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Eurasiascience» ХII Международная Научно-Практическая Конференция «EurasiaScience» ХII Международная научно-практическая конференция 15 декабря 2017 Научно-издательский центр «Актуальность.РФ» СБОРНИК СТАТЕЙ ЧАСТЬ 1 Сollected Рapers Part I XII International Scientific-Practical conference “EurasiaScience” Research and Publishing Center “Actualnots.RF”, Moscow, Russia December, 15, 2017 Moscow 2017 УДК 00, 1, 33, 34, 36, 37,39, 50, 51, 57, 60, 61, 62, 63, 67, 68, 7 ББК 1 E91 EurasiaScience Сборник статей XII международной научно-практической конференции, часть I, E91 Москва: «Научно-издательский центр «Актуальность.РФ», 2017. – 288 с. ISBN 978-5-9500959-2-4 Книга представляет собой первую часть сборника статей XII международной научно- практической конференции «EurasiaScience» (Москва, 15 декабря 2017 г.). Представленные доклады отражают наиболее значительные достижения в области теоретической и прикладной науки. Книга рекомендована специалистам, преподавателям и студентам. Сборник рецензируется членами оргкомитета. Издание включено в РИНЦ согласно лицензионного договора 930-03/2015K. Организатор конференции: Научно-издательский центр «Актуальность.РФ» При информационной поддержке: Пензенского государственного университета Федерального государственного унитарного предприятия «Информационное телеграфное агентство России (ИТАР-ТАСС)» Федерального государственного бюджетного учреждения науки «Российская книжная палата» Научной электронной библиотеки eLIBRARY.RU Московского государственного университета им. М. В. Ломоносова ISBN 978-5-9500959-2-4 © ООО «Актуальность.РФ» УДК 619:616.9-07 К ВОПРОСУ СОЗДАНИЯ ИММУНОХИМИЧЕСКОГО ЭКСПРЕСС-МЕТОДА ДИАГНОСТИКИ ИНФЕКЦИОННЫХ ЗАБОЛЕВАНИЙ ПТИЦ Дмитриева М. Е., Новикова О. Б., Проккоева Ж. А. Всероссийский научно-исследовательский ветеринарный институт птицеводства, Ломоносов, Россия В промышленном и племенном птицеводстве актуальна проблема биобезопасности. Заболевания птиц вирусной и бактериальной этиологии успешно диагностируются экспрессными методами. Реакция гидрозольной агглютинации позволяет количественно определять патогенного возбудителя в исследуемом материале. Авторы описывают результаты экспериментальных серий иммунохимических гидрозольных тест-систем на основе полимера (ПВК), инактивированных антигенов и комплиментарных антител. Ключевые слова: агглютинация, вирус ньюкаслской болезни, вирус синдрома снижения яйценоскости, сальмонеллез, птицеводство Благодаря научно-производственному обеспечению биобезопасности птицеводческой отрасли, реализации комплекса ветеринарно-санитарных мер на птицефабриках, при этом основными являются диагностика инфекционных заболеваний птиц и вакцинопрофилактика, птицеводство развивается успешно. При диагностике заболеваний используются эпизоотологические данные, клиническая картина, патологоанатомические изменения, бактериологические исследования, но составляющими являются серологические исследования (РНГА, ККРНГА, ИФА, ПЦР и др.), позволяющие прогнозировать эпизоотическую ситуацию на птицефабрике [1]. Методы лабораторной диагностики совершенствуются, в том числе в связи с возросшей потребностью в экспресс-тестах, практическое применение которых доказало их удобство, быстроту, экономичность, эффективность применения, позволяющее сократить в три раза количество анализов, используемой лабораторной посуды и питательных сред, снизить затраты труда на постановку диагноза, стандартизировать трудоемкий этап идентификации патогенных микроорганизмов. На практике постановка своевременного и точного диагноза бывает затруднена при субклинической или латентной форме инфекции, такой как инфекционный бронхит, синдром снижения яйценоскости, инфекционный энцефаломиелит кур [2]. В таких случаях оперативную информацию дают дополнительные диагностические тесты, например, иммунохимические гидрозольные, позволяющие исследовать широкий диапазон тестируемых объектов. Высокая чувствительность и специфичность универсального метода РГЗА – реакции гидрозольной агглютинации, учет результатов в течение 3-5 минут, возможность применения в «полевых» условиях, а также количественное определение антигенов и антител в исследуемом материале (кровь, сыворотка крови, желточные фолликулы птиц, белок и желток яиц, пробы жидкости организма, гомогенат внутренних органов и тканей, комбикорм, питьевая вода, смывы с поверхности стен, пола и технологического оборудования) позволяют использовать РГЗА для бесприборной экспресс-диагностики отдельных вирусных и бактериальных болезней птиц. В задачу наших исследований входило изучение экспериментальных серий иммунохимических гидрозольных тест- систем на основе полимера поливинил-капролактама (ПВК), инактивированных антигенов и комплиментарных антител – маркеров определенных болезней птиц: ньюкаслской болезни (НБ), синдрома снижения яйценоскости кур (ССЯ-76), сальмонеллеза (SE). В качестве комплиментарных антител использовали моноспецифическую сыворотку крови птиц, содержащую иммуноглобулины G с известным уровнем антител к каждому из антигенов. Синтез гидрозоля осуществили при комнатной температуре на основе стабилизированного резорцином 2 % водного раствора ПВК, диоксида титана. Контрольными методами и тест-системами для определения достоверной соразмерности явились: реакция торможения гемагглютинизации РТГА, «Набор компонентов для выявления антител к вирусу ньюкаслской болезни в РТГА», «Набор компонентов для выявления антител к вирусу синдрома снижения яйценоскости кур (ССЯ-76) в РТГА». Контроль проб сыворотки крови на наличие антител в SE проводили в ККРНГА с эритроцитарным антигеном [3]. Применяли стандартные методы химического анализа. РГЗА ставили в микропанелях и на предметном стекле, тестируемые пробы сыворотки крови в разведении 1:10-1:40 наносили в объеме 10мкл на подготовленный твердофазный носитель гидрозоля. Специфические белки-маркеры с полимерными носителями гидрозоля создают стабильно устойчивую ультрадисперсную коллоидную систему, которая позволяет определять результаты взаимодействия антигенов и антител, как сорбированных биолигандов. В исследовании использовали серии иммунохимических гидрозольных тест-систем: №1 – раствор ПВК+резорцин+физраствор, №2 – раствор ПВК+метилоранж+резорцин, №3 – супернатант при гидротермальном синтезе диоксида титана 3 в присутствии ПВК и ПВП из прекурсора сульфата титанола, №4 – супернатант №3+метилоранж. Гидрозольные препараты в качестве бесприборного иммунохимического экспресс-метода применяли для индикации специфических антител в пробах сыворотки крови, полученной от птиц, иммунизированных инактивированным химическим методом антигеном: возбудителя НБ с активностью в РГА не ниже 9 log2 1:512, вируса ССЯ-76 с активностью в РГА 13 log 2 1:8192, культуры Salmonella enteritidis SE (АГᶧ SE). Титр антигемагглютинирующих антител в монорецепторной сыворотке крови, содержащей специфические антитела к каждому из использованных в работе возбудителей, составил в РТГА 1:256 к НБ, 1:1024 к ССЯ-76, в ККРНГА к СЕ 1:80. На примере SE установили через 5-8 минут с момента постановки реакции визуальные различия результатов РГЗА с положительной сывороткой крови к SE (осадок из хлопьев агглютинина) и отрицательной сывороткой крови (однородный мутный раствор). Также и в отношении НБ и ССЯ-76 наблюдали либо формирование хорошо различимого осадка агглютината по центру капли или лунки планшета, либо реакционная смесь по всему объёму была однородно мутная, без формирования осадка. На результаты влиял рН гидрозоля - отличия более четкие при увеличении кислотности раствора. В изученных сериях оптимальное количество и соотношение исходных компонентов РГЗА составило: гидрозоль № 3 – 0,05 см³, АГᶧ (НБ) - 0,02 см³, АТ ᶧ (НБ) - 0,02 см³. Аналогично в отношении ССЯ-76 гидрозоли № 1 и № 2. Таким образом, задача изучения экспериментальных серий иммунохимических гидрозольных тест-систем, где в качестве иммобилизированных биолигандов использованы маркеры ньюкаслской болезни, синдрома снижения яйценоскости кур, сальмонеллеза, выполнена в виде проведенных комиссионных испытаний. Создание гидрозольного экспресс-метода для применения в «полевых» условиях является перспективным и обоснованным решением. Список цитируемой литературы 1. Lancaster J.E. Newcastle disease. Virus Disease of Food Animals / v.11 - 1981. – 43-465. 2. Хохлачев О.Ф., Терюханов А.Б. Ассоциированное течение инфекционного бронхита и синдрома снижения яйценоскости кур / 20 лет на благо промышленного птицеводства: НПП АВИВАК, Санкт- Петербург. - 2010. – С. 36. 3. Мешандин А.Г. и др. Серологическая диагностика сальмонеллезов / А.Г.Мешандин, Ю.В.Золотарев Л.В.Золотарева // Клиническая лабораторная диагностика. М.: Медицина. - 2001.- № 1. - C. 52-53. TO THE QUESTION OF THE CREATING OF IMMUNOCHEMICAL DIAGNOSTIC RAPID METHOD FOR THE DIAGNOSIS AVIAN INFECTIOUS DISEASES Dmitrieva M. E., Novikova O. B., Prokkoeva Zh. A. All-Russian Research Veterinary Institute of Poultry Science, Lomonosov, Russia In industrial and breeding poultry farming biosafety problem is relevant. Diseases of birds of a virus and bacterial etiology are successfully diagnosed by express methods. Reaction of a hydrocindery agglyutinization allows to define quantitatively the pathogenic activator in the studied material. Authors describe results of experimental series of immunochemical hydrocindery test systems on the basis of polymer (PVK), the inactivated antigens and complementary antibodies. Keywords: agglutination, virus Newcastle Disease, virus Egg Drop Syndrome'76, Salmonella Enteritidis Infection, Poultry farming 4 УДК: 619:616.9-07 ИДЕНТИФИКАЦИЯ ВИРУСА ИНФЕКЦИОННОЙ АНЕМИИ ЦЫПЛЯТ МЕТОДОМ ЭЛЕКТРОННОЙ МИКРОСКОПИИ Дмитриева М. Е., Занько М. А., Балендор Е. В. Всероссийский
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