vniigisРоссийский паразитологический журналRussian Journal of Parasitology1998-84352541-7843ВНИИП – филиал ФГБНУ ФНЦ ВИЭВ РАН10.31016/1998-8435-2024-18-3-264-273vniigis-1199Research ArticleБИОХИМИЯ, БИОТЕХНОЛОГИЯ И ДИАГНОСТИКАBIOCHEMISTRY, BIOTECHNOLOGY AND DIAGNOSTICSВозможности использования «вложенной» ПЦР-ПДРФ для таксономической идентификации личинок L3 семейства Trichostrongylidae, Leiper, 1912Possibilities of using nested PCR-RFLP for taxonomic identification of L3 larvae of the family Trichostrongylidae, Leiper, 1912https://orcid.org/0009-0001-8712-6073ПименовИ. А.PimenovI. A.

Пименов Илья Александрович, аспирант

117218 Москва, ул. Б. Черемушкинская, 28

Pimenov Ilya A., Postgraduate Student

28, Bolshaya Cheremushkinskaya st., Moscow, 117218

mr.pimenov123@yandex.ruhttps://orcid.org/0000-0002-3644-5592ОдоевскаяИ. М.OdoevskayaI. M.

Одоевская Ирина Михайловна, кандидат биологических наук

117218 Москва, ул. Б. Черемушкинская, 28

Odoevskaya Irina M., Candidate of Biological Sciences

28, Bolshaya Cheremushkinskaya st., Moscow, 117218

odoevskayaim@rambler.ruПлиеваА. М.PlievaA. M.

Плиева Айшет Магомедовна, доктор биологических наук, член-корреспондент МАНЭБ

386001, Республика Ингушетия, г. Магас, пр-кт И. Б. Зязикова, 7

Plieva Aishet M., Doctor of Biological Sciences, Corresponding Member of the International Academy of Ecology & Life Protection Sciences

I. B. Zyazikova Ave., Magas, Republic of Ingushetia, 386001

aishet57@mail.ruhttps://orcid.org/0000-0001-8364-5055ВарламоваА. И.VarlamovaA. I.

Варламова Анастасия Ивановна, доктор биологических наук

117218 Москва, ул. Б. Черемушкинская, 28

Varlamova Anastasiya I., Doctor of Biological Sciences

28, Bolshaya Cheremushkinskaya st., Moscow, 117218

arsphoeb@mail.ruВсероссийский научно-исследовательский институт фундаментальной и прикладной паразитологии животных и растений – филиал Федерального государственного бюджетного научного учреждения «Федеральный научный центр – Всероссийский научно-исследовательский институт экспериментальной ветеринарии имени К. И. Скрябина и Я. Р. Коваленко Российской академии наук» (ВНИИП – фил. ФГБНУ ФНЦ ВИЭВ РАН)РоссияAll-Russian Scientific Research Institute for Fundamental and Applied Parasitology of Animals and Plant – a branch of the Federal State Budget Scientific Institution “Federal Scientific Centre VIEV” (VNIIP – FSC VIEV)Russian FederationФГБОУ ВО «Ингушский государственный университет»ГДРFSBEI HE Ingush State UniversityGDR202408092024183264273Copyright © Пименов И.А., Одоевская И.М., Плиева А.М., Варламова А.И., 20242024Пименов И.А., Одоевская И.М., Плиева А.М., Варламова А.И.Pimenov I.A., Odoevskaya I.M., Plieva A.M., Varlamova A.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vniigis.elpub.ru/jour/article/view/1199

Цель исследования – применить молекулярно-генетические методы исследований для выявления таксономической принадлежности паразитических нематод желудочно-кишечного тракта овец сем. Trichostrongylidae, используя «вложенную» ПЦР с последующим анализом полиморфизма длин рестрикционных фрагментов (ПДРФ).Материалы и методы. Паразитические нематоды: личинки стронгилят L3, полученные в результате инкубирования проб фекалий овец. Выделение геномной ДНК проводили с использованием коммерческого набора для экстракции ДНК из микроколичеств тканей (фирма «Синтол», Москва), согласно рекомендациям производителя. Для амплификации ДНК использовали термоциклер T-100 Bio-Rad и коммерческий набор реактивов Master Mix, Евроген. Режим проведения ПЦР осуществляли согласно руководству WAAVP, 2006. Реакцию рестрикции эндонуклеазой RsaI амплифицированных фрагментов трихостронгилид проводили согласно рекомендациям фирмы-производителя фермента («Сибэнзим», г. Новосибирск).Результаты и обсуждение. Для определения таксономической принадлежности личинок стронгилят, выделенных после инкубации фекалий, полученных от овец, были проведены молекулярно-генетические исследования методом «вложенной» ПЦР с последующим анализом полиморфизма длин рестрикционных фрагментов (ПДРФ). Данный метод дает возможность с наименьшими трудозатратами идентифицировать генотипы трех видов стронгилят: Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta на стадии личинки.

The purpose of the research is to apply molecular genetic research methods to identify the taxonomic affiliation of gastrointestinal parasitic sheep nematodes of the family Trichostrongylidae using nested PCR followed by the restriction fragment length polymorphism (RFLP) analysis.Materials and methods. Parasitic nematodes, L3 Strongylata larvae obtained from incubated fecal samples of sheep. The genomic DNA was isolated using a commercial kit for DNA extraction from micro-quantities of tissues (Synthol, Moscow) as per the manufacturer’s guidelines. For DNA amplification, a T-100 Bio-Rad thermal cycler and a commercial Eurogen Master Mix reagent kit were used. The PCR regime was performed according to the WAAVP guidelines, 2006. The restriction endonuclease Rsa I of amplified Trichostrongylidae fragments was performed according to guidelines of the enzyme manufacturer (Sibenzyme, Novosibirsk).Results and discussion. To determine the taxonomic affiliation of Strongylata larvae isolated after incubation of feces from sheep, molecular genetic studies were performed using nested PCR followed by the restriction fragment length polymorphism (RFLP) analysis. This method makes it possible to identify, with the least effort, the genotypes of three species of Strongylata Haemonchus contortus, Trichostrongylus colubriformis, and Teladorsagia circumcincta at the larval stage.

Teladorsagia circumcinctaHaemonchus contortusTrichostrongylus colubriformisпаразитические нематодытрихостронгилидыПЦР-ПДРФрестриктазаTeladorsagia circumcinctaHaemonchus contortusTrichostrongylus colubriformisparasitic nematodesTrichostrongylidaePCR-RFLPrestriction enzymeИсследование выполнено при финансовой поддержке Гранта РНФ №23-26-00220.The study was conducted with financial support from the Russian Science Foundation Grant No. 23- 26-00220.ReferencesИвашкин В. М., Орипов А. О., Сонин М. Д. Определитель гельминтов мелкого рогатого скота. М.: Наука, 1989. 255 с.Ivashkin V. M., Oripov A. O., Sonin M. D. Identification guide to helminths of small ruminants. M.: Science, 1989; 255. (In Russ.)Никитин В. Ф., Павласек И. Aвторское свидетельство CCCР № 1095908, кп. A 61 В 10/10, 1981. SU 1 391 625 A1Nikitin V. F., Pavlasek I., Copyright certificate of the USSR No. 1095908, pc. A 61 В 10/10, 1981. SU 1 391 625 A1Пименов И. А., Кузнецов Д. Н., Одоевская И. М., Афанасьев А. Д., Варламова А. И., Архипов И. А. К фауне нематод пищеварительного тракта овец в Европейской части России // Российский паразитологический журнал. 2023. Т. 17. № 2. С. 206–213. https://doi.org/10.31016/1998-8435-2023-17-2-206-213Pimenov I. A., Kuznetsov D. N., Odoevskaya I. M., Afanasyev A. D., Varlamova A. I., Arkhipov I. A. To the fauna of gastrointestinal nematodes of sheep in the European part of Russia. Rossiyskiy parazitologicheskiy zhurnal = Russian Journal of Parasitology. 2023; 17 (2): 206–213. (In Russ.) https:// doi.org/10.31016/1998-8435-2023-17-2-206-213Álvarez-Sánchez M. A., Pérez-García J., Cruz-Rojo M. A., Rojo-Vázquez F. A. Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep. Veterinary Parasitology. 2005; 129 (3–4): 291-298. https://doi.org/10.1016/j.vetpar.2005.02.004Álvarez-Sánchez M. A., Pérez-García J., Cruz-Rojo M. A., Rojo-Vázquez F. A. Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep. Veterinary Parasitology. 2005; 129 (3–4): 291-298. https://doi.org/10.1016/j.vetpar.2005.02.004Baltrušis A., Claude L. Halvarsson P., Mikko S., Höglund J. Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus. International Journal for Parasitology: Drugs and Drug Resistance. 2021; 15. 168-176. https://doi.org/10.1016/j.ijpddr.2021.03.002Baltrušis A., Claude L. Halvarsson P., Mikko S., Höglund J. Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus. International Journal for Parasitology: Drugs and Drug Resistance. 2021; 15. 168-176. https://doi.org/10.1016/j.ijpddr.2021.03.002Baltrušis A., Halvarsson P., Höglund J. Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR. International Journal for Parasitology: Drugs and Drug Resistance. 2018; 8 (3): 411-419. https://doi.org/10.1016/j.ijpddr.2018.09.003Baltrušis A., Halvarsson P., Höglund J., Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR. International Journal for Parasitology: Drugs and Drug Resistance. 2018; 8 (3): 411-419. https:// doi.org/10.1016/j.ijpddr.2018.09.003Charlier J., Rinaldi L., Musella V., Ploeger H. W., Chartier C., Vineer H. R., Hinney B., Samson-Himmelstjerna G., Ba ̆cescu B., Mickiewicz M. Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe. Preventive Veterinary Medicine. 2020; 182. 105103. https://doi.org/10.1016/j.prevetmed.2020.105103Charlier J., Rinaldi L., Musella V., Ploeger H. W., Chartier C., Vineer H. R., Hinney B., Samson-Himmelstjerna G., Ba ̆cescu B., Mickiewicz M. Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe. Preventive Veterinary Medicine. 2020; 182. 105103. https://doi.org/10.1016/j.prevetmed.2020.105103Coles G. C., Jackson F., Pomroy W. E., Prichard R. K., von Samson-Himmelstjerna G., Silvestre A., Taylor M. A., Vercruysse J. The detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary Parasitology. 2006; 31; 136 (3-4): 167-185. https://doi.org/10.1016/j.vetpar.2005.11.019Coles G. C., Jackson F., Pomroy W. E., Prichard R. K., von Samson-Himmelstjerna G., Silvestre A., Taylor M. A., Vercruysse J. The detection of anthelmintic resistance in nematodes of veterinary importance. Veterinary Parasitology. 2006; 31; 136 (3-4): 167- 185. https://doi.org/10.1016/j.vetpar.2005.11.019Dilks C. M., Hahnel S. R., Sheng Q., Long L., McGrath P. T., Andersen E. C. Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta- tubulin alleles. International Journal for Parasitology: Drugs and Drug Resistance. 2020; 14. 28–36. https://doi.org/10.1016/j.ijpddr.2020.08.003Dilks C. M., Hahnel S. R., Sheng Q., Long L., McGrath P. T., Andersen E. C. Quantitative benzimidazole resistance and fitness effects of parasitic nematode beta- tubulin alleles. International Journal for Parasitology: Drugs and Drug Resistance. 2020; 14. 28–36. https://doi.org/10.1016/j.ijpddr.2020.08.003Dobson R. J., Hosking B. C., Jacobson C. L., Cotter J. L., Besier R. B., Stein P. A., Reid S. A. Preserving new anthelmintics: a simple method for estimating faecal egg count reduction test (FECRT) confidence limits when efficacy and/or nematode aggregation is high. Veterinary Parasitology. 2012; 186. 79–92. https://doi.org/10.1016/j.vetpar.2011.11.049Dobson R. J., Hosking B. C., Jacobson C. L., Cotter J. L., Besier R. B., Stein P. A., Reid S. A. Preserving new anthelmintics: a simple method for estimating faecal egg count reduction test (FECRT) confidence limits when efficacy and/or nematode aggregation is high. Veterinary Parasitology. 2012; 186. 79–92. https://doi.org/10.1016/j.vetpar.2011.11.049Gasser R. B. PCR-based technology in veterinary parasitology. Veterinary Parasitology. 1999. 84. 229–258. https://doi.org/10.1016/s0304-4017(99)00036-9Gasser R. B. PCR-based technology in veterinary parasitology. Veterinary Parasitology. 1999. 84. 229–258. https://doi.org/10.1016/s0304-4017(99)00036-9Hinney B., Wiedermann S., Bosco A., Rinaldi L., Hofer M., Joachim A., Krücken J., Steinborn R. Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus. International Journal for Parasitology: Drugs and Drug Resistance. 2023; 22. 88-95. https://doi.org/10.1016/j.ijpddr.2023.06.001Hinney B., Wiedermann S., Bosco A., Rinaldi L., Hofer M., Joachim A., Krücken J., Steinborn R. Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus. International Journal for Parasitology: Drugs and Drug Resistance. 2023; 22. 88-95. https://doi.org/10.1016/j.ijpddr.2023.06.001Humbert J. F., Elard L. A simple PCR method for rapidly detecting defined point mutations. Journal: Technical Tips Online. 1997; ISSN: 1366-2120.Humbert J. F., Elard L. A simple PCR method for rapidly detecting defined point mutations. Journal: Technical Tips Online. 1997; ISSN: 1366-2120.Kaplan R. M. Biology, epidemiology, diagnosis, and management of anthelmintic resistance in gastrointestinal nematodes of livestock. Veterinary Clinics: Food Animal Practice. 2020; 36 (1): 17–30. https://doi.org/10.1016/j.cvfa.2019.12.001Kaplan R. M. Biology, epidemiology, diagnosis, and management of anthelmintic resistance in gastrointestinal nematodes of livestock. Veterinary Clinics: Food Animal Practice. 2020; 36 (1): 17–30. https://doi.org/10.1016/j.cvfa.2019.12.001Kaplan R. M., Denwood M. J., Nielsen M. K., Thamsborg S. M., Torgerson P. R., Gilleard J. S., Dobson R. J., Vercruysse J., Levecke B. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine. Veterinary Parasitology. 2023; 318. https://doi.org/10.1016/j.vetpar.2023.109936Kaplan R. M., Denwood M. J., Nielsen M. K., Thamsborg S. M., Torgerson P. R., Gilleard J. S., Dobson R. J., Vercruysse J., Levecke B. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) guideline for diagnosing anthelmintic resistance using the faecal egg count reduction test in ruminants, horses and swine. Veterinary Parasitology. 2023; 318. https://doi.org/10.1016/j.vetpar.2023.109936Kotze A. C., Gilleard J. S., Doyle S. R., Prichard R. K. Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance. International Journal for Parasitology: Drugs and Drug Resistance. 2020; 14. 264–273. https://doi.org/10.1016/j.ijpddr.2020.11.005Kotze A. C., Gilleard J. S., Doyle S. R., Prichard R. K. Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance. International Journal for Parasitology: Drugs and Drug Resistance. 2020; 14. 264–273. https://doi.org/10.1016/j.ijpddr.2020.11.005Kotze A. C., Prichard R. Anthelmintic resistance in Haemonchus contortus: history, mechanisms and diagnosis. Advances in Parasitology. 2016; 93. 397–428. https://doi.org/10.1016/bs.apar.2016.02.012Kotze A. C., Prichard R. Anthelmintic resistance in Haemonchus contortus: history, mechanisms and diagnosis. Advances in Parasitology. 2016; 93. 397–428. https://doi.org/10.1016/bs.apar.2016.02.012Lancaster M. B., Hong C. Differentiation of third stage larvae of “ovine Ostertagia” type and Trichostrongylus species. The Veterinary Record. 1987; 120. 503. http://doi.org/10.1136/vr.120.21.503, PMid:3604011Lancaster M. B., Hong C. Differentiation of third stage larvae of “ovine Ostertagia” type and Trichostrongylus species. The Veterinary Record. 1987; 120. 503. http://doi.org/10.1136/vr.120.21.503, PMid:3604011O’Callaghan M. G. Observations on the sheath extension of the third stage, infective larvae of Trichostrongylus rugatus. Veterinary Parasitology. 2004; 126. 397–402. http://doi.org/10.1016/j.vetpar.2004.07.027O’Callaghan M. G. Observations on the sheath extension of the third stage, infective larvae of Trichostrongylus rugatus. Veterinary Parasitology. 2004; 126. 397–402. http://doi.org/10.1016/j.vetpar.2004.07.027Prichard R. Application of molecular biology in veterinary parasitology. Veterinary Parasitology. 1997; 71. 155–175. https://doi.org/10.1016/s0304-4017(97)00029-0Prichard R. Application of molecular biology in veterinary parasitology. Veterinary Parasitology. 1997; 71. 155–175. https://doi.org/10.1016/s0304-4017(97)00029-0Rajagopal A., Sabu L., Radhika R., Devada K., Jain Jose K., Thomas N., Aravindakshan T. V. Development of PCR-RFLP for the detection of benzimidazole resistance polymorphisms in isotype 1 β-tubulin gene of Trichostrongylus colubriformis. Small Ruminant Research. 2023. 222. 106954. https://doi.org/10.1016/j.smallrumres.2023.106954.Rajagopal A., Sabu L., Radhika R., Devada K., Jain Jose K., Thomas N., Aravindakshan T. V. Development of PCR-RFLP for the detection of benzimidazole resistance polymorphisms in isotype 1 β-tubulin gene of Trichostrongylus colubriformis. Small Ruminant Research. 2023. 222. 106954. https://doi.org/10.1016/j.smallrumres.2023.106954.Rossanigo C. E., Gruner L. The length of strongylid nematode infective larvae as a reflection of developmental conditions in faeces and consequence on their viability. Parasitology Research. 1996; 82. 304–311. http://doi.org/10.1007/s004360050118, PMid:8740545Rossanigo C. E., Gruner L. The length of strongylid nematode infective larvae as a reflection of developmental conditions in faeces and consequence on their viability. Parasitology Research. 1996; 82. 304–311. http://doi.org/10.1007/s004360050118, PMid:8740545Santos J. M. L., Vasconcelos J. F., Frota G. A., Freitas E. P., Teixeira M., Vieira L. S., Bevilaqua C. M. L., Monteiro J. P. Quantitative molecular diagnosis of levamisole resistance in populations of Haemonchus contortus. Experimental Parasitology. 2019; 205. 107734. https://doi.org/10.1016/j.exppara.2019.107734Santos J. M. L., Vasconcelos J. F., Frota G. A., Freitas E. P., Teixeira M., Vieira L. S., Bevilaqua C. M. L., Monteiro J. P. Quantitative molecular diagnosis of levamisole resistance in populations of Haemonchus contortus. Experimental Parasitology. 2019; 205. 107734. https://doi.org/10.1016/j.exppara.2019.107734Silvestre A., Humbert J.-F. A Molecular Tool for Species Identification and Benzimidazole Resistance Diagnosis in Larval Communities of Small Ruminant Parasites. Experimental Parasitology. 2000; 95 (4): 271–276. https://doi.org/10.1006/expr.2000.4542Silvestre A., Humbert J.-F. A Molecular Tool for Species Identification and Benzimidazole Resistance Diagnosis in Larval Communities of Small Ruminant Parasites. Experimental Parasitology. 2000; 95 (4): 271–276. https://doi.org/10.1006/expr.2000.4542Smits H. L., Hartskeerl R. A. PCR amplification reactions in parasitology. Journal of Microbiological Methods. 1995; 23. 41–54.Smits H. L., Hartskeerl R. A. PCR amplification reactions in parasitology. Journal of Microbiological Methods. 1995; 23. 41–54.Van Wyk J. A., Cabaret J., Michael L. M. Morphological identification of nematodes of small ruminants and cattle simplified. Veterinary Parasitology. 2004; 119. 277–306. http://doi.org/10.1016/j.vetpar.2003.11.012Van Wyk J. A., Cabaret J., Michael L. M. Morphological identification of nematodes of small ruminants and cattle simplified. Veterinary Parasitology. 2004; 119. 277–306. http://doi.org/10.1016/j.vetpar.2003.11.012Van Wyk J. A., Mayhew E. Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide. Onderstepoort Journal of Veterinary Research. 2013; 80 (1): 14. http://doi.org/10.4102/ojvr.v80i1.539Van Wyk J. A., Mayhew E. Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide. Onderstepoort Journal of Veterinary Research. 2013; 80 (1): 14. http:// doi.org/10.4102/ojvr. v80i1.539Weiss J. B. DNA probes and PCR for diagnosis of parasitic infections. Clinical Microbiology Reviews. 1995; 8. 113–130. https://doi.org/10.1128/CMR.8.1.113Weiss J. B. DNA probes and PCR for diagnosis of parasitic infections. Clinical Microbiology Reviews. 1995; 8. 113–130. https://doi.org/10.1128/CMR.8.1.113

The authors declare that there are no conflicts of interest present.