Mosquito (Diptera: Culicidae) surveillance for arboviruses in an area endemic for West Nile (Lineage Rabensburg) and Tahyna viruses in Central Europe.

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.

Presence of the mosquito Anopheles hyrcanus in South Moravia, Czech Republic.

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.

[Borrelia burgdorferi sensu lato in ixodid ticks from Ostrava slag heaps]. / Borrelia burgdorferi s.l. v klíst'atech na ostravských haldách.

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.

West Nile virus investigations in South Moravia, Czechland.

Seven virus isolates were obtained from 11,334 mosquitoes after the 1997 Morava River flooding in South Moravia (Czech Republic): 6 strains of Tahyna bunyavirus, California antigenic group (5 from Aedes vexans, 1 from Ae. cinereus), and 1 strain of West Nile flavivirus (WNV) from Culex pipiens. In 1999, one isolate of Tahyna virus from Ae. vexans and one isolate of WNV from Cx. pipiens were recovered from a total of 14,354 mosquitoes examined in the same area, whereas no virus was detected there in 1,179 overwintering mosquitoes (mostly Cx. pipiens) in March 2000. The infection rate of mosquitoes with arboviruses was significantly higher in 1997, the year of the flood and an enormously high population density of mosquitoes. Antibodies neutralizing WNV were detected in 13 of 619 (2.1%) hospitalized patients or persons seeking outpatient clinics of the area in 1997. Five of the seroreactors revealed clinical symptoms compatible with West Nile fever: in 2 of them (children), recent infection with WNV was confirmed by a significant increase of antibody titer between acute and convalescent serum samples.

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

We investigated the use of a TaqMan 5' nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (<100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.

Growth temperature ranges of Borrelia burgdorferi sensu lato strains.

Three strains of Borrelia burgdorferi sensu lato, representing three human pathogenic genomospecies (B31, B. burgdorferi sensu stricto; BR14, B. garinii; BR75, B. afzelii) were grown in BSK-H medium at different temperatures and the spirochaetal cells were counted by dark-field microscopy after 0, 4, 8, 16 and 48 days. Approximate optimum (minimum-maximum) temperatures for the in-vitro growth were found to be 33 degrees C (22-39 degrees C) in strain B31, 35 degrees C (20-40 degrees C) in strain BR75 and 37 degrees C (20-41 degrees C) in strain BR14. Maximum, optimum and minimum growth temperatures seem to be important characteristics of B. burgdorferi s.l. strains, with relevance for the symptomatology, epidemiology and epizoology of Lyme borreliosis.

Mosquitoes (Diptera: Culicidae), in contrast to ticks (Acari: Ixodidae), do not carry Francisella tularensis in a natural focus of tularemia in the Czech Republic.

In total, 9,167 mosquitoes (Aedes spp.) and 1,987 ixodid ticks--1,423 Dermacentor reticulatus (F.), 504 Ixodes ricinus (L.), and 60 Haemaphysalis concinna Koch--were examined in an active enzootic focus (floodplain forest-meadow ecosystem) of tularemia in South Moravia. Czech Republic. Although no F. tularensis was detected in mosquitoes or H. concinna, 30 isolates were recovered from D. reticulatus (infection rate, 2.1%) and 1 isolate from I. ricinus (infection rate, 0.2%). Ixodid ticks, especially D. reticulatus, but not mosquitoes serve as vectors (and perhaps reservoirs) of F. tularensis at this natural focus.

Borreliae in immature Ixodes ricinus (Acari:Ixodidae) ticks parasitizing birds in the Czech Republic.

Of 411 forest birds of 33 species examined near Valtice, Czech Republic, 29% were infested with Ixodes ricinus (L.); 2.2% were parasitized by Haemaphysalis concinna Koch. Borreliae were detected in 5.1 and 11.7% of larval and nymphal I. ricinus, respectively. None of the 13 H. concinna tested was infected. In total, 3.2% of the birds examined were parasitized by I. ricinus immatures infected by borreliae. Borreliae-containing ticks parasitized European robin, Erithacus rubecula (L.); Eurasian blackbird, Turdus merula L.; Blackcap, Sylvia atricapilla (L.); Eurasian chiffchaff, Phylloscopus collybita (Vieillot); Great tit, Parus major L.; and Eurasian jay, Garrulus glandarius (L.). The isolate BR-34 from a nymphal I. ricinus off a Eurasian blackbird had a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein pattern, reactions to monoclonal antibodies, and fragments of HindIII digested DNA probed with fla and ospA genes that suggested to us that it belongs to the genospecies Borrelia garinii. Free-living birds may be involved in the circulation of B. burgdorferi sensu lato principally as disseminators of infected ixodid ticks to new area.

Francisella tularensis from ixodid ticks in Czechoslovakia.

A total of 26,478 ixodid ticks (935 pools) were examined by intracerebral inoculation of suckling mice. Six species of ticks were tested: Ixodes ricinus (23,470 individuals), I. trianguliceps (12), Haemaphysalis punctata (831), H. concinna (39), Dermacentor reticulatus (69) and D. marginatus (2,057). The ticks were collected largely by flagging vegetation, a substantial minority (4%) from animals. Three strains of Francisella tularensis were isolated, one each from I. ricinus (males, district Breclav, southern Moravia), D. reticulatus (males, district Breclav) and D. marginatus (engorged females collected from sheep in Roznava district, eastern Slovakia). D. marginatus and D. reticulatus represent new vector species for Czechoslovakia.

Investigation of haematophagous arthropods for borreliae--summarized data, 1988-1996.

Blood-sucking arthropods, collected in South Moravia, Czech Republic, were examined by darkfield microscopy for borreliae from 1988 to 1996. Among host-seeking ixodid ticks (8481 Ixodes ricinus (L.), 372 Dermacentor reticulatus (Fabr.), 167 Haemaphysalis concinna Koch), borreliae were only observed in adult (23.2%), nymphal (17.2%) and larval (6.3%) I. ricinus. The prevalence of borreliae in I. ricinus did not vary considerably among habitats except for lower values in agroecosystems, xerothermic oak woods and grasslands. The frequency of intensity of spirochaetal infection (log10 counts of borreliae per tick) in I. ricinus approximated the negative binomial distribution. The proportions of host-seeking female and nymphal ticks containing > 100 borreliae were 5.0% and 1.7%, respectively. Among preimaginal ticks (749 I. ricinus, 222 D. reticulatus, 82 H. concinna) parasitizing free-living forest birds and small mammals, borreliae were detected in 6.1% of larval and 10.3% of nymphal I. ricinus, and in one larval H. concinna; 3.2% of the birds and 19.4% of the mammals carried infected ticks. Among 3464 female mosquitoes (Culicidae) of 6 species, 4.1% contained spirochaetes: 1.4% of Aedes vexans Meig., 1.3% of A. cantans (Meig.), 2.2% of A. sticticus (Meig.), 2.2% of Culex pipiens pipiens L. and 5.9% of C. p. molestus Forskal. Borreliae were also detected in 8.4% of 142 fleas (Siphonaptera, largely Ctenophthalmus agyrtes Heller and Hystrichopsylla talpae Curtis) collected from small mammals. Twelve isolates of B. burgdorferi sensu lato have been identified to genospecies: 6 strains from I. ricinus (4 Borrelia garinii Baranton et al., 1 B. afzelii Canica et al. and 1 B. lusitaniae Le Fleche et al.), 1 strain from A. vexans (B. afzelii), 2 strains from C. agyrtes (B. afzelii), and 3 strains from host rodents (B. afzelii).

Isolation of a rickettsial organism (Eperythrozoon) from mice during virological testing of ixodid ticks.

A strain of Eperythrozoon (Rickettsiales: Anaplasmataceae) was isolated from suckling SPF mice (SM) inoculated with a homogenate of nymphal Ixodes ricinus (L.) ticks. The strain, passaged in the SM brain, was partially filterable through a 220-nm Millipore membrane, moderately sensitive to diethylether, and pathogenic for SM while no symptoms were observed in inoculated adult mice or male guinea pigs. A conspicuous cytopathic effect was produced in PS and SPEV (embryo pig kidney) stable cell lines. Both the cell cultures and SM could be used for the infectivity titration of the agent.

Results of arbovirological examination of birds of the family Hirundinidae in Czechoslovakia.

Migratory birds (swallow, Hirundo rustica; sand martin, Riparia riparia; house martin, Delichon urbica) caught in southern Moravia (Czechoslovakia) in 1984-87 were examined for arbovirus infections. Isolation experiments were carried out using blood samples of 183 birds (52 swallows, 107 sand martins, and 24 house martins). The results were negative. Serological examinations of 136 birds (36 swallows, 86 sand martins, and 14 house martins) were made by haemagglutination-inhibition test (HIT) using 6 arboviral antigens of the genera Alphavirus (Sindbis--SIN) and Flavivirus (tick-borne encephalitis--TBE, West Nile--WN) and of the family Bunyaviridae (Tahyna--TAH, Calovo, CVO, and Bhanja--BHA). Antibodies against all of the tested viruses were detected at different rates: SIN 2.9%, TBE 1.5%, WN 1.5%, TAH 4.4%, CVO 1.5%, and BHA 2.2%. The titres ranged from 1.20 to 1.80.

Haemagglutination-inhibiting antibodies against arboviruses of the families Togaviridae and Bunyaviridae in birds caught in southern Moravia, Czechoslovakia.

A total of 295 birds belonging to 19 species of 7 families of wild Passeriformes were examined by haemagglutination-inhibition test. The birds were caught for an international research program "Balt" at the time of autumn migration (August-September 1984). Their blood sera were examined for antibodies against 6 arbovirus antigens of the genera Alphavirus (Sindbis-SIN) and Flavivirus (tick-borne encephalitis-TBE, West Nile-WN) and family Bunyaviridae (Tahyna-TAH, Calovo-CVO and Bhanja-BHA). Antibodies against all studied viruses were detected at different frequencies: SIN 6.4%, TBE 7.1%, WN 9.7%, TAH 16.3%, CVO 12.1%, and BHA 1.0%.

Prevalence of borreliae in Ixodes ricinus ticks from southern Moravia, Czechoslovakia.

A total of 378 adult Ixodes ricinus ticks were collected by flagging vegetation in four localities of two districts (Breclav, Znojmo) in south Moravia and examined microscopically. Borreliae were identified in Giemsa-stained midgut smears from 32 (i.e. 8.5%) ticks (9.4% females, 7.2% males); the infection rate varied between 0.0 and 11.4% in the four localities examined. Among female ticks, significantly more were found to be infected in autumn (19.7%) than in spring (5.8%). Dark-field (DF) and Giemsa-stained smears (GS) examinations were compared for their sensitivity in detecting borreliae in 128 field-collected ticks; GS method showed a little higher sensitivity (11.7% ticks were positive) than DF procedure (9.4% ticks positive). Two strains of Borrelia burgdorferi were isolated from a total of 150 adult I. ricinus ticks cultured in BSK medium.

Persistence of Clostridium botulinum type C toxin in blow fly (Calliphoridae) larvae as a possible cause of avian botulism in spring.

Diverse samples were examined at a site of water-bird mortality, caused by Clostridium botulinum type C toxin in southern Moravia (Czechoslovakia). The toxin was detected in high concentrations in mute swan (Cygnus olor) carcasses (less than or equal to 1 x 10(6) LD50/g) as well as in necrophagous larvae and pupae of the blow flies Lucilia sericata and Calliphora vomitoria (less than or equal to 1 x 10(5) LD50/g) collected from them. It was detected in lower concentrations (less than or equal to 1 x 10(3) LD50/g) in other invertebrates (ptychopterid fly larvae, leeches, sow-bugs) associated with these carcasses, and occasionally in water samples (8 LD50/ml) close to the carrion. The toxin was not detected in the samples of water, mud or invertebrates collected at a distance greater than or equal to 5 m from the carcasses. The toxin-bearing larvae of L. sericata and C. vomitoria, containing 80,000 LD50/g of type C toxin, were exposed in the mud at the study site for 131 days from November to March. Although the toxin activity decreased 25-fold and 40-fold in the two samples of maggots exposed during this period, it remained very high (less than or equal to 3,200 LD50/g). Birds ingesting a relatively low number of these toxic larvae (or pupae) in the spring could receive a lethal dose of the toxin.

A serologic survey for some bacterial and viral zoonoses in game animals in the Czech Republic.

Between 1986 and 1991, sera were collected from 33 roe deer (Capreolus capreolus), 24 red deer (Cervus elaphus), four fallow deer (Dama dama), two mouflon (Ovis musimon), 34 wild boars (Sus scrofa), and 48 hares (Lepus europaeus) shot in two areas of the Czech Republic. Collectively, the sera contained antibodies to Coxiella burnetii (prevalence of 12%), Francisella tularensis (4%), Brucella spp. (2%), Central European tick-borne encephalitis virus (8%), Tahyna (California serogroup) virus (36%), and Calovo (= Batai) virus (23%). We propose that these mammals may play a role in maintaining natural foci of Q-fever, Tahyna fever and Calovo virus infection.

Francisella tularensis in Dermacentor reticulatus ticks from the Czech Republic and Austria.

Host-seeking adult Dermacentor reticulatus ticks were examined for the prevalence of Francisella tularensis in an active natural focus of tularemia along the lower reaches of the Dyje (Thaya) river in South Moravia (Czech Republic) and adjacent Lower Austria, in four localities of the flood plain forest-meadow ecosystem during the spring of 1996. The ticks were pooled (10 male or female ticks per group) and inoculated subcutaneously in 4-week-old SPF mice. Dead mice were sectioned, the spleens were homogenized in PBS and passaged in mice, and impression smears from the spleens, liver and heart blood were stained by Giemsa. Twenty-five isolates of F. tularenis were recovered from 1098 pooled D. reticulatus: the minimum infection rate (MIR) is 2.3%. MIRs for 629 female and 469 male D. reticulatus were 2.4% and 2.1%, respectively. The prevalence varied according to locality, but did not significantly differ between the Moravian (2.2%) and Austrian (2.8%) sites. The monitoring of D. reticulatus for F. tularensis might be a valuable contribution to the surveillance of tularemia in Europe.

Species identification of Borrelia burgdorferi sensu lato from tick and human isolates in Styria (Austria) by PCR-RFLP analysis.

Seventy-one isolates of Borrelia burgdorferi sensu lato (B.b.s.l.) derived from Ixodes ricinus ticks (50 strains) and patients (21 strains) were characterised by PCR-RLFP analysis. In four cases the human isolates were obtained from the cerebrospinal fluid (CSF) of patients with clinical symptoms of neuroborreliosis and in 17 cases from skin biopsies of patients with dermatological manifestation of Lyme borreliosis. Ixodes ricinus isolates originated from 14 localities in three regions (Mur valley, eastern and western Styria) in Styria. Thirty six strains of B.b.s.l. were isolated from nymphal ticks, nine strains from female and five strains from male ticks. Species identification of human isolates revealed three B. garinii and one B. afzelii isolates in CSF. In the PCR-RFLP analysis of 17 skin specimens a pattern for B. afzelii was found in ten cases, while six could be identified as B. garinii and one as a mixed infection of B. afzelii and B. garinii. Genetic characterisation of tick isolates resulted in 24 strains of B. afzelii (48%), 11 strains of B. garinii (40%) and 5 strains of B. burgdorferi s.st. (10%); one isolate showed a mixed infection of B. afzelii and B. garinii. Our findings indicate that B. afzelii and B. garinii predominate over B. burgdorferi s.str. in Ixodes ricinus ticks from Styria, which is similar to findings in neighbouring countries. This also reflects the occurrence of different pathogenic Borrelia strains in human samples.

Effect of pH on the stability of type-C toxin of Clostridium botulinum.

Stability of type-C botulinum toxin at pH 1.8-12.0 and during exposure to 5 and 28 degrees C for 20 and 16 h, respectively, was tested by titration on adult mice. The toxin was found in the samples kept at pH of 2.7-10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.

Enzyme activities of Borrelia burgdorferi sensu lato.

Activities of 19 enzymes were tested by the API ZYM system in 13 strains of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, B. valaisiana) grown in liquid BSK-H medium supplemented with rabbit serum. All strains produced acid phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Nine strains also produced alkaline phosphatase, and three strains produced alpha-glucosidase. The API ZYM system probably cannot be used for differentiation between B. burgdorferi sensu lato genomospecies.