Cyathostomins are the
most common and important group of large intestine nematodes, infecting horses
worldwide. The current control strategy is associated with the development of
anthelmintic resistance, which has been reported worldwide. Therefore,
experiments with this family of parasites have become progressively important
to provide their monitoring and control strategies. The aim of the present
study was to propose a faster and more economic assay for isolation of genomic
DNA from the adult stage of Cyathostomin parasites than reported. Adult
parasites were collected from a single horse from a farm in São José dos
Pinhais, PR, Brazil, and were identified. Genomic DNA was isolated from ten
individual female adult parasites using a standardized procedure developed.
Then, extraction from ten individual female was carried out by another DNA
extraction method. DNA concentration from both methods were measured and
compared. We obtained a good DNA quality with this standardized procedure. As a
result of this analysis, we propose a modified phenol-chloroform method, which
will contribute to assays that require DNA extraction from adult worms for
genomic DNA sequences of cyathostomin, or species-specific identification.
D., Molento, M.B., 2013. Lack of Cyathostomin sp. reduction after anthelmintic treatment in horses in Brazil. Veterinary Parasitology 194, 35-39.
Chagas, A.C.S., Niciura, S.C.M., Molento, M.B., 2011. Manual Prático: metodologias de diagnóstico da resistência e de detecção de substâncias ativas em parasitas de ruminantes. Embrapa Informação Tecnológica, Brasília, D.F., p. 153.
Fontenele, J.A., Freitas, E.P., Santos, J.M.L., Bezerra, A.C.D.S., Bevilaqua, C.M.L., Vieira, L. da S., Monteiro, J.P., 2013. Extração de DNA de Haemonchus Contortus In: Hispanic Biotechnology of Northeast Symposium, Parnaíba, Brazil, p. 649.
Kaplan, R.M., Vidyashankar, A.N., 2012. An inconvenient truth: Global warming and anthelmintic resistance. Veterinary Parasitology 186, 70-78.
Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infection. Veterinary Parasitology 85, 113-122.
Lyons, E.T., Tolliver, S.C., Drudge, J.H., 1999. Historical perspective of cyathostomes: prevalence, treatment and control programs. Veterinary Parasitology 85, 97-112.
Matthews, A.G., Morris, J.R., 1995. Cyathostomiasis in horses. Veterinary Record 136, 52.
Molento, M.B., Antunes, J., Bentes, R.N., Coles, G.C., 2008. Anthelmintic resistant nematodes in Brazilian horses. Veterinary Record 162, 384-385.
Molento, M.B., Nielsen, M.K., Kaplan, R.M., 2012. Resistance to avermectin/milbemycin anthelmintics in equine cyathostomins - Current situation. Veterinary Parasitology 185, 16-24.
Peregrine, A.S., Molento, M.B., Kaplan, R.M., Nielsen, M.K., 2014. Anthelmintic resistance in important parasites of horses: Does it really matter? Veterinary Parasitology 201, 1-8.
Tandon, R., Lyons, E.T., Tolliver, S.C., Kaplan, R.M., 2005. Effect of moxidectin selection on the genetic variation within Cylicocyclus nassatus based on amplified fragment length polymorphism (AFLP). International Journal of Parasitology 35, 813-819.
Tolliver, S.C., 2000. A practical method of identification of the North American cyathostomes (small strongyles) in equids in Kentucky. University of Kentucky, Department of Veterinary Science.
Truett, G.E., Heeger, P., Mynatt, R.L., Truett, A.A., Walker, J.A., Warman, M.L., 2000. Preparation of PCR-Quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques. 29, 52-54.
Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites
Year 2017,
Volume: 43 Issue: 2, 146 - 148, 09.06.2017
Cyathostomins are the most common and important group of large intestine nematodes, infecting horses worldwide. The current control strategy is associated with the development of anthelmintic resistance, which has been reported worldwide. Therefore, experiments with this family of parasites have become progressively important to provide their monitoring and control strategies. The aim of the present study was to propose a faster and more economic assay for isolation of genomic DNA from the adult stage of Cyathostomin parasites than reported. Adult parasites were collected from a single horse from a farm in São José dos Pinhais, PR, Brazil, and were identified. Genomic DNA was isolated from ten individual female adult parasites using a standardized procedure developed. Then, extraction from ten individual female was carried out by another DNA extraction method. DNA concentration from both methods were measured and compared. We obtained a good DNA quality with this standardized procedure. As a result of this analysis, we propose a modified phenol-chloroform method, which will contribute to assays that require DNA extraction from adult worms for genomic DNA sequences of cyathostomin, or species-specific identification.
D., Molento, M.B., 2013. Lack of Cyathostomin sp. reduction after anthelmintic treatment in horses in Brazil. Veterinary Parasitology 194, 35-39.
Chagas, A.C.S., Niciura, S.C.M., Molento, M.B., 2011. Manual Prático: metodologias de diagnóstico da resistência e de detecção de substâncias ativas em parasitas de ruminantes. Embrapa Informação Tecnológica, Brasília, D.F., p. 153.
Fontenele, J.A., Freitas, E.P., Santos, J.M.L., Bezerra, A.C.D.S., Bevilaqua, C.M.L., Vieira, L. da S., Monteiro, J.P., 2013. Extração de DNA de Haemonchus Contortus In: Hispanic Biotechnology of Northeast Symposium, Parnaíba, Brazil, p. 649.
Kaplan, R.M., Vidyashankar, A.N., 2012. An inconvenient truth: Global warming and anthelmintic resistance. Veterinary Parasitology 186, 70-78.
Love, S., Murphy, D., Mellor, D., 1999. Pathogenicity of cyathostome infection. Veterinary Parasitology 85, 113-122.
Lyons, E.T., Tolliver, S.C., Drudge, J.H., 1999. Historical perspective of cyathostomes: prevalence, treatment and control programs. Veterinary Parasitology 85, 97-112.
Matthews, A.G., Morris, J.R., 1995. Cyathostomiasis in horses. Veterinary Record 136, 52.
Molento, M.B., Antunes, J., Bentes, R.N., Coles, G.C., 2008. Anthelmintic resistant nematodes in Brazilian horses. Veterinary Record 162, 384-385.
Molento, M.B., Nielsen, M.K., Kaplan, R.M., 2012. Resistance to avermectin/milbemycin anthelmintics in equine cyathostomins - Current situation. Veterinary Parasitology 185, 16-24.
Peregrine, A.S., Molento, M.B., Kaplan, R.M., Nielsen, M.K., 2014. Anthelmintic resistance in important parasites of horses: Does it really matter? Veterinary Parasitology 201, 1-8.
Tandon, R., Lyons, E.T., Tolliver, S.C., Kaplan, R.M., 2005. Effect of moxidectin selection on the genetic variation within Cylicocyclus nassatus based on amplified fragment length polymorphism (AFLP). International Journal of Parasitology 35, 813-819.
Tolliver, S.C., 2000. A practical method of identification of the North American cyathostomes (small strongyles) in equids in Kentucky. University of Kentucky, Department of Veterinary Science.
Truett, G.E., Heeger, P., Mynatt, R.L., Truett, A.A., Walker, J.A., Warman, M.L., 2000. Preparation of PCR-Quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques. 29, 52-54.
Ishii, J. B., Arenal, A., Chicoy, Y., Molento, M. B. (2017). Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites. İstanbul Üniversitesi Veteriner Fakültesi Dergisi, 43(2), 146-148. https://doi.org/10.16988/iuvfd.320203
AMA
Ishii JB, Arenal A, Chicoy Y, Molento MB. Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites. iuvfd. July 2017;43(2):146-148. doi:10.16988/iuvfd.320203
Chicago
Ishii, Juliana Bana, Amilcar Arenal, Yanise Chicoy, and Marcelo Beltrão Molento. “Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites”. İstanbul Üniversitesi Veteriner Fakültesi Dergisi 43, no. 2 (July 2017): 146-48. https://doi.org/10.16988/iuvfd.320203.
EndNote
Ishii JB, Arenal A, Chicoy Y, Molento MB (July 1, 2017) Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites. İstanbul Üniversitesi Veteriner Fakültesi Dergisi 43 2 146–148.
IEEE
J. B. Ishii, A. Arenal, Y. Chicoy, and M. B. Molento, “Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites”, iuvfd, vol. 43, no. 2, pp. 146–148, 2017, doi: 10.16988/iuvfd.320203.
ISNAD
Ishii, Juliana Bana et al. “Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites”. İstanbul Üniversitesi Veteriner Fakültesi Dergisi 43/2 (July 2017), 146-148. https://doi.org/10.16988/iuvfd.320203.
JAMA
Ishii JB, Arenal A, Chicoy Y, Molento MB. Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites. iuvfd. 2017;43:146–148.
MLA
Ishii, Juliana Bana et al. “Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites”. İstanbul Üniversitesi Veteriner Fakültesi Dergisi, vol. 43, no. 2, 2017, pp. 146-8, doi:10.16988/iuvfd.320203.
Vancouver
Ishii JB, Arenal A, Chicoy Y, Molento MB. Comparison of Two Methods for the Isolation of Genomic DNA from Cyathostomin Adult Parasites. iuvfd. 2017;43(2):146-8.