Correlation with Infection of Ixodes ricinus Ticks by ... - CiteSeerX

VECTOR-BORNE AND ZOONOTIC DISEASES Volume 7, Number 4, 2007 © Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2006.0633

Lyme Borreliosis Incidence in Two French Departments: Correlation with Infection of Ixodes ricinus Ticks by Borrelia burgdorferi Sensu Lato J. BEYTOUT,1 J.C. GEORGE,2 J. MALAVAL,3 M. GARNIER,4 M. BEYTOUT,1 G. BARANTON,4 E. FERQUEL,4 D. POSTIC4

ABSTRACT We conducted a prospective study to estimate the Lyme borreliosis incidence in two rural French departments, Meuse and Puy-de-Dôme. Concurrently, we investigated the prevalence of ticks infected with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. The incidence of Lyme borreliosis decreased from 156 to 109/100,000 inhabitants in Meuse and from 117 to 76/100,000 inhabitants in Puy-de-Dôme in 2004 and 2005, respectively, corresponding to a decrease in the density of Ixodes ricinus nymphs infected with B. burgdorferi sl. During the same period, the density of adult ticks increased. Interestingly, B. valaisiana, a nonpathogenic species, infected adult ticks more often than nymphs. These results confirmed the correlation between the Lyme borreliosis incidence and the density of infected nymphs, a stage preferentially infected with B. afzelii. In contrast, we found a low rate of infection by A. phagocytophilum, ranging from 0% to 0.4% in Puy-de-Dôme and from 0.8% to 1.4% in Meuse, suggesting a low risk for humans. Key words: Lyme borreliosis incidence—Ixodes ricinus—Ticks— Density—B. burgdorferi sl—France.

INTRODUCTION

L

(LB) is an emerging infectious disease transmitted by the bite of Ixodes ricinus ticks carrying pathogenic species within the Borrelia burgdorferi sensu lato (sl) complex, B. burgdorferi sensu stricto (ss), B. afzelii, B. garinii, and B. spielmanii. In recent years most European countries have reported a high—even an increasing—LB incidence (Bormane et al. 2004, Jensen et al. 2000, Kampen et al. 2004, Mehnert and Krause 2005, Tomao et al. 2005). In France, only one recent study was conducted at a national scale, and it reported a mean incidence of 9.4 cases per 100,000 inhabitants, with a range from 0 cases in Auvergne to 86 cases in Alsace (Letrilliart et YME BORRELIOSIS

al. 2005). Indeed, the most important and welldocumented focus of LB was located in the northeastern part of the country, more precisely in the Alsace region, where the incidence was estimated at around 200 cases/100,000 inhabitants (www.invs.sante.fr/recherche/index2. asp?txtQueryLyme, 2005). However, localized studies revealed a rather high incidence in Berry (Christiann et al. 1996) in central France. Epidemiological data on B. burgdorferi sl infected ticks are available from Brittany (Anderson et al. 1986), Ile de France (Zhioua et al. 1996), and the Lyon region (Quessada et al. 2003). Less is known about the incidence and the epidemiology of LB in other French areas. Moreover, in contrast with the high interest that is usually directed toward LB, other tick-

1Department

3Department

of Infectious Diseases, Clermont Ferrand, France; 2Department of Infectious Diseases, Souilly, France; of Infectious Diseases, Pleaux, France; 4Laboratoire des Spirochètes, Institut Pasteur, Paris, France.

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borne pathogens have received little attention. Such is the case for human granulocytic ehrlichiosis, a flu-like illness due to Anaplasma phagocytophilum, a pathogen that can be cotransmitted with B. burgdorferi sl (Loebermann et al. 2006). Our objectives in the present study were to estimate the incidence of Lyme borreliosis in the human population of two rural French departments, the Meuse in the Lorraine region, northeastern France, and the Puy-de-Dôme in the Auvergne region, at the center of the country. This tick-transmitted zoonose is closely associated with the density of ticks (Hubálek et al. 2003, Stafford et al. 1998), and with the Borrelia species infecting ticks. Therefore, during the same period, we estimated the density of the tick vector I. ricinus, and finally the prevalence of the tick infection by two pathogens, B. burgdorferi sl and A. phagocytophilum in these two departments. We attempted to establish a correlation between the LB incidence and the density of ticks infected with B. burgdorferi sl.

MATERIALS AND METHODS LB incidence Physicians in the two departments, 763 in Puy-de-Dôme and 392 in Meuse, were first contacted by mail. They were asked to report all suspected LB cases to the National Reference Centre (NRC). For this purpose, we provided a questionnaire that included epidemiological, clinical, and biological data for each case. This prospective study was conducted from 2003 to 2005 in Meuse and from 2004 to 2005 in Puyde-Dôme. We reviewed all reported cases and included in the analysis only those that satisfied the European Union Concerted Action on Lyme Borreliosis (EUCALB) inclusion criteria (Stanek et al. 1996). Diagnosis of the early stage of the disease, erythema migrans (EM), was based on clinical examination only, whereas diagnosis of late stages of LB is hampered by a nonspecific clinical presentation. Therefore either serological confirmation or confirmation by pathogen isolation or positive DNA amplification was required to confirm and include the late cases. The annual incidence in each de-

partment, assigned as the number of confirmed cases, was estimated taking into account the local physician population, the actual local physician participation rate, and the population size of each department (INED). Tick collection The methodology used in this study was described previously (Ferquel et al. 2006). Three sampling sites were selected randomly, by drawing of lots among forests in each of the two departments. Twelve and 48 sub-samplings, each designating a 10-m-long distance, were investigated in Meuse and Puy-de-Dôme, respectively, during the two consecutive years 2004–2005. Questing ticks were sampled by dragging a white 1 1 m blanket over the vegetation of each sub-sampling, after which ticks attached to the cloth were removed and identified. Only adult and nymph I. ricinus ticks were counted and collected. Ticks were maintained alive in tubes containing grass to maintain humidity. Examination for infection was performed within 4–5 days of collection. Infection of I. ricinus by pathogens The detection of pathogens was performed monthly in at least 40 ticks (20 nymphs and 20 adults) randomly picked in each sampling site, when available. The presence of B. burgdorferi sl was assessed either by culture in BSK-H medium, followed by polymerase chain reaction (PCR) performed on the culture medium or directly by PCR on DNA extracted from ticks, as described previously (Ferquel et al. 2006). The target used for PCR was the intergenic rrf-rrl spacer, and species identification was based on the MseI restriction pattern of amplified products (Postic et al. 1994). Sequencing was performed to confirm the identification of atypical restriction patterns. Specific detection of A. phagocytophilum was performed by PCR targeting 932-bp in the rrs gene and using primers Ge3A and Ge10R, eventually followed by a nested-PCR using Ge9F and Ge2 (Massung et al. 1998), leading to a 546-bp DNA fragment. The specificity of amplified products as well as genetic diversity of all amplified products was confirmed by sequencing.

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Statistical analysis To estimate the tick density in each forest, we used a two-level sampling design, as described previously (Ferquel et al. 2006). A 95% confidence interval was calculated for each collection. The density of infected ticks was obtained by multiplying the tick infection rate by the tick density per 100 m2. The Spearman rank correlation coefficient rS was used to estimate correlations between the tick density and the density of infected ticks, and between the density of infected ticks and the Lyme borreliosis incidence. A p value less than 0.05 was considered to be significant. RESULTS

neurology (2%), and rheumatology (2%) also participated. Moreover, 7% of participants were hospital practitioners, experts in either internal medicine or infectious diseases. From 2003 to 2005 332 cases were reported to the NRC for the two departments. Only 261 were validated according to the EUCALB criteria (Stanek et al. 1996), 109 cases in Meuse from 2003 to 2005 and 152 cases in Puy-de-Dôme in 2004–2005. The annual incidence rate observed for each department is reported in Table 1. Despite large annual variations in each department, the difference in the incidence rates observed in 2004 and 2005 was not significant, either in Meuse or in Puy-de-Dôme, because of the broad confidence intervals.

LB incidence rate

Clinical presentation

The participation rate of physicians in the LB surveillance ranged from 13% to 17%, slightly higher in Meuse than in Puy-de-Dôme (Table 1), and it remained constant in each department throughout the study. General practitioners represented 73% of participants, but experts in the field of dermatology (5%), pediatrics (5%),

The most common clinical presentation of the disease was erythema migrans (EM) (Table 1), reported in 232 cases (89%). No significant difference in the incidence of different manifestations was observed in the two departments studied. Erythema migrans was the only symptom observed in 71% of patients, and it

TABLE 1. LYME BORRELIOSIS INCIDENCE AND CLINICAL CHARACTERISTICS OF LYME BORRELIOSIS CASES IN TWO FRENCH DEPARTMENTS. FOR EACH CLINICAL MANIFESTATION, THE SEX OF PATIENTS IS INDICATED, WHEN AVAILABLE. Meuse

No. physicians participating to the surveillance network (%) No. cases satisfying the EUCALB criteria Incidence of LB/100 000 inhabitants [95% confidence interval] Cutaneous manifestations EM only EM with no specific symptoms ACA LBC Neuroborreliosis Facial palsy Radiculitis Arthritis Cardiac involvement

Puy-de-Dôme

2003

2004

2005

2004

2005

52 14.4% 25 84 [47–121]

59 16.3% 49 156 [85–227]

62 17.2% 35 106 [55–157]

97 12.7% 90 117 [76–159]

103 13.5% 62 76 [38–114]

16 (8F, 7M) 6 (4F, 2M) 1 (F) —

37 (20F, 17M) 8 (4F, 3M) 1 (M) —

28 (12F, 15M) 5 (4F, 1M) 2 (F) —

51 (29F, 22M) 29 (16F, 13M) 4 (F) 1 (F)

33 (19F, 8M) 19 (12F, 7M) 1 (F) 1 (F)

1 (M)

1* (M)

1* (M) 4 (3*) (3F, 1M) 3 (2F, 1M) 1 (M)

4 (1F, 2M) 2** (M)

— 1 (M) —

EM: Erythema migrans. F: Female, M: Male *Cases with concomitant erythema migrans. **Cases with both radiculitis and facial palsy.

2 (1F, 1M) 1 (M)

— —

4 (M) —

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was associated with general manifestations in 29%, mostly arthralgia and myalgia, then fatigue and fever (Table 1). The size of EM was reported in only 50 cases, between 5 and 10 cm in 10 patients, between 10 and 15 cm in 26 patients, and 15 cm in 14 patients. Disseminated manifestations were more rarely reported. Unexpectedly, neuroborreliosis was noted in only 4.2% of LB patients (n 11). However, we have to underline that 17 clinically suspected neuroborreliosis cases (12 radiculitis, 4 facial palsy, and 1 cranial neuritis), for which the Lyme serology was positive, have not been validated because lumbar puncture was not performed. Thus it was not possible to demonstrate lymphocytosis and/or the synthesis of specific antibodies in the cerebrospinal fluid (CSF). Arthritis and acrodermatitis chronica atrophicans (ACA) occurred in 4% and 3.5% of cases, respectively. Cardiac involvement (third-degree atrioventricular block) and lymphocytoma each occurred in less than 1% of patients (Table 1). Erythema migrans occurred months before arthritis in three cases, and it occurred concomitantly with two cases of facial palsy and three cases of radiculitis. Dates of presumed tick bite, dates of the on-

set of the disease, and dates of the diagnosis were known for 68 patients suffering from EM. The median lag between the presumed contaminating tick bite and the onset of EM was 12 days (range: 1–66 days), and the median lag between the erythema onset and the diagnosis was seven days (range: 1–48 days). In contrast, for seven arthritis and four ACA cases, disease onset occurred 3–14 months before the diagnosis for the former and from 1 to 6 years for the latter. Age and sex distribution The female/male ratio was 1.3. As shown in Table 1, cutaneous manifestations occurred more often in females than in males. The highest incidence was found in people between 40 and 70 years of age. Among seven facial palsy cases, however, three occurred in children younger than 15 years of age, whereas only 15 EM among 232 cases occurred in this age range. Epidemiological features Although LB cases occurred throughout the year, a seasonal case distribution is clearly from Figure 1. No significant difference in the sea-

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25

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FIG. 1.

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35

Seasonal repartition of Lyme borreliosis cases in the two French departments studied.

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LYME BORRELIOSIS INCIDENCE AND IXODES RICINUS INFECTION TABLE 2.

IXODES RICINUS TICKS COLLECTED IN THE TWO FRENCH DEPARTMENTS OF MEUSE, PUY-DE-DÔME IN 2004–2005. NUMBER OF TICKS COLLECTED ACCORDING TO THE STAGE OF TICKS AND INFECTION RATE BY B. BURGDORFERI SL.

AND

Meuse Adult ticks

Puy-de-Dôme Nymph ticks

Adult ticks

Nymph ticks

Number of ticks collected

Infection ratea


Infection ratea


Infection ratea


Infection ratea

Apr-04 May-04 Jun-04 Jul-04 Aug-04 Sep-04 Oct-04

ND ND 7 10 16 4 2

ND ND 1/7 (14)0 1/9 (11)0 5/13 (38) 0/4 0/2

ND ND 262 117 66 29 22

ND ND 5/60 (8)0 2/60 (3)0 6/48 (12) 0/23 2/19 (10)

39 94 73 34 ND 13 ND

10/38 (26) 11/48 (23) 18/48 (37) 11/34 (32) ND 0/13 ND

83 260 347 302 ND 67 ND

6/58 (10) 12/60 (20) 5/60 (8) 10/60 (17) ND 4/14 (29) ND

Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05b Nov-05

19 59 47 40 19 7 13 ND

5/19 (26) 7/29 (24) 5/31 (16) 4/30 (13) ND 2/7 (29)0 1/13 (8)0 ND

40 69 62 64 42 60 22 ND

3/20 (15) 2/52 (4)0 6/49 (12) 3/47 (6)0 ND 2/37 (5)0 2/19 (10) ND

11 153 59 78 ND 75 31 2

3/11 (27) 24/89 (27) 21/59 (36) 22/78 (28) ND 19/75 (25) 12/31 (39) 1/2

10 111 78 106 ND 116 58 2

4/10 (40) 8/49 (16) 10/39 (26) 9/60 (15) ND 13/60 (22) 10/52 (19) 1/3

aInfection rate: Number of ticks infected with B. burgdorferi sl/number of ticks tested for the presence of B. burgdorferi sl (percentage). bIn October 2005, due to bad climatic conditions, only one sub sampling was investigated in Meuse. ND, Not done.

sonal distribution was observed between the two regions (data not shown). Most patients lived in rural areas; 53% recalled previous tick bites and 66% reported a tick bite before the onset of symptoms. The profession or occupation was known for 213 patients (82%). Most had no professional risk, 39% (84/213) had a non-exposed profession, and 49% (104/213) were students, had no occupation, or were retired. Only 12% (25/213) of patients had professional exposure (foresters or farmers). The anatomical location of the tick bite was reported in 149 cases: on the lower limbs in 94 patients (63%); on the neck, trunk, or abdomen in 35 patients (23%); and on the upper limbs in 20 patients (13%). Tick density Number of ticks collected and density of both nymph and adult I. ricinus ticks per 100 m2 collected in the two departments are shown in Table 2 and Figure 2. It is clear that, in Meuse

as in Puy-de-Dôme, the tick density in 2005 was lower than in 2004. However, the decreasing density concerned the nymphs more than the adult ticks. The adult density as estimated in 2005 was higher than in 2004, reaching or even exceeding the nymph density observed in May in Puy-de-Dôme. If the mean values of tick density are compared in this latter department, the nymph density decreased from 37/100 m2 in 2004 to 15/100 m2 in 2005, while the adult density increased from 9 to 12/ 100 m2. In Meuse, the mean density of ticks collected during five months in 2004 was 82 nymphs per 100 m2 and 6 adults per 100 m2, whereas an average of 49 nymphs/100 m2 and 25 adults/100 m2 was recorded during the corresponding months in 2005.The tick density recorded in 2005 differed from that recorded in 2004 in two major points. First, in 2005, there was no significant difference between the nymph tick density and the adult tick density. Second, the tick density was stable throughout 2005, without seasonal variations, and no evident peak density was observed. The contrast, in the tick distribution ob-

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Density of infected ticks /100 m

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Oct-05

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Month of tick collection Nymphs Nymphs infected by B. burgdorferi sl Adults infected by B. burgdorferi sl

Adults Nymphs infected by pathogenic B. burgdorferi Adults infected by pathogenic B. burgdorferi

FIG. 2. Density of questing nymph and adult Ixodes ricinus ticks per 100 m2 and density of ticks infected by Borrelia burgdorferi sl or pathogenic species of B. burgdorferi (B. afzelii, B. garinii, B. burgdorferi ss, and B. spielmanii) per 100 m2 (obtained by multiplying the tick density by the infection rate) determined in Meuse (A), and in Puy-de-Dôme (B).

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served between 2004 and 2005 was particularly marked in Meuse (Fig. 2). Tick infection Altogether, 598 ticks in Meuse, and 1051 ticks in Puy-de-Dôme were studied for infection by B. burgdorferi sl in 2004–2005. Comparable infection rates were observed every year in the same region. Because of large variations in both the tick density and infection rate, wide monthly variations were observed for the density of ticks infected with B. burgdorferi sl in the two departments studied (Fig. 2). An average of 17% of nymphs and 29% of adults collected in Puy-de-Dôme were infected, whereas only 8% of nymphs and 19% of adults collected in Meuse were infected in 2004 and 2005. As a result of the high adult density, the peak density of infected adults in 2005 was about two times higher than in 2004 in the two departments, and it was above the peak density of infected nymphs (Fig. 2). There was a positive

correlation between the tick density and the density of infected adult ticks in Meuse (rS 0.90, p 0.001) as in Puy-de Dôme (r 0.94, p 0.001). There was a positive correlation for nymph ticks in Puy-de Dôme, (rS 0.89, p 0.001) but no correlation in Meuse (r 0.50, p 0.11). As shown in Figure 3, B. afzelii B. garinii, and B. valaisiana were found in, respectively, 42%, 22%, and 18% of infected ticks. However, although B. afzelii was the predominant species infecting nymphs in Puy-de-Dôme (65%) as in Meuse (60%), adults were almost equally infected with B. garinii and B. afzelii in Puy-deDôme, whereas they were mostly infected with B. valaisiana in Meuse (Fig. 3). B. burgdorferi ss was rare, particularly in Meuse. A. phagocytophilum was not detected in 2004 and was detected in only two ticks among 455 collected in Puy-de-Dôme in 2005. In Meuse, two ticks among 245 collected in 2004 and five ticks among 349 collected in 2005 were infected with A. phagocytophilum.

70

60

Adult ticks Meuse ( N=31) Adult ticks Puy-de-Dôme (N=152) 50

% of infected ticks

Nymph ticks Meuse (N=33) Nymph ticks Puy-de-Dôme (N=92) 40

30

20

10

0 B.afzelii

B.garinii

B.b ss

B.valaisiana

B.lusitaniae

B. spielmanii

Coinfection

FIG. 3. Borrelia burgdorferi sl species involved in tick infection, according to tick development stage and sampling sites.

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DISCUSSION Lyme borreliosis is not a notifiable disease in France; Therefore the objectives of our prospective study were achieved through the set-up of a volunteer physician network to transmit clinical and epidemiological data for Borrelia to the NRC. Such a prospective study, costly in terms of time and resources, can be more easily conducted at a restricted regional scale. This is why we focused on two French departments, the Meuse department, in the Lorraine region, where the incidence ranged from 20 to 50 cases per 100,000 inhabitants, and the Puy-de-Dôme, in the Auvergne region, where no cases were reported in a previous national study (Letrilliart et al. 2005). The incidence estimated in the present study was markedly higher than in the Letrilliart et al. study and was in the same range for the two French departments, and was similar to incidence rates usually reported in neighboring European countries (Huppertz et al. 1999, Mehnert and Krause 2005, Nygard et al. 2005). However, the LB incidence rate estimated in Meuse was far lower than in Alsace, a geographically close region, where the incidence rate was previously estimated around 200 cases/100,000 inhabitants (http://www. invs.sante.fr/recherche/index2.asp?txtQuery Lyme 2005). Interestingly, in Alsace, we previously reported a very high density of infected nymphs, reaching more than 100 ticks/100 m2 (Ferquel et al. 2006). For comparison, in eastern Europe as in Slovenia, the LB incidence recorded in 1997 was 155/100,000 inhabitants (Strle 1999), reaching up to 2.3% in forestry workers (Rojko et al. 2005), while in the USA, the highest annual incidence amounted to 600 cases per 100,000 inhabitants in some counties (Chow et al. 2003). Despite an acceptable participation rate of practitioners in our study, we are aware that some of our results could be compromised, particularly by the lack of biological data concerning late cases of LB. Indeed, according to the EUCALB criteria, strong laboratory support is required for confirmation of late LB cases. Particularly, lymphocytic pleocytosis and specific antibodies in the CSF are essential to confirm the diagnosis of neuroborreliosis. In a

rural and private practice, however, lumbar puncture is not always feasible, and these data were often lacking. Indeed, 17 neuroborreliosis cases were clinically suspected but not included in the study. If we hypothesize that the cases with positive serology but without available data on the CSF were actual neuroborreliosis, then the frequency of this manifestation would increase from 4% to 10% of LB cases. This finding would be more consistent with that reported in France in private practice (Letrilliart et al., 2005). Thus, the incidence of neuroborreliosis was probably underestimated in our study. In contrast, although we have included in the study only acute objective joint swelling associated with high level of specific IgG serum antibodies, we are also aware that in endemic regions, people in high-risk groups may have high seroprevalence rates related to former infections. Previous studies in central France in a region adjacent to the Puy-deDôme, revealed a seropositivity of 3% of blood donors, whereas 15% of hunters were seropositive (Christiann et al. 1997a, b). In the present study, however, we reported 4% of patients suffering from articular involvement, a frequency consistent with that reported in private practice in France (Letrilliart et al. 2005). In contrast, reports from hospital practice in France, indicate that arthritis occurred in about 20% of patients (Dhôte et al. 2000, Lipsker et al. 2001). Otherwise, arthritis was reported more frequently than neuroborreliosis in a surveillance study conducted in Central Germany (Huppertz et al. 1999). The relative frequency of each clinical presentation associated with borreliosis is consistent with previous studies conducted in Europe (Mehnert and Krause 2005, Nygard et al. 2005), although different case definitions or inclusion criteria were used in the various studies. We found a very high incidence of EM, the early manifestation of LB (reported in 89% of included cases), a result within the upper range reported in comparable studies in Europe (Berglund et al. 1995, Huppertz et al. 1999) as in the United States (Chow et al. 2003). This manifestation is more often observed in private practice. Because few physicians indicated the size of the cutaneous manifestation, we in-


cluded all cases considered clinically confirmed by the physician. The difficulty in correctly identifying EM among erythematous dermatoses has been emphasized previously (Lipsker et al. 2004). However, no means other than clinical examination was available. These data demonstrate the need to engage in continuous medical education in the field of clinical LB and to reinforce to physicians the need to make good use of laboratory investigation, particularly serological testing. Because LB is the most frequent tick-borne disease, it was of interest to determine how the LB incidence correlated to the density of ticks and, more precisely, to the density of ticks infected with B. burgdorferi sl. This is the first time that local variations in tick densities and tick infection rates have been provided concurrently with clinical LB incidence data in France. Considerable monthly as well as annual variations in the density of ticks infected with B. burgdorferi sl occurred concurrently with variations in I. ricinus density in the two French departments studied. The reasons for such variability probably lay in diverse competing environmental factors (Schauber et al. 2005), mainly climatic conditions (McCabe and Bunnell 2004), vegetation type, and abundance of wild hosts (LoGiudice et al. 2003). Tick density was higher in Meuse than in Puy-de-Dôme; however, because of higher infection rates in the latter, mean densities of infected ticks were similar in the two departments. Only one annual tick density peak was observed during the course of our study, usually occurring between April and June. However, the exposure of the human population, peaked in July and August, when outdoor activities are highest. This is likely the reason for the lag between the peak of tick activity and the peak of disease incidence. Although the density of ticks infected by pathogenic B. burgdorferi sl species and the LB incidence were positively associated, the correlation between these two factors was not significant. Moreover, this correlation increased, although it still was not significant, when we considered the LB incidence and the density of infected ticks two months previously. A longer-term study could help to elucidate such correlations. One strik-

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ing observation should be noted: in 2005, in Meuse as in Puy-de-Dôme, the density of I. ricinus nymphs was significantly lower than in previous years, contrasting with a high adult density. Consequently, the density of nymphs infected with B. burgdorferi sl was particularly low, whereas the density of infected adults was high, largely exceeding, as in May 2005, the values usually observed in nymphs. Concurrently, the LB incidence decreased, although not significantly due to a large confidence interval, from 2004 to 2005, suggesting that the risk of LB is principally linked to infected I. ricinus nymphs. These results are consistent with previous studies in Europe (Jensen et al. 2000, Jouda et al. 2004, Robertson et al. 2000). Moreover, B. valaisiana, a species of uncertain pathogenicity, more often infected adult ticks (42% in Meuse and 20% in Puy-de-Dôme). In England, high densities of ticks, most of which were infected with B. valaisiana, did not lead to any LB cases (Robertson et al. 2000). The period June-August presents the highest contamination risk because the highest levels of outdoor activity occur in conjunction with peak tick activity. This was demonstrated by the higher incidence among people without any professional exposure. Our data suggest that LB incidence is determined primarily by variations in densities of infected I. ricinus nymphs. The sampling sites selected for our study in the two French departments were forested areas that included footpaths heavily used for recreational activities. The high proportion of ticks infected with pathogenic B. burgdorferi sl in these areas suggested a high risk of acquiring LB, confirmed by a high incidence of the disease in the general population of the concerned departments. In contrast with the high prevalence of tick infection by B. burgdorferi sl, we observed a very low rate of infection by A. phagocytophilum in Meuse, and an even lower rate in Puy-deDôme, suggesting a low risk for humans. Long-term studies involving the trend of both LB incidence and density of infected ticks, will be necessary to determine the actual correlation between these parameters. This should help to promote interventions from health care providers for public information and prevention.

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