Rapid identification of Corynebacterium diphtheriae clonal group associated with diphtheria epidemic, Russian Federation.

We used 199 Corynebacterium diphtheriae isolated from 1995 to 1997 in Russia to evaluate the ability of random amplified polymorphic DNA (RAPD) to identify the unique clonal group that emerged there in 1990. Our data show that RAPD can reliably, reproducibly, and rapidly screen a large number of strains to identify the epidemic clonal group

Simulation of Bubonic Plague in BSL-2 Laboratory

The causative agent of plague, Yersinia pestis, is classified as pathogenicity (hazard) group I agent, which means that the work with “wild type” strains should be carried out in BSL-3 facilities. Y. pestis EV NIIEG is a Δpgm strain, allowing experimental studies to be carried out in BSL-2 laboratories. However, the disease and its progression elicited by such strain do not entirely mirror the infection observed with fully virulent strains. Residual virulence of Y. pestis EV NIIEG strain for mice can be increased under in vivo iron supplementation. The aim of the study was to optimize methodological approaches to modeling experimental plague in laboratory animals following administration of attenuated Δpgm Y. pestis strains with iron dextran. Materials and methods. Simulation of plague infection in outbred mice was carried out through subcutaneous inoculation of Y. pestis EV NIIEG strain with iron dextran supplementation. The animal condition was assessed on a daily basis. In the course of the experiment, the pathological presentation and bacterial content in organs of mice were evaluated. Results and discussion. Mice inoculated subcutaneously with Y. pestis EV NIIEG strain in the presence of iron dextran developed a bubonic plague that resulted in lethal outcome with pathological changes of internal organs, characteristic of plague infection. In case of daily administration of iron, LD50 of Y. pestis EV NNIEG strain for the mice significantly exceeded the same one with a single injection. Differences in the survival rate among animals in the groups with a single and multiple administration of iron compared to the control group were statistically valid. Thus, attenuated Δpgm Y. pestis strains in the presence of iron dextran can be used to model experimental plague in mice with marked pathological changes and lethality in BSL-2 laboratories

Evaluation of the long-term memory T cell in mice after immunization with a live tularemia vaccine

The vaccine strain F. tularensis 15 NIIEG induces long-lived cell-mediated immunity but exhibits a certain reactogenicity and genetic instability. Progress in development of a vaccine against tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. The BALB/c mouse is the most commonly used animal to study tularemia due to its relatively low cost, well-characterized genetics, available immunological tools and mouse infection with virulent F. tularensis recapitulates human disease.CD4+ and CD8+T cells are known to be critical for the formation of protective immunity but the relative roles of memory T cell subpopulations in long lived protection against virulent strains of F. tularensis are not well established. We hypothesized that this immunity depends on central (TCM) and effector memory (TEM) T cells and their functional activity. In this study we have dissected the T cell immune response in BALB/c mice 30, 60 and 90 days after subcutaneous vaccination with 15 NIIEG.Multiparametric flow cytometry were used to characterize in vitro recall responses of splenocytes to F. tularensis antigen. TEM cells were identified as CD3+CD4+CD44+CD62L- and CD3+CD8+CD44+CD62L-, TCM cells as CD3+CD4+CD44+CD62L+ and CD3+CD8+CD44+CD62L+, respectively. The functional activity of memory T cells was assessed by the following parameters: the level of expression of the activation marker CD69 and cytokine-producing activity by staining with the intracellular cytokines IFNg and TNFa.Thus, development of a long-lived vaccine directed against F. tularensis is dependent on identifying not only the correlates of immunity present early after vaccination, but also those that persist in the host after the effector phase has ended. The maintenance of long-term protective immunity initiated by vaccination with F. tularensis strain 15 NIIEG has been shown to require the presence of antigen-specific CD4+ and CD8+ memory T cells producing IFNg and TNFa and expressing the activation marker CD69. A decrease in count and functional activity of CD8+TCM and CD8+TEM was detected in the long term after vaccination. The detected parameters of functional activity of memory T cells can be used as criteria for evaluation of protective immunity against virulent strains of F. tularensis

Infectious Sensitivity of BALB/c Mice to Infestation with <I>Photorhabdus asymbiotica</I> and <I>Photorhabdus temperata</I>

) KOE). Identified are the patho-morphological changes in the organs of animals, inoculated with Photorhabdus spp. strains

Dynamics of Antibody Response to <i>Yersinia pestis</i> Proteins in Plague Affected Guinea Pigs

Designing of new means for the specific prevention of plague, especially protein subunit vaccines, is impossible without studying the role of individual antigens in the manifestation of the pathogenic and immunogenic properties of Yersinia pestis. The aim of the present study was to determine the antibody levels to Y. pestis antigens in guinea pigs that survived infection with sub-lethal doses of virulent plague agent strains using enzyme immunoassay (ELISA). Materials and methods. Guinea pigs were inoculated subcutaneously with 30 CFU of the wild type Y. pestis subsp. Pestis strain 231 or non-capsular Y. pestis subsp. pestis Caf1-negative strain 358/12. Blood samples from sick or recovered guinea pigs were collected on day 15, 30, 60, and 90 after infection. The antibody response was assessed by 18 recombinant Y. pestis proteins in ELISA. Results and discussion. Heterogeneity of the antibody responses to the majority of the antigens with variation of IgG titers from animal to animal has been revealed. We observed increase in antibody titers by day 90 for the most analyzed antigens in the sera of the guinea pigs injected with wild type Y. pestis 231. On the contrary we found reduction in antibody titers by day 90 in case of inoculation with Y. pestis 358/12. The preservation of antibodies to Y. pestis proteins of different localization in the organism of the guinea pigs, as well functional activity, and the degree of representation on the surface of bacterial cell for a prolonged period of time indicates the multiplex nature of the plague immunity formation. Our findings are significant for the future design and development of effective vaccines against plague and the search for new targets for diagnostics of this disease

РАСПРЕДЕЛЕНИЕ ЭЯКУЛЯТОВ ПО ЧИСЛУ СПЕРМАТОЗОИДОВ У БЫКОВ ГОЛШТИНСКОЙ ПОРОДЫ В ВОЗРАСТЕ 6–7 ЛЕТ

The paper highlights the results of the system analysis carried out in order to explore the primary characteristics of native seed from Holstein servicing bulls of Black-and-White breed aged 6-7 years. The analysis was carried out to rank ejaculates by the total number of spermatozoa. The experiment was carried out at OAO “Moskovskoye on pedigree activities” in 2011 - 2013. The researchers tested 5964 ejaculates, obtained from 17 bulls during three years, in relation to the season. The researchers explored the number of ejaculates (n), defect of the native sperm when taking (%), and the seasonal dynamics of the total number of sperm cells in the ejaculate. The keeping technology, feeding and maintenance, sperm collection, its assessment and cryopreservation was carried out in accordance with the requirements of the “National technology of producing and application of servicing bulls’ sperm “. Ejaculates were distributed into three groups according to the total number of spermatozoa: - up to 3 billion; - 3.1 - 5.0 billion; - 5.1 billion and more. The researchers found out that the number of ejaculates of good quality selected for cryopreservation in the mature reproductive aged bulls (6 - 7 years) is 76% on average with a variability of 73-82%, depending on the season and year of maintenance. The highest number of the ejaculates of good quality was obtained in autumn and it was 82.3%, which is 7-10% higher than that in other seasons (p&lt;0.001). The analysis of ejaculates over a three-year period showed that the first group contained the main mass of ejaculates, where the number of spermatozoa was up to 3 billion. This parameter varied within 40-48% from season to season; the researchers observed the ejaculates that have total number of spermatozoa equal to 3.1 - 5.0 billion were 28.5-33.4%, the ejaculates that have the highest number of spermatozoa (5.1 billion and more) were 23-29%.Был проведен системный анализ массивов первичных характеристик нативного семени, полученного от быков-производителей голштинской породы черно-пестрой масти в зрелом репродуктивном возрасте (6–7 лет) для ранжирования эякулятов по общему числу выделенных сперматозоидов. Опыт проводился на базе ОАО «Московское» по племенной работе в 2011–2013 гг. Всего было проанализировано 5964 эякулята, полученных от 17 быков в течение трех лет в зависимости от сезона года. Изучено число эякулятов, брак нативного семени при взятии, сезонная динамика общего числа сперматозоидов в эякуляте. Взятие семени, его оценка и криоконсервация проводились согласно требованиям Национальной технологии получения и использование спермы племенных быков-производителей. Эякуляты были распределены на три группы по общему числу сперматозоидов: до 3 млрд; 3,1–5,0 млрд; 5,1 млрд и выше. Установлено, что у быковпроизводителей зрелого репродуктивного возраста (6–7 лет) количество качественных эякулятов, отобранных для криоконсервации, составляет в среднем 76 % с вариабельностью 73–82 % в зависимости от сезона и года эксплуатации. Наибольшее количество качественных эякулятов было получено в осенний сезон и составило 82,3 %, что на 7–10 % выше по сравнению с остальными сезонами года (Р&lt;0,001). Анализ эякулятов за трехлетний период показал, что основной массив распределен в первой группе, где число сперматозоидов составило до 3 млрд. Данный показатель в зависимости от сезона года варьировал между 40–48 %. Эякуляты, имеющие общее количество сперматозоидов от 3,1 до 5,0 млрд, составили 28,5–33,4 %, эякуляты с наивысшим числом сперматозоидов (5,1 млрд и выше) – 23–29 %

Диагностическая ценность метода ИФА при коклюше у детей

Because of low effectiveness of laboratory methods for diagnosing pertussis it is important to look for new ways of verification of this infection. The article presents the analysis of the diagnostic value of ELISA method, which involves the identification of antibodies of different isotypes (IgM, IgG, IgA) to pertussis toxoid (PT) and filamentous haemagglutinin (FHA). The study included 279 children: 114 were under 1 year of age, 165 — older than 1 year. The pertussis was confirmed in 74.3 ± 2.6% of patients by using ELISA method. A significant proportion of seronegative patients (46.1 ± 6.2 per cent) was revealed in the group of patients under 1 year. The pattern of production of antibodies in unvaccinated children was different. It depended on the age of the children and timing of illness. A low proportion of diagnostically significant indicators of IgM-antibodies at 2—3 weeks of illness was typical for patients under 1 year of age (e.g. 6.7 ± 6.5% as compared to 20.0 ± 7.9% and 50.0 ± 15.3 — 1—3 and 4—6 years of age). The diagnosis of pertussis in children under 1 year of age was confirmed mainly by the detection of IgG, starting from the 4th week of the disease. In the examination of vaccinated children diagnostically significant levels of IgA and IgG were identified (even in the late stages of the disease). Thus, the results of the analysis show special significance of using ELISA method for the diagnosis of pertussis in vaccinated children.Недостаточная эффективность используемых методов лабораторной диагностики коклюша в практическом здравоохранении диктует необходимость расширения спектра средств верификации этой инфекции. В статье представлен анализ диагностической ценности метода ИФА, который предусматривает выявление антител различных изотипов (IgM, IgG, IgA) к коклюшному токсину и филаментозному гемагглютинину. Всего обследовано 279 детей, среди которых 114 были в возрасте до 1 года, 165 — старше 1 года. Использование метода ИФА в целом позволило подтвердить диагноз коклюша у 74,3 ± 2,6% больных. При проведении сравнительного анализа его эффективности у детей различного возраста установлено, что в группе больных в возрасте до 1 года выявлен значительный удельный вес серонегативных пациентов, составивших 46,1 ± 6,2%. Анализ частоты выявления антител различных классов у непривитых детей разного возраста выявил отличия в характере динамики продукции антител, превышающих пороговый уровень, в зависимости от сроков болезни и возрастных групп. Для пациентов раннего возраста был характерен низкий удельный вес больных с диагностически значимыми показателями IgM-антител на 2—3-й неделях болезни (например, на 2-й неделе у 6,7 ± 6,5% против 20,0 ± 7,9% и 50,0 ± 15,3 — 1—3 и 4—6 лет соответственно). Диагноз коклюша у детей до 1 года был подтвержден преимущественно выявлением антител IgG класса, начиная с 4 недели болезни. Сравнительный анализ частоты обнаружения антител различных классов у привитых детей показал значительный удельный вес больных с диагностически значимыми уровнями IgA наряду с высоким уровнем продукции IgG, причем и на поздних сроках болезни, что позволяет принимать это во внимание как важный серологический критерий диагностики коклюша у привитых детей

Определение противококлюшных антител у школьников с длительным кашлем

Objective: to assess anti-pertussis immunity in schoolchildren aged 7–17 who complained of a prolonged cough during the 11-year follow-up period.  Materials and methods. The study included 1046 patients aged 7 to 17 years who applied to the Consultative and Diagnostic Center of the G.N. Gabrichevsky Research Institute of Epidemiology and Microbiology with complaints of prolonged cough in the period from 2010 to 2020. Blood serums were examined in ELISA with the determination of IgM, IgG, IgA antibodies using RIDASCREEN test system (Germany).  Results. An active infection with the detection of IgM and/ or IgA, IgG antibodies above threshold levels was detected in 51,3% of children with prolonged cough, while annually in a fairly high percentage throughout the follow-up period. Active pertussis infection, established based on the detection of IgM, IgG, IgA antibodies above thresholds in blood serum samples, prevailed in children 12–15 years old, accounting for more than 60% in children with prolonged cough. Antipertussis immunity as a result of childhood vaccination or previous disease was detected in 16.1-20.2% of people in the period 2010–2014 and in 12,8-20,9% in 2015–2020.  Conclusion. The results obtained by us on the study of anti-pertussis immunity in schoolchildren confirm the presence of active latent circulation of the pathogen whooping cough among children of this age cohort and, therefore, the presence of unaccounted for cases of the disease. This confirms the importance of timely diagnosis of pertussis, isolation of children for the period of active infection and justifies the need for the widespread introduction of a second revaccination against pertussis. Цель: оценка противококлюшного иммунитета у школьников 7–17 лет, обратившихся с жалобами на длительный кашель, в течение 11-летнего периода наблюдения.  Материалы и методы. В исследование включено 1046 пациентов в возрасте от 7 до 17 лет, обратившихся в консультативно-диагностический центр Московского научно-исследовательского института эпидемиологии и микробиологии им. Г.Н. Габричевского с жалобами на длительный кашель в период с 2010 по 2020 г. Сыворотки крови исследовали в ИФА с определением IgM, IgG, IgA антител с помощью тест-системы «RIDASCRЕЕN» (Германия). Результаты. Активная инфекция с выявлением антител классов IgM и (или) IgA, IgG выше пороговых уровней выявлена у 51,3% детей с длительным кашлем, при этом ежегодно в достаточно высоком проценте на протяжении всего периода наблюдения. Активная коклюшная инфекция, установленная на основании выявления в образцах сывороток крови антител IgM, IgG, IgA выше пороговых значений, преобладала у детей 12–15 лет, составляя выше 60% у детей с длительным кашлем. Противококлюшный иммунитет в результате проведенной в детстве вакцинации или перенесенного заболевания выявили у 16,1–20,2% лиц в период 2010–2014 гг. и у 12,8–20,9% – в 2015–2020 гг.  Заключение. Полученные нами результаты по изучению противококлюшного иммунитета у школьников подтверждают наличие активной скрытой циркуляции возбудителя коклюша среди школьников и, следовательно, наличие недоучтенных случаев заболевания. Это подтверждает важность своевременной диагностики коклюша, изоляции детей на период активной инфекции и обосновывает необходимость повсеместного введения второй ревакцинации против коклюша.

Features of developing SARS-CoV-2 nucleocapsid protein population-based seroprevalence during the first wave of the COVID-19 epidemic in the Russian Federation

The novel coronavirus (SARS-CoV-2) pandemic, dubbed COVID-19, has become one of the most serious challenges for human populations in the vast majority of countries worldwide. Rapid spreading and increased mortality related to it required new approaches to manage epidemic processes on a global scale. One of such approaches was based on analyzing SARS-CoV-2 seroprevalence associated with COVID-19. Our aim was to summarize the results on assessing seroprevalence to the SARS-CoV-2 nucleocapsid antigen (Nc) in residents from 26 regions of the Russian Federation, carried out during the first wave of the COVID-19 epidemic.Materials and methods. Seroprevalence distribution was examined in 26 model regions of the Russian Federation according to the unified method developed by the Rospotrebnadzor with the participation of the Federal State Institution Saint Petersburg Pasteur Research Institute of Epidemiology and Microbiology. Such approach implied formation of a group of volunteer subjects in model geographic region who were tested by ELISA for anti-Nc serum antibody level in peripheral blood. Analyzed primary data obtained in separate regions were either accepted for publication or released.Results. The current paper finalizes the data obtained in all 26 regions of the Russian Federation. The total SARS-CoV-2 seroprevalence was 19.5 (10.0–25.6)% with the maximum and minimum value found in the Kaliningrad Region and the Republic of Crimea, respectively (50.2% vs. 4.3%). A pattern of age-related seroprevalence distribution indicates insignificant predominance of seroprevalence among subjects of 1–17 years old: 22.1 (13.1–31.8)%. Among COVID-19 convalescents positive for SARS-CoV Nc antibodies it reached 60.0 (40.0–73.3)%. The number of contact persons comprised 6285 subjects or 8.5% of total volunteer cohort, with the level of seroprevalence reaching up to 25.3 (17.95–35.8)%. A direct correlation was revealed between levels of seroprevalence in convalescent and contact volunteers. In addition, the reproductive number for SARS-CoV was calculated comprising 5.8 (4.3–8.5) suggesting that one convalescent subject can infect at least 4 healthy individuals. A high level of asymptomatic forms of COVID-19 among seropositive subjects was confirmed empirically comprising up to 93.6 (87.1–94.9)%.Conclusion. A single cross-sectional study performed during 2020 June–August timeframe allowed to assess pattern of sex- and agerelated COVID-19 seroprevalence for general population in 26 Russian Federation regions. The data obtained may serve as a basis for the longitudinal cohort investigation with serial subject sampling. The timing and duration of study will be determined by dynamics of ongoing COVID-19 epidemic