Epidemiol. Infect. (2003), 131, 1139–1147. f 2003 Cambridge University Press DOI: 10.1017/S0950268803001328 Printed in the United Kingdom Acarological risk of exposure to agents of tick-borne zoonoses in the first recognized Italian focus of Lyme borreliosis A. MANNELLI1*, G. BOGGIATTO1,E.GREGO1,M.CINCO2,R.MURGIA2, S. STEFANELLI3, D. DE MENEGHI1 AND S. ROSATI1 1 Dipartimento di Produzioni Animali, Epidemiologia, Ecologia, Facolta` di Medicina Veterinaria, Universita` degli Studi di Torino, Italy 2 Laboratorio sulle Spirochete, Dipartimento di Scienze Biomediche, Universita` degli Studi di Trieste, Italy 3 Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Pisa, Italy (Accepted 3 July 2003) SUMMARY Acarological risk was calculated as the probability of encountering at least one host-seeking Ixodes ricinus tick infected by the pathogen Borrelia burgdorferi sensu lato, in 100 m transects in the province of Genoa, Italy. The seasonal pattern of I. ricinus was studied using generalized estimating equations (GEE) with negative binomial error, to consider overdispersion of tick counts and repeated sampling of the same dragging sites from April 1998 to March 1999. Prevalence of infection by B. burgdorferi s.l. was evaluated by PCR and hybridization with genospecies-specific probes. Acarological risk (R) peaked in April (R=0.2, 95% CI 0.13–0.26) and November (R=0.29, 95% CI 0.10–0.46). Borrelia garinii and B. valaisiana were the most common genospecies at our study site suggesting a major role of birds as reservoirs. DNA from Anaplasma phagocytophilum, the agent of granulocytic ehrlichiosis in humans and animals, was amplified from an adult I. ricinus. INTRODUCTION regions [4, 5]. Anaplasma phagocytophilum, the aetio- logical agent of human granulocytic ehrlichiosis The first Italian case of Lyme borreliosis, a tick-borne (HGE), was identified in ticks in Italy in 1998 [6], and zoonosis caused by the spirochaete Borrelia burgdor- specific antibodies were detected in dogs from Liguria feri sensu lato (s.l.) [1], was reported in 1984 from the [7]. Under these circumstances, the risk of exposure village of Borzonasca, in the province of Genoa, in to pathogen-infected I. ricinus for people and other the northwestern region of Liguria [2]. The disease’s susceptible animals (acarological risk) should be as- annual incidence rate in the same area was estimated sessed in the Borzonasca area. Such a risk measure to be 17 human cases per 100 000 inhabitants [3]. In can be estimated as the mean number of infected, Italy, the intensity of transmission of B. burgdorferi host- seeking ticks per 100 m2 of ground [8]. s.l. by the tick vector Ixodes ricinus varies geographi- Borrelia burgdorferi s.l. can be further classified in cally, with the highest prevalences of spirochaetes in genospecies based upon genetic and phenotypic dif- host-seeking ticks being reported from northeastern ferences [1]. Three genospecies are presently known to cause the disease in Europe: B. garinii causes neuro- * Author for correspondence: A. Mannelli, Dipartimento di Pro- borreliosis and is mostly associated with birds as duzioni Animali, Epidemiologia, Ecologia, Facolta` di Medicina Veterinaria, Universita` degli Studi di Torino, via Leonardo da reservoir hosts; whereas B. afzelii which causes cu- Vinci, 44, 10095, Grugliasco (To), Italy. taneous lesions, and B. burgdorferi sensu stricto (s.s.) Downloaded from https://www.cambridge.org/core. IP address: 170.106.202.226, on 30 Sep 2021 at 15:47:42, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268803001328 1140 A. Mannelli and others which causes joint disease, are found in small and March 1999, were provided by the Hydrographic and medium-sized mammals [9]. Therefore, geographical Mareographic Office (Ufficio Idrografico e Mare- variations in the relative frequency of genospecies grafico) of Genoa. may be associated with ecological and clinical pat- terns of Lyme borreliosis. Detection of B. burgdorferi s.l. in ticks In this study, we collected host-seeking I. ricinus Borrelia burgdorferi s.l. isolation was attempted from in Liguria, and applied statistical models for over- 10 adult and 18 nymphal I. ricinus from Borzonasca dispersed, correlated data to obtain valid estimates of and from 10 adult and 12 nymphs from Chiavari, tick abundance and seasonality [10]. We also carried in pools of 4–10, as previously described [12]. For out laboratory analyses to identify B. burgdorferi the characterization of isolates by genospecies, re- s.l., as well as A. phagocytophilum,inI. ricinus, and striction fragment length polymorphism (RFLP) was characterized these agents by genetic techniques. Tick employed as described by Postic et al. [13]. abundance data and laboratory results were integrated For pathogen detection by PCR, host-seeking to obtain point estimates and confidence intervals I. ricinus (115 nymphs and 55 adults) were individu- (CIs) of an acarological risk index. ally homogenized with a pestle in microcentrifuge tubes. After extraction by Isoquick (Orca Research, Bothwell, WA, USA), DNA was suspended in 50 ml METHODS of nuclease-free water and utilized (5 ml per reaction) Tick collection in a nested PCR as described by Rijpkema et al. [14], using primer pairs specific for the intergenic spacer Host-seeking ticks were collected during 11 monthly region comprised between genes codifying for sub- sessions, from April 1998 to March 1999, excluding units 5S and 23S of ribosomal RNA. The variability February 1999. Six inland sites were selected, based of the amplified fragment allows characterization of on accessibility, which encircled the inhabited centre B. burgdorferi s.l. species. The cycling profile de- of the village of Borzonasca (2000 inhabitants, ap- scribed in the original paper was used [14]. As positive proximately 50 km east of Genoa, and 13 km from controls, we alternatively used DNA from a B. garinii the sea). Three sites were in the immediate vicinity strain isolated within this study, B. burgdorferi s.s. of housing and three within 1 km. The area is charac- (Alcaide strain), B. afzelii (Nancy strain), B. valai- terized by the presence of other villages of approxi- siana VS 116 strain. Distilled water and I. scapularis mately the same size as Borzonasca, resulting in a nymphs from a colony maintained at the Yale School population density of 114 people per km2 [infor- of Medicine (kindly provided by Professor D. Fish), mation provided by the Consortium of Municipalities and DNA extracted from bacteria (Escherichia coli, (Comunita` Montana) of Aveto, Graveglia, Sturla]. Staphylococcus intermedius, Streptococcus spp.) were Five additional tick collection sites were within 2 km used as negative controls. Amplified DNA underwent from the sea, on the northern slopes of hills sur- electrophoresis on agarose gel, and was visualized in rounding the coastal town of Chiavari (27 500 inhabi- ethidium bromide. tants). Here, the high density of houses and enclosed private properties limited sampling, and four out of Hybridization with DNA probes five sites were within 3 km of each other. All sites were on hills covered by deciduous woods, dominated by In order to characterize amplified DNA by species, chestnuts (Castanea sativa), oaks (Quercus cerris), and we carried out hybridization with oligonucleotide black locust (Robinia pseudacacia). probes designed by Rijpkema et al. [14]. One probe At each site, ticks were collected by dragging on was specific for B. burgdorferi s.l., whereas the re- 50 m transects by two operators. One of them used maining four were specific for B. burgdorferi s.s., a tick drag comprised of 12 flannel strips, the other a B. garinii, B. afzelii and B. valaisiana. Probes were 1m2 cotton cloth. Ticks that were collected on a site 3k-tailed, labelled with digoxigenin-11-dUTP and by the two operators were pooled together and quantified. Amplicons were denatured at 95 xC for maintained alive in humidified vials or preserved in 5 min with 0.1 vol. of 4 M NaOH, 0.1 M EDTA, 70% ethanol and subsequently identified using keys spotted (2 ml/spot) on a positively charged nylon from Manilla and Iori [11]. Data on rainfall and membrane and cross-linked at 120 xC for 30 min. Pre- temperature in Borzonasca, from April 1998 to hybridization was carried out for 1 h at 55 xC while Downloaded from https://www.cambridge.org/core. IP address: 170.106.202.226, on 30 Sep 2021 at 15:47:42, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268803001328 Risk of tick-borne zoonoses in Italy 1141 the hybridization solution, containing 10 pmol/ml of log-linear models using the GENMOD procedure in each labelled oligoprobe was allowed to react over- the SAS1 system (Exp option in the ESTIMATE night at the same temperature. After post-hybridiz- statement, SAS version 8.2 [19]). Negative binomial ation washes and immunostaing procedures, positive error was used in the statistical models in order to spots were identified by colorimetric detection. All take into account the potential overdispersion of the procedures were carried out according to the digoxi- distribution of host-seeking ticks among dragging genin system user’s guide (Boehringer–Mannheim, sites. Germany). Amplicons of positive controls for each To estimate tick seasonal patterns and the effect B. burgdorferi s.l. genospecies that were included on of proximity to the sea (inland vs. coastal sites) on each membrane allowed checking for cross reactions. the numbers of collected ticks, we used generalized estimating equations (GEE) for counted responses with negative binomial error for larvae, nymphs and Detection of A. phagocytophilum in host-seeking ticks adult I. ricinus (PROC GENMOD) [19]. Using GEE DNA from 19 I. ricinus adults and 41 nymphs was (exchangeable correlation structure), we accounted used in PCR using primers EHR521 and EHR747 for correlation arising from repeatedly collecting ticks that amplify a 247-bp fragment of 16S rDNA [15].