The myxosporean parasite Myxobolus aeglefini is a marine species, which can be found in the cartilage of mainly gadid fish species. The parasite has, however, been recorded in the flatfish plaice (Pleuronectes platessa) and dab (Limanda limanda). It is not clear if isolates from unrelated hosts represent the same species. Therefore a molecular study was conducted to reveal differences at the DNA level between these isolates. PCR was successfully conducted on three different isolates of Myxobolus aeglefini sampled from cod (Gadus morhua), plaice and dab respectively, using 18S rDNA as template. A PCR product of approx. 1600 base pairs was obtained and RFLP (Restriction Fragment Length Polymerase) was conducted on the fragment with the restriction enzymes Hinf I, Msp I and Hae III. No differences between the isolates were found, suggesting that the three isolates represent the same species

Buchmann, K.

Koie, M.

Nielsen, C. V.

Székely, Csaba

English

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Bull. Eur. Ass. Fish Pathol., 22(3) 2002,  201Comparative analysis of 18S rRNA genes fromMyxobolus aeglefini Auerbach, 1906 isolatedfrom cod (Gadus morhua), plaice (Pleuronectesplatessa) and dab (Limanda limanda), usingPCR-RFLPC.V. Nielsen1*, M. Køie2, Cs. Székely3 and K. Buchmann11 Section of Fish Diseases, Department of Veterinary Microbiology, The Royal Veterinary and Agricul-tural University, Stigbøjlen 4, DK-1870 Frederiksberg C. Denmark. 2 Marine Biological Laboratory,University of Copenhagen, Helsingør. 3Veterinary Medical Research Institute, Budapest, HungaryAbstractThe myxosporean parasite Myxobolus aeglefini is a marine species, which can be found in thecartilage of mainly gadid fish species. The parasite has, however, been recorded in the flatfishplaice (Pleuronectes platessa) and dab (Limanda limanda). It is not clear if isolates from unrelatedhosts represent the same species. Therefore a molecular study was conducted to reveal differ-ences at the DNA level between these isolates. PCR was successfully conducted on three differ-ent isolates of Myxobolus aeglefini sampled from cod (Gadus morhua), plaice and dab respectively,using 18S rDNA as template. A PCR product of approx. 1600 base pairs was obtained and RFLP(Restriction Fragment Length Polymerase) was conducted on the fragment with the restrictionenzymes Hinf I, Msp I and Hae III. No differences between the isolates were found, suggestingthat the three isolates represent the same species.IntroductionThe marine myxozoan M. aeglefini was origi-nally described from the whitingMelanogrammus aeglefini (L.). It parasitizes thecartilage of other gadid fishes including codGadus morhua  (L.) and hake Merlucciusmerluccius (L.) and has also been found inplaice Pleuronectes platessa (L.) (Kabata, 1957;Karasev, 1988; Lom & Dyková, 1992).Mellergaard & Nielsen (1984) recorded it fromdab Limanda limanda (L.). However,Gaevskaya & Kovaleva (1976) noticed that M.aeglefini from gadid fishes differs from speci-mens described from plaice in a number ofmorphological details. Morphological distinc-tion of myxospores isolated from hosts (themost widely used diagnostic method) is dif-ficult, but it has been suggested that parasitesfrom distantly related hosts could representdifferent species (Molnár, 1994; Molnár &Székely, 1999). New methods for moleculardiagnosis of myxosporeans have been intro-duced (Andree et al. 1998; 1999a,b;Eszterbauer et al., 2000; Kent et al., 2001).Primers specific for ribosomal genes in theMyxobolidae were developed by Andree etal. (1997). These primers amplify fragmentsof the 18S rRNA genes, and thus enabledRFLP or sequencing of a diverse range ofMyxobolus species (Andree et al., 1998;1999a,b; Eszterbauer et al., 2000; Kent et al.,2001).Bull. Eur. Ass. Fish Pathol., 22(3) 2002, 202The aim of the present paper was to investi-gate whether M. aeglefini from a gadid fish(cod) was identical to specimens described asM. aeglefini from plaice and dab. And furtherto investigate the 18S rRNA genes of three iso-lates of M. aeglefini isolated from cod, dab andplaice, respectively. Thus by using molecularmethods (PCR and RFLP) a comparativeanalysis was conducted in order to detect dif-ferences between the isolates, which couldindicate taxonomic differentiation.Materials and MethodsSamplingCartilage samples were taken from infectedcod (Gadus morhua), plaice (Pleuronectesplatessa), and dab (Limanda limanda) caught inthe Kattegat. Parasite infected tissue was thenpreserved in 96% ethanol. Single sporocysts(approx. 2-3 x106 myxospores) were freedfrom fish tissue under a dissection microscope(7-40x magnification) and subsequentlyplaced in 0.5 ml microfuge tubes.Myxobolus macrocapsularis and M. pseudodisparreference samples were taken from the gill ofbream Abramis brama (L.) and from the mus-cle of roach Rutilus rutilus (L.) caught in LakeBalaton, Hungary. The plasmodia filled withmature spores were ruptured by a needle un-der dissection microscope (7-40x magnifica-tion) and the spore contents (approx. 1-2 x106myxospores) were collected into microfugetubes and preserved in 96% ethanol.DNA extractionGenomic DNA from myxospores (three iso-lates of putative Myxobolus aeglefini and iso-lates of M. macrocapsularis and M.pseudodispar) were extracted using DNA af-finity columns from a DNeasy Tissue Kit(Qiagen).  Briefly, myxospores were lysed byadding lysis-buffer containing Proteinase K(20 mg/ml) and subsequently incubating thesample at 55 ∞C overnight. Myxospore walldegradation was observed under microscope(400x magnification) before proceeding. Thelysed sample was added to the DNA affinitycolumn and subjected to a series ofcentrifugations at 7000x g after which the sam-pled DNA was eluted in 25 ml distilled waterand stored at –20 ∞C before further process-ing.DNA yieldThe size and amount of DNA extracted fromthe samples were measured by agarose gelelectrophoresis (2% agarose, 100V, 1 h) bycomparing the result with a Low DNA MassLadder (Invitrogen). Only samples yieldingDNA concentrations greater than 5 ng/mlwere used for PCR.PCRThe oligonucleotide primers MX3 and MX5,developed by Andree et al. (1997) specific for18S rRNA genes in myxobolidae, were used.The sequence of the primers are as follow:Forward primer (MX5); 5´-CTGCGGACGGCTCAGTAAATCAGT-3´;and reverse primer (MX3); 5´-CCAGGACATCTTAGGGCATCACAGA-3´.The PCR reaction was conducted in PCR tubeswith a total volume of 50 ml using Ready-To-Go PCR beads (Amersham PharmaciaBiotech). The reaction volume comprisedminimum 25 ng in 5 ml of DNA sample tem-plate, distilled water (41 ml), Primer MX5 (1mM), Primer MX3 (1 mM) and 1.5 U Taq DNApolymerase, 10mM Tris-HCl pH 9.0, 50 mMKCl, 1.5 mM MgCl, 200 mM dNTP. PCR wasconducted using a program of denaturationBull. Eur. Ass. Fish Pathol., 22(3) 2002,  203at 95 ∞C for 5 min followed by 30 cycles of 95∞C (1 min), 60 ∞C (2 min) and 72∞C (2 min)and an extended elongation period of 72 ∞C(10 min).Gel purificationThe PCR product (10 ml) was electrophoresedon a 2% agarose gel (150V, 2 h). The products(approx. 1600 bp) were cut and isolated fromthe gel and subsequently purified using a GFXGel Band Purification Kit (AmershamPharmacia Biotech) following the manufactur-ers manual. The purified DNA fragment waseluted in 50 ml of distilled water.RFLPThe PCR products of the three isolates ofMyxobolus aeglefini and the three referencespecies were subjected to a RFLP analysis us-ing the restriction enzymes Hinf I and Msp I(New England Biolab) (Eszterbauer et al.,2000) and the enzymes Hae III (Gibco). Ap-proximately 2 units of enzyme were added to8 ml of PCR product including 1 ml of buffer(10x) and 0.8 ml distilled water and incubatedat 37 ∞C for 2 h. The digested product wasloaded on a 1.5% agarose gel for electrophore-sis (150V, 2h).Results and DiscussionThe resulting product of M. aeglefini DNA af-ter PCR and gel purification was a fragmentof approx. 1600 bp in length. The referencespecies M. macrocapsularis and M. pseudodisparyielded similar fragments of approx. 1600 bp,which correspond well with the fragmentsfound by Eszterbauer et al. (2000).  Figures 1,2 and 3 show the results of the enzymatic di-gestion by the restiction enzymes Hinf I, MspI and Hae III, respectively. DNA fragmentlengths were calculated by linear regressionanalysis for each digestion (Table 1). The com-parative RFLP analysis of DNA from the threespecies of M. aeglefini showed no differencewhen using the restriction enzymes Hinf I,Msp I and Hae III (Table 1). The reliability ofthe procedure was confirmed by comparingwith the RFLP from the species M.macrocapsularis and M. pseudodispar using thesame enzymes as in the marine isolates (Ta-ble 1). The specific restriction enzymes wereFigure 1.  The approx. 1600 bp PCR fragment cutby the restriction enzyme Hinf I. L2 to L4 are M.aeglefini  isolates from plaice, cod and dabrespectively. L5 is M. macrocapsularis and L6 is M.pseudodispar. L1 is the marker.Figure 2. The approx. 1600 bp PCR fragment cutby the restriction enzyme Msp I. L2 to L4 are M.aeglefini  isolates from plaice, cod and dabrespectively. L5 is M. macrocapsularis and L6 is M.pseudodispar. L1 is the marker.Bull. Eur. Ass. Fish Pathol., 22(3) 2002, 204chosen in accordance with other authors,where Hinf I and Msp I were defined as en-zymes for distinguishing the species M.macrocapsularis and M. pseudodispar(Eszterbauer et al., 2000). The enzyme Hinf Iwas also successfully used by Xiao & Desser(2000) to distinguish eighteen differentMyxozoan parasites. However, Xiao & Desser(2000) used different primers in their experi-ments and the subsequent restriction digestsare therefore not directly comparable to therestriction digests found in this paper. In ad-dition, we used the enzyme Hae III and thisenzyme successfully distinguished our refer-ence species, but not the three M. aeglefini iso-lates. As indicated there is no justification tosuggest that the three M. aeglefini isolates rep-resent different species. In addition, the threehost species inhabit the same marine habitat(demersal fish in Kattegat), which suggeststhat they all are exposed to infection withthese myxozoans. If the isolates are all M.aeglefini, it could indicate relative low hostspecificity. However, this is not unique amongmyxozoans (Lom & Dykova, 1992). In addi-tion, metazoan parasites from these hosts; e.g.Hysterothylacium aduncum and Anisakis simplex(Nematoda), Echinorhynchus gadi,Pomphorhynchus laevis (Acanthocephala),Cryptocotyle lingua (Digenea) do also success-fully infect all three host species (own obser-vations).It could alternatively be suggested that marinemyxozoans are more closely related than is thecase for freshwater forms. However, future ad-vances, such as sequencing of the approx. 1600bp DNA fragment and subsequent RFLP, arenecessary in order to obtain more conclusiveevidence of the relationship within and betweenmarine and freshwater myxozoans.elpmaS )tsoh(seicepSezis.xorppAtucnufo)pb(tcudorp)pb(ezistnemgarfxorppAIfniH IpsM IIIeaH1 inifelgea.M )ecialp( 0061 05/052/0031 054/0511 052/05312 inifelgea.M )doc( 0061 05/052/0031 054/0511 052/05313 inifelgea.M )bad( 0061 05/052/0031 054/0511 052/05314 siraluspacorcam.M 0061 05/051/054/059 051/053/054/056 051/052/573/524/0055 rapsidoduesp.M 0061 051/054/0001 002/003/0511 003/0031Table 1. Approximate sizes of fragments derived from restriction digests. Products were digested with therestriction enzymes of either Hinf I, Msp I or Hae III.Figure 3. The approx. 1600 bp PCR fragment cutby the restriction enzyme Hae III. L2 to L4 are M.aeglefini isolates from plaice, cod and dabrespectively. L5 is M. macrocapsularis and L6 is M.pseudodispar. L1 is the marker.Bull. Eur. Ass. Fish Pathol., 22(3) 2002,  205AcknowlegementsThis work was supported by The Elisabethand Knud Petersen Foundation and the Dan-ish Agricultural and Veterinary ResearchCouncil and the Danish Ministry of Food,Agricuture and Fisheries by a grant to the re-search network SCOFDA (Sustainable Controlof Fish Diseases in Aquaculture) and partiallyby the Hungarian Scientific Research Fund(OTKA) project No. T31755.ReferencesAndree, K., Gresoviac, S. & Hedrick, R. (1997).Small subunit ribosomal RNA sequencesunite alternate actinosporean andmyxosporean stages of Myxobolus cerebralisthe causative agent of whirling disease insalmonid fish. J Eukaryot Microbiol. 44(3):208-15Andree, K., MacConnell, E. & Hedrick, R.,(1998). A nested polymerase chain reaction forthe detection of genomic DNA of Myxoboluscerebralis in rainbow trout Oncorhynchusmykiss. Dis Aquat Org. 34:145-154.Andree, K., El-Matbouli, M. & Hoffmann, R.(1999a). Comparison of 18S and ITS-1 rDNAsequences of selected geographic isolates ofMyxobolus cerebralis. Int J Parasitol. 29(5): 771-775.Andree, K., Székely, Cc., Molnár, K., Gresiviac,S. & Hedrick, R. (1999b). Relationships amongmembers of the genus Myxobolus (Myxozoa:Bivalvidae) based on small subunit ribosomalRNA sequences. J Parasitol. 85(1):68-74.Eszterbauer, E., Benk?, M., Dán, Á. & Molnár,K. (2001). Identification of fish-parasiticMyxobolus (Myxosporea) species using a com-bined PCR-RFLP method. Dis Aquat Org.44:35-39.Gaevskaya, A & Kovaleva, A. (1976). On dis-ease of blue whiting from the Northeast At-lantic, caused by the myxosporidianMyxobolus aeglefini . Biologicheskierybokhozyaistvennye issledovaniya vAtlanticheskom okeane. Kaliningrad, 164-167(In Russian)Kabata, Z. (1957). Note on a new host ofMyxobolus aeglefini. Parasitology. 47:165-168.Karasev, A. (1988). Myxosporidian Myxobolusaeglefini (Cnidospora: Myxosporea) – bluewhiting tag from the Celtic Sea and adjacentwaters. 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Molecular charac-terization of Myxozoan Parasites from LakeSasajewun, Algonquin Park, Ontario, byriboprinting. J Eukaryot Microbiol. 47(1):85-89.

Comments on the host, organ and tissue specificity of fish myxosporeans and on the types of their intrapiscine development. Parasitol Hung.

Comparison of 18S and ITS-1 rDNA sequences of selected geographic isolates of Myxobolus cerebralis.

Identification of fish-parasitic Myxobolus (Myxosporea) species using a combined PCR-RFLP method. Dis Aquat Org.

Molecular characterization of Myxozoan Parasites from Lake Sasajewun, Algonquin Park, Ontario, by riboprinting.

Myxobolusinfection of the gill of the common bream (Abramis brama L.)

Myxosporidian Myxobolus aeglefini (Cnidospora: Myxosporea) – blue whiting tag from the Celtic Sea and adjacent waters.

On disease of blue whiting from the Northeast Atlantic, caused by the myxosporidian Myxobolus aeglefini. Biologicheskie rybokhozyaistvennye issledovaniya v Atlanticheskom okeane.

Preliminary investigations on the eastern North Sea and the Skagerrak dab (Limanda limanda) populations and their diseases.

Protozoan parasites of fishes.

Recent advances in our knowledge of Myxozoa.

Relationships among members of the genus Myxobolus (Myxozoa: Bivalvidae) based on small subunit ribosomal RNA sequences.

Small subunit ribosomal RNA sequences unite alternate actinosporean and myxosporean stages of Myxobolus cerebralis the causative agent of whirling disease in salmonid fish.

Comparative analysis of 18S rRNA genes from Myxobolus aeglefini Auerbach, 1906 isolated from cod (Gadus morhua), Plaice (Pleuronectes platessa) and dab (Limanda limanda), using PCR-RFLP

Comparative analysis of 18S rRNA genes from Myxobolus aeglefini Auerbach, 1906 isolated from cod (Gadus morhua), Plaice (Pleuronectes platessa) and dab (Limanda limanda), using PCR-RFLP

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