View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositório Científico do Instituto Nacional de Saúde Microb Ecol DOI 10.1007/s00248-017-0994-y INVERTEBRATE MICROBIOLOGY Importance of Common Wall Lizards in the Transmission Dynamics of Tick-Borne Pathogens in the Northern Apennine Mountains, Italy Laura Tomassone1 & L. A. Ceballos1,2 & C. Ragagli3 & E. Martello1 & R. De Sousa4 & M. C. Stella1 & A. Mannelli1 Received: 20 February 2017 /Accepted: 5 May 2017 # Springer Science+Business Media New York 2017 Abstract During the investigations on ticks and tick-borne new findings in the study area, and R. helvetica infection in pathogens (TBP) range expansion in the Northern a tail tissue indicates a systemic infection. R. hoogstraalii is Apennines, we captured 107 Podarcis muralis lizards. Sixty- reported for the first time in I. ricinus ticks. Lizards seem to eight animals were infested by immature Ixodes ricinus, favour the bacterial exchange among different tick species, Haemaphysalis sulcata and H. punctata. Borrelia burgdorferi with possible public health consequences. s.l. was detected in 3.7% of I. ricinus larvae and 8.0% of nymphs. Together with the species-specific B. lusitaniae,we Keywords Podarcis muralis . Northern Apennines . Ixodid identified B. garinii, B. afzelii and B. valaisiana. Rickettsia ticks . Zoonoses . Borrelia burgdorferi s.l. SFG Rickettsiae spp. (18.1% larvae, 12.0% nymphs), namely R. monacensis, R. helvetica and R. hoogstraalii, were also found in I. ricinus. R. hoogstraalii was detected in H. sulcata nymphs as well, Introduction while the two H. punctata did not harbour any bacteria. One out of 16 lizard tail tissues was positive to R. helvetica.Our Like other small vertebrates, lizards are suitable hosts for the results support the hypothesis that lizards are involved in the immature stages of different tick species across Europe and epidemiological cycles of TBP. The heterogeneity of the Mediterranean basin, including Ixodes ricinus, the major B. burgdorferi genospecies mirrors previous findings in vector of tick-borne diseases (TBD) in Europe [45](Table1). questing ticks in the area, and their finding in attached Recently, studies have investigated the possible role of liz- I. ricinus larvae suggests that lizards may contribute to the ards as reservoir of TBD agents. The infection by Borrelia maintenance of different genospecies. The rickettsiae are burgdorferi s.l. and Rickettsia spp. in tissues and attached ticks was shown in several lizard species (Table 2). Lizards Electronic supplementary material The online version of this article are considered reservoir of Borrelia lusitaniae [42], and some (doi:10.1007/s00248-017-0994-y) contains supplementary material, authors also suggest that they may be reservoir of Spotted which is available to authorized users. Fever Group (SFG) rickettsiae, R. helvetica and R. monacensis in particular [7, 21, 55]. Interestingly, multiple * Laura Tomassone pathogens (B. burgdorferi s.l., SFG rickettsiae, Anaplasma [email protected] phagocytophilum) have been shown to co-infect immature I. ricinus ticks feeding on lizards [14, 56]. 1 Dipartimento di Scienze Veterinarie, University of Turin, In the Tuscan-Emilian Apennine National Park, Italy, liz- Largo Braccini 2, Grugliasco Turin, Italy ards are among the small vertebrate species inhabiting dry and 2 Present address: Istituto Zooprofilattico Sperimentale del Piemonte, sunny rocky habitats. Our previous studies showed the exis- Liguria e Valle d’Aosta, Turin, Italy tence of a complex vertebrate-tick-microbial community in 3 UTCB Lucca- Comando Unità per la Tutela Forestale Ambientale e the area. Indeed, I. ricinus, which recently colonized the ter- Agroalimentare Carabinieri, Lucca, Italy ritory, coexists with I. trianguliceps, Dermacentor 4 Centro de estudos de Vectores e Doenças Infecciosas, Instituto marginatus, Haemaphysalis sulcata and H. punctata [30, Nacional de Saude Dr. Ricardo Jorge, Águas de Moura, Portugal 40]. A focus of transmission of Rickettsia slovaca and Tomassone L. et al. Table 1 Bibliographic reports of ixodid tick species feeding on lizards 12′ N, 10° 22′ E) [40]. Podarcis muralis and Lacerta viridis in Europe and Northern Africa (Laurenti 1768) are the two Lacertidae reported in the study Tick species Lizard species Country area [2]. Lizards capture sites (n = 12) were located from 800 to1600 Ixodes ricinus Lacerta agilis Germany [42], Netherlands [55], meters above sea level (m a.s.l.) and were specifically chosen Hungary [15], Poland [11, 14, 18], Romania and Slovakia [27] to be an optimal habitat for lizards, having a good sun expo- sure and abundant refuges. Sites were characterized by differ- Lacerta viridis Hungary [15], Slovakia [56] ent vegetation typologies: open meadows with rocks and Lacerta Italy [48] bilineata bushes; hiking trails with stone walls and tall grass; areas of Lacerta Portugal [21, 35], Spain [21] exposed rocks and mixed deciduous woods dominated by hop schreiberi hornbeam (Ostrya carpinifolia) and Turkey oaks (Quercus Podarcis taurica Hungary [15] cerris); and, in the upper part of the study area, gravelly soil Podarcis Germany [42], Italy [1; this study] areas with scarce vegetation at the border of beech (Fagus muralis Podarcis Portugal [35] sylvatica) woods (Online Resource 1). hispanica Podarcis Algeria [52] Lizard and Tick Sampling vaucheri Timon lepidus Portugal [35] Lizards were captured by a noose affixed to a stick during six Teira dugesii Portugal [7] sampling sessions in spring and summer (April–August) from Psammodromus Algeria [52], Portugal [35], Spain algirus [29]; Tunisia [10] 2011 to 2013. Animals were identified by species, age class Timon pater Algeria [52] (adult, young) and sex, according to Vanni and Nistri [57]. Dermacentor Lacerta viridis Slovakia [56] Attached ticks were removed with forceps and stored in marginatus 70% ethanol, and were identified by species by using keys Haemaphysalis Apathya Turkey [20] from Manilla [28]. In the case the lizard tail detached via tail sulcata cappadocica fracture (a natural escape mechanism in lizards), it was stored Lacerta media Turkey [20] in 70% ethanol. Afterwards, each lizard was released in its Psammodromus Spain [29] algirus capture site. Animal capture and sampling protocols were ap- Podarcis This study proved by the Commission for Bioethics and Animal Welfare muralis of the University of Turin. Haemaphysalis Psammodromus Spain [29] punctata algirus Podarcis This study Laboratory Analyses muralis DNA from ticks was extracted by using the DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany), while R. raoultii is present [49], involving wild boars [50] and small DNA extraction from tail tissues was carried out with rodents [31]. Moreover, B. burgdorferi s.l. infects questing MagCore HF16 Automated DNA/RNA purification System I. ricinus, and tissues and ticks from small rodents [32, 40]. and MagCore genomic DNA tissue kit (RBC Bioscience, Due to the variety of tick species and TBD agents in the New Taipei City, Taiwan). Negative controls (distilled water) area, and that previous studies of our group in a close park had were added during the extraction to verify for possible cross- shown lizards’ involvement in the maintenance of contaminations. B. lusitaniae [1], we investigated if lizards play a role in the Tested ticks included all attached I. ricinus larvae and maintenance of ticks and transmitted pathogens. We present nymphs, all H. punctata,andasampleofH. sulcata nymphs, here the results of the evaluation of tick infestation and infec- the size of which was determined in order to detect the tion by B. burgdorferi s.l. and Rickettsia spp. in attached ticks presence/absence of Rickettsia spp. infection (considering a and lizard tissues. 95% confidence level and 20% expected prevalence). All DNA tick and tissue samples were analysed for B. burgdorferi s.l. and Rickettsia spp. The infection by Materials and Methods B. burgdorferi s.l. was studied by a PCR protocol targeting the intergenic spacer (IGS) region as previously described Study Area [44]. Detection of Rickettsia spp. in ticks was performed by targeting the citrate synthase (gltA)[22], OmpA [41]and The research was carried out on the Tuscan side of the Tuscan- OmpB genes [46]. Rickettsia spp. detection in lizard tissues Emilian Apennine National Park, Lucca province, Italy (44° was performed by a nested-PCR targeting the OmpB gene [4]. Lizards and Tick-Borne Pathogens, Italy Table 2 Bibliographic reports of the infection by B. burgdorferi s.l. Lizard species Pathogens infecting Pathogens infecting attached Ixodes ricinus Reference and Rickettsia spp. in lizard lizards tissues tissues and attached I. ricinus in Europe and Northern Africa Lacerta agilis B. lusitaniae B. lusitaniae, B. burgdorferi s.s., [14] B. burgdorferi s.l. B. lusitaniae Negative to B. burgdorferi s.l. [15] N.I. B. afzelii, B. garinii, B. burgdorferi s.s. [18] N.I. B. lusitaniae, B. valaisiana [27] N.I. B. afzelii, B. burgdorferi s.s., R. helvetica [55] N.I. B. lusitaniae [42] Lacerta N.I. R. monacensis, R. helvetica [21] schreiberi Negative to B. lusitaniae [35] B. burgdorferi s.l. Lacerta viridis B. lusitaniae B. lusitaniae, B. afzelii, B. burgdorferi s.s. [15] Podarcis B. lusitaniae B. lusitaniae [1] muralis N.I. B. lusitaniae, B. valaisiana [42] R. helvetica B lusitaniae , B. afzelii, B. valaisiana, B. garinii, This Negative to R. monacensis, R. helvetica, study B. burgdorferi s.l. R. hoogstraalii Podarcis Negative to B. lusitaniae [35] hispanica B. burgdorferi s.l. Podarcis taurica B. lusitaniae B. lusitaniae, B. afzelii, B. burgdorferi s.s. [15] Psammodromus B. lusitaniae B. lusitaniae [10] algirus B. lusitaniae B. lusitaniae [35] Teira dugesii B. lusitaniae, B. lusitaniae, R. helvetica, R. monacensis [7] R. helvetica, R. monacensis Timon lepidus Negative to B. lusitaniae [35] B. burgdorferi s.l. N.I. not investigated In all PCR reactions, 2.5 μl of DNA sample was tested. In 1999). Prevalence of infestation by ticks, in young and adult each PCR run, distilled water was added as negative control; lizards and between sexes, and between lizards and small ro- DNA from B.