Laboratory inv est iga tion Borrelia Burgdo1:f"eri genospecies 
K E Y 
WORDS 
Borrelia 
burgdorferi, 
Borre/ia 
afzelii, 
isolation of 
strains, 
ticks, 
detection of 
DNA, 
lxodes 
ricinus, 
epidemiology, 
RFLP profiles, 
genospecies 
Borrelia burgdorferi ge,wspecies in 
humans afld ticks in theAlpeAdria region 
M. Cinco 
SUMMARY 
In this paper we report on the genospecies prevalence of Borrelia burgdorferi sensu stricto we found in 
human Lyme borreliosis and in the tick vector lxodes ricinus in two distinct but restricted areas of the 
Alpe Adria reg ion, the Karst and Belluno territory. Different rates of genospecies prevalence were found 
in the two areas studied, in humans as well as in ticks, with a net predominance of Borrelia afzelii in 
human infection in the Belluno area. 
Introduction 
Lyme borreliosis (LB) is enclemic in North-Eastern 
Italy, affecting humans mainly in the Friuli Venezia 
Giulia ancl Veneto regions. About 50 cases per year have 
been recorclecl at the Hospital of Cattinara, Trieste (De-
partment of Dermatology ), and 119 patient with LB have 
been recordecl at the Hospital of Belluno during the 
1993-98 period. 
Investigations on the presence of Borrelia burg-
dorferi (Bb) sensu lato, the etiological agent of LB in 
Italy have been carriecl out mainly in the Karst territory 
by isolating strains and by detecting Borrelia DNA in 
the tick vector I. ricinus by PCR (1,2), leading to the 
iclentification of the circulating genospecies and the risk 
locations. Since the prevalence ofthe circulating geno-
species of Bb is important for epidemiological reasons 
and for basic knowledge on the biology of the organ-
ism, both in humans ancl ticks, we report here the com-
parative data obtained from the identification ofthe Bb 
strains isolated from patients with genospecies found 
in ticks, in the Karst and Belluno areas. 
Methods 
Clinical samples and isolation oj Bb 
Samples of skin biopsies, blood, heart biopsies, or 
cerebrospinal fluicl were taken from patients with vari-
ous manifestations ofLB ancl put into BSK medium con-
taining 5% normal rabbit serum. Ali specimens were 
incubated at 32°C for at least two months, and samples 
of each culture were examined for spirochetes weekly 
Acta Dermatoven APA Vol 10, 2001, No 4 - -------------------------------131 
Borre/ia Burgdo1feri genospecies 
by dark-field microscopy. 
Classijication ojisolated Bb strains 
Bb strains were classified by PCR-RFLP analysis of 
the rr/A-rr!B intergenic spacer (1) and by chromosomal 
analysis w ith pulse field electrophoresis (PFGE). This 
latter method was performed by Dr. Lorenzo Ciceroni 
at Istituto Superiore di Sanita (Rome). Genomic DNAs 
were prepared as described by Taylor et al. (2) with 
slight modifications. 
Detection oj B. burgdorjeri Dl\TA in Ixodes 
ricinus. 
Ticks collected in different areas of the Karst and Belluno 
territo1y were processed for DNA extraction and Borre-
lia DNA detection by PCR. Different levels of PCR sen-
sitivity and specificity were used in our Laboratory, 
ancl finally we eh ose the technique of Reverse Blot Line 
Hybridization as reported (3-7), which allowed us to 
individuate the genospecies of the infecting Borreliae. 
Mixed infections were often reported. 
Results and discussion 
The sam ples of the patients from the Karst area were 
from erythema migrans , disseminatecl erythema 
migrans, arthritis, and myocarditis: the isolatecl strains 
belonged to all three Bb genospecies known to be hu-
man pathogens, Borrelia garinii, Borrelia burgdor/eri 
sensu stricto and Borrelia afzelii, with a prevalence of 
40%, 30% ancl 30%, respectively. Strains isolatecl from 
e1ythema migrans patients of the Belluno area predomi-
nantly belonged to the Borrelia afzelii genospecies. The 
RFLP profiles w ere obtainecl after MSEI cligestion of the 
amplicon. The majority of the profiles were B. afzelii 
(85%). Only two strains (5%) belongecl to the B. garinii 
genospecies ancl two strains (5%) were B.burgdorferi 
sensu stricto. These data were confirmed by the PFGE 
analysis performed at the Istituto Superiore di Sanita, 
Rome. 
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Laborator y investigation 
The prevalence of Borrelia genospecies in I. rici-
nus was determined by PCR and Reverse Blot Line Hy-
bridization. Four genospecies were detected, B. afzelii, 
B . garinii, B . burgdorferi sensu stricto, and B. 
valaisiana, very often in combination; most prevalent 
was B. garinii alone (30%), followed by the associa-
tion between B. garinii and B. burgdo1/eri sensu stricto 
(32%); less prevalent were other combinations and only 
2% and 4% of the ticks were infected with either B. 
afzelii or B. valaisiana. 
In the Belluno area the genospecies B. burgdor/eri 
s.s ., B. valaisiana and B. afzeliiwere detected in ticks 
(Grazioli et al. , personal communication), but the rate 
of infection of B. afzelii was not as high as found in 
hmnans. 
In conclusion, different rates of genospecies circu-
lation exist in the two areas examined. Of note is the 
higher prevalence of isolates belonging to the geno-
species B. afzelii among humans. Similar epidemiologi-
cal findings have been reported from Slovenia (8) . The 
dominant prevalence of B.a/zelii in humans in the Be-
lluno area may be clue to the fact that the isolates were 
obtained from patients with e1ythema migrans; there is 
general agreement that skin manifestations are most 
often caused by this genospecies . Moreover, our data 
suggest that genospecies prevalence in ticks does not 
reflect the genospecies prevalence in infected humans. 
It may be hypothesised that this finding is clue to a clif-
ferent selective pressure p erformed by the different 
animal hosts and ticks on Borrelia strains having which 
have a clifferent complement resistance. 'rt has been 
demonstratecl that strains of B. a:fzelii are resistant to 
human complement (9) , but many strains of B. garinii 
are not. Therefore this latter genospecies is more favor-
ite to maintain the infection in ticks than in mammalian 
hosts. 
Acknowledgments 
We thank C. Zasio and G. Bertiato, Microbiology 
Laborato1y, Hospital ofBelluno, for isolation and sencl-
ing of B. burgdor/eri strains from Belluno. 
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132 
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Laboratory investigation 
A U T H O R 'S 
ADDRESS 
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Marina Cinca, PhD, Department oj Biochemistry Microbiology Section, 
Lab Spirochetae, Via Fleming 22, Universita di T'rieste, 34 12 7 'I'rieste, Italy 
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