Detection and Identification of Fish Pathogens: What Is the Future?

The Israeli Journal of Aquaculture Ð Bamidgeh 60(4), 2008, 213-229. 213

Detection and Identification of Fish Pathogens: What is the Future?

A Review

I. Frans1,2†, B. Lievens1,2*†, C. Heusdens1,2 and K.A. Willems1,2 1 Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium 2 Research Group Process Microbial Ecology and Management, Department Microbial and Molecular Systems, Katholieke Universiteit Leuven Association, De Nayer Campus, B-2860 Sint-Katelijne-Waver, Belgium, and Leuven Food Science and Nutrition Research Centre (LfoRCe), Katholieke Universiteit Leuven, B-3001 Heverlee-Leuven, Belgium

(Received 1.8.08, Accepted 20.8.08)

Key words: biosecurity, diagnosis, DNA array, multiplexing, real-time PCR

Abstract Fish diseases pose a universal threat to the ornamental fish industry, aquaculture, and public health. They can be caused by many organisms, including bacteria, fungi, viruses, and protozoa. The lack of rapid, accurate, and reliable means of detecting and identifying fish pathogens is one of the main limitations in fish pathogen diagnosis and disease management and has triggered the search for alternative diagnostic techniques. In this regard, the advent of molecular biology, especially polymerase chain reaction (PCR), provides alternative means for detecting and identifying fish pathogens. Many techniques have been developed, each requiring its own protocol, equipment, and expertise. A major challenge at the moment is the development of multiplex assays that allow accurate detection, identification, and quantification of multiple pathogens in a single assay, even if they belong to different superkingdoms. In this review, recent advances in molecular fish pathogen diagnosis are discussed with an emphasis on nucleic acid-based detection and identification techniques. Major features and applications of current predominant methods and promising methods likely to impact future fish disease control and prevention are outlined.

Introduction Diseases caused by fish pathogens, including that address aquatic animal pathogens and bacteria, fungi, viruses, and protozoa, can diseases are becoming an increasingly impor- cause considerable economic losses to the tant focus of these industries (Scarfe et al., ornamental fish and aquaculture industries. 2006). Nevertheless, the lack of rapid, accu- Biosecurity (pathogen preventing) programs rate, and reliable means by which fish

* Corresponding author. Tel.: +32-15-305590, fax: +32-15-305599, e-mail: [email protected] † These authors contributed equally to this work. 214 Frans et al. pathogens can be timely detected and identi- specific, more sensitive, and more accurate fied has been one of the main limitations in (Cunningham, 2002). fish pathogen diagnosis, fish disease man- One of the most common serological iden- agement, and biosecurity policies. tification techniques is the enzyme-linked Conventional diagnosis methods often rely immunosorbent assay (ELISA; Clark and on interpretation of clinical and histological Adams, 1977) and its variations, which are all signs, culturing pathogens in or on a suitable based on the binding between diagnostic anti- medium, and analysis of morphological, phe- bodies and specific antigens of the target. notypic, or biochemical characteristics of the Because of their versatility, simplicity, speed, presumptive pathogen. Although these meth- and possibility to quantify the target pathogen, ods are fundamental to the development of ELISA assays have been frequently used in any alternative diagnostic method, the accura- pathogen diagnosis, especially for the detec- cy and reliability of these techniques largely tion of viruses and bacteria (Martinez-Govea depend on competent (taxonomical) expertise. et al., 2001; Wagner et al., 2001; Adkison et Further, diagnosis requiring a culturing step is al., 2005; Reschova et al., 2007). Highly spe- generally time-consuming and labor intensive. cific assays can be developed using mono- For example, assays for Flavobacterium or clonal antibodies that recognize a specific epi- Mycobacterium species may require several tope of the pathogen. However, to detect the days for growth with specialized media and different strains of a given virus, for example, growth conditions (Nematollahi et al., 2003; polyclonal antibodies that target multiple epi- Van Trappen et al., 2003). Detection of topes of the pathogen are needed. Renibacterium salmoninarum, the causative Nevertheless, major limitations for the devel- agent of bacterial kidney disease in salmonids, opment of serological assays include that the can take up to 12 weeks (Benediktsdottir et al., required antiserum for detection of a 1991). Moreover, these techniques rely on the pathogen be accessible and affordable and ability of the organism to be cultured in vitro. that the required degree of sensitivity and This aspect considerably limits the applicabili- specificity is often difficult to reach (Adkison et ty of these methods since possibly less than al., 2005). 1% of the microorganisms in an environmental On the other hand, nucleic acid-based sample may be cultured (Rappe and techniques, especially if they make use of Giovannoni, 2003). Viruses are usually detect- polymerase chain reaction (PCR; Mullis and ed by designated virology laboratories using Faloona, 1987), have the advantage of being isolation, electron microscopy, in vitro viral cul- exceedingly sensitive and specific and requir- ture or, if available, serological assays to ing reagents that are easily available. As a detect viral antigens or test for the immune result, PCR-based techniques have increas- response to a given virus (Leong, 1995; ingly been developed for (fish) pathogen diag- Lightner and Redman, 1998; Storch, 2000). nosis (Cunningham, 2002). This trend is stim- Indeed, disadvantages associated with ulated by the continuously growing availability traditional identification techniques have trig- of sequence data in databases such as gered the search for alternative culture-inde- GenBank (http://www.ncbi.nlm.nih.gov/Gen pendent detection and identification tech- bank/; Benson et al., 2004) and the increasing niques, such as those based on the detection availability of microbial full genome sequences of antigenic determinants (serological tech- (e.g., http://www.sanger.ac.uk/Projects/Microbes/). niques) or nucleic acids (nucleic acid-based Nevertheless, although most of these techniques). Compared to traditional assays, methods are convenient for the detection of a these molecular techniques can avoid prob- single pathogen, screening for large numbers lems in investigating organisms for which no of different pathogens relies on a significant culture medium, cell lines (for viruses), or number of parallel tests, often using different detection method is available. In addition, technologies (Evangelopoulos et al., 2001; these techniques are generally faster, more Lievens et al., 2005a). Consequently, testing Molecular identification of fish pathogens 215 multiple targets using these methods is ineffi- sequence domains, (c) its high discriminatory cient, laborious, and expensive. Therefore, a potential over a wide range of taxonomical number of methods have recently been devel- levels, (d) its, often, multiple-copy nature, oped that can be used for the simultaneous resulting in more sensitive analyses, and (e) detection of multiple pathogens, encompass- the extensive availability of ribosomal DNA ing multiplex PCR, DNA arrays, and PCR sequences in public databases. These exten- arrays (Elnifro et al., 2000; del Cerro et al., sive sequence data allow comparison of 2002; Wang et al., 2002, 2003; Gonzalez et sequences and, in turn, determination of diag- al., 2004; Mata et al., 2004; Warsen et al., nostic regions that can be used to design spe- 2004; Lievens et al., 2005a). cific primers, probes, or oligonucleotides. In this manuscript some recent advances Nevertheless, ribosomal DNA sequences do in fish pathogen diagnosis are described, with not always reflect sufficient variation to dis- an emphasis on nucleic acid-based detection cern particular species (Mollet et al., 1997; and identification of the two major fish patho- Blackwood et al., 2000; Thompson et al., genic groups for which classical detection 2004; Kupfer et al., 2006). Therefore, other may be problematic: bacteria and viruses. housekeeping genes showing intertaxa Major features and applications of the most sequence variation are becoming more inten- predominant methods used nowadays and sively studied, including the DNA gyrase sub- some methods that look promising for the unit B gene (gyrB; Watanabe et al., 2001; future are outlined. Yanez et al., 2003), genes encoding the RNA polymerase subunits A and B (rpoA and rpoB; Nucleic Acid-Based Identification of Fish Dahllof et al., 2000; Thompson et al., 2005; Pathogens Tarr et al., 2007), the recombinase subunit A Choice of target sequences. The first stage in gene (recA; Thompson et al., 2004), genes the development of nucleic acid-based diag- encoding heat shock proteins (hsp60, hsp65 nostic assays is the selection of specific and dnaJ; Nhung et al., 2007), the elongation sequences that can be used to identify factor-Tu encoding (tuf) gene (Mignard and pathogens. There are some generally applic- Flandrois, 2007), and the gene encoding a able techniques for bacteria and fungi, but manganese-dependent enzyme (sodA; viruses usually need different approaches. Adekambi and Drancourt, 2004). However, in Regarding bacteria, the most common strate- comparison with ribosomal DNA sequences, gy for selecting target sequences involves the databases for these gene sequences general- use of ubiquitously conserved genes that har- ly contain a small number of sequences, bor specific sequences. At present, the bacte- necessitating extensive experimental screen- rial ribosomal RNA (rRNA) operon, encom- ing to ensure specificity of a diagnostic assay passing a 16S rRNA and 23S rRNA gene as based on these genes. well as an intergenic spacer (IGS) region, is In contrast to bacteria, viruses may con- most frequently used in the development of tain a DNA- or RNA-based genome, repre- molecular bacterial diagnostics (Ludwig and senting DNA or RNA [single (ss) or double Schleifer, 1994; Call et al., 2003; Sachse, stranded (ds)] viruses, respectively. In gener- 2004; Toranzo et al., 2005). In particular, the al, viral genomes are relatively small and in 16S rRNA gene is commonly targeted many cases data on complete virus genomes (Drancourt et al., 2000; Hongoh et al., 2003; are available in sequence databases. Warsen et al., 2004; Osborne et al., 2005). Currently, one of the most common targets for There are several reasons why ribosomal virus diagnostics is the coat protein gene, but sequences are such widely used targets for other regions such as the DNA or RNA poly- diagnostic development, including (a) its uni- merase gene are also being used (Culley et versal abundance, (b) its evolutionary and al., 2003; Ishioka et al., 2005). In fact, any part phylogenetic properties, reflected by the pres- of the genome could be suitable depending on ence of both variable and highly conserved how much sequence data is available from 216 Frans et al. target and related virus strains in the same plementary DNA strand (cDNA), followed by a region of the genome. Nevertheless, since (real-time) PCR assay. Many reports describe viral genomes, especially those of RNA-virus- the development of specific (RT-) PCR es, are prone to mutation, there are many assays in the ornamental fish industry and groups of viruses for which no conserved aquaculture (Cunningham, 2002; Toranzo et sequences are available that can be used for al., 2005). the design of genus- or group-specific primers Clinical laboratories are increasingly using or probes. As a result, detection of emerging PCR to complement or replace classic diag- or uncharacterized viruses remains a great nostic assays, often in the context of biosecu- challenge in molecular virology. rity (preventing) programs or to ensure the Nucleic acid-based detection techniques identity of a pathogen. Several PCR assays can be divided into DNA- and RNA-based (Gibello et al., 1999; LeJeune and techniques; some of the most common are Rurangirwa, 2000; Altinok et al., 2001) have addressed below. Efficient extraction proto- been developed for the specific detection of cols and commercially available extraction kits Yersinia ruckeri, the causative agent of are available for both types of genetic materi- enteric redmouth disease (ERM) or yersinio- al, rendering highly purified DNA or RNA from sis that can cause high morbidity and mortali- biological samples such as water or fish tissue ty rates in fish farms. To minimize economic (e.g., Filter Service S.A., Eupen, Belgium; Mo losses, rapid, specific, and sensitive detection Bio Laboratories, Solana Beach, CA, USA; of this pathogen is needed and can be met by Qiagen, Inc., Valencia, CA, USA; Fahle and PCR. Spring viraemia of carp virus (SVCV) is Fischer, 2000), favoring their use in scientific an RNA virus responsible for a severe hemor- research as well as routine diagnosis. rhagic disease in farmed cyprinids. Fast and Polymerase Chain Reaction (PCR). Using timely detection of the virus is necessary PCR, millions of copies of specific DNA because currently no vaccine against SVCV is sequences are generated in a thermocyclic commercially available. To avoid transmission process consisting of repetitive cycles of DNA of the virus, a specific RT-PCR assay employ- denaturation, primer annealing, and elonga- ing a nested PCR has been successfully used tion using a thermostable DNA polymerase to identify SVCV in fish tissue (Oreshkova et (Mullis and Faloona, 1987). If a DNA al., 1999; Koutna et al., 2003; Sanders et al., sequence unique to a particular organism is 2003). Another contagious viral disease is determined, specific PCR primers can be caused by koi herpes virus (KHV), a DNA designed that allow determination of the pres- virus responsible for significant morbidity and ence or absence of that sequence and, thus, massive mortality in common carp (Cyprinus of the corresponding organism. The presence carpio), koi carp (C. carpio koi), and C. carpio of amplified DNA is traditionally detected by gio. Because of its huge impact on the orna- gel electrophoresis, but alternative detection mental fish industry, increasingly sensitive methods exist as well (e.g., Mutasa et al., detection techniques like PCR are being 1996; Fraaije et al., 1999). Any pathogen hav- developed to detect this virus in an early stage ing a DNA genome can potentially be detect- of infection (Gilad et al., 2002, 2004; ed in this way. Bercovier et al., 2005; Ishioka et al., 2005; El- In addition, by inclusion of a step employ- Matbouli et al., 2007; Matsui et al., 2008). ing the reverse transcriptase enzyme, RNA Depending on the primers and the detec- targets such as RNA viruses can also be tion method used, minute quantities of detected. This technique is referred to as pathogen DNA can generally be detected reverse transcriptase PCR (RT-PCR; Raineri using PCR (Gibello et al., 1999; Gilad et al., et al., 1991; Tan and Weis, 1992). Typically, 2002; Bader et al., 2003). Nevertheless, to RT-PCR consists of an annealing step for a increase sensitivity (and specificity) nested reverse primer or a mixture of random primers PCR or immunocapture PCR (IC-PCR) may and an extension step to synthesize a com- be used. Nested PCR involves two sets of Molecular identification of fish pathogens 217 primers, used in two successive PCR reac- ucts. This method has successfully been used tions. The second reaction uses primers that to quantify, for instance, the bacterium hybridize to a sequence within the DNA frag- Piscirickettsia salmonis, the causative agent ment that is generated in the first reaction of Piscirickettsiosis (Heath et al., 2000). (Arias et al., 1995; Alonso, 1999; Wiklund et However, designing an appropriate competi- al., 2000; Welker et al., 2005). tor might be problematic to ensure accurate IC-PCR makes use of immobilized anti- DNA quantification. In addition, one should be bodies to isolate the target pathogen from a careful to ensure detection at the exponential sample prior to PCR amplification (Sharman phase of the PCR reaction. et al., 2000; Peng et al., 2002). Alternatively, Quantitative real-time PCR. More recently, specific probes may be used to improve sen- quantitative real-time PCR (Heid et al., 1996) sitivity and specificity (Greisen et al., 1994; has been proven to be reliable with regard to Leon et al., 1994). The advantage of the high- both pathogen detection and quantification. er sensitivity obtained by these methods can This technology is more sensitive, more accu- be exemplified for one of the most important rate, and less time-consuming than conven- pathogens in salmonid aquaculture, namely tional end-point PCR because it monitors Flavobacterium psychrophilum, the causative PCR products as they accumulate during the agent of the rainbow trout fry syndrome and reaction. This allows template quantification bacterial cold-water disease. Although Urdaci during the exponential phase of the reaction, et al. (1998) had developed a specific PCR for before reaction components become limiting. this bacterium, the detection limit of the assay In addition, since there is no need to open the appeared too low to detect the pathogen at tubes in which the amplification takes place, low densities, resulting in “false negatives” for the likelihood of post-PCR carry-over contam- subclinical or covert infections (Cipriano and ination is greatly reduced. Typically, DNA Holt, 2005). Since even low pathogen con- amplification is monitored each cycle based centrations can lead to considerable econom- on the excitation of fluorescent dyes and ic losses, more sensitive, nested PCR assays detection of fluorescent emissions (Heid et al., have been developed, enabling detection of 1996; Mackay et al., 2002). In general, the ini- this pathogen at low densities in fish tissue tial amount of target DNA is related to a and water samples (Wiklund et al., 2000; threshold cycle, which is defined as the cycle Baliarda et al., 2002; Izumi et al., 2005; number at which fluorescence significantly Madsen et al., 2005; Crumlish et al., 2007). increases above the background level. Target PCR is also used to quantify the amount of DNA is quantified using a calibration curve pathogen DNA. Although it is relatively easy that relates threshold cycles to a specific to quantify the amount of PCR products, it is amount of template DNA. more difficult to relate this quantity to the orig- As extensively discussed in other reviews inal amount of target DNA. For certain (Mackay et al., 2002; Hanna et al., 2005; pathogens, however, this information may be Lievens et al., 2005a; Espy et al., 2006), accu- necessary to make suitable disease manage- mulating amplicons can be detected using ment decisions and for monitoring the effects either amplicon specific or non-specific detec- of these decisions. Though challenging, tion methods, i.e., sequence-specific probes pathogen DNA may be quantified using com- or DNA-intercalating dyes, respectively. The petitive PCR (Siebert and Larrick, 1992), use of a DNA-binding dye like SYBR¨ Green which involves co-amplification of the target is more straightforward and less expensive DNA and known quantities of competitor DNA then using probes but is less specific since the amplifiable by the same primer pair that yield fluorogenic molecule binds to all double a product of a different length. The amount of stranded DNA (dsDNA) present in the sam- initial target DNA is then determined on ple. Further, interpretation of the analysis may agarose gel by comparison of the relative be disturbed by the formation of primer- amounts of target and competitor PCR prod- dimers or aspecific PCR products. However, 218 Frans et al. the risk of the latter can be reduced by using single nucleotide polymorphisms (SNP), inde- highly specific primers and stringent reaction pendent of the detection chemistry (Livak, conditions and the accuracy (specificity) of the 1999; Papp et al., 2003). Taking all these reaction can be checked by a melt curve advantages together, this technology offers analysis at the end of the PCR run (Bustin, many opportunities in fish pathogen diagno- 2000; Mackay et al., 2002). sis. In contrast to amplicon non-specific detec- Real-time PCR assays have been devel- tion chemistries, probe-based assays like oped for accurate detection and/or quantifica- those based on a TaqMan¨ probe offer the tion of specific fish pathogens, including advantage of increased specificity, certainly in Aeromonas spp., Flavobacterium spp., Vibrio combination with specific primers (Livak et al., spp., and DNA and RNA viruses (Overturf et 1995). These probes consist of a single al., 2001; Gilad et al., 2004; Balcazar et al., stranded short oligonucleotide labeled with a 2007; Kamimura et al., 2007). Examples reporter fluorophore at the 5’ end and a fluo- include a real-time RT-PCR combining rogenic quencher at the 3’ end. Because of reverse transcription for the detection and the proximity of both groups, the fluorescent quantification of the infectious haematopoietic signal is quenched. During the annealing necrosis virus (IHNV), an RNA virus affecting phase of each PCR cycle the probe hybridizes various salmonid species. The assay, using a to a specific region within the amplified frag- TaqMan¨ probe, was 103 times more sensi- ment. During the elongation phase, the probe tive than the standard RT-PCR (Overturf et is degraded by the 5’-exonuclease activity of al., 2001). Another example includes the the DNA polymerase causing the release of development of real-time PCR assays for koi the reporter from the quencher, resulting in a herpes virus (KHV), e.g., the TaqMan¨ probe- fluorescent signal. based assay of Gilad et al. (2004). Kamimura A drawback for this technique is that no et al. (2007) developed a QProbe-based melting curve analysis can be performed and assay for identification and quantification of thus, in theory, false positive results can be KHV in fish tissues. Although the sensitivity of obtained. This potential limitation may be cir- this assay was similar to that of a TaqMan¨ cumvented by the use of a quenching probe probe PCR, the accuracy of KHV identification (QProbe). A QProbe contains cytosine at its 5’ and quantification was slightly better with the or 3’ end which is labeled with a guanine QProbe in cases of low KHV concentrations quench fluorophore. When a QProbe (Kamimura et al., 2007). hybridizes with a target sequence, its fluores- Loop-mediated isothermal amplification cence is quenched by the guanine in the tar- (LAMP). Apart from PCR, other techniques get that is complementary to the modified have been developed for the amplification of cytosine. Consequently, in contrast to a nucleic acids (Andras et al., 2001). One tech- TaqMan¨ probe-based assay, a reduction in nique increasingly used in fish pathogen diag- fluorescence is measured as the PCR prod- nostics includes the loop-mediated isothermal ucts accumulate during the reaction. In addi- amplification method (LAMP) which rapidly tion, no DNA polymerase is needed to obtain amplifies genomic DNA with high specificity fluorescence. As a result, a DNA polymerase and amplification efficiency under isothermal without 5’-exonclease activity can be used, conditions, avoiding the need of a thermocy- which, in turn, enables the formation of a melt- cler (Notomi et al., 2000). In combination with ing curve, allowing a specificity check of the an additional reverse transcription step, this reaction (Kurata et al., 2001). technique may also be used for RNA viruses Depending on the target gene selected, (reverse transcription-coupled LAMP; closely related microbial species may differ in Gunimaladevi et al., 2005; Soliman and El- only a single (or few) base(s) of the investi- Matbouli, 2006; Shivappa et al., 2008). gated gene. The high degree of specificity of LAMP typically relies on an auto-cycling real-time PCR technology allows detection of strand displacement DNA synthesis, per- Molecular identification of fish pathogens 219 formed by a DNA polymerase with high strand successfully developed a LAMP assay for the displacement activity and a set of four specif- detection of Flavobacterium columnare, ic primers, as described and well illustrated by causative agent of columnaris in many fish Notomi et al. (2000). Although a high speci- species. As an example regarding RNA virus- ficity is obtained using this method, the selec- es, Shivappa et al. (2008) developed an RT- tivity of the technique can be increased by coupled LAMP assay that can be used under using six primers (Soliman and El-Matbouli, field conditions for diagnosis of spring 2005). The entire procedure can be complet- viraemia of carp virus (SVCV), a considerable ed in one hour and results in a large amount pathogenic agent which causes systemic ill- of stem loop DNAs (Notomi et al., 2000; ness and high mortality in cyprinids, especial- Savan et al., 2005). Subsequently, these ly in common carp. products can be detected by gel electrophore- Multiplex detection. One limitation of most sis resulting in several bands of different sizes detection procedures, whether serological or for a single sample. nucleic acid-based, is that only a single or a Another method for detection involves few targets can be detected and identified in a real-time detection. As in LAMP, a large single assay. As most fish can be infected by amount of DNA is synthesized. Accordingly, a a multitude and wide variety of pathogens, large amount of pyrophosphate ion by-product with new pathogens being recorded regularly, is generated, yielding an insoluble salt of a comprehensive pathogen screening pack- magnesium pyrophosphate. The presence or age would require an endless number of indi- absence of target DNA may be judged visual- vidual tests, making routine screening of mul- ly by the appearance of a white precipitate tiple targets inefficient, laborious, and expen- (Caipang et al., 2004) or by turbidity mea- sive. In addition, fish symptoms often result surement in the reaction mixture (Mori et al., from infection by several pathogens rather 2001). Alternatively, SYBR¨ Green may be than a single pathogen, complicating classical added to the reaction causing a color change diagnosis. Therefore, multiplex detection from orange to green in case target DNA is enabling detection of numerous pathogens in detected (Soliman and El-Matbouli, 2005). a single assay has been a major challenge in The high correlation between turbidity at the fish disease diagnostics. end of the reaction and the initial concentra- In theory, multiplex detection can be tion of target DNA makes both qualitative and achieved by multiplex PCR (Wilton and semi-quantitative diagnosis possible (Caipang Cousins, 1992) using several primer sets in et al., 2004). the same reaction targeting discrete The detection limit of this technique is sim- pathogens (del Cerro et al., 2002; Gonzalez et ilar or better to that of PCR. As a result, LAMP al., 2004). However, the development of accu- is a rapid, highly specific, sensitive, and cost- rate multiplex formats is often difficult, leads to effective alternative for PCR which can be less sensitive assays, and requires extensive used for detection, even on-site detection, of optimization of reaction conditions in order to specific fish pathogens (Caipang et al., 2004; discriminate at least a few amplicons per Gunimaladevi et al., 2004, 2005; Kono et al., reaction. Further, amplicon sizes should be 2004; Soliman and El-Matbouli, 2005, 2006; different enough to ensure clear discrimina- Yeh et al., 2005, 2006; Sun et al., 2006; tion of the amplicons by gel electrophoresis Shivappa et al., 2008; Wei et al., 2008). As an (Henegariu et al., 1997). This latter limitation example, a LAMP-based method has been does not apply to real-time PCR using specif- developed to rapidly and specifically detect ic probes which are labeled with different fluo- the fish pathogenic bacterium Edwardsiella rescent dyes but the limited availability of dif- ictaluri, one of the most important pathogens ferent fluorophores and the common use of in the aquaculture of channel catfish causing monochromatic light in real-time PCR instru- enteric septicemia (Yeh et al., 2005). ments limit the total amount of PCR reactions Likewise, the same authors (Yeh et al., 2006) that can be performed in a single run (Mackay 220 Frans et al. et al., 2002). As a result, the maximum num- geting different markers, PCR products can ber of pathogens detectable in a single assay be combined at hybridization. In general, is currently relatively small using these strate- short oligonucleotides of approximately 20 gies. nucleotides are used to obtain a high speci- Apart from multiplex PCR, degenerate ficity. Indeed, the discriminative power of this primers (e.g., for members of a class) have approach is very high since even microorgan- been used, for example, for unambiguous isms whose target sequences differ by a sin- identification of unknown viruses. This strate- gle nucleotide can be discriminated if specific gy is complicated by the existence of highly criteria are taken into account (Lievens et al., homologous relatives, making additional pro- 2006). This approach is efficient for rapid cedures such as restriction enzyme analysis, detection and identification of various microor- blotting analysis, or cloning and sequencing ganisms including bacteria (Call et al., 2003), necessary (Oppegaard and Sorum, 1996; fungi (Lievens et al., 2003), and some virus Lilley et al., 1997; Talaat et al., 1997; groups (Chizhikov et al., 2002). Warsen et al. Heidelberg et al., 2000; Chen et al., 2003). (2004) developed a DNA microarray based on So far, the most promising technology for 16S rDNA sequences for the simultaneous the development of multi-pathogen detection discrimination between 15 economically systems has been the advent of DNA array important fish pathogenic bacterial species technology. In theory, an unlimited number of among which are several species of target organisms can be simultaneously Aeromonas, Flavobacterium, Mycobacterium, detected and identified using low-density and Streptococcus. macroarrays (e.g., on a nylon membrane) or There are many groups of organisms, high density microarrays (e.g., on a glass especially viruses, for which no effective uni- slide). In addition, DNA arrays may allow versal primers are available. For these, pathogen detection over a wide range of tax- sequence-nonspecific amplification methods onomic levels, even across superkingdom such as strategies based on the random borders (Wilson et al., 2002). Apart from phy- primed amplification method of Bohlander et logenetic markers, other biomarkers such as al. (1992) may be used in combination with virulence genes or antibiotic resistance arrays of 70-mer oligonucleotides to identify markers can also be implemented on the sequences of numerous unrelated targets array. (Wang et al., 2002; Agindotan and Perry, Although DNA arrays were originally 2007). The most remarkable application of designed to study gene expression, gene dis- this technology has been the development of covery, SNP analysis, and DNA sequencing a microarray for the detection and genotyping (Schena et al., 1996; Ramsay, 1998), taxono- of over 1000 viruses, including those from fish mists and diagnosticians quickly recognized (Wang et al., 2003). the potential of this technology for identifying Major advantages of the first approach pathogens. With this technology, specific over the second one are its higher sensitivity, detector oligonucleotides are immobilized on which is comparable to the sensitivity of other a solid support, essentially allowing reverse molecular techniques (Lievens et al., 2003), dot blot hybridization. and its higher specificity (Levesque et al., In general, two approaches have been 1998). The advantage of the second used for signal amplification. The most com- approach is that non-related sequences can mon involves the use of universal primers that be simultaneously amplified in a relatively anneal to conserved sequences flanking diag- simple and cost-effective way. nostic domains in housekeeping genes such Another strategy of multiplexing with high as the ribosomal rRNA gene. In this way, levels of specificity (and sensitivity) is the use numerous targets can be amplified with a sin- of multiplex PCR primers that amplify discrete gle primer pair, while target discrimination is targets followed by amplicon discrimination performed afterwards on the array. When tar- using DNA arrays (Gonzalez et al., 2004). Li et Molecular identification of fish pathogens 221 al. (2001) showed that this detection technique of such assays, however, is relatively low sen- is also possible for discriminating RNA viruses sitivity, limiting its widespread use. and, in particular, to type multiple strains of the Nevertheless, since these tests are relatively influenza virus. Nevertheless, the develop- inexpensive, take little time to perform, and do ment of an efficient reaction in which all targets not require specialized equipment or knowl- are amplified with the same efficiency is not edge, there is growing interest in the use of always straightforward and requires extensive these tests for on-site, front-line pathogen optimization of the reaction conditions. screening. Nucleic acid-based detection platforms Discussion and Future Directions are also becoming available for on-site Laboratories that provide diagnostic services pathogen diagnosis. One example includes and inspection agencies are increasingly the development of portable real-time PCR searching for fast routine methods that pro- instruments such as the SmartCycler vide rapid detection and reliable identification (Cepheid, Sunnyvale, CA, USA), which of pathogenic organisms, including fish enables parallel testing of 16 samples under pathogens. In this regard, PCR-based meth- different conditions. ods such as (real-time) PCR are more and These new developments pose new chal- more implemented in practice. Nevertheless, lenges for sample processing as several limi- to increase efficiency and reduce costs, time, tations inherent to field-testing need to be cir- and labor, multiplex detection assays are cumvented. For example, PCR reagents need desirable. Currently, DNA array technology is to be stable at ambient temperature the most suitable technique to simultaneously (Tomlinson et al., 2005). detect numerous targets. This technology can Regarding multiplex technologies, progress also be used for pathogen quantification since can be expected from PCR arrays that com- hybridization signals are proportional to the bine the advantages of DNA arrays and real- quantity of target DNA (Lievens et al., 2005b), time PCR, resulting in high throughput capaci- making this technique even more attractive for ty and accurate quantification (Belgrader et al., fish pathogen diagnosis and disease manage- 1998). Typically, PCR arrays provide a plat- ment decisions. However, as the amount of form on which spatially separated PCR reac- material necessary for analysis becomes less tions can be performed simultaneously. This with the development of more sensitive tech- can be exemplified by the OpenArrayTM technologies such as those based on PCR, devel- nology of BioTrove (Woburn, MA, USA) in opment of appropriate sampling strategies which a few thousand real-time PCR assays and knowledge of the disease development (48 x 64 reactions) can be performed at the will become more challenging. same time in minuscule reaction holes. So far, molecular diagnostics are relative- However, the sensitivity and accuracy of quan- ly expensive in terms of investment and facili- tification may suffer from the ultralow reaction ties. Consequently, they are pertinent only for volumes used. Another interesting develop- well-equipped laboratories. The next chal- ment is the lab-on-chip instrument (Anderson lenge is to simplify molecular diagnostics and et al., 2000; Wang, 2000), which combines bring them into the field, enabling on-site several handlings (from DNA extraction to DNA pathogen diagnosis. Antibody-based lateral analysis) on a single, portable, and fully auto- flow devices, originally developed for preg- mated device. nancy testing, can meet these demands Which technologies will eventually be (Smits et al., 2001) and are increasingly being implemented in fish pathogen diagnosis developed for on-site diagnosis of fish-related remains unclear, but obviously only those diseases. A recent example includes a lateral assays that become available at an affordable flow assay for rapid detection of the infectious price. Taking into account the unlimited salmon anaemia virus, ISAV (Aquatic expansion possibilities of DNA arrays to Diagnostics Ltd., Stirling, UK). A disadvantage include oligonucleotides for all markers of 222 Frans et al. interest and current technical and economic (www.DNAmultiscan.com; Lievens and requirements for routine diagnostics, we Thomma, 2005). believe that as soon as DNA array-based Likewise, we are now developing a DNA detection procedures become more automat- macroarray for a comprehensive set of eco- ed, DNA macroarrays may become the new nomically important fish pathogens for the orna- benchmark in fish pathogen diagnosis. mental fish industry. In Fig. 1, a first generation Compared to DNA microarrays, macroarrays DNA array containing 16S rDNA oligonu- are cheaper, more sensitive, and can be cleotides for the identification of 15 fish patho- reused many times (Lievens and Thomma, genic bacterial species is shown. The new diag- 2005), favoring the use of this type of DNA nostic assay will eventually contain detector arrays. oligonucleotides for a diverse set of fish Even in its current format, DNA macroar- pathogens, including multiple species from rays are routinely used by several diagnostic Aeromonas, Edwardsiella, Flavobacterium, laboratories. For example, laboratories are Mycobacterium, Pseudomonas, Renibacterium, increasingly using routine DNA arrays for Vibrio, and Yersinia, and a selection of viral plant pathogen detection, using for example pathogens, e.g., koi herpes virus (KHV), carp the DNA Multiscan¨, a membrane-based DNA pox virus (CPV), channel catfish virus (CCV), array to detect and identify over 75 plant path- white spot syndrome virus (WSSV), spring ogenic fungi, oomycetes, and bacteria viraemia of carp virus (SVCV), viral haemor-

AB CDE

Fig. 1. Identification of a fish pathogenic bacterium (Pseudomonas anguilliseptica, the causative agent of pseudomonadiasis) using a 16S rDNA sequence-based DNA macroarray. Each detector oligonucleotide is spotted in duplicate on a nylon membrane. Specificity of the analysis is enhanced by using multiple oligonucleotides for each target species. In addition to the immobilized target-specific sequences, the array contains control oligonucleotides for hybridization (13A, 14A, 15A, 16A, 13E, 14E, 15E, and 16E) and a ref- erence for detection and calibration (1A, 1E, 17A, and 17E). PCR-labeled amplicons generated with universal primers hybridize to species-specific oligonucleotides for P. anguillispetica (7A, 8A, 9A, 10A, 11A, 7B, 8B, 9B, 10B, and 11B). Based on the location of the signals, identification is performed. Results are shown for different amounts of genomic DNA that have been amplified (ranging 5 ng to 50 fg) and hybridized to the array. 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