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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.

A total of 1935 migratory birds from 104 different species were captured in southeastern Sweden in 2005-2006 and tested for antibodies against West Nile virus (WNV). Overall, 46 birds (2.4%; binomial confidence limits, 1.8-3.2) were positive by blocking-ELISA, but only 2 (0.10%; binomial confidence limits, 0.0-0.4) had antibodies detectable by both blocking-ELISA and WNV neutralization test. ELISA-positive birds included long- and short-distance migrants likely exposed to WNV while wintering in or migrating through areas enzootic for WNV. Exposure to a cross-reactive Flavivirus was suspected for short-distance migrants of the Turdidae family, but no cross-neutralization with tick-borne encephalitis and Usutu viruses was observed.

Mosquito collections with CDC light traps using dry ice and pigeon-baited traps were carried out in south Moravia (Czech Republic) from April to October in 2007 and 2008 at two study sites. In 2007, 11 two-day captures were carried out in two-week intervals, and 1,490 female mosquitoes of nine species were caught. In 2008, 15 two-day trappings of mosquitoes were carried out: 6,778 females of 22 species of mosquitoes were trapped. The results showed marked differences in abundance and species composition of mosquitoes between both study sites and between the trapping methods. In the floodplain forest ecosystem of the Soutok study area, Aedes vexans predominated. The species composition in the Nesyt study site was more varied and the most common species was Culex pipiens. At the latter study site, Anopheles hyrcanus (var. pseudopictus) and Uranotaenia unguiculata, mosquito species with largely southern Eurasian distribution, were repeatedly demonstrated. The largest capture of mosquitoes was in traps with CO2 placed at a height 1 m above the ground. The capture of mosquitoes in the pigeon-baited traps as well as in the traps with CO2 placed in the canopy of trees was markedly lower in both study sites, with the predominant species being Culex pipiens.

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.

Six viral isolates were obtained from 23,243 female mosquitoes (examined in 513 pools) belonging to 16 species and collected along the lower reaches of the Dyje River in South Moravia (Czech Republic, central Europe) during 2006-2008: five isolates of Orthobunyavirus Tahyna (TAHV, California group, family Bunyaviridae: three isolations from Aedes vexans (Meigen), one from Ae. sticticus (Meigen), one from Culex modestus Ficalbi); and one isolation of Flavivirus West Nile (WNV, Japanese encephalitis group, family Flaviviridae)-strain Rabensburg (proposed lineage 3 of WNV) from Ae. rossicus (Dolbeshkin et al). All viral isolates were recovered from mosquitoes collected in 2006 (15,882 mosquitoes examined), while no virus was isolated from mosquitoes trapped in 2007 and 2008, when 1,555 and 5,806 mosquitoes were examined, respectively. The population density of local mosquitoes was very low in 2007 and 2008 because of warm and dry summer including a considerably low water table, compared with environmental conditions favorable for mosquito development in 2006. The virus isolation procedure was based on intracerebral inoculation of newborn mice. In parallel, more than one-third of the samples (183 pools consisting of 8,470 individual mosquitoes) were also examined by inoculating Vero cell cultures in Leighton tubes. However, the latter method detected only three of the six virus isolates (including WNV-Rabensburg). Ae. rossicus is a new potential vector for WNV-Rabensburg. This species feeds mostly on mammals including man; this raises the question whether this virus lineage is not adapted to an alternative mosquito-mammal cycle in the South-Moravian natural focus.

A total of 151 bacterial isolates were recovered from different developmental stages (larvae, nymphs and adults) of field-collected ticks (67 strains from Ixodes ricinus, 38 from Dermacentor reticulatus, 46 from Haemaphysalis concinna). Microorganisms were identified by means of 16S rRNA gene sequencing. Almost 87 % of the strains belonged to G(+) bacteria with predominantly occurring genera Bacillus and Paenibacillus. Other G(+) strains included Arthrobacter, Corynebacterium, Frigoribacterium, Kocuria, Microbacterium, Micrococcus, Plantibacter, Rhodococcus, Rothia, and Staphylococcus. G(-) strains occurred less frequently, comprising genera Advenella, Pseudomonas, Rahnella, Stenotrophomonas, and Xanthomonas. Several strains of medical importance were found, namely Advenella incenata, Corynebacterium aurimucosum, Microbacterium oxydans, M. schleiferi, Staphylococcus spp., and Stenotrophomonas maltophilia. Data on cultivable microbial diversity in Eurasian tick species D. reticulatus and H. concinna are given, along with the extension of present knowledge concerning bacterial flora of I. ricinus.

During a survey of mosquitoes in the South Moravian lowland area, the mosquito Anopheles hyrcanus (Pallas) (Diptera: Culicidae) was found breeding in an ancient fishpond (Nesyt). It is not clear whether this southern Palaearctic species, a known vector of malaria in Asia which has not been recorded in the Czech Republic until this year, has gone undetected in the past or whether it has recently moved into the region as a result of climate change.

A total of 178 free-living birds of 14 species of 7 families of Passeriformes sampled in a freshwater reedswamp habitat in southern Moravia in July 2006 were examined for hemagglutination-inhibiting (HI) antibodies to Alphavirus Sindbis (SINV), and bunyaviruses Tahyna (TAHV) and Batai (BATV). Hemagglutination-inhibiting antibody was detected against all three viruses, but at different frequencies: SINV 0.7%, TAHV 14.0%, and BATV 6.8%. The survey indicates circulation of mosquito-borne viruses TAHV and BATV and very low, if any, SINV activity in the area.

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.

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.

Questing Ixodes ricinus L. (Acari: Ixodidae) ticks were collected on a forest trail that had been completely cleared of shrubs and ground vegetation in winter 2002 and on a nearby control uncleared forest transect in South Moravia (Czech Republic). Samples were collected each May in 2003, 2004 and 2005. Nymphal ticks were 3.4 times, 1.9 times and 1.2 times less frequent on cleared forest than on uncleared forest trails in the three respective years, whereas adult tick abundance was 27.2 times, 4.0 times and 2.2 times lower, respectively. The ticks were examined for borreliae by dark-field microscopy: prevalence of nymphal ticks infected with Borrelia burgdorferi sensu lato (12.6% to 20.0%) did not differ significantly between the cleared and uncleared trail during the 3 years. In conclusion, the habitat modification appeared to result in a decreased abundance of I. ricinus as well as a reduced frequency of infected ticks (and thus indirectly a lower potential risk of Lyme borreliosis), which lasted, however, for only 2 years. Eight cultures of borreliae isolated from the ticks were all identified as the 'ornithophilic' genomic species Borrelia garinii, possibly indicating a greater role of forest birds than that of forest rodents as the hosts of immature I. ricinus in the tick (and borrelial) colonization of the cleared part of the forest.

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.

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.

Aim of this study was to evaluate cellular fatty acid analysis for characterization of spirochetes. Strains were isolated from arthropods collected in South Moravia, Czech Republic. Fatty acid methyl esters (FAME) profile was determined for five Borrelia burgdorferi sensu lato (s.l.) strains isolated from Ixodes ricinus ticks, one "Spironema culicis" strain recovered from mosquito Culex pipiens and seven spirochetal strains (not identified yet) isolated from mosquitoes and blackflies. Analysis was performed using a gas chromatography column in conjunction with Microbial Identification System Sherlock (MIDI Inc., Newark, DE, USA). Results obtained on the basis of cluster analysis of FAME profiles showed, that the B. burgdorferi sensu lato isolates could be well separated from other spirochetal isolates. We recommended method used in this study as a useful tool for preliminary identification of spirochetes isolated from ticks and dipterans.

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.