ORIGINAL ARTIGLESAnn Agric Environ Med 1997, 41 26Y269

BORRELIA BURGDORFERT SENSU LATO IN TIJE IXODES RICINAS TICKS IN

SOUTHERNPOLAND

Branislav Pet'kor, Krzysztof Siuda2, Michal Stanko3,,Gabriela Tresov6l, Grzegorz Karbowiaka,Jana Fridov6'

l lnstitute of Parasitology, Slovak Academy of Sciences, KoSice, Slovakia2Department

of Biology and Parasitology, Silesian Medical Academy, Katowice, Poland3lnstitute of Zoology, Slovak Academy of Sciences, Ko5ice, Slovakia

aW. Stefafrski Institute of Parasitology, Polish Academy of Sciences, Warsaw, Poland

'' Pet'ko B, Siuda K, Stanko M, Tresov6 G, Karbowiak G, Fridov6 J: Borrelia burgdorferi

sensu lato in the lxodes ricinus ticks in southern Poland. Ann Agric Environ Med 1997,4 . 2 6 3 - 2 6 9 .

Abstract: In May 1996,164 pooled and single samples containing 913 larvae, nymphsand adults of lxodes ricinus from urban, suburban and wooded biotopes of southern '.

' Poland were examined for the presence of Borrelia burgdorferi sensu lato. Ticks werecollected by draggin g a flag over the vegetation and B. burgdorferi was detected inpooled tick samples by darkfield microscopy or isolated by cultivation in BSK-IImedium. Minimum infection rates of nymphs, males and females in the area were 8.2,

'

9.8 and l0.3Vo, respectively. Infection of adult I. ricinus ticks on l0localities fluctuatedfrom 4.0 to 15.0o/o and over IOTo of adult ticks in forests and in some urban andsuburban parks of the city Katowice were infected. Five Borrelia strains were isolatedfrom the adult L ricinus ticks. Relative density of ticks ranged from 20-65 ticks per onecollecting hour and from 3.3- 10.8 ticks per 100 m2. respectively. Frequent occurrence ofticks was also observed in urban parks and residential districts of the city Katowice. Theresults refer to the risk of human and animal infections bv borreliae in the areas of bigcities in southern Poland.

Address for correspondence: Pet'ko Branislav, MVD., Ph.D., Institute of Parasitology,Slovak Academy of Sciences, Hlinkova 3,040 01 Ko5ice, Slovakia.E-mail: petko@ saske.sk

Key words: Bonelia burgdorferi. Lyme borreliosis, ticks, Ixodes ricinus, WestCarpathians, Upper Silesia, Malopolska Region, Poland.

INTRODUCTION Eurasia, L pacificzs Cooley et Kohls and /. scapularis Sayin North America [1, 7, 23]. Infected ticks frequently

Since its discovery in 1981, Borrelia burgdorfei sensu occur in peripheral md central town parks in the

lato [ 3], etiological agent of Lyme boneliosis (LB), has teritories oflarge cities in Central Europe. Therefore, we

been the subject of many epidemiological and were interesied in the occunence md overinfestation of

epizootiological studies. This polysystemic disease of ticks by the agent ofthis dangerous zoonosis in the area of

humms and animals has been recently regmded as the southemPolandwithhighconcentrationof lagecities.

most common human tick-borne disease in the Holarctic. Little is known about the prevalence of Botelia

In natural foci, LB is transmitted primarily by blood- spirochetes in ticks in Poland. The most complete data

sucking arthropods, especially by ticks of the genus was reported from the northeast provinces by the Institute

Ixodes: I. ricinus (L) in Europe, L persulcatus Schulze in of Maritime and Tropical Medicine in Gdynia | 6, 24,25,

Received: 14February 1997Accepted: 24 Apnl1997

=AAEM

II| 264 Pet'ko B. Siuda K. Stanko M. Tresovd G. Karbowiak G. Fridov6 J

Figure 1. The locations of tick collection in South Poland: A) In

Katowice and vicinity, B) In the northwest region of Tarn6w province.

261. Ticks of the species I. ricinus and Dermacentorreticulatu.r were examined. Most of positive results were

obtained from L ricinus; the spirochetes Borrelia sp. havebeen observed recently also in D. reticulatus (Wegner,

personal information).

Moreover, Siriski et al. [20] published the results of astudy on the Borrelia burgdorferi infectton of ticks inUrwitalt in Masuria Region and several places in thesoutheast Poland (including Katowice and Szczyghce near

Krak6w). Tylewska - Wierzbanowska et al. l22lexamined ticks collected from vegetation, humans andanimals in different provinces all over Poland. L ricinus

and D. reticulatus mostly and occasionalll' ,Argas re.fle.ruswere examined in the above cited works. Positive resultswere obtained only for I. ricinus.

The occurrence of Borrelia burgdorferi in ticks in theclosely neighbouring area of the Carpathians in Slovakia

was studied by Kmety et al. [3], Prokopd6kor'6 er ql.

[19], Petko et aI. [15] and in the bordering area of the

West Carpathians in Moravia by Hubiilek er ul. 19. l0l

and Chmela [4].This paper presents the studies on the occurrence of

Lyme borreliosis agent in ticks in the areas of large citiesin southern Poland over the vear 1996.

J

MATERIAL AND METHODS

Studied area and collection of ticks. The density oftick populations and their overinfestation with LB agent,spirocheta Borrelia burgdorferi sensu lato. u'as studied inten localities of southern Poland in May 1996 (Fi-e. l).Ticks were collected by dragging a light woolen flag(60 x 90 cm) over the plant and bush ve-eetation. Thecollections were carried out primarily in the urban andsuburban biotopes of the city Katowice (Fig. 2). in theplaces most frequently visited by people, e.g. in parks,sporting areas and peripheral villa districts. Some tickswere collected in wooded areas at places with lowintensity of anthropogenic damaging (Chobot, Wal Ruda -

Fig. 3). The area dragged with one flag in one hourmeasured approximately 600 m' in pastures and forestswith shrubs, 800 m- in meadows and parks. Tickscollected from the particular localities were placed in test

tubes with a few blades of grass and preserved at 4oCuntil investigation. The relative density of active ticks wasexpressed as the number of ticks collected with one flagper one hour, as well as a number of ticks per 100 m-(Tab. 1).

Y

tF

Table 1. Material of lxodes ricinus ticks collected in southern Poland in Mav 1996.

Locality Date No. of ticks Time Areadragged" draggedb

RD.No./ hour

males females nymphs total

1

2aJ

4

)6

7

8

9

1 0

May 24

May 24

May 29

May 30

May 30

May 30

May 30

May 25

May 25

May 25

45

7T

37

52

3 1

32

30

1 3

35

39

5 5

5 6

36

47

39

4 I

43

7

30

52

3

5

52d

20

5

27

10

r03

r32

t25

l l 9

75

100

83

20

65

9 l

3

3

3

3aJ

2.25

2.25

I

I

2

2400

1800

I 800

34

.t-1

40

-r0

l5

-lJ

39

20

65

16

2m

l-r00

I _150

l 350

600

600

1200

Total

uhours, bm2

913 23.50 l59m -r0.3

Borrelia burgdorferi in the ticks in southern Poland

Table 2. Borrelia species in lxodes ricinus ticks collected in southern Poland.

Stage Pool samples Single samples

No. of Infection

rate (Vo)"

No. of Infection

rate (Vc)pools positive pools positive

0

16.7

28.3

t2 .5

l 0

6 l

46 13

4 0 5

0

8.2

10.3

9.8

5 1

110 22

360 72

345 69

0

9 (40.97o)

37 (5l.4%o)

34 (49.37o)

Larvae (n = 6)

N y m p h s ( n = 1 1 6 )

Females (n = 406)

Males (n = 385)

u calculated minimum infection rate.

Localities:1. Mikol6w near Katowice, in the "Barbara" coal mine

area, meadow with mixed shrubs and trees.2. Katowice, Ligota, oak-hornbeam forest and surrounding

pastures with shrubs and dewberry.3. Katowice, Kokociniec, forest and pastures with shrubs

near railway.4. Katowice, Wojew6dzki Park Kultury i Wypoczynku

Gen. J. ZiEtka, park in surroundings of tennis-court.5. Katowice, Park Tadeusza KoSciuszki, old park with

local occurrence of shrubs.6. Katowice, Ochojec, forest near railway and

residential district.l. Katowice, Murcki, forest in surroundings of sports

afea.8. Chobot (Krak6w province), Niepolomicka Forest,

old oak-hornbeam forest.9. BorzEcin (Tarn6w province), pastures with mixed

shrubs and trees.10.Wal Ruda (Tarn6w province), pastures and

meadows near forest with predominance of oaks andpines.

Investigation of ticks. Nymphs, males and femaleswere investigated in pools of 5 specimens each. Moreover,some specimens were examined individually. Ticks wereexamined for the presence of Borrelia spp. by dark fieldmicroscopy, according to Wilske et al. I27l and bycultivation according to Preac-Mursic et al. tl8l andTresov6 et al. [211. The samples were examined with aNikon microscope at 400 x magnification. Minimum infectionrate of pools was calculated according to Kahl et aI. f.l2l.

A total of 164 pooled samples containing 820specimens of L ricinus were examined for the presence ofBorrelia burgdorferi sensu lato. Another 93 ticks wereinvestigated individually (Tab. 2).

RESIILTS

In 1996,913 larvae, nymphs and adult ticks I. ricinuswere collected during a 23.5-hour vegetation flagging insouthern Poland. Their relative density in l0 examinedlocalities ranged between 20-65 ticks per one flag in onehour, or 2.8-12.3 ticks per 100 mt, ,espectively (Tab. l).Higher relative densities were observed on pastures in a

Table 3. Examination of pooled samples of lxodes ricinus adults for Borrelia species in the localities of southern Poland.

Locality No. of pools u Positive pools b Infection rate (Vof Isolatedd

Borrelia strainsMales Males Females Males

I

2

3

4

)6

7

8

9

1 0

9

t 2

7

8

6

6

6

2

7

6

1 7 . 8

1 0

2.9

5

13.3

0

1 0

0

t 7 . l

1 3 . 3

t2 .7

1 5 . 5

14.3

2 .9

l t .4

12.5

5

0

6.9

l 0

l 1 P153

y2t0

Pl75

Pt37.Pl42

266 petko B. siuda K, Stanko M. Tresovd G. Karbowiak G, Fridov6 J

+ +

i . lr tuclrl' 4!

ii i', t iI t l, l !

I

L,-"-1

::=J

-ililtr:lr[]!<trT*

r : n Q :l r v r d

iMYQ])itft

1 l r

;\lk/ f tr \

i.qgI r > 4 l

\

o-I3BI

effir--

i- t

o

+'-, ,4 \-.j / Li.iR

Borrelia burgdorferi in the ticks in southern Poland

t % ,

#j*"=** ./ PrzybYsla'1ic"\

" 1 ' " ) ' x

"4:.-. Ma-nfnf31ic'e,:+b -<l f.*,

(1@4

olor... /-lifal-Rudai

f ) (

Figure 3. Tick collection localities in the northwest region of Tarn6w province: 8) Chobot. 9) Borzqcin, l0) Wat Ruda.

\Jadowr ik i i

Mokre

1. t

$:-\I

? /- .-1 *'\i

.-,t -€ s* , ra -4 , ,

' r

$\ffif, - "a'il'\,, iF"le ( r . l \ t " p

wooded area (loc. 9 and 10). Relatively high densitieswere also recorded in some urban and suburban parks andresidential districts in the city Katowice (loc. 2,3, 4,6 and j).

Moving spirochetes, morphologically resemblingbacteria from Borrelia cultures, were observed in 49.3%oof male and 5l.4Vo of female I. ricinus pools. The lowervalues for nymphs (40.9vo) were statistically insignificant(t = 0.690 and t = 0.866, respectively). Minimum infection

1 6

1 4

, r =f

10 = 'c

38 = '

+o

6 g- of

4 do

2 80

1 2 3 4 5 6 7 8 9 1 0

Locality

I Relative density f Minimum infection rate

Figure 4. Relative density and minimum infection rate of lxodes ricinusadults with Borrelia species in southern poland.

ru

rate of I. ricinus nymphs, males and females was balanced(8.2-10.37o) in the entire studied area. Individuallyinvestigated ticks showed the proportion of infectedfemales to be more than twice as high as for males. Singlesamples of larvae and nymphs were not evaluated becauseof a too smal l set (Tab. 2).

Borreliae were found in ticks from 9 localities (Tab. 3).The absence of infected ticks on locality 8 is probably dueto the small number of examined specimens. Minimuminfection rate of I. ricinus adult ticks at studied localitiesfluctuated from 4.0-r5.ovo without correlation withrelative density of ticks (Fig. 4). A relative highinfestation was observed in some suburban meadows andforests of the cities Katowice and Mikol6w (loc. 1,2), aswell as in the urban park (loc. 5), which was higher thanthat in wooded rural areas (loc. 9 and l0).

Minimum infection rate of males and females at studiedlocalities showed differences. At localities 2, 3 and 6,females were infected more frequently, at other localitiesit was males. Five strains of Borrelia spp. were isolatedfrom tick pooled samples at localities I, 4, 5 and 9. Thestrain marked as Pl42 comes from males. the others fromfemales (Tab. 3). Immunochemical investigations of theisolated strains are published in other paper l2l).

DISCUSSION

Relatively high densities of ticks were found in May1996 in the central and peripheral parks of the city ofKatowice. Finding of ticks in central parks of large cities

'.J4rfl

\=.

60

50

40

30

20

1 0

0

L

f

o\o)o

qU'-:<o

v

=aCo

E^ l gI EJ &

I

268

(parks, gardens etc.) is a common phenomenon in the

Central European region l2l.In the urban parks of Prague,

Cerny and Daniel [5] ascertained 23.5-139.5 nymphs and

adults per flag in one hour of collecting, with variable

density during a year and also between different years. In

a suburban park of the city Ko5ice, from 9-212 nymphs

and adults of L ricinus (flaglhour) were collected at 8

localit ies t151.The occurrence of ticks in towns is connected with

establishing new parks in original forest areas or with

building new residential districts on the peripheries of

towns close to forests. Persistence of ticks is determined

by their possibility to accomplish their life cycle, which

depends on the presence of suitable hosts for all

developmental stages. The presence of hosts is important

for adult ticks. In Central Europe these hosts are represented

mainly by squirrels (Sciurus vulgaris), both species of

hedgehog s (Erinaceus europaeus and E. concolor) and

local stray dogs and cats. A sporadical occuffence of ticks

in isolated parks can be attributed to the transmission by

birds.The occurrence of ticks has a mosaic-like character and

their infection with borreliae at different places of an area

with the presence of LB is highly variable, depending on

the place and time of investigation t281. Cases with

seasonal alteration were observed in local populations of

t icks [9, 13, 15].Long-term observations under Central European conditions

showed an impressive local fluctuation in the infestation

of t icks in the individual years of study: 6.5-11.47o [10],19-22.0%o [16]; 0.9-18.67o lI l l ; 2.l4l.7Vo | 151. These

variations in tick infestation are most likely reflecting the

periodical occurrence of borreliae in ticks, which was

pointed out in Slovakia by Kmety et al. t131. In the

Carpathian region of Slovakia this appears as a 4-5 year

cycle, with the last minimum recorded in 1994 and

maximum in 1992-1993 t151. In comparing the values of

the LB prevalence at different places of this area, the

phase of epizootiological cycle should also be considered.

The high variability of Borrelia infection in ticks at

individual developmental stages is probably associated

with the periodicity of LB. Adult ticks become generally

more frequently infected than nymphs, females more

frequently than males. Kahl et al. ll2l reported a

minimum 2.57o infection rate of nymphs, 5.3Vo of males

and I0.27o of females from the territory of Berlin in 1986.

Percentage of ticks infected by spirochetes of Borrelia

spp. examined in Poland varied. In Olsztyn province it has

ranged from 2.9-35.77o (average II.57o) 1251, in Urwitalt

in Masuria region 3.5Vo and in examined localities in

southeast Poland from l3-58.3Vo (in Szczyghce near

Krak6w l9.2%o) t201. Among I. ricinus specimens from

different parts of Poland tested by Tylewska-Wierzbanowska

et al. t22) only 0.75Vo of ticks were infected by B.

burgdorferi. In southern Germany, Wilske et al. l27lfound an average l%o infection in larvae, l07o in nymphs

and 207o in adult ticks, with no difference observed

between the infection rate in males and females. Nymphs

are less frequently infected than adult ticks 120,25,261. as

was observed at several localities in 1992 t8. l5l. A

variation in the prevalence of infected adults is generally

preceded by the change in the infection rate of nymphs in

the previous year. In both mentioned cases, an increased

prevalence of infected adults occurred during the next

year or the next two years. These differences may reflect

the presence of Boruelia donors (animals at the stage of

bacteraemia) and of naturally immunized hosts of I-

ricinus, which may cause either the increase in the

occurrence of borreliae [11] or their loss in ticks tl4l.The proportion between the prevalence of boneliae in

nymphs and adults and the absolute prevalence values in

May 1996 may serve as a basis for estimating the phase of

epizootiological cycle and for prognosing the epidemiological

situation for LB in the region. A significant local

variability of infected tick prevalence on a relatively small

territory points out the wide spectrum of ecological

factors influencins the occurrence of borreliae in nature.

CONCLUSION

The preliminary results indicate the hazard of human

infection elicited by the agent of Lyme borreliosis - a

grave natural focal zoonosis in the urban and suburban

verdure of large cities in southern Poland, which should

not be underestimated. Relatively high prevalence of

infected ticks and the balanced proportion of nymph and

adults infestation with borreliae in southern Poland

suggests the culmination of cycle and its possible

persistence in the year 1997.

Acknowledgement

This work was supported in part by grants No.2/577196 and2t2006196 from the Science Grant Agency of the Ministry ofEducation of the Slovak Republic and Slovak Academy ofSciences, and Grant NN - 4-012196 from the Silesian MedicalAcademy. The authors are grateful to Dr. Jin Panc6k fortechnical assistance.

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t , '

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