Hostname: page-component-78c5997874-fbnjt Total loading time: 0 Render date: 2024-11-10T12:54:44.610Z Has data issue: false hasContentIssue false
  • English
  • Français

New insights into sequence variation in the IGS region of 21 cyathostomin species and the implication for molecular identification

Published online by Cambridge University Press:  13 April 2012

K. CWIKLINSKI ,
F. N. J. KOOYMAN ,
D. C. K. VAN DOORN ,
J. B. MATTHEWS  and
J. E. HODGKINSON
K. CWIKLINSKI*
Affiliation:
Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L69 7ZJ, UK
F. N. J. KOOYMAN
Affiliation:
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands
D. C. K. VAN DOORN
Affiliation:
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands
J. B. MATTHEWS
Affiliation:
Moredun Research Institute, Penicuik, Midlothian EH26 0PZ, Scotland
J. E. HODGKINSON
Affiliation:
Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L69 7ZJ, UK
*
*Corresponding author: Veterinary Parasitology, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool L69 7ZJ, UK. Tel: +44 (0)151 7941586. Fax: +44 (0)151 7941519. E-mail: K.Cwiklinski@liverpool.ac.uk
Get access Rights & Permissions [Opens in a new window]

Summary

Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3–62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means†.

Keywords

cyathostominsIGSintra-specificinter-specificRLB hybridization
Type
Research Article
Information
Parasitology , Volume 139 , Issue 8 , July 2012 , pp. 1063 - 1073
Copyright
Copyright © Cambridge University Press 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Appels, R. and Dvorak, J. (1982). Relative rates of divergence of spacer and gene sequences within the rDNA region of species in the Triticeae: implications for the maintenance of homogeneity of a repeated gene family. Theoretical and Applied Genetics 63, 361–365. doi: 10.1007/BF00303907.Google Scholar
Baker, S. M. and Platt, T. (1986). Pol I transcription: which comes first, the end or the beginning? Cell 47, 839–840. doi: 10.1016/0092-8674(86)90795-6.CrossRefGoogle ScholarPubMed
Bucknell, D. G., Gasser, R. B. and Beveridge, I. (1995). The prevalence and epidemiology of gastrointestinal parasites of horses in Victoria, Australia. International Journal for Parasitology 25, 711–724. doi: 10.1016/0020-7519(94)00214-9.CrossRefGoogle ScholarPubMed
Cernanská, D., Paoletti, B., Kralova-Hromadova, I., Iorio, R., Cudekova, P., Milillo, P. and Traversa, D. (2009). Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic. Veterinary Parasitology 160, 171–174. doi: 10.1016/j.vetpar.2008.10.078.Google Scholar
Chapman, M. R., French, D. D. and Klei, T. R. (2002). Gastrointestinal helminths of ponies in Louisiana: a comparison of species currently prevalent with those present 20 years ago. The Journal of Parasitology 88, 1130–1134. doi: 10.1645/0022-3395(2002)088[1130:GHOPIL]2.0.CO;2.CrossRefGoogle ScholarPubMed
De Winter, R. F. and Moss, T. (1986). The ribosomal spacer in Xenopus laevis is transcribed as part of the primary ribosomal RNA. Nucleic Acids Research 14, 6041–6051. doi: 10.1093/nar/14.15.6041.Google Scholar
Elder, J. J. and Turner, B. J. (1995). Concerted evolution of repetitive DNA sequences in eukaryotes. The Quarterly Review of Biology 70, 297–320.Google Scholar
Eysker, M. and Pandey, V. S. (1989). Small strongyle infections in donkeys from the Highveld in Zimbabwe. Veterinary Parasitology 30, 345–349. doi: 10.1016/0304-4017(89)90104-0.Google Scholar
Flavell, R. B., O'Dell, M., Thompson, W. F., Vincentz, M., Sardana, R. and Barker, R. F. (1986). The differential expression of ribosomal RNA genes. Philosophical Transactions of the Royal Society of London, B 314, 385–397. doi: 10.1098/rstb.1986.0060.Google Scholar
Gasser, R. B. and Newton, S. E. (2000). Genomic and genetic research on bursate nematodes: significance, implications and prospects. International Journal for Parasitology 30, 509–534. doi: 10.1016/S0020-7519(00)00021-7.Google Scholar
Giles, C. J., Urquhart, K. A. and Longstaffe, J. A. (1985). Larval cyathostomiasis (immature trichonema-induced enteropathy): a report of 15 clinical cases. Equine Veterinary Journal 17, 196–201. doi: 10.1111/j.2042-3306.1985.tb02469.x.Google Scholar
Harrington, C. A. and Chikaraishi, D. M. (1987). Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene. Molecular and Cellular Biology 7, 314–325. doi: 10.1128/MCB.7.1.314.Google ScholarPubMed
Hodgkinson, J. E., Freeman, K. L., Lichtenfels, J. R., Palfreman, S., Love, S. and Matthews, J. B. (2005). Identification of strongyle eggs from anthelmintic-treated horses using a PCR-ELISA based on intergenic DNA sequences. Parasitology Research 95, 287–292. doi: 10.1007/s00436-004-1289-z.CrossRefGoogle ScholarPubMed
Hodgkinson, J. E., Lichtenfels, J. R., Mair, T. S., Cripps, P., Freeman, K. L., Ramsey, Y. H., Love, S. and Matthews, J. B. (2003). A PCR-ELISA for the identification of cyathostomin fourth-stage larvae from clinical cases of larval cyathostominosis. International Journal for Parasitology 33, 1427–1435. doi: 10.1016/S0020-7519(03)00140-1.Google Scholar
Hodgkinson, J. E., Love, S., Lichtenfels, J. R., Palfreman, S., Ramsey, Y. H. and Matthews, J. B. (2001). Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses. International Journal for Parasitology 31, 197–204. doi: 10.1016/S0020-7519(00)00161-2.Google Scholar
Hung, G. C., Gasser, R. B., Beveridge, I., and Chilton, N. B. (1999 b). Species-specific amplification by PCR of ribosomal DNA from some equine strongyles. Parasitology 119, 69–80.Google Scholar
Hung, G. C., Chilton, N. B., Beveridge, I., Zhu, X. Q., Lichtenfels, J. R. and Gasser, R. B. (1999 a). Molecular evidence for cryptic species within Cylicostephanus minutus (Nematoda: Strongylidae). International Journal for Parasitology 29, 285–291. doi: 10.1016/S0020-7519(98)00203-3.Google Scholar
Ionita, M., Howe, D. K., Lyons, E. T., Tolliver, S. C., Kaplan, R. M., Mitrea, I. L. and Yeargan, M. (2010). Use of a reverse line blot assay to survey small strongyle (Strongylida: Cyathostominae) populations in horses before and after treatment with ivermectin. Veterinary Parasitology 168, 332–337. doi: 10.1016/j.vetpar.2009.11.021.Google Scholar
Kaye, J. N., Love, S., Lichtenfels, J. R. and McKeand, J. B. (1998). Comparative sequence analysis of the intergenic spacer region of cyathostome species. International Journal for Parasitology 28, 831–836. doi: 10.1016/S0020-7519(98)00031-9.Google Scholar
Kharchenko, V., Kuzmina, T., Trawford, A., Getachew, M. and Feseha, G. (2009). Morphology and diagnosis of some fourth-stage larvae of cyathostomines (Nematoda: Strongyloidea) in donkeys Equus asinus L. from Ethiopia. Systematic Parasitology 72, 1–13. doi: 10.1007/s11230-008-9152-8.Google Scholar
Kornaś, S., Cabaret, J., Skalska, M. and Nowosad, B. (2010). Horse infection with intestinal helminths in relation to age, sex, access to grass and farm system. Veterinary Parasitology 174, 285–291. doi: 10.1016/j.vetpar.2010.09.007.CrossRefGoogle ScholarPubMed
Labhart, P. and Reeder, R. H. (1986). Characterization of three sites of RNA 3′ end formation in the Xenopus ribosomal gene spacer. Cell 45, 431–443. doi: 10.1016/0092-8674(86)90329-6.Google Scholar
Lichtenfels, J. R., Kharchenko, V. A. and Dvojnos, G. M. (2008). Illustrated identification keys to strongylid parasites (Strongylidae: Nematoda) of horses, zebras and asses (Equidae). Veterinary Parasitology 156, 4–161. doi: 10.1016/j.vetpar.2008.04.026.Google Scholar
Lichtenfels, J. R., Kharchenko, V. A., Krecek, R. C. and Gibbons, L. M. (1998). An annotated checklist by genus and species of 93 species level names for 51 recognized species of small strongyles (Nematoda: Strongyloidea: Cyathostominea) of horses, asses and zebras of the world. Veterinary Parasitology 79, 65–79. doi: 10.1016/S0304-4017(98)00149-6.Google Scholar
Love, S., Murphy, D. and Mellor, D. (1999). Pathogenicity of cyathostome infection. Veterinary Parasitology 85, 113–121; discussion 121–2, 215–25. doi: 10.1016/S0304-4017(99)00092-8.Google Scholar
Lyons, E. T., Tolliver, S. C. and Drudge, J. H. (1999). Historical perspective of cyathostomes: prevalence, treatment and control programs. Veterinary Parasitology 85, 97–111; discussion 111–2, 215–25. doi: 10.1016/S0304-4017(99)00091-6.Google Scholar
Lyons, E. T., Tolliver, S. C., Drudge, J. H., Stamper, S., Swerczek, T. W. and Granstrom, D. E. (1996). A study (1977–1992) of population dynamics of endoparasites featuring benzimidazole-resistant small strongyles (population S) in Shetland ponies. Veterinary Parasitology 66, 75–86. doi: 10.1016/S0304-4017(96)00998-3.CrossRefGoogle Scholar
Matthee, S., Krecek, R. C. and Gibbons, L. M. (2002). Cylicocyclus asini n.sp. (Nematoda: Cyathostominae) from donkeys Equus asinus in South Africa. Systematic Parasitology 51, 29–35. doi: 10.1023/A:1012989810087.CrossRefGoogle ScholarPubMed
Matthee, S., Krecek, R. C. and McGeoch, M. A. (2004). A comparison of the intestinal helminth communities of Equidae in Southern Africa. Journal of Parasitology 90, 1263–1273. doi: 10.1645/GE-3353.Google Scholar
Matthee, S., Krecek, R. C. and Milne, S. A. (2000). Prevalence and biodiversity of helminth parasites in donkeys from South Africa. Journal of Parasitology 86, 756–762. doi: 10.1645/0022-3395(2000)086[-756:PABOHP]2.0.CO;2.Google Scholar
Moss, T. and Stefanovsky, V. Y. (1995). Promotion and regulation of ribosomal transcription in eukaryotes by RNA polymerase I. Progress in Nucleic Acid Research and Molecular Biology 50, 25–66. doi: 10.1016/S0079-6603(08)60810-7.Google Scholar
Ogbourne, C. P. (1976). The prevalence, relative abundance and site distribution of nematodes of the subfamily Cyathostominae in horses killed in Britain. Journal of Helminthology 50, 203–214. doi: 10.1017/S0022149X00027760.Google Scholar
Reid, S. W., Mair, T. S., Hillyer, M. H. and Love, S. (1995). Epidemiological risk factors associated with a diagnosis of clinical cyathostomiasis in the horse. Equine Veterinary Journal 27, 127–130. doi: 10.1111/j.2042-3306.1995.tb03048.x.CrossRefGoogle ScholarPubMed
Traversa, D., Iorio, R., Klei, T. R., Kharchenko, V. A., Gawor, J., Otranto, D. and Sparagano, O. A. (2007). New method for simultaneous species-specific identification of equine strongyles (Nematoda, Strongylida) by reverse line blot hybridization. Journal of Clinical Microbiology 45, 2937–2942. doi: 10.1128/JCM.00714-07.Google Scholar
Traversa, D., Iorio, R., Otranto, D., Giangaspero, A., Milillo, P. and Klei, T. R. (2009). Species-specific identification of equine cyathostomes resistant to fenbendazole and susceptible to oxibendazole and moxidectin by macroarray probing. Experimental Parasitology 121, 92–95. doi: 10.1016/j.exppara.2008.10.001.Google Scholar
Traversa, D., Milillo, P., Barnes, H., Von Samson-Himmelstjerna, G., Schurmann, S., Demeler, J., Otranto, D., Lia, R. P., Perrucci, S., Frangipane Di Regalbono, A., Beraldo, P., Amodie, D., Rohn, K., Cobb, R. and Boeckh, A. (2010). Distribution and species-specific occurrence of cyathostomins (Nematoda, Strongylida) in naturally infected horses from Italy, United Kingdom and Germany. Veterinary Parasitology 168, 84–92. doi: 10.1016/j.vetpar.2009.10.006.Google Scholar
van Doorn, D. C., Kooyman, F. N., Eysker, M., Hodgkinson, J. E., Wagenaar, J. A. and Ploeger, H. W. (2010). In vitro selection and differentiation of ivermectin resistant cyathostomin larvae. Veterinary Parasitology 174, 292–299. doi: 10.1016/j.vetpar.2010.08.021.Google Scholar
van Loon, G., Deprez, P., Muylle, E. and Sustronck, B. (1995). Larval cyathostomiasis as a cause of death in two regularly dewormed horses. Zentralblatt für Veterinarmedizin.Reihe A 42, 301–306. doi: 10.1111/j.1439-0442.1995.tb00381.Google Scholar