Problems of Epidemiological Surveillance of West Nile Fever in Ukraine

ObjectiveTo define the problems of epidemiological surveillance of West Nile fever (WNF) in Ukraine.IntroductionFlaviviridae are one of the most widespread arboviruses in Ukraine. Mosquitoes are vectors of WNF in a majority of cases due to bites during swimming, fishing, work in suburban areas and outdoor recreation without use of individual protection from mosquitoes.A study of the species composition of bloodsucking mosquitoes is conducted in Ukraine. Existence of natural foci of WNF viruses has been well-proven all over the territory of Ukraine by testing IgG antibodies in different groups of population, including children [1]. Also, infection of mosquitoes (RNA found in Culex pipiens (including Culex pipiens f. molestus, Culiseta annulata)) was registered. Infection of I. ricinus and D. reticulates was also determined, and it acts as a factor for circulation of virus in the wild too [2].MethodsStatistical, serological and epidemiological methods were used during the study. Serological tests included reactions with IgM and IgG antibody in human serum performed using immunofluorescent and ELISA methods.ResultsIn Ukraine, the causative agent of WNF is detected in all landscapes. It is the main arboviral infection in the forest-steppe zone (53.1 % among all arboviral infections). Enzootic territories are located in 18 regions, 47 administrative districts, and 63 settlements.The majority of natural foci of WNF is located in the Dnieper left-bank steppes, and also in North-Western and Western forest-steppes. The enzootic territories are located on the East of steppe zone and on the East of forest-steppes. The smallest number of natural foci is registered in the Dnieper right-bank part of the steppes. Enzootic territories are absent in Chernivtsi, Chernihiv, Sumy, Ternopil, Luhansk, Kirovohrad Oblasts and Kyiv. Most of them are located in Zaporizhzhia with 9 administrative districts and 16 settlements; in Rivno Oblast - 7 and 9; in Kherson - 5 and 4, and in Poltava Oblasts - 2 and 4 respectively [3].During the period from 2007 to 2016, 86 cases of WNF were registered. WNF was registered in 7 oblasts (Zaporizhzhya - 40 cases, Poltava - 24, Donetsk - 16, Mykolaiv- 3, Kherson, Kharkiv, Zhytomyr Oblasts - one case in each) [4].Registration of WNF cases separately from other viral hemorrhagic fevers has been conducted in the country since 2010 (official registration of total amount of viral hemorrhagic fevers has been performed since 2005).In enzootic territories, 2 cases of the diseases were registered and were associated with ticks bites. The strains of WNV were detected in bloodsucking mosquitoes in Rivne and Zaporizhzhia Oblasts and in tick samples of Ixodes genus collected in Lviv Oblast (probably may be found in other species of tick (Argasidae and Gamazoidea) where the causative agent is kept in natural foci under unfavorable conditions).Laboratory diagnostics was conducted (mainly retrospectively) in Zaporizhzhia, Poltava, Donetsk Oblastss. All diagnoses (exception Mykolaiv Oblast in 2011, data is absent) were laboratory confirmed, including 10 cases confirmed in the State Institution Lviv Research Institute of Epidemiology and Hygiene of the Ministry of Health of Ukraine, and 3 more cases were confirmed by a private laboratory [2].In total, 129 samples of blood sera collected from patients with clinical manifestations of a fever of unknown origin were delivered to the Laboratory of Virology of Ukrainian Center for Diseases Control and Monitoring during 2016-2017. Samples were investigated using the immunofluorescent and enzyme immunoassay methods including immunoblot. West Nile virus markers such as IgM/IgG antibodies have been detected in 4 cases (Poltava oblast) [4].ConclusionsMainly, single cases were registered. It is caused by insufficient level of diagnostics in most of the regions, as a result, diseases pass under other diagnoses. Migratory birds (3 flyways of migratory birds pass through Ukraine) and local animals (crows, jackdaws, doves and other) may be the possible reservoirs of causative agent of WNF. Laboratory diagnostics need to be improved and more attention should be paid to testing of samples of blood serum from patients with suspected WNF.References[1] Rusev I.T., Zakusilo V.M., Vinnuk V.D. Bloodsucking mosquitoes of urbanized biocenosis and their role are in circulation of viruses of West Nile fever. Series are "Biology, chemistry". issue 24 (63). 2011. No. 2. p. 240-248.[2] Lozinskyi I.M., Beletska G.V., Drul O.S., Fedoruck V.I., Kozlovskyi M.M., Rogochiy E.G., Sholomey M.V., Ben I.I., Shulgan A.M./Epidemic situation of Western Nile fever in Ukraine. Magazine of infectology, issue 6, No. 2, 2014 Appendix 66-65.[3] Official data of state statistic form of the Ministry of Health.[4] Data of the State Institution Ukrainian center for Diseases Control and Monitoring of the Ministry of Health of Ukraine

Genotyping of pathogenic leptospira by Multiple Locus Variable-number Tandem Repeat Analysis (MLVA)

ObjectiveTo introduce the method of molecular genotyping (MLVA) to determine the genotype of field isolates of leptospira.IntroductionLeptospirosis (ictherohemoglobinuria, Leptospirosis biliousness) is a natural focal and zoonotic infectious disease dangerous for humans and farm animals. It is important to identify specific leptospira strains isolated from rodents or sick and suspicious animals by the serotype or genotype. In comparison with serotyping using micro agglutination test (MAT), molecular genotyping makes it possible to accurately identify a specific pathogen strain. The genetic classification now becomes more significant than the phenotypic classification.MethodsSpecific oligonucleotide primers, which flank fragments of the genome locus of pathogenic leptospira varies in terms of the number of tandem repeats VNTR-4, -7, -10 specific for L.interrogans, L.kirschneri, and L.borgpetersenii were used. The amplification products were detected using agar gel electrophoresis with the following identification of the fragment length with a molecular weight marker and comparison with the collection of VNTR profiles of the strains described in the literature.ResultsIt was established that the method of leptospira molecular genotyping by determining the number of variable tandem repeats of a locus (VNTR-variable number tandem repeats analysis) is suitable for molecular epizootology studies in Ukraine. The advantages of the method are the simplicity of performance and availability for diagnostic and research laboratories in Ukraine compared to other pathogen genome sequencing based genotyping methods, in particular Multilocus sequence typing (MLST) or Multispacer Sequence Typing (MST), which require complex equipment and operating conditions. The reference strain of Leptospira M20 serotype Copengageni serogroup Icterohaemorrhagiae from the NAAS IVM collection of was studied and its VNTR profile was identified with the genotype of the strain Fiocruz L1-130 that is described in the literature as a serotype of Copengageni serogroup Icterohaemorrhagiae. The genotype of the leptospira field isolate obtained from a rat in Lviv Oblast of Ukraine was specified and its identity was established in the aforementioned genotype. The obtained data support the prospects of using MLVA genotyping method to study the distribution of different genotypes of leptospira. The research will continue to study the specificities of molecular epizootology of leptospirosis in Ukraine.ConclusionsThe method of leptospira molecular genotyping by multilocus analysis of the number of variable tandem repeats has been tested in the Leptospirosis Research Laboratory in collaboration with the Museum of Microorganisms at the National Academy of Sciences, the Ukraine Institute of Veterinary Medicine, the ELISA and PCR Research Laboratory, and the Bila Tserkva National Agrarian University. The genotype of the reference strain has been correlated with its serological profile; identification of the genotype of the field isolate pathogenic leptospira has been completed. The tested method is planned to be implemented in surveillance and control over leptospirosis spreading in Ukraine, and aimed to help in development and improvement of leptospirosis vaccine formulations. Additionally, method of Multiple-Locus Variable number tandem repeat Analysis will be used for molecular epidemiology research in Ukraine.ReferencesSalaün L, Mérien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006;44(11):3954-3962. doi:10.1128/JCM.00336-06.Caimi K, Repetto SA, Varni V, Ruybal P. Infection , Genetics and Evolution Leptospira species molecular epidemiology in the genomic era. Infect Genet Evol. 2017;54(July):478-485. doi:10.1016/j.meegid.2017.08.013.Ayral F, Zilber AL, Bicout DJ, Kodjo A, Artois M, Djelouadji Z. Distribution of leptospira interrogans by multispacer sequence typing in urban Norway rats (Rattus norvegicus): A survey in France in 2011-2013. PLoS One. 2015;10(10):1-14. doi:10.1371/journal.pone.0139604

Volume-surface barrier discharge in dried air in three-electrode system fed by impulse high voltage with nanosecond rise time

Results of experimental investigation of a volume-surface barrier discharge in a three-electrode system under periodic impulse voltage applied to the surface discharge (SD) electrodes and a d.c. potential applied to an additional third electrode are presented. It is shown that there is a strong influence of polarity and amplitude of the d.c. potential on the direct current “extracted” out of the surface discharge plasma layer by electric field of the third electrode. The amount of charged positive species that constitute the “extracted” current prevails under positive impulse voltage for low values of the negative d.c. potential of the third electrode. The amount of negative species prevails with higher values of the positive d.c. positive of the third electrode

Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92–95% nucleotide or 99–100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine

Dermacentor reticulatus – a tick on its way from glacial refugia to a panmictic Eurasian population

International audienceThe ornate dog tick (Dermacentor reticulatus) shows a recently expanding geographic distribution. Knowledge on its intraspecific variability, population structure, rate of genetic diversity and divergence, including its evolution and geographic distribution, is crucial to understand its dispersal capacity. All such information would help to evaluate the potential risk of future spread of associated pathogens of medical and veterinary concern. A set of 865 D. reticulatus ticks was collected from 65 localities across 21 countries, from Portugal in the west to Kazakhstan and southern Russia in the east. Cluster analyses of 16 microsatellite loci were combined with nuclear (ITS2, 18S) and mitochondrial (12S, 16S, COI) sequence data to uncover the ticks’ population structures and geographical patterns. Approximate Bayesian computation was applied to model evolutionary relationships among the found clusters. Low variability and a weak phylogenetic signal showing an east–west cline were detected both for mitochondrial and nuclear sequence markers. Microsatellite analyses revealed three genetic clusters, where the eastern and western cluster gradient was supplemented by a third, northern cluster. Alternative scenarios could explain such a tripartite population structure by independent formation of clusters in separate refugia, limited gene flow connected with isolation by distance causing a “bipolar pattern”, and the northern cluster deriving from admixture between the eastern and western populations. The best supported demographic scenario of this tick species indicates that the northern cluster derived from admixture between the eastern and western populations 441 (median) to 224 (mode) generations ago, suggesting a possible link with the end of the Little Ice Age in Europe