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.

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.

Antibodies to alphavirus, flavivirus, and bunyavirus arboviruses in house sparrows (Passer domesticus) and tree sparrows (P. montanus) in Poland.

Sparrows from central Poland were examined by a hemagglutination-inhibition test (titer > or = 20) for the presence of antibodies to arboviruses, between 1995 and 1996. In house sparrows (Passer domesticus) (n = 179), antibodies to Sindbis, West Nile, tick-borne encephalitis, Tahyna, and Calovo viruses were detected at seroprevalences of 1.1%, 2.8%, 1.1%, 2.8%, and 1.1%, respectively. In tree sparrows (P. montanus) (n = 33), antibodies to the Sindbis, West Nile, and Tahyna viruses were detected at seroprevalences of 9.1%, 12.1%, and 3.0%, respectively.

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).

Circulation of mosquito-borne viruses in large-scale sheep farms in eastern Slovakia.

Samples of blood sera collected from 608 sheep in large farms of the districts of Trebisov, Michalovce, Vranov nad Toplou and Humenné were investigated in hemagglutination-inhibition test for the presence of mosquito-borne viruses Sindbis, West Nile, Tahyna and Calovo. Antibodies to arboviruses Tahyna (11.5%) and Calovo (10.9%) were detected most frequently, while a lower sero-positivity was recorded to the Sindbis (2.3%) and West-Nile (1.0%) viruses. Certain differences were found in the infection rates in the respective districts: the highest frequency of antibodies to Tahyna and Calovo viruses was recorded in sheep of the district of Trebisov and the lowest one in animals of the district of Humenné.

Experimental infection of foxes with Tahyna virus.

Three foxes about 18 weeks old were each subcutaneously inoculated with approximately 200 LD50 dose of extraneurally passaged strain of the Tahyna virus "236". Viremia was demonstrated in all animals during the 48-96 hours interval after infection. The maximum value was 1.31 dex LD50/0.02 ml. Three weeks after infection a seroconversion was revealed by means of hemagglutination-inhibition test (from titre less than 1 : 10 to 1 : 320) and plaque-reduction neutralization test (from titre less than 1 : 4 to titre 1 : 4096 or 1 : 8192).

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.

A serological survey for Bhanja and tick-borne encephalitis viruses in sheep of eastern Slovakia.

755 sheep from 41 farms in the districts of Trebisov, Michalovce, Humenné and Roznava in the East Slovakian region were investigated on the presence of antibodies to Bhanja (neutralization test) and tick-borne encephalitis (haemagglutination-inhibition test) viruses. In respective districts 2.2%, 0.4%, 1.7% and 26.7% of animals (total 5.3%) were found to be positive on Bhanja virus, 4.4%, 1.5%, 1.7% and 5.0% (total 2.6%) on tick-borne encephalitis (TBE) virus. The considerable infection rate in sheep with Bhanja virus in the district of Roznava (mean 26.7%-on one farm as high as 63.9% serologically positive animals) can be explained by the local occurrence of the tick Haemaphysalis punctata, the main vector of this virus in Europe. The serologic screening conducted indicates a low activity of natural foci of TBE in eastern Slovakia today, and confirms the existence of a natural focus of infection with Bhanja virus in the district of Roznava. Recent circulation of Bhanja virus is indicated by antibodies detected in several one-year-old sheep.

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.

Isolation of Bhanja virus from ticks of the genus Haemaphysalis in southeast Bulgaria and presence of antibodies in pastured sheep.

Bhanja virus was isolated from ticks of the genus Haemaphysalis (H. punctata and H. sulcata) collected from sheep pastured in the surroundings of Akhtopol in southeast Bulgaria, and simultaneously blood sera of 58 sheep were investigated in plaque-reduction neutralization test with B lranja arbovirus. All sera contained antibodies to this virus in titres from 1:32 to 1:4096 (geometrical mean titre 1:494). The district of Akhtopol proved to be a natural focus of Bhanja virus.

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.

Prevalence of antibodies to tickborne encephalitis and West Nile flaviviruses and the clinical signs of tickborne encephalitis in dogs in the Czech Republic.

Blood sera from 151 dogs from areas of the Czech Republic endemic for human tickborne encephalitis (TBE) were examined for the presence of antibodies to TBE and West Nile (WN) flaviviruses by the haemagglutination-inhibition test Antibodies to TBE virus at titres equal to or exceeding 40 were found in five dogs. Antibodies to WN virus were detected in only one dog that also had a high antibody titre to TBE, suggesting this was a cross-reaction between the two closely related viruses. Three of the dogs (all rottweilers) with a TBE titre of 320 had clinical signs of meningoencephalitis or encephalitis. They all survived after treatment for the clinical signs. It was proved by seroconversion that the disease was caused by the TBE virus in one of these three dogs, and it seems very likely that the virus was responsible for the disease in the other two.

Eperythrozoon--a lesson for arbovirologists.

Eperythrozoon was detected in suckling mice inoculated with a suspension of nymphal Ixodes ricinus ticks. The organism was filterable through 220 nm Millipore membranes, moderately sensitive to diethyl ether, pathogenic to suckling but not to adult mice when given intracerebrally, intraperitoneally or subcutaneously, and mimicked an arbovirus. Eperythrozoon should be considered as an agent potentially interfering with experiments performed on laboratory mice.

Isolation and characterization of Sedlec virus, a new bunyavirus from birds.

A pathogenic agent designated AV 172 was isolated from the blood of a Reed Warbler (Acrocephalus scirpaceus) out of 767 samples from birds belonging to 35 species and 14 families. The birds (largely wetland passerines) were captured in the reed-belt littoral of Nesyt fishpond in southern Moravia, Czechoslovakia, during the years 1984 to 1987. Virus AV 172 has been found to represent probably a new species (designated virus "Sedlec") of family Bunyaviridae. Sedlec virus is pathogenic to suckling and adult mice when inoculated intracerebrally (i.c.) but not intraperitoneally (i.p.) and its ether-sensitive spherical particles measures 90-100 nm.

[Borrelia in ticks biting humans and prevention of Lyme borreliosis]. / Borrelie v klíst'atech parazitujících na cloveku a profylaxe lymské borreliózy.

We examined by dark field microscopy 195 of 209 Ixodes ricinus ticks that were removed from humans in the Czech Republic (mainly in southern Moravia) during 1997-2001. The majority of the ticks were nymphs (62%), 31% were females and 7% larvae; 10.7% of the nymphs, 20.3% of the females but no larvae were found to be infected with B. burgdorferi s.l., the agent of Lyme borreliosis (LB). This observation supports for Central Europe the conclusions drawn from North America about nymphal ixodid ticks as the most important vector stage in transmission of LB. In the course of the year, we found the highest proportion of infected nymphal and female ticks taken off humans in June. The detection of borreliae in a human-biting tick is an important step that enables the general practitioner to prescribe prophylactic antimicrobial treatment and to reduce significantly the risk of Lyme disease in the patient.

[Analysis of the incidence of tularemia in the Breclav District 1994-1999]. / Analýza výskytu tularémie na okrese Breclav v letech 1994-1999.

Southern Moravia is one of the areas where there are long-term natural foci of tularaemia. In 1994 in the Breclav district an exacerbation of the disease was recorded in hares. During autumn hunts a positive reaction was recorded in 5.75% of examined hares. An elevated seropositivity persisted also in subsequent years and at the same time a higher human morbidity in the mentioned region. The objective of the investigation was to assess the causes and circumstances which led to the exacerbation of old known but in recent years quiescent foci in the mentioned region. To this end 350 small terrestrial mammals were examined. In one case it proved possible to detect the causal agent in Microtus arvalis. By examination of arthropods it proved possible to isolate 33 strains, i.e. 32 strains from ticks Dermacentor reticulatus and one strain from Ixodes ricinus. The results of the examination revealed that tularaemia as a classical infection with a natural focus may persist in areas where there are prerequisites for survival of the causal agent in the environment. One of the important factors of the environment are susceptible individuals (hares, small terrestrial mammals) as well as blood sucking arthropods as vectors of the disease.