Incidence of Spirochetes (Borrelia sp.) in the Tick Ixodes ricinus in the Urban Environment (Capital of Prague) between 1994-1997

Zent.bl. Bakteriol. 289, 79-88 (1999) © Urban & Fischet Verlag

Zentralblatt fUr

81kteriolqle

Incidence of Spirochetes (Borrelia sp.) in the Tick Ixodes ricinus in the Urban Environment (Capital of Prague) between 1994-1997 JiH Plch and Jifi Basta Nationallnsritutc of Public Health, Prague, Czech Republic

Received Match 2, 1998· Revision received Jul y 25, 1998 . Accepted September 15, 1998

Summary A IOtal of 12287 ticks were collected from 10 localities of the capital of Prague between 1994 and 1997 (2978 ticks in 1994, 5612 ticks in 1995-1996, 3686 ticks in 1997). The presence of Borrelia was investigated by the indirect immunofluorescence assay using hyperimmune rabbit sera. The tick infestation ranged between 3.8 % and 9.7% (1994),7.9% and 11.3% (1995),3.6% and 6.2% (1996) and 4.8% and 9.2% (1997). The results obtained are indicative of a relati ve ly high opportunity for exposure to the causative agent in Prague - the dynamics of the spirochete posi tivity rates in ticks in different years corresponds with the dynamics recorded in the incidence of Lyme borreliosis in the same region.

Introduction Ticks of the genus Ixodes were identified as the main vector of Borrelia burgdorferi sensu lato (1), the causative agent of the multisystemic disorder called Lyme borreliosis (LB) (2). The Czech Republic with its geographical position represents a unique area for natural foclls infections transmitted by the tick Ixodes ricinus (9). An uprising trend is observed in the incidence of LB between 1988 and 1995 when 6320 cases were reported. In 1996, the LB incidence decreased to 4192 cases (EPIDAT, Nationa l Institute of Public Health). The occurrence of spirochetes found in ticks in the Czech Republic ranges between 17.2 %, 18.6 %, and 16.3 % in females, males, and nymphae, respectively (7) . 0934-8840/99/289/1-079 S 12.0010

80

J. rich and J. Basta

The goals of this study were to find Out about the rates of infected ticks in the conurbation areas used for recreation and to consider the risk to population of acquiring LB in this conurbation.

Materials and Methods Ticks were collected by flagging (white flag, 50 em x 50 em, cotton) between 9.00 a.m. and 12.30 p.m. when humidity in different localities ranged between 60 % and 85 % and temperature between 14 ·C and 23·C (April, May, September, October) or between 16·C and 31 ·C (june, July, August). Ticks were collected in the following loca lities:

Petrin, Vitkov, and Stromovka belong to the category of parks. Petern and Strornovka represent biocypes where beeches (genus Fagus) predominate, less frequent are oaks (genus Querqus /. ) and rare are conifers (genus Pinus, Picea ). In Stromovka, also chest-nut trees (genus Castanea) and decorative exotic trees are relatively abundant. Shruhwood is rarher rare, flagging was performed on lower vegetation and open grass areas. In the Vfrkoy locality, a tree cover (spruce - Picea, oak) is present only at the periphery. Ticks were flagged from shrubwood and open areas. The Krc wood is a park-like wood with a predominating beech and oak cover and a sporadic presence of birches (genus Betula) and spruces. The shrubwood layer is

formed by bramble and raspberry bushes. The Sarka locality is also a park-like wood. Its periphery is covered with oaks and witch hazels (genus Caprimus), in the vic inity of the stream, alders (genus Almus) predominate. The Hanspaulka locality is a mixed

park-like wood (beech, spruce, pine) with an abundant shrubwood cover; flagging was performed in grass areas. The Troja loca lity, characterised as a wood because of the higher presence of the tree species, is very similar in its biotype to Stromovka but the exotic trees are less fre-

quent there. The Dablice locality shows a higher share of conifers (spruce, pine) and a lower quantity of beeches and oaks. A rather dense cover is outlined by large grass areas. The Klanovice locality was the only summer forest with predominating oak and

beech and a limited shrubwood layer. Between 1994 and 1997, a total of 12276 ticks (males, females, and nymphae) was collected. The ticks were dipped into 70 % alcohol and then washed with sterile PBS, the dorsum was removed and the midgut portion was extracted, transferred onto a slide and smeared. When air-dried, the specimens were fixed with acetone. The indirect immunofluorescence assay (3) was used for the detection of spirochetes.

Hyperimmune rabbit serum prepared in our laboratory (immunization with B 31 strain, obtained by courtesy of Dr. Harbour A. C., and Kc90 strain, National Reference Laboratory for Lyme borreliosis, National Institute of Public Health, Prague) and

F1TC-labelied swine anti-rabbit immunoglobulin (Sevac, Praha) were used for this purpose. The specimens were investigated under the fluorescence microscope (Olympus

BH-2) at a magnification of 200 to 400 x. Statistica l method: The study is based on the quantitative description of a population group. The percentage of frequency of each feature (P) was ca lcu lated together with its 9S % confidence interval (95 % CI, i. e. its upper and lower limits - UP, LP). The Poisson distribution of the actual number of positive findings (x) was ca lculated

for all the groups assessed (N). Graphical representation of the confidence intervals

enabled a comparison between the data. If these intervals did not overlap, the differ-

Incidence of Spirochetes in Ticks

81

ence is statistically significant (at least at a 5 % significance level) while overlapping intervals are indicative of a statistically insignificant difference between the frequen -

cies compared, at a 95 % confidence interval (10).

Results For the 10 localities, the infestation of ticks with Borrelia was expressed in percentages (see Tables). Collections of up to 50 ticks from one locality (Lhotka, Satalice, Pruhonice) were not considered to be sufficiently significant. The Borrelia positivity rates in ticks ranged between 3.8 % and 9.7%, 7.9 % and 11.3%,3.6% and 6.2%, and 4.8% and 9.2% in 1994, 1995, 1996 and 1997, respectively (Fig. 1 - Incidence by yearly means of confidence values). The following highest and lowest positivity percentages in different seasons were found in the localities studied: 1994: 1995: 1996: 1997:

Hvezda (9.7%) vs. Sarka (3.8%) Stromovka (11.3% ) vs. Krc (8.0%) Vitkov (6.2 % ) vs. t":l:iblice (3.6%) Stromovka (9.2 % ) vs. Hvezda (4.8 %)

Localities negative for the presence of spirochetes were not found. The tick females showed a fairly higher Borrelia positivity rate as compared to males and nymphae (see Tables). The overall incidence of ticks harbouring Borrelia was similar in all localities during the four seasons studied. The mean total of the ticks collected was 270. The absolute number of the infected ticks was, as a rule, lower than 50, nevertheless, the percentages of the infected ticks in the selected localities were similar within the same season. This is suggestive of a high reliability of the estimate of the infection incidence and of a homogeneous distribution of the infection in allioealities studied. The infection percentages are equal and the confidence intervals are rather small.

Discussion The findings of ticks in large cities including Prague have been confirmed by several authors (5, 8, 14). The fact that some small mammals and birds harbour not only ticks but also spirochetes has been reported repeatedly. The infestation of ticks with the causative agent of LB varies widely, between 2 % and 45 % (6, 13, 15, 11 ), which is consistent with the results of this study. This is true also of other Czech studies (7), even though the mean positivity found in this study is a little lower. Another difference consists in a higher Borrelia infection rate in tick females compared to that in males. A higher infection rate in adult ticks recorded in the urban environment is consistent with the data found in other studies (12) and is due to the inter-stage transmission of the pathogen. I>

Zent.hl. Rakterio l. 18911

Klanovice Hostivar

Dablice

Stromovka

Hanspaulka Hvezda

Modrany Kunraricky les Motol Sarka

Perrin Vitkov

Locality

189 312 151 112 174 79 180 205 643 310 357 266

total

10 60 83 120 120 145 103

45 39 85 60 81 125 32 28 84 36 67 50 164 74 114 46 72

359 116 98 117

63 148 34 24 90 33 53

nymphs males . females

Number of ticks

5,3 5,1 4,6 8,0 5,7 3,8 6,6 9,7 4,6 4,8 6,2 4,5

findings % of total

Positive

10,0 3,3 7,3 4,2 4,2 4,1 6,0

6,6 5,1 3,5 6,6

4,9 4,0 6,3 10,7 4,7 2,7 5,9 9,8 4,3 8,1 6,1 6,5

nymphs males

4,0 6,0 5,9 8,3 6,6 3,0 9,4 12,5 5,0 5,2 9,2 2,5

females

Positive findings %

2,54 2,93 1,86 3,68 2,76 0,78 3,44 5,96 3,02 2,71 3,86 2,33

LPF

95%

9,73 8,33 9,55 15,25 10,57 11,10 11,64 15,07 6,48 7,98 9,33 7,88

UPF

CI

P P W W WP WP WP WP P W W WP

Locality

Type of

WI> ::;: wooded park, W = wood .

CI::;: 95% confidence interval, PF = percentage of frequency of each feature, LPF::;: irs lower limits, UPF::;: its upper limits, P = park,

1 3 4 4 5 6 6 6 7 8 9 10

quarter

Prague

Table 1. Positive findings in ticks from various localities of Prague in 1994

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1 3 4 5 6 6 7 7 8 9 10

Khinovice Hostivar

Dablice

Hvezda Hanspaulka Troja Stromovka

Kri:sky les Prokopske udoli

Periin Vi"tkov

Prague Locality quarter

281 163 176 213 2ll 251 237 673 290 384 273

total

110 48 58 71 73 94 82 380 141 124 131 71 41 45 65 59 60 71 91 59 lOO 75

nymphs males

Number of ricks

100 74 73 77 79 97 84 202 90 160 67

females

11,0 9,2 7;9 10,8 10,5 9,2 8,0 11,3 10,0 11 ,2 8,0

Positive findings % of rota I

Table 2. Posirive findings in ricks from various localities of Prague in 1995

8,2 6,3 5,2 11,3 8,2 7,5 6,1 9,7 7,1 10,5 7,6

12,7 7,3 8,8 7,7 10,2 11,6 7,2 7,7 11,9 10,0 9,3

nymphs males

13,0 12,2 9,6 13,0 12,7 9,3 10,7 15,8 13,3 12,5 J 1,9

females

Posirive findings %

P P WP W WP WP W W W WI'

14,36 15,08 12,20 6,70 8,10 5,05

P

Locality

15,66 15,18 13,10 16,20 15,79 13,75 12,52

UPF

LPF

Type of

7,49 5,15 4,35 6,85 6,54 5,81 4,83

C[

95 %

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1 3 4 4 6 6 6 7 8 9 10

Hostivar

Saralice

Dablice

Stromovka

Hanspaulka Hvezda

Sarka

Kunraticky les

Krtsky Ie,

Virkov

Petrin

Prague Locality quarter

169 229 253 113 289 63 436 295 194 237 186

total

20 60 82 40 105 18 193 31 74 72 56 25 140 150 75 95 70

111

82 100 101 44

nymphs males

N urn ber of ticks

67 69 70 39 73 20 103 114 45 70 60

females

4,2

5,4

4,1 6,2 4,7 4,4 3,8 4,8 4,6 4,1 3,6

Positive findings % of total

Table 3. Positive findings in ticks from various localities of Prague in 1996

2,1 6,5 1,4 1,4 5,4

1,9 .

3,3 3,6 1,3

4,9 6,0 4,9 6,8 5,4 8,0 6,4 4,0 5,3 6,3 5,7

nymphs males

4,5 8,9 5,7 3,3 4,2 5,1 6,8 3,5 4,4 4,3 5,0

females

Positive findings %

1,66 3,34 2,45 1,43 1,90 0,98 2,80 2,10 1,45 2,03 2,58

LPF

95%

8,53 10,07 8,28 10,33 6,81 13,92 7,08 7,11 7,43 7,76 9,89

UPF

CI

P P WP W WP WP WP P W W WP

Type of Locality

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1 3 4 6 6 6 7 8 9 10

Perrin Vitkov Kri'sky les Sarka Hanspaulka Hvezda Stromovka IHblice Klanovice Hostivar

Prague Locality quarter

351 337 348 339 411 482 546 251 311 310

total

101 81 110 118 120 77 151 65 103 50

nymphs

90 110 102 97 150 180 175 95 98 115

males

Number of ticks

160 146 136 124 141 225 220 91 110 145

females

6,0 5,0 8,6 6,8 6,7 4,8 9,2 4,8 5,8 6,1

Positive findings % of total

Table 4. Positive findings in ticks from various localities of Prague in 1997

5,9 6,2 10,0 7,6 6,7 3,9 9,9 4,6 6,8 8,0

7,8 4,5 8,8 8,2 5,3 6,7 10,5 6,3 5,1 6,1

nymphs males

5,1 4,8 7,3 4,8 8,5 3,5 7,7 3,3 5,4 5,5

females

Positive findings % CJ

UPF 9,15 8,08 12,31 10,18 9,85 7,16 12,07 8,35 9,15 9,57

95%

LPF 3,70 2,94 5,82 4,30 4,53 3,02 6,80 2,47 3,43 3,69

W W WP

P

WP WP WP WP

P P

Type of Locality

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J. Plch and J. Basta Incidence (%) 20 ,------------ ,---------------. I 95% CI. - mean

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10

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oL------- - -- --....J 1994

1995

1996

1997

Year Fig. 1. Incidence of spirochetes Borrelia sp. in the tick Ixodes ricinus in the urban environment (capital of Prague) between 1994-1997.

In all localities, the ticks were infected with a roughly equal probability. Higher differences were found between different seasons. Any difference in the infection incidence in ticks depending on the type of the wooded area has not been demonstrated. Most infected ticks were found in 1995, the lowest share of infected ticks was reported in 1996. In contrast to 1996 with its extremely low temperatures and to 1997 with its high total rainfall, the climatic conditions were rather favourable in 1995. Juntilla et al. (1994) offered a hypothesis that a low incidence of LB in humans would correspond to a low share of infected ticks. Nevertheless, they did not confirm this hypothesis in the study. Anyway, if we compare the shares of infected ticks in different years with the incidence of LB reported in Prague, we find the same dynamics in development of both factors. The highest number of LB cases in the capital of Prague (21.7 caseS/100000 population) was reported in 1995 when the highest share of infected ticks was

Incidence of Spirochetes in Ticks

87

found while the lowest LB incidence (15.3 cases/100000 population) reported in 1996 was concomitant with the lowest share of infected ticks. The results of this study over several years are indicative of possible contacts of the urban population with the causative agent of LB directly in the city area. The presence of spirochetes together with persisting wrong habits as related to tick removal and frequent outdoor activities (4) are the factors increasing the risk of infection for LB in the urban environment. Acknowledgements. We thank D. Hulinskti for her valuable remarks and criticism and

E. Svandova for statistical analysis of the data.

References 1. Anderson, A.: Epizootiology of Borrelia burgdorferi in Ixodes ticks vectors and reservoir hosts. Rev. Inf. Dis. II (Suppl. 6) (1989 ) 1451-1459 2. Barbour. A. C.: Isolation and cultivation of Lyme disease spirochetes, Yale J. BioI. ~ed.57(1984)521-525

3. Burgdorfer, w., R. S. Lane, A. G. Barbour, R. A. Gresbrink, and J. R. Anderson: The western black-legged tick, Ixodes pacificus: a vector of Borrelia burgdorferi. Am . ./. Trop. ~ed. Hyg. 34 (1985) 925-930 4. BaSta, J. , D.Janovskd, and M. Daniel: Knowledge of the population about Lyme borrdiosis and experience of the attack of ticks. (Pfispevek k informovanosti obyvatelstva 0 Lymeske borelioze a zkusenosti s napadenim kliSt'aty- pilotni studie). E.~.l. 2 (1998) 52-55 5. Cerny, V. and M. Daniel: Occurrence of Ticks under Condirions of Big Towns and their Epidemiological Importance (Vyskyt Klist'at v podrninkach vclkych mest a jejich epidemiologicky vyznam). Cs. Epidemiologie 34 (1985 ) 239-243 6. Gem, L., W. Burgdorfer, A. Aeschliman, and H. E. Krampitz: The ecology of Lyme borreliosis in Europe. In: Aspt:cts of Lyme borreliosis. (Weber, K., Burgdorfer, w., eds.), pp. 59-69, Berlin, Heidelberg: Springer-Verlag: (1993 ) 7. Hubdlek, Z., J. Halouzka, Z.Jufj(,ovd, and S. Svohodova: Seasonal Distrihution of Borreliae in Ixudes ricinus ticks. Zbl. Bakt. 280 (1994) 423-431 8. HubdJek, Z., E. I. Korenherg, and Z.Juricovd et al.: Prevalence of Borrelia in Ixodes ricinus ticks from souther Morava, Czechoslovakia. Folia Parasite!' 37 (1990) 359-362 9. Hulinskd, D., D. Janovskd, and J. Basta: Lyme Borreliosis- Epidemiological Survey in the Czech Republic in 1994. Report of WHO workshop on Lyme borreliosis diagnosis and surveillance, WHO (1995) 53-75 10. Janko, J.: Statistic table-book (Statisticke tabulky), Academia press, Prague (1958) I!. ]unttila, J., R. Tanskanen, and J. Tuomi: Prevalence of Borrelia burgdorferi in Selected Tick Populations in Finland. Scand. J. Infect. Dis. 26 (1994 ) 349-355. 12. Kahl, 0., K. Schmidt, A. Schonberg, U. Laukamm-Josten, W. Knulle, and U. Rienzle: Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in Berlin (West ). Zbl. Bakt. Hyg. A270 (1989 ) 434-440 13. Milanese, R., M. Marin. and Antonmini et al.: Borreliosis Risk after Ticks Bite in Noeth-Easte," Iraly. ~icrobiologica 14 (1991) 257-259

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]. Plch and]. Basta

14. Pokorny, P.: Borrelia sp. in Ixodes ricinus ticks in the area of the city Prague (Borrelia sp. v. kliSteti obecncm (Ixodes ricinus) na uzemf mesta Prahy). Epidem. mikrob. imun. 30 (1990) 1 15. Preac Mursic v., B. Wi/ske, and C. Schierz: European Borrelia burgdorferi isolated from human and ticks: culture conditions and antibiotic susceptibility. Zbl. Bakt. Hyg. 263 (1986) 112-118

es.

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Corresponding author: Jifi Basta J National Institute of Public Health, ~robarova 48, CZ-l 0042 Praha 10, Czech Republic, Fax: ++420/2/67311188