Medical und Veterinary Entomology (1992) 6 , 165- 167

Borrelia burgdorferi infection in lxodes ricinus from habitats in Denmark A N N E S . L A N D B O and P E R T . F L O N G

Instituteof Biology,

University of Aarhus, Denmark

Abstract. A total of 2647 ticks of the genus Zxodes was sampled by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. A11 ticks were identified as Zxodes ricinus Linnaeus. A total of 317 ticks (202 nymphs and 115 adults) from three different sites were examined for the spirochaete Borrefia burgdorferi Johnson et al. by indirect fluorescent antibody staining; the frequency of infection varied from 7% to 22%. It is concluded that I . ricinus, known to be the most common tick in Denmark, is the vector largely responsible for the transmission of B. burgdorferi in this country. Key words. Zxodes ricinus, Borrelia burgdorferi, Lyme borreliosis, tick abundance, tick infection, Acan.

Introduction

Materials and Methods

Lyme borreliosis is a tick-borne human disease caused by infection with the spirochaete Borrelia burgdorferi Johnson et ul. (Burgdorfer et al., 1982). Ticks belonging to the genus Ixodes are the vectors responsible for transmission of the spirochaete (Stanek et al., 1988; Aeschlimann et ul., 1986; Kahl, 1991). Ticks are infected while bloodfeeding on vertebrate hosts. In North America the white-footed mouse (Peromyscus leucopus Rafinesque) appears to be the most important reservoir host of B.burgdorferi. In Europe the main reservoir hosts of Borrefiu are unknown, but various small rodents are suspected (Kahl, 1991). A wide distribution of Lyme borreliosis has been recorded. Cases have been reported from North America, Africa, Asia, Australia and Europe (Stanek et al., 1988; Nohlmans et al., 1990). In 1989,433 Danish patients were found serologically positive to B. burgdorferi. t>f these, 264 patients were clinically diagnosed as suffering from Lyme borreliosis (Hansen & Lebech, 1990). It is estimated that in Denmark approximately 1000 cases of Lyme disease occur each year. In several parts of Europe, e.g. Sweden, Germany, France, Switzerland, B . burgdoferi has been detected in ticks of the species lxodes ricinus but there have been no studies in Denmark. The objective of this study was to determine the occurrence and abundance of I.ricinus in different habitats in eastern Denmark and their infection with B.burgdorferi.

Sites and sampling methods. In autumn 1990 and spring 1991 adult and nymphal ticks were collected by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. Different habitats were represented, including deciduous forest, mainly old beech (Fagus sifvatzca),old oak (Quercus robur), ash (Fraxinus exelsior) and birch (Berula sp.), mixed deciduous forests, young and old mixed spruce forests and a site in marshy meadow and dry grassland respectively. A stripflag of blanket material with the dimensions of 1.0 x 0.7m and attached to a 1.5m batten was used as sampling equipment. An area of 12 x 10m2 was flagged at each site and the abundance of and species of ticks was determined (Arthur, 1963). In three sites, Frijsenborg, Hestehaven and Strandkaer, the incidence of ticks infected with B.burgdorferi was studied. Larval ticks were neglected since transovarial transmission of B. burgdorferi is considered to be insignificant (Aeschlimann etal., 1986). The three sites represented areas with reported cases of human Lyme borreliosis, and were further characterized by a high tick population density. By flagging the vegetation, three to nine ticks were sampled per lorn2. The three sites varied in habitat characteristics: Frijsenborg is a mixed deciduous forest with a well-developed field layer while Hestehaven is dominated by old beech (Fagus sylvaticu), with a sparse field layer. Wildlife is abundant in both sites including roe deer (Capreol:,s cupreofus Linnaeus), bank vole (Clethrionomys gfareolus Schreber) and yellow-necked field-mouse (Apodemus jlavicollis Melchior) (Flong, unpubl). Strandkaer is an open habitat with marshy meadows bordered by mixed deciduous

Correspondence: Dr A. S. Landbo, Institute of Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.

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forest. The meadows were grazcd by cattle. Wildlife was ctominatctl by small rodents. mainly C'.glureolirs and ,4 .jlot.iu)l(i.\ (Fiiing, unpubl. ). 1)twcrioti of Borrelia iri ticks. All ticks were kept in vials :it 18'C' and Y2% r.h. until they werc tested for Rorrrlici byindirect imniunofluoresccnce antibody method (IFA). In the test il R.hurgdorfiw DK-ECM-1 antigen w;is used (donated by Klaus tlansen. Statens Serum Institute. Copenhagen). 12 pooled rabbit antiserum was used ;is the first antibodv and ;i fluorescein conjugated swine anti-rabbit immunoglobulin (DAK(>-patt F205) were used as second antibody in the test. Before testing the ticks, a chessboard titration was made with antiserum and conjugate in dilutions with phosphate buffered saline (PRS).p H = 7.4. 'The highest dilutions giving maximum titres werc 1:100 for the rabbit antiseruni and 1:400 for the conjugate. These dilutions were chosen as standard dilutions in all test\. Tick5 were surfaced-cleansed with 70% alcohol prior to diswction. The midgut was removed and homogenized in PHS. Glass slides degreased with 96% ethanol were coated with 2.5 gl honiogenated suspension. air dried. fixed i n methanol tor lSmin, and air dried again. The slides were covered with 25 yl 1:200 rabbit antiserum diluted in f'BS and incubated for 3Oniin at 37°C in a moist chamber. After washing with PRS, the slides were covered with 25 111 fluorescein conjugated swine anti-rabbit immunoglohulin diluted 1:400 in I'RS and incubated for another 30min at 37°C' i n a moist chamber (Hansen Xr Dietz. 1989). A positive and a negative serum control were carried out.

Results A total of 2647 ticks (1456 adults and 1191 nymphs) were collectcd from thirty-one sites and identified. All ticks belongcd to the species, 1.ricitiits being recorded from all sites except for old spruce forests and dry grassland. The abundance of 1. ricitiit.r varied considerably between sites (Table 1). Ticks were most common in deciduous beech forests. mixed deciduous forests and in marshy meadows. In sprtice forests, ticks were absent or few in number. Of the 2647 ticks collected, 202 nymphs and 115 adults from the sites Frijsenborg. Hestehaven and Strandkaer were investigated by IFA-method. The presence of Borreliu was coiifirnmt i n 1.riciriu.s from all three sites. In adults Table 1. Mean iiuinbcr o f tick.; collected i n seven hahitat types in Denmark. No. of

sites

Habitat type .-

Mean no ( ISD) of ticks collected per 12 x 10 m'

______

...~

Hzccti tc.rcst Oak forz\l

Birch foresi Mixed tlcciduous forest Spruce t t m s t

Marsh!, mcadou I>r! grassland

16 62 2 1 05 4.33 = 2 96 23 0 23 3 3 5 11 28 233COIW

58 0 00

Table 2. Infection ratc of H.6uugdorfiri in Danish I,ric.inri.c licks as dcrnonstrated by thc IFA method.

Ticks cxamiried

NO

"4) positive

Adult

81 82 167

19.8 23.2 21.5

Nymph 'rotai

Fri jscnborg

_ _ __

Adult

18

Nymph Total

7s 93

1 I .o 24.0 21.5

Adult

16 45 61

0.0 8.9 0.h

Nymph Tot ii I

and nymphs from Hcstehavrn and Strandkaer an average infection incidence of 22% was found. I n f;rijsenborp art average of 7% of the ticks were infected (Table 2).

Discussion This study confirms that 1.ricintc.T is a common tick found on vegetation in Denmark. in agreement with Stanek r t ul. (1988). It is remarkable that n o other species were found. A similar result was obtained in investigaiions in seventy-six sites in Switzerland (Kaltenrieder et ul.. 1 OM). The thirty-one sites investigated represent different habitat types; 1.ricitzu.s was found in all habitats, except for old spruce forests with very sparse field layer and dry grassland. These habitats are probably too dry for survival of I.ricirzus, being unable to survive in microhahitats in which the relative humidity declines below 8094, for prolonged periods (Kahl RC Kniille. 1988). It is noticeable that the three sites, Frijsenborg. f-lestehaven and Strandkaer, in which very high tick population densities were found, represent different habitat types. O n the other hand. the three sites were characteri7ed by humid field layers with dense undergrowths or plenty of litter. The actual composition and physical structure of plant species seem to be of minor importance for maintenance of high tick densities. More likely presence and abundance of I.riciizu.y are positively influenced by high humidity and presencr of larger vertehratc hosts (Flijng Kr Landbo, 1992). Adult I. riciirus primarily feed on larger vertebrate hosts (Gray, 1991) for successful reproduction. The three sites of investigation were characterized by an abundant wildlife of larger mammals. The population densities o f C.capreoli~sin Hestehaven and in Frijsenborg are rinusu:illy high. At the Strandkaer site the abundance of 1.ricir11rs clearly depends on the presence of cattle in the summer (Flong & Landbo, unpubl.). In North America, the distribution of I.duinrniiii, one of the American vectors of Lyme borreliosis, is found t o be correlated with distribution of the white-tailed deer. Odocoilms virgiriimiu Zirnmerman (Wilson et al., 1990). The infection of 7-22% is i n agreement with similar investigations in other parts of Western Europe, e.g. in Sweden, Germany. Austria, Swit7erlanrl and Holland

H . hurgdorferi itzfectiotz itz I . ricitzus (Stanek et ul.; 1988; Aeschlimann et al., 1986; Hovmark ef a / . , 1988; Nohlmans et al., 1990). In our investigation nymphal ticks seemingly had a higher infection percentage than adults. This is in disagreement with the wellestablished fact of transstadial transmission in ticks leading to an accumulation of Borrelia infections in later stages. Our results differ from those of Nohlmans et al. (1990) and Kahl (1991) from Western Europe but are in accordance with the results of Hubalek et al. (1991) from Czechoslovakia and reports from France and Germany cited by Hubalek. Further studies in factors regulating survival and population growth rates of B . burgdorferi in ticks may explain the deviations observed. Survival of B . burgdorferi may be critical if time between repletion and moulting in the ticks is too short (Kahl, 1991). The later the post-repletion females of the species I.damrnirzi were examined, the higher rate of Borrelia was observed (Kahl, 1991). More than half of the ticks analysed in our investigation were collected in August -September 1990. In countries with mild, oceanic climates large numbers of newly moulted adults are abundant in early autumn, deriving from nymphs that fed in spring of the same year. These nymphs do not develop a morphogenetic diapause, but have a short engorged phase (Gray, 1991). This may reduce or even prevent population growth of B. burgdorferi in these ticks and may explain our indications of low transstadial accuinulation between nymphs and adults. We conclude that /.ricinz~.sis the vector largely responsible for the transmission of B. hurgdorferi in Denmark. The rodents, Cglareolus and A.flavicollis, trapped in the sites Frijsenborg, Hestehaven and Strandkaer, were serologically tested and found to contain high titres of Barrelia antibodies (Flong, unpubl.). More than 20% of the animals captured were seropositive. These results are consistent with the infectivity found in ticks from the same sites. This indicates that C.glareolus and A.pavicollis harbour Borrelia and may be possible reservoirs for the agent causing Lyme borreliosis (Hovmark et al., 1988; Flong, unpubl.). Most cases of human Lyme borreliosis in Denmark are observed from June to October (Hansen, 1986), when activity of ticks and man coincide in natural foci. In another investigation it was found that the activity of ticks peaked from May to October (Flong & Landbo, 1992). The high infectioii rate of B . hurgdorferi in 317 Z.ricinus ticks examined from Frijsenborg, Hestehaven and Strandkaer is in good agreement with the presence of Lyme borreliosis patients living in the same area ( D r L. Halkjaer, Aarhus, pers. comm.). All three sites are used by the public for recreation.

Acknowledgments We thank D r K . Hansen, Statens Serum Institut, Copenhagen, for providing the Borrelia antigens (ECM-DK-1) and for introduction to the IFA method; D r H. Erno, Institute of Medical Microbiology, University of Aarhus, where the inrmunofluorescence testings were performed,

167

for help and advice, and K. Peterslund for technical assistance. A special thank to D r B. Overgaard Nielsen , Institute of Biology, University of Aarhus, for supervision and comments on the manuscript. This work was supported in part by the Fond of 17 December 1981.

References Aeschlimann, A., Chamot, E., Gigon, F., Jcanncrct, J . P . . Kesseler, D. & Walther, C . (1986) Borreliu Durgdorferi in Switzerland. Zentrulblutt fur Bakteriologie und Hygiene. 263, 450 -458. Arthur, D.R. (1963) British Ticks. Buttcnvorths. London. Burgdorfer, W., Barbour, A.G., Haycs, S.F., Bcnach, J . L . . Grundwaldt, E. & Davis, J.P. (1982) Lyme discasc: a tickborne spirochetosis? Science, 216, 18. Flong, P. & Landbo, A.S. (1992) Abundance and occurrcncc of Ixodes ricinus in different habitats, Jutland, Dcnmark. (Undersogelser of skovflaatens forekomst paa forskclligc biotoper i Ostjylland). Speciulerapport. Institutc af Biology, University of Aarhus, Denmark. Gray, J.S. (1991) The development and scasonal activity of thc tick Ixodes ricinus: a vector of Lyme borrcliosis. Review of’ Medical and Veterinury Entomolog.y, 79, 323-333. Hansen, K. (1986) Ixodes ricinus transmitted Borreliu-infcction. (Skovflaat-overfort Borreliu-infektion). EPl-NYT, 22 wcck. Borrelia Laboratory, Department of Treponematoscs, Statens Serum Institut, Copenhagen. Hansen, K. & Dietz, H.H. (1989) Serosurvey for antibodies to Borrelia burgdorferi in Danish dogs. Actu Puthologicu. Microbioiogica et Immunologica Scandinuvica, 97, 281-285. Hansen, K. & Lebech, A.M. (1990) Lyme borreliosis (Lyme borreliose), EPI-NYT, 38 week. Borrelia Laboratory, Dcpartment of Treponematoses, Statens Serum Institut, Copcnhagcn. Hovmark, A,, Jaenson, T.G.T., Aasbrink, E., Forsman. A. & Jansson, E. (1988) First isolations of Borrelia burgdorferi from rodents collected in Northern Europe. Actu Putlzologica. Mircrobiologica et Immunologica Scundinuvicu, 96, 917- 920. Hubalek, Z., Halouzka, J. & Juricova, Z. (1991) A comparison of occurrence of Borreliue in nymphal and adult 1.rodes ricinus Ticks. Zentralblatt fur Bakteriologie und Hygiene, 275. 133- 137. Kahl, 0. (1991) Lyme borreliosis: an ecological perspcctive of a tick-borne human disease. Anzeiger fur Schudlingskunde, 26, 81-84.

Kahl, 0. & Kniille, W. (1988) Water vapour uptake from subsaturated atmospheres by engorged immature ixodid ticks. Experimental and Applied Acarology, 4, 73-83. Kaltenrieder, M., Hess, E. & Aeschlimann, A. (1985) Zum Vorkommen der Zecke Ixodes ricinus L. (Ixodoidea, Ixodidac) in der Schweitz. Revue Suisse Zoologique, 92, 685-692. Nohlmans, M.K.E., De Boer, R.R., Van den Bogaard, A.E.J .M Blaauw, A.A.M. &Van Boven, C.P.A. (1990) Voorkommen van Borrelia burgdorferi in Ixodes ricinus in Nederland. Nederlntids Tijdschrift voor Geneeskunde, 134, (27), 1300- 1303. Stanek, G., Pletschette, M., Flamm, H., Hirschl, A.M., Abcrcr. E., Kristoferitsch, W. & Schmutzhard, E. (1988) Europcan Lyme borreliosis. Annals of the New York Academy of Sciences-, 539, 274-282. Wilson, M.L., Ducey, A.M., Litwin, T.S., Gavin, T.A. & Spielman, A. (1990) Microgeographic distribution of inimaturc Ixodes dummini ticks correlated with that of deer. Medical und Veterinary Entomology, 4, 151-159.

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Accepted 6 February 1992