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Lack of intraspecific variation in the second Internal Transcribed Spacer (ITS-2) of Trichostrongylus colubriformis ribosomal DNA
lnrernorionol Journu/forParosirology Primed in Grrut Britain
002&7519/93 IE6.M) + 0.00 Prrgemon Press Lid Ausrralian Socrrfy for Parasirology
Vol. 23. No. 8, pp. 1069-1071. 1993
RESEARCHNOTE LACK OF INTRASPECIFIC VARIATION IN THE SECOND INTERNAL TRANSCRIBED SPACER (ITS-2) OF TRZCHOSTRONG YLUS COLUBRZF'ORMZS RIBOSOMALDNA HERV~ HOSTE,*~ ROBIN B. GASSER,~ NEIL B. CHILTON,~ *INTRA-CR SDepartment
SERGE MALLET*
and IAN BEVERIDGE~
Tours, Station de Pathologie aviaire et de Parasitologie, Nouzilly F37380, France of Veterinary Science, University of Melbourne, Parkville, Victoria 3052, Australia (Received
IS June 1993; accepted 13 July 1993)
Abstract-HosTE H., GASSER R. B., CHILTONN. B., MALLET S. and BEVERIDGEI. 1993. Lack of intraspecific variation in the second internal transcribed spacer (ITS-2) of Trichostrongylus colubriformis ribosomal DNA. International Journal for Parasitology 23: 1069-1071. The nucleotide sequence of the second internal transcribed spacer (ITS-2) was determined for three populations of the parasitic nematode Trichostrongylus colubriformis which differed in their susceptibility to benzimidazole anthelmintics and/or in their geographical origin. No intraspecific variation was found in the ITS-2 sequence, indicating that this region of rDNA is inadequate to discriminate between resistant and susceptible populations of T. colubriformis, but it may prove useful for distinguishing between species of Trichoslrongylus. INDEX KEY WORDS: nematode; benzimidazole
Ribosomal resistance.
DNA;
internal
DISEASES due to trichostrongyle nematodes of ruminants are of major animal health and economic importance. Mixed infections with different nematodes (e.g. Trichostrongylus, Ostertagia, Cooperia, Haemonchus and Nematodirus) are common in sheep. It is, however, difficult to differentiate on morphological grounds between the eggs of these species, except for Nematodirus [see Georgi & McCulloch (1989)], and hence time-consuming larval cultures are required for species identification. Developing rapid and sensitive molecular techniques for the specific diagnosis of these infections therefore has major significance for their control. Recent reports have indicated that the second internal transcribed spacer (ITS-2) of the ribosomal DNA may be useful for delineating between closely-related species of parasitic trematodes (Luton, Walker & Blair, 1992; Adlard, Barker, Blair & Cribb, 1993; Michot, Despres, Bonhomme & Bachellerie, 1993), but no comparable studies have been conducted on the ITS2 of parasitic nematodes. However, before speciesspecific DNA probes can be developed it is of paramount importance to establish that there is little
t To whom all correspondence
should be addressed.
transcribed
spacer;
Trichostrongylus colubriformis;
within-species variation in the rDNA sequence. Therefore, in this paper we examine the level of intraspecific variation in the ITS-2 sequence of T. colubriformis, which is one of the most common trichostrongyles in sheep. The three populations of T. colubriformis used in this study originated from disparate geographical areas and differed in their level of resistance to benzimidazole anthelmintics. Nematodes of the “Weybridge strain” from the United Kingdom (LD,, = 0.036 ppm) and the “McMaster strain” from Australia (LD,, = 0.06 ppm) (Lacey, Redwin, Gill, Demargheriti & Waller, 1990) are susceptible to thiabendazole, whereas the population from France is resistant (LD,, = 0.496 ppm) (Hubert & Kerboeuf, 1992). DNA was extracted and purified from a pool of 1520 male worms from each population as described in Gasser, Chilton, Hoste & Beveridge, 1993. The ITS-2 of each sample was amplified and sequenced from both the 5’ and 3’ ends using two oligonucleotide primers (NC 1: S’ACGTCTGGT”TCAGGGTTGTT3 and NC2: STTAGTT”XTTTTCCTCCGCT3’) as described in Gasser et al. (1993). For each sample, at least two sequence gels were run (for 2 and 4 h). The start and end of the ITS-2 region was determined by comparison with the sequence of the
HERVCHOSTE et al.
1070
TABLE I. ITS-2 OF Trichostrongylus colubriformis rDNA* AATGAATTTCTACAGTGTGGCTAACTCTAACACTGTTTGTCGAAT TaqI GGTCATTGTCAAATATTGTGATGATTCCCATTTCAGTTCAAGAAT SSPI Hittf7 AATACATGCAACATGATGTTAATGTTGTAATGACATTAATGTTCC Asel TGTATGATGTGAACGTGTTGTTACTGTTTGAATGTACTCAGTGAA MaelI Rsai TTTGAGATTGATTTAAACAGGGACATGTATAACAATAATGTTCAA DraI TTATCATTTGTAT *Recognition
sites of restriction
45 90 135 180 225 238
enzymes are underlined.
free-living nematode Caenorhabditis elegans (GenBank accession code: X03680). No differences were detected in the ITS-2 sequences of the three T. colubriformis samples irrespective of their geographical origin or level of resistance to benzimidazole anthelmintics. The 238 nucleotide sequence of the ITS-2 of T. colubriformis (Table I) is considerably shorter in size than those of other helminths including C. elegans [383 bases (Ellis, Sulston & Coulson, 1986)], and the parasitic trematodes Fasciola heputica [366 bases (Michot et al., I993)], Schistosoma mansoni, S. haematobium, S. intercalatum and S. japonicum [308-348 bases (Michot ct al., 1993)], but similar to that of the parasitic trematode Dolichosaccus symmetrus [247 bases (Luton et al., 1992)]. The CC content of the ITS-2 sequence of T. colubrijivmis is 3 I. I %, which is considerably lower than that recorded for C. elegans [48% (Ellis et al., 1986)], D. symmetrus [47% (Luton et al., 1992)], F. hepatica [48% (Michot et al., 1993)] and several species of Schistosoma [45- 5 I % (Michot et al.. 1993)]. The ITS-2 sequence of T. colubriformis also has little homology with the free-living nematode C. elegans. Currently, there are no other ITS-2 sequences available for parasitic nematodes to compare with T. colubriformis. Recognition sites for several restriction enzymes, which are 4-6 base cutters, were detected on the ITS-2 sequence of T. colubr~formis (Table I) using the mapping program on GenBank. The significance of these restriction enzymes as a diagnostic tool for distinguishing between species of Trichostrongylus is currently under investigation. The results of the present study clearly demonstrate that the ITS-2 region is not suitable for delineating between resistant and susceptible populations of T. colubriformis. Nevertheless, the apparent lack of intraspecific variation in the sequence of the ITS-2 shows promise for developing species-specific diagnostic tools, providing sufficient
sequence
variation
exists between
different
species of
Trichostrongylus.
Acknowledgements-Dr Hervk Hoste, a visiting scientist at Melbourne University, is a grateful recipient of a Fellowship from the French Ministry of Research and Space Technology. Financial support was provided by the Australian Research Council, Ian Potter Foundation, ANZ Executors and Trustees, Australian Academy of Science and United Film Completions Ltd.
REFERENCES ADLARD R. D., BARKER S. C., BLAIR D. & CRIBB T. H. 1993. Comparison of the second internal transcribed spacer (ribosomal DNA) from populations and species of Fasciolidae (Digenea). International Journal ,for Parasitology 23: 423426. ELLIS R. E., SULSTON J. E. & COULSON A. R. 1986. The rDNA of C. elegans: sequence and structure. Nucleic Acids Research 14: 2345-2364. GASSER R. B.. CHILTON N. B., HOSTE H. & BEVERIDGE1. 1993. Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Research 21: 252552526. GEORGI .I. R. & MCCULLOCH C. E. 1989. Diagnostic morphometry: identification of helminth eggs by discriminant analysis of morphometric data. Proceedings of the Helminthological Society qf Washington 56: 44-57. HUBERT J. & KERBOEUP D. 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Veterinary Record 130: 442446. LACEY E., REDWINJ. M.. GILL J. H., DEMARGHERITIV. M. & WALLER P. J. 1990. A larval development assay for the simultaneous detection of broad spectrum anthelmintic resistance. In: Resistance of Parasites to Antiparasite Drugs (Edited by BORAY J. C., MARTIN P. J. & ROUSH R. T.), pp. 177-184. Round Table Conference, VII
Research
International Congress of Parasitology, Paris, August 1990. LUTON K., WALKER D. & BLAIR D. 1992. Comparisons of ribosomal internal transcribed spacers from two congeneric species of flukes (Platyhelminthes : Trematoda : Digenea).
Note
1071
Molecular and Biochemical Parasitology 56.323-328. MICHOT B., DESPRES L., BONHOMMEF. & BACHELLERIEJ.-P. 1993. Conserved secondary structures in the ITS2 of trematode pre-RNA. Federation qf European Biochemical Sciences Letters 316: 247-252.
002&7519/93 IE6.M) + 0.00 Prrgemon Press Lid Ausrralian Socrrfy for Parasirology
Vol. 23. No. 8, pp. 1069-1071. 1993
RESEARCHNOTE LACK OF INTRASPECIFIC VARIATION IN THE SECOND INTERNAL TRANSCRIBED SPACER (ITS-2) OF TRZCHOSTRONG YLUS COLUBRZF'ORMZS RIBOSOMALDNA HERV~ HOSTE,*~ ROBIN B. GASSER,~ NEIL B. CHILTON,~ *INTRA-CR SDepartment
SERGE MALLET*
and IAN BEVERIDGE~
Tours, Station de Pathologie aviaire et de Parasitologie, Nouzilly F37380, France of Veterinary Science, University of Melbourne, Parkville, Victoria 3052, Australia (Received
IS June 1993; accepted 13 July 1993)
Abstract-HosTE H., GASSER R. B., CHILTONN. B., MALLET S. and BEVERIDGEI. 1993. Lack of intraspecific variation in the second internal transcribed spacer (ITS-2) of Trichostrongylus colubriformis ribosomal DNA. International Journal for Parasitology 23: 1069-1071. The nucleotide sequence of the second internal transcribed spacer (ITS-2) was determined for three populations of the parasitic nematode Trichostrongylus colubriformis which differed in their susceptibility to benzimidazole anthelmintics and/or in their geographical origin. No intraspecific variation was found in the ITS-2 sequence, indicating that this region of rDNA is inadequate to discriminate between resistant and susceptible populations of T. colubriformis, but it may prove useful for distinguishing between species of Trichoslrongylus. INDEX KEY WORDS: nematode; benzimidazole
Ribosomal resistance.
DNA;
internal
DISEASES due to trichostrongyle nematodes of ruminants are of major animal health and economic importance. Mixed infections with different nematodes (e.g. Trichostrongylus, Ostertagia, Cooperia, Haemonchus and Nematodirus) are common in sheep. It is, however, difficult to differentiate on morphological grounds between the eggs of these species, except for Nematodirus [see Georgi & McCulloch (1989)], and hence time-consuming larval cultures are required for species identification. Developing rapid and sensitive molecular techniques for the specific diagnosis of these infections therefore has major significance for their control. Recent reports have indicated that the second internal transcribed spacer (ITS-2) of the ribosomal DNA may be useful for delineating between closely-related species of parasitic trematodes (Luton, Walker & Blair, 1992; Adlard, Barker, Blair & Cribb, 1993; Michot, Despres, Bonhomme & Bachellerie, 1993), but no comparable studies have been conducted on the ITS2 of parasitic nematodes. However, before speciesspecific DNA probes can be developed it is of paramount importance to establish that there is little
t To whom all correspondence
should be addressed.
transcribed
spacer;
Trichostrongylus colubriformis;
within-species variation in the rDNA sequence. Therefore, in this paper we examine the level of intraspecific variation in the ITS-2 sequence of T. colubriformis, which is one of the most common trichostrongyles in sheep. The three populations of T. colubriformis used in this study originated from disparate geographical areas and differed in their level of resistance to benzimidazole anthelmintics. Nematodes of the “Weybridge strain” from the United Kingdom (LD,, = 0.036 ppm) and the “McMaster strain” from Australia (LD,, = 0.06 ppm) (Lacey, Redwin, Gill, Demargheriti & Waller, 1990) are susceptible to thiabendazole, whereas the population from France is resistant (LD,, = 0.496 ppm) (Hubert & Kerboeuf, 1992). DNA was extracted and purified from a pool of 1520 male worms from each population as described in Gasser, Chilton, Hoste & Beveridge, 1993. The ITS-2 of each sample was amplified and sequenced from both the 5’ and 3’ ends using two oligonucleotide primers (NC 1: S’ACGTCTGGT”TCAGGGTTGTT3 and NC2: STTAGTT”XTTTTCCTCCGCT3’) as described in Gasser et al. (1993). For each sample, at least two sequence gels were run (for 2 and 4 h). The start and end of the ITS-2 region was determined by comparison with the sequence of the
HERVCHOSTE et al.
1070
TABLE I. ITS-2 OF Trichostrongylus colubriformis rDNA* AATGAATTTCTACAGTGTGGCTAACTCTAACACTGTTTGTCGAAT TaqI GGTCATTGTCAAATATTGTGATGATTCCCATTTCAGTTCAAGAAT SSPI Hittf7 AATACATGCAACATGATGTTAATGTTGTAATGACATTAATGTTCC Asel TGTATGATGTGAACGTGTTGTTACTGTTTGAATGTACTCAGTGAA MaelI Rsai TTTGAGATTGATTTAAACAGGGACATGTATAACAATAATGTTCAA DraI TTATCATTTGTAT *Recognition
sites of restriction
45 90 135 180 225 238
enzymes are underlined.
free-living nematode Caenorhabditis elegans (GenBank accession code: X03680). No differences were detected in the ITS-2 sequences of the three T. colubriformis samples irrespective of their geographical origin or level of resistance to benzimidazole anthelmintics. The 238 nucleotide sequence of the ITS-2 of T. colubriformis (Table I) is considerably shorter in size than those of other helminths including C. elegans [383 bases (Ellis, Sulston & Coulson, 1986)], and the parasitic trematodes Fasciola heputica [366 bases (Michot et al., I993)], Schistosoma mansoni, S. haematobium, S. intercalatum and S. japonicum [308-348 bases (Michot ct al., 1993)], but similar to that of the parasitic trematode Dolichosaccus symmetrus [247 bases (Luton et al., 1992)]. The CC content of the ITS-2 sequence of T. colubrijivmis is 3 I. I %, which is considerably lower than that recorded for C. elegans [48% (Ellis et al., 1986)], D. symmetrus [47% (Luton et al., 1992)], F. hepatica [48% (Michot et al., 1993)] and several species of Schistosoma [45- 5 I % (Michot et al.. 1993)]. The ITS-2 sequence of T. colubriformis also has little homology with the free-living nematode C. elegans. Currently, there are no other ITS-2 sequences available for parasitic nematodes to compare with T. colubriformis. Recognition sites for several restriction enzymes, which are 4-6 base cutters, were detected on the ITS-2 sequence of T. colubr~formis (Table I) using the mapping program on GenBank. The significance of these restriction enzymes as a diagnostic tool for distinguishing between species of Trichostrongylus is currently under investigation. The results of the present study clearly demonstrate that the ITS-2 region is not suitable for delineating between resistant and susceptible populations of T. colubriformis. Nevertheless, the apparent lack of intraspecific variation in the sequence of the ITS-2 shows promise for developing species-specific diagnostic tools, providing sufficient
sequence
variation
exists between
different
species of
Trichostrongylus.
Acknowledgements-Dr Hervk Hoste, a visiting scientist at Melbourne University, is a grateful recipient of a Fellowship from the French Ministry of Research and Space Technology. Financial support was provided by the Australian Research Council, Ian Potter Foundation, ANZ Executors and Trustees, Australian Academy of Science and United Film Completions Ltd.
REFERENCES ADLARD R. D., BARKER S. C., BLAIR D. & CRIBB T. H. 1993. Comparison of the second internal transcribed spacer (ribosomal DNA) from populations and species of Fasciolidae (Digenea). International Journal ,for Parasitology 23: 423426. ELLIS R. E., SULSTON J. E. & COULSON A. R. 1986. The rDNA of C. elegans: sequence and structure. Nucleic Acids Research 14: 2345-2364. GASSER R. B.. CHILTON N. B., HOSTE H. & BEVERIDGE1. 1993. Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Research 21: 252552526. GEORGI .I. R. & MCCULLOCH C. E. 1989. Diagnostic morphometry: identification of helminth eggs by discriminant analysis of morphometric data. Proceedings of the Helminthological Society qf Washington 56: 44-57. HUBERT J. & KERBOEUP D. 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Veterinary Record 130: 442446. LACEY E., REDWINJ. M.. GILL J. H., DEMARGHERITIV. M. & WALLER P. J. 1990. A larval development assay for the simultaneous detection of broad spectrum anthelmintic resistance. In: Resistance of Parasites to Antiparasite Drugs (Edited by BORAY J. C., MARTIN P. J. & ROUSH R. T.), pp. 177-184. Round Table Conference, VII
Research
International Congress of Parasitology, Paris, August 1990. LUTON K., WALKER D. & BLAIR D. 1992. Comparisons of ribosomal internal transcribed spacers from two congeneric species of flukes (Platyhelminthes : Trematoda : Digenea).
Note
1071
Molecular and Biochemical Parasitology 56.323-328. MICHOT B., DESPRES L., BONHOMMEF. & BACHELLERIEJ.-P. 1993. Conserved secondary structures in the ITS2 of trematode pre-RNA. Federation qf European Biochemical Sciences Letters 316: 247-252.