OspC gene variations among Borrelia burgdorferi isolates 
INVESTIGATION OF TICK-DERIVED 
L YME DISEASE BORRELIA STRAINS 
ISOLATED IN STYRIA, AUSTRIA 
K. Pierer, D. Stiinzner, I. Livey, C. P. Gibbs, 
H. H. Kessler and E. Marth 
ABSTRACT 
In Styria, Austria, several areas are known to be endemic far Lyme disease. The Borrelia spirochetes which 
are the causative agent far this illness are transmitted by the tick vector Ixodes ricinus. The present study was 
undertaken to survey the tick population of Styria far the presence of Borrelia and to characterize the strains 
isolated. Ticks were collected in biotopes known to be natural faci of tick borne encephalitis, and Borrelia 
were cultivated from tick extracts. Each isolate was characterized by species and, from several strains, the 
sequence of the ospC gene was determined. Sixteen Lyme disease Borrelia strains were isolated, ten of which 
were further characterized. These included three Borrelia burgdorferi sensu stricto, three Borrelia afzelii and 
faur Borrelia garinii isolates. Sequence analysis of two of the Borrelia afzelii isolates and three of the Borrelia 
garinii isolates indicated that two of the strains have ospC genes identical to alleles previously described 
(RFLP types 20 and 34), and three of the genes were novel variants. In conclusion, it was demonstrated for 
the first tirne that the three major Lyme disease Borrelia species are present in the tick population of Styria. 
The ospC genes of the analyzed strains showed a high degree of variability. 
KEY WORDS 
Lyme disease, borrelia strains, ospC gene, RFLP types, tick isolates, Styria 
INTRODUCTION 
Lyme disease (LD) is a vector-transmitted infection 
caused by spirochetes of the genus Borrelia (1,2). In 
the Austrian province of Styria, the primary vector 
transmitting the infective agent is the tick Ixodes 
ricinus (3,4). In humans, LD is a multi-systemic 
illness with a wide range of symptoms ( dermatological, 
neurological, articular or cardiological) (5-8). Corre-
acta derrnatovenerologica A.P.A. Vol 5, 96, No 3-4 
lations between clinical manifestations of LD and 
Borrelia species have been reported (9,10). The 
major species responsible for LD are Borrelia 
burgdorferi (Bb) sensu stricto, B. afzelii and B. garinii. 
LD Borrelia are extremely heterogeneous (11-13); 
one of the most variable proteins expressed by 
Borrelia is the outer surface protein OspC (14-i6). 
Pior to this study, no data was available regarding 
LD Borrelia strains present in the tick population of 
93 
OspC gene variations among Borrelia burgdorferi isolates 
Table I. Collection areas, number of collected ticks and number of isolates. 
Tobelbad 284 
Fernitz 500 
Grambach 71 
St. Veit / Vogau 53 
Kleinsemmering 98 
Styria. The aim of the present study was to survey 
ticks from this region for the presence of Borrelia 
and to perform a preliminary characterization of the 
spirochete strains isolated. Ticks were therefore 
collected from five different areas. Borrelia were 
isolated from the ticks, and the species of each 
spirochete isolate identified. Additionally, far severa! 
isolates, the nucleotide sequence of the ospC gene 
was determined. 
MATERIALS AND METHODS 
Five biotopes were selected for tick collection 
(Tab. I and II). These areas are endemic far human 
manifestations of LD and natura! foci of tick borne 
encephalitis. A total of 1004 ticks, including adults, 
nymphs and larvae, were collected by the flag dragging 
method; all were identified as Ixodes ricinus. Ticks 
were pooled (2-5 per pool) and cultivated in BSK-
II- medium (Sigma Aldrich Chemie, Deisenhofen, 
Germany) with 12 mg/ml SXT at 33°C. After three 
to five passages, cultures were harvested in the 
logarithmic growing period. The protein profile was 
examined by SDS-PAGE using the PHAST-Gel-
94 
5 
6 
1 
1 
3 
Zl (5 nymphs) 
Z2 (5 nymphs) 
Z12 (3 nymphs) 
Z13 (2 nymphs) 
Z14 (2 females) 
Z3 (2 females) 
Z4 (5 nymphs) 
Z5 (5 nymphs) 
Z6 (5 nymphs) 
Z7 (5 nymphs) 
Z16 (2 nymphs) 
Z15 (2 nymphs) 
ZS (2 females) 
Z9 (5 nymphs) 
ZlO (2 males) 
Zll (2 males) 
Gradient 8-25 in a Phast System Separation and 
Development Unit (Pharmacia, Uppsala, Sweden). 
The protein patterns were visualized by silver staining 
(data not shown). 
Species determination was performed for isolates 
(n = 10) with less than six passages in culture. 
Species-specific primers were used in the polymerase 
chain reaction (PCR) to amplify 16S rRNA sequences 
from the strains as described (17). 
The ospC gene was amplified from five of the 
strains and the nucleotide sequence of the PCR 
product determined as recently described (16). 
Sequences were analyzed using GNASIS and PROSIS 
software. 
RESULTS AND DISCUSSION 
Cultivation of pools of the ticks resulted in the 
isolation of sixteen Borrelia isolates (Tab. I). Species-
specific identification of those isolates with less than 
six passages in culture (n = 10) revealed that three 
isolates were Bb sensu stricto, three isolates B. 
afzelii, and four isolates B. garinii. More than one 
acta dennatovenerologica A.P.A. Vol 5, 96, No 3-4 
OspC gene variations among Bo1Te/ia burgdorferi isolates 
Table II. Species-specific identified isolates of B. burgdo,feri s.l. in 5 Styrian regions endemic for bo1Telial infections . 
. COLLECTIO 
c(n = 5) 
Tobelbad 
Fernitz 
Grambach 
St. Veit/Vogau 
Kleinsemmering 
species was found in two of the five collection areas 
(Tab. II). The ospC gene was sequenced from five 
of the isolates. Bach strain examined had a different 
ospC allele; one of the B. garinii strains had RFLP 
type 20, another had RFLP type 34 and the remaining 
three ospC genes from the isolates Z5, Z13 and 
Z16 were novel alleles. 
In the present study it is shown for the first tirne 
that the three major LD Bolielia species Bb sensu 
stricto, B. afzelii and B. garinii are present in the 
tick population of Styria. In two of the five collection 
areas, more than one Bo1Telia species could be 
isolated, indicating co-existence of multiple species. 
Z12 
Z13 
zs 
Z6 
Z16 
Z15 
Z8 
Z9 
ZlO 
Zll 
B. garinii 
B. garinii 
B. afzelii 
B. burgdo,feri s.s. 
B. afzelii 
B. afzelii 
B. garinii 
B. burgdo,feri s.s. 
B. garinii 
B. burgdo,feri s.s. 
Among the Styrian isolates, the ospC gene is highly 
variable. Three of the five determined ospC RFLP 
types have not been described until now. The other 
two RFLP types were described in Bomlia strains 
originating from varying geographical locations. The 
ospC gene of isolate Z8 showed RFLP type 20, 
which has been found in the Czech Republic, France 
and Switzerland. !solate ZlO possessed an ospC 
gene with RFLP type 34, also found in France and 
Finland. It is concluded that the Bo1Telia strains 
infecting Ixodes ricinus ticks in the Austrian province 
of Styria are genetically highly diverse. 
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AUTHORS' ADDRESSES 
Karen Pierer, MD, Institute of Hygiene, Karl-Franzens-University Graz, 
Universitatsplatz 4, A-8010 Graz, Austria 
Doris Stiinzner, PhD, Institute of Hygiene Graz, same address 
lan Livey, PhD, Immuno AG, Biomedical Research Center, UferstraBe 15, A-2304 Orth/Donau, Austria 
Carol P. Gibbs, PhD, Immuno AG, Biomedical Research Center, same address 
Harald H. Kessler, MD, Institute of Hygiene Graz, same address 
Egon Marth, MD, professor, chairman, Institute of Hygiene Graz, same address 
96 acta dennatovenerologica A.P A. Vol 5, 96, No 3-4