Prevalence of Borrelia burgdorferi in Ixodes ricinus Ticks in Belgrade Area

Abstract

Objective: Lyme borreliosis is vector-borne zoonosis. The causative agent of Lyme borreliosis is a spirochete of Borrelia burgdorferi (Bb) sensu lato complex, which is transmitted by ticks of the Ixodes ricinus complex. The aim of our paper is to estimate the prevalence of I. ricinus ticks, the level of their infectivity by Bb, and the prevalence of certain genospecies of Bb sensu lato in ixodide ticks inhabiting Belgrade. Materials and Methods: An estimate of the tick population density was expressed by the value of flag/hour. For isolation and cultivation of Borrelia, selective Barbour-Stonner-Kelly II media was used. Typization of Borrelia was made by applying the species-specific polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism analysis. In statistical analysis, X² test was used. Results: Values of flag/hour have varied in relation to year observed and type of habitat: The lowest values were recorded in the city parks (17.9). The values were higher in parks-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 differences by years of investigation. We found the dominance of Borrelia afzelii (75%). Bb sensu stricto (22.2%) as well as Borrelia garinii (2.8%) was much less present. Statistically significant difference was established in the prevalence of the above-mentioned genospecies in relation to the examined localities. Conclusions: We have established the prevalence of all three genospecies in the city of Belgrade. Bb sensu lato was found, with the dominance of B. afzelii.

Introduction

A lthough great results were achieved in XX century in controlling communicable diseases, these diseases still remain a major challenge. Certain known contagious diseases have been re-emerging and some new ones have appeared. Lyme disease (LD) is in the group of communicable diseases whose causative agents have been identified in the past three decades. In the ecological/epidemiological sense, LD is vector-borne zooanthroponosis. Its causative agent is a spirochete 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 established between LD in humans and three genospecies of this complex. Natural foci of LD were established in the zones of the Northern hemisphere experiencing mild climate. LD has been registered in more than 40 countries of the United States and in almost all European and Far Eastern countries (Yanagihara and Masuzawa 1997, Steere 2001).

Since 1987, LD has been monitored in Serbia. In the first few years of our research, we defined the primary vector (Ixodes ricinus tick) and performed isolation of Bb from the ticks and mouse-like rodents (Apodemus flavicolis genus), the reservoir of Lyme borreliosis causative agent, therefore establishing the natural foci of the disease in our country (unpublished data).

In Europe, the prevalence of all three pathogens of the Bb genospecies was established. Uneven geographical distribution of these genospecies as well as different prevalence of clinical manifestations of the LD has been noted (Ciceroni et al. 2001, Barral et al. 2002, EUCALB 2005).

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

Materials and Methods

Field investigations

Between 1996 and 2000, we collected ticks in 10 localities from 5 municipalities of Belgrade (Rakovica, Cukarica, Zvezdara, Savski Venac, and Vozdovac). The ticks were collected between March and October each year, whereas the choice of localities was based on epidemiological and ecological indications.

According to the type of habitat, the examined localities may be defined as parks (in the municipalities of Cukarica and Savski Venac), parks-woods (Rakovica and Zvezdara), and wooded areas (Vozdovac).

To establish the prevalence of individual ticks in the field, we used flag. The estimate of tick population density was expressed as flag/hour (FH). The determination of collected ticks was performed to the level of the species, using the Pomerancevs key methodology. The developmental stage of each tick and its sex was determined by the Furmann and Catts method (Pomerancev 1950, Furman and Katts 1982).

Detection of Bb in the ticks

The infection of the ticks by Bb was established by microscopy of native media in a moist and dark field, under 400 × magnifications, according to the Kovalevskij method (Kovalevskij et al. 1990). It was done in all developmental forms of ticks. In a number of natively positive media, we performed confirmation of Bb by direct immunofluorescence test (under 1.200 × magnification).

Isolation and cultivation of Bb

Preparation for the isolation of Bb from ticks consisted of the choice of pools of five individual ticks taken from the explored localities. Macerates of the gut content of the ticks were then inoculated in 5 mL selective Barbour-Stonner-Kelly (BSK II) medium. The media were then incubated in a thermostat at 34°C. The content of the vials with Bb growth was inoculated in 1 mL to an ordinary BSK II medium. Passage was continued every 3 to 4 days until we obtained a rich and clean culture.

Borrelia typization

For the typization of Borrelia that had been cultivated in BSK medium, we used the genospecies-specific polymerase chain reaction (PCR). However, typization of Borrelia directly from the ticks was performed using PCR–restriction fragment length polymorphism. The ticks were processed by modification method applied by Persing et al. (1990). Primer nucleotide sequence was done according to Postic et al. (1994).

Statistical analysis

Subsequent statistical analysis was performed using Statistica Program, version 5.0. for Windows. We used X² test in the analysis. Statistically significant difference was accepted for p < 0.05 level.

Results

Density of I. ricinus ticks in the examined localities

Between 1996 and 2005, from the chosen localities in five municipalities of Belgrade, we collected (using FH method) 10,317 individual I. ricinus ticks. Collection of the ticks lasted 390 FHs, with average value of an FH being 26.5 individual ticks. Average collection time per year of examination was 39 FHs, ranging from 32 FHs in 1996 to 49.5 FHs in 2004.

In relation to the examined municipalities, greatest prevalence of I. ricinus was recorded in the localities of Vozdovac. The smallest prevalence of ticks was registered in Savski Venac and Cukarica (city parks). Collection of the ticks lasted from 73.5 FHs in Vozdovac (wooded area) to 81 FHs in Rakovica (park-wood) municipality (Table 1).

Table 1.

Number of Collected Ticks, Flag/Hours Spent, and Values of Flag/Hour in the Municipalities of Belgrade

	Rakovica ^a	Zvezdara ^a	Cukarica ^b	Savski Venac ^b	Vozdovac ^c	Total
CT	2703	1588	1393	1103	3530	10,317
FHS	81	80.5	78.5	76.5	73.5	390
F/H	33.4	19.7	17.7	16.3	48.0	26.5

Park-wood.

City park.

Wooded area.

CT, collected ticks; FHS, flag/hours spent; F/H, values of flag/hour.

FH values ranged from 16.3 in Savski Venac to 48.0 in Vozdovac municipality. The differences we observed were statistically significant (X ² = 26.96; p < 0.001).

FH values of I. ricinus ticks in four of the five examined municipalities, by years of investigation, showed statistically significant differences (Rakovica, X ² = 20.22, p = 0.017; Zvezdara, X ² = 17.64, p = 0.040; Cukarica, X ² = 17.95; p = 0.036; Vozdovac, X ² = 39.88, p < 0.001). Only in the localities of Savski Venac municipality, the calculated differences were not statistically significant (X ² = 5.74; p = 0.766) (Table 2).

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
Rakovica^a	47.1	15.8	53.5	36.0	32.6	54.2	35.8	21.9	27.5	16.6	33.4
Zvezdara^a	22.4	7.5	24.0	39.8	20.4	35.4	20.8	11.0	12.0	15.7	19.7
Čukarica^b	26.3	7.5	12.3	14.8	28.9	19.5	18.3	17.0	22.9	8.0	17.7
Savski Venac^b	16.3	10.4	18.0	16.2	15.1	13.5	9.3	14.0	18.0	12.5	16.3
Voždovac^c	45.6	32.5	59.3	57.0	63.1	73.7	44.9	46.9	35.0	26.3	48.0

Park-wood.

City park.

Wooded area.

Infectivity of I. ricinus ticks

Out of 10,317 individual I. ricinus ticks collected in the chosen localities, 10,158 (98.5%) were examined for the prevalence of Bb. Positive findings were established in 2222 (21.9%) of them. By the year of examination, the percentage of ticks infected by Bb ranged from 19.4% in 2003 to 24.4% in 2005, although the differences were not statistically significant (X ² = 1.18; p = 0.999).

In relation to the examined municipalities, the greatest percentage of tick infestation by Bb was established in the ticks collected in the localities of Rakovica (park-wood) municipality (23.0%) whereas the smallest (19.6%) one was found in the localities of Cukarica (city park) municipality (Table 3). The observed differences were not statistically significant (X ² = 0.27; p = 0.992).

Table 3.

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

	Rakovica ^a	Zvezdara ^a	Cukarica ^b	Savski Venac ^b	Vozdovac ^c	Total
Examined	2650	1588	1348	1042	3530	10,158
Positive	609	357	264	233	759	2222
% of positive	23.0	22.5	19.6	22.4	21.5	21.9

Park-wood.

City park.

Wooded area.

Analysis of Bb tick infectivity, by months, in the observed period showed that the greatest infectivity of the ticks was recorded in March (23.5%) whereas the smallest one was recorded in October (18.9%). The differences were not statistically significant (X ² = 0.11; p = 0.988).

Prevalence of Bb sensu lato genospecies in I. ricinus ticks

Between 2002 and 2005, we obtained 18 Bb isolates sensu lato by cultivation in the BSK medium. Through their typization (by PCR) and the typization of 17 individual tick gut contents, in which we found naturally present Bb, we established the prevalence of Borrelia afzelii in 27 samples. Bb sensu stricto was found in seven samples whereas Borrelia garinii was found in only one sample. In the localities of Rakovica municipality, we established prevalence of B. afzelii in three samples. Bb sensu stricto and B. garinii were each identified in one sample. In the chosen localities of Zvezdara municipality, we established the prevalence of B. afzelii in five ticks and Bb sensu stricto in four of the examined ticks. Out of 10 isolates, Bb sensu lato obtained from the ticks in Vozdovac localities, 8 were identified as B. afzelii and two as Bb sensu stricto. Out of the samples of ticks collected in the localities of Cukarica and Savski Venac, we established only B. afzelii. The established differences in prevalence of B. afzelii in relation to the remaining two genospecies are statistically significant in the examined municipalities. The calculated statistical significance is X ² = 50.537; p < 0.000 (Table 4).

Table 4.

Prevalence of Pathogenic Genospecies of Borrelia burgdorferi Sensu Lato, by Municipalities

	Borrelia afzelii		Borrelia burgdorferi sensu stricto		Borrelia garinii		Total
Municipality	N°	%	N°	%	N°	%	N°	%
Rakovica^a	3	60.0	1	20.0	1	20.0	5	100.0
Zvezdara^a	5	55.5	4	44.5	—	—	9	100.0
Cukarica^b	5	100.0	—	—	—	—	5	100.0
Savski Venac^b	6	100.0	—	—	—	—	6	100.0
Vozdovac^c	8	80.0	2	20.0	—	—	10	100.0
Total	27	77.1	7	20.0	1	2.9	35	100.0

Park-wood.

City park.

Wooded area.

Discussion

According to our results, the tick population density estimated by FH values was smallest in the parks (Savski Venac, 17.9), somewhat greater in parks-woods (Zvezdara, 19.7; Rakovica, 33.4), and greatest in the localities of the forest type (Vozdovac, 48.0), like in a Czech study (Daniel and Černi 1986). The average value of FH was 26.5. By years of investigation, it ranged from 13.5 (in 1997) to 39.4 (in 2001). One of the reasons for such a small figure in 1997 was the introduction of systematic pest control in the examined localities in June 1996. The only exception was the municipality of Vozdovac. Differences in the obtained FH values for the entire research period are statistically significant in relation to years and months as well as to the chosen localities. In relation to the FH values per year of investigation, analysis of each locality did not show statistical significance only in the municipality of Savski Venac. This can be explained by the fact that the chosen localities in that municipality are defined as “parks”; the areas covered by grass are regularly maintained and trimmed. Maintenance workers regularly clear away brushwood and remove and burn plant remnants. Both maintenance and spatial structure of these areas do influence the conditions in tick microhabitat. Changing moisture and light conditions in these areas do have an impact upon the ticks' density.

There are great differences in infectivity of ixodide ticks with Bb in relation to their species and geographical distribution. The Ixodes dammini tick from the American continent is more intensively infected with Bb than the European-living I. ricinus. In a natural habitat, there is a difference between the species in respect to the percentage of Bb infectivity. The infectivity of I. dammini ranges between 10% and 90%; in I. ricinus, it ranges between 3% and 40%; and in Ixodes pacificus, it is between 1% and 3% (Karlsson 1996). This feature is also known to change by the year, depending on the type of habitat and developmental stages of ticks. Slovak researchers have presented that they have registered, in four consecutive years, different values of tick infectivity with Bb: 4.8% in 1994; 17.2% in 1995; 15.5% in 1996; and 14.2% in 1997. Depending on the habitat, infectivity of the ticks ranged from 2.1% to 23.5% (Lane 1990).

Between 1996 and 2000, in the areas of Northern Moravia, the prevalence of tick infection by Bb had been varying. It was 6.8% in 1996; 8.4% in 1997; and 12.3% in 1998. The smallest value was recorded in 1999 (3.6%) and 2000 (4.0%), with the mean value of 6.5%. In various localities in Southern Bohemia (the Czech Republic), during 1997 and 1998, the prevalence of tick 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 among regions in Switzerland. Infectivity of nymphs ranged from 9% to 40%. In adult ticks, it ranged from 22% to 47%. Total average infectivity of adult ticks was 35% and total average infectivity of nymphs was 20%. Another study was conducted in the Valais region of Switzerland, which lasted 5 years. It encompassed 727 individual I. ricinus ticks and showed that Bb had been detected in 30.4% of them. Another research, conducted in five other regions of Switzerland, showed a very low infectivity of the larvae (3.1%) in relation to nymphs (12.8%) and adult ticks (14.5%). In 12 cities of the Ticino Canton, researchers established differences in Bb infection prevalence 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). Their results also showed significant variations in tick infectivity with Bb, related to the areas and developmental stages of the ticks themselves. In our study, we established that an average tick Bb infectivity was 21.9%. However, differences in the prevalence of infectivity were statistically significant neither in relation to years/months of research nor in relation to the chosen municipalities. In relation to the results obtained from other European studies, we concluded that there is a balance in the value of tick infectivity in Belgrade. Greatest average infectivity was established in March, and that was the case in all municipalities. This may be explained by the dominance of adult ticks (both males and females) and their greater infectivity in relation to nymphs and larvae that are prevalent in the summer months and infected to a lesser degree.

FH values and infectivity of the ticks with Bb, according to our results, are not correlated. It means that the number of ticks does not influence the frequency with which they are infected. This finding stresses the complexity of Bb ecology. Numerous vectors, a great diversity of host vectors as primary and 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 infectivity and prevalence of Bb sensu lato genospecies. In Northern Moravia, between 1996 and 2000, the estimated density of B. afzelii was 77.0% and of B. garinii was 23.0%. In Southern Bohemia, during 1997 and 1998, Bb sensu stricto was found in 6 out of 10 isolates. B. garinii was detected in two isolates, 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 and density of Bb genospecies in Switzerland showed that out of 50 isolates, 26 (52%) were identified as Bb sensu stricto; 19 (38%) as B. garinii; 3 (6%) as B. afzelii; and 2 as Borrelia valaisiana. 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 seems that B. valaisiana and B. lusitaniae might be connected with LD in humans (Collares-Pereira et al. 2004). Research done in the localities 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 the nymphs of 8.2% was established in the commune of Rambuille (France). Prevalence of B. afzelii was 58.6%; B. garinii, 31.1%; and Bb sensu stricto, 10.3% (Pichon et al. 1999). In the northwestern parts of Croatia, at five different localities, prevalence of Bb infection of 45% was established. Prevalence of B. afzelii was 46.4%; B. garinii, 8.9%; B. valaisiana, 8.9%; and Bb sensu stricto, 3.6% (Golubic et al. 1998). In four localities within a region in Belgium, total tick infectivity was 23%. B. garinii was present in 53% of the cases; Bb sensu stricto, in 38%; and B. afzelii, in 9% (Misonne et al. 1998).

According to our results related to prevalence of Bb genospecies in LD vectors in the area of Belgrade, there was a strong dominance of B. afzelii (75%). Bb sensu stricto was present in a lesser degree (22.2%) and B. garinii was seen only in 2.8% of the cases. There were statistically significant differences in the prevalence of these genospecies in relation to the examined localities, although the number of identified strains of certain genospecies (except B. afzelii) was relatively small for more indicative conclusions. Therefore, the presence of only B. afzelii was established in the municipalities of Cukarica and Savski Venac, and the presence of B. afzelii and Bb sensu stricto was established in the municipalities of Zvezdara and Vozdovac. In the municipality of Rakovica, we found 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 Europe), different prevalence of certain genospecies of Bb sensu lato was found. It has also been noted that prevalence of B. afzelii increases when going southward from the north of Europe. In Finland and Sweden, it is 18.3% and 28%, respectively; 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 our environment represents an expected value.

Our results showed that the numerousness of tick population (F/H values) does vary, in relation to years of research and type of the ticks' habitat. Infectivity of the ticks by Bb has maintained approximately equal in all research periods. We have proved that all three pathogens of the genospecies Bb sensu lato do exist in the area of Belgrade. However, B. afzelii has been dominant.

Footnotes

Acknowledgment

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

Disclosure Statement

No competing financial interests exist.

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