Search

Hantavirus disease is the most common rodent-borne viral infection in the Czech Republic, with a mean annual incidence of 0.02 cases per 100 000 population and specific antibodies detected in 1% of the human population. Four hantaviruses (Puumala, Dobrava-Belgrade, Tula, and Seewis) circulate in this country, of which Puumala virus (responsible for a mild form of hemorrhagic fever with renal syndrome called nephropathia epidemica) and Dobrava-Belgrade virus (causing haemorrhagic fever with renal syndrome) have been proven to cause human disease. The aim of this study is to provide a comprehensive review of the hantaviruses occurring in the Czech Republic, based on the literature published during the past three decades, including their geographical distribution and clinical symptoms. The recent detection of Tula virus in an immunocompromised person as well as reports of Seoul virus infections in Europe highlight the possible emergence of neglected hantavirus infections in the foreseeable future.

We examined 146 common coots (Fulica atra) on fishponds in central Moravia, Czech Republic, for antibodies to West Nile (WNV) and Usutu (USUV) flaviviruses. Eighteen birds reacted in the plaque-reduction neutralization test against WNV; these WNV seropositive samples were then titrated in parallel against USUV and tick-borne encephalitis virus (TBEV) to exclude flavivirus cross-reactivity. Two birds (1.4% overall) had the highest titers against WNV while 9 birds (6.2% overall) were seropositive for USUV, and in 7 birds the infecting flavivirus could not be differentiated with certainty. Our results indicate that both WNV and USUV infections occur in common coots; these birds might serve as a 'sentinel' species indicating the presence of these viruses at fishpond and wetland habitats in Central Europe., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The detection of West Nile virus (WNV) nucleic acid in a blood donation from Vienna, Austria, as well as in Culex pipiens pupae and egg rafts, sampled close to the donor's residence, is reported. Complete genomic sequences of the human- and mosquito-derived viruses were established, genetically compared and phylogenetically analyzed. The viruses were not identical, but closely related to each other and to recent Czech and Italian isolates, indicating co-circulation of related WNV strains within a confined geographic area. The detection of WNV in a blood donation originating from an area with low WNV prevalence in humans (only three serologically diagnosed cases between 2008 and 2014) is surprising and emphasizes the importance of WNV nucleic acid testing of blood donations even in such areas, along with active mosquito surveillance programs.

Introduction and Objective: Ixodes ricinus is the most common tick species occurring in Central Europe and it serves as a principal vector of emerging human pathogens. The aim of this study was to determine the prevalence of Babesia spp. in host-seeking I. ricinus in urban and natural habitats., Materials and Methods: PCR was applied on samples to assess prevalence of Babesia spp. in questing ixodid ticks. Sequencing was used for Babesia species determination., Results: 1,473 I. ricinus ticks (1,294 nymphs, 99 males and 80 females) were examined for the presence of Babesia spp. at the two study sites. Minimum infection rate for Babesia spp. was found to be 0.5% (infected I. ricinus nymphs were only detected in the natural ecosystem). Two Babesia species were identified by sequencing: B. venatorum (formerly called Babesia sp. EU1) and B. capreoli., Conclusions: The results obtained represent the first evidence of the occurrence of B. venatorum and B. capreoli in host-seeking I. ricinus ticks in the Czech Republic.

Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis," Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.

West Nile virus (WNV) is becoming more widespread and markedly effecting public health. We sequenced the complete polyprotein gene of a divergent WNV strain newly detected in a pool of Uranotaenia unguiculata mosquitoes in Austria. Phylogenetic analyses suggest that the new strain constitutes a ninth WNV lineage or a sublineage of WNV lineage 4.

The surveillance of vectors for arthropod-borne pathogens is nowadays an important tool in surveillance programmes throughout Europe. Whereas many studies have been performed to screen arthropods for viruses or bacterial pathogens, only limited information is available concerning the geographical distribution and vector range of pathogenic filariae in Central Europe. To consider the prevalence of filarial parasites in mosquito vectors, we performed a molecular survey of mosquitoes for filarial DNA. Mosquito collection was conducted at six study sites in the South Moravian region (Czech Republic) close to the borders with Slovakia and Austria from 2009 to 2011. Molecular screening of mosquitoes was conducted using conventional PCR with primers designed to amplify the mitochondrial cytochromoxidase subunit I gene as well as the partial 5.8S ribosomal RNA gene. A total of 13,222 mosquitoes belonging to six species were captured and distributed into 237 pools with different numbers of individuals. Overall, four pools were positive for Dirofilaria repens (a minimum infection rate 0.03%) at two study sites (both natural and urban). Another filarial parasite detected during a study into Aedes vexans mosquitoes revealed the closest homology to Setaria spp. We detected specific D. repens DNA in Ae. vexans mosquitoes for the first time in the Czech Republic and confirmed the circulation of Dirofilaria spp. in a natural focus of infection providing an epidemiological link between autochthonous canine cases and mosquito vectors in the area studied.

A total of 1279 field-collected Ixodes ricinus ticks were screened for Borrelia burgdorferi sensu lato (s.l.) in a natural and an urban ecosystem of Ostrava city (Czech Republic) by using molecular methods. Minimal prevalence rate for Borrelia burgdorferi s.l. in ticks for the urban park Bělský les was found to be 13.8% (17.6% in males, 17.8% in females and 11.7% in nymphs), similarly for the natural site Proskovice was minimal prevalence 15% (12.5% in males, 20% in females and 14.9% in nymphs). Six proven human pathogenic genomic species have been recorded in the study: B. afzelii, B. garinii, B. burgdorferi s.s., B. valaisiana, B. lusitaniae, and B. spielmanii. Emerging B. spielmanii was detected for the first time in Ixodes ricinus ticks in the region. Our results highlight the need for surveillance of zoonotic tick-borne pathogens even in urban areas.

Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1-1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1-542) or intermediate (TcdA1102-1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

The central European lineage of Usutu virus was isolated from a blackbird (Turdus merula), which was found dead in the city of Brno, Czech Republic, in 2011. The virus RNA was detected in two other dead blackbirds in Brno during 2012., (© 2012 Blackwell Verlag GmbH.)

The aim of this work was to retrospectively examine serum samples from 230 people living in two districts in the Czech Republic in year 1989 (Znojmo and Jihlava) for the IgG antibodies against hepatitis E virus (HEV). For examination, commercial ELISA kit DIA.PRO was used and 13 seropositive persons were found, i.e. 5.7% of those examined. The seropositivity rate was higher in women (6.8%) than in men (3.7%), but the difference was not statistically significant. The age of examined persons did not reveal a significant effect on the seropositivity rate: mean age of seropositive subjects was 38.0 years while that of seronegative persons was 39.4 years.

Growth temperature range, resistance to selective antibiotics, activities of 23 enzymes, protein fingerprints and fatty acids composition of the spirochaetal strain BR91, isolated from the Culex pipiens mosquito, were tested. The spirochaetes were grown in BSK-H Complete liquid medium. The optimal in vitro growth temperature of the strain was 33 °C. Strain BR91 was sensitive to trimethoprim, nalidixic acid, 5-fluorouracil, and tolerated phosphomycin. The strain produced acid and alkaline phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and α-fucosidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay revealed several major proteins in the size range of 13-16 kDa, 22-30 kDa and 37-131 kDa. Fatty acid methyl ester (FAME) analysis showed that C₁₄:₀, C₁₆:₀, C₁₈:₁ ω9c and summed feature 5 (C₁₈:₂ ω6,9c and/or C₁₈:₀ anteiso) are major fatty acids. This study highlights certain phenotypic differences between strain BR91 and the Lyme disease spirochaete Borrelia burgdorferi, and supports the hypothesis that strain BR91 represents a unique taxonomical entity in a system of spirochaetal species., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Tick-borne rickettsiae are an important topic in the field of emerging infectious diseases. In the study, we screened a total of 1473 field-collected Ixodes ricinus ticks (1294 nymphs, 99 males, and 80 females) for the presence of human pathogenic rickettsiae (Rickettsia helvetica, R. monacensis, 'Candidatus Neoehrlichia mikurensis', and Anaplasma phagocytophilum) in natural and urban ecosystems using molecular techniques. The minimum infection rate (MIR) for Rickettsia spp. was found to be 2.9% in an urban park and 3.4% in a natural forest ecosystem; for 'Candidatus Neoehrlichia mikurensis', we observed MIRs of 0.4% in the city park and 4.4% in the natural habitat, while for A. phagocytophilum the MIR was 9.4% and 1.9%, respectively. Our study provides the first data on the occurrence of human pathogenic rickettsiae in questing I. ricinus ticks in the Czech Republic., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

The objective of this chapter is to provide an updated and concise systematic review on taxonomy, history, arthropod vectors, vertebrate hosts, animal disease, and geographic distribution of all arboviruses known to date to cause disease in homeotherm (endotherm) vertebrates, except those affecting exclusively man. Fifty arboviruses pathogenic for animals have been documented worldwide, belonging to seven families: Togaviridae (mosquito-borne Eastern, Western, and Venezuelan equine encephalilitis viruses; Sindbis, Middelburg, Getah, and Semliki Forest viruses), Flaviviridae (mosquito-borne yellow fever, Japanese encephalitis, Murray Valley encephalitis, West Nile, Usutu, Israel turkey meningoencephalitis, Tembusu and Wesselsbron viruses; tick-borne encephalitis, louping ill, Omsk hemorrhagic fever, Kyasanur Forest disease, and Tyuleniy viruses), Bunyaviridae (tick-borne Nairobi sheep disease, Soldado, and Bhanja viruses; mosquito-borne Rift Valley fever, La Crosse, Snowshoe hare, and Cache Valley viruses; biting midges-borne Main Drain, Akabane, Aino, Shuni, and Schmallenberg viruses), Reoviridae (biting midges-borne African horse sickness, Kasba, bluetongue, epizootic hemorrhagic disease of deer, Ibaraki, equine encephalosis, Peruvian horse sickness, and Yunnan viruses), Rhabdoviridae (sandfly/mosquito-borne bovine ephemeral fever, vesicular stomatitis-Indiana, vesicular stomatitis-New Jersey, vesicular stomatitis-Alagoas, and Coccal viruses), Orthomyxoviridae (tick-borne Thogoto virus), and Asfarviridae (tick-borne African swine fever virus). They are transmitted to animals by five groups of hematophagous arthropods of the subphyllum Chelicerata (order Acarina, families Ixodidae and Argasidae-ticks) or members of the class Insecta: mosquitoes (family Culicidae); biting midges (family Ceratopogonidae); sandflies (subfamily Phlebotominae); and cimicid bugs (family Cimicidae). Arboviral diseases in endotherm animals may therefore be classified as: tick-borne (louping ill and tick-borne encephalitis, Omsk hemorrhagic fever, Kyasanur Forest disease, Tyuleniy fever, Nairobi sheep disease, Soldado fever, Bhanja fever, Thogoto fever, African swine fever), mosquito-borne (Eastern, Western, and Venezuelan equine encephalomyelitides, Highlands J disease, Getah disease, Semliki Forest disease, yellow fever, Japanese encephalitis, Murray Valley encephalitis, West Nile encephalitis, Usutu disease, Israel turkey meningoencephalitis, Tembusu disease/duck egg-drop syndrome, Wesselsbron disease, La Crosse encephalitis, Snowshoe hare encephalitis, Cache Valley disease, Main Drain disease, Rift Valley fever, Peruvian horse sickness, Yunnan disease), sandfly-borne (vesicular stomatitis-Indiana, New Jersey, and Alagoas, Cocal disease), midge-borne (Akabane disease, Aino disease, Schmallenberg disease, Shuni disease, African horse sickness, Kasba disease, bluetongue, epizootic hemorrhagic disease of deer, Ibaraki disease, equine encephalosis, bovine ephemeral fever, Kotonkan disease), and cimicid-borne (Buggy Creek disease). Animals infected with these arboviruses regularly develop a febrile disease accompanied by various nonspecific symptoms; however, additional severe syndromes may occur: neurological diseases (meningitis, encephalitis, encephalomyelitis); hemorrhagic symptoms; abortions and congenital disorders; or vesicular stomatitis. Certain arboviral diseases cause significant economic losses in domestic animals-for example, Eastern, Western and Venezuelan equine encephalitides, West Nile encephalitis, Nairobi sheep disease, Rift Valley fever, Akabane fever, Schmallenberg disease (emerged recently in Europe), African horse sickness, bluetongue, vesicular stomatitis, and African swine fever; all of these (except for Akabane and Schmallenberg diseases) are notifiable to the World Organisation for Animal Health (OIE, 2012)., (© 2014 Elsevier Inc. All rights reserved.)

Sedlec virus (SEDV) was isolated from the blood of a reed warbler (Acrocephalus scirpaceus) in July 1984 in South Moravia, Czech Republic. In this study first genetic data of SEDV are presented which allow an estimate on its phylogenetic and taxonomic positioning within the genus Orthobunyavirus. The phylogenetic analysis of a 369 nt long stretch within the S segment (nucleocapsid protein gene and non-structural S protein gene) indicates genetic relatedness of SEDV to Leanyer virus and Simbu group viruses, while the phylogenetic tree based on 1796nt long sequences of the L segment (RNA-dependent RNA polymerase gene) demonstrates genetic relationship of SEDV to two yet unclassified orthobunyaviruses: I612045 virus (isolated in India in 1961) and Oyo virus (isolated in Nigeria in 1964). Considering the genetic distances and the phylogenetic analyses, SEDV might represent a novel serogroup of the Orthobunyavirus genus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this study, we screened field-caught mosquitoes for presence of Dirofilaria spp. by using a polymerase chain reaction (PCR) assay. Potential occurrence of Dirofilaria repens and Dirofilaria immitis microfilariae was examined in 3,600 mosquitoes of eight species (Aedes vexans, Aedes cinereus, Aedes rossicus, Culex pipiens, Culiseta annulata, Ochlerotatus sticticus, Ochlerotatus cantans and Ochlerotatus caspius) collected from five locations in two districts (Kosice and Trebisov) of Eastern Slovakia, endemic region of canine dirofilariasis. Collection of mosquitoes was performed between May and August 2012 in premises known to be inhabited by Dirofilaria-infected dogs. PCR assays were performed on 72 pools, each pool containing 50 mosquitoes of the same species, collected on the same location. Each pool was examined separately for the presence of D. immitis and D. repens, respectively. A positive finding of D. repens was recorded in one pool of A. vexans mosquitoes collected in Košické Olšany village. Minimum infection rate in A. vexans was 1:1,750, i.e. 0.57 per 1,000 mosquitoes. The identity of D. repens was confirmed by direct sequencing of PCR product which has shown 100% homology with sequence attributed to D. repens (GenBank accession number AJ271614). This study represents the first molecular evidence of D. repens microfilariae in mosquitoes in Slovakia and highlights a need for better surveillance of zoonotic dirofilariasis in central Europe.

A serological survey for West Nile virus (WNV) infection involved 395 horses from 43 administrative districts of the Czech Republic (163 animals) and 29 districts of Slovakia (232 animals), sampled between 2008 and 2011. Using a plaque-reduction neutralization microtest, antibodies to WNV were not detected in any horse from the Czech Republic, whereas 19 nonvaccinated horses from Slovakia had specific antibodies to WNV (no cross-reactions were observed with tick-borne encephalitis and Usutu flaviviruses in those animals). The seropositivity rate of nonvaccinated horses in Slovakia was 8.3% (95% confidence interval [CI] 4.7-11.9%), and autochthonous local infection with WNV occurred at least in 11, i.e., 4.8% (95% CI 2.0-7.6%) of the animals. All seropositive horses lived in six lowland districts of southern Slovakia; overall, 15.1% (95% CI 8.8-21.4%) of 126 nonvaccinated horses were seropositive in those districts, situated relatively closely to the border with Hungary, i.e., the country where WNV disease cases have been reported in birds, horses and humans since 2003.

A set of 25 urease-producing, yellow-pigmented enterococci was isolated from environmental sources. Phenotypic classification divided the isolates into two phena. Both phena were characterized using 16S rRNA gene sequence analysis, DNA base composition, rep-PCR fingerprinting and automated ribotyping. The obtained data distinguished the isolates from all members of the genus Enterococcus with validly published names and placed them in the Enterococcus faecalis species group. DNA-DNA hybridization experiments, pheS and rpoA sequencing and whole-cell protein electrophoresis provided conclusive evidence for the classification of each phenon as a novel species of the genus Enterococcus, for which the names Enterococcus ureilyticus sp. nov. (type strain CCM 4629(T)  = LMG 26676(T)  = CCUG 48799(T)), inhabiting water and plants, and Enterococcus rotai sp. nov. (type strain CCM 4630(T)  = LMG 26678(T)  = CCUG 61593(T)), inhabiting water, insects (mosquitoes) and plants, are proposed.

The aim of this review is to present briefly background information on 27 tick-borne viruses ("tiboviruses") that have been detected in Europe, viz flaviviruses tick-borne encephalitis (TBEV), louping-ill (LIV), Tyuleniy (TYUV), and Meaban (MEAV); orthobunyaviruses Bahig (BAHV) and Matruh (MTRV); phleboviruses Grand Arbaud (GAV), Ponteves (PTVV), Uukuniemi (UUKV), Zaliv Terpeniya (ZTV), and St. Abb's Head (SAHV); nairoviruses Soldado (SOLV), Puffin Island (PIV), Avalon (AVAV), Clo Mor (CMV), Crimean-Congo hemorrhagic fever (CCHFV); bunyavirus Bhanja (BHAV); coltivirus Eyach (EYAV); orbiviruses Tribec (TRBV), Okhotskiy (OKHV), Cape Wrath (CWV), Mykines (MYKV), Tindholmur (TDMV), and Bauline (BAUV); two thogotoviruses (Thogoto THOV, Dhori DHOV); and one asfivirus (African swine fever virus ASFV). Emphasis is laid on the taxonomic status of these viruses, range of their ixodid or argasid vectors and vertebrate hosts, pathogenicity for vertebrates including humans, and relevance to public health. In general, three groups of tibovirus diseases can be recognized according to main clinical symptoms produced: (i) febrile illness-usually with a rapid onset, fever, sweating, headache, nausea, weakness, myalgia, arthralgia, sometimes polyarthritis and rash; (ii) the CNS affection-meningitis, meningoencephalitis or encephalomyelitis with pareses, paralysis and other sequelae; (iii) hemorrhagic disease. Several "European" tiboviruses cause very serious human (TBEV, CCHFV) or animal (LIV, ASFV) diseases. Other arboviruses play definite role in human or animal pathology though the disease is usually either less serious or infrequently reported (TYUV, BHAV, AVAV, EYAV, TRBV, DHOV, THOV). The other European arboviruses are "orphans" without a proven medical or veterinary significance (BAHV, MTRV, MEAV, GAV, PTVV, ZTV, SAHV, UUKV, SOLV, PIV, AVAV, CMV, OKHV, CWV, MYKV, TDMV, BAUV). However, certain arbovirus diseases of free-living vertebrates (but also those of domestic animals and even man) may often pass unnoticed or misdiagnosed and eventually, they might potentially appear as emerging diseases. Active search for new tiboviruses or for new, pathogenic variants of the known tiboviruses in Europe should therefore continue.

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK-H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host., (((c) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).)

During the years 1999-2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark-field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK-H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.

Purpose: To investigate the radiofrequency (RF) effects on the nonlactating and lactating ex vivo bovine udder as a model for normal breast tissue., Materials and Methods: RF ablation in three lactating and three nonlactating ex vivo bovine udders (ie, six udders) was performed. The opening of the electrodes was 3 cm. The temperature was applied in 10 degrees C increasing steps between 60 degrees C and 100 degrees C and each temperature was maintained for 15 minutes. The experiment was repeated three times for each temperature step in the lactating and nonlactating udder. Resected specimens were assessed histologically. The maximum diameter of the ablation zone and maximum width of the transition zone with respect to the temperature applied were measured., Results: In the nonlactating tissue, there was a correlation of the temperature and diameter of tissue damage. There was a narrow transition zone of 0.10 cm in all cases except at 100 degrees C, when it was 0.17 cm. In the lactating udder, no correlation was seen. The transition zone was not well visualized at temperatures less than 80 degrees C, ranging in overall size between 0.15 cm and 0.20 cm., Conclusions: The results of the study, with clear demarcation of the ablation zones and transition zones in the normal breast tissue, support the potential of breast thermal ablation as a viable treatment for further study. Lactating tissue does not seem ideal for thermal ablation. The discrepancy of the extent of ablation and the length of the electrodes is an important finding in this study. Further in vivo studies in normal glandular tissue and tumor are necessary.

In 2005 and 2006, Ixodes ricinus ticks were collected on two slag (waste rock) heaps from coal mines in the Ostrava area (North Moravia/Silesia, Czech Republic), Oskar (site A) and Emma (site B), partially covered by vegetation including trees, and at a control forest site near Hlucín (site C). The mean numbers of L. ricinus nymphs and imagoes flagged per person-hour were high: 35.3 nymphs and 12.7 imagoes, at site A, 23.3 and 26.0, respectively, at site B, and 25.4 and 16.8, respectively, at control site C. Using dark-field microscopy, 100 nymphs and 100 imagoes (50 females and 50 males) from each site were examined for borreliae. The mean prevalence rates of Borrelia burgdorferi sensu lato in nymphs and imagoes were 10.0% and 12.0%, respectively, at site A, 10.0% and 24.0%, respectively, at site B, and 13.0% and 17.0%, respectively, at site C. Differences in the prevalence of borreliae in nymphal and adult ticks from the slag heaps and control site were insignificant, but adult ticks from site B compared to site A contained borreliae significantly more frequently. The mean numbers of nymphs and imagoes infected with borreliae flagged per person-hour were 3.3 and 1.2, respectively at site A, 1.5 and 2.9, respectively, at site B, and 3.1 and 2.6, respectively, at site C. Isolation experiments for borreliae were carried out only in 16 ticks containing higher numbers of borreliae, with eight of these being culture-positive. The cultured borreliae were identified by PCR-RFLP as B. garinii (3 isolates: two from site B, one from site C), B. afzelii (4 isolates: one from site A, three from site B) and B. burgdorferi s.s. (one isolate from site A). Surprisingly, the results suggest that slag heaps, when covered by woody vegetation and frequented by humans, could theoretically pose roughly the same LB transmission risk to humans as common forest biotopes.

Blood sera collected from 400 domestic animals (260 cattle, 100 Merino sheep, and 40 Hutzul horses) in northeastern Hungary in 2005 were examined for antibodies against two tick-borne viruses, tick-borne encephalitis flavivirus (TBEV) and Bhanja bunyavirus (BHAV). Using ELISA as screening test and plaque-reduction neutralization as confirmatory test, seropositivity to TBEV was found to be 26.5% in cattle, 7.0% in sheep, and 0.0% in horses. Among cattle, the animals up to 3 years old had significantly lower seroprevalence rate than those in older age groups. Natural foci of tick-borne encephalitis are obviously present in northeastern Hungary. On the other hand, no antibodies neutralizing BHAV were detected in the domestic animals.

A serosurvey for West Nile virus (WNV) was carried out in 54 domestic birds (geese and ducks bred on fishponds) and 391 wild birds representing 28 migratory and resident species, using a plaque-reduction neutralization microtest with Vero cells and Egyptian topotype Eg-101 strain as test virus. The birds were sampled in the South-Moravian fishpond ecosystem between 2004 and 2006. Antibodies to WNV were not detected in domestic waterfowl, but 23 (5.9%) free-living birds of 10 species showed a positive response. These were the common coot (Fulica atra, 5 positive/18 examined), common kingfisher (Alcedo atthis, 1/1), reed warbler (Acrocephalus scirpaceus, 2/80), sedge warbler (A. schoenobaenus, 3/80), marsh warbler (A. palustris, 2/28), Savi's warbler (Locustella luscinioides, 3/12), reed bunting (Emberiza schoeniclus, 1/28), blackcap (Sylvia atricapilla, 2/11), penduline tit (Remiz pendulinus, 1/14), blue tit (Parus caeruleus, 1/1), and starling (Sturnus vulgaris, 2/4). The antibody titers were comparatively low (1:20-1:40), and the only high titer (1:160) was found in an adult marsh warbler. When 14 of the sera reacting with WNV were titrated in parallel with Usutu Flavivirus, 12 were interpreted as having specific antibodies to WNV, one coot had a higher titer against Usutu virus, and another one could not be attributed to either of the two viruses. In conclusion, 13 (3.3%) of 391 wild birds had specific antibodies to WNV. The results indicate that WNV activity in southern Moravia was limited during 2004-2006.

Central European encephalitis is the most common arthropod-borne virus disease in the Czech Republic, with the mean annual incidence of 6 cases per 100 000 population. However, seven less known arboviruses (Flavivirus West Nile, Bunyavirus Tahyna, Bunyavirus Batai, Bunyavirus Sedlec, Bunyavirus Lednice, Orbivirus Tribee, Uukuvirus Uukuniemi) also circulate in this country, of which West Nile, Tahyna, Tribec and possibly Batai have been reported to cause human disease. Moreover, antibodies against two other pathogenic viruses found in Europe, i.e. Alphavirus Sindbis and Coltivirus Eyach, have been detected in the Czech Republic. The aim of this study is to review briefly the less known arboviruses found in the Czech Republic with emphasis on the taxonomic status, identification of their hosts and vectors, and pathogenicity to humans. These arboviruses can cause febrile illness to aseptic (meningo)encephalitis of unclear etiology. The review points out the possible emergence of these neglected arboviruses in the foreseeable future and provides diagnostic guidance.

A survey for antibodies to West Nile virus (WNV; genus ,Flavivirus) was carried out by plaque-re-duction neutralization microtesting in 78 horses, 20 domestic chickens, and 97 wild birds belonging to 10 species from different areas in Poland. Specific antibodies were detected in five juvenile (hatching-year) birds collected in 2006: three white storks (Ciconia ciconia) in a wildlife rehabilitation center (5.4% of all examined storks; the antibody titers in each bird were 1:320, 1:160, and 1:20), one free-living mute swan (Cygnus olor; the titer was 1:20), and one hooded crow (Corvus corone cornix; the titer 1:20) in a wildlife rehabilitation center; thus the overall seropositivity to WNV was 5.2% among all the birds sampled. These data do not rule out the presence of WNV activity in Poland with 100% certainty, but they indicate a significant trace that demands verification. In addition, one black-headed gull (Larus ridibundus) had neutralizing antibodies for the Usutu Flavivirus, the first case recorded in Poland.

Usutu virus (USUV), family Flaviviridae, has been responsible for avian mortality in Austria from 2001 to 2006. The proportion of USUV-positive individuals among the investigated dead birds decreased dramatically after 2004. To test the hypothesis that establishment of herd immunity might be responsible, serological examinations of susceptible wild birds were performed. Blood samples of 442 wild birds of 55 species were collected in 4 consecutive years (2003--2006). In addition, 86 individuals from a birds of prey rehabilitation centre were bled before, at the peak, and after the 2005 USUV transmission season in order to identify titre dynamics and seroconversions. The haemagglutination inhibition test was used for screening and the plaque reduction neutralization test for confirmation. While in the years 2003 and 2004 the proportion of seropositive wild birds was <10%, the percentage of seroreactors raised to >50% in 2005 and 2006. At the birds of prey centre, almost three quarters of the owls and raptors exhibited antibodies before the 2005 transmission season; this percentage dropped to less than half at the peak of USUV transmission and raised again to almost two thirds after the transmission season. These data show a from year to year continuously increasing proportion of seropositive wild birds. The owl and raptor data indicate significant viral exposure in the previous season(s), but also a number of new infections during the current season, despite the presence of antibodies in some of these birds. Herd immunity is a possible explanation for the significant decrease in USUV-associated bird mortalities in Austria during the recent years.

In the presented work, six Borrelia burgdorferi sensu lato isolates were recovered from Ixodes ricinus ticks and one strain of spirochete with uncertain taxonomic position isolated from Culex pipiens mosquito collected in the Valtice area (South Moravia, Czech Republic), as well as representative type strains, were further characterized by ribotyping and whole-cell protein fingerprinting. The ribotype patterns obtained with HindII and EcoRV ribotype patterns showed good correlations with the recently proposed genotypes and grouped each genospecies in a well-separated cluster. The whole-cell protein profiles of genospecies were more heterogeneous than the ribotype patterns. The non-identified spirochetal strain was clearly separated from the Borrelia isolates in both methods thus proving their good capacity to characterize Borrelia burgdorferi sensu lato strains.

A total of 150 nymphal Ixodes ricinus (L., 1758) (Acari: Ixodidae) from the Czech Republic were examined for Anaplasma phagocytophilum (Foggie, 1949) Dumler et al., 2001 by PCR using EHR521/747 primers: 22 of 50 pools were positive (minimum prevalence, 14.7%). However, sequencing of the PCR products did not show complete homology with A. phagocytophilum (91%) while the closest relationship (95%) was found to "Candidatus Ehrlichia walkefii". The results indicate a need for care in interpretation of Anaplasma PCR results and for PCR optimization for detecting A. phagocytophilum in ticks.

Background: Epidermal cyst of the breast is a rare benign intramammary lesion; however, a common finding in other parts of the body and most often located in the scalp, back, and neck. Only a few cases of epidermal cysts of the breast have been reported in literature., Case Report: The patient presented with a small movable left breast lump. Mammography and ultrasonography did not show features of a benign lesion. Ultrasound-guided core biopsy was undertaken. Histology showed an epidermal cyst. The patient returned with an inflammation at the puncture site and a small abscess seen on ultrasound. The abscess was removed surgically., Conclusion: Mammographic and sonographic features of an epidermal cyst may mimick a malignant lesion. Biopsy can result in complications, such as inflammation. In addition, an association between epidermal cyst and squamous carcinoma has been reported. Therefore, it is recommended that these lesions are resected.

A total of 350 nymphs of the common tick Ixodes ricinus (Linnaeus, 1758) were collected in an endemic focus of Lyme borreliosis (South Moravia, Czech Republic) and examined for the presence of the protozoan Babesia microti (França, 1909) by polymerase chain reaction (PCR), using primers specific for the B. microti gene encoding small subunit rRNA. The assay revealed five positive pools (out of 70 pools examined); the corresponding prevalence rate was about 1.5%. Sequence analysis of the PCR products confirmed their 100% homology with that of B. microti. The study represents the first evidence of B. microti in ixodid ticks in the Czech Republic.

A flavivirus (strain 97-103) was isolated from Culex pipens mosquitoes in 1997 following floods in South Moravia, Czech Republic. The strain exhibited close antigenic relationship to West Nile virus (WNV) prototype strain Eg-101 in a cross-neutralization test. In this study, mouse pathogenicity characteristics and the complete nucleotide and putative amino acid sequences of isolate 97-103, named Rabensburg virus (RabV) after a nearby Austrian city, were determined. RabV shares only 75%-77% nucleotide identity and 89%-90% amino acid identity with representative strains of WNV lineages 1 and 2. Another RabV strain (99-222) was isolated in the same location 2 years later; it showed >99% nucleotide identity to strain 97-103. Phylogenetic analyses of RabV, WNV strains, and other members of the Japanese encephalitis virus (JEV) complex clearly demonstrated that RabV is either a new (third) lineage of WNV or a novel flavivirus of the JEV group.

Specimens from residents (N = 497) of an area affected by the 2002 flood were examined serologically for mosquitoborne viruses. Antibodies were detected against Tahyna (16%), Sindbis (1%), and Batai (0.2%) viruses, but not West Nile virus. An examination of paired serum samples showed 1 Tahyna bunyavirus (California group) infection.

In the Central-Bohemian area affected by the flood of 2002, 497 residents were screened for antibodies against the mosquito-borne viruses Tahyna (TAHV), West Nile (WNV), Sindbis (SINV) and Batai (BATV; syn. Calovo) using the haemagglutination-inhibition (HIT) and plaque-reduction neutralization (PRNT) tests. Blood samples were collected in September 2002 when the mosquito populations showed the maximum density following the flood. Antibodies against TAHV (16.5% persons in PRNT, 14.9% in HIT), SINV (1.4% in HIT) and BATV (1.4% in HIT, 0.2% in PRNT) were detected. Although 6.8% and 1.2% of the subjects tested reactive with WNV in HIT and PRNT, respectively, the results were interpreted as cross-reactivity with tick-borne encephalitis virus. The seroprevalence of TAHV (both in PRNT and HIT) showed no association with gender (15.8% of males, 16.9% of females), increased with age (1.4% of persons younger than 20 years, 11.2% of persons aged between 20 and 50 years, and 26.2% of persons older than 50 years were positive), and correlated with the mosquito peri-residential challenge (5.0% residents seropositive in a mosquito-free control zone D--mostly Prague, 14.7% in a mild-risk zone C, 20.5% in a moderate-risk zone B, and 28.0% in the most heavily mosquito-infested risk zone A). The highest TAHV seropositivity rate (> 25%) was found amongst the inhabitants of the villages Obríství, Kozly, Tuhan, Chrást, Chlumín and Hostín. Paired blood samples were obtained from 150 of the persons at a 6-month interval: an infection episode with TAHV during or after the flood was clearly evidenced in one person living in Obríství, and less convincing findings of recent TAHV infections were found in other three residents of Chlumín and Obríství (seroconversion and/or significant antibody titres increase detected in HIT only). This serosurvey indicated the existence of an active natural focus of Valtice fever (TAHV infection) stretched along the river Labe nearby Neratovice (Obríství, Chlumín, Tuhan; Kozly, Tisice, Chrást), and a low TAHV activity area along the lower reaches of the river Vltava between Zloncice and Bukol/Zálezlice. An increased population density of mosquitoes after the flood may have boosted the incidence of mosquito-borne virus diseases, particularly Valtice fever, in Central Bohemia. An optimum prophylactic strategy to control these diseases would be epidemiological surveillance (including monitoring of both the density of mosquitoes and their rate of infection with viruses in natural foci) on the basis of which antiepidemic measures such as integrated mosquito control can be taken.

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis., (Copyright 2003 Wiley-Liss, Inc.)

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu(2+) centre with the following EPR parameters: g(z) 2.24, g(x,y) 2.06, A(Cu)(z) 19.0 mT and A(Cu)(x,y) 1.0 mT. Simulation of the Cu(2+) spectrum yielded a best match by using four equivalent nitrogens (A(N)=1.5 mT, 42 MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu(2+), in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron-nuclear double resonance (CW ENDOR; 9.4 GHz, 5-20 K) experiments at g( perpendicular) of the Cu(II) spectrum show a weak coupling to protons with an A(H) of 2.9 MHz that corresponds to a distance of 3.8 A (1 A identical with 0.1 nm), assuming that it is a purely dipolar coupling. Incubation in (2)H(2)O leads to a significant decrease in these (1)H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu(2+) by diethyldithiocarbamic acid. The (1)H and (14)N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu(2+) enzyme, dopamine beta-mono-oxygenase (DbetaM). For DbetaM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and (2)H(2)O-exchangeable protons (2.9 MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40 MHz) from the co-ordinating N of the histidines in DbetaM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

An in situ hybridization method was applied to the identification of marine cyanobacteria assignable to the genus Prochlorococcus using horseradish-peroxidase-labelled 16S rRNA-targeted oligonucleotide probes in combination with tyramide signal amplification (TSA). With this method very bright signals were obtained, in contrast to hybridizations with oligonucleotides monolabelled with fluorochromes, which failed to give positive signals. Genotype-specific oligonucleotides for high light (HL)- and low light (LL)-adapted members of this genus were identified by 16S rRNA sequence analyses and their specificities confirmed in whole-cell hybridizations with cultured strains of Prochlorococcus marinus Chisholm et al., 1992, Prochlorococcus sp. and Synechococcus sp. In situ hybridization of these genotype-specific probes to field samples from stratified water bodies collected in the North Atlantic Ocean and the Red Sea allowed a rapid assessment of the abundance and spatial distribution of HL- and LL-adapted Prochlorococcus. In both oceanic regions the LL-adapted Prochlorococcus populations were localized in deeper water whereas the HL-adapted Prochlorococcus populations were not only distinct in each region but also exhibited strikingly different depth distributions, HLI being confined to shallow water in the North Atlantic, in contrast to HLII, which was present throughout the water column in the Red Sea.

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6-8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.