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319 Borrelia-infection rates in tick and insect vectors accompanying human risk of acquiring Lyme borreliosis in a highly endemic region in Central Europe Petr Zeman Regional Center of Hygiene, Dittrichova 17, 120 07 Prague 2, Czech Republic Key words: Lyme borreliosis, retrospective epidemiological data, statistical mapping, arthropod vectors, direct immunofluorescence assay, correlation, Central Bohemia, geographical information system (GIS) Abstract. The methods of spatial statistics were applied to assess the geographical pattern of risk of Lyme borreliosis in Central Bohemia, the Czech Republic, based on retrospective data on disease contractions. The statistical risk was then compared at 15 selected localities with the infection challenge presented by ticks and insects carrying borreliae. Over 5,000 Ixodes ricinus (L.) ticks and 390 haematophagous dipterans were screened by direct immunofluorescence method, and the spatial and seasonal variance of infection rates were studied. Infected ticks were found at each locality throughout the warm season; in nymphs, sample infection rates ranged from 4.9% to 23.1% with a mean of 14.5% in spring, from 7.7% to 28.7% with a mean of 16.1% in summer, and from 7% to 20.6% with a mean of 13.6% in autumn. The statistical risk was found to correlate well with an average nymphal infection challenge, i.e. I. ricinus nymphal abundance × infection rate, at a given locality. Statistically significant cumulation of insect-history recalling patients into several, generally wetland, areas was ascertained; borreliae were revealed in 0.5% of the dipterans examined. Lyme borreliosis (LB) has recently been recognized as a prevalent zoonosis throughout Europe, though its incidence varies considerably with geographical position (Schmid 1985). Modulating factors involve the respective pathogen, reservoir and/or vector abun- dances, human access etc., which collectively depend on peculiar environmental conditions changing from site to site. The immediate threat to people would primarily be associated with the host-questing ticks in vegetation, predominantly nymphal and adult stages of Ixodes ricinus (L.), carrying borreliae (Anderson 1991, Jaenson 1991, Lane et al. 1991). Although both geographical and seasonal variability of the vector abundances and infection rates have been extensively studied in various biotopes, much less is elucidated how these factors influence an actual risk of acquiring LB at a given place. The aim of this study is to parallel local variations of the vectorial challenge with the concomitant risk of human infections as reflected in epidemiological data across a highly endemic region in Central Europe, and thus to contribute an insight into the geographical pattern of this disease. MATERIALS AND METHODS Epidemiological map. Retrospective data on patients contracting LB within the Central Bohemian region, the Czech Republic, between 1987-91 were obtained from an epidemiological register contributed to by clinicians; 867 reported places of acquisition (i.e. tick/insect bite) were pin- pointed in a digital map in a geographic information system (GIS), and a risk map of LB was computed using the methods of spatial statistics. A non-homogeneous Poisson model was applied that assumes the density of emerging disease cases l*(x) in vicinity of a location x to be a product of a local disease risk l(x) and the human population density h(x): l*(x) = l(x)h(x); while l(x) is the subject of estimation. It was performed by means of a non-parametric kernel smoothing method (Zeman1997), and the risk has been expressed relative to a regional mean; thus, values >1 indicate case densities higher than the mean (i.e. total No. of cases/ total area), and vice versa. The estimates were computed with a 0.5-km resolution and arranged in a cellular map (i.e. physically a GIS grid file). Furthermore, spatial variation of the mode of transmission was analyzed making distinction between ‘tick’ and ‘insect’ history; 632 and 235 patients recalled that the respective vector’s bite had preceded the disease onset. The located places of acquisition were split correspondingly into two separate point-pattern data-layers, and a map of log relative risk was computed by means of the method of Kelsall and Diggle (1995). It enables both testing the null hypothesis of constant ratio of the two reported vector types over the whole region, and, alternatively, to identify areas of unusually high or low involvement of either of them in the disease transmission (responsible for causing rejection of the null hypothesis). The same 0.5 × 0.5 km grid system as in the foregoing map was applied. Experimental localities. Five remote areas, which differ in mean levels of LB-risk on the epidemiological map and/or in natural conditions, were selected. Each area was represented by a triplet of localities ca. 3-4 km apart; to cover local habitat diversity, each of which, again, represented by a triplet of stations ca. 200 m apart adding up to about 10,000 m 2 of total area per locality. The first polygon (Křivoklát, Tři prameny, Adress for correspondence: P. Zeman, Regional Center of Hygiene, Dittrichova 17, 120 07 Prague 2, Czech Republic. Phone: ++420 2 884941; Fax: ++420 2 888287; E-mail: [email protected] FOLIA PARASITOLOGICA 45: 319-325, 1998

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Page 1: Borrelia-infection rates in tick and insect vectors accompanying …folia.paru.cas.cz/pdfs/fol/1998/04/08.pdf · 2021. 2. 28. · Key words: Lyme borreliosis, retrospective epidemiological

Zeman: Lyme borreliosis

319

Borrelia-infection rates in tick and insect vectors accompanying human risk of acquiring Lyme borreliosis in a highly endemic region in Central Europe

Petr Zeman

Regional Center of Hygiene, Dittrichova 17, 120 07 Prague 2, Czech Republic

Key words: Lyme borreliosis, retrospective epidemiological data, statistical mapping, arthropod vectors, direct immunofluorescence assay, correlation, Central Bohemia, geographical information system (GIS)

Abstract. The methods of spatial statistics were applied to assess the geographical pattern of risk of Lyme borreliosis in Central Bohemia, the Czech Republic, based on retrospective data on disease contractions. The statistical risk was then compared at 15 selected localities with the infection challenge presented by ticks and insects carrying borreliae. Over 5,000 Ixodes ricinus (L.) ticks and 390 haematophagous dipterans were screened by direct immunofluorescence method, and the spatial and seasonal variance of infection rates were studied. Infected ticks were found at each locality throughout the warm season; in nymphs, sample infection rates ranged from 4.9% to 23.1% with a mean of 14.5% in spring, from 7.7% to 28.7% with a mean of 16.1% in summer, and from 7% to 20.6% with a mean of 13.6% in autumn. The statistical risk was found to correlate well with an average nymphal infection challenge, i.e. I. ricinus nymphal abundance × infection rate, at a given locality. Statistically significant cumulation of insect-history recalling patients into several, generally wetland, areas was ascertained; borreliae were revealed in 0.5% of the dipterans examined.

Lyme borreliosis (LB) has recently been recognized as a prevalent zoonosis throughout Europe, though its incidence varies considerably with geographical position (Schmid 1985). Modulating factors involve the respective pathogen, reservoir and/or vector abun-dances, human access etc., which collectively depend on peculiar environmental conditions changing from site to site. The immediate threat to people would primarily be associated with the host-questing ticks in vegetation, predominantly nymphal and adult stages of Ixodes ricinus (L.), carrying borreliae (Anderson 1991, Jaenson 1991, Lane et al. 1991). Although both geographical and seasonal variability of the vector abundances and infection rates have been extensively studied in various biotopes, much less is elucidated how these factors influence an actual risk of acquiring LB at a given place. The aim of this study is to parallel local variations of the vectorial challenge with the concomitant risk of human infections as reflected in epidemiological data across a highly endemic region in Central Europe, and thus to contribute an insight into the geographical pattern of this disease.

MATERIALS AND METHODS

Epidemiological map. Retrospective data on patients contracting LB within the Central Bohemian region, the Czech Republic, between 1987-91 were obtained from an epidemiological register contributed to by clinicians; 867 reported places of acquisition (i.e. tick/insect bite) were pin-pointed in a digital map in a geographic information system

(GIS), and a risk map of LB was computed using the methods of spatial statistics. A non-homogeneous Poisson model was applied that assumes the density of emerging disease cases l*(x) in vicinity of a location x to be a product of a local disease risk l(x) and the human population density h(x): l*(x) = l(x)h(x); while l(x) is the subject of estimation. It was performed by means of a non-parametric kernel smoothing method (Zeman1997), and the risk has been expressed relative to a regional mean; thus, values >1 indicate case densities higher than the mean (i.e. total No. of cases/ total area), and vice versa. The estimates were computed with a 0.5-km resolution and arranged in a cellular map (i.e. physically a GIS grid file). Furthermore, spatial variation of the mode of transmission was analyzed making distinction between ‘tick’ and ‘insect’ history; 632 and 235 patients recalled that the respective vector’s bite had preceded the disease onset. The located places of acquisition were split correspondingly into two separate point-pattern data-layers, and a map of log relative risk was computed by means of the method of Kelsall and Diggle (1995). It enables both testing the null hypothesis of constant ratio of the two reported vector types over the whole region, and, alternatively, to identify areas of unusually high or low involvement of either of them in the disease transmission (responsible for causing rejection of the null hypothesis). The same 0.5 × 0.5 km grid system as in the foregoing map was applied.

Experimental localities. Five remote areas, which differ in mean levels of LB-risk on the epidemiological map and/or in natural conditions, were selected. Each area was represented by a triplet of localities ca. 3-4 km apart; to cover local habitat diversity, each of which, again, represented by a triplet of stations ca. 200 m apart adding up to about 10,000 m2 of total area per locality. The first polygon (Křivoklát, Tři prameny,

Adress for correspondence: P. Zeman, Regional Center of Hygiene, Dittrichova 17, 120 07 Prague 2, Czech Republic. Phone: ++420 2 884941; Fax: ++420 2 888287; E-mail: [email protected]

FOLIA PARASITOLOGICA 45: 319-325, 1998

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Zbečno) was situated on a plateau ca. 300 m a.s.l. intersected by a few deep valleys of rivers and brooks; it was largely wooded by an autochthonous, conserved deciduous forest composed mainly of beech and oak with ample undergrowth and scattered intrusions of spruce, larch and other conifers. The second polygon (Borek, Záryby, Dřísy) represents a mosaic of meadowy woods in the Elbe-river lowland composed mainly of alders, poplars, birches and ashes, with moderate undergrowth, growing on alluviums drained readily at mounds and elevated grounds where pine-trees dominate. The rest of localities (Pečice-Bohostice, Dalskabáty, and Horní Líšnice, in area 3; Vraník, Malovidy, and Rataje, in area 4; Adamov-Tupadly, Zbudovice, and Tisá skála, in area 5) encompass clearings and edges of cultivated spruce or mixed forests as well as interspersed secondary deciduous woods mainly of oak, hornbeam, maple and birch, mostly with abundant bushy undergrowth, in a moderately hilly tract of the region.

Entomological survey. Each experimental locality was inspected three times a year during the spring (late April-May), summer (late June-July) and autumn (September) of 1991. Ticks were collected by ‘flagging’ the vegetation with a flannel cloth; tick density was measured by No. of ticks caught per flag and hour. Since one-hundred specimen size per season and locality was adopted as a sine qua non for any reasonable statistical inference, the necessary time of flagging had been adequately extended at tick-deficient sites. Haematophagous dipterans were captured into a collapsable canopy-trap as well as caught individually in a net whenever

attacking humans. Extremely hot, cold or rainy days were avoided.

Laboratory assay. Extracted tick intestines were homogenized in a drop of PBS, let dry on a slide, methanol-fixed, and frozen. Dipterans were treated in the same way as ticks except mosquitoes, which underwent a ‘malariological’ dissection to split salivary glands and intestine into separate slide wells. Direct immunofluorescence assay (DFA) employing a commercial, goat polyclonal Borrelia burg-dorferi-affinity-purified conjugate (Kirkegaard & Perry Ltd., Gaithersburg, MD) for the detection of borreliae was then applied; the smears were co-stained with the conjugate and methylene-blue in the respective final dilutions of 0.006% and 15 ppm in PBS at 37°C for 45 min., washed thoroughly, closed in buffered glycerol, and examined under a fluorescent microscope. The conjugate’s working dilution was adjusted using the typical B-31 B. burgdorferi strain, and proved with laboratory strains of B. garinii, B. afzelii, and B. valaisiana, respectively; cross-reactivity with a strain of B. recurrentis was also observed.

Statistical evaluation. Tick numbers and DFA findings were pooled at each locality combining the trios of stations together. Comparison-of-Poisson-rates procedure was applied for testing differences between relative counts. A linkage of trends, i.e. whether the growing abundance of ticks was also accompanied by a growth of Borrelia-infection rate, or the other way round, was investigated by means of a contingency table.

Table 1. Yearly summary of Borrelia-infection rates in I. ricinus ticks in five different regions of Central Bohemia; a-g: signif. contrast at p = 0.05.

Locality ADULTS positive/examined

Percent positive

95%-fiducial interval

NYMPHS positive/examined

Percent positive

95%-fiducial interval

Tři prameny 5/27 18.5 3.9-33.2 48/305 15.7 11.7-19.8 Křivoklát 7/64 10.9 3.3-18.6 53/312 17.0 12.8-21.2 Zbečno 7/29 24.1 8.6-39.7 45/271 16.6 12.2-21.0

Subtotal area 1 19/120 15.8 9.3-22.4 146/888 16.4 14.0-18.9 Borek 12/49 24.5 12.5-36.5 54/253 21.3 16.3-26.4 Dřísy 3/34 8.8 > 0.0-18.4 27/252 14.7 10.3-19.1 Záryby 1/7 14.3 > 0.0-40.2 36/256 14.1 9.8-18.3

Subtotal area 2 16/90 17.8 9.9-25.7 127/761 16.7 14.0-19.3 H. Líšnice 5/44 11.4 2.0-20.7 49/308 15.9 11.8-20.0 Dalskabáty 6/35 17.1 4.7-29.6 32/307 10.4 7.0-13.8 Pečice-Bohostice 5/43 11.6 2.1-21.2 30/308 9.7 6.4-13.1

Subtotal area 3 16/122 13.1 7.1-19.1 111/923 12.0 9.9-14.1 Malovidy 35/108 32.4a 23.6-41.2 59/306 19.3c 14.9-23.7 Vraník 12/51 23.5b 11.9-35.2 67/310 21.6d 17.0-26.2 Rataje 1/51 2.0ab > 0.0- 5.8 21/301 7.0cd 4.1-9.9

Subtotal area 4 48/210 22.9 17.2-28.5 147/917 16.0 13.7-18.4 Adamov-Tupadly 4/28 14.3 1.3-27.3 35/309 11.3 7.8-14.9 Tisá skála 7/62 11.3e 3.4-19.2 47/314 15.0 11.0-18.9 Zbudovice 21/55 38.2ef 25.3-51.0 37/305 12.1f 8.5-15.8

Subtotal area 5 32/145 22.1g 15.3-28.8 119/928 12.8g 10.7-15.0 Grand total 131/687 19.1 16.1-22.0 650/4417 14.72 13.67-15.76

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Fig. 1. Geographical pattern of risk of LB in Central Bohemia as appeared from the statistical analysis of registered places of acquisition; clustering in highlighted areas meets the indicated thresholds of confidence. Encircled are the localities where the statistical and entomological risk estimates were compared. The blank area in the center is the city of Prague. (Reproduced with permission from International Journal of Epidemiology.) Fig. 2. Log relative risk of acquiring LB through the tick or insect bite according to self-reported data in cases from Central Bohemia; test of non-constant ratio gave p-value of 0.023, and the respective solid and dashed lines indicate where the 95%-tolerance limits of either insect or tick relative predominance are surpassed.

Fig. 3. An overlay of tick/insect log relative risk zonation from Fig.2 with hydrology (Inst. of Military Topography, Dobruška) in Central Bohemia; shown are ponds and lakes (>5ha), dams, and meandering lowland rivers (Elbe, Moldau). Notice that 75% of the shallow ponds (historically founded in wetlands) join the insect zone. RESULTS

Statistical map of the risk of LB is shown in Fig. 1; it also indicates the disposition of the experimental localities. It is seen that LB foci are scattered ± evenly almost over the whole region, mainly within its southern, well wooded part. In addition, there also emerged a few areas of decidedly raised risk (e.g., a belt along the Moldau river south of Prague, the Křivoklát forest stretched by the mid-west boundary, and several others). Figure 2 illustrates the relative contribution of the ‘tick’ and ‘insect’ reports to the statistical risk. The null hypothesis of overall uniformity must be rejected (p = 0.026), and several zones of significant dissim-ilarity can be delimited. The two aforementioned hyperendemic areas plus another, less wooded suburban area east of Prague, correspond with the zone where the ‘tick’ history quite prevail. In turn, some areas show excessive ‘insect’ cases; Fig. 3 documents that this zone largely corresponds with wetlands. In these areas, as much as 1/3-1/2 patients stated the insect bite history, and in places they even predominated (e.g. in a lowland-meadowy area close to the mid-eastern boundary).

Ixodes ricinus was the only tick species recorded. Infected ticks were proven at each locality throughout the study; in nymphs, sample infection rates ranged from 4.9% to 23.1% with a mean of 14.5% in spring, from 7.7% to 28.7% with a mean of 16.1% in summer,

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Fig. 4. Plot of prevalence of borreliae in nymphal I. ricinus against number of nymphs flagged per hr at 15 experimental localities in Central Bohemia (cumulative numbers for the whole year). Fig. 5. Plot of assumed (statistical, human cases-derived) risk of LB against I. ricinus-nymphal infection challenge (which=No. nymphs flagged per hr × Borrelia-infection rate) at 15 experimental localities in Central Bohemia (cumulative numbers for the whole year).

and from 7% to 20.6% with a mean of 13.6% in autumn. Generally, the infection rates increased from spring to summer and decreased again in autumn, however, all the differences but one were short of statistical significance; the exception is the nymphal data representing the whole third polygon (Malovidy, Vraník, Rataje) which significantly differ between summer (22.0%) and autumn (12.0%). An average tick questing activity (measured by nymphal abundances) at the experimental localities declined from spring through autumn, apart from three localities (namely Vraník, Dřísy, and Záryby) approaching rather the bi-modal activity course intermitted by a summer depression. A test for homogeneity within a 3 × 3 contingency table of grad-uating abundances of ticks vs. coincident Borrelia-infection rates (both ranked: minimum, medium, and maximum) failed to show any link between their trends. In general, there has not been evidenced any exclusive

Table 2. Haematophagous dipterans screened for borreliae.

Species Positive / examined Culicidae:

Aëdes cantans 1/44 Aëdes cinereus 0/1 Aëdes geniculatus 0/1 Aëdes vexans 0/9 Aëdes sp.(communis-rusticus) 0/6 Aëdes sp. 0/33 Anopheles plumbeus 0/1

Ceratopogonidae: Culicoides sp. 0/23

Simuliidae: Eusimulium latipes 0/1 Eusimulium sp. 0/12 Prosimulium sp. 0/1 Wilhelmia equina 0/86 Wilhelmia sp. 1/38

Tabanidae: Chrysops caecutiens 0/4 Chrysops relictus 0/2 Chrysops viduatus 0/5 Haematopota crassicornis 0/2 Haematopota pluvialis 0/77 Haematopota subcylindrica 0/4 Hybomitra bimaculata 0/2 Hybomitra distinguenda 0/3 Hybomitra lundbecki 0/1 Tabanus autumnalis 0/1 Tabanus cordiger 0/2 Tabanus maculicornis 0/3

Stomoxidae: Stomoxis calcitrans 0/17

Hippoboscidae: Lipoptena cervi 0/22 Lipoptena fortisetosa 0/5

Dipterans Total: 2/397 (0.5%)

dynamic pattern which distinguishes any locality from its neighbours or any area from the others.

Overall infection rates in ticks are summarized in Table 1; of 687 imagoes and 4,417 nymphs examined in total, the respective proportions 19.1% and 14.7% harbored borreliae. In spite of landscape diversity, no statistical differences of infection rates of ticks was proven between the five areas, however, some adjacent localities within them differed significantly (namely: Zbudovice/Tisá-skála in adults, and Malovidy/Rataje and Vraník/Rataje in both adults and nymphs). A plot of nymphal abundances vs. Borrelia-infection rates, both averaged for the whole year, shows no interdependence of these factors (Fig. 4). Paradoxically, the most tick-infested locality (Rataje) displays the least proportion of infected nymphs; an overall negative correlation, however, lacks statistical significance. The plot also indicates that the range of variation of tick abundances is almost twice as broad as that of Borrelia-infection rates (the respective coeffs. of variance were 50.8 and 27.3). Figure 5 plots an ‘infection challenge’, calculated as the product of nymphal abundance and Borrelia-infection rate, against the epidemiological risk estimates; their correlation manifests clearly. It proves

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that sole exposure to infected nymphs - disregarding other stages and vector species - may be significative enough for an overall public risk at a locality.

Finally, Table 2 documents the insect species examined; only two specimens - Aëdes cantans (Meig.) (Borek, spring) and Wilhelmia sp. (Malovidy, autumn) out of 397 dipterans (i.e. 0.5%) were found positive for spirochetes. In A. cantans, the positive specimen was the smear of salivary glands. The local compositions of the fly species attacking humans obviously harmonized with the particular landscape type: the black-flies chiefly predominated in the first, stream-crisscrossed area; the hematophagous fly association of the second, lowland-meadowy area was the most abundant dominated by mosquitoes and horse-flies; while the rest of areas exhibited a mediocre collection of sylvan species.

DISCUSSION

The epidemiological map of LB has already been discussed in detail in Zeman (1997). The hyperendemic areas of LB, particularly those along the Moldau river and in the Křivoklát forest, correspond well with early recognized foci of tick-borne encephalitis; the ‘tick’ anamnesis prevails in these co-endemic areas as seen in Fig. 2.

The prevalences of borreliae in Ixodes ricinus ticks recorded in this study accord with numbers already reported from this and neighbouring territories by other authors (Burger et al. 1985, Kméty et al. 1990, Kahl et al. 1989, 1992, Pokorný 1990, Pokorný and Zahrádková 1990, Hubálek et al. 1991, 1993, Pospíšil et al. 1992, Gupta et al. 1994, Siński et al. 1994, Wegner at al. 1994, etc.). Apart from one regional disparity between summer and autumn, the differences between seasonal infection rates in Central Bohemia were not statistically significant. Several studies throughout Europe were done to trace seasonal variation of Borrelia-infection rates in ticks (Hubálek et al. 1994, Kurtenbach et al. 1995, Wegner et al. 1994, Doby et al. 1995, Tälleklint and Jaenson 1996a); the two former surveys returned uniformity, but the latter ones showed some seasonal contrasts. While the estimates of statistical risk varied conspicuously from place to place and the local densities of ticks differed similarly, the prevalences of borreliae showed only minor geographical variation. So, one could assume that a risk assessment might be simplified to mere tick-density estimation, as has already been suggested for some localities in Scandinavia (Tälleklint and Jaenson 1996a). A close analysis revealed, however, that no strict correlation (p > 0.05) exists between the nymphal densities and the statistical risk (data not given). It seems, that such a risk assessment would be over-simplified. There was also no significant correlation (p > 0.05) evidenced between the respective tick densities and Borrelia-infection rates,

though the data might indicate a slight negative regression. This observation is, in turn, quite in harmony with findings in Scandinavia, where the infection rate in sparse tick populations moderately grows with nymphal density to dwindle again in dense populations (Tälleklint and Jaenson 1996b). Although no attempt to examine local animal associations was done in this study, there is no doubt that relative abundances of host species conditioned both tick densities and Borrelia-infection rates (Gray et al. 1992, 1995, Mejlon and Jaenson 1993, etc.).

Significant correlation of the statistical risk with the nymphal ‘infection challenge’ corresponds with the importance of Ixodes ricinus nymphs in the epidemiology of human LB (Matuschka et al. 1992). If the nymphal ‘infection challenge’-index is plotted against its epidemiological counterpart in Central Bohemia (Fig. 5), it is seen that magnitudes of 30-40 could be met on the risk ceiling, a range of 20-30 signifies serious risk responsible for numerous LB cases yearly, magnitudes between 10-20 are commonplace and betray moderate risk, and values below 10 mean low risk suggesting only sporadic cases. A rough comparison with similar estimates from Europe indicates that they would generally fall within the low or moderate risk grade in terms of this scale however some numbers, e.g. from South Sweden, suggest that the upper limit applicable for Central Bohemia may be passed significantly elsewhere (Tälleklint and Jaenson 1996a,b).

The epidemiological data might suggest an involvement of hematophagous insects in the trans-mission of LB in some areas, yet the role of insects in a circulation of borreliae remains in dispute. There have been published a few reports on cases of LB and/or related symptoms acquired through an insect bite (e.g. Balban 1911, Hård 1966, Doby et al. 1986, Luger 1990). Several retrospective epidemiological studies of LB also admit that some arthropods other than ticks have been contributory to disease transmission (e.g. Stanek et al. 1986); about 19% of ‘insect’ cases have been reported from the Czech Republic, as well (Markvart et al. 1988, Pazdiora et al. 1994, Bartůněk et al. 1996, etc.). Examinations of diverse field-collected dipterans actually revealed the carried borreliae (Magnarelli et al. 1986, Magnarelli and Anderson 1988, Halouzka 1993, Doby et al. 1994), however, it is expected that LB can be transmitted in a ‘shared dirty needle’-way only on insect mouthparts; experiments with mosquitoes failed to prove their vectorial potential (Magnarelli et al. 1986). Although low infection rates in the screened dipterans can be interpreted as a contamination from bloodmeal, the detection of numerous borreliae in salivary glands of Aëdes cantans is surprising in this context. This finding notably resembles that made by Sinton and Shute (1939) in

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English anophelines; it indicates that mosquitoes in Europe might occasionally transmit some Borrelia-like organisms in a biological way. Limited specificity of the polyclonal conjugate, however, thwarts decision whether they might belong to the B. burgdorferi s.lat. complex or whether they represent an antigenically related, but dissimilar spirochete. Overall, ticks have been found to be quite predominant as well as important vectors of borreliae in Central Bohemia, however

dipterans, spec-ifically mosquitoes, should not be omitted unless carefully reinvestigated. Acknowledgements. Mrs. E. Marečková, Mrs. E. Malá, Mrs. A. Kloudová, and Dr. V. Hanzal are thanked for their assistance in the field. The author is indebted to fy. Hoechst-Shering, AgrEvo, for contributing to this study. The Borrelia strains were kindly provided by Prof. E. Kméty (Bratislava), Dr. O. Kahl and Dr. B. Kissig (Berlin), and Dr. J. Chalupský (Prague).

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DOBY J.M., BIGAIGNON G., DOBY-DUBOIS M. 1995: Risque de contamination par Borrelia burgdorferi s. lato en milieu forestier. Suivi pendant 13 mois de l’abondance de la tique Ixodes ricinus et de niveau d’infestation par l’agent de borréliose de Lyme en Bretagne. Bull. Soc. Pathol. Exot. 88: 61-65.

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Received 9 April 1997 Accepted 22 January 1998