involved by biochemical identification (e.g. bacteria), microscopy (e.g. parasites) or electron microscopy (e.g. viruses). Histology and histopathology are also routinely used in disease diagnosis.
The different immunological methods routinely used in fish diagnostics allow the specific detection of pathogens without first having to isolate the pathogen. Monoclonal antibodies (MAbs) provide ideal standardized reagents for use in these methods, and many such products are now commercially available (Adams & Thompson 2008). The antibody-based test used is normally selected on its merits and disadvantages. Direct and indirect fluorescent antibody techniques (FAT and IFAT), for example, are methods that are widely used in fish diagnostics because they are simple, sensitive and rapid to perform, with results obtained within 2 h of taking the sample. A fluorescent or a confocal microscope, however, is needed to visualize the results, and staff need to be familiar with differentiating specific and non-specific staining to read the results of the test. These techniques are used to detect pathogens in samples cultured from infected fish, but also for visualizing viruses and bacteria directly in formalin-fixed tissue sections, or imprints prepared from infected tissue. Such methods and are thus especially useful for visualizing viruses and bacteria that are difficult to culture, or for fungal pathogens such as Aphanamyces invadans, as shown in Fig. 2a (Miles, Thompson, Lilley & Adams 2003).
Immunohistochemistry (IHC) is an extension of traditional histology where formalized, paraffin wax-embedded tissue is sectioned and incubated with a pathogen-specific antibody (Adams & Marin de Mateo 1994). The method is simple to perform, has the advantage of being able to visualize the pathology associated with the infection and only requires a light microscope for analysis. However, IHC is considered to be less sensitive than IFAT, although amplification methods such as those based on biotin–streptavidin can increase the sensitivity of the reaction (Hsu & Raine 1981) as shown in Fig. 2b for the detection of the viral haemorrhagic septicaemia virus. Antigen retrieval is sometimes also used to help expose and improve the detection of the antigens (Jung, Thompson, Adams, Morris & Snedden 2001) (Fig. 2c).
The enzyme-linked immunosorbent assay (ELISA) can be used to measure either the pathogen or the host antibody response (i.e. serology as described later) depending on how the assay has been set up (e.g. a sandwich ELISA is generally used to detect pathogens) (Adams 1992; Adams & Thompson 2008). It has the advantage of high throughput, and can be automated (and is quantitative for which standards are required). This assay is particularly useful for detecting and quantifying pathogens during clinical disease, but is less useful for sub-clinical infections due to the sensitivity limits of the assay.
Western blot and dot blot are methods not routinely used in fish diagnostics, but their application can be useful in certain situations. Western blot, for example, is used in mammalian diagnostics as the definitive test for bovine spongiform encephalopathy (World Organisation for Animal Health 2008), while we use western blotting in our laboratory to serotype Flavobacterium psychrophilum isolates (unpublished data).
Sometimes, immunological methods are unable to detect the pathogen because the amount of pathogen present in the sample is below the sensitivity threshold of the assay (as is often the case of pathogens in water samples) or the antigens on the pathogen have been altered and no longer recognized by the antibody (either due to being denatured during sample processing or because a different life cycle stage is present as occurs with parasites). Generally, molecular methods offer increased sensitivity over immunological methods and are not affected by changes or inappropriate expression of antigens as DNA (or RNA) is detected by these methods rather than proteins.
Molecular methods are routinely used to detect pathogens in tissue samples and confirm the identity of a pathogenic agent. Molecular methods are applied to detect pathogens in a diverse range of environmental samples including water, soil and food samples, and enable very low levels of aquatic pathogens to be detected and identified (Cunningham 2004; Adams 2009). They are also very useful for detecting micro-organisms that are difficult to culture, exist in a dormant state or are involved in zoonosis. They are also valuable in epidemiological studies to identify individual strains and for the differentiation of closely related strains.
The most common molecular method, the polymerase chain reaction (PCR), results in the amplification of very small amounts of defined sequences of DNA so that the amplicons produced can be detected. Many variations of this assay exist, including a two-step or a nested PCR or nested reverse transcriptase-PCR (RT-PCR), random amplified polymorphic DNA, reverse cross blot PCR, real-time PCR and an RT–enzyme hybridization assay (reviewed by Adams 2009). Alternative amplification methods to PCR include nucleic acid sequence based amplification, transcription-mediated amplification for the amplification of RNA under isothermal conditions and loop-mediated amplification (LAMP). Another molecular method used widely in the diagnosis of virus infections in shrimp is in situ hybridization. This is a hybridization method in which complementary DNA or RNA (labelled for identification) acts as a probe to locate a specific DNA or RNA sequence of the pathogen, in infected tissue sections for example. This method is distinct from immunohistochemisty, where proteins rather than nucleic acids are detected in the tissues. A wide variety of formats are available for hybridization of pathogen DNA with a specific oligonucleotide probe, e.g. dot blot, Southern blot and reverse hybridization.
Different PCR methods have different levels of sensitivities; for example nested PCR is more sensitive than one-round (conventional) PCR. Real-time PCR [or quantitative PCR (qPCR)] is also more sensitive than conventional PCR and faster to perform. The detection and quantification of the increase in fluorescent reporter molecules in real time during PCR amplification is the principle for detecting the target amplified by qPCR, and several chemistries are available, such as SYBR Green and molecular beacons (Bustin 2000). Products are monitored as they are amplified during each reaction cycle, and no post-reaction processing is needed. The initial amount of target DNA is related to a threshold cycle, and can be quantified by means of a standard curve. Conventional PCR is prone to contamination particularly from previously amplified products, but this can be overcome by good laboratory practice and separating the extraction, amplification and electrophoresis processes. The potential for contamination is significantly reduced in real-time PCR because of the closed tube system used during the amplification and post amplification analysis.
Standard PCR may be sufficient to detect the pathogen, and identification of the PCR products by sequencing is the gold standard for ultimately characterizing the pathogen. Traditionally, this was seen as too expensive in routine diagnostics in aquaculture, but over the last decade, sequencing has become affordable and commercial sequencing is now offered widely. Where there is a need to identify the pathogen to the species level, differentiate between closely related species or distinguish live pathogen from dead pathogen, modifications to the PCR or additional analyses are required. Ribosomal RNA genes are often used as the target DNA for bacterial pathogens because they contain highly conserved regions and variable species-specific regions. This can confirm closely related pathogens; however, polygenic sequencing is sometimes used to confirm the species where a number of genes are amplified for a particular pathogen rather than just one gene. High-throughput sequencing technologies (such as 454 sequencing) developed recently allow large amounts of DNA to be sequenced rapidly at low costs (Ansorge 2009). Other DNA profiling methods used for bacterial identification and speciation include restriction fragment length polymorphism and density gradient gel electrophoresis, both of which are simple, rapid and economical methods to perform, but the results can sometimes be difficult to interpret.