The collection of adult ticks over a three-year period combining the use of permanent and temporary sampling sites provided relevant information on the occurrence of pathogens in the area under investigation. Up to 11 pathogens were detected in about 40% of I. ricinus individuals sampled from north-eastern Italy, with one or more pathogens occurring in 14 collection sites. The pathogens detected in the present study had already been identified from 1989 to date in I. ricinus collected in the same area [3, 21–32], with the exception of B. lusitaniae, which was detected once in nymphs , and B. divergens which was isolated from cattle only . However, this study reports a comprehensive survey of TTPs occurring at one time in this area.
LB agents and Rickettsia species were the most prevalent pathogens in ticks and are therefore regarded as the most likely transmissible agents to animals and humans in this area. The study monitored and confirmed the occurrence of other emergent pathogens, such as A. phagocytophilum, and Babesia EU1. Interestingly, it also ascertained the presence and the distribution of "Ca. N. mikurensis" for the first time in Italy. The relevant prevalence of ticks positive to "Ca. N. mikurensis" (more than 10%) is of particular interest considering the role of this pathogen as the aetiological agent of human infections in Germany, Switzerland, and Sweden [35–37] and in a dog in Germany . Indeed, following the primary isolation from rats (Rattus norvegicus) and Ixodes ovatus ticks  in Japan, this bacterium has been included in the list of emerging pathogens in Europe . TBEv and A. phagocytophilum were detected in a few sites of those monitored (Table 3). The low prevalence and the scattered distribution patterns recorded for these agents, which often occur in local foci of transmission [40, 41], complicates monitoring of tick vectors, calling for the use of other tools, such as serological methods and clinical case reports, for supporting surveillance strategies. Bartonella spp. was also detected in I. ricinus and, in spite of the increasing number of infections reported in ticks [42, 43], the role played by I. ricinus in the transmission of this pathogen to animals and humans is disputable. However, recent laboratory evidence showed that the transmission of Bartonella birtlesii by I. ricinus ticks may occurr in naive mice .
Twenty-seven percent of positive ticks displayed co-infections by two or even three pathogens. Co-infections have been frequently reported in Europe not only in questing ticks [45–47, 43, 48], but also in ticks removed from humans , as well as domestic and wild animals [50, 51].
Co-infections in questing I. ricinus confirm the wide host range of this tick species and the role played by mammals, such as small rodents, or birds, as reservoirs of several pathogens simultaneously. The frequent finding of co-infections in adult ticks should stimulate an increased awareness of physicians and veterinarians of potential multiple infections in vertebrate hosts, leading to different or atypical clinical presentations .
The present study indicates that screening of adult ticks is a successful strategy to maximize the probability of pathogen detection. The rationale for monitoring adult ticks is that the pathogen rate of infection in adult questing ticks is usually higher than in nymphs, as a consequence of the transtadial transmission of agents accumulated during the blood meal on different hosts .
However, despite the fact that the original screening strategy was focussed on a relatively small number of adult ticks, this strategy had considerable costs (table 7). Hence, other sampling strategies were hypothesized a-posteriori, in order to evaluate their effeciency in terms of data collected and reduction of costs. Reducing the sampling time to three months (strategies B and C) instead of the whole year, decreased costs consistently (i.e., travel and staff costs), by reducing the draggings from the initial 146 to 71. Nonetheless, strategy C resulted in a loss of data, especially at local level (provinces and sites).
Specific screening of female ticks (strategies A and C) was justified by the higher pathogen rate of infection found in I. ricinus females compared to males. Nevertheless, the screening of females only resulted in the fact that sporadic pathogens were not detected.
Strategy B (processing of all adult ticks from April to June) was the most cost-effective choice, and represented the best compromise for both cost reduction and reliability of results (Table 8). Therefore, this strategy is recommended as basis for circulation studies of TTPs in this specific context. However, other areas characterized by different climate, tick dynamics, and pathogen prevalence may need modifications in terms of sample size and time of tick collection.