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Differences in a ribosomal DNA sequence of lungworm species (Nematoda: Dictyocaulidae) from fallow deer, cattle, sheep and donkeys
Research in Veterinary Science 1997, 62, 17-21
I ~ ' ~
Differences in a ribosomal DNA sequence of lungworm species (Nematoda: Dictyocaulidae) from fallow deer, cattle, sheep and donkeys C. EPE, G. V. SAMSON-HIMMELSTJERNA, T. SCHNIEDER, Institute of Parasitology, School of Veterinary Medicine, Biinteweg 17, D-30559 Hannover, Germany
SUMMARY The second internal transcribed spacer (ITS2)* of the ribosomal DNA of Dictyocaulus viviparus from cattle, D eckerti from fallow deer, Dfilaria from sheep and D arnfieldi from donkeys has been sequenced to investigate the genetic relationships among lungworm species, especially between D viviparus and D eckerti, because the latter is not generally accepted as a separate species. The length of the ITS2 varied between 403 and 481 bases, and its GC content ranged from 25 to 33 per cent. Intraspecific variations in D viviparus (0 to 1.5 per cent) and D eckerti (0.6 to 3.3 per cent) were slight; sequence homology between the species ranged from 50.3 to 76.7 per cent. Some sequence differences occurred at restriction sites of endonucleases leading to characteristic restriction fragment length patterns. The interspecific differences between D viviparus and D eckerti far exceeded the intraspecific variation, thus providing additional evidence that the two species are genetically distinct.
LUNGWORMS of the genus Dictyocaulus are common parasites in countries with temperate climates. They affect animals on pasture and cause economic losses due to respiratory disease and death. Except in endemic areas, the occurrence of lungworm infections is not predictable. The parasites may be spread by numerous vectors, such as birds, beetles or free-living ruminants like roe or fallow deer. Deer are generally regarded as a reservoir for bovine lungworms, although a separate lungworm species, D eckerti (Skrjabin 1931), has been described. This species description has been based on minor differences in the shape of the buccal ring between D eckerti and the bovine lungworm D viviparus. These morphological differences have been doubted by several authors and D eckerti is not generally accepted as a separate species (Cameron 1933, Dikmans 1936, Bacinsky 1973). The results of transmission trials have been as confusing as the morphological studies. Some investigators successfully transmitted lungworm larvae from roe deer to cattle but others did not (Hildebrandt 1962, Kutzer 1988). It is therefore still uncertain whether D eckerti is identical to D viviparus. The aim of this investigation was to compare the DNA sequences of the ribosomal second internal transcribed spacer (ITS2) of four lungworm species to obtain information about the genetic relationship between lungworms in general and between D viviparus and D eckerti in particular.
MATERIALS AND METHODS
Parasite material Two field isolates of D viviparus from cattle that origi* The nucleotide sequences reported here have been deposited in the GenBank/EMBL data bank under accession numbers U37715 for Dictyocaulus arnfieldi-ITS2, U37716 for Dictyocaulus eckerti-ITS2, U37717 for Dictyocaulus filaria-ITS2 and U37718 for Dictyocaulus
viviparus-ITS2
0034-5288/97/010017 + 05 $18.00/0
nated from different areas of northern Germany, about 250 km apart, were examined. In addition, one D arnfieldi isolate from a donkey, one D filaria isolate from sheep and one isolate of lungworms from fallow deer that was collected at the Institute of Parasitology of the University of Leipzig and which showed the morphological characteristics described for D eckerti were examined. This last isolate is referred to as D eckerti. DNA isolation, amplification and sequencing
For each set of polymerase chain reactions (PER), genomic DNA from individual worms was prepared by using a QIAamp Tissue Kit (Qiagen) according to the manufacturer's protocol. DNA concentrations were measured by means of a DNA Dipstick (Invitrogen). ITS2 DNA was amplified with a thermal cycler (Trio Thermoblock; Biometra) in a 100 gl PCR mix containing 15 ng DNA, 5 ~1 20X reaction buffer (Biozym), 1.5 mM magnesium chloride, dNTP (40pM each; Boehringer Mannheim), 1 U Tfl-Polymerase (Biozym), 50 pmol ITS2-forward-primer, and 50 pmol ITS2-reverse-primer under the following conditions: 1 x 94°C 3', 35 x (94°C 1', 55°C 1", 72°C 1"), 1 x 72°C 5'. DNA sequences of the ITS2-forward-primer (5'ACGTCTGGTTCAGGGTTGTT-3') and ITS2-reverseprimer (5'-TTAGTTTCTTTTCCTCCGCT-3') correspond to regions at or near the 3' and 5' ends of the 5.8S and 28S rDNA genes, respectively, of Caenorhabditis elegans (Ellis et al 1986, Gasser et al 1993). The PeR products of each lungworm species were separated on an ethidium bromide (0.1 gg/m1-1) stained 2 per cent TAg (0.04 M Tris-acetate, 0-001 M EDTA) agarose gel (Sambrook et al 1989). Bands were eluted from the agarose by using the Wizard PCR Preps DNA Purification System (Promega) and the isolated DNA was cloned into the plasmid vector pCR II (TA Cloning Kit; Invitrogen). To sequence the cloned fragments, plasmid DNA preparations were made from 50 ml cultures with the Nucleobond Kit (Macherey and Nagel). Between 70 and 100 ~g DNA were © 1997 W. B. Saunders Company Ltd
C. Epe, G. V. Samson-Himmelstjerna, T. Schnieder
18
isolated from each culture, of which 3 /ag were digested with EcoRI and separated on an agarose gel again to check for successful cloning. Each insert was sequenced according to the manufacturer' s protocol by using the Sequenase 2.0 sequencing kit (USB) and [35S]dATP (Redivue; Amersham) at least three times in both directions to confirm the sequence. The 5' and 3' ends of the ITS2 sequence were determined by comparison with those of C elegans. The sequences were aligned and genetic distances were calculated by using the CLUSTAL V programme (Higgins et al 1992). Restriction maps of the different ITS2 were determined by using the computer programme SEQUAID IX version 3.81 (Kansas State University).
RESULTS The sequence analysis of the four Dictyocaulus species determined that the sizes of the PCR products were between 451 and 530 bp including 403 to 481 bp of the ITS2 (Table 1) and 48 bp (D filaria and D arnfieldi) or 49 bp (D viviparus and D eckerti) of the 5' end of the 28S rDNA gene. The GC content of the ITS2 of all four species ranged from 25 to 33 per cent.
TABLE 1: Pairwise comparison of the number of homologous nucleotides in the ITS2 sequence among the four Dictyocaulusspecies
D viviparus (cattle) D eckerti (fallow deer) D amfieldi (donkey) D filaria (sheep)
Number of bases
D viviparus
D eckerti
D amfieldi
454 481 403 469
369 (76.7%) 243 (53.5%) 248 (52.9%)
256 (53.2%) 251 (52.2%)
236 (50.3%)
of between 52-2 and 53.5 per cent (Table 1). A diagrammatic presentation of the sequence shows that lungworms of ruminant species are more closely related to each other than to D arnfieldi from equine species (Fig 2). Several alignment gaps were present in D viviparus, compared with the other species, ranging from 1 to 17 bases. The difference in length between D viviparus and D eckerti (27 bases) consisted mainly of additional AT repeats at alignment positions 15 and 221 of the D eckerti sequence. Single base substitutions occurred at 160 alignment positions, of which 62 per cent were transitions (55 purines, 44 pyrimidines) and 38 per cent were transversions between a purine and a pyrimidine. The sequence length of the PCR products including the flanking regions (451 to 530 bp) was in accordance with the size of the amplificates determined by agarose gel electrophoresis (not shown).
Intraspecific variations Intraspecific variations were found in some individual worms of the two D viviparus isolates. A sequence comparison of all the sequenced individual worms of D viviparus revealed between three and seven GAT repeats at alignment position 145 in all the individuals of both isolates (Fig la). Additional differences occurred as single base substitutions in either of the sequenced D viviparus individuals at positions 4, 56, 106, 119, 380, 401,416, 418, 433 and 456, exclusively as transitions between either purines in seven cases or pyrimidines in three (Fig lb). Individual worms of both isolates differed in up to six bases (0 to 1-5 per cent intraspecific variation). For the detection of intraspecific variation in D eckerti, five worms were sequenced. Intraspecific variations occurred as single base substitutions in any of the five at positions 17, 75, 158, 162, 168, 220, 224, 228, 260, 366, 367, 429 and 467 as transitions between purines in eight cases or pyrimidines in two or as transversions between them in three cases (Fig lc). An insertion of ATAT was present between position 24 and 25 in one individual. Different gaps were detected: in one worm between position 242 and position 260, in another between positions 248 and 260, and in another two worms between positions 249 and 259. The second gap occurred at the positions 236 to 238 in four of the five individual worms, and the third gap was at position 377 also in four of the five worms. The individual worms differed in up to 13 bases and three gaps (0-6 to 3.3 per cent intraspecific variation). Intraspecific variation was not determined for the other species.
Interspecific differences ITS2 sequence differences in all the Dictyocaulus species ranged from 112 to 221 bases. Shared bases were found at 102 alignment positions. D viviparus and D eckerti showed the highest sequence similarity (76-7 per cent) and these species had similarities with D filaria and D arnfieldi
DISCUSSION Species definitions are generally based on morphological criteria. If the morphological differences are small, as in D eckerti for example, the taxonomic classification may be doubtful. Sequence data of the ribosomal ITS2 have proved to be a valuable tool in the definition of species. They are highly species-specific and are flanked by conserved regions of the ribosomal DNA which make it possible to design universal primers which bind to the 5-8S and 28S rDNA genes of many nematodes. The aim of this study was to compare the ITS2 of four lungworm species and to examine whether D viviparus and D eckerti are genetically distinct and therefore separate species. The intraspecific variations D viviparus (0 to 1.5 per cent) and in D eckerti (0-6 to 3.3 per cent) were very slight, in accordance with the results of studies on other parasites. Intraspecific variations in Strongylus species from horses varied from 0 to 0.9 per cent (Campbell et al 1995), and in Haemonchus species and Trichostrongylus coIubriformis no intraspecific variations were found (Hoste et al 1995, Stevenson et al 1995), whereas in other TrichostrongyIus species variations up to 0.4 per cent were detected (Hoste et al 1995). In Fasciola species, intraspecific variations were only 0 to 0.4 per cent. These results indicate that intraspecific variations within the ITS2 are generally absent or slight, probably as the result of a concerted evolution of repeated DNA sequences, such as those in the ribosomal DNA gene cluster, which leads to homogeneity of sequences among individuals and among populations (Dowling et al 1990). As a result, although in the present study intraspecific variations could be determined only for D viviparus and D eckerti, it may be assumed that variations within the other Dictyocaulus species are also slight. Its species-specific nature, together with the convenience of using universal primers from the conserved flanking regions, make the ITS2 PeR an ideal tool for the definition of species.
Differences in ribosomal DNAof Iungworms
i0
vlviparus eckerti arnfieldi filaria
AATTAAGAAT
vlviparus D. e c k e r t i D. a r n f i e l d i D. f i l a r i a
ATTTGATATA
vl v l p a r u s eckerti arnfieldi filaria
CGGTTATCGT
vlviparus eckerti arnfieldi filaria
GATTACCGTT
m. v ] v i p a r u s D. e c k e r t i D. a r n f i e l d i D. f i l a r l a
GTGTAATGTT
vlviparus D. e c k e r t i D. a r n f i e l d i D, f i l a r i a
---ATACGTG
m.
D. D. D.
20
ATGTG
D. D.
D. D, D.
A.ATATAT
ATATT
.............
-.ATGAAT
ACAGT
.....
. . . .
..C,.TT.T
. . . .
-ATGAAT
.C..G
• .A.A.G.--
.TG...ATC.
..-TAG.
--G..G,T..
AA...C.T..
---.
.......
CAG..---.
TTA.A..--.
.A.C..TT•G
C..-
.,.TG
D. D. D.
..CG.TTTGC
AC ..... G..
TA..GCGTG.
D. D. D.
Vl v i p a ru s eckerti arnfieldi filarla
..T.A.C... 191]
220
G..TC
.....
A--.... •G .... -.G.
.G.GCG-.T.
CG•..A.A.-
230
CATC
200
TATGTGATAT
.G..TGTGTG
240
.................
CACATA
250
TATGTATATG
ACAACATATA
TATAT
A...G..---
-..CCG.TTC
A .... GTATA
GAAAA..GAT
.,CA-.A..T
- - A .G C .T C .
CGTC
ATAACCGTTC
TAGTG
. - - - .G A . A A
. T .A C G
TAT...T
270
.........
........
.T G C .
280
TATTGAC
--TA-TATATATT
A ..... A
290
ATTAACGACA
300
TATTATGCTA
GA..'CG.GT
...G.TT.T.
..A..A.•G.
TTC..CAT.A
.G...-CATT
G-..-.-..T
..-..T..TG
A ..... -.G.
GAA.G.AA..
CGA.CGTATC
GA..ACG..T
..-G•T.GT.
320
330
GCTAATGTTC
TTA-ATCGTT
GATCATCATC
CA•
.T.A.G.
..CG.G.T.C 340
GATGACTGAA
350
GCAGTATTTA
.A.TG.TC..
A...G--...
.T ..... A ............
A--.G..A.T
. .-T..TA.A
A.GT..AT.A
T..AG..---
-T.T
AT--G..AAT
.A.A ......
A..ACAG.AA
TGCG.G.ACT
AT.T...AC.
360
m.
.....
A ..... .----..Ac.
.C.G..AT
- ........
G.GA...-.T
310
vlviparus eckerti arnf i eldi filaria
.CA.AGC..G
TTTACAAAAT
..A..----.
150
TGGAGATGAT
180
ATGAAGAAAT
260
m.
140
........
........................
,AC.GTT-
, .A ........... ,
]70
.T ........ C..
GTGTGCTATA
AGTG...T.
TTAGTATAGA
.... G ...........
.CTG
GAG...--C. 130
--...A.-A.
i0C TGTTGTCAAA
------..h . . . . . . . . . . .
.AACA.
TGACATGCGT
.... A .... C
CA.GT
90
.-T
..........
GT
.... A..---
......
TGCTTTATGA
120
CGTTATCCGA
GAATTAT
.
80 TGACGTCAGC
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.CA..
....
G-..GT.TA
70
5¢
ATATGTGT-A .........
-...-
ACGGT...GT
CACATATATA
210
m.
ATGCAGTAGT
.--i---.
160
D°
4C
AATAA
..... GT .....
ii0
m. D,
30
..........
60
D.
19
TGTTGGTTT.
.
380
TGATA--TAA
T ..... CG ......
. - . A . . . -
.A...T.C.•T C A A A .
370
GACTAT--AA
T.TG..C--.
CGA
C.TCGCTAT.
410
GATCG, .TG.TGTC.G .
390
400
ATATTGCT--
TGACTTGATC
G.. AAAAAAA
CAGTA.
G ...........
.TCGAA.GT
420
.... AT
G.-.
..TGTA
430
TGCT
..ACA..T ..GTG..T.A
440
450
vlviparus D. e c k e r t i D. a r n f i e l d i D, f i l a r i a
ATAATGAT-C
CGATGTAAGG
CAGT-ATATA
TGAATATCAT
CAATACATGT
-CA-TATCGA
D. D. D.
v~viparus eckerti arnfieldi filaria
• .G.AGC..T
ACAC-,.
.A ..... T-.
T .........
A..A
TAG.A.
ATA.-,..CT
.T.-...-..
G• . .-...A.
T---..
.G• •---.A.
-.GTCG..-.
D, D, D. D,
viviparus eckerti arnfieldi filaria
TTG-TAT
m.
GAT-GTCGAT
T .......
--
i . . - . . . i
------. . . . .
-T
T..-.-
.CG..T..GT
.
A..A.A.AGA
T .TT
•.T.A.TC.T
.
.
.
. . . . .
46O
m.
GATTGACGTG .
.
.
.
m___
.
.
.
.
.
.A..
.C. •GA.
-
. . . .
- ....
A .... GT..A 470
• T..C•
TCGACGACGA .
. . . . . . . . . .
.TAG
.T---.TTA.
480
CA.TCG.T.
TATAAGAAGA
•
--.
..G.GA.CAG ..AG.A.C..
490
500
...... •TA.
507
.
.
. G . . .
A.A--.. C.AAA.
,
FIG 1: Alignment of the ITS2 sequences (5' to 3') of D viviparus, D eckerti, D amfieldiand D filaria. (.) = same base as D viviparus, (-) = alignment gap, (b) intraspecific base changes and their alignment positions in the sequences of the two D viviparus isolates, (c) intraspecific base changes and their alignment positions in the D eckerti sequence of five individual worms, (b) and (c) are illustrated overleaf
Some of the sequence differences between species occur at specific recognition sites for restriction endonucleases. This explains the results of a previous study (Schnieder et al 1996) in which different restriction fragment length patterns (RFLP) were found in all four Dictyocaulus species after digestion with a range of restriction enzymes. Most of the sequence differences between the four Dictyocaulus species are single base substitutions. As Hoste et al (1995) pointed out, if such changes occurred at random, the chances of a substitution between purines, between pyrimidines or between a purine and a pyrimidine would be 25, 25 and 50 per cent, respectively. However, in
the four Dictyocaulus species examined, the substitutions did not occur at random (in the D viviparus sequence the proportions were 34, 28 and 38 per cent, and in D eckerti they were 61, 16 and 23 per cent, respectively). This is in accordance with the results observed with other parasitic nematodes, in which a trend for substitutions between purines or between pyrimidines rather than between a purine and a pyrimidine has been described (Hoste et al 1995, Chilton et al 1996). This type of substitution has been related to the maintenance of the secondary structure of the rDNA of these organisms (Hoste et al 1995). The authors have demonstrated that Dictyocaulus species
C. Epe, G. V Samson-Himmelstjerna, T. Schnieder
20
(b)
(c)
Intraspecific base changes in D. viviparus ITS2
Intraspecific base changes in D. eckerti ITS2 Isolates 2-5 compared to reference isolate No. 1)
Alignment position
Nucleotides
4
T/C A/G
56 106 119 145 380 401 416 418 433 456
C/T G/A 4 addit. G/A
Alignment position
IndividualNo.
17 24/25 75
3-5 2 2-5 2 2,5 5 2 3-5 2-5
158 162 168 220 224 228 242
GAT
T/C G/A A/G G/A A/G
4
248 249
2,5
260 236-238 366 367 377 429 467
2,5 2-5 3 2-5 2-5 2-5 2-5
Nucleotides
G/A 2x AT A/G A/G G/A G/A
C/T G/T
T/C gap until 260 gap until 260 gap until 259 A/T gap G/A A/G gap A/G
C/A
FIG 1 (continued)
D. viviparus
D. eckerti D. filaria D. arn.fieldi
1
[ I
FIG 2: Diagrammatic representation of the sequence similarity relationships among the four Dictyocaulus species calculated with CLUSTALV (Sneath and Soka11973)
can reliably be differentiated by their ITS2 sequences and RFLP patterns (Schnieder et al 1996). The ITS2 sequence differences between D viviparus and D eckerti confirmed the results of previous RAPD-PCR (Epe et al 1995) and PCRRFLP (Schnieder et al 1996) studies. The taxonomic classification of species is most accurately based on the ability of all members of the same species to reproduce. This implies that the gene flow between all its members is a major characteristic of a species. The magnitude of the sequence differences between D viviparus and D eckerti far exceeds the intraspecific variations, which are generally slight in ITS2 (as described above), indicating that there is no gene flow between them and that the classification of D eckerti as a separate species is justified. Transmission trials showed that both species develop in roe deer or fallow deer (Bienioschek 1995). Sequence differences within the ITS2 allow the development of pCR-based species-specific diagnostic tests which can be used for epidemiological studies of the prevalence of the two species in deer and their role in the transmission of D viviparus to cattle. ACKNOWLEDGEMENTS This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Schn 267/8-1). The authors wish to thank S. Bienioschek and S. Rehbein from the Institute of Parasitology, University of Leipzig, for providing D eckerti.
REFERENCES BACINSKY, A. (1973) The morphological evaluation of Dictycaulus found in red deer and cattle. In Russian. Folia Veterinaria 17, 209-219 BIENIOSCHEK, S. (1995) ExpefimenteUe Wechselinfektionen zwischan Zerviden und Hauswierderk~iuem mit groBen Lungenwtirmen (Dictyocaulus spp.) Thesis. Veterinary Faculty, University of Leipzig CAMERON, T. W. M. (1933) Some notes on the parasitic worms of the Scottish red deer. Proceedings of the Royal Physiological Society of Edinburgh 22, 91-97 CAMPBELL, A. J. D., GASSER, R. B. & CHILTON, N. B. (1995) Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs. International Journalfor Parasitology 25, 359-365 CHILTON, N. B , GASSER, R. B. & BEVERIDGE, I. (1996) Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). International Journal for Parasitology 25, 647-651 DIKMANS, (3. (1936) A note on Dictyocaulus from domestic and wild ruminants. Journal of the Washington Academy of Science 26, 298-303 DOWLING, T. E., MORITZ, C. & PALMER, J. D. (1990) Nucleic acids n: restriction site analysis. In Molecular systematics. Eds. D. M. Hillis and C. Moritz. Massachusetts, Sinauer. pp 250-317 ELLIS, R. E., SULSTON, J. E. & COULSTON, A. R. (1986) The rDNA of C elegans: sequence and structure. Nucleic Acids Research 14, 2345-2364 EPE, C., BIENIOSCHEK, S., REHBEIN, S. & SCHNIEDER, T. (1995) Comparative RAPD-PCR analysis of lungworms (Dictyocaulidae) from fallow deer, cattle, sheep and horses. Journal of Veterinary Medicine B 42, 187-191 GASSER, R. B., CHILTON, N. B., HOSTE, H. & BEVERIDGE, L (1993) Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Research 21, 2525-2526 HIGG1NS, D. G., BLEASBY, A. L & FUCHS, R. (1992) Clustal V: improved software for multiple sequence alignment. Computer Applications in the Biosciences 8, 189-191 HILDEBRANDT, J. (1962) Die Empf'inglichkeit der Hanswiederk~iuer und des Hochwildas fiir Dictyocaulus viviparus und Dictyocaulus filaria (Strongylata). Drmedvet, Thesis, Hanover School of Veterinary Medicine
Differences in ribosomal DNA o f l u n g w o r m x
HOSTE, H., CHILTON, N, B., GASSER, R, B. & BEVERIDGE, I. (1995) Differences in the second internal transcribed spacer (ribosomal DNA) between five species of Trichostrongylus (Nematoda: Trichostrongylidae). International Journal for Parasitology 25, 75-80 KUTZER, E. (1988) Bedeutung parasitgrer Wechselinfektionen bei Haus- und Wildwiederk~iuern. Monatsschriftfiir Veteriniirmedizin 43, 577-580 SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989) Molecular Cloning: a Laboratory Manual. New York, Cold Spring Harbor Laboratory Press SCHNIEDER, T., EPE, C. & V. SAMSON-HIMMELSTJERNA, G. (1996) Species differentiation of lungworms (Dictyocaulidae) by PCR-RFLP of ITS2 ribosomal DNA. Parasitology Research (In press)
21
SKRJABIN, K. I. (1931) Dictyocauliasis of cattle in the USSR. In Russian. Veterinarnyi Spetsialist na Sotsialistischeskoj Stroike 19, 23-32 SNEATH, P. H. A. & SOKAL, R. R. (1973) Numerical Taxonomy. San Francisco, Freeman. STEVENSON, L. A., CHILTON, N. B. & GASSER, R. B. (1995) Differentiation of Haemonchus placei from H contortus (Nematoda: Trichostrongylidae) by the ribosomal DNA second internal transcribed spacer. International Journal for Parasitology 25,483-488
Received February 6, 1996 Accepted May 24, 1996
I ~ ' ~
Differences in a ribosomal DNA sequence of lungworm species (Nematoda: Dictyocaulidae) from fallow deer, cattle, sheep and donkeys C. EPE, G. V. SAMSON-HIMMELSTJERNA, T. SCHNIEDER, Institute of Parasitology, School of Veterinary Medicine, Biinteweg 17, D-30559 Hannover, Germany
SUMMARY The second internal transcribed spacer (ITS2)* of the ribosomal DNA of Dictyocaulus viviparus from cattle, D eckerti from fallow deer, Dfilaria from sheep and D arnfieldi from donkeys has been sequenced to investigate the genetic relationships among lungworm species, especially between D viviparus and D eckerti, because the latter is not generally accepted as a separate species. The length of the ITS2 varied between 403 and 481 bases, and its GC content ranged from 25 to 33 per cent. Intraspecific variations in D viviparus (0 to 1.5 per cent) and D eckerti (0.6 to 3.3 per cent) were slight; sequence homology between the species ranged from 50.3 to 76.7 per cent. Some sequence differences occurred at restriction sites of endonucleases leading to characteristic restriction fragment length patterns. The interspecific differences between D viviparus and D eckerti far exceeded the intraspecific variation, thus providing additional evidence that the two species are genetically distinct.
LUNGWORMS of the genus Dictyocaulus are common parasites in countries with temperate climates. They affect animals on pasture and cause economic losses due to respiratory disease and death. Except in endemic areas, the occurrence of lungworm infections is not predictable. The parasites may be spread by numerous vectors, such as birds, beetles or free-living ruminants like roe or fallow deer. Deer are generally regarded as a reservoir for bovine lungworms, although a separate lungworm species, D eckerti (Skrjabin 1931), has been described. This species description has been based on minor differences in the shape of the buccal ring between D eckerti and the bovine lungworm D viviparus. These morphological differences have been doubted by several authors and D eckerti is not generally accepted as a separate species (Cameron 1933, Dikmans 1936, Bacinsky 1973). The results of transmission trials have been as confusing as the morphological studies. Some investigators successfully transmitted lungworm larvae from roe deer to cattle but others did not (Hildebrandt 1962, Kutzer 1988). It is therefore still uncertain whether D eckerti is identical to D viviparus. The aim of this investigation was to compare the DNA sequences of the ribosomal second internal transcribed spacer (ITS2) of four lungworm species to obtain information about the genetic relationship between lungworms in general and between D viviparus and D eckerti in particular.
MATERIALS AND METHODS
Parasite material Two field isolates of D viviparus from cattle that origi* The nucleotide sequences reported here have been deposited in the GenBank/EMBL data bank under accession numbers U37715 for Dictyocaulus arnfieldi-ITS2, U37716 for Dictyocaulus eckerti-ITS2, U37717 for Dictyocaulus filaria-ITS2 and U37718 for Dictyocaulus
viviparus-ITS2
0034-5288/97/010017 + 05 $18.00/0
nated from different areas of northern Germany, about 250 km apart, were examined. In addition, one D arnfieldi isolate from a donkey, one D filaria isolate from sheep and one isolate of lungworms from fallow deer that was collected at the Institute of Parasitology of the University of Leipzig and which showed the morphological characteristics described for D eckerti were examined. This last isolate is referred to as D eckerti. DNA isolation, amplification and sequencing
For each set of polymerase chain reactions (PER), genomic DNA from individual worms was prepared by using a QIAamp Tissue Kit (Qiagen) according to the manufacturer's protocol. DNA concentrations were measured by means of a DNA Dipstick (Invitrogen). ITS2 DNA was amplified with a thermal cycler (Trio Thermoblock; Biometra) in a 100 gl PCR mix containing 15 ng DNA, 5 ~1 20X reaction buffer (Biozym), 1.5 mM magnesium chloride, dNTP (40pM each; Boehringer Mannheim), 1 U Tfl-Polymerase (Biozym), 50 pmol ITS2-forward-primer, and 50 pmol ITS2-reverse-primer under the following conditions: 1 x 94°C 3', 35 x (94°C 1', 55°C 1", 72°C 1"), 1 x 72°C 5'. DNA sequences of the ITS2-forward-primer (5'ACGTCTGGTTCAGGGTTGTT-3') and ITS2-reverseprimer (5'-TTAGTTTCTTTTCCTCCGCT-3') correspond to regions at or near the 3' and 5' ends of the 5.8S and 28S rDNA genes, respectively, of Caenorhabditis elegans (Ellis et al 1986, Gasser et al 1993). The PeR products of each lungworm species were separated on an ethidium bromide (0.1 gg/m1-1) stained 2 per cent TAg (0.04 M Tris-acetate, 0-001 M EDTA) agarose gel (Sambrook et al 1989). Bands were eluted from the agarose by using the Wizard PCR Preps DNA Purification System (Promega) and the isolated DNA was cloned into the plasmid vector pCR II (TA Cloning Kit; Invitrogen). To sequence the cloned fragments, plasmid DNA preparations were made from 50 ml cultures with the Nucleobond Kit (Macherey and Nagel). Between 70 and 100 ~g DNA were © 1997 W. B. Saunders Company Ltd
C. Epe, G. V. Samson-Himmelstjerna, T. Schnieder
18
isolated from each culture, of which 3 /ag were digested with EcoRI and separated on an agarose gel again to check for successful cloning. Each insert was sequenced according to the manufacturer' s protocol by using the Sequenase 2.0 sequencing kit (USB) and [35S]dATP (Redivue; Amersham) at least three times in both directions to confirm the sequence. The 5' and 3' ends of the ITS2 sequence were determined by comparison with those of C elegans. The sequences were aligned and genetic distances were calculated by using the CLUSTAL V programme (Higgins et al 1992). Restriction maps of the different ITS2 were determined by using the computer programme SEQUAID IX version 3.81 (Kansas State University).
RESULTS The sequence analysis of the four Dictyocaulus species determined that the sizes of the PCR products were between 451 and 530 bp including 403 to 481 bp of the ITS2 (Table 1) and 48 bp (D filaria and D arnfieldi) or 49 bp (D viviparus and D eckerti) of the 5' end of the 28S rDNA gene. The GC content of the ITS2 of all four species ranged from 25 to 33 per cent.
TABLE 1: Pairwise comparison of the number of homologous nucleotides in the ITS2 sequence among the four Dictyocaulusspecies
D viviparus (cattle) D eckerti (fallow deer) D amfieldi (donkey) D filaria (sheep)
Number of bases
D viviparus
D eckerti
D amfieldi
454 481 403 469
369 (76.7%) 243 (53.5%) 248 (52.9%)
256 (53.2%) 251 (52.2%)
236 (50.3%)
of between 52-2 and 53.5 per cent (Table 1). A diagrammatic presentation of the sequence shows that lungworms of ruminant species are more closely related to each other than to D arnfieldi from equine species (Fig 2). Several alignment gaps were present in D viviparus, compared with the other species, ranging from 1 to 17 bases. The difference in length between D viviparus and D eckerti (27 bases) consisted mainly of additional AT repeats at alignment positions 15 and 221 of the D eckerti sequence. Single base substitutions occurred at 160 alignment positions, of which 62 per cent were transitions (55 purines, 44 pyrimidines) and 38 per cent were transversions between a purine and a pyrimidine. The sequence length of the PCR products including the flanking regions (451 to 530 bp) was in accordance with the size of the amplificates determined by agarose gel electrophoresis (not shown).
Intraspecific variations Intraspecific variations were found in some individual worms of the two D viviparus isolates. A sequence comparison of all the sequenced individual worms of D viviparus revealed between three and seven GAT repeats at alignment position 145 in all the individuals of both isolates (Fig la). Additional differences occurred as single base substitutions in either of the sequenced D viviparus individuals at positions 4, 56, 106, 119, 380, 401,416, 418, 433 and 456, exclusively as transitions between either purines in seven cases or pyrimidines in three (Fig lb). Individual worms of both isolates differed in up to six bases (0 to 1-5 per cent intraspecific variation). For the detection of intraspecific variation in D eckerti, five worms were sequenced. Intraspecific variations occurred as single base substitutions in any of the five at positions 17, 75, 158, 162, 168, 220, 224, 228, 260, 366, 367, 429 and 467 as transitions between purines in eight cases or pyrimidines in two or as transversions between them in three cases (Fig lc). An insertion of ATAT was present between position 24 and 25 in one individual. Different gaps were detected: in one worm between position 242 and position 260, in another between positions 248 and 260, and in another two worms between positions 249 and 259. The second gap occurred at the positions 236 to 238 in four of the five individual worms, and the third gap was at position 377 also in four of the five worms. The individual worms differed in up to 13 bases and three gaps (0-6 to 3.3 per cent intraspecific variation). Intraspecific variation was not determined for the other species.
Interspecific differences ITS2 sequence differences in all the Dictyocaulus species ranged from 112 to 221 bases. Shared bases were found at 102 alignment positions. D viviparus and D eckerti showed the highest sequence similarity (76-7 per cent) and these species had similarities with D filaria and D arnfieldi
DISCUSSION Species definitions are generally based on morphological criteria. If the morphological differences are small, as in D eckerti for example, the taxonomic classification may be doubtful. Sequence data of the ribosomal ITS2 have proved to be a valuable tool in the definition of species. They are highly species-specific and are flanked by conserved regions of the ribosomal DNA which make it possible to design universal primers which bind to the 5-8S and 28S rDNA genes of many nematodes. The aim of this study was to compare the ITS2 of four lungworm species and to examine whether D viviparus and D eckerti are genetically distinct and therefore separate species. The intraspecific variations D viviparus (0 to 1.5 per cent) and in D eckerti (0-6 to 3.3 per cent) were very slight, in accordance with the results of studies on other parasites. Intraspecific variations in Strongylus species from horses varied from 0 to 0.9 per cent (Campbell et al 1995), and in Haemonchus species and Trichostrongylus coIubriformis no intraspecific variations were found (Hoste et al 1995, Stevenson et al 1995), whereas in other TrichostrongyIus species variations up to 0.4 per cent were detected (Hoste et al 1995). In Fasciola species, intraspecific variations were only 0 to 0.4 per cent. These results indicate that intraspecific variations within the ITS2 are generally absent or slight, probably as the result of a concerted evolution of repeated DNA sequences, such as those in the ribosomal DNA gene cluster, which leads to homogeneity of sequences among individuals and among populations (Dowling et al 1990). As a result, although in the present study intraspecific variations could be determined only for D viviparus and D eckerti, it may be assumed that variations within the other Dictyocaulus species are also slight. Its species-specific nature, together with the convenience of using universal primers from the conserved flanking regions, make the ITS2 PeR an ideal tool for the definition of species.
Differences in ribosomal DNAof Iungworms
i0
vlviparus eckerti arnfieldi filaria
AATTAAGAAT
vlviparus D. e c k e r t i D. a r n f i e l d i D. f i l a r i a
ATTTGATATA
vl v l p a r u s eckerti arnfieldi filaria
CGGTTATCGT
vlviparus eckerti arnfieldi filaria
GATTACCGTT
m. v ] v i p a r u s D. e c k e r t i D. a r n f i e l d i D. f i l a r l a
GTGTAATGTT
vlviparus D. e c k e r t i D. a r n f i e l d i D, f i l a r i a
---ATACGTG
m.
D. D. D.
20
ATGTG
D. D.
D. D, D.
A.ATATAT
ATATT
.............
-.ATGAAT
ACAGT
.....
. . . .
..C,.TT.T
. . . .
-ATGAAT
.C..G
• .A.A.G.--
.TG...ATC.
..-TAG.
--G..G,T..
AA...C.T..
---.
.......
CAG..---.
TTA.A..--.
.A.C..TT•G
C..-
.,.TG
D. D. D.
..CG.TTTGC
AC ..... G..
TA..GCGTG.
D. D. D.
Vl v i p a ru s eckerti arnfieldi filarla
..T.A.C... 191]
220
G..TC
.....
A--.... •G .... -.G.
.G.GCG-.T.
CG•..A.A.-
230
CATC
200
TATGTGATAT
.G..TGTGTG
240
.................
CACATA
250
TATGTATATG
ACAACATATA
TATAT
A...G..---
-..CCG.TTC
A .... GTATA
GAAAA..GAT
.,CA-.A..T
- - A .G C .T C .
CGTC
ATAACCGTTC
TAGTG
. - - - .G A . A A
. T .A C G
TAT...T
270
.........
........
.T G C .
280
TATTGAC
--TA-TATATATT
A ..... A
290
ATTAACGACA
300
TATTATGCTA
GA..'CG.GT
...G.TT.T.
..A..A.•G.
TTC..CAT.A
.G...-CATT
G-..-.-..T
..-..T..TG
A ..... -.G.
GAA.G.AA..
CGA.CGTATC
GA..ACG..T
..-G•T.GT.
320
330
GCTAATGTTC
TTA-ATCGTT
GATCATCATC
CA•
.T.A.G.
..CG.G.T.C 340
GATGACTGAA
350
GCAGTATTTA
.A.TG.TC..
A...G--...
.T ..... A ............
A--.G..A.T
. .-T..TA.A
A.GT..AT.A
T..AG..---
-T.T
AT--G..AAT
.A.A ......
A..ACAG.AA
TGCG.G.ACT
AT.T...AC.
360
m.
.....
A ..... .----..Ac.
.C.G..AT
- ........
G.GA...-.T
310
vlviparus eckerti arnf i eldi filaria
.CA.AGC..G
TTTACAAAAT
..A..----.
150
TGGAGATGAT
180
ATGAAGAAAT
260
m.
140
........
........................
,AC.GTT-
, .A ........... ,
]70
.T ........ C..
GTGTGCTATA
AGTG...T.
TTAGTATAGA
.... G ...........
.CTG
GAG...--C. 130
--...A.-A.
i0C TGTTGTCAAA
------..h . . . . . . . . . . .
.AACA.
TGACATGCGT
.... A .... C
CA.GT
90
.-T
..........
GT
.... A..---
......
TGCTTTATGA
120
CGTTATCCGA
GAATTAT
.
80 TGACGTCAGC
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.CA..
....
G-..GT.TA
70
5¢
ATATGTGT-A .........
-...-
ACGGT...GT
CACATATATA
210
m.
ATGCAGTAGT
.--i---.
160
D°
4C
AATAA
..... GT .....
ii0
m. D,
30
..........
60
D.
19
TGTTGGTTT.
.
380
TGATA--TAA
T ..... CG ......
. - . A . . . -
.A...T.C.•T C A A A .
370
GACTAT--AA
T.TG..C--.
CGA
C.TCGCTAT.
410
GATCG, .TG.TGTC.G .
390
400
ATATTGCT--
TGACTTGATC
G.. AAAAAAA
CAGTA.
G ...........
.TCGAA.GT
420
.... AT
G.-.
..TGTA
430
TGCT
..ACA..T ..GTG..T.A
440
450
vlviparus D. e c k e r t i D. a r n f i e l d i D, f i l a r i a
ATAATGAT-C
CGATGTAAGG
CAGT-ATATA
TGAATATCAT
CAATACATGT
-CA-TATCGA
D. D. D.
v~viparus eckerti arnfieldi filaria
• .G.AGC..T
ACAC-,.
.A ..... T-.
T .........
A..A
TAG.A.
ATA.-,..CT
.T.-...-..
G• . .-...A.
T---..
.G• •---.A.
-.GTCG..-.
D, D, D. D,
viviparus eckerti arnfieldi filaria
TTG-TAT
m.
GAT-GTCGAT
T .......
--
i . . - . . . i
------. . . . .
-T
T..-.-
.CG..T..GT
.
A..A.A.AGA
T .TT
•.T.A.TC.T
.
.
.
. . . . .
46O
m.
GATTGACGTG .
.
.
.
m___
.
.
.
.
.
.A..
.C. •GA.
-
. . . .
- ....
A .... GT..A 470
• T..C•
TCGACGACGA .
. . . . . . . . . .
.TAG
.T---.TTA.
480
CA.TCG.T.
TATAAGAAGA
•
--.
..G.GA.CAG ..AG.A.C..
490
500
...... •TA.
507
.
.
. G . . .
A.A--.. C.AAA.
,
FIG 1: Alignment of the ITS2 sequences (5' to 3') of D viviparus, D eckerti, D amfieldiand D filaria. (.) = same base as D viviparus, (-) = alignment gap, (b) intraspecific base changes and their alignment positions in the sequences of the two D viviparus isolates, (c) intraspecific base changes and their alignment positions in the D eckerti sequence of five individual worms, (b) and (c) are illustrated overleaf
Some of the sequence differences between species occur at specific recognition sites for restriction endonucleases. This explains the results of a previous study (Schnieder et al 1996) in which different restriction fragment length patterns (RFLP) were found in all four Dictyocaulus species after digestion with a range of restriction enzymes. Most of the sequence differences between the four Dictyocaulus species are single base substitutions. As Hoste et al (1995) pointed out, if such changes occurred at random, the chances of a substitution between purines, between pyrimidines or between a purine and a pyrimidine would be 25, 25 and 50 per cent, respectively. However, in
the four Dictyocaulus species examined, the substitutions did not occur at random (in the D viviparus sequence the proportions were 34, 28 and 38 per cent, and in D eckerti they were 61, 16 and 23 per cent, respectively). This is in accordance with the results observed with other parasitic nematodes, in which a trend for substitutions between purines or between pyrimidines rather than between a purine and a pyrimidine has been described (Hoste et al 1995, Chilton et al 1996). This type of substitution has been related to the maintenance of the secondary structure of the rDNA of these organisms (Hoste et al 1995). The authors have demonstrated that Dictyocaulus species
C. Epe, G. V Samson-Himmelstjerna, T. Schnieder
20
(b)
(c)
Intraspecific base changes in D. viviparus ITS2
Intraspecific base changes in D. eckerti ITS2 Isolates 2-5 compared to reference isolate No. 1)
Alignment position
Nucleotides
4
T/C A/G
56 106 119 145 380 401 416 418 433 456
C/T G/A 4 addit. G/A
Alignment position
IndividualNo.
17 24/25 75
3-5 2 2-5 2 2,5 5 2 3-5 2-5
158 162 168 220 224 228 242
GAT
T/C G/A A/G G/A A/G
4
248 249
2,5
260 236-238 366 367 377 429 467
2,5 2-5 3 2-5 2-5 2-5 2-5
Nucleotides
G/A 2x AT A/G A/G G/A G/A
C/T G/T
T/C gap until 260 gap until 260 gap until 259 A/T gap G/A A/G gap A/G
C/A
FIG 1 (continued)
D. viviparus
D. eckerti D. filaria D. arn.fieldi
1
[ I
FIG 2: Diagrammatic representation of the sequence similarity relationships among the four Dictyocaulus species calculated with CLUSTALV (Sneath and Soka11973)
can reliably be differentiated by their ITS2 sequences and RFLP patterns (Schnieder et al 1996). The ITS2 sequence differences between D viviparus and D eckerti confirmed the results of previous RAPD-PCR (Epe et al 1995) and PCRRFLP (Schnieder et al 1996) studies. The taxonomic classification of species is most accurately based on the ability of all members of the same species to reproduce. This implies that the gene flow between all its members is a major characteristic of a species. The magnitude of the sequence differences between D viviparus and D eckerti far exceeds the intraspecific variations, which are generally slight in ITS2 (as described above), indicating that there is no gene flow between them and that the classification of D eckerti as a separate species is justified. Transmission trials showed that both species develop in roe deer or fallow deer (Bienioschek 1995). Sequence differences within the ITS2 allow the development of pCR-based species-specific diagnostic tests which can be used for epidemiological studies of the prevalence of the two species in deer and their role in the transmission of D viviparus to cattle. ACKNOWLEDGEMENTS This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Schn 267/8-1). The authors wish to thank S. Bienioschek and S. Rehbein from the Institute of Parasitology, University of Leipzig, for providing D eckerti.
REFERENCES BACINSKY, A. (1973) The morphological evaluation of Dictycaulus found in red deer and cattle. In Russian. Folia Veterinaria 17, 209-219 BIENIOSCHEK, S. (1995) ExpefimenteUe Wechselinfektionen zwischan Zerviden und Hauswierderk~iuem mit groBen Lungenwtirmen (Dictyocaulus spp.) Thesis. Veterinary Faculty, University of Leipzig CAMERON, T. W. M. (1933) Some notes on the parasitic worms of the Scottish red deer. Proceedings of the Royal Physiological Society of Edinburgh 22, 91-97 CAMPBELL, A. J. D., GASSER, R. B. & CHILTON, N. B. (1995) Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs. International Journalfor Parasitology 25, 359-365 CHILTON, N. B , GASSER, R. B. & BEVERIDGE, I. (1996) Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea). International Journal for Parasitology 25, 647-651 DIKMANS, (3. (1936) A note on Dictyocaulus from domestic and wild ruminants. Journal of the Washington Academy of Science 26, 298-303 DOWLING, T. E., MORITZ, C. & PALMER, J. D. (1990) Nucleic acids n: restriction site analysis. In Molecular systematics. Eds. D. M. Hillis and C. Moritz. Massachusetts, Sinauer. pp 250-317 ELLIS, R. E., SULSTON, J. E. & COULSTON, A. R. (1986) The rDNA of C elegans: sequence and structure. Nucleic Acids Research 14, 2345-2364 EPE, C., BIENIOSCHEK, S., REHBEIN, S. & SCHNIEDER, T. (1995) Comparative RAPD-PCR analysis of lungworms (Dictyocaulidae) from fallow deer, cattle, sheep and horses. Journal of Veterinary Medicine B 42, 187-191 GASSER, R. B., CHILTON, N. B., HOSTE, H. & BEVERIDGE, L (1993) Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Research 21, 2525-2526 HIGG1NS, D. G., BLEASBY, A. L & FUCHS, R. (1992) Clustal V: improved software for multiple sequence alignment. Computer Applications in the Biosciences 8, 189-191 HILDEBRANDT, J. (1962) Die Empf'inglichkeit der Hanswiederk~iuer und des Hochwildas fiir Dictyocaulus viviparus und Dictyocaulus filaria (Strongylata). Drmedvet, Thesis, Hanover School of Veterinary Medicine
Differences in ribosomal DNA o f l u n g w o r m x
HOSTE, H., CHILTON, N, B., GASSER, R, B. & BEVERIDGE, I. (1995) Differences in the second internal transcribed spacer (ribosomal DNA) between five species of Trichostrongylus (Nematoda: Trichostrongylidae). International Journal for Parasitology 25, 75-80 KUTZER, E. (1988) Bedeutung parasitgrer Wechselinfektionen bei Haus- und Wildwiederk~iuern. Monatsschriftfiir Veteriniirmedizin 43, 577-580 SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989) Molecular Cloning: a Laboratory Manual. New York, Cold Spring Harbor Laboratory Press SCHNIEDER, T., EPE, C. & V. SAMSON-HIMMELSTJERNA, G. (1996) Species differentiation of lungworms (Dictyocaulidae) by PCR-RFLP of ITS2 ribosomal DNA. Parasitology Research (In press)
21
SKRJABIN, K. I. (1931) Dictyocauliasis of cattle in the USSR. In Russian. Veterinarnyi Spetsialist na Sotsialistischeskoj Stroike 19, 23-32 SNEATH, P. H. A. & SOKAL, R. R. (1973) Numerical Taxonomy. San Francisco, Freeman. STEVENSON, L. A., CHILTON, N. B. & GASSER, R. B. (1995) Differentiation of Haemonchus placei from H contortus (Nematoda: Trichostrongylidae) by the ribosomal DNA second internal transcribed spacer. International Journal for Parasitology 25,483-488
Received February 6, 1996 Accepted May 24, 1996