20 Molecular Approaches and Techniques

Home , Anisakis simplex, Ceratomyxa yokoyamai, Hymenostome, Ichthyophonida, Myxobolus cerebralis, Myxozoa

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 unravelling 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 particular 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 tissues 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 purified 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. A PCR-based test that amplifies a 104 bp Myxozoa SSU rDNA fragment from Henneguya ictaluri has proved extremely useful in detecting Analyses of SSU rDNA sequences have the proliferative gill disease agent of chan- been particularly informative in studies of nel catfish (Ictalurus punctatus). The prim- the Myxozoa (Chapter 8). Classification ers used for this test are highly specific schemes based on SSU rDNAs separate for H. ictaluri and do not amplify detectable these organisms into two classes, the Myxo- fragments from genomic DNA of other sporea, which alternate between vertebrate Myxozoa (including Henneguya exilis, 728 T.G. Clark

Fig. 20.3. (Top panel) SSU rRNA gene primer map for C. shasta. Predicted amplicon sizes for different combinations of the primers (Cs1, Cs2, R, Cs3, Cs4, Cs5, 18SUNIF and 18SUNIR are shown in base pairs. Bottom panel shows specificity of different primer pairs for C. shasta DNA. Amplicons of the predicted size were obtained with DNA from C. shasta spores (lanes a, d, g, j and m), but not rainbow trout (lanes b, e, h, k and n) or Henneguya salminicola (c, f, i, l and o). Primer pairs were Cs1–Cs5 (lanes a–c); Cs1–Cs3 (lanes d–f); Cs2–Cs5 (lanes g–i); R–Cs4 (lanes j–l); and r–Cs5 (lanes m–o). (From Palenzuela et al., 1999 with permission from Diseases of Aquatic Organisms.)

Aurantiactinomyxon mississippiensis and than the more standard pepsin–trypsin Helioactinomyxon sp.) shed by the alterna- digest method, which relies on microscopic tive invertebrate host, Dero digitata (Hanson detection of parasites released from enzy- et al., 2001). In addition to H. ictaluri, SSU matically digested tissue (Andree et al., rDNA sequences have been determined for 1997, 1998; Schisle et al., 2001). PCR-based several other freshwater and marine species tests also provide definitive diagnoses of of Henneguya (Kent et al., 2001). M. cerebralis in host tissues, in contrast to Both single-round and nested PCR tests less specific microscopic methods (Frasca have been developed for detection of et al., 1999). Single-round PCR tests con- the whirling disease agent, Myxobolus cere- ducted on large numbers of free-ranging bralis, in salmonids. Both tests have been and hatchery-reared fish have suggested shown to be more reliable indicators of that this agent may be present in locations infection (particularly at low parasite load) once thought to be free of the parasite Molecular Approaches and Techniques 729

(Schisle et al., 2001). Nested PCR, which is found worldwide, and SSU rDNA analysis typically more sensitive than single-round has permitted definitive diagnosis of parti- reactions owing to successive rounds of cular species (for example, K. thyrsites and amplification with different sets of primer K. amamiensis) across a broad geographical pairs, can detect the equivalent of one range, including waters off eastern Australia sporoplasm of M. cerebralis per sample of (Whipps et al., 2003). At least six species of infected tissue. The 415 bp amplicon gener- Kudoa have been inferred based on SSU ated by this method can also be detected in rDNA analysis (K. thyrsites, K. paniformis, tissue samples as early as 1 week following K. ciliatae, K. crumena, K. amamiensis, and exposure of rainbow trout to the actino- K. miniauriculata) (Kent et al., 2001). The sporean stage (Andree et al., 1998). In com- total number of species remains unknown, parative studies of SSU and ITS rDNA although analyses of infected tissues of a sequences from parasites collected in dif- bullseye puffer (Sphoeroides annulatus) ferent geographical locations, relatively lit- and sweeper (Pempheris ypsilychnus) have tle sequence divergence was found among suggested the existence of at least two more isolates from the USA and Germany species, namely, Kudoa dianae and Kudoa (Andree et al., 1999a). This lack of diver- minithyrsites, respectively (Dykova et al., gence provides support for the suggestion 2002; Whipps et al., 2003). that M. cerebralis was introduced relatively PCR-based diagnostic tests have been recently into the USA from Europe. The applied to less prevalent myxozoan species, parasite was first reported in North America such as Parvicapsula minibicornis and in 1958. Similar analyses of 18S rDNA from Enteromyxum scophthalmi. Epidemiologi- ten different species of Myxobolus placed cal studies in migrating sockeye salmon in these into three distinct clusters that were the Fraser River (British Columbia, Canada) independent of spore morphology, spore found that the infection rate with P. mini- size and host specificity. The phenotypic bicornis increased dramatically as fish trait most closely correlated with the entered their spawning grounds, with prev- rRNA-based clusters appeared to be tissue alence reaching ≥ 95% while it was 0% for tropism, as these organisms infect muscle, fish while at sea. Infections were also detected neuronal or enteric sites (Andree et al., in other salmon species (Jones et al., 2003). 1999b). Additional applications of SSU In the case of E. scopthalmi, SSU rDNA rDNA analysis to the Myxobolidae include analysis has suggested that this parasite rep- the identification of distinct species among resents an entirely new genus (Palenzuela the morphologically similar types Myxobolus et al., 2002). E. scophthalmi has been iden- elegans (Kashkovsky) and Myxobolus tified as an enteric pathogen that can be hungaricus (Jaczo) (Eszterbauer, 2002). lethal to cultured turbot (Psetta maxima). PCR-based (SSU rDNA) tests have also Based on rDNA analysis, it appears taxo- been developed for Kudoa spp., an emerg- nomically related to Myxidium leei, which ing group of disease-causing agents of marine had previously been ascribed to the genus fishes. Such tests have permitted the diag- Myxidium on morphological grounds. It has nosis of Kudoa thyrsites in net-pen-reared been suggested that M. leei be renamed Atlantic salmon, where it causes myolique- Enteromyxum leei and grouped with faction and problems with flesh quality. E. scopthalmi as members of the new genus, PCR-based tests have suggested that tube- Enteromyxum, consisting of histozoic and snouts (Aulorhynchus flavidus) are reser- enteric pathogens of marine fin fishes voirs for infection of commercial salmon (Palenzuela et al., 2002). fisheries (Shaw et al., 1997). Tube-snouts In addition to Myxosporea, PCR-based were often found in the vicinity of net tests have been developed for Malaco- pens, and a high prevalence of K. thyrsites sporea, in particular Tetracapsuloides bryo- infection (up to 100%) was shown in fish salmonae. This is the causative agent of collected near Vancouver, British Columbia proliferative kidney disease (PKD) in (Shaw et al., 1997). The genus Kudoa is salmonids (Saulnier and de Kinkelin, 1997; 730 T.G. Clark

Kent et al., 1998; Hedrick et al., 2004). present in a range of bryozoan host species Such tests have been used to detect para- collected in diverse habitats, with temporal sites in infected fish and to distinguish and spatial variations in the incidence of T. bryosalmonae from other myxozoa within infection (Okamura et al., 2001). the class Malacosporea, including the worm- Finally, classification of the Myxozoa like parasite Buddenbrokia plumatellae and in respect of higher-order taxonomy has an as yet unidentified species belonging to relied heavily on gene sequence data. Based what may be a third genus (Canning et al., on morphological criteria alone, the phylum 2002; Hedrick et al., 2004). was difficult to classify, and the Myxozoa In addition to their practical applica- were originally placed with the cnidarians. tion in diagnostics, molecular genetic While there is some support for this analyses have played critical roles in hypothesis based on SSU rDNA sequence unravelling the complex life cycles of analysis, a number of studies suggest that myxosporeans. Comparisons of 18S rDNA they either fall within or are sister to the sequences provided the first direct evidence Bilateria (see Kent et al., 2001; Canning and that organisms previously considered to be Okamura, 2004). This view is bolstered by members of different taxonomic classes the analysis of protein-coding genes, in par- (that is, Myxosporea and Actinosporea) ticular Hox genes whose sequences in were in fact different forms of the same Myxozoa align with those of bilaterians organism alternating between vertebrate (Ferrier and Holland, 2001; Finnerty, 2001). and invertebrate hosts (Andree et al., 1997; However, a more recent analysis of a gene Bartholomew et al., 1997). This was demon- that encodes a cathepsin-like protease has strated at about the same time for both suggested that the Myxozoa may actually C. shasta and M. cerebralis. In the case of predate the bilaterians (Kelley et al., 2003). C. shasta, primers specific for the parasite were able to detect relevant rDNA sequences in Manayunkia speciosa, a polychaete worm that was also capable of transmitting Microspora Ceratomyxa infection to susceptible fish (Bartholomew et al., 1997). Annelids that The microsporidia are an extremely inter- could not transmit infections had no esting group of intracellular parasites that detectable C. shasta sequences. Similarly, infect vertebrate and invertebrate hosts 18S rDNA sequences from M. cerebralis (Chapter 7). More than 150 species are known were detectable using PCR in both infected to infect fishes (Lom and Nilsen, 2003). fish (harbouring the myxosporean stage) Microsporidia may cause either destruction and aquatic oligochaete worms (harbouring of the host cell cytoplasm or hypertrophy the triactinomyxon stage of the parasite) and the formation of an enlarged host cell/ (Andree et al., 1997). Further studies using parasite complex referred to as a xenoma. In this technique showed that 2–3% of tubifex fish, microsporidia are found in different worms in areas that are endemic for whirl- host tissues, including gills, muscle, nerve, ing disease actually carry the parasite blood, gonads, liver and intestine, where (Rognlie and Knapp, 1998). Such worms they cause severe disease. Although mor- (Tubifex tubifex) appear to be persistently phological criteria have been used to clas- infected, and can release the triactinomyxon sify the microsporidia, SSU rDNA sequence stage periodically into the environment analyses have been far more informative in (Gilbert and Granath, 2001). Primers specific respect of phylogenetic relationships within for rDNA sequences of H. ictaluri detected this group. While a detailed phylogenetic the actinosporean stage of the parasite tree for microsporidia is still emerging, (Aurantiactinomyxon ictaluri) in the aquatic sequence data are available for 12 of the 15 oligochaete D. digitata (Pote et al., 2000). microsporidian genera known to infect fish. Analogous PCR-based studies have shown SSU sequence comparisons suggest that that the malacosporean T. bryosalmonae is these cluster into five taxonomic groups, Molecular Approaches and Techniques 731

four of which form a single clade. The out- highly sensitive and can detect parasites in lying group (Nucleospora) is only distantly host tissues at the pre-spore stage, and as related and probably represents an entirely early as 3 days post-infection (Hung et al., different taxonomic order (Nilsen, 2000). 1998; Bell et al., 1999). In N. salmonis, diag- Supporting this argument is the fact that nostic PCR tests have been used to look SSU ribosomal genes from Nucleospora for divergence among parasites infecting salmonis and species belonging to the main salmonids and other fish hosts in different clade share only ∼ 60% sequence identity geographical locations. Sequences derived overall (Lom and Nilsen, 2003). from amplified rRNA genes (along with The high degree of conservation of SSU their respective ITS domains) have been sequences sometimes obscures relationships shown to be highly conserved, suggesting between closely related species within the that N. salmonis forms a relatively homoge- individual microsporidian group. However, neous group across a broad geographical ITS regions (which vary in length by a fac- range (Gresoviac et al., 2000). A rapid assay tor of 10 among different microsporidia), as for differentiating rDNA sequences from well as protein-coding genes, often provide microsporidia of fish, insects and shrimps the necessary data to resolve the relation- was developed by Pomport-Castillon et al. ships between individual species (Lom and (1997). In this assay, fragments of SSU and Nilsen, 2003). Interestingly, within the ITS sequences, amplified using PCR, are principal clade of microsporidia infecting subjected to restriction endonuclease diges- teleosts, a number of species appear to be tion to generate RFLPs, which are screened specific for hosts other than fish (including visually on agarose gels. The technique, crustaceans, insects and humans). If this alternatively known as riboprinting or PCR- host specificity can be verified, similarities RFLP (Graham, 1997), avoids the require- in the SSU sequences between fish-specific ment for sequencing DNA fragments from parasites and the non-fish species would individual isolates (Fig. 20.4). suggest that the switch to non-fish hosts is a fairly recent event (Lom and Nilsen, 2003). At the higher taxonomic level, initial Ciliates studies of SSU rDNA sequences suggested that microsporidia evolved early in the SSU rDNA sequence analysis has been eukaryotic lineage and were considered to applied to the two most important parasitic be primitive protozoans (Vossbrinck et al., ciliates of commercially raised fish, namely, 1987). Nevertheless, accumulating data from Ichthyophthirius multifiliis, the causative both SSU and protein-coding genes, includ- agent of white-spot disease in freshwater ing tubulin (Edlind et al., 1996; Keeling and fish, and Cryptocaryon irritans, its saltwater Doolittle, 1996), HSP70 (Germot et al., 1997; counterpart (Chapter 4). These species were Hirt et al., 1997; Peyretaillade et al., 1998) long considered to be taxonomically related and RNA polymerase II (Cheney et al., 2001), because of their similar life cycles and have indicated that the microsporidia are morphologies. Nevertheless, rDNA sequence related to fungi and have evolved more analysis has indicated that I. multifiliis and recently than previously thought. C. irritans are highly diverged. I. multifiliis Apart from their use in phylogenetic is one of two genera within the group studies, PCR-based analyses of SSU rDNAs Ophryoglenina that is sister to the tetra- have been useful as diagnostic tools for the hymenines (class Oligohymenophorea) detection of microsporidian infections in (Wright and Lynn, 1995). C. irritans, on the fishes. Single and nested PCR tests have other hand, is only distantly related and is been developed for Microsporidium seriolae in a different class, the Prostomatea (order (Bell et al., 1999), Pleistophora anguillarum Prorodontida) (Diggles and Adlard, 1995; (Hung et al., 1998), Loma salmonae (Docker Wright and Colorni, 2002). Analysis of et al., 1997), and N. salmonis (Barlough et al. rDNA sequences from individual isolates of 1995; Gresoviac et al., 2000). These tests are Cryptocaryon from Australia (Diggles and 732 T.G. Clark

Fig. 20.4. Riboprinting. Specific primers (in this example for SSU rDNA) are used to amplify regions of DNA by PCR. PCR products are then digested with a given restriction endonuclease. Sequence polymorphisms among individual organisms, strains or species are manifested as different patterns of bands on agarose gels. (From Clark, 1997. With permission from Journal of Eukaryotic Microbiology.)

Adlard, 1997) and Taiwan (Yambot et al., SSU rDNAs of these organisms (Pugovkin 2003) suggests the existence of strains that et al., 2001). The test is designed to ensure may occupy different ecological niches. that I. multifiliis maintained in artificial Because C. irritans appears capable of chang- media is free of contaminating T. corlissi. ing its morphological and developmental characteristics, rDNA analysis is likely to provide the most reliable marker for strain Kinetoplastids differences in this species (Yambot et al., 2003). The kinetoplastids (Chapter 3) are a group of In the case of Ichthyophthirius, rDNA flagellated protozoa (phylum Euglenozoa) sequence analysis has been used to estimate that share a common structural feature, the the age of the ciliate phylum and, by infer- kinetoplast, which lies within the single ence, the ‘crown’ group eukaryotes as a mitochondrion at the base of the flagellum(a). whole. The rate of nucleotide substitutions This structure comprises a network of cir- within the rDNA of ciliates was estimated cular DNA molecules containing genes for by comparison of SSU sequences of Ichthyo- mitochondrial proteins. However, unlike phthirius and its closest free-living relative, standard protein coding genes, those within Ophryolgena, using the earliest emergence the kinetoplast undergo an unusual pro- of freshwater fish in the fossil record as a cess of post-transcriptional RNA editing in reference point. Based on this, the ciliates order that their sequences can be trans- are predicted to have arisen in the lated in an appropriate fashion (Maslov Palaeoproterozoic, roughly 2 billion years et al., 1994; Vickerman and Coombs, ago (Wright and Lynn, 1997). The predic- 1999). The kinetoplastids consist of two or tion is based on a number of assumptions, more suborders, the most widely recog- and suggests that the ‘crown’ group of nized being Trypanosomatina, a group of eukaryotes (Knoll, 1992) evolved much exclusively parasitic forms that have a sin- earlier than previously thought. gle flagellum (including the well-known A more practical application of sequence Trypanosoma and Leishmania species), and data has been the development of a PCR Bodonina, which include free-living hetero- assay that distinguishes I. multifiliis from trophic, ectocommensal and parasitic forms Tetrahymena corlissi in mixed cultures, with two flagella. A number of species using primers specific for regions of the within each group are parasitic on/in Molecular Approaches and Techniques 733

fishes. With the inception of SSU rDNA primarily of protozoan parasites of fishes sequence analysis, the taxonomic relation- and crustaceans. Known alternatively as ships of such species within the larger DRIPs (an acronym representing the genera kinetoplastid order have changed dramati- Dermocystidium, the rosette agent (recently cally relative to the more classical scheme Sphaerothecum destruens Arkush et al., based on morphological features alone 2003), Ichthyophonus and Psorospermium), (Maslov et al., 1996, 2001; Hollar et al., this group maintained an enigmatic stand- 1998; Callahan et al., 2002). Nevertheless, ing prior to phylogenetic analyses of rDNA even molecular approaches have left a and mitochondrial protein-coding genes. comprehensive phylogenic tree for the kin- Based on SSU rDNA sequencing, these etoplastids in doubt, due in part to the pau- organisms (along with additional genera, city of data for many members of the group such as Rhinosporidium) are clearly members (Maslov et al., 2001). In this regard, a recent of a distinct class of organisms that arose study that included Ichthyobodo necator near the metazoan–protozoan boundary and a number of other important fish para- (Ragan et al., 1996; Herr et al., 1999). sites has suggested a novel taxonomic order Indeed, their position in the descent lead- for the kinetoplastids (Callahan et al., 2002). ing from single-celled protists to metazoa is While previous work had indicated the extremely interesting from an evolutionary existence of two well-defined suborders standpoint, and prompted the recent (consisting of Trypanosomatina and sequencing of the entire mitochondrial Bodonina), the inclusion of SSU rDNA genome of Amoebidium parasiticum,an sequences from I. necator splits the order ichthyosporean that can be readily cultured. into two different sublineages, one consist- Analysis of the content and architecture of ing of Ichthyobodo itself, and the other con- the mitochondrial genome of A. parasiticum, taining all other kinetoplastid species when compared with mitochondrial DNA (Callahan et al., 2002). Whether this alterna- (mtDNA) from choanoflagellates (repre- tive view prevails remains to be determined. sented by Monosiga brevicollis) and Metazoa, However, even for the previous trees, 18S indicates that these three lineages comprise rDNA sequence analysis clearly indicates a monophyletic assemblage (termed Holozoa) that several genera (as defined morphologi- separate from the other major eukaryotic cally) within the suborders Trypanosomatina clades, including fungi. Based on likeli- and Bodonina are polyphyletic, indicating hood tests of tree topologies generated from that a reordering of these taxa will almost comparisons of 11 conserved mitochondrial certainly occur (Maslov et al., 2001). proteins, the Holozoa appear to have Classification schemes for the kineto- evolved from a common ancestor, with plastids are interesting not just from the Ichthyosporea emerging first, and Metazoa standpoint of taxonomy, but in terms of and choanoflagellates later as branching how parasitism evolved. In the bonodids, sister taxa (Lang et al., 2002; Burger et al., free-living and parasitic species intermingle 2003). A few Ichthyosporea have evolved within distinct lineages, and current schema into fish parasites, thus falling within the (which include the trypanosomatids) sug- lineage that led directly to the formation of gest that a parasitic life style may have sponges and other extant animals, and evolved independently a number of times were among the first organisms to separate during the evolution of the kinetoplastids this lineage from the fungi. Given their (Maslov et al., 2001). phylogenetic position, Ichthyosporea have been renamed Mesomycetozoea (Herr et al., 1999), and have been further subdivided Ichthyosporea (Mesomycetozoea) into the orders Ichthyophonida and Dermo- cystida based on gene sequence, life style Molecular genetic analyses have been and morphological criteria (see Mendoza instrumental in the classification of et al., 2002). Although computational analy- Ichthyosporea, a discrete taxon consisting ses of sequence data for the nuclear genes 734 T.G. Clark

encoding elongation factor 1-α from Ichthyo- been classified into 400 species on mor- sporea, animals and fungi do not provide a phological grounds, although the true clear picture of the relationships between number may exceed 20,000 (Zietara and these organisms, they clearly group all three Lumme, 2002). A number of these species within a separate clade, known formally as are pathogenic to fishes and have serious the Opisthokonta (Ragan et al., 2003). effects on wild populations, particularly Analyses of the mtDNAs of the Holozoa those of Norwegian Atlantic salmon. Genetic provide additional, interesting information markers that can distinguish species and/or regarding the evolution of the genome intraspecific (i.e. strain) differences that structure of this organelle. While Metazoa relate to pathogenicity have long been have relatively uniform compact (13–19 kb) sought. Because of its economic impact, mitochondrial genomes, consisting of closed considerable emphasis has been placed on circular DNAs present as monomers and Gyrodactylus salaris. The parasite has been concatenated oligomers, mtDNAs in other identified in rivers and streams across eukaryotic clades (including plants, fungi Northern Europe, where it infects both and protozoa) vary greatly in terms of their Atlantic salmon (Salmo salar L.), and size and overall structures. Interestingly, farmed rainbow trout. Disease outbreaks in the circular mtDNAs of choanoflagellates specific locations have suggested that are up to four times as large as those in pathogenic strains of G. salaris may exist. Metazoa and have roughly twice the num- However, pathogenicity has not been linked ber of protein-coding genes. Mitochondrial to any particular morphological trait(s). DNAs of Ichthyosporea are even larger (up Indeed, while intraspecific differences in to ten times the size), and consist of several host adaptation appear to exist, the genus as hundred linear chromosomes that share a whole is morphologically quite similar. At elaborate terminal repeat structures (Burger the molecular level, neither SSU nor ITS et al., 2003). The differences in mtDNA rDNA sequence analysis has been parti- structure within various branches of the cularly useful for discriminating strains. Holozoa would clearly suggest that compac- For example, ITS sequences are identical tion of the mitochondrial genomes seen in between G. salaris isolates obtained from animals occurred at or near the time of different host species across a wide geo- emergence of a multicellular body plan. graphical range and fail to discriminate G. salaris from Gyrodactylus thymalli, its avirulent sister group that is specific for Helminths European grayling (Thymallus thymallus) (Cunningham, 1997; Zietara and Lumme, Nucleotide sequence analyses of rDNA and 2002). However, analyses of the intergenic protein-coding genes have been widely spacer (IGS) regions of rDNA, as well as the applied in epidemiological and phylogen- mitochondrial cytochrome c oxidase I (COI) etic studies of parasitic helminths of fish. gene, have been far more revealing (Sterud These approaches are particularly useful in et al., 2002; Cunningham et al., 2003; uncovering cryptic or sibling species whose Hansen et al., 2003; Meinilä et al., 2004). members are more or less morphologically While these studies have failed to yield mark- similar. In addition, they relate to important ers for pathogenic strains, they provide con- issues in parasite evolution, including the siderable insight into the phylogeny of idea that host switching plays a role in the G. salaris and its sister group G. thymalli, speciation of at least some taxa. Examples and suggest an interesting hypothesis of the use of these techniques are summa- regarding mechanisms of speciation within rized briefly below, with primary emphasis the genus. Analysis of IGS rDNA sequences on species that have an impact on wild and of Gyrodactylus collected at different loca- commercial fisheries. tions from different types of fish show clear Monogenean parasites (Chapter 9) differences in the number and sequence of belonging to the genus Gyrodactylus have individual 23 bp repeats within the two Molecular Approaches and Techniques 735

repetitive regions of the IGS domain (Sterud (1997a,b) employed two complementary et al., 2002; Cunningham et al., 2003). While approaches (namely, DNA/DNA hybridiza- IGS elements were too conserved to make tion and ITS rDNA sequence analysis) to inferences regarding the geographical spread determine phylogenetic relationships among of the parasite, analysis of these regions seven marine bothriocephalid species, and showed unequivocal separation of G. salaris compared these with the lineages of their and G. thymalli and presumptive diver- fish hosts. The study showed little congru- gence of G. salaris into forms that infect ence between the branching patterns of the either salmon or trout (Cunningham et al., different bothriocephalid species and their 2003). Two independent studies of the host species. Indeed, it appears that the mitochondrial COI gene were equally majority of the parasites (six of seven spe- informative. While they differed in their cies) have diverged recently, and it seems precise conclusions, each study identified clear that switching events (leading to multiple clades of both G. salaris and speciation) have occurred since the emer- G. thymalli (Hansen et al., 2003; Meinilä gence of at least some host groups. et al., 2004). Furthermore, consistent with Along with marine bothriocephalids, IGS sequence analysis, phylogenetic trees sequence analysis of the ITS rDNA domains based on mitochondrial COI data showed of the freshwater tapeworm Bothriocephalus that these clades are specific for either acheilognathi has revealed the existence of salmon (G. salaris), both trout and salmon multiple genotypes (distinguished on the (G. salaris) or grayling exclusively basis of microsatellite repeats within the (G. thymalli) (Hansen et al., 2003; Meinilä ITS2 region) that exhibit degrees of host et al., 2004). The fact that neither G. salaris specificity as well (Luo et al., 2002). The nor G. thymalli appears to be monophyletic apparent increase in the number of fish leaves open the question of whether they species infected by B. acheilognathi has sug- constitute: (i) a single polytypic species; gested that colonization and host switching (ii) two polytypic species; or (iii) a complex may have occurred recently (Luo et al., 2002). of more than two sibling species (Hansen The absence of widespread co-speciation et al., 2003). Irrespective of the alterna- during the evolution of Lamellodiscus tives, the linkage between parasite clades (Monogenea) and their fish hosts, the and host species suggests that host switch- Sparidae, has also been inferred based on ing (either in addition to or separate from molecular genetic data. In this case, phylo- geographical isolation) may underlie genetic trees built on SSU rDNA sequences speciation within the gyrodactylids (Brooks (for Lamellodiscus) and both 16S and and McLennan, 1993; Cribb et al., 2002; cytochrome-b mitochondrial gene sequences Huyse and Volckaert, 2002; Poulin, 2002; (for the Sparidae) were examined for Meinilä et al., 2004). co-evolution using computational methods, Molecular genetic analyses of other all of which agreed that widespread parasitic helminths of fishes also suggest co-speciation in this host–parasite system is co-speciation of parasites and their hosts. unlikely to have occurred (Desdevises et al., For example, tapeworms (Chapter 11) of the 2002). In contrast, comparative phylogenies genus Bothriocephalus infect a broad range of helminths and their fish hosts have sug- of fish hosts that belong to distantly related gested co-speciation in evolution in at least taxa. Nevertheless, individual parasite spe- two cases, namely, the genus Diplozoon,a cies appear to be restricted to a given fish class of monogeneans that interacts with host. Since host species in this case extend cyprinid species (Sicard et al., 2001), and across a broad taxonomic range, it becomes the Tetraphyllidea, a group of tapeworms possible to examine whether particular that infect elasmobranchs (Olson et al., 1999). hosts and their parasites evolved in parallel In addition to its use in systematics and (through co-evolution) or whether switch- evolutionary biology, PCR amplification ing to different hosts led to the emergence of rDNA sequences has served as an impor- of different bothriocephalids. Verneau et al. tant tool for identifying species and for 736 T.G. Clark

determining fish hosts parasitized by worms the possible involvement of a second such as digenean trematodes (Chapter 10). unnamed species termed Bolbophorus sp. In this regard, ITS2 rDNA sequences were type 2 (Levy et al., 2002). Using specific used to infer a three-host life cycle for primers that generate 820 and 420 bp Bivesicula claviformis, a digenean found in amplicons from B. damnificus and Bolbo- the intestines of warmwater fishes (Cribb phorus sp. type 2, respectively, both species et al., 1998). Amplification of ITS2 rDNA were shown to be present in the American from immature bivesiculids found in labrid white pelican, Pelecanus erythrorhynchos species, such as Thalassoma lunare, identi- suggesting that the pelican is the definitive fied these parasites as B. claviformis. Previ- host (Levy et al., 2002; Overstreet et al., ously, this trematode had been found (as 2002). Similarly, both species were shown adults) only in large carnivorous serranids to be present in field-collected ramshorn (namely, Epinephelus fasciatus) in the snails (Planorbella trivolvis), considered the Great Barrier Reef, and was assumed to first intermediate host (Levy et al., 2002). have a two-host life cycle. Since the diet of Interestingly, only B. damnificus was identi- E. fasciatus includes labrid fish, it is likely fied in field isolates of infected channel cat- that the larger carnivorous fish become fish, which presumably acts as a second infected by feeding on the intermediate host intermediate host. Although Bolbophorus (T. lunare)(Cribbet al., 1998). In other stud- sp. type 2 has been shown to be pathogenic ies, ITS1 rDNA sequences have been used to to a variety of fish species in direct chal- identify encysted metacercariae in molluscs lenge studies, it remains to be determined (Cerastoderma edule) collected from the whether it causes disease in the commer- Atlantic Ocean off the Iberian coast as those cially important catfish, I. punctatus (Levy of Monorchis parvus. The adult stage infects et al., 2002). Given the extreme differences Diplodus sargus and other fish species in the in morphologies of the different stages of Mediterranean Sea. In the absence of rDNA these parasites, molecular genetic analyses sequence data, the encysted form had been were indispensable in correctly matching identified only as Cercaria cerastodermae I parasite species with their respective hosts. (Bartoli et al., 2000). The study also pro- Dzikowski et al. (2003) have used a similar vided a means to distinguish M. parvus from approach to distinguish the North American the related species Monorchis monorchis,on species B. damnificus and Bolbophorus the basis of ITS1 rDNA sequence data (Cribb sp. type 2 from Old World species that et al., 1998). affect cyprinid fish, namely, Bolbophorus Application of PCR and DNA sequence confusus and Bolbophorus levantinus. analysis has been useful in identification Using species-specific primers that ampli- and epidemiological studies of other fied 820 and 420 bp fragments of SSU rDNA trematodes, most notably Bolbophorus,which from B. confusus and B. levantinus, respec- can be highly pathogenic to farm-raised fish. tively, it was possible to generate a prelim- Since the late 1990s, catfish farms in south- inary phylogenetic tree relating the Old and western Louisiana and the Mississippi delta New World species based on sequence com- region of the USA have experienced epi- parisons. In this analysis, B. damnificus was demics of metacercarial infections with more closely related to B. levantinus than to high mortalities, especially among young Bolbophorus sp. type 2. It is not understood fish. These mortalities were originally why two species from different continents thought to have a viral etiology but have (and different hosts) are more closely now been ascribed to Bolbophorus species, related than species from the same conti- which can be distinguished on the basis of nent. More in-depth sequencing of rDNA rDNA and mitochondrial COI gene sequence and protein-coding genes is clearly war- analysis (Levy et al., 2002; Overstreet et al., ranted to determine whether the inferred 2002). The principal agent responsible for relationship is correct. these outbreaks is probably Bolbophorus Marine and freshwater nematodes damnificus (Overstreet et al., 2002), with (Chapter 12) have also been targeted for Molecular Approaches and Techniques 737

genetic analysis. A number of anisakid spe- gels. The complexity of eukaryotic genomes cies (in particular, Anisakis simplex) are ensures that multiple fragments (RAPD recognized as zoonotic agents in humans markers) are generated with most arbitrary who consume undercooked seafood. Sim- primer pairs, and that the patterns of bands ple PCR-based assays that can be used to in each case are distinct. Such patterns detect and distinguish species of anisakids establish a fingerprint of DNA for an indi- infecting marine and freshwater fishes, vidual, with certain (fixed) markers being mammals and fish-eating birds have now indicative of species. RAPD PCR provides a been developed (Zhu et al., 1998; D’Amelio nimble way to study genetic diversity et al., 2000; Kijewska et al., 2002). These within populations, and has been particu- assays rely on RFLPs in a region of the larly useful in uncovering sibling or cryptic rDNA containing ITS1, the 5.8S rRNA gene, species. Its advantages include the fact that ITS2 and ∼ 70 bp of the 28S rRNA gene. it does not require prior knowledge of the Upon amplification of this region with nucleotide sequence of any gene and that it broadly specific primers spanning the ITS1 can generate a large number of genetic and ITS2 domains of anisakid rDNA, batter- markers for establishing the population ies of restriction enzymes generate discrete structure for a given species. RAPD analysis patterns of fragments from different parasite has been applied to two different types of species, including possible cryptic species helminths infecting fish, namely, G. salaris of A. simplex (Zhu et al., 1998; D’Amelio and Hysterothylacium fabri (an anisakid et al., 2000; Kijewska et al., 2002). These nematode). In the case of G. salaris, certain types of PCR-RFLP assays will undoubtedly primers revealed clear polymorphisms in be extremely useful in future diagnostic and populations of worms taken from Atlantic epidemiological studies of marine and salmon in different Norwegian rivers freshwater ascarids. (Cunningham and Mo, 1997). In a more detailed study with H. fabri, RAPD PCR was used to generate more than 400 genetic Arbitrary primed PCR: RAPD analysis markers, of which > 90% were polymorphic (Martín-Sánchez et al., 2003). An analysis In addition to the direct sequencing of of genetic diversity and population struc- rDNA and protein-coding genes, intra- and ture using this approach suggested that interspecific differences within and H. fabri represents a species complex com- between taxa can also be detected using prising at least three sister groups having arbitrary primed PCR. Referred to alterna- little host specificity (Martín-Sánchez et al., tively as random amplified polymorphic 2003). RAPD PCR analysis has also suggested DNA (RAPD) analysis, this relatively sim- the existence of bottlenecks of genetic ple method relies on the ability of arbitrary diversity in I. multifiliis grown in different PCR primers to generate visibly different species of host fish (M. Gray, D.M. Cassidy- patterns of amplified DNA from templates Hanley and T.G. Clark, unpublished; representing different species (or strains of Fig. 20.5). the same species) following electrophoresis on agarose gels. Initially either single or pairs of random PCR primers are used to Solid-phase hybridization techniques amplify total DNA from a given organism, under conditions of low annealing tempera- Along with solution-based assays, such as ture, which allow some mismatch between PCR, solid-phase methods, which rely on the primers and their target sequences nucleic acid hybridization with labelled within the genome. Following the initial probes, provide additional means for rounds of PCR, the annealing temperature is detection and identification of pathogens raised and additional cycles are carried out (Fig. 20.6). As with PCR, such methods are to generate PCR fragments that can be visu- highly specific, owing to the discrete inter- alized using ethidium staining on agarose action of nucleic acid probes with their 738 T.G. Clark

Fig. 20.5. RAPD PCR analysis of Ichthyophthirius multifiliis. DNA from individual (cloned) parasites collected from rainbow trout following a natural outbreak of ‘white spot’ disease in upstate New York was amplified,using a single pair of random PCR primers (lanes 1–10). The infection was passaged on channel catfish and individual parasites were again harvested and analysed by RAPD PCR with the same set of random primers (lanes 11–19). While DNA fingerprints from parasites collected from the initial infection on rainbow trout were quite varied, the patterns generated with parasites taken from channel catfish were largely homogeneous. The lane on the extreme right represents a DNA size standard.

cognate sequences. With most solid-phase to the probe. Following hybridization, sig- techniques, DNA or RNA is purified from nals generated by the probe are used to the target organism prior to annealing with detect and localize the pathogen (where the a labelled probe that is specific for a given probe is labelled with a fluorochrome, the gene or sequence. In such cases, the puri- method is referred to as fluorescence in situ fied DNA/RNA is either directly bound to a hybridization (FISH)). support (dot blots, slot blots, etc.) or frac- ISH techniques for the detection of tionated using gel electrophoresis prior to fish parasites have been applied to both immobilization on nylon or nitrocellulose Myxozoa and Microsporea. Antonio et al. membranes (Southern and Northern blot- (1998) used them to detect M. cerebralis in ting analysis). Once attached, the target fixed tissues of rainbow trout and its inter- DNA/RNA is reacted with the probe under mediate host, T. tubifex. Three different conditions that favour specific hybridiza- oligonucleotide primers intended originally tion and discourage non-specific binding to for PCR amplification of M. cerebralis SSU non-target sequences or to the support itself. rDNA were end-labelled with the steroid A second approach involves in situ hybrid- hapten digoxigenin (DIG) and hybridized ization (ISH), in which pathogen-specific to fixed tissue sections that had been probes are reacted directly with histological dewaxed and proteinase K-treated to expose sections suspected to contain a given agent parasite DNA. After high-stringency washing, (Wilcox, 1993). Prior to screening, the sec- probes were detected using alkaline tioned material is treated so that endo- phosphatase-conjugated anti-DIG antibodies genous nucleic acids are available for binding and appropriate colorimetric (NBT/BCIP) Molecular Approaches and Techniques 739

Fig. 20.6. Solid-phase hybridization with labelled nucleic acid probes. DNA probes are constructed from either PCR-generated or cloned fragments of DNA. Probes are labelled with tagged deoxyribonucleotides (dNTPs), using any of a number of different methods. Shown here are end-labelling with terminal deoxynucleotide transferase (TdT) and continuous labelling of both DNA strands in synthesis reactions with either the Klenow fragment of DNA polymerase or with thermostable Taq DNA polymerase (that is, by PCR). Commonly used tags include the steroid Fig. 20.7. Localization of M. cerebralis in host hapten digoxigenin, biotin and radioactive 32P. tissues by in situ hybridization. Panels (A) and (B) Once labelled, probes are denatured to separate the show sections of cranial cartilage from rainbow DNA strands and then hybridized to immobilized trout infected with M. cerebralis and processed for RNA (Northern blot), genomic DNA (Southern blot) in situ hybridization 3 months post-exposure. or histological sections (ISH) under low stringency Oligonucleotide probes were labelled at their conditions that favour annealing. The probe is then 3′ ends with dUTP–digoxigenin. Preincubation of washed off the substrate under high-stringency sections with excess unlabelled probe eliminated conditions, which disrupt non-specific interactions tissue staining (panel A). Strong staining of (including mismatched hybrids), leaving only the developmental stages of the parasite was visible probe bound to its complementary target sequence. in host tissues using the labelled probe by itself Radioactive hybrids are detected using X-ray film, (arrows, panel B). (From Antonio et al., 1998 with while biotin and digoxigenin probes are detected permission from Journal of Aquatic Animal Health.) with streptavidin–enzyme conjugates and anti-digoxigenin antibody conjugates, respectively. detected with anti-DIG monoclonal antibodies linked to alkaline phosphatase. substrates. Developmental and sporogonic A modification of this method, which stages of the parasite were easily detected in reduces the workup time from 2 days to fish and oligochaete tissues (Fig. 20.7). The about 5 h, involves the use of biotinylated probes were specific for M. cerebralis and probes and a streptavidin peroxidase conju- did not generate positive signals in tissues gate. The ability to detect the parasite in tis- from fish infected with other Myxozoa, sues suggests that the gill is an important including C. shasta, T. briosalmonae and portal of entry for T. bryosalmonae and Myxobolus sp. (Antonio et al., 1998). Morris that the parasite spreads directly to the et al. (2000) applied similar methods for kidneys prior to the involvement of other detection of the PKX agent, T. bryosalmonae, tissues (Morris et al., 2000). using oligonucleotide probes that were also Detection of the microsporidia developed for PCR. T. bryosalmonae-specific L. salmonae and Tetramicra brevifilum has probes were end-labelled with DIG and also been described using ISH techniques 740 T.G. Clark

(Sánchez et al., 1999, 2001; Leiro et al., 2001). 28S rDNA. After the addition of poly-dT In the case of Loma, a 272 bp single-stranded tails, the probes were covalently attached to probe corresponding to the ITS and por- the wells of microtitre plates, where they tions of the small- and large-subunit rDNA became available for hybridization with was constructed by PCR and labelled during labelled probes. The probes consisted of synthesis with digoxigenin-11-deoxyuridine amplicons generated from single copepods triphosphate (dUTP) in reactions that con- (adult and naupliar stages) labelled during tained only a forward primer (Sánchez synthesis with biotin. In all cases, amplicons et al., 1999, 2001). When applied to sections were produced using the same (universal) of fixed tissue from infected rainbow trout, primers that recognize conserved sequences the probe detected parasite xenomas with delimiting the variable D1/D2 region. Fol- high sensitivity and specificity (Sánchez lowing hybridization, the probe was et al., 1999, 2001). Using similar methods, a detected with a streptavidin–horse radish 1.2 kb digoxigenin-tagged probe, generated peroxidase conjugate. Because a single set using PCR from SSU rDNA of Tetramicra; of primers was used to amplify all unknown allowed definitive diagnosis of this agent in samples, the requirement for a large number xenomas of infected turbot (Scophthalmus of PCR reactions for each sample, using maximus)(Leiroet al., 2001). Although pat- multiple species-specific primers, was ent infections with T. brevifilum are easily avoided (Kiesling et al., 2002). While the visualized in host tissues using conven- copepods identified in this study were not tional light microscopy, identification and necessarily pathogenic, the procedure could localization of immature stages (or mild easily be adapted and used as a means of infections) are more difficult. ISH, on the identifying parasitic species within the other hand, has permitted detection of environment. L. salmonae during its earliest stages of infection where it is localized to the gut epi- thelium of infected fish. With time, parasites are visible in the lamina propria and Gene Cloning then blood cells within the heart, where they appear to undergo merogony prior to their The isolation of genes encoding specific final development in the gills (Sánchez proteins has become an indispensable tool et al., 2001). This is the first description of for studying the biology of living species early stages of infection in fish exposed to and is often a key step in the development L. salmonae. of therapeutic and prophylactic reagents Applications of Southern and Northern against pathogens. Procedures for gene puri- blotting analyses in fish parasitology are fication generally take one of two routes. The described below in connection with the first involves construction and plating of development of vaccines against I. multifiliis. libraries of genomic or complementary However, another solid-phase technique, DNA (cDNA) from the pathogen, followed which combines PCR with hybridization of by the identification and isolation of clones target and probe sequences in a microtitre of interest by hybridization with specific plate format, has been developed for marine nucleic acid probes (or by cross-reactivity copepods (Kiesling et al., 2002). The tech- with antibody probes in the case of expres- nique, which was developed to distinguish sion libraries). The alternative approach copepod species within the marine envi- involves amplification of known (or sus- ronment, significantly reduced the number pected) protein-coding regions by PCR, of individual PCR reactions that would be using genomic DNA as the template and required using more conventional methods. degenerate oligonucleotide primers that tar- With this streamlined approach, a series of get conserved regions of coding sequence. species-specific oligonucleotide probes was Once desired genes have been isolated, cor- constructed against the highly variable responding protein products can be synthe- D1/D2 region at the 5′ end of copepod sized using any of a number of heterologous Molecular Approaches and Techniques 741

protein expression systems (e.g. Esche- Trypanosoma carassii (a kinetoplastid that richia coli (Swartz, 2001), yeast (Gellissen, infects cyprinid fishes) (Aguero et al., 2002), 2000), Tetrahymena (Gaertig et al., 1999), Lepeophtheirus salmonis (an ectoparasitic insect (Jarvis et al., 1996) and mammalian copepod that affects salmonids) (Johnson tissue culture cells (Fussenegger et al., et al., 2002) and I. multifiliis (the causative 1999)). The resulting recombinant products agent of ‘white spot’ in freshwater fishes) can then be used for a variety of purposes, (T.G. Clark, unpublished). The GenBank ranging from X-ray crystallography and dbEST database (http://www.ncbi.nlm.nih. drug design to the development of recombi- gov/dbEST/) currently lists 1921 EST nant subunit vaccines. In systems that sequences for T. carassii, 512 for I. multi- allow reverse genetics, gene isolation often filiis and 248 for C. sinensis. An additional permits one to conduct functional assays of 392 ESTs have been sequenced for L. sal- specific proteins by ‘knocking out’ the cor- monis (Johnson et al., 2002). Of the 1921 responding gene in vivo, using either ESTs for T. carassii, the vast majority targeted deletion via homologous recombi- (> 90%) had no significant match to other nation (Gong and Rong, 2003) or interfering known protein-coding sequences at the time RNA technology (RNAi) (Zanmore, 2002). they were submitted to GenBank (Aguero The creation of ‘knockout’ strains can also et al., 2002). Similarly, ∼ 33% of the EST serve in the construction of rationally atten- sequences identified in the C. sinensis uated vaccine strains, particularly in cases library did not have significant database where gene deletion results in altered viru- matches (Lee et al., 2003). Such results lence (Kit et al., 1985; Charles and Dougan, clearly attest to the power of this approach 1990; Uzonna et al., 2004). for gene discovery. While the numbers of Along with conventional gene isolation gene and EST sequences for fish parasites methods, robotic systems are now available are relatively low (particularly when com- that permit high-throughput sequencing of pared with agents that infect mammalian large numbers of cloned inserts from DNA hosts), one can only expect that they will libraries. This approach has opened up an increase dramatically as work in this area entirely new method for gene discovery, in expands and as the cost of high-throughput which computational methods can be applied sequencing continues to decline. on a genome-wide scale. High-throughput, Apart from large-scale data collection, single-pass sequencing of cloned cDNAs (to the construction of genomic and/or cDNA create expressed sequence tags (ESTs)) (Gill libraries has made possible in-depth analy- and Sanseau, 2000) offers further opportu- ses of a number of important gene products nities, along with microarrays (Freeman, from agents that parasitize fish. These 2003), to examine patterns of gene expres- include several trypsin-like enzymes from sion at different stages of parasite life cycles L. salmonis (Johnson et al., 2002), trans- or in response to changes in environmental sialidase-like and mucin-like proteins from conditions. High-throughput methods are T. carassii (Aguero et al., 2002) and a family particularly useful in systems that do not of surface membrane antigens involved in allow straightforward genetics, and they protective immunity against I. multifiliis offer a powerful approach towards the dis- (Clark et al., 1992, 1999; Lin et al., 2002b). covery of novel targets for diagnosis, drug The latter case provides an example of how treatment and vaccine development. While gene cloning can be used in vaccine devel- the application of genomics to fish disease opment and is discussed in detail below. is still in its infancy, a number of pilot EST The use of expression libraries for the clon- projects are under way for a diverse group ing of genes from the PKX agent of sal- of parasitic agents that affect wild and monids (T. bryosalmonae) has also been farm-raised fish. These include Clonorchis described (Saulnier et al., 1996). sinensis (a digenean trematode that is an The alternative approach of gene important zoonotic agent in China, Korea cloning using PCR has been applied to and South East Asia) (Lee et al., 2003), a variety of fish parasites. For example, 742 T.G. Clark

full-length coding sequences for genes with numerous other phylogenetic studies. encoding abundant glycosyl phosphatidyl As indicated above, these include full or par- inositol (GPI)-anchored surface proteins tial sequences for homeobox-containing pro- from Ichthyophthirius and a cathepsin teins, tubulins, ribosomal proteins, heat-shock Z-like enzyme from M. cerebralis have been proteins and mitochondrial proteins from described (Lin et al., 2002b; Kelley et al., Myxozoa, microsporidia, flatworms and a 2003). In both instances, fragments of the variety of other parasitic forms. relevant coding sequences are amplified using PCR. The degenerate primers are designed against conserved regions of the Molecular Parasitology and Vaccine corresponding proteins. After sequencing Development the truncated fragments, missing regions are obtained using rapid amplification of Fish are capable of mounting effective cDNA ends (RACE), a procedure that allows immune responses to parasitic protozoa, amplification of the 5′ and 3′ regions of the including Ichthyophthirius (Chapter 4). As coding sequences of genes, using cDNA as a with a variety of other parasites, however, template (Frohman, 1990). In RACE, a first- Ichthyophthirius cannot be easily cul- strand cDNA is synthesized from mRNA, tured, and it has long been recognized using oligo-dT as the primer, and missing 3′ that development of an effective vaccine sequences are amplified in PCR reactions, against this agent will require the expres- which utilize a forward primer based on a sion of protective antigens in recombinant known sequence (derived from the previ- form. Immunological studies have shown ously amplified gene fragment) and oligo-dT that immunity against I. multifiliis is as the reverse primer. Missing 5′ sequences largely humoral and involves an antibody are obtained after addition of a homopoly- response to abundant parasite membrane meric tail (e.g. poly-G) to the 5′ end of the proteins, known as immobilization antigens first-strand cDNA, using terminal deoxynu- (i-antigens) (Clark et al., 1995; Clark and cleotidyl transferase. The relevant 5′ Dickerson, 1997). Antibodies against these sequence is obtained by PCR, using a reverse proteins immobilize swimming (theront primer of known sequence and a forward and tomont) stages of the parasite in vitro primer complementary to the homopoly- and offer passive protection against infec- meric tail. A variation on 5′ RACE, known as tion when administered parenterally to SMART (switching mechanism at 5′ end of naïve fish. Purified i-antigens confer active RNA transcript) RACE, allows the addition immunity as well (Wang et al., 2002). How- of a specific sequence rather than a homo- ever, inability to grow the parasite on a polymeric tail at the 5′ end of the cDNA and large-scale precludes the use of the native strongly favors the production of full-length antigens for commercial vaccine develop- products (Chenchik et al., 1998). A variety ment. This has led to efforts to express of modifications of these procedures are these proteins as recombinant antigens in also available (Schaefer, 1995; Das et al., heterologous systems. 2001). In the case of the cathepsin Z-like These efforts began with the isolation gene from M. cerebralis, a phylogenetic of a 1.2 kb cDNA containing the preponder- analysis comparing its deduced sequence ance of the coding sequence for a 48 kDa with cathepsin proteases of other eukaryotic i-antigen from parasite isolate G1 (i-antigen species has suggested that the myxozoan serotype A). This sequence was obtained protein is among the earliest of the Z-group by screening a trophont cDNA library with enzymes to have evolved. This analysis has an oligonucleotide probe based on the implications for the origins of the Myxozoa N-terminal sequence of the purified antigen (see above and Chapter 8) (Kelley et al., itself (Clark et al., 1992). The library was 2003). constructed using a standard approach, in Additional protein-coding sequences which poly-A+ trophont mRNA was used have been amplified using PCR in connection as the template for first-strand cDNA Molecular Approaches and Techniques 743

synthesis, together with oligo-dT as the primer and AMV reverse transcriptase to make the first strand. Second-strand synthesis was carried out with DNA polymerase, and the resulting double-stranded product was introduced into a lambda phage vector. After plating on E. coli, the library was screened with the 32P-labelled probe, and a phage plaque containing the 1.2 kb cDNA was isolated. This cDNA had the deduced sequence for a nearly full-length protein, 394 amino acids in length, that was rich in cysteine and had five tandemly repetitive amino acid sequence domains of ∼ 80 resi- dues each. Northern and Southern hybridization studies were then carried out to examine the expression and organization of the corresponding gene (Fig. 20.8). In Northern blotting analysis, either total or poly-A+ Fig. 20.8. Northern and Southern blotting analysis. RNA was fractionated on agarose gels and In the Northern blot in panel (A), poly-A+ RNA from blotted onto nitrocelluose or nylon filters I. multifiliis was size-fractionated on a 1.2% agarose under denaturing conditions. Membranes gel, blotted onto nylon and probed with a 32P-labelled cDNA for the 48 kDa i-antigen of were then incubated with a labelled nucleic parasite isolate G1 (serotype A). The probe acid probe specific for the gene (cDNA) of recognized two distinct RNA transcripts of 1.6 and interest. The probe was permitted to anneal 1.9 kb. In the Southern blot in panel (B), genomic with its target sequence under conditions of DNA from Ichthyophthirius strain G1 was digested low stringency (high salt, low temperature) with EcoRI (lane 1), Hind III (lane 2) or SmaI (lane and the filter was washed at high stringency 3), then fractionated on a 0.8% agarose gel and (low salt, high temperature) to remove blotted as above for Northern analysis. When weakly bound probe. As shown in Fig. 20.8, screened with the same cDNA probe used in (A), when the 1.2 kb cDNA was used to screen two major bands were seen in each of the Northern blots of I. multifiliis poly-A+ RNA, restriction digests. Consistent with the presence of two transcripts in (A), the Southern blot suggests that the probe recognized two transcripts of 1.6 two i-antigen genes with strong homology to the and 1.9 kb. This was consistent with probes are present in the G1 parasite strain. Western blotting studies, which revealed two closely related i-antigens of 48 and 60 kDa in the G1 parasite strain. Steady- 1.2 kb cDNA revealed two prominent bands state levels of RNA transcripts for these in I. multifiliis DNA cut with different proteins were shown to be very high (equiv- restriction enzymes, indicating the presence alent to ∼ 6% of total poly-A+ RNA) and of two, closely related, i-antigen genes in the were developmentally regulated through G1 parasite strain (Clark et al., 1992). the parasite life cycle (with maximal levels With the 1.2 kb cDNA in hand, it of expression occurring at the infective became possible to isolate the gene for the theront stage) (Clark et al., 1992). Southern 48 kDa i-antigen (Clark et al., 1999). The blotting analysis, using the 1.2 kb cDNA as gene, designated IAG48[G1], was obtained probe, also revealed two closely related from a lambda phage library prepared from genes (Fig. 20.8). In Southern blotting, genomic DNA fragments cut with the genomic DNA fragments produced by restric- restriction enzymes SwaI and probed with tion endonuclease digestion were screened the 1.2 kb cDNA. The gene was found to with labelled probes for specific genes contain a single, uninterrupted, reading (Sambrook et al., 1989). In this case, the frame that predicts a full-length (that is, 744 T.G. Clark

preprocessed) protein of 442 amino acids. adjuvant was reported to provide signifi- Interestingly, the coding sequence of the cant protection in terms of parasite load and gene differed from the cDNA at its 3′ end. The overall mortality (although the challenge gene (in contrast to the cDNA) predicted a dose itself killed fewer than half the fish) protein with a hydrophobic C-terminus (He et al., 1997). This result is clearly inter- having all the hallmarks of a signal peptide esting and deserves further investigation, for a GPI-anchored protein. Since the particularly in light of studies indicating i-antigens are known to be GPI-anchored that protection afforded by purified subunit (Clark et al., 2001), the 1.2 kb cDNA repre- antigens is serotoype-specific. As shown by sented an alternatively spliced transcript or Wang et al. (2002), channel catfish injected was the product of a cloning artefact. The with i-antigens purified directly from the latter seems to be the case since: (i) primers parasite confer protection against only flanking the coding region of the gene those strains from which the antigens are amplify a fragment identical to the gene obtained. Similarly, passive protection using itself when RNA is used as a template (i.e. i-antigen-specific mouse monoclonal anti- by reverse-transcriptase PCR (RT-PCR)) bodies is serotype-specific, with the conform- (Clark et al., 1999); (ii) the 3′ sequence of ational epitopes recognized by these the 1.2 kb cDNA recognizes an entirely dif- antibodies being unique to a given sero- ferent transcript on Northern blots than logical strain (Clark and Dickerson, 1997). In does a probe from the 5′ end of the gene the studies of He et al. (1997), the parasite (Clark et al., 1999); and (iii) no other strain used for challenge was undefined, as i-antigen-specific cDNA clones encoding was the nature of the epitope(s) associated the anomalous 3′ end have been identified with the recombinant fusion protein. (in > 100 independent clones) (T.G. Clark, Recently, paralogous surface antigen unpublished). genes have been isolated from a different Analysis of the coding region of the I. multifiliis strain (G5), representing a dis- gene for the 48 kDa antigen made it clear that tinct i-antigen serotype (namely, D) (Lin Ichthyophthirius, like other hymenostome et al., 2002b). These genes, designated ciliates, utilizes a non-standard genetic code, IAG52A[G5] and IAG52B[G5], encode sur- in which the normal UAA and UAG stop face antigens of 468 and 460 amino acids, codons specify glutamine instead (Clark respectively. As with the 48 kDa protein et al., 1992, 1999). This has important impli- from the G1 isolate, the serotype D proteins cations in respect of vaccine development, are predicted to contain hydrophobic signal since the native gene sequence would yield peptides at their N- and C-termini and a truncated proteins when produced in con- series of imperfect tandem repeats of ~ 80 ventional heterologous protein expression amino acids each spanning their length. systems, such as E. coli or yeast. The gene Antibodies against affinity-purified i-antigens for the 48 kDa antigen contains 18 such of serotype D recognize a broad band of ‘stop’ codons, including several near the 52/55 kDa on Western blots, which resolves N-terminus itself (Clark et al., 1992, 1999). into four or more spots on two dimensional To overcome this problem, He et al. (1997) gels. Along with IAG48, the IAG52A and used overlapping oligonucleotides (prim- IAG52B genes have recently been cloned ers) to synthesize a 315 nucleotide fragment andexpressedinTetrahymena thermophila, (corresponding to roughly one tandem a non-pathogenic, freshwater ciliate that is repeat near the N-terminus of the 48 kDa taxonomically related to Ichthyophthirius protein) lacking seven potential UAA (Shang et al., 2002; T.G. Clark, Y. Bisharyan glutamine codons. The synthetic gene frag- and D.M. Cassidy-Hanley, unpublished). ment was then expressed as a GST fusion Following their expression, T. thermophila protein in E. coli and tested as a subunit cell lines are rapidly immobilized by spe- vaccine in goldfish. In a single vaccine trial, cific antibodies against the parasite proteins 15 µg of the fusion protein administered in a manner almost identical to that seen intraperitoneally in Freund’s complete with I. multifiliis (T.G. Clark, Y. Bisharyan Molecular Approaches and Techniques 745

and D.M. Cassidy-Hanley, unpublished). say, similar methods are being used against Expression of the native I. multifiliis genes other fish parasites, particularly those of in Tetrahymena was possible because economic importance within the aqua- T. thermophila and Ichthyophthirius share culture industry. In this regard, a large-scale identical codon usage with respect to UAA effort to identify potential targets for vaccine and UAG triplets. Synthetic genes in which development against sea lice and Neopara- the native sequences of the 48 and moeba pemaquidensis (the causative agent 52/55 kDa antigens were replaced with the of amoebic gill disease in Atlantic salmon) preferred codon usage of channel catfish are now well under way (Raynard et al., 2002; have also been constructed. However, B. Nowak, personal communication; Inter- unlike the recombinant antigens expressed national Association of Biologicals, 3rd Inter- in Tetrahymena, the synthetic gene prod- national Symposium on Fish Vaccinology, ucts expressed in E. coli and mammalian Bergen, Norway, 2003). tissue-culture cells do not bind to protective monoclonal antibodies, indicating that the proteins did not fold correctly in these Conclusions heterologous (non-ciliate) systems (Lin et al., 2002a). Consistent with this observa- Over the past 15 years, molecular genetics tion, plasmid constructs encoding these techniques have proved exceedingly useful proteins elicit only weak protection against in studies of fish parasites and have revealed parasite challenge when administered as novel aspects of the biology, evolution and DNA vaccines (Lin, 2002). life styles of these organisms. In many Although to date, DNA vaccines (also instances, such studies have gone beyond known as genetic immunization) have proved fish parasites and shed important light on less than successful with Ichthyophthirius, evolutionary biology as a whole. In more this general approach has shown that it has practical terms, molecular cloning tech- enormous potential against a variety of micro- niques provide sensitive and specific meth- bial agents and may serve an important role ods for diagnosing parasite infections of in the prevention of parasitic diseases of fish. fish. They have contributed significantly A DNA vaccine is constructed by introducing towards our understanding of the epidemi- the gene for a protective antigen into a ology of parasite disease, and are beginning plasmid expression vector that contains a to play a role in the development of effica- promoter element, which can drive transcrip- cious vaccines for the prevention of para- tion of the cloned sequence in host cells sitic infections in farm-raised fish. Despite (Donnelly et al., 1997; Heppell and Davis, this explosion of new information, continued 2000). Promoters from viruses normally asso- studies at the molecular level will be needed ciated with mammalian infection (for exam- to unravel species differences and the pre- ple, cytomegalovirus) have been shown to be cise evolutionary relationships among and highly efficient even in fish. The vaccine, in between taxa for a variety of parasite lin- the form of naked DNA, is introduced into an eages. One would imagine that molecular animal by parenteral injection, where it is approaches will continue to be applied in taken up by host cells. Once expressed, the the arena of vaccine development, since these vector-encoded antigen is recognized by the offer the best hope for protecting farm- immune system as a foreign substance, lead- raised fish against a number of the most ing to the production of humoral and cell- important disease-causing agents of commer- mediated immune responses. cial aquaculture. Finally, the bioinformatics Apart from genetic immunization, recom- revolution spawned by genomic and binant subunit antigens are also effective as proteomics-based approaches to biology vaccines, and pilot studies with i-antigens have begun to find their way into the study expressed in Tetrahymena have been quite of fish parasites and will almost certainly promising (X. Wang, T.G. Clark and provide the next wave of understanding of H. Dickerson unpublished). Needless to these complex and fascinating organisms. 746 T.G. Clark

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