20 Molecular Approaches and Techniques Ted G. Clark Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Introduction tissues. Finally, gene cloning methods and their use in the rapidly emerging fields of Molecular genetic techniques have in the genomics and vaccine development are last 15 years gained widespread use in the described. study of fish parasites. These techniques have been particularly effective in unravel- ling the complex life histories of various parasitic forms, and have played a key role PCR and DNA Sequence Analysis: an in determining the precise taxonomic rela- Overview tionships between parasite species that are difficult to ascertain using traditional mor- PCR is among the most widely used meth- phological techniques. In some instances, ods in biology today. Briefly, the technique these studies have provided key insights into utilizes short oligonucleotide primers that evolutionary biology as a whole. In addition, flank a given region of DNA to amplify large molecular tools have contributed to our amounts (µg–mg quantities) of a specific tar- understanding of the cellular biology of para- get sequence in successive rounds of primer sites, and have proved indispensable in the annealing and strand elongation (DNA syn- more practical realm of disease diagnosis and thesis) catalysed by thermostable DNA vaccine development. polymerase enzymes. Once made, amplified The present chapter focuses primarily DNA fragments are purified, using agarose on the use of two methods, namely, the gel electrophoresis, and either directly polymerase chain reaction (PCR) and DNA sequenced or introduced into a standard sequencing, in the analysis of fish parasites. plasmid cloning vector for subsequent This emphasis reflects the widespread use analysis (Sambrook et al., 1989; Fig. 20.1). of these techniques in diagnostics, epidemi- Perhaps the most common targets for ology and systematics. Specific applica- amplification in both diagnostic testing and tions are described for Myxozoa, Microspora, phylogenetic studies are the 18S small sub- Ciliates, kinetoplastids, Icthyosporea and unit ribosomal RNA genes (SSU rDNAs) and helminths. Along with PCR and DNA the internally transcribed spacer regions of sequence analysis (which rely on DNA–DNA the rDNA loci (ITS domains) (Sogin and interactions in solution), solid-phase hybri- Silberman, 1998; Van de Peer et al., 2000; dization methods are also discussed, with Fig. 20.2). These rDNA genes evolve at a particular emphasis on in situ hybridization slow rate in eukaryotes and thus serve as a means of identifying parasites in host as reliable markers for the identification of CAB International 2006. Fish Diseases and Disorders Vol. 1 (ed. P.T.K. Woo) 725 726 T.G. Clark differences within species. For some types of analyses, rDNA sequences alone are not sufficient and amplification of protein-coding genes becomes necessary. For diagnostic testing, genomic DNA from the parasite in question is used as a template for PCR amplification, with primers that recognize sequences common to parti- cular species or conserved amongst broader taxonomic groups. The choice often depends on the likelihood that a particular organism is present. In most instances, amplification does not require a highly purified template, owing to the sensitivity and specificity of PCR. Indeed, bulk DNA from infected tis- sues is often used as starting material for these reactions, since contaminating DNA from the host generally does not interfere Fig. 20.1. Applications of PCR and DNA with amplification of target sequences from sequencing in molecular parasitology. Amplification the infecting agent. Resulting products of a DNA fragment from small subunit ribosomal (referred to as amplicons) are then fraction- RNA genes (SSU rDNA) is illustrated. Genomic ated using agarose gel electrophoresis and DNA from infected host tissue (or from the agent visualized under ultraviolet (UV) light, fol- itself) is used as a template for PCR amplification, lowing staining with the fluorescent dye along with forward and reverse primers ethidium bromide. The presence of an complementary to regions of the rDNA sequence. amplicon of the expected size is generally Primers can be directed towards regions that are sufficient to identify a particular agent, conserved among broad classes of organisms or towards sequences found only in particular species. assuming proper controls are included. More Production of an amplicon of the expected size, stringent diagnostic tests include additional following agarose gel electrophoresis and staining steps, such as digestion of purified amplicons with ethidium bromide, is often diagnostic for the with restriction endonucleases that recognize presence of a particular agent. A DNA size standard specific sequences within the target DNA is depicted in the left lane of the gel. Definitive (see below, PCR-restriction fragment length diagnosis can be achieved by sequencing of the polymorphism (RFLP)). Restriction digests purified PCR fragment (either directly or after result in a complex pattern of bands (on insertion into a plasmid DNA cloning vector). agarose gel electrophoresis), which lend Alternatively, the sequence can be used to infer added confidence to the presumptive diag- evolutionary relatedness between the identified organism and other species. In the case of nosis. Clearly, the most definitive test and protein-coding genes, amplified PCR fragments can the one used in most phylogenetic studies be used for a variety of other purposes, including involves the sequencing of amplicons to the production of subunit vaccine antigens in determine their primary structures. Follow- heterologous cell types (typically yeast or ing agarose gel electrophoresis, individual Escherichia coli). bands containing amplified DNA fragments are cut from the gel. The DNA is then puri- fied on silica-based resins and either species. Indeed, the conserved nature of directly sequenced or introduced into SSU rDNAs makes them ideal molecular plasmid cloning vectors for subsequent ‘clocks’ for estimating evolutionary dis- analysis (Fig. 20.1). tances between widely diverged taxa. In Although sequencing is still performed contrast, the more rapidly evolving ITS within individual laboratories, a more com- domains are used to discriminate closely mon practice is to outsource the DNA to a related species or, in some instances, strain commercial or academic centre that employs Molecular Approaches and Techniques 727 Fig. 20.2. Organization of ribosomal RNA loci. Small subunit (18S) and large subunit (28S) rRNA genes are separated by internally transcribed spacer regions (ITS1 and ITS2). The rDNA genes are expressed as a single large transcript that extends from the externally transcribed spacer region (ETS) just proximal to the small subunit gene, through the end of the large subunit gene. The transcript is processed into separate 18S, 5.8S and 28S rRNAs, which are then incorporated into the ribosome. The variable region at the proximal end of the large subunit gene (D1/D2), as well as the intergeneic non-transcribed spacer regions (IGS1 and IGS2) are sometimes used as targets for phylogenetic analyses. standard dideoxy chain termination methods (primarily fish) and annelid hosts, and the of sequence analysis (Sambrook et al., 1989). Malacosporea, which alternate between fish Sequences are provided for the end-user in and bryozoan hosts (Kent et al., 2001). Par- electronic form, which are then used to tial or full-length 18S rDNA sequences are search comprehensive databases (e.g. Gen- now available for a wide range of myxozoan Bank; http://www.ncbi.nlm.nih.gov/) using species (Kent et al., 2001) and have been standard algorithms such as the basic local used in the development of PCR-based diag- alignment search tool (BLAST) (Altschul nostic tests for detection of these organisms et al., 1990). An exact (or near) match is in fishes and in the aquatic environment. considered diagnostic for a given species. In Such tests have clear advantages in terms of the event that a novel sequence is found, a speed, sensitivity and specificity relative to variety of computational methods (cluster more classical methods of detection, and analysis, maximum parsimony, maximum have been applied to a number of species likelihood, neighbour-joining, etc.) can be that are of importance to commercial used to define the taxonomic relationship(s) aquaculture. For example, primer pairs that between the new or presumptive species specifically amplify rDNA sequences of the and its closest relatives (see Swofford et al., enteropathogen Ceratomyxa shasta have 1996). Comparisons of many sequences within been used to develop a simple diagnostic and among taxonomic groups allow the test for this pathogen in salmonids establishment of phylogenetic trees. Inferred (Fig. 20.3). One such pair can be used to relationships are further strengthened by the detect as little as 50 fg of purified C. shasta inclusion of protein-coding gene sequences genomic DNA. The test is able to detect (in addition to rDNA) in such comparisons. organisms in gill tissues of rainbow trout (Oncorhynchus mykiss) within 2 days of exposure of naïve fish to the actinosporean stage of the parasite (Palenzuela et al., 1999). Applications of PCR and DNA Sequence Diagnostic tests for several other important Analysis in Diagnostics, Epidemiology myxozoans affecting farm-raised fish are and Systematics of Fish Parasites reviewed briefly.