The Israeli Journal of Aquaculture – 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 iden- tifying 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 detec- tion and identification techniques. Major features and applications of current predominant meth- ods and promising methods likely to impact future fish disease control and prevention are out- lined. 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