Selected phenotypic features of BR91, a unique spirochaetal strain isolated from the Culex pipiens mosquito.

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.

Serologic survey of humans for flavivirus West Nile in Southern Moravia (Czech Republic).

A serosurvey for West Nile virus (WNV) was carried out in 525 persons, using a plaque-reduction neutralization microtest (PRNmicroT) with Vero cells and Egyptian topotype Eg-101 strain as test virus. The blood sera were collected in four South-Moravian districts (Hodonin 44 persons, Breclav 102 persons, Znojmo 170 persons, Jihlava 209 persons) of the Czech Republic in the years 1988 and 1989, and stored at -20 degrees C. Antibodies to WNV were detected in only three humans (0.6% seropositivity): one person each in the districts of Hodonin (2.3% persons positive), Breclav (1.0% positive) and Jihlava (0.5% positive), with the titres of 1:64, 1:32, and 1:32, respectively. All the three sera were negative for antibody to tick-borne encephalitis virus. The results indicate that activity of WNV in southern Moravia was very low before 1990.

Novel spirochetes isolated from mosquitoes and black flies in the Czech Republic.

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.

Ribotyping and whole-cell protein analysis of spirochetes isolated from arthropods in the Czech Republic.

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.

Serologic survey of potential vertebrate hosts for West Nile virus in Poland.

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.

Isolation, cultivation and identification of Borrelia burgdorferi genospecies from Ixodes ricinus ticks from the city of Brno, Czech Republic.

A total of 305 ticks (21 larvae, 243 nymphs, 19 females and 22 males) were collected by flagging of vegetation in suburban woods of Pisarky Park (city of Brno) from July to October 2002. The midgut of each tick was dissected out and transferred individually into BSK-H medium. After cultivation, all specimens were examined by dark-field microscopy (DFM) for the presence of borreliae. Out of 305 tick samples, 45 were (14.8%) DFM positive. The following polymerase chain reaction (PCR) then revealed 37 (12.1%) samples positive for the presence of Borrelia burgdorferi sensu lato DNA. All 37 samples were further analysed by restriction fragment length polymorphism (RFLP) method. PCR-RFLP analysis revealed 14 strains of B. afzelii (37.8%), 15 strains of B. garinii (40.5%) and 2 strains of B. burgdorferi sensu stricto (5.4%). Four samples (10.8%) showed a mixed population of these genospecies. Two samples produced atypical RFLP pattern which were detected by sequence analysis as B. valaisiana (5.4%). Isolation attempts resulted in 21 spirochaetal strains (including two stains of B. valaisiana). The results show the diversity of B. burgdorferi s.l. in tick population and refer the risk of infection by pathogenic borreliae in Brno.

Prevalence of Borrelia burgdorferi sensu lato in the tick Ixodes ricinus in the Styrian mountains of Austria.

A total of 691 Ixodes ricinus (22 male, 39 female, 501 nymphs and 129 larvae), the tick vector of Lyme borreliosis, were collected by flagging from vegetation in 11 areas at altitudes between 789 m and 1350 m above sea level in mixed woodland with pasture land (cattle) in the province of Styria in Austria. The ticks were individually examined for presence of Borrelia burgdorferi sensu lato by dark-field microscopy and 107 of them by real-time PCR. Attempts to cultivate borreliae were made in BSK-H medium. The overall positivity rate of all collected ticks (excepting larvae) was 10.9%: 9.1% in males, 17.9% in females and 10.4% in nymphs. The 129 larvae examined showed no presence of B. burgdorferi s.l. The mean infection rate of I. ricinus collected at the highest altitude in this study, Gaberl at 1350 m a.s.l.--and at the same time the highest one reported in Europe--was 6.4%: 1/9 males, 2/18 females and 6/114 (5.3%) nymphs were positive. Culture attempts were positive in 12 cases and species identification showed eight isolates were B. afzelii and four B. garinii. Three additional positive results found by PCR (negative by culture) were identified twice as B. afzelii and once as B. garinii. This study shows that the risk of acquiring Lyme borreliosis in habitats at higher altitudes is limited, because of the lower density of I. ricinus and its lesser infection rate than at lower altitudes in central Europe, but nevertheless the risk does exist.

Import of West Nile virus infection in the Czech Republic.

We report West Nile virus infection of the central nervous system in a 69-year-old man, residing in North Moravia (Czech Republic), who visited the USA from 6 July to 31 August 2002. He developed fever with fatigue at the end of his US stay, and was hospitalized in Ostrava after his return on 3 September with fever (up to 39.5 degrees Celsius), fatigue, anorexia, moderate laryngotracheitis, dizziness, insomnia, blurred speech, and a marked bradypsychism. EEG demonstrated a slow bifrontal theta-delta activity, and CT of the brain a slight hydrocephalus. A significant increase of antibodies neutralizing West Nile virus was detected between the first (1:16) and second (1:256) blood serum sample. The patient recovered gradually and was released from hospital on 16 September. This is the first recorded human case of West Nile fever (WNF) imported to the Czech Republic. Nine similar cases of WNF import from the USA have already been reported in other European countries - France, Denmark, the Netherlands, and Germany.

Isolation of Borrelia afzelii from overwintering Culex pipiens biotype molestus mosquitoes.

A total of 662 samples (winter period: 469; summer period: 193 specimens) of female mosquitoes of the genus Culex, Aedes and Anopheles were collected during the period March 2000-April 2001 from the locality of Vysoke Myto (Eastern Bohemia, Czech Republic). They were examined by dark field microscopy for the presence of spirochetes. The motile spirochetes were observed in 4.2 % of all species of investigated mosquitoes. One spirochetal strain out of the 8 isolation attempts (BRZ14) was obtained (cultivation rate was 12.5 %) and the spirochetal strain was then successfully cultivated and identified using PCR for the presence of Borrelia burgdorferi s.l., and subsequently with the RFLP as genomospecies Borrelia afzelii. This strain was derived from overwintering Culex (Culex) pipiens biotype molestus female mosquitoe. This is apparently one of the sporadic cases of the occurrence of pathogenic borreliae in haematophagous arthropods, other than Ixodes ricinus complex ticks.

Mosquitoborne viruses, Czech Republic, 2002.

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.

Characterization of spirochetal isolates from arthropods collected in South Moravia, Czech Republic, using fatty acid methyl esters analysis.

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.

Occurrence of Borrelia afzelii and Borrelia garinii in Ixodes ricinus ticks from southern Moravia, Czech Republic.

During the years 1996-2000, a total of 2398 Ixodes ricinus ticks were collected in three areas of southern Moravia and eastern Bohemia, Czech Republic, and examined by dark-field microscopy for the presence of spirochetes. The prevalence of B. burgdorferi sensu lato in Ixodes ricinus ticks varied and depended upon the year. In 1996, the prevalence 6.8% was observed, during 1997 and 1998, it increased to 8.4% and 12.3%, respectively. The lowest prevalence was observed in 1999 (3.6%), and in 2000 it increased to 4.0%. The mean rate of infection was 6.5%, and the proportions of infected ticks were 12.2% in 263 male ticks, 8.3% in 289 female ticks, 6.0% in 1621 nymphs, and 1.3% in 225 larvae. From the total of 156 highly infected ticks (>100 spirochetes per sample) transferred into BSK-H medium for isolation attempts, 13 isolates were obtained. PCR-RFLP and electrophoresis (SDS-PAGE) were used for the identification and characterization of Borrelia strains. Ten tick isolates were identified as Borrelia afzelii, and the other three isolates were found to be Borrelia garinii. The results indicate the epidemiological importance of B. afzelii and B. garinii in central Europe, and emphasize the role of I. ricinus in the ecology of B. burgdorferi and in the epidemiology and epizootiology of Lyme disease.

Comparison of assays for the detection of West Nile virus antibodies in chicken serum.

Six tests for the detection of West Nile virus (WNV) antibodies in the serum of experimentally infected chickens were compared. The tests included the hemagglutination-inhibition test (HIT), immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (ELISA) with WNV-infected mouse brain antigen, immunoglobulin G (IgG) indirect ELISA with tickborne encephalitis viral antigen, the microtitre virus neutralization test, the standard plaque reduction neutralization test (PRNT), and the microtitre PRNT (micro-PRNT). Thirty adult chickens, intravenously and intramuscularly inoculated with 10(7) plaque-forming units (PFU) of WNV strain Egypt 101, were bled and given a booster of 10(7) PFU at 7,15, and 21 d postinoculation; the final blood collection was on day 28. Although the micro-PRNT is capable of detecting the highest antibody titres during both early and late infection, because of the technical complexity and time requirements of this test a combination of IgM and IgG ELISAs is recommended for serologic screening. Serum samples that give positive results in the ELISAs can then be tested by the micro-PRNT to determine the specificity of antibodies to WNV.

Prevalence of borreliae in ixodid ticks from a floodplain forest ecosystem.

Host-seeking ixodid ticks were sampled in a floodplain forest ecosystem along the lower reaches of the Thaya (Dyje) river in South Moravia (Czech Republic) and Lower Austria during the period 1989-2002. The ticks were examined by dark-field microscopy for borreliae (Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis), and attempts were made to culture the spirochetes in BSK-H medium from preparations containing their high numbers. Isolated borreliae were identified by PCR-RFLP analysis using probes directed against ribosomal spacer genes. A total of 797 nymphal and 719 adult (391 female, 328 male) Ixodes ricinus were examined: 16.2% of nymphs, 28.6% of females and 29.0% of males were positive. Dermacentor reticulatus (70 females, 30 males) and Haemaphysalis concinna (12 nymphs, 8 females, 2 males) were negative for spirochetes. The overall prevalence rate of borreliae in I. ricinus from the floodplain forest is slightly higher than the mean European data (i.e., 14% for nymphs, 21% for adults). The difference in infection rate between nymphal and adult ticks was significant, including the proportion of heavily infected (with > 100 borreliae) nymphs (2.1%) vs. adults (7.6%). Prevalence of borreliae in I. ricinus showed a significant decrease during autumn in this ecosystem. Three strains of spirochetes, all of the Borrelia afzelii genomic group, were isolated from female I. ricinus. Moreover, Trypanosoma/Crithidia sp. protozoa and Dipetalonema rugosicauda nematodes were detected in 0.4% and 1.0%, respectively, of all I. ricinus.

Host-seeking activity of ixodid ticks in relation to weather variables.

Ixodid ticks were monitored in a temperate deciduous broad-leaved forest in South Moravia (Czech Republic). Relative abundance of the ticks collected before noon (10.00-12.00 h) was compared to several weather variables (air and soil temperatures, relative humidity, precipitation, wind speed, and derived values) using the Pearson correlation coefficient. The tick numbers were found to be most closely related to the amplitude of the soil (-5 cm) temperature between 07 h and 14 h (TSamp, in Ixodes ricinus), and the soil temperature (TS) at noon (in Haemaphysalis concinna) or in the morning (Dermacentor reticulatus). While a growing amplitude in TSamp caused an increased host-seeking activity of I. ricinus and H. concinna, it suppressed the activity of D. reticulatus, a tick species mainly occurring in colder seasons of the year in Central Europe. The air temperature (TA) and relative humidity (RH) were also closely related to the tick activity, whereas rainfall and wind speed remained largely uncorrelated with the activity of the three tick species. Multiple linear regression on several variables (TSamp, TA, TS, TA-TS, RH) explained 48% of the variance in I. ricinus, 47% in H. concinna, and 38% in D. reticulatus. Predictive two-variable regression models of relative abundance in host-seeking ticks were based on morning temperature (TA or TS) and morning RH as the most important environmental factors: they explained 32% (I. ricinus), 39% (H. concinna), and 35% (D. reticulatus) of the variance.