Jiří Halouzka

Distribution of Borrelia burgdorferi sensu lato genomic groups in Europe, a review

The survey is based on a total of 1263 records (738 isolations and 525 molecular DNA detections) of five Borrelia burgdorferi s.l. genomic groups available from 26 European countries: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana (= VS116) and B. lusitaniae (= PoTiB2). It shows the geographic distribution, the source (ixodid ticks 802 records, fleas 2 records, mosquitoes 2 records, wild mammals 66 records, human patients 391 records) and the association of the genomic groups with particular clinical manifestations of Lyme borreliosis in humans (B. afzelii significantly prevails in skin lesions whereas B. garinii is more often associated with neuroborreliosis). The most frequent genomic groups in Europe are B. garinii (501 records) and B. afzelii (469 records). They occur across the continent and islands, whereas the third frequent genomic group, B. burgdorferi s.s. (201 records), has only rarely been isolated in eastern Europe. The remaining genomic groups, i.e. B. valaisiana (85 records) and B. lusitaniae (7 records) have only been isolated from, or detected in, Ixodes ricinus ticks in a few European countries.

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe.

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0–11%), 10.8% (2–43%) and 17.4% (3–58%) for larvae (n = 5699), nymphs (n = 48 804) and adults (n = 41 666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17–20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed.

Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia.

Abstract. A total of 924 questing Dermacentor reticulatus (Fabricius), 504 Ixodes ricinus (L.), sixty Haemaphysalis concinna Koch and 718 mosquitoes (Aedes spp.) were examined in a floodplain forest ecosystem during the 1994‐95 outbreak of tularaemia in South Moravia, Czech Republic. Francisella tularensis was not isolated from H.concinna ticks or Aedes spp. mosquitoes, whereas twenty‐one isolates were recovered from the other haematophagous arthropods. Dermacentor reticulatus revealed a significantly higher infection rate (2.6%) than I.ricinus (0.2%). This tick species acts as principal vector for tularaemia in the enzootic focus. Monitoring of D.reticulatus for F.tularensis thus seems to be a very efficient approach in the surveillance of tularaemia in the flood‐plain forest ecosystems of Europe.

Serologic survey of birds for West Nile Flavivirus in southern Moravia (Czech Republic).

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), Savis 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.

Mosquito (Diptera: Culicidae) Surveillance for Arboviruses in an Area Endemic for West Nile (Lineage Rabensburg) and Ťahyňa Viruses in Central Europe

ABSTRACT 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 Ťahyňa (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.

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.

Longitudinal surveillance of the tick Ixodes ricinusfor borreliae

Host‐seeking Ixodes ricinus (L.) (Acari: Ixodidae) were monitored for borreliae (Borrelia burgdorferi s.l.) using dark‐field microscopy in South Moravia (Czech Republic) each May from 1991 to 2001 (150 nymphs, 100 females and 100 males each year). This survey revealed a mean annual percentage of infected ticks of 16.8% (range, 11.7–24.2) in nymphs, 24.9% (range, 16.5–33.6) in females and 26.1% (range, 17.1–37.3) in males. Annual incidence of Lyme borreliosis in humans of the area in the same period (range, 8.7–41.7 per 100 000) correlated significantly with the frequency (number of ticks per flag per hour) of nymphs infected with >50 borreliae or all nymphal ticks, but not with the frequency of females, infected females or the infection rate (% of ticks infected) of either nymphal or female ticks. A prediction of the annual incidence of Lyme borreliosis, based on the frequency of heavily infected or all nymphal I. ricinus ticks, is feasible. The infection rate in I. ricinus correlated significantly with the North Atlantic Oscillation winter index of the last year (in nymphs) or of the year before last (in adults).

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.

Isolation of the spirochaete Borrelia afzelii from the mosquito Aedes vexans in the Czech Republic.

During the years 1993–1995, a total of 3580 culicine mosquitoes of six species were collected in South Moravia, Czech Republic, and examined by dark‐field microscopy for the presence of borreliae. Females of Aedes cantans, Ae. sticticus, Ae. vexans, Culex pipiens and Cx pipiens biotype molestus (but not Ae. geniculatus or Culiseta annulata) harboured spirochaetes, the frequencies ranging from 0.7% to 7.8%. One isolate (BR‐53) from Ae. vexans was identified as Borrelia afzelii genospecies. The potential role of mosquitoes in the epidemiology of Lyme borreliosis should be investigated.

Isolation of Borrelia afzelii from overwintering Culex pipiens biotype molestus mosquitoes

SummaryDuring the years 1995–1996, a total of 1,743 overwintering Culex pipiens biotype molestus female mosquitoes were tested for the presence of spirochetes in several localities in South Moravia, Czech Republic. The spirochetes were observed in 5% of the mosquitoes investigated. One of the five isolated strains of spirochetes (BR-84) was identified as Borrelia afzelii. The potential role of mosquitoes in the ecology and epidemiology of Lyme disease (LD) borreliae should be further investigated.