179 Phylogeny of Dictyocaulus (lungworms) from eight species of ruminants based on analyses of ribosomal RNA data J. HO¨ GLUND1*,D.A.MORRISON1, B. P. DIVINA1,2, E. WILHELMSSON1 and J. G. MATTSSON1 1 Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agriculture Sciences, 751 89 Uppsala, Sweden 2 College of Veterinary Medicine, University of the Philippines Los Ban˜os, College, Laguna, 4031 Philippines (Received 8 November 2002; revised 8 February 2003; accepted 8 February 2003) SUMMARY In this study, we conducted phylogenetic analyses of nematode parasites within the genus Dictyocaulus (superfamily Trichostrongyloidea). Lungworms from cattle (Bos taurus), domestic sheep (Ovis aries), European fallow deer (Dama dama), moose (Alces alces), musk ox (Ovibos moschatus), red deer (Cervus elaphus), reindeer (Rangifer tarandus) and roe deer (Capreolus capreolus) were obtained and their small subunit ribosomal RNA (SSU) and internal transcribed spacer 2 (ITS2) sequences analysed. In the hosts examined we identified D. capreolus, D. eckerti, D. filaria and D. viviparus. However, in fallow deer we detected a taxon with unique SSU and ITS2 sequences. The phylogenetic position of this taxon based on the SSU sequences shows that it is a separate evolutionary lineage from the other recognized species of Dictyocaulus. Furthermore, the analysis of the ITS2 sequence data indicates that it is as genetically distinct as are the named species of Dictyocaulus. Therefore, either this taxon needs to be recognized as a new species, or D. capreolus, D. eckerti and D. viviparus need to be combined into a single species. Traditionally, the genus Dictyocaulus has been placed as a separate family within the superfamily Trichostrongyloidea. The present molecular phylogenetic analyses support the placement as a separate family, but the current data do not support the placement of the Dictyocaulidae within the Trichostrongyloidea without a reassessment of the placement of the superfamily Strongyloidea. While D. eckerti has been regarded as the one and only lungworm species of cervids, this study showed that 4 host species including 3 members of Cervidae (moose, reindeer, red deer) and 1 Bovidae (musk ox) were infected with this parasite. Host ranges of D. viviparus (cattle), D. filaria (sheep) and D. capreolus (moose and roe deer) were more restricted. No clear pattern of co-evolution between the dictyocaulid taxa and their bovid and cervid hosts could be determined. Key words: Dictyocaulus, Strongylida, genetic diversity, genotypes, phylogentic analysis, SSU rRNA, ITS2, ruminants, Scandinavia. INTRODUCTION exclusively of artiodactylids were included (Railliet & Henry, 1907). Many years later, Skrjabin, Shi- Parasitic nematodes of the genus Dictyocaulus (family khobalova & Schultz (1954) reviewed the taxonomy Dictyocaulidae, superfamily Trichostrongyloidea) of the genus Dictyocaulus. They made 2 important are found as adults in the bronchial branches of a contributions to the taxonomy of the genus. Firstly, range of domestic and wild ruminants (Anderson, D. noerneri of cervids was considered an invalid 1992). In many host species lungworms are potential name because no formal morphological description causative agents of parasitic bronchitis and, particu- was available. Second, D. eckerti was described from larly in cattle, sheep and semi-domesticated red deer, reindeer. More recently, Gibbons & Khalil (1988) they are regarded as important pathogens (Urquhart revised the genus and concluded that it contained et al. 1996). However, for a long time the taxonomy 6 species: D. africanus, D. arnfeldi, D. cameli, D. has been very confused, both with reference to the eckerti, D. filaria and D. viviparus. Collectively these number of species and to the phylogeny within the species are all very similar, with a paucity of dis- order Strongylida. tinctive morphological characters. The controversies Although bovine lungworms were first recognized on the composition of the genus Dictyocaulus have in 1782 as Gordius viviparus, the genus Dictyocaulus continued over the years, particularly in relation to was not described until the start of the 20th century. Dictyocaulus of cervids. For example, (Durette- In the original description of the genus, 4 lungworms Desset, Hugonnet & Chabaud, 1988) questioned whether D. noerneri (Railett & Henry, 1907) should be dismissed as a valid species. However, as pointed out * Corresponding author: Department of Parasitology, D. noerneri Swedish University of Agriculture Sciences, 751 89 by Jansen & Borgsteede (1990), was not Uppsala, Sweden. Tel: +46 18674156. Fax: +45 based on sufficient information to confirm its ident- 18309162. E-mail: [email protected] ity, and according to their opinion it should be Parasitology (2003), 127, 179–187. f 2003 Cambridge University Press DOI: 10.1017/S0031182003003366 Printed in the United Kingdom J. Ho¨glund and others 180 considered as a nomen dubium. Instead, these authors animals slaughtered for other reasons and sent for were of the opinion that lungworms of cervids are autopsy at the National Veterinary Institute in identical to D. eckerti. Uppsala, Sweden. Lungworms from reindeer were The taxonomy and identification of lungworms of collected from a herd in northern Norway, fixed in the genus Dictyocaulus have increasingly been ana- 70% alcohol and then sent to our laboratory. The lysed with various molecular techniques. In most specimens from musk ox came from Alaska, USA. studies, results are based on information of the in- Genomic worm DNA was extracted either from ternal transcribed spacer 2 (ITS2) of nuclear ribo- fresh-frozen (x70 xC) or ethanol-fixed individual somal RNA (Divina et al. 2000; Epe et al. 1995; specimens by using the QIAamp tissue kit, accord- Ho¨glund et al. 1999; Schnieder, Epe & Samson- ing to the manufacturer’s protocol (QIAgen, Hilden, Himmelstjerna, 1996; Von Samson-Himmelstjerna Germany). et al. 1997). Sequence analysis of the ITS2 con- firmed that lungworms from fallow deer are geneti- cally different from those of donkey, sheep and cattle DNA amplification (Epe, Samson-Himmelstjerna & Schnieder, 1997). The ITS2 was amplified by PCR with the primers Accordingly, the ITS2 sequence of lungworm from ITS2F (5 -ACG TCT GGT TCA GGG TTG fallow deer in Germany was deposited in GenBank k TT-3 ) and ITS2R (5 -TTA GTT TCT TTT as D. eckerti (op. cit.). With a similar analytical ap- k k CCT CCG CT-3 ) (Ho¨glund et al. 1999). The SSU proach we could not only confirm the identity of k rRNA genes were amplified in two parts, overlapping lungworms from cattle, but we could also show that by B60 bp. The 5 ends of the SSU rRNA genes a novel Dictyocaulus species infects roe deer and k were amplified with the primer pair OP150 (5k- moose (Ho¨glund et al. 1999). Specifically, as a result, AAG ATT AGG CCA TGC ATG-3 ) and OP151 we determined the identity of nearly 300 lungworms k (5 -TCT TGG CAA ATG CTT TCG-3 ). The am- with an ITS2 PCR combined with a DNA hy- k k plification of the 3 ends of the SSU rRNA genes was bridization assay using species-specific oligonucleo- k performed using the primer pair OP152 (5 -AGA tide probes (Divina et al. 2000). Using this assay, we k GGT GAA ATT CKT AGA-3 ) and OP153 demonstrated that Swedish cattle harboured a mono- k (5 -ACC TTG TTG TTA CGA CTT-3 ). For the specific D. viviparus infection. In contrast, infections k k PCR, each 50 ml reaction mixture contained 10 mM composed entirely of the new Dictyocaulus species Tris–HCl, pH 8.3, 50 mM KCl, 1.5mM MgCl , were found in roe deer, whereas in moose, mixed 2 0.4 mM of each primer, 200 mM of each deoxynucleo- infections containing this species and D. eckerti were tide, 1 ml of template and 1 U of AmpliTaq DNA recorded (op. cit.). The morphology of the new polymerase (Applied Biosystems, Foster City, species has now been described, and it has been USA). The amplifications were carried out in a PE formally named as D. capreolus (see Gibbons & 2400 thermocycler (Applied Biosystems). After an Ho¨glund 2002). initial 2 min incubation at 93 xC, the DNA was The aim of the present study was to investigate amplified for 30 cycles of 45 s of denaturation at the genetic diversity and phylogeny of a range of 94 xC, for 30 s of annealing at 42 xC and for 2 min of Dictyocaulus taxa from 8 species of ruminant host, all extension at 72 xC. The PCR ended with a final ex- of which are represented in the Swedish fauna. We tension of 7 min at 72 xC. PCR products were veri- employed not only ITS2 sequence data but also fied on 1.5% agarose gels with 0.5 mg/ml ethidium new small subunit ribosomal RNA (SSU rRNA) bromide. The amplicons were purified over spin sequence data for all taxa. The inclusion of the SSU columns (QIAquick PCR purification kit from rRNA sequence data enabled the evolutionary re- QIAgen) and eluted with 30 mlofHO. lationships of the members of Dictyocaulus to other 2 nematodes to be explored. For both data sets we also performed a preliminary assessment of host–parasite Sequence analysis relationships. The 5k end of the SSU rRNA gene was sequenced using primers OP150, OP151, OP188 (5k-GCA MATERIALS AND METHODS GGC GCG AAA CTT ATC CAA-3k) and OP189 (5 -CAT TGA AAT AAC CGT TCC ATA GG- Sampling parasites and DNA extraction k 3k). The 3k end of the SSU rRNA gene was Adult worms were obtained from the bronchi and sequenced using primers OP152, OP153, OP154 trachea following dissection of lungs from cattle (Bos (5k-GCG GTG TTT AGC CGC ACG AG3k) and taurus), domestic sheep (Ovis aries), European fallow OP155 (5kCTG ATT CTT CCA TTG TAG CG-3k). deer (Dama dama), moose (Alces alces), musk ox BigDye chemistry (Applied Biosystems) was used (Ovibos moschatus), red deer (Cervus elaphus), rein- for the DNA sequencing reactions, and the samples deer (Rangifer tarandus) and roe deer (Capreolus were analysed on an ABI 377 DNA sequencer capreolus).