Detection and genetic characterization of a putative novel Borrelia genospecies in Ixodes apronophorus / Ixodes persulcatus / Ixodes trianguliceps sympatric areas in Western Siberia.

Four genospecies from the Borrelia burgdorferi sensu lato complex were detected in Ixodes persulcatus and Ixodes pavlovskyi ticks from Siberia and genetically characterized. The presence of Borrelia spp. in Ixodes apronophorus and Ixodes trianguliceps ticks found in Asia has never been studied. In this study, genetic diversity of B. burgdorferi s.l. was investigated in three I. persulcatus / I. trianguliceps / I. apronophorus sympatric habitats in Western Siberia. Three groups of samples were examined: (i) ticks that were taken from rodents and molted in a laboratory; (ii) non-molted ticks collected from rodents; (iii) specimens from small mammals. Expectedly, Borrelia afzelii and Borrelia bavariensis were detected in I. persulcatus and in small mammals from the studied locations. Borrelia bavariensis was first found in molted I. apronophorus and I. trianguliceps. Identical genovariants of B. bavariensis were found in I. apronophorus, I. trianguliceps, and I. persulcatus. In addition, a new Borrelia genovariant was discovered in non-molted and molted I. apronophorus and non-molted I. persulcatus and I. trianguliceps, as well as in small mammals. This new genovariant was genetically characterized using MLST and single locus sequence analysis, which indicated that the new Borrelia genovariant significantly differs from all known Borrelia species. We propose the name "Candidatus Borrelia sibirica" for this putative new species.

Ixodes persulcatus/pavlovskyi natural hybrids in Siberia: Occurrence in sympatric areas and infection by a wide range of tick-transmitted agents.

Ixodes persulcatus and Ixodes pavlovskyi ticks, two closely related species of the I. ricinus - I. persulcatus group, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatus and I. pavlovskyi ticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyi hybrids in several locations with different ratios of parental tick species and to investigate the prevalence and genetic variability of a wide range of infectious agents in these hybrids compared to the parental tick species. Natural hybrids of I. persulcatus and I. pavlovskyi ticks were identified in all examined locations in Altai and Novosibirsk, Western Siberia, Russia. The abundance of hybrids varied from 7% to 40% in different locations and was maximal in a location with similar proportions of I. persulcatus and I. pavlovskyi ticks. For the first time, it was shown that hybrids can be infected with the same agents as their parental tick species: tick-borne encephalitis and Kemerovo viruses, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Rickettsia raoultii, Rickettsia sibirica, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis", and Babesia microti. The prevalence of most bacterial agents in hybrids was intermediate compared to their parental tick species. Most genetic variants of the identified agents have been previously found in the parental tick species. Wide distribution of I. persulcatus/pavlovskyi natural hybrids implies that I. persulcatus, I. pavlovskyi and their hybrids coexist in all I. persulcatus - I. pavlovskyi sympatric areas.

Dog survey in Russian veterinary hospitals: tick identification and molecular detection of tick-borne pathogens.

BACKGROUND: Species of Canidae in Russia can be infested with up to 24 different tick species; however, the frequency of different tick species infesting domestic dogs across Russia is not known. In addition, tick-borne disease risks for domestic dogs in Russia are not well quantified. The goal of this study was to conduct a nationwide survey of ticks collected from infested dogs admitted to veterinary clinics in Russian cities and to identify pathogens found in these ticks. METHODS: Ticks feeding on dogs admitted to 32 veterinary clinics in 27 major cities across Russia were preserved in ethanol and submitted to a central facility for examination. After identification, each tick was evaluated for infection with known tick-borne pathogens using PCR. RESULTS: There were 990 individual ticks collected from 636 dogs. All collected ticks belonged to the Ixodidae (hard ticks) and represented 11 species of four genera, Dermacentor, Ixodes, Rhipicephalus and Haemaphysalis. Four most common tick species were D. reticulatus, followed by I. persulcatus, I. ricinus and R. sanguineus. Ixodes persulcatus ticks were found to be infected with 10 different pathogens, and ticks of this species were more frequently infected than either D. reticulatus or I. ricinus. Ixodes persulcatus females were also more frequently co-infected with two or more pathogens than any other tick. Pathogenic species of five genera were detected in ticks: Anaplasma centrale, A. phagocytophilum and A. marginale; Babesia canis, B. microti, B. venatorum, B. divergens, B. crassa and B. vogeli; Borrelia miyamotoi, B. afzelii and B. garinii; Ehrlichia muris, E. canis and E. ruminantum; and Theileria cervi. Anaplasma marginale, E. canis, B. crassa, B. vogeli and T. cervi were detected in I. persulcatus, and Babesia canis in D. marginatum, for the first time in Russia. CONCLUSIONS: Multiple ticks from four genera and 11 species of the family Ixodidae were collected from domestic dogs across Russia. These ticks commonly carry pathogens and act as disease vectors. Ixodes persulcatus ticks present the greatest risk for transmission of multiple arthropod-borne pathogens.

Detection and genetic characterization of a wide range of infectious agents in Ixodes pavlovskyi ticks in Western Siberia, Russia.

BACKGROUND: The Ixodes pavlovskyi tick species, a member of the I. persulcatus/I. ricinus group, was discovered in the middle of the 20th century in the Russian Far East. Limited data have been reported on the detection of infectious agents in this tick species. The aim of this study was to investigate the prevalence and genetic variability of a wide range of infectious agents in I. pavlovskyi ticks collected in their traditional and recently invaded habitats, the Altai Mountains and Novosibirsk Province, respectively, which are both located within the Western Siberian part of the I. pavlovskyi distribution area. RESULTS: This study reports the novel discovery of Borrelia bavariensis, Rickettsia helvetica, R. heilongjiangensis, R. raoultii, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis" and Babesia microti in I. pavlovskyi ticks. In addition, we confirmed the previous identification of B. afzelii, B. garinii and B. miyamotoi, as well as tick-borne encephalitis and Kemerovo viruses in this tick species. The prevalence and some genetic characteristics of all of the tested agents were compared with those found in I. persulcatus ticks that were collected at the same time in the same locations, where these tick species occur in sympatry. It was shown that the prevalence and genotypes of many of the identified pathogens did not significantly differ between I. pavlovskyi and I. persulcatus ticks. However, I. pavlovskyi ticks were significantly more often infected by B. garinii and less often by B. bavariensis, B. afzelii, "Ca. R. tarasevichiae", and E. muris than I. persulcatus ticks in both studied regions. Moreover, new genetic variants of B. burgdorferi (sensu lato) and Rickettsia spp. as well as tick-borne encephalitis and Kemerovo viruses were found in both I. pavlovskyi and I. persulcatus ticks. CONCLUSION: Almost all pathogens that were previously detected in I. persulcatus ticks were identified in I. pavlovskyi ticks; however, the distribution of species belonging to the B. burgdorferi (sensu lato) complex, the genus Rickettsia, and the family Anaplasmataceae was different between the two tick species. Several new genetic variants of viral and bacterial agents were identified in I. pavlovskyi and I. persulcatus ticks.

Occurrence and genetic variability of Kemerovo virus in Ixodes ticks from different regions of Western Siberia, Russia and Kazakhstan.

Kemerovo virus (KEMV), a member of the Reoviridae family, Orbivirus genus, is transmitted by Ixodes ticks and can cause aseptic meningitis and meningoencephalitis. Recently, this virus was observed in certain provinces of European part of Russia, Ural, and Western and Eastern Siberia. However, the occurrence and genetic diversity of KEMV in Western Siberia remain poorly studied. Therefore, the aim of this work was to investigate the prevalence and genetic variability of KEMV in Ixodes ticks from Western Siberia. A total of 1958 Ixodes persulcatus, I. pavlovskyi ticks and their hybrids from Novosibirsk and Omsk provinces, Altai Republic (Russia) and East Kazakhstan province (Kazakhstan) were analyzed for the presence of KEMV and tick-borne encephalitis virus (TBEV) RNA. It was observed that the KEMV distribution area in Western Siberia was wider than originally thought and included Northern and Northeastern Altai in addition to the Omsk and Novosibirsk provinces. For the first time, this virus was found in Kazakhstan. The occurrence of KEMV was statistically lower than TBEV in most locations in Western Siberia. KEMV was found both in I. persulcatus and I. pavlovskyi ticks and in their hybrids. Notably, KEMV variants observed in the 2010s were genetically different from those isolated in the 1960s, which indicated the ongoing process of evolution of the Kemerovo virus group. Moreover, the possibility of reassortment for KEMV was demonstrated for the first time.

Genetic diversity of Ixodes pavlovskyi and I. persulcatus (Acari: Ixodidae) from the sympatric zone in the south of Western Siberia and Kazakhstan.

The most epidemiologically significant tick species in Siberia involved in transmission of a large number of pathogens causing human infectious diseases is Ixodes persulcatus. Ixodes pavlovskyi, being more active, also poses epidemiological threats. These tick species share morphology, activity seasons and geographic distribution range. In this paper, we characterize the geographic and genetic structures of I. persulcatus and I. pavlovskyi populations inhabiting the southern part of Western Siberia (Russia and Kazakhstan)--the western part of I. pavlovskyi distribution range. The data are based on six distinct Ixodes tick populations. Analysis of the concatenated mitochondrial marker sequences (16S rRNA and COI) and the nuclear sequence (ITS2) showed genetic polymorphisms in both I. persulcatus and I. pavlovskyi ticks inhabiting the sympatric zone. We could not determine the phylogeographic structure of I. pavlovskyi populations whereas for I. persulcatus significant within-region variance was shown. Notably, the abundance of I. persulcatus ticks negatively correlates with nucleotide and haplotype diversity in the concatenated sequence of mitochondrial gene (16S rRNA and COI) fragments. This is the first description of the genetic polymorphism of I. persulcatus and I. pavlovskyi ticks coexisting in a sympatric zone based on analysis of mitochondrial and nuclear markers.

Cooperative self-association of phosphorylase kinase from rabbit skeletal muscle.

Ca(2+)- and Mg(2+)-induced association of phosphorylase kinase (PhK) from rabbit skeletal muscle has been studied at the magnitudes of the ionic strength close to the physiological values (40 mM Hepes, pH 6.8, containing 0.1 M NaCl, 0.1 mM Ca(2+), 10 mM Mg(2+); 25 degrees C) and under the molecular crowding conditions produced by high concentrations (1 M) of the natural osmolyte, trimethylamine N-oxide (TMAO). In the presence of 0.1 M NaCl two forms of PhK were registered, namely the "basic form" and "highly associated form", suggesting that PhK association may be treated as an example of cooperative association. According to the data on dynamic light scattering the average hydrodynamic radii of these forms were 16 and 144 nm. The addition of 1 M TMAO produces the time dependent increase in the light scattering intensity caused by the conversion of the basic form into the highly associated form. According to the data of the sedimentation analysis the basic form of PhK comprises a hexadecamer (M(r)=1320 kDa) and its small associates. The removal of Ca(2+) by addition of EGTA results in the reverse conversion of the highly associated form into the basic form suggesting reversibility of self-association of PhK. FAD, the ligand that is specifically bound to PhK, blocks the conversion of the basic form of PhK into the highly associated form.

Effect of molecular crowding on self-association of phosphorylase kinase and its interaction with phosphorylase b and glycogen.

Self-association of phosphorylase kinase (PhK) and its interaction with glycogen (M=5500 kDa) and phosphorylase b (Phb) has been studied using analytical ultracentrifugation and turbidimetry under the conditions of molecular crowding arising from the presence of high concentrations of osmolytes. In accordance with the predictions of the molecular crowding theory, trimethylamine N-oxide (TMAO) and betaine greatly favor self-association of PhK induced by Mg2+ and Ca2+ and PhK interaction with glycogen. In contrast, proline suppresses these processes, probably, due to its specific interaction with PhK. All osmolytes tested prevented the complex formation between PhK and its physiological substrate, Phb. The specific interactions of PhK and Phb with glycogen, in the living cell, presumably is a factor allowing the negative effect of crowding on the recognition of Phb by PhK to be overcome.