Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs

International

002&751!3(94)00116-2

Journalfor Parasitology. Vol. 25. No. 3, pp. 359 -3hS. 1995 Copyright 0 1995 Australian Society for Parasitology ELwvicr Scimce Ltd Printed in Great Britain. All rigtrts nscrved cozo-7519/95 $9 50 + 0.00

in a s spies Singk Eggs ANGUS

J. D. CAMPBELL,

ROBIN

B GASSER*

and NEIL

B. CHILTON

University of Melbourne, Department of Veterinary Science, Princes Highway, Werribee, Victoria 3030, Australia (Received 19 April 1994; accepted 14 June 1994)

AlMract-CpppBen

A. J. D., Gamer R. R, and Ckbm 8uowsklat&ka~of

N. R 1995.

FcRacd dtkeribotmdspaeersasgene~mukersforsgecies horse faeees. Key words: Strongylus, egg; species identification; diagnosis; rDNA; internal lranscribed spacer, ITS-2; sequencing; PCR-linked RFLP.

INTRODUCTION

There are over 50 speciesof strongylid nematode which infect horses,many of which are of major veterinary importance (e.g., Arundel, 1985; Ogbourne & Duncan, 1985;Uhlinger, 1991;Huntington, Gasser,Campbell, Blanch & Bidstrup, 1993).These parasitescomprisetwo subfamilies,the Strongylinae (large strongyles) and the Cyathostominae (small strongylesor cyathostomes)(Lichtenfels, 1975). Accurate diagnosisof strongyle infections is an important adjunct to parasite control and surveillance, especially since the intensive use of anthelmintics has had seriousconsequenceson parasite populations,such as drug resistanceand an increase * To whom all correspondence should be addressed.

in the incidenceof cyathostomeinfections {reviewed by Herd, 199Oa,199Oh,1993;Slocombe,1992).The methodscurrently usedifor the diagnosisof strongyle from infections involve flotation of nematode faecescomplementedwith larval culturing (B&4 & Supperer,1992).Differentiation of eggsof the different speciesof strongyleis not possibleusingmorphological criteria. Eggs are therefore cuhured to third-stage larvae to allow their identification under This procedure takes 7 days for a microscope.

cyathostomesand 10-14days for largestrongyies.In addition, the techniquemay be unreliable for determining the relative abundanceof eggsof different speciespresent in a faexal sample, Wause their survival rates vary under certain culture conditions (e.g., Dobson, Barnes, Birclijin & GilI, 1997).

359

360

A. J. D. Campbell et al

There have been attempts to develop alternative diagnosticsystemsfor strongyle infectionsin horses. For instance,indirect biochemicaland immunological tests have been evaluated (Schulze, Bergfeld & Wall, 1983;Nichol & Masterson, 1987;Soule, 1990; Weiland, Hasslinger,Mezger & Pallein, 1991), but have been shown to be unreliable for species-or genus-specific diagnosis. DNA technology has provided accurate tools for the speciesidentification of different developmental stagesof various parasites(Barker, 1989;McManus, 1990;Zarlenga& Barta, 1990;Wilson, 1991;Bowles & McManus, 1993; Gasser, Chilton, Hoste & Beveridge, 1993).Recent studieshave demonstrated the suitability of ribosomal(r) RNA and rDNA for this purposebecauseof its abundancein the organism and little or no sequencevariation within a species(Hillis & Mortiz, 1990;Hillis & Dixon, 1991; Hoste, Gasser,Chilton, Mallet & Beveridge, 1993). In particular, the sequenceof the secondinternal transcribedspacerrDNA (ITS-2) has beenshownto distinguishreliably betweenclosely-relatedspeciesof trematodesand arthropods (Adlard, Barker, Blair & Cribb, 1993;Porter & Collins, 1991;Luton, Walker & Blair, 1992; Wesson, Porter & Collins, 1992). Recently, the ITS-2 hasalsobeenusedto distinguish betweenclosely-relatedand morphologically similar species of parasitic nematode from Australian macropodid marsupials (Chilton, Gasser & Beveridge,in press)and domesticruminants(Gasser, Chilton, Hoste 8c Stevenson,1994;Hoste, Chilton, Gasser& Beveridge, in press).A similar approach may therefore be applicablefor the identification of eggsand larval stagesof equine strongyles, which cannot be identified to the specieslevel using morphologicalcharacters. The first aim of the presentstudy wasto examine the extent of DNA sequencedifferencesin the ITS-2 between members of the genus Strongylus. This genus, which contains only 4 species(Strongylus edentatus, S. equinus, S. vulgaris and S. asini), was chosen becausemale and female worms of each species are morphologically well-defined. The secondaim was to evaluate the feasibility of establishing a technique for the specificidentification of singlestrongyle eggs. MATERIALS AND METHODS Parasite material. Adult worms of Strongylus edentatus, S. equintrs and S. vutgaris were collected from horses from

differentgeographical regionsin Australiaandthe U.S.A. (Table 1). Individual worms were collected from the large intestine at necropsy, washed extensively in phosphatebuffered saline, pH 7.3 (PBS), identified morphologically to the species-level (according to Lichtenfels, 1975) and

Table I-DNA Species

Isolate

S. edentatus

Sed.1 Sed.2 Sed.3 Sed.4

S. equines

S. vulgaris

Sed.5 Sed.6 Sed.7 Seq. 1 Seq.2 Seq.3 Seq.4* SCXJ.5 Seq.6 sv.1 sv.2 sv.3 sv.4* sv.5

isolates used in this study Origin Dandenong, Victoria, Australia Omeo, Victoria, Australia Geelong, Victoria, Australia Echuca,Victoria, Australia Echuca, Victoria, Australia Louisiana, U.S.A. Louisiana, U.S.A. Echuca, Victoria, Australia Echuca, Victoria, Australia Echuca, Victoria, Australia Townsville, Queensland, Australia Townsville, Queensland, Australia Victoria, Australia Omeo, Victoria, Australia Geelong, Victoria, Australia Pakenham, Victoria, Australia Echuca, Victoria, Australia Louisiana, U.S.A.

DNA from single worms, or* from several worms. frozen at -70°C until required for PCR and sequencing. Eggs of each species were dissected from the uterusof gravid female worms under a microscope and washed five times by agitation and centrifugation (200 g) in 50 ml distilled H,O. Single eggs were pipetted in 6 pl H,O into separate 0.5 ml Eppendorf tubes for direct use in PCR (Mow). DNA isolation, amplification and sequencing. Techniques follow those described recently (Gasser et al., 1993) with slight modifications. In brief, genomic DNA was isolated from single or poofed worms of each species (Table I). The ITS-2 plus primer flankingsequence (definedhereinasITS2+), which comprises 64 bp at the 3’ end of the 5.8s gene and 68 bp at the 5’ end of the 28s gene, was amplified by PCR from worm DNA (10-20 ng template) or directly

from singleeggs.For eachsetof PCR reactions,negative (i.e. no-DNA) and positive controls were included. Conserved oligonucleotide primers NC4: 5’TGAAATTKGAACGAAT-3’ (forward) and NC2: STTAGlTTCTTTTCCTCCGCT-3’ (reverse), designed to flanking regions of the ITS-2 sequence of Caenorhabditb elegans rDNA (GenBank accession no X036801 Ellis, Sulston & Coulson, 1986), were used for PCR under the following conditions: 94”C, 1 min (denaturation); 55”C, 1 min (annealing); 72’C, 1 min (extension) for 30 cycles. A proportion (10%) of each PCR product and 50% of the noDNA control samples were examined by eletrophoresis on 1.5% TBE (89 mM Tris-HCl, 1 mM EDTA, pH 7.4) agarose gels (Sambrook, Fritsch & Maniatis, 1989), stained with ethidium bromide and photographed. After purification using an anion-exchange spin c&mm (Qiagen, Diagen), 1 pl (2.5-5.0 ng DNA) of the ITS-2+ PCR product was sequenced with NC4 and NC2 primers using the following cycles: 95°C for 5 min (initial time delay), then 95”C, 30 s (denaturation); 48°C (NC4) or 55°C (NC2), 30 s (annealing); 7O”C, 30 s (extension) for 20 cycles,

Ribosomal DNA of Strongylus

361

followedby 9YC, 30s; 70°C,60 s for 10cycles.TheITS-2 accessionnumbers: X77807 (S. eqzukusj, X77808 of each isolate was sequenced at least 3 times in both direcand X77863 (S. vu&&). The length of the ITS-2 tions to confirm the sequence. The 5’ and 3’ ends of the sequenceof S. edentatus, S. equirws and S. vulguris ITS-2 sequence were determined by comparison with those was235 bp, 229bp and 217 bp, respectively(Fig. I). of C. elegant. Sequence alignments were performed manuThe GC content of the ITS-2 of the 3 speciesranged ally following the use of the Clustal V program (Higgins, Bleasby 62 Fuchs, 1992), and a consensus sequence was from 30.9 to 38.3%. determined for each species. Restriction mapping and PCR-linked RFLP. Restriction maps were determined using the “Map” programme available on GenBank. For PCR-linked RFLP, unpurified ITS2-t PCR products (10 ~1) from worms or single eggs were restricted directly with 8-12 units (1 ~1) of endonuclease (Promega) in 20 ~1 for 4-12 h according to the manufacturer’s protocols. Digests (3-S pl) were separated by agarose gel electrophoresis. Size comparisons were made to pUClP Hpa II (Bresatec) or PhiX 174-Hue III markers(Promega). RESULTS

sequencedata reported in this paper appear in the EMBL, GenBank and DDBJ Nucleotide SequenceDatabasesunder the following The

nucleotide

10

Intraspecl$c

variation

The degreeof intraspecificvariation in the ITS-2 sequence differed between the 3 species of Strongylus. No vasiation was detectedamong the S. edentatus isolates,while a low level of intraspecific variation wasdetectedin S. equininus (o-0.4%) and S. vulgaris(O-0.9%). Sequencevariation in S. vt.&ris occurred at alignment positions112 and 231 (Table I; Fig. 1). All S. vulgaris isolates,except Sv.2, had either a G or C at position 112. Isolate Sv.2 had both basesat this position. Similarly, 2 bases(C and T) werepresentat alignmentposition 231for isolates Sv.2 and Sv.4, while the other 3 S. vulgarisisolates had only a singlebase(C or T) at this position. For

30

20

40

50

S. edentatus S. equinus S. vulgaris

TATATACATA --.....G.. --....TG..

S. edentatus S. equinus S. vulgaris

TTGCATTCAG TTGTAATCCC . . . . . . . . . A . . . C...... A.A.. ..A.A . . ..GTC...

CATTCTAGAA AAGAATAATA . . . . . . . . . . ..I...--.. . . . . . . . . . . ..,....-,.

ATTGCAACAT . . . . . . . . . . -.........

S. edentatus S. equinus S. vulgaris

GTATGTTAGC . . . .* . . . . . . . . A...-T

TGGGTGGTAA ..TTCAT.GG .*---AA...

TACTGGCTAA --..AA...-------..-

TGGCATCACA . . . . ..A... ..T...GGAT

S. edentatus S. equinus S. vulgaris

TCGTTATC-T . . . . . . . . A. .TA..C..--

GCTGCTAAAT A,........ A..A..T...

TGTTTACCGA CTTATTAACA --TTTAGCAG . . . . . . . . . . .t,....... TA........ . . . ..CG... . . . . . . . . . . -A.....T..

S. edentatus S. equinus S. vulgaris

GGCCTGTTCG . . . . . . . AT. A......-..

AGGATAACGT ,.-....... GA-.G..TCA

TGTTCAGTGC TATTTGCAA . . . . . . . . . . . . . . . . . AA.CT.A..A l . . . . . .

CTACAATGTG GCCTGTCAA. . . . . . . . . . . . . . ..TG.. . . . . . . ..A . . . . ..T..A

CATTGTTTGT .I........ . . . . ..CG..

CTACACXAA -......... -.......T.

CGAATGGTGC . . . . . . . . . . . . . . . . . ..T

Alignment position

Bases(Isolate)

*105 *112 *231

G andA (Seq.4);G (all other Seqisollltes) G and C (Sv.2); G or C (all other Sv isolates) T andC (Sv.2 and Sv.4); T or C (all other Sv isolates)

Fig. 1. Alignment of the ITS-2 sequences of Strongyh edentatw, S. equine and S. vulgaris. (.) = same base as that of S. edentutus, and (*) = position of intraindividual/intras~iti variation, where one of the two bases present is the same as that in the sequence of S. edentutus (see below).

A. J. D. Campbell ef al,

362

S. equinus, 2 bases(G and A) were presentat alignment position 105 for isolate Seq.4, whereasthe other 5 isolateshad only a single base(G) at this position. InterspeciJic differences

Comparisonsbetweenspecieswere made over an alignment length of 239 bp (Fig. 1). The ITS-2 sequences of S. edentatus and S. equinus were more similar (87.4%) to each other than S. edentatus was to S. vulgaris (71.3%), or S. equinus was to S. vulgaris (73.6%). The 3 speciessharedbasesat 161 (67.4%) alignment positions. Most differenceswere observedin 3 regions(alignment positions 109-131, 147-161, and 213-231; Fig. 1). Smaller regions of sequencedifferenceswere also present (e.g., alignment positions 64-67 and 87-91). The length of alignment gaps ranged from 1 to 7 nucleotides. Single base substitutions between all species occurred at 51 alignment positions for which there were no alignment gaps.Of these,62.7%were transitionsbetweeneither purines(n=19) or pyrimidines (n=13), and 37.3% were transversions(i.e. substitutions betweena purine and pyrimidine). The ITS-2+ of the Strongylus speciesconsistedof the ITS-2 (217-235 bp) plus 132 basesof flanking sequence5’ and 3’ of the spacer.This is in accordancewith the sizeof ITS-2+ ranging from = 350to 370 bp for Strongyhs speciesdeterminedby agarose gel electrophoresis(Fig. 3, panelsA&B). Basedon the ITS-2 sequences,restriction mapping identified endonucleases that could be usedto delineatethe 3 Strongylus species(Fig. 2). All 3 specieshad restriction sitesfor Taq I, Xba I and Mae I, except that

S.vulgaris

2 :a5 se TR$ , 11

DC

I s

I %

I I 2

FTa as ...i

I ;. ‘. P,a

S. edentatus had two Tag I sitescomparedto a single

site in the other two species.Each speciesalso had restriction sitesfor Mse I, but the number of sites varied betweenspecies(Fig. 2). An Ah I site and 2 Hae III siteswere presentin S. equinus and S. edentatus but not in S. vulgaris. There were 2 restriction sitesfor Mae II in S. equinus and one sitein S. edentatus, but nonein S. vulgaris. The Rsa I site at the 5’ end of the ITS-2 of S. vulgaris and S. equinus was absentin S. edentatus. A restriction site for Hinf I wasonly presentin S. vulgaris, while S. equinus was the only specieswith a restriction site for Vsp I. The endonucleases Alu I and Vsp I were chosen for usein the PCR-linked RFLP analysisof ITS-2+. At least 5 singleworm isolatesand 5 singleeggsof eachspecieswereexamined,and no differenceswere detectedin their individual restriction patterns (data not shown). RepresentativePCR-linked RFLP’s are shown in Fig. 3, panel C. As predicted by the sequencedata, restriction of ITS-2+ PCR products of the 3 specieswith Alu I produced 3 restriction fragmentsof = 50, 140and 190bp in sizefor S. edentatus and S. equinus, while the ITS-2+ of S. vulgaris remainedunrestricted.The band of 50 bp in S. edentatus and S. equinus was prodcued by restriction at the Alu I site(AGCT) located9-12 nucleotidesfrom the 5’ of the 28s rDNA flanking sequence,which is absentin S. vulgaris due to a basesubstitution (i.e. AACT). Restriction of ITS-2+ of S. equinus with Vsp I produced 2 fragmentsof = 140 and 220 bp, while that of the other 2 speciesremained unrestricted. The 3 speciesof Strongylus could also be distinguished using the endonuclease Tru 91 (Promega;isoschizomerof Mse I) (data not shown),

g7 3’

$$ / 0 z

I s-

I

1

5

Fig. 2. Schematic representation of restrictionmapsof the ITS-2 of Strongylw edentam, S. equines andS. vulgaris.

Ribosomal DNA of Strongylus

Uncut

363

A/l/I

Fig. 3. Results of PCR and PCR-linked RFLP of ITS-2+ of 3 Strongyh species. Panel A: ITS-2+ amplified from genomic DNA from worms of S. vdguris (lane 2), S. edentutus (lanes 3824) and S. equhus lanes 5&j). Panel B: ITS-2+ amplified directly from single eggs of S. ehntutus (lane 2), S. equinus (lane 3) and S. vulgaris (lanes 4&S). Panel C: ITS-2+ PCR products (uncut) restricted using endonucleases Ah I or Vsp I. S. edentam (lanes 1, 4 & 7), S. equinus (lanes 2, 5 8c 8) and S. vulgaris (lanes 3. 6 & 9). Positive (Panels A&B, lane 1) and no-DNA (PanelA, lane7; PanelB, lanes 6&7) controls used in PCR. Ordinate values indicate approximate size of fragments in base pairs.

however, the low restriction efficiency of this enzyme position in the ITS-2 sequenceof the pool&-worm resultedin partial digestionof the PCR products. isolatesof S. vulgaris (Sv.4) and S. equimrsfSeq-4j could also representintraindividual variation. However, it could alsobe attributed to diff&enoesbetween DISCUSSION The ITS-2 sequences of all 7 S. edentutusisolates individual worms making up the pool (i.e. intraapexaminedwere identical, whereasintraspecificvaria- cific variation). Intraindividual variation of &WA has been confirmed in Aeaks mosquitoes tion wasdetectedamongisolatesof S’.equinus (0.4%) sequences and S. vulgaris (0.9%). For S. vulgaris, 2 baseswere using cloning procedures(Wessonet al., 1992)and recorded at alignmentpositions 112 and 231 in the may be the result of “slippagemutations” produ& ITS-2 of an individual worm (isolateSv.2,Fig. 2). It during DNA replication (Levinson& Gutman, 1987; is unlikely that this is a resultof an experimentalarte- Schloetterer& Tautz, 1992;Wessonet al., 1992). The level of differences in the ITS-2 sequence fact becausethese“double bases”werepresentin all sequences obtained (irrespectiveof the primer used betweenthe three speciesof Strungylus extamjned in and day on which sequencingwas conducted).It is this study (13-29%) is markedly greater than the possible that sequencedifferences occur in the degreeof intraspecific or intraindividual variation multiple copiesof the rDNA transcriptionalunit of a (cl%), which implies that the ITS-2 is useful for singlenematode(= intraindividual variation), how- speciesdelineation.Associatedwith the interspecific ever, this needsto be confirmed usingDNA cloning differencesin ITS-2 sequenceare diagnostic endoprocedures.The 2 nucleotidespresent at a single nucleaserestriction sites (e.g. Ah I and Vsp 1).

A. J. D. Cam tpbellet

364

Restriction analysis of this spacer using these endonucleases demonstratedthe potential of PCRlinked RFLP as an experimental tool to reliably distinguishbetweendifferent life cycle stagesof the 3 Strongylus species.This technique has reduced the time requirementsto identify the speciesof single eggsfrom 10 to 14 days for larval culturing to less than 1 day. However, a technique for routine use requiresfurther development. The magnitude of the sequence differences betweenthe 3 Strongylus speciesis alsoof taxonomic significance.For instance,Skrjabin (1993;cited in Skrjabin, Shikhobalova, Schulz, Popova, Boev & Delyamure, 1952)divided the 4 speciesof Srrongylus into 3 genera (Alfortia, Delafondia and Strongylusj basedon the number of buccal teeth present,whereasLichtenfels(1975, 1980)proposedto retain the 4 specieswithin the one genus.The level of sequence differencesbetween S. edentam, S. equims and S. vulgaris (13-29%) is similar to that detectedbetween sibling species of Hypodontus (25-280/o), which belong to a related family of nematodes(Chilton et al., in press). This evidence,supports the view of Lichtenfels(1975, 1980)that Alfortia and Defafondia are synonymsof Strongylus. In this study, it has beendemonstratedthat the 3 species of Strongyh can be distinguishedreliably basedon their ITS-2 sequence,and that singleeggs of each speciescan be identified by PCR-linked RFLP. Given that the eggsof over 50 speciesof equinestrongylid are morphologically indistinguishable, the ability to usegeneticmarkers in the ITS-2 to identify individual eggs and larval stages to specieshasimportant implicationsfor studying their life cyle, pathogenesis and population biology. While the differentiation at the specieslevel is useful from an experimental point of view, the potential of a molecular assay for epidemiological applications currently liesin the rapid distinction of singleeggsof small strongyles(cyathostomes)from those of large strongylesin faecal samples.

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