Mycoplasmosis control remains urgent in view of wide spread of bovine mycoplasmoses in the countries with intensive animal farming and trade relations between the Russian Federation and foreign partners including import of pedigree livestock and stud bull semen. Results of testing 1,186 biomaterial samples (blood, sera, nasal swabs, milk, preputial swabs, vaginal swabs, aborted and stillborn fetuses) collected from animals that demonstrated clinical signs of respiratory and reproductive disorders in 34 different regions of the Russian Federation for 2015–2018 are presented in the paper. The samples were tested with real-time polymerase chain reaction (rtPCR) for genomes of the following mycoplasmosis agents: Mycoplasma bovis, Mycoplasma bovigenitalium, Mycoplasma dispar. As a result, M. bovis genome was detected in 10.1% of the samples, M. bovigenitalium genome was detected in 8.6% of the samples and М. dispar genome was detected in 37.15% of the samples. Also, 927 semen samples submitted from Russian and foreign breeding farms were tested with PCR. Test results showed presence of M. bovis and M. bovigenitalium genomes in semen samples collected from native bull population. Presented data support Russian scientists’ conclusions on wide mycoplasmoses occurrence in cattle in the Russian Federation territory and risk of the disease agent introduction through semen import. All of these highlight the need for control of semen products as a source for mycoplasmosis spread as well as insufficiency of single testing of semen for granting the disease-free status to the breeding farm for genetic material marketing.
Porcine reproductive and respiratory syndrome (PRRS) being endemic and reported in the most countries in the world remains one of the most challenging diseases in pig industry. The main disease control measures include preventive vaccination and animal movement control within and outside the country as well as diagnostic testing of pigs in the population. Live and inactivated vaccines are used for specific prevention of porcine reproductive and respiratory syndrome. Complete and irreversible infectious agent inactivation with maximum epitope preservation and protective immunity in immunized animals are the main requirements for inactivated vaccines. Therefore, continuous improvement of methods for vaccine quality control at various vaccine production stages is of current importance. Results of development of the test system based on indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) for PRRS virus antigen detection and activity testing in infectious and inactivated virus-containing cell cultures at intermediate stages of the vaccine production process are described in the paper. The test-system development process included purified and concentrated virus antigen as well as hyperimmune rabbit sera preparation. Specificity of purified and concentrated virus antigen was confirmed with real-time polymerase chain reaction. The developed test-system was shown to detect the virus antigen at initial infectivity titre of 4.87–7.21 lg TCID50/cm3 corresponding to ELISA titre (dilution) of 1:4 up to 1:64. Methodical Guidelines for detection of porcine reproductive and respiratory syndrome virus antigen with indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) (2019) were developed based on the work results, commissioned and approved by the FGBI “ARRIAH” Scientific Board.
Lumpy skin disease is an economically significant disease as it results in decrease in weight gain and milk yield, abortions, mastitis, reproduction disorders, animal emaciation, lesions of respiratory organs and in some cases – death. Today the disease is included in the OIE list and is subject to obligatory notification. The emergence and spread of the disease in the Russian Federation necessitated performance of tests in the framework of laboratory diagnosis method improvement. The test-system based on the indirect “Sandwich” ELISA for diagnosis of lumpy skin disease allowing performance of biomaterial tests within 24 hours was for the first time developed in Russia. The test-system development included antibody preparation in laboratory animals (rabbits and guinea pigs), selection of optimal dilution of capture and detection antibodies, composition of a buffer solution and conditions of the reaction procedure. 50 samples of initial culture antigens of the lumpy skin disease virus as well virus-containing suspensions collected on different stages of purification and concentration were tested using this technique. To confirm the ELISA results all analyzed samples were tested using RT-PCR. Besides, the virus infectivity titer was determined by titration in YaDK-04 (Goat gonad cells). The test specificity was 100%, and analytical sensitivity – 3.5 lg TCD50. The developed “Sandwich” ELISA allows performing tests of 24 antigen samples at 1:2–1:16 dilution simultaneously using 96-well plate and it can be used for lumpy skin disease diagnosis.
Cattle respiratory diseases are some of the most spread pathologies that can cause economic damage, resulting from fi nancial losses and costs of treatment and diagnostics. One of the major factors contributing to respiratory pathology development is bovine respiratory syncytial infection. The analysis of serological testing, performed by the FGBI “ARRIAH” Reference Laboratory for Cattle Diseases in 2017–2018, showed that respiratory syncytial virus seroprevalence in animals of dairy farms is 60%. Herewith, it was noted that the most susceptible animals to this infection are calves under one year of age. The eff ectiveness of bovine respiratory syncytial infection control measures depends on timely diagnosis; that is why reliable and accurate diagnostic tools are needed, including optimal techniques of virus isolation from pathological material. For successful virus isolation from clinical samples, it is necessary to adhere strictly to optimal parameters of this agent cultivation. This paper presents data on study of bovine respiratory syncytial virus strain AB 1908 cultural properties. The tests performed showed that a continuous bovine turbinate (BT) cell line, continuous bovine fetal trachea (FBT) cell line and continuous bovine calf kidney (RBT) cell line are sensitive for cultivation of this agent and can be used to prepare viral suspension, needed for further research. Virus titre in BT cell culture was 4.33 ± 0.16 – 4.66 ± 0.12 lg TCID50/ cm3, in RBT cell culture – 4.33 ± 0.33 – 4.7 ± 0.36 lg TCID50/cm3 and in FBT cell culture – 4.13 ± 0.20 – 4.78 ± 0.17 lg TCID50/cm3. The following virus cultivation optimal parameters were also determined during this study: the age of the culture for virus inoculation should be 1–2 days and multiplicity of inoculation should be 0.1 TCID50/cell.
Интенсификация животноводства неизбежно повышает риски заноса и распространения инфекционных заболеваний, из которых наиболее опасны трансграничные. Инфекции крупного рогатого скота, вызываемые каприпоксвирусами, в частности вирусом заразного узелкового дерматита (ЗУД, Lumpy skin disease, LSD), поражают крупный и мелкий рогатый скоту, нанося существенный экономический ущерб. До 2015 года территория Российской Федерации была благополучна по ЗУД, однако из-за локальных конфликтов на Ближнем Востоке и изменений климата заболевание распространяется в северном направлении (масштабные вспышки LSD в Турции, странах Балканского полуострова, ЕС и в России). Широкое использование живых гомологичных вакцин против LSD в соседних странах также актуализирует разработку методов диагностики этого возбудителя, позволяющих выявлять геномы полевых изолятов и дифференцировать вакцинный вирус ЗУД КРС. Нами впервые в России разработан и валидирован комплекс методов ПЦР в режиме реального времени для однорежимного тестирования проб на наличие генома каприпоксвирусов, полевых изолятов вируса заразного узелкового дерматита и вакцинного вируса ЗУД КРС типа Neethling. На основе выравнивания полногеномных последовательностей идентифицированы сайты, наиболее пригодные для специфичной амплификации при выявлении генома полевых изолятов, вакцинных штаммов и каприпоксвирусов. Для амплификации фрагмента генома полевых изолятов мы выбрали участок ORF126 гена EEV вируса LSD-вставку размером 27 п.н., отсутствующую у других представителей рода Capripoxvirus и вакцинных штаммов типа Neethling. Для амплификации фрагмента генома вакцинных штаммов использовали участок ORF008, в котором присутствуют уникальные замены, характерные только для таких штаммов, для каприпоксвирусов-участок гена P32, консервативный для всех представителей рода Capripoxvirus. Предложенные методы показали высокую чувствительность (98 %), специфичность (99 %) и адекватную повторяемость (коэффициент вариации не более 2 %). Апробацию тест-систем проводили на панели из 596 проб биологического материала, собранного при вспышках каприпоксвирусных инфекций в России,-стабилизированной крови, сыворотки крови, соскобов кожи (нодулы), назальных и окулярных смывов, молока, тканей лимфатических узлов, легких, трахеи, селезенки и абортплодов, отобранных от животных, инфицированных в естественных условиях. Следует отметить, что с помощью разработанного комплекса тест-систем ПЦР-РВ в рамках мониторингового исследования на заразный узелковый дерматит в ряде регионов Российской Федерации отмечены случаи выявления генома вакцинного вируса ЗУД КРС. Это может свидетельствовать либо о нелегальном использовании запрещенной к ввозу в Россию аттенуированной живой вакцины типа Neethling, либо о естественном распространении вакцинного Neethling. На основе разработанных методов оформлено два патента РФ на изобретения, что подтверждает оригинальность и значимость предложенных методик для диагностических исследований. Ключевые слова: заразный узелковый дерматит, нодулярный дерматит, вакцинный штамм, каприпоксв...
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