Ticks Infesting Humans in North-Western Italy and Associated Pathogens: A Cross-Sectional Study in a Three-Year Period (2017-2019) in North-Western Italy.

Background. Tick-borne diseases are widespread in many European Countries and high incidence has been reported in the past few years. Ticks are able to transmit several pathogens to the host while feeding and are considered the most important vectors of infectious agents worldwide together with mosquitos. As tick-bite events has remarkably increased in North-Western Italy and information about the prevalence of tick-borne pathogens in ticks removed from humans in Italy are scarce, this study aimed to identify the species of ticks biting humans and the tick-borne pathogens they can transmit. Methods. Ticks collected from bitten humans during 2017-2019 were morphologically identied and screened by biomolecular essays. A total of 1,290 ticks were analysed. Most of the ticks belonged to the Ixodes genus: 1,009 ticks (78.0%) were classied as Ixodes ricinus. Some Ixodes ticks (n = 158; 12.3%) were identied only at genus level due to lack of morphological features. Overall, 500 ticks were PCR tested for the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. in at-risk population (elderly and children). Pathogen identity was conrmed by Sanger sequencing. Results. The overall prevalence was 22.8%; (n = 114; 95%CI 19.19-26.73%), meaning that at least one pathogen was detected. Molecular analysis was carried out on a subset of 500 ticks showing the presence of R. helvetica (n=31), R. monacensis (n=35), R. slovaca (n=3), R. aeshlimannii (n=1), Rickettsia spp. (n=6); B. afzelii (n=11), B. burgdorferi sensu stricto (n=1), B. garinii (n=3), B. lusitaniae (n=4), and B. valaisiana (n=2), Borrellia spp. (n=11); Anaplasmaphagocytophilum(n=6).


Results
During 2017-2019, a total of 1,290 ticks were collected from humans ( Table 2). Ticks were taxonomically assigned to 11 species belonging to the genera Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Rhipicephalus (Table 3). Most ticks were identi ed as Ixodes ricinus (78.0%). Some ticks (12.3%), belonging to the genus Ixodes, resulted to have been damaged during collection (e.g. the rostrum was absent) making it impossible to classify them at the species level). These ticks came from North-West of Italy. We identi ed one Amblyomma parvum, belonging to a man travelling abroad and bitten in Brazil, and one Hyalomma marginatus belonged to a man bitten in Greece. The other ticks coming from mans who travelled abroad were identi ed as Ixodes ricinus.
The life stages most frequently collected were nymphs (59.8%) and adult female (28.9%) followed by larvae (2.3%) and males (0.7%) ( Table 4).  Table 5 and Fig. 1 show a map with the geographic distribution (%) of ticks collected in North-Western Italy. Legend for province  Table 6). As regards Borrelia burgdorferi s.l., 32 samples were positive for Borrelia spp.  Table 7 shows the distribution of positive samples by province of Piedmont region. Finally, six ticks were positive for Anaplasma phagocytophilum (n = 6 Ix. ricinus nymphs; 1.2%; 95%CI 0.44-2.59%). Table 8 shows the distribution of positive samples by province. With regard to Italy, the province is a local authority with jurisdiction over a group of municipalities, not necessarily contiguous, and at the same time a peripheral constituency of state o ces.

Discussion
This study describes a 3-year survey of ticks and tick-borne pathogens circulating in different areas of North-Western Italy. The results revealed a wide distribution of tick-borne pathogens in this area. Ixodes ricinus, one of the most abundant ticks in Italy, con rms to play an important role as a source of infection: this species was the most frequently detected and also the most common species found on humans. This nding is in accordance with a previous study reporting that I. ricinus was widespread in woodland areas of North-Western Italy, where Ixodes ticks nd optimal conditions in terms of temperature (i.e. 20-23 °C) and relative humidity (i.e. 85-98%) for its development (Tagliapietra et al. 2011). Due to of their small size, the majority of ticks collected from people in the present study were nymphs (60%), which might be easily overlooked (Wilhelmsson et al. 2013).
These Rickettsiae are part of the Spotted Fever Group; R. monacensis was rst isolated in Germany and it is widespread throughout Europe in I. ricinus tick vectors. Its prevalence in ticks reaches 34.6% in some European countries (Oteo and Portillo 2012). To date, it has been identi ed in human cases with symptoms similar to MSF (Mediteranean Spotted Fever-like), in Spain (Jado et al. 2007) and in Sardinia island (Italy), where the disease is now considered endemic (Madeddu et al. 2012). In the past, R. helvetica has been associated with patients with perimyocarditis (Nilsson et al. 1999), fever and skin rash (Nilsson 2009) and, more recently, with subacute meningitis (Nilsson et al. 2010). Its main carrier is I. ricinus and its prevalence varies in European countries (Oteo and Portillo 2012).
Regarding Borrelia burgdorferi s.l., our study con rmed the presence of the complex in ticks from Piedmont even though the number of positive ticks was lower (32 out of 500) with a prevalence of 6,4% Ecological and climatic conditions of North-Western Italy are favourable to the persistence and spread of ticks and this has certainly favoured the growing exposure of the population to the bite of these parasites. In our study, the number of ticks collected yearly was variable, ranging from 239 to 624 samples, and this likely re ect climatic conditions, considering the fact that sampling has not been systematic in the three years. In fact, while 2017 was quite a dry year, the period between spring and summer 2018 was characterized by abundant rainfall, so much to rank May 2018 as the seventh wettest month since 1958 (source ARPA Piedmont -Forecast Systems). The hot temperatures recorded in the subsequent summer, which however remained average (source ARPA Piedmont -Natural Risks), thus created the ideal conditions for an escalation of the parasitic environmental pressure. As a consequence, there was a considerable increase in episodes of tick bites in humans in Piedmont between May and July 2018. Also in 2019, the highest number of ticks was registered in May and June.
Most of the patients in our study were bitten during a walk in the woods, which represents the ideal habitat of I. ricinus, also named "wood tick". However, high tick presence, even in non-wooded environments, emerged from the anamnestic data collected from physicians who conferred ticks for analysis. Overall, the nding of pathogens in 24% of the analyzed ticks underlines the importance of surveillance, prevention, and correct diagnosis in the human sector. Increase in tick-borne disease prevalence and transmission are important public health issues. The geographic spread of tick species caused by micro-and macroclimate changes, human behaviour, land use, vector population growth, and many other factors has allowed tick-borne bacterial diseases to follow in their wake. As we continue to discover new species of bacteria, it is important to monitor the emergence of both new and existing pathogens. Tick surveillance and tracking enhance our understanding of tick spread and ecology helping to identify areas of risk for disease transmission.
It is important to raise awareness of the population; personal protective strategies can help in the prevention of tick-borne diseases. Exposure to ticks may also result from exposure to domestic and companion animals that bring ticks into the house. Tick prevention in pets using repellents and tick checks after domestic animal exposure may assist in prevention. As tick-transmitted pathogens are discovered and emerge in new geographic regions, our ability to detect, describe, and understand the growing public health threat must also grow to meet the challenge.

Conclusions
The risk of tick-borne diseases in humans is associated with local tick abundance, infection prevalence, density of vertebrate reservoir hosts, climate changes.
Moreover, important is the local information campaign. Our results show that humans bitten by ticks in North-Western Italy are at risk of infection by different pathogens in agreement with data by Otranto et al.  Prevalence of positive ticks conferred / month.

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