Prevalence of Borrelia burgdorferi Sensu Lato and Anaplasmataceae Members in Ixodes ricinus Ticks in Alsace, a Focus of Lyme Borreliosis Endemicity in France

Abstract

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in
this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105
and 114 per 100 m2, respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m2), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2006, p. 3074–3078 Vol. 72, No. 4
0099-2240/06/$08.00⫹0 doi:10.1128/AEM.72.4.3074–3078.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Prevalence of Borrelia burgdorferi Sensu Lato and Anaplasmataceae
Members in Ixodes ricinus Ticks in Alsace, a Focus of
Lyme Borreliosis Endemicity in France
Elisabeth Ferquel,
1
Martine Garnier,
1
Je´roˆme Marie,
1
† Claire Berne`de-Bauduin,
2,3
Guy Baranton,
1
Claudine Pe´rez-Eid,
1
and Danie`le Postic
1
*
Laboratoire des Spiroche`tes,
1
Centre de Ressources en Biostatistiques, Epide´miologie et Pharmaco-Epide´miologie,
2
and
INSERM U657,
3
Institut Pasteur, 75724 Paris, France
Received 6 December 2005/Accepted 9 February 2006
Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in
2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by
Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi
sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest
reported in Europe (105 and 114 per 100 m
2
, respectively). In contrast, the peak density of infected nymphs
was low in the canton of Dannemarie (5/100 m
2
), where the disease incidence was low. The two main
species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent
in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and
adults, respectively.
Lyme borreliosis (LB) is a worldwide disease due to bacteria
belonging to the complex Borrelia burgdorferi sensu lato (31)
and transmitted in Europe by the bite of Ixodes ricinus ticks
(10), which is also involved in the transmission of other patho-
gens, particularly members of the Anaplasmataceae family (7).
A phytoecological mapping of I. ricinus in France revealed a
very large distribution of this tick (12). Moreover, the infesta-
tion of ticks by B. burgdorferi sensu lato has been reported in
the whole French territory (11) and more precisely in restricted
areas (1, 29, 30, 32, 39). A single study in Alsace revealed the
presence of B. burgdorferi sensu lato in 11% of 2,223 nymphs
collected in August 1989 (6). This northeastern region of
France is reported as a region where LB is highly endemic (24).
Moreover, a prospective study conducted in Alsace from 2001
to 2003 (18) estimated the LB incidence between 180 and 232
cases per 100,000 inhabitants per year and recorded high local
variations between cantons (administrative divisions of a de-
partment in France). On the basis of this study (18), we se-
lected two cantons in the Haut-Rhin department where the LB
incidence was high and one canton where the incidence was
low, in order to establish a correlation between the LB inci-
dence and the density of infected ticks. We conducted a two-
year survey in 2003 and 2004 of the I. ricinus population density
and the B. burgdorferi sensu lato infection rate. Because
Anaplasma phagocytophilum is maintained in the same tick, the
infection rate of I. ricinus ticks by members of Anaplasmata-
ceae was also determined.
The study was carried out in the Parc Re´gional des Ballons
des Vosges, in northeastern France. By the drawing of lots, five
and three sampling sites were designated, respectively, in the
north part of the Parc (Munster canton) and in the south part
(Guebwiller canton), where a high LB incidence of 250 cases/
100,000 inhabitants was recorded (18). These sites are at an
altitude of 400 to 700 m, largely covered by dense and contin-
uous forests, and the climate is defined as continental. One
sampling site was designated a “negative control site” in the
Dannemarie canton, where the LB incidence was 36 cases/
100,000 inhabitants. This site is at a lower level (altitude 230 m),
the woods are small and individualized, interspersed with fields,
and the climate is typical of hills in the area.
Questing ticks were collected monthly by three or four
collectors, by dragging a 1-m
2
white cotton cloth on the
vegetation and litter. Each 10 m, designated a subsampling,
ticks attached on the cloth were removed, identified, and
counted. A minimum of 64, 48, and 16 subsamplings were
investigated at Munster, Guebwiller, and Dannemarie, re-
spectively. Adult and nymph ticks were transported still
alive to the laboratory, where they were analyzed for infec-
tion by B. burgdorferi sensu lato. We proceeded using either
culture or PCR on DNA directly extracted from ticks as
described previously (31, 38), after having checked the con-
cordance between the two methods. Identification at the
species level was assessed by PCR-restriction fragment
length polymorphism of the intergenic spacer as described
previously (31, 38). For the detection of members of
Anaplasmataceae, we used primers Ehr521 and Ehr747 (27)
to amplify 247 bp of the rrs gene. The identification was
systematically confirmed by sequencing of PCR products.
For all analyses, ticks were processed individually. To mon-
itor any contamination during the extraction process, one
extraction control (distilled water) was added in each ex-
traction experiment for every 20 ticks.
For the survey, we used a two-level sampling design with
forest sampling sites as the first level and the 10-m-long sub-
* Corresponding author. Mailing address: Laboratoire des Spiro-
che`tes, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15,
France. Phone: 33 1 45 68 83 37. Fax: 33 1 40 61 30 01. E-mail:
dpostic@pasteur.fr.
† Present address: Institut Louis Malarde´, 98713 Papeete, Tahiti.
3074

samplings as the second level, both randomly selected. Tick
density (d) was estimated from the total number of ticks col-
lected in each forest as follows:
d⫽
1
St
ˆy⫽
1
S
M
m
冘
i⫽1
mNi
ni
冘
j⫽1
ni
yij
where Sis the surface of the forest, t
ˆythe estimated total
number of ticks, mthe number of sampling sites randomly
selected, Mthe total number of sampling sites, n
i
the number
of subsamplings randomly selected in the sampling site i,N
i
the
total number of subsamplings in the sampling site i, and y
ij
the
number of ticks collected in subsampling jin the sampling site
i. A 95% confidence interval was calculated for each collection.
The chi-square test was used for between-group comparisons.
The Spearman rank correlation coefficient (r
S
) was calculated
between the tick density and the density of infected ticks and
between the tick density and the infection rate. A Pvalue less
than 0.05 was considered significant.
Densities of I. ricinus ticks, expressed as the number of ticks
per 100 m
2
, are shown in Table 1. The seasonal pattern of tick
activity was unimodal in Alsace, where nymphs and adults
reached a peak at the end of spring. However, the questing
period was spread over a large period of the year. Nymph
activity drastically declined from September and disappeared
in November. The adult activity showed a drop as early as June
in 2004, and the density remained more or less stable until
November, when adults also disappeared (Table 1). Seasonal
patterns of nymph and adult ticks were very similar in the two
cantons of Munster and Guebwiller. The mean nymph density
deduced from the whole tick collection at Munster and
Guebwiller from April to October 2004 (201/100 m
2
) was
two times higher than in 2003 (100 nymphs/100 m
2
). Such a
distribution has been previously observed in a British wood-
land (5). Annual variations in tick density, as observed in
Alsace between 2003 and 2004, have been reported by other
authors (22, 35, 36).
High tick infection rates were recorded (Table 1) in Munster
and Guebwiller. Overall 18% of nymphs (406/2,296) and 25%
of adults (361/1,459) were infected. Similar infection rates
were found in male (26% [88/334]) and female (30% [100/
333]) ticks in 2004. Percentages of infected ticks differed
widely, ranging from 7 to 33% for nymphs and from 8 to 42%
for adult ticks. We did not find any seasonal variation in the
prevalence of infection in I. ricinus (Table 1). Despite monthly
variations, the infection prevalences were regularly and often
significantly higher in Munster and Guebwiller than in Danne-
marie, where ticks were less abundant (Table 1). Particularly,
significantly more nymphs were infected in Munster (P⫽0.01)
and Guebwiller (P⫽0.03) than in Dannemarie (Table 1).
In contrast with the large continuous forests of Munster and
Guebwiller, the smallness of the investigated Gildwiller wood
in Dannemarie, which is interspersed with open fields, could
hamper the maintenance of an abundant wild fauna which
could be used as hosts for ticks. Consequently, the likelihood
of acquiring an infected tick bite in this forest is lower than in
TABLE 1. Density of I. ricinus ticks collected in three cantons of Alsace in 2003–2004 and prevalence of B. burgdorferi
sensu lato infection of ticks
Month or parameter
Density per 100 m
2
(CI) and infection rate (%) for indicated ticks or parameter value
a
Nymphs Adults
Munster Guebwiller Dannemarie Munster Guebwiller Dannemarie
2003
April 51 (34–69), 10.3 NC NC 4 (0–9), 27.6 NC NC
May 103 (78–129), 21.1 248 (54–442), 8.2 NC 7 (5–9), 29.8 18 (5–31), 28.3 NC
June 110 (56–164), 13.3 308 (53–563), 9.3 NC 12 (5–18), 16.2 19 (17–22), 17.6 NC
July 117 (84–150), 17.3 203 (97–308), 6.9 29 (28–31), 5.1 12 (7–17), 21.6 8 (6–11), 13.3 0
September 31 (4–58), 21.2 39 (20–58), 11.7 6 (5–8), 33.3 3 (0–6), 25 6 (3–8), 35.7 0
October 30 (24–35), 20.3 53 (9–97), 15.9 NC 5 (1–8), 27.6 6 (6–6), 21.7 NC
Total no. of infected 119/697 (17.1) 47/487 (9.6) 5/48 (10.4) 129/557 (23.2) 44/209 (21)
ticks/examined
ticks (%)
2004
March 80 (12–149), 21.6 109 (38–180), 14.7 4 (1–6), 0 5 (1–9), 18.4 16 (4–29), 31.6 0
April 246 (38–455), 30.9 308 (156–459), 20 40 (29–51), 9.5 17 (3–30), 23.7 33 (7–58), 36 6 (2–10), 0
May 398 (26–770), 26.2 488 (303–673), 23.3 41 (24–58), 10 34 (5–63), 24.3 51 (14–89), 39.6 6 (2–10), 0
June 177 (0–384), 23.7 238 (101–375), 21.7 35 (19–51), 15 11 (3–18), 23.5 8 (2–13), 29.7 2 (0–4), 0
July 215 (0–471), 20 255 (63–447), 25 34 (21–47), 15 14 (0–29), 20 14 (1–28), 40 2 (0–4), 0
September 34 (3–65), 17.8 59 (10–107), 22.8 21 (12–31), 15 4 (0–8), 30.8 8 (1–15), 42.1 0
October 14 (0–28), 28.6 16 (0–31), 30.2 NC 4 (1–6), 8.3 7 (0–14), 32.3 NC
November 0 1 (0–2) NC 0 1 (0–2) NC
Total no. of infected 130/540 (24.1) 92/420 (21.9) 13/104 (12.5) 82/376 (21.8) 106/291 (36.4) 0/26
ticks/examined
ticks (%)
a
NC, no tick collection was carried out during this month.
VOL. 72, 2006 INFECTION OF IXODES RICINUS IN ALSACE, FRANCE 3075

neighboring forests, as was confirmed by the low incidence of
the disease at Dannemarie.
The human threat is linked to the frequency of encountering
an infected tick and therefore is correlated to the density of
infected ticks (Fig. 1), the most important parameter for eval-
uating the infection risk for humans. Our results confirm that
in Alsace, the LB incidence is correlated to the density of
infected nymphs, as previously shown in Europe as in the
United States (14, 33). Due to their abundance, their small-
ness, the fact that they are not easily visible on the skin, and
their more pronounced anthropophily, nymphs represent the
tick stage the most threatening for humans. However, usually,
less nymphs are infected than adult ticks, which have ingested
a supplementary potentially infected meal. In Alsace, nymphs
are a particular risk for humans because of their high density
and especially because of their high infection rate, often very
close to that of adult ticks (Table 1). We failed to quantitatively
correlate the climatic parameters and the density of infected
ticks observed in Alsace (Fig. 1). High temperatures during the
summer of 2003 (Fig. 1A) could have contributed to a loss of
water by ticks and an interruption of their questing behavior
(28). Moreover, higher hygrometry values and abundant rain-
falls in January (Fig. 1A) could be one of the reasons of the rise
of density in 2004. Otherwise, it has been shown that climatic
parameters of the last year or the year before last had more
influence on tick density (15).
There was a positive correlation between the nymph density
and the density of infected nymphs (r
S
⫽1, P⬍0.001) or adult
ticks (r
S
⫽0.97, P⬍0.001) in 2004, while in 2003, there was a
positive significant correlation for adults (r
S
⫽0.94, P⫽0.005)
but not for nymphs. In contrast, there was no correlation be-
tween the tick density and the B. burgdorferi sensu lato infec-
tion rate in 2003 as in 2004. Although the density of nymphs
was higher in Guebwiller than in Munster, the high infection
rate in the latter canton resulted in similar values for the
density of infected nymphs in the two cantons (Fig. 1C). The tick
density and the density of infected ticks observed in Alsace are
among the highest reported in Europe, where considerable
variations have been recorded (16). In foci of endemicity of
Switzerland, the highest density of infected ticks was 30/100 m
2
(20), values lower than those found in Alsace where the peak
of infected ticks reached near 120 ticks/100 m
2
. One could
argue that the large confidence interval of estimated density
values in Alsace (Table 1) could be at least partly related to
different performances of different collectors. However, no
significant difference was recorded, and our observations
showed that large differences in the numbers of ticks collected
occurred for the same operator from one subsampling to an-
other one, even in the same sampling site. Instead, the very
heterogeneous distribution of ticks, both in time and space,
should account for this large confidence interval. Various B.
burgdorferi sensu lato infection rates were reported from dif-
ferent European countries (3, 8, 17, 19, 21). In the Lyon region
of France, the global infection prevalence was 13%, without
any significant difference in the infection rate of nymphs and
adults (32). In contrast in the Ile de France region, nymphs
were significantly more infected than adult ticks (39). A high
variability of infection levels occurred according to sites (even
those very close together) and time of collection but also be-
tween batches of ticks. Therefore, a large vegetation surface
should be sampled and a large number of ticks should be
included in infection studies in order to obtain more robust
results of the density of infected ticks.
The prevalences of different B. burgdorferi sensu lato species
detected in I. ricinus ticks vary widely. However, as usually
reported in Europe, Borrelia afzelii and B. garinii were by far
the species most frequently encountered in Alsace, each in-
fecting 36% (275/767) of ticks tested. Prevalence of B. burg-
dorferi sensu stricto was rather low (8%) in Alsace, although it
was highly variable in different studies (19, 21, 34). Borrelia
valaisiana infected 16% of ticks (125/767) collected in Alsace.
A similar species distribution was reported in the region of
Lyon (32), France. “Borrelia spielmanii” (which is still awaiting
standing in nomenclature) was identified in only one nymph
collected in Munster in 2004. No Borrelia lusitaniae was found
in Alsace during the two-year survey, while it is the most
prevalent species found in I. ricinus ticks in Portugal (2) and
North Africa (38). We did not observe any difference in the
distribution of B. burgdorferi sensu lato species in ticks accord-
ing to site or time of tick collection. In contrast, the distribu-
tion of B. burgdorferi sensu lato species varied widely according
to the tick stages. B. afzelii was significantly more common in
nymphs (212/406 infected nymphs, i.e., 52%) than in adults
(63/361 infected adults, i.e., 17%). In contrast, B. garinii was
more common in adults (164/361, i.e., 46%) than in nymphs
(112/406, i.e., 28%). In both cases, the difference was highly
significant (P⬍0.001). Similarly B. valaisiana was more com-
mon in adults (71/361, i.e., 20%) than in nymphs (54/406, i.e.,
13%) (P⫽0.02). This fact could be due to different feeding
preferences of ticks according to their development stages.
Nymphs are issued from larvae, which frequently feed on small
rodents, which have been identified as a major reservoir of B.
afzelii (23). In contrast, adults are issued from nymphs, which
frequently feed on birds, a major reservoir of B. garinii and B.
valaisiana (9). The most frequent mixture of species found in
ticks, which associated B. garinii and B. valaisiana (17 ticks/30
ticks infected by two or three species), is consistent with this
hypothesis.
Few data are available concerning the incidence of anaplas-
mosis in Europe, mostly documented on serological evidence
(4, 26). Therefore, 1,065 nymphs and 171 adult ticks, collected
in the three cantons of Alsace, were investigated in 2004 for
infection by Anaplasmataceae. DNA from A. phagocytophilum
was detected in four nymphs and two adults collected at Mun-
ster and Dannemarie, yielding very low infection rates of 0.4%
in nymphs and 1.2% in adult ticks, in the range usually re-
ported in Europe (13, 25, 37). Additionally, DNA of Ehrlichia
sp. was amplified from 44 nymphs and 1 adult tick, whereas
DNA of Wolbachia sp. was amplified from 30 nymphs. No
DNA from Ehrlichia chaffeensis was ever amplified from ticks
collected in Alsace.
In conclusion, the results of our study show that the density
of I. ricinus infected by B. burgdorferi sensu lato in Alsace is
among the highest reported in Europe. Therefore, these re-
sults are in total accordance with the known high endemicity of
LB in Alsace. The demonstration of the infection of ticks by A.
phagocytophilum reveals their possible role in human infec-
tions. However, further investigations, particularly epidemio-
logical studies, are needed to provide evidence of human
3076 FERQUEL ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 1. (A) Climatic parameters provided by Me´te´o France at the Munster meteorological station, monthly average temperature (line) and
maximum temperatures (vertical bars). (B) Density of adult I. ricinus ticks infected by B. burgdorferi sensu lato (obtained by multiplying the tick
density/100 m
2
by the infection rate) in the three sampling sites. (C) Density of nymph ticks infected by B. burgdorferi sensu lato.
VOL. 72, 2006 INFECTION OF IXODES RICINUS IN ALSACE, FRANCE 3077

anaplasmosis in Alsace and to know the actual significance, in
terms of public health, of symbionts in I. ricinus ticks.
We thank Didier Guillemot for his expertise in statistical analysis.
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