Prevalence of Borrelia burgdorferi in Ixodes ricinusTicks in Belgrade Area

Radovan Cekanac,1 Nevenka Pavlovic,2 Zorana Gledovic,3 Anita Grgurevic,3 Novica Stajkovic,1

Zorica Lepsanovic,2 and Elizabeta Ristanovic1

Abstract

Objective: Lyme borreliosis is vector-borne zoonosis. The causative agent of Lyme borreliosis is a spirochete ofBorrelia burgdorferi (Bb) sensu lato complex, which is transmitted by ticks of the Ixodes ricinus complex. The aimof our paper is to estimate the prevalence of I. ricinus ticks, the level of their infectivity by Bb, and the prevalence ofcertain genospecies of Bb sensu lato in ixodide ticks inhabiting Belgrade. Materials and Methods: An estimate of thetick population density was expressed by the value of flag=hour. For isolation and cultivation of Borrelia, selectiveBarbour-Stonner-Kelly II media was used. Typization of Borrelia was made by applying the species-specificpolymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphismanalysis. In statistical analysis, X2 test was used. Results: Values of flag=hour have varied in relation to yearobserved and type of habitat: The lowest values were recorded in the city parks (17.9). The values were higher inparks-woods (19.7 and 33.4, respectively). The highest values were detected in localities similar to wooded areas(48.0). The estimated average infestation of ticks with Bb was 21.9%, excluding statistically significant differencesby years of investigation. We found the dominance of Borrelia afzelii (75%). Bb sensu stricto (22.2%) as well asBorrelia garinii (2.8%) was much less present. Statistically significant difference was established in the prevalence ofthe above-mentioned genospecies in relation to the examined localities. Conclusions: We have established theprevalence of all three genospecies in the city of Belgrade. Bb sensu lato was found, with the dominance of B. afzelii.

Key Words: Belgrade—Borrelia burgdorferi—Ixodes ricinus—Ticks.

Introduction

Although great results were achieved in XX centuryin controlling communicable diseases, these diseases still

remain a major challenge. Certain known contagious diseaseshave been re-emerging and some new ones have appeared.Lyme disease (LD) is in the group of communicable diseaseswhose causative agents have been identified in the past threedecades. In the ecological=epidemiological sense, LD isvector-borne zooanthroponosis. Its causative agent is a spi-rochete of the Borrelia burgdorferi (Bb) sensu lato complex,which is transmitted by ticks of Ixodide ricinus complex(Steere et al. 2004). Until today, the connection was estab-lished between LD in humans and three genospecies of thiscomplex. Natural foci of LD were established in the zones ofthe Northern hemisphere experiencing mild climate. LD hasbeen registered in more than 40 countries of the United States

and in almost all European and Far Eastern countries (Yana-gihara and Masuzawa 1997, Steere 2001).

Since 1987, LD has been monitored in Serbia. In the firstfew years of our research, we defined the primary vector(Ixodes ricinus tick) and performed isolation of Bb from theticks and mouse-like rodents (Apodemus flavicolis genus), thereservoir of Lyme borreliosis causative agent, therefore es-tablishing the natural foci of the disease in our country (un-published data).

In Europe, the prevalence of all three pathogens of the Bbgenospecies was established. Uneven geographical distribu-tion of these genospecies as well as different prevalence ofclinical manifestations of the LD has been noted (Ciceroniet al. 2001, Barral et al. 2002, EUCALB 2005).

The aim of our study is to estimate the prevalence ofI. ricinus ticks, degree of their infestation by Bb, and preva-lence of certain genospecies of Bb in them in Belgrade.

1Military Medical Academy, Institute of Epidemiology, Belgrade, Serbia.2Department of Epidemiology, Institute of Public Health of Belgrade, Belgrade, Serbia.3Institute of Epidemiology, School of Medicine, University of Belgrade, Belgrade, Serbia.

VECTOR-BORNE AND ZOONOTIC DISEASESVolume 10, Number 5, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089=vbz.2009.0139

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Materials and Methods

Field investigations

Between 1996 and 2000, we collected ticks in 10 localitiesfrom 5 municipalities of Belgrade (Rakovica, Cukarica,Zvezdara, Savski Venac, and Vozdovac). The ticks were col-lected between March and October each year, whereas thechoice of localities was based on epidemiological and eco-logical indications.

According to the type of habitat, the examined localitiesmay be defined as parks (in the municipalities of Cukarica andSavski Venac), parks-woods (Rakovica and Zvezdara), andwooded areas (Vozdovac).

To establish the prevalence of individual ticks in the field,we used flag. The estimate of tick population density wasexpressed as flag=hour (FH). The determination of collectedticks was performed to the level of the species, using the Po-merancevs key methodology. The developmental stage ofeach tick and its sex was determined by the Furmann andCatts method (Pomerancev 1950, Furman and Katts 1982).

Detection of Bb in the ticks

The infection of the ticks by Bb was established by mi-croscopy of native media in a moist and dark field, under 400�magnifications, according to the Kovalevskij method (Kova-levskij et al. 1990). It was done in all developmental forms ofticks. In a number of natively positive media, we performedconfirmation of Bb by direct immunofluorescence test (under1.200�magnification).

Isolation and cultivation of Bb

Preparation for the isolation of Bb from ticks consisted ofthe choice of pools of five individual ticks taken from theexplored localities. Macerates of the gut content of the tickswere then inoculated in 5 mL selective Barbour-Stonner-Kelly(BSK II) medium. The media were then incubated in a ther-mostat at 348C. The content of the vials with Bb growth wasinoculated in 1 mL to an ordinary BSK II medium. Passagewas continued every 3 to 4 days until we obtained a rich andclean culture.

Borrelia typization

For the typization of Borrelia that had been cultivated inBSK medium, we used the genospecies-specific polymerasechain reaction (PCR). However, typization of Borrelia directlyfrom the ticks was performed using PCR–restriction fragmentlength polymorphism. The ticks were processed by modifi-

cation method applied by Persing et al. (1990). Primer nucle-otide sequence was done according to Postic et al. (1994).

Statistical analysis

Subsequent statistical analysis was performed using Sta-tistica Program, version 5.0. for Windows. We used X2 test inthe analysis. Statistically significant difference was acceptedfor p< 0.05 level.

Results

Density of I. ricinus ticks in the examined localities

Between 1996 and 2005, from the chosen localities in fivemunicipalities of Belgrade, we collected (using FH method)10,317 individual I. ricinus ticks. Collection of the ticks lasted390 FHs, with average value of an FH being 26.5 individualticks. Average collection time per year of examination was 39FHs, ranging from 32 FHs in 1996 to 49.5 FHs in 2004.

In relation to the examined municipalities, greatest preva-lence of I. ricinus was recorded in the localities of Vozdovac.The smallest prevalence of ticks was registered in SavskiVenac and Cukarica (city parks). Collection of the ticks lastedfrom 73.5 FHs in Vozdovac (wooded area) to 81 FHs in Ra-kovica (park-wood) municipality (Table 1).

FH values ranged from 16.3 in Savski Venac to 48.0 inVozdovac municipality. The differences we observed werestatistically significant (X2¼ 26.96; p< 0.001).

FH values of I. ricinus ticks in four of the five examinedmunicipalities, by years of investigation, showed statisticallysignificant differences (Rakovica, X2¼ 20.22, p¼ 0.017; Zvez-dara, X2¼ 17.64, p¼ 0.040; Cukarica, X2¼ 17.95; p¼ 0.036;Vozdovac, X2¼ 39.88, p< 0.001). Only in the localities ofSavski Venac municipality, the calculated differences werenot statistically significant (X2¼ 5.74; p¼ 0.766) (Table 2).

Infectivity of I. ricinus ticks

Out of 10,317 individual I. ricinus ticks collected in thechosen localities, 10,158 (98.5%) were examined for theprevalence of Bb. Positive findings were established in 2222(21.9%) of them. By the year of examination, the percentage ofticks infected by Bb ranged from 19.4% in 2003 to 24.4% in2005, although the differences were not statistically significant(X2¼ 1.18; p¼ 0.999).

In relation to the examined municipalities, the greatestpercentage of tick infestation by Bb was established in theticks collected in the localities of Rakovica (park-wood) mu-nicipality (23.0%) whereas the smallest (19.6%) one was foundin the localities of Cukarica (city park) municipality (Table 3).

Table 1. Number of Collected Ticks, Flag=Hours Spent, and Values of Flag=Hour

in the Municipalities of Belgrade

Rakovicaa Zvezdaraa Cukaricab Savski Venacb Vozdovacc Total

CT 2703 1588 1393 1103 3530 10,317FHS 81 80.5 78.5 76.5 73.5 390F=H 33.4 19.7 17.7 16.3 48.0 26.5

aPark-wood.bCity park.cWooded area.CT, collected ticks; FHS, flag=hours spent; F=H, values of flag=hour.

448 CEKANAC ET AL.

The observed differences were not statistically significant(X2¼ 0.27; p¼ 0.992).

Analysis of Bb tick infectivity, by months, in the observedperiod showed that the greatest infectivity of the ticks wasrecorded in March (23.5%) whereas the smallest one was re-corded in October (18.9%). The differences were not statisti-cally significant (X2¼ 0.11; p¼ 0.988).

Prevalence of Bb sensu lato genospeciesin I. ricinus ticks

Between 2002 and 2005, we obtained 18 Bb isolates sensulato by cultivation in the BSK medium. Through their typi-zation (by PCR) and the typization of 17 individual tick gutcontents, in which we found naturally present Bb, we estab-lished the prevalence of Borrelia afzelii in 27 samples. Bb sensustricto was found in seven samples whereas Borrelia gariniiwas found in only one sample. In the localities of Rakovicamunicipality, we established prevalence of B. afzelii in threesamples. Bb sensu stricto and B. garinii were each identified inone sample. In the chosen localities of Zvezdara municipality,we established the prevalence of B. afzelii in five ticks and Bbsensu stricto in four of the examined ticks. Out of 10 isolates,Bb sensu lato obtained from the ticks in Vozdovac localities, 8were identified as B. afzelii and two as Bb sensu stricto. Out ofthe samples of ticks collected in the localities of Cukarica andSavski Venac, we established only B. afzelii. The establisheddifferences in prevalence of B. afzelii in relation to the re-maining two genospecies are statistically significant in theexamined municipalities. The calculated statistical signifi-cance is X2¼ 50.537; p< 0.000 (Table 4).

Discussion

According to our results, the tick population density esti-mated by FH values was smallest in the parks (Savski Venac,17.9), somewhat greater in parks-woods (Zvezdara, 19.7; Ra-kovica, 33.4), and greatest in the localities of the forest type

(Vozdovac, 48.0), like in a Czech study (Daniel and Cerni1986). The average value of FH was 26.5. By years of investi-gation, it ranged from 13.5 (in 1997) to 39.4 (in 2001). One ofthe reasons for such a small figure in 1997 was the introduc-tion of systematic pest control in the examined localities inJune 1996. The only exception was the municipality of Voz-dovac. Differences in the obtained FH values for the entireresearch period are statistically significant in relation to yearsand months as well as to the chosen localities. In relation to theFH values per year of investigation, analysis of each localitydid not show statistical significance only in the municipality ofSavski Venac. This can be explained by the fact that the chosenlocalities in that municipality are defined as ‘‘parks’’; the areascovered by grass are regularly maintained and trimmed.Maintenance workers regularly clear away brushwood andremove and burn plant remnants. Both maintenance andspatial structure of these areas do influence the conditions intick microhabitat. Changing moisture and light conditions inthese areas do have an impact upon the ticks’ density.

There are great differences in infectivity of ixodide tickswith Bb in relation to their species and geographical distri-bution. The Ixodes dammini tick from the American continent ismore intensively infected with Bb than the European-livingI. ricinus. In a natural habitat, there is a difference between thespecies in respect to the percentage of Bb infectivity. Theinfectivity of I. dammini ranges between 10% and 90%; inI. ricinus, it ranges between 3% and 40%; and in Ixodes pacifi-cus, it is between 1% and 3% (Karlsson 1996). This feature isalso known to change by the year, depending on the type ofhabitat and developmental stages of ticks. Slovak researchershave presented that they have registered, in four consecutiveyears, different values of tick infectivity with Bb: 4.8% in 1994;17.2% in 1995; 15.5% in 1996; and 14.2% in 1997. Dependingon the habitat, infectivity of the ticks ranged from 2.1% to23.5% (Lane 1990).

Between 1996 and 2000, in the areas of Northern Moravia,the prevalence of tick infection by Bb had been varying. It was

Table 2. Values of Flag=Hour for Ixodes ricinus Ticks in the Examined Municipalities, by Year

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Total

Rakovicaa 47.1 15.8 53.5 36.0 32.6 54.2 35.8 21.9 27.5 16.6 33.4Zvezdaraa 22.4 7.5 24.0 39.8 20.4 35.4 20.8 11.0 12.0 15.7 19.7Cukaricab 26.3 7.5 12.3 14.8 28.9 19.5 18.3 17.0 22.9 8.0 17.7Savski Venacb 16.3 10.4 18.0 16.2 15.1 13.5 9.3 14.0 18.0 12.5 16.3Vozdovacc 45.6 32.5 59.3 57.0 63.1 73.7 44.9 46.9 35.0 26.3 48.0

aPark-wood.bCity park.cWooded area.

Table 3. Number of Examined Ixodes ricinus Ticks, Borrelia burgdorferi Positive, by Municipalities

Rakovicaa Zvezdaraa Cukaricab Savski Venacb Vozdovacc Total

Examined 2650 1588 1348 1042 3530 10,158Positive 609 357 264 233 759 2222% of positive 23.0 22.5 19.6 22.4 21.5 21.9

aPark-wood.bCity park.cWooded area.

PREVALENCE OF Borrelia burgdorferi 449

6.8% in 1996; 8.4% in 1997; and 12.3% in 1998. The smallestvalue was recorded in 1999 (3.6%) and 2000 (4.0%), with themean value of 6.5%. In various localities in Southern Bohemia(the Czech Republic), during 1997 and 1998, the prevalence oftick infectivity with Bb was 4.8%. In 1998, it was 11.04%(Stepanova-Tresova et al. 2000, Janouskovcova et al. 2004).There are huge differences in the recorded prevalence amongregions in Switzerland. Infectivity of nymphs ranged from 9%to 40%. In adult ticks, it ranged from 22% to 47%. Total av-erage infectivity of adult ticks was 35% and total average in-fectivity of nymphs was 20%. Another study was conductedin the Valais region of Switzerland, which lasted 5 years. Itencompassed 727 individual I. ricinus ticks and showed thatBb had been detected in 30.4% of them. Another research,conducted in five other regions of Switzerland, showed a verylow infectivity of the larvae (3.1%) in relation to nymphs(12.8%) and adult ticks (14.5%). In 12 cities of the TicinoCanton, researchers established differences in Bb infectionprevalence that ranged from 5% to 19% (Zhioua et al. 1994,Peter et al. 1995, Jouda et al. 2003, 2004).

Similar investigations have been done in Slovenia (Ruzic-Sabljic et al. 1993), The Netherlands (Nohlmans et al. 1990),Italy (Santino et al. 2003), Denmark (Landbo and Flong 1992),and other European countries (Gustafson et al. 1995). Theirresults also showed significant variations in tick infectivitywith Bb, related to the areas and developmental stages of theticks themselves. In our study, we established that an averagetick Bb infectivity was 21.9%. However, differences in theprevalence of infectivity were statistically significant neitherin relation to years=months of research nor in relation to thechosen municipalities. In relation to the results obtained fromother European studies, we concluded that there is a balancein the value of tick infectivity in Belgrade. Greatest averageinfectivity was established in March, and that was the case inall municipalities. This may be explained by the dominance ofadult ticks (both males and females) and their greater infec-tivity in relation to nymphs and larvae that are prevalent inthe summer months and infected to a lesser degree.

FH values and infectivity of the ticks with Bb, according toour results, are not correlated. It means that the number ofticks does not influence the frequency with which they areinfected. This finding stresses the complexity of Bb ecology.Numerous vectors, a great diversity of host vectors as primaryand secondary reservoirs, are the important part of its ecology(Goodwin et al. 2001, Anderson et al. 2006, Vourc’h et al.2007).

All around the world, many studies are related to tick in-fectivity and prevalence of Bb sensu lato genospecies. InNorthern Moravia, between 1996 and 2000, the estimateddensity of B. afzelii was 77.0% and of B. garinii was 23.0%. InSouthern Bohemia, during 1997 and 1998, Bb sensu stricto wasfound in 6 out of 10 isolates. B. garinii was detected in twoisolates, whereas B. afzelii was detected in the remaining two(Stepanova-Tresova et al. 2000, Janouskovcova et al. 2004).The results of the first study related to the prevalence anddensity of Bb genospecies in Switzerland showed that out of50 isolates, 26 (52%) were identified as Bb sensu stricto; 19(38%) as B. garinii; 3 (6%) as B. afzelii; and 2 as Borrelia va-laisiana. B. garinii was dominant in the Ticino Canton. In 2003,Borrelia lusitaniae was detected for the first time (Peter et al.1995, Jouda et al. 2003). According to most recent data, it seemsthat B. valaisiana and B. lusitaniae might be connected with LDin humans (Collares-Pereira et al. 2004). Research done in thelocalities of Bonn established the prevalence of B. afzelii (39.5%),B. garinii (27.9%), Bb sensu stricto (15.6%), and B. valaisiana(8.6%) (Maetzel et al. 2005). Prevalence of Bb infection in thenymphs of 8.2% was established in the commune of Ram-buille (France). Prevalence of B. afzelii was 58.6%; B. garinii,31.1%; and Bb sensu stricto, 10.3% (Pichon et al. 1999). In thenorthwestern parts of Croatia, at five different localities,prevalence of Bb infection of 45% was established. Prevalenceof B. afzelii was 46.4%; B. garinii, 8.9%; B. valaisiana, 8.9%; andBb sensu stricto, 3.6% (Golubic et al. 1998). In four localitieswithin a region in Belgium, total tick infectivity was 23%. B.garinii was present in 53% of the cases; Bb sensu stricto, in38%; and B. afzelii, in 9% (Misonne et al. 1998).

According to our results related to prevalence of Bb gen-ospecies in LD vectors in the area of Belgrade, there was astrong dominance of B. afzelii (75%). Bb sensu stricto waspresent in a lesser degree (22.2%) and B. garinii was seen onlyin 2.8% of the cases. There were statistically significant dif-ferences in the prevalence of these genospecies in relation tothe examined localities, although the number of identifiedstrains of certain genospecies (except B. afzelii) was relativelysmall for more indicative conclusions. Therefore, the presenceof only B. afzelii was established in the municipalities ofCukarica and Savski Venac, and the presence of B. afzelii andBb sensu stricto was established in the municipalities ofZvezdara and Vozdovac. In the municipality of Rakovica, wefound all three pathogenic genospecies of Bb sensu lato.

In the study that was conducted in 26 countries of Europe(without participation of the countries from South Eastern

Table 4. Prevalence of Pathogenic Genospecies of Borrelia burgdorferi Sensu Lato, by Municipalities

Municipality

Borrelia afzelii Borrelia burgdorferi sensu stricto Borrelia garinii Total

N8 % N8 % N8 % N8 %

Rakovicaa 3 60.0 1 20.0 1 20.0 5 100.0Zvezdaraa 5 55.5 4 44.5 — — 9 100.0Cukaricab 5 100.0 — — — — 5 100.0Savski Venacb 6 100.0 — — — — 6 100.0Vozdovacc 8 80.0 2 20.0 — — 10 100.0Total 27 77.1 7 20.0 1 2.9 35 100.0

aPark-wood.bCity park.cWooded area.

450 CEKANAC ET AL.

Europe), different prevalence of certain genospecies of Bbsensu lato was found. It has also been noted that prevalence ofB. afzelii increases when going southward from the north ofEurope. In Finland and Sweden, it is 18.3% and 28%, respec-tively; in Germany, 39.7%; in Austria, 49.2%; in Slovenia,60.4%; and in Croatia, 63.3% (Hubalek and Halouzka 1997).Based on the results, abundance of B. afzelii of 75.0% in ourenvironment represents an expected value.

Our results showed that the numerousness of tick popu-lation (F=H values) does vary, in relation to years of researchand type of the ticks’ habitat. Infectivity of the ticks by Bb hasmaintained approximately equal in all research periods. Wehave proved that all three pathogens of the genospecies Bbsensu lato do exist in the area of Belgrade. However, B. afzeliihas been dominant.

Acknowledgment

Supported by the Ministry of Science, Technology, andDevelopment through contract No. 145084=2006 and No.145045=2006.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Zorana Gledovic

Institute of EpidemiologySchool of Medicine

University of BelgradeVisegradska 26aBelgrade 11000

Serbia

E-mail: glzo@med.bg.ac.rs

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