Development of diagnostics for aquaculture: challenges and opportunities

Authors


Correspondence: A Adams, Institute of Aquaculture, University of Stirling, Scotland, UK. E-mail: alexandra.adams@stir.ac.uk

Abstract

The application of biotechnology in aquaculture has enabled the development and improvement of a wide range of immunodiagnostic and molecular technologies, and reagents and commercial kits have become more generally available. Recently, method development has increased exponentially as techniques used for clinical and veterinary medicine are adapted and optimized for use in aquaculture. Careful consideration needs to be given to selecting which rapid diagnostic methods to take forward and apply in aquaculture – pathogen detection methods need to be robust yet sensitive, and in many cases, capable of detecting a high degree of heterogeneity. There are numerous innovative techniques that may fulfil these criteria and provide valuable diagnostic tools. It is also important, however, that useful diagnostic methods already developed are standardized and fully validated, and that new technologies do not supercede these just because they are novel methods. The cost, speed, specificity and sensitivity of assays are all extremely important to end-users. This paper looks at some of the opportunities and challenges for the development of rapid diagnostics for aquaculture.

Introduction

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.

Figure 1.

 Different methods used to detect the salmonid alpha virus (SAV-1): (a and b) culture on Chum salmon heart -1 (CHH-1) cells for virus isolation [(a) non-infected cells, (b) cytopathic effect in cells caused by the virus 6 days post inoculation] (Herath et al. 2009); (c) agarose gel (1% w/v) of reverse transcriptase-polymerase chain reaction (RT-PCR) bands of kidney tissue sampled at day 3 p.i., lanes: (1) 100 bp ladder, (2–6) uninfected fish, (7–11) infected samples, (12) non-template negative control and (13, 14) positive control (Herath et al. 2009); (d) real-time PCR analysis of salmonid alpha virus1-infected samples.

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).

Figure 2.

 Common immunodiagnostics methods (a) IFAT on a muscle section of striped snakehead experimentally infected with Aphanomyces invadans (scale bar indicates 50 μm) (Miles et al. 2003); (b) biotin–streptavidin-amplified immunohistochemistry (IHC) on rainbow trout tissue infected with viral haemorrhagic septicaemia) (scale bar indicates 500 μm); (c) antigen retrieval IHC on spleen tissue from sea bass artificially challenged with Photobacterium damselae subsp. piscicida (magnification × 400) (Jung et al. 2001). H, hyphae; P, pinpoint sources of fluorescence.

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.

Challenges and opportunities

Exponential development of new methods for use in aquaculture has taken place by adapting techniques used in clinical and veterinary medicine. These new technologies should not supercede existing methods just because they are novel methods but because clear advantages can be identified. Advances in biotechnology have improved the immunological and molecular tests available with respect to both the sensitivity that can be achieved and the time to perform the test. These tests enable rapid specific detection of pathogens where there is no need to first isolate the pathogen, and complement and enhance existing traditional methods.

A number of challenges are faced with the introduction of these methods for use in routine diagnostics for aquaculture. We need to establish which of these methods are suitable for use in aquaculture, and ensure that they have the appropriate accuracy, specificity and sensitive required. The time required to perform an assay is important, together with the technical complexity of the test. The cost and availability of tests are also important considerations. Furthermore, new methodologies require to be fully validated before they can be used in diagnostics and recommended for use by the Office International des Epizooties (OIE).

Sensitivity of methods

Tests that are used in diagnostics need to be accurate, specific and sensitive. Test accuracy is the proportion of all tests' results, both positive and negative, that are correct, and describes the overall performance of a test. In general, as a test becomes more accurate, it becomes more tedious, more invasive and more costly. Useful methods should be standardized and validated and compared with a gold standard test, which is the definitive test where you can determine that the sample is positive and the disease is truly present. The Gold Standard is a quality control device and provides the basis for determining the value of diagnostic tests, treatments and prognosis.

The specificity and sensitivity of a test have different meanings for different individuals. For an immunologist or a molecular biologist, the sensitivity refers to the analytical sensitivity, i.e. the detection limits of the assay, and the specificity refers to the analytical specificity, i.e. that the test only reacts with the pathogen causing the disease, while to an epidemiologist, the sensitivity refers to the operational sensitivity, i.e. the likelihood of a positive result in animals known to have the disease and specificity to the operational specificity, i.e. the likelihood of a negative result in animals known to be free of the disease.

Novel methods in development

Novel methods are being developed all the time and recent advancements in diagnostics for aquaculture include a range of reagents and commercial kits, some of which are based on either nano- or multiplex technology.

Lateral flow kits (rapid kits)

The detection of pathogens using the lateral flow immunoassay system (based on immunochromatography) is now widely used in the diagnosis of infection agents in animals and humans (Al-Yousif, Anderson, Chard-Bergstrom & Kapil 2002; Bautista, Elankumaran, Arking & Heckert 2002; Dunn, Gordon, Kelley & Carroll 2003; Gatta, Perna, Ricci, Osborn, Tampieri, Bernabucci, Miglioli & Vaira 2004; Bai, Sakoda, Mweene, Kishida, Yamada, Minakawa & Kida 2005; Bai, Sakoda, Mweene, Fujii, Minakawa & Kida 2006). The attractiveness of this technique is that it allows a very rapid and sensitive detection of pathogens and, because of its simplicity of use, can be used as a pond-side test. The principle of lateral flow and how it has been applied to aquaculture was reviewed recently (Adams & Thompson 2008; Adams 2009). Commercially available ISAV rapid kits have a sensitivity equivalent to a one-round PCR (Adams & Thompson 2008). This technology has many advantages over traditional immunoassays; it is simple to use, rapid (with results in <10 min), cheap to perform and does not require skilled operators or expensive equipment.

Bluspot technology

Another immunological-based pond-side test under development for aquaculture is the Bluspot kit. Such tests are already available for Salmonella in food (BIO ART NV/SA, Belgium; http://www.bio-art.be). In this assay, the sample flows through a membrane that has been coated with a MAb specific against the pathogen of interest by vertical filtration. This has the effect of concentrating the pathogen on the membrane to improve the sensitivity of the reaction. Antigen–antibody complexes are then detected with a second antibody labelled with an enzyme. The reaction of this enzyme with its substrate produces a permanent blue-coloured spot when the reaction is positive. Results are obtained in 5 min, no equipment is required and the results are easy to interpret.

Nanotechnology

An advanced immunonanotechnology platform based on immunomagnetic reduction is currently being developed for use in aquaculture, modified from the test used for bird flu (Yang, Chieh, Wang, Yu, Lan, Chen, Horng, Hong & Yang 2008). Magnetic beads (coated with antibody) rotate under a magnet via magnetic induction. When the antibodies on the beads bind to the pathogen, the beads agglutinate. The movement of the agglutinated clumps of nanoparticles is reduced within the magnetic field compared with the individual unbound nanoparticles, and this leads to a reduction in the magnetic signal measured by the magnetic detector (i.e. immunomagnetic reduction occurs). The assay procedure is simple to use, with no wash steps required, and offers a high specificity and sensitivity.

Luminex technology (xMap) xMap technology

Luminex xMap technology is becoming more widespread in diagnostics for human diseases (Mohamed & McCoy 2006) and has a similar potential for fish diagnostics. It consists of a bead array containing a number of different bead sets, each uniquely identifiable from the ratio of two internal fluorochrome dyes, allowing them to be individually identified by a laser within the analyser. The bead can be coated with different fragments of DNA or antibodies to detect pathogens, and an appropriate reporter fluorochrome conjugate (i.e. to detect the test molecule) coupled with either R-phycoerythrin, Alexa 532 or Cy3 is then reacted with the bead. This label is then detected by a second laser.

The method is ideal for multiplexing, i.e. the detection of several pathogens in one sample (Dunbar & Jacobson 2005; Dunbar 2006). The system allows high-throughput detection and quantification (in a 96-well format) of both proteins and nucleic acids, and can therefore be used in molecular and immunodiagnostics to detect pathogens directly from tissue samples or culture, or can be used in serology to measure fish antibodies. The current drawback of this method is that it is expensive to perform.

New molecular methods with potential for use in aquaculture

Multiplex-PCR assays

Multiplex-PCR methods are becoming more popular as a diagnostic tool. They do have the advantage that a sample can be screened for a variety of pathogens within a single reaction, but standardization of the assay can be very difficult. The assay is designed using multiple primer sets to detect specific pathogen DNA sequences within a single PCR reaction, and the amplicons of these reactions are visualized as bands of varying sizes on an agarose gel. Optimization of the assay can be time-consuming because the annealing temperatures must be determined for each primer set, the size of the products must be sufficiently different to be distinguished from each other by gel electrophoresis and the specificity and sensitivity of the multiplex test need to be determined for each pathogen under investigation. Real-time PCR multiplex assays have also been developed for the detection of a variety of pathogens, including viral pathogen of shrimp (Xie, Xie, Pang, Lu, Xie, Sun, Deng, Liu, Tang & Khan 2008).

LAMP

Loop-mediated isothermal amplification is a novel method of amplifying DNA under isothermic conditions, and has been used by a number of authors to detect bacterial, parasitic and viral fish pathogens. It is faster and more sensitive than conventional PCR, and capable of detecting as few as six copies of DNA in the reaction mixture (Notomi, Okayama, Masubuchi, Yonekawa, Watanabe, Amino & Hase 2000). The complete LAMP procedure can be performed in 90 min and as it is carried out under isothermal conditions, it can be performed without the use of a thermocycler, making it suitable as a field test if a small oven is available (Soliman & El-Matbouli 2005). A number of LAMP kits are now available on the market for aquaculture. The method relies on the autocycling strand displacement DNA synthesis, using Bst DNA polymerase and a set of at least four specially designed primers (two inner and two outer primers) to recognize a total of six distinct sequences on the template DNA (Notomi et al. 2000). The reaction time can be reduced using two further primers. Products of LAMP amplification can be visualized by eye with the addition of SYBR Green I to the mixture, changing from orange to green in colour if the reaction is positive, or can be detected by photometry for turbidity caused by increasing the quantity of magnesium pyrophosphate in solution. Some commercial LAMP kits use an enzyme substrate system to visualize the reaction on a membrane.

DNA microarrays

The use of DNA microarrays as a diagnostic tool for aquaculture is in its infancy (Kostic, Francois, Bodrossy & Schrenzel 2008). The method involves hybridizing samples of DNA fragments (amplicons), amplified by PCR, onto specific DNA detector fragments spotted onto a solid support. The advantage of this technology is that a large number of DNA spots from different pathogens can be included on a single slide, allowing multiplexing for different pathogens. Fluorescence is the most common method of detection for microarrays. When combined with generic amplifications during the nucleic acid labelling step, the method allows for a high degree of sensitivity and specificity, and the high throughput can reduce the cost and increase the speed of a comprehensive disease screen.

Colony hybridization

Colony hybridization is another hybridization-based method using labelled polynucleotide probes complementary to a unique sequence of DNA of the suspected pathogen. The method is used in human medicine on pathogens isolated from clinical samples by culture. The resulting bacterial colonies are lysed, and the DNA is denatured and fixed onto an inert support, which is then subsequently hybridized with the DNA probe. The method allows specific pathogen DNA to be detected in mixed cultures of bacteria and there is no need to isolate the pathogen DNA before performing the assay. However, the method is time consuming and the method can only be performed on samples where it has been possible to culture the micro-organism.

Serology

The diagnostic methods presented so far have been direct diagnostic methods, while serology offers an alternative, indirect approach to pathogen detection. Serology, screening for the presence of specific antibodies in the serum of animals, can be a useful indicator of previous exposure to a pathogen, especially for viruses, and this type of screening is used routinely in clinical and veterinary medicine (Palmer-Densmore, Johnson & Samara 1998; Yüce, Yücesoy, Genç, Sayan & Uçan 2001; Fournier & Raoult 2003).

The advantage of serology is that it is able to indicate infection before it is possible to detect the pathogen by culture or other methods. In addition, pathogen-specific host antibody responses have been reported to be more persistent than other methods (Graham, Fringuelli, Wilson, Rowley, Brown, Rodger, Mclouchlin, Mcmanus, Casey, Mccarthy & Ruane 2010). It also has the advantage of being non-destructive, as only a blood sample is required. Normally, the serum is screened using an indirect antibody capture ELISA to measure the fish's antibody response. The specificity of the test is dictated by the pathogen used to coat the ELISA plate, and the fish species screened by the availability of anti-fish species antibody (Adams & Thompson 2006). The lateral flow kit can be set up to detect pathogen-specific antibodies, but commercial application of the kits in this format may be restricted because of the need to include pathogen (antigen) in the kit. This could be overcome by using recombinant proteins or peptides to render the kits safe for transportation.

Because of insufficient development of serological methodology, the detection of antibodies to pathogens in fish has not been established thus far as a routine method for assessing the health status of fish populations (with the exception of SAV, where a virus-neutralizing test is well established; Graham, Jewhurst, Rowley, Mclouchlin & Todd 2003), or been widely accepted for use in the OIE manual (mainly due to a lack of knowledge about the antibody responses of fish to viral infections), but it does have the potential for future use once validated, particularly for detecting pre-exposure of fish to viral pathogens. The OIE manual says of serology for ISAV for example ‘The test is not standardized for surveillance or diagnostic use, but may be used as a supplement to direct virus detection and pathology in obscure cases’. Furthermore, the level and distribution of sero-conversion in an ISAV-infected population may provide some information about the spread of infections.

Serological tests do not distinguish between infected and vaccinated fish unless vaccines (in the future) are specifically designed to incorporate markers that can be identified. This should be considered, particularly for recombinant vaccines, as it would widen the availability of serology in diagnostic screening.

OIE manual and novel diagnostics

The OIE Manual of Diagnostic Tests for Aquatic Animals 2009 contains a variety of diagnostic methods (traditional, immunological and molecular) for the identification of pathogens from aquatic animals, and includes up-to-date methods validated for use in the identification of disease agents in farmed fish. The manual does not cover all diseases/pathogens, however, and focuses on those that resist or respond poorly to therapy, have a restricted geographical range, are of high socio-economic importance or occur in species involved in international trade.

The aim of the manual is to bring a uniform approach to disease diagnosis for aquaculture, and conventional isolation and characterization techniques remain the methods of choice for the diagnosis of many diseases in the manual. There have been rapid developments in a wide range of molecular and immunodiagnostic techniques in recent years (Cunningham 2004; Adams & Thompson 2006; Adams & Thompson 2008) and a number of new reagents and commercial kits are now marketed for use in aquaculture. However, their addition to the manual is not a straightforward matter. Rigorous validation is necessary to confirm that the test is ‘fit for propose’ to demonstrate that it has been optimized and performance characteristics such as sensitivity and specificity can be demonstrated.

Future prospects

Rapid diagnosis and prompt removal of infected fish are needed to implement effective control strategies during disease outbreaks. Traditional diagnostic methods tend to be costly, labour intensive, slow and might not lead to a definitive diagnosis being made, even when complemented with histological evidence. A number of opportunities exist to overcome the challenges that lie in the path of developing and implementing diagnostics in aquaculture. Firstly, there is a need to fully exploit the existing methods currently used. Secondly, biotechnology can provide many opportunities for developing new and refining existing diagnostic methods, to improve the accuracy, sensitivity and specificity of rapid tests, as well as enable multiplex testing. There is a need to validate these new methods and make reagents and standardized methods available quickly. More reagents and kits are becoming commercially available for testing both in the laboratory and in the field. Lateral-flow immunoassays, for example, allow quick and sensitive detection of a pathogen, thus providing time to implement early control measures to avoid the spread of the disease. There is a need to include such methods in the OIE manual as soon as the new technologies have been validated. Improved rapid diagnostics is crucial for the sustainable future for aquaculture. These tests will be invaluable for rapid reliable diagnosis applied to regular screening programmes for aquaculture, such as sensitive broodstock/egg screening (to create disease-free broodstock and pathogen-free eggs) and for monitoring the disease status of fish in the field. Formal evaluation of diagnostic sensitivity and specificity is crucial for new methods. The diagnostic process covers everything from sample collection in the field through to processing in the lab. This is the best way to truly understand diagnostic sensitivity and specificity and thus develop the most efficient approach to diagnostics.

Acknowledgments

The authors would like to thank Tharangani K. Herath and Aquatic Diagnostics for providing photographs of the real-time analysis and the VHS IHC respectively.

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