A Geographical Information System Based Approach for Integrated Strategies of Tick Surveillance and Control in the Peri-Urban Natural Reserve of Monte Pellegrino (Palermo, Southern Italy)

Ticks (Acari: Ixodidae) are bloodsucking arthropods involved in pathogen transmission in animals and humans. Tick activity depends on various ecological factors such as vegetation, hosts, and temperature. The aim of this study was to analyse the spatial/temporal distribution of ticks in six sites within a peri-urban area of Palermo (Natural Reserve of Monte Pellegrino) and correlate it with field data using Geographical Information System (GIS) data. A total of 3092 ticks were gathered via dragging method from June 2012 to May 2014. The species collected were: Ixodes ventalloi (46.09%), Hyalomma lusitanicum (19.99%), Rhipicephalus sanguineus (17.34%), Rhipicephalus pusillus (16.11%), Haemaphisalis sulcata (0.36%), Dermacentor marginatus (0.10%), and Rhipicephalus turanicus (0.03%). GIS analysis revealed environmental characteristics of each site, and abundance of each tick species was analysed in relation to time (monthly trend) and space (site-specific abundance). A relevant presence of I. ventalloi in site 2 and H. lusitanicum in site 5 was observed, suggesting the possible exposure of animals and humans to tick-borne pathogens. Our study shows the importance of surveillance of ticks in peri-urban areas and the useful implementation of GIS analysis in vector ecology; studies on temporal and spatial distribution of ticks correlated to GIS-based ecological analysis represent an integrated strategy for decision support in public health.


Introduction
Ticks (Acari: Ixodidae) are the most common vectors of infectious animal diseases and they pose a serious threat to humans, pets, wild animals, and livestock worldwide. Ticks are involved in the transmission of several tick borne pathogens (TBPs), such as Rickettsia, Babesia, Theileria, Borrelia, and Coxiella; some of them can also be agents of zoonosis [1]. Effective vaccines are not yet available for the majority of tick-borne pathogens [2][3][4]. Prevention methods against vectors are, therefore, to date, the most effective tools against tick borne pathogens. Monitoring of tick distribution and identification of the greatest risk environments is one of the most useful strategies to prevent the risk of tick-borne diseases. Tick questing activity, reproduction, and survival depend on several factors, including vegetation coverage, host availability, moisture, and temperature [5,6].
This study was aimed at the analysis, through GIS application, of spatial and temporal distribution of free-living ticks in the Natural Reserve of Monte Pellegrino, in Palermo (Italy), a periurban area of the city used by families, walkers, and pets for recreational and sportive activities.

Collection Sites
Monitoring was carried out in the Natural Reserve of Monte Pellegrino, a regional natural reserve established in 1996, situated in the northern part of Palermo (Sicily, Italy, Figure 1). Monte Pellegrino extends over an area of about 1300 hectares (ha), and it strongly characterizes the image of Palermo, bordering the city in the north and extending out over the Mediterranean Sea. Its expanse is 1050 ha and includes the whole mountain of Monte Pellegrino (Zone A-Reserve) and the Real Tenuta Favorita (Zone B-Pre-reserve). The Reserve, with its Mediterranean climate, hosts a rich fauna and flora; it is characterized by a diffuse artificial forest, but it also includes a wide variety of natural environments, with a considerable biodiversity and the presence of some endemic species [30][31][32]. The Reserve constitutes an important area for recreation activities of many inhabitants of Palermo. Six monitoring sites were selected ( , and some species of Canidae. In site n° 3 natural forest with shadow areas were present, while site n° 4 was characterized by shrubs, herbaceous plants, and the presence of goats. The last two sites showed the presence of artificial forest with pine and cypress in site n° 5 and pine, eucalyptus, and a little lake in site n° 6. Geographical coordinates of each site were recorded by Global Positioning System (GPS) with Roma1940 reference system, using East-North coordinate pairs.

Ticks Collection and Identification
Ticks were collected by dragging method every two weeks for two years from June 2012 to May 2014 and stored in 70% ethyl alcohol. Collected arthropods were identified according to morphological keys [33][34][35][36].

Ecological Analysis
Analysis of environmental characteristics of tick collection sites was carried out by processing and overlapping different information levels through Environmental Systems Research Institute (ESRI) ArcGIS 9.3 software [37]. Analysed levels were the digital elevation model (DEM), regional technical maps, land cover (Corine land cover), and forest vegetation.

Data Analysis
Data were processed taking into account the sampling month, collection site, and tick species in order to define how the density of the collected ticks varied in time (during the months of the year) and in space (in the different collection sites). Charts were drawn showing the percentage of the species identified at each site.

Ticks Collection and Identification
Ticks were collected by dragging method every two weeks for two years from June 2012 to May 2014 and stored in 70% ethyl alcohol. Collected arthropods were identified according to morphological keys [33][34][35][36].

Ecological Analysis
Analysis of environmental characteristics of tick collection sites was carried out by processing and overlapping different information levels through Environmental Systems Research Institute (ESRI) ArcGIS 9.3 software [37]. Analysed levels were the digital elevation model (DEM), regional technical maps, land cover (Corine land cover), and forest vegetation.

Data Analysis
Data were processed taking into account the sampling month, collection site, and tick species in order to define how the density of the collected ticks varied in time (during the months of the year) and in space (in the different collection sites). Charts were drawn showing the percentage of the species identified at each site.

Processing of Maps with Proportionate Circles
Monthly maps related to the two years of monitoring were drawn using ESRI ArcGIS 9.3 (Redlands, CA, USA). Maps provide epidemiological information with circles, whose size is proportional to the number of ticks collected in the monitored sites.

Ecological Analysis
Environmental characteristics of tick collection sites were analysed according to the land use (Corine land cover), forest vegetation, and DEM, and the obtained maps are showed in Figures 3-5, respectively. Corine land cover analysis showed that sites n • 1, 4, and 5 fall within the Transitional woodland-shrub area, with sites n • 1 and n • 4 at the border with Discontinuous Urban Fabric Area and, for site n • 1, also with a Green Urban Area. Site n • 2 is characterized by Sport/Leisure Facilities, site n • 3 is within a Green Urban Area, and site n • 6 entirely falls in a Coniferous Forest. In particular, the Forest Vegetation map of Monte Pellegrino showed that sampling sites fell into areas of natural vegetation (woodlands, site n • 2), artificial vegetation (pine and cypress/eucalyptus, site n • 3, n • 5, and n • 6), and other kind of vegetation (mainly herbaceous vegetation, sites n • 1 and n • 4).
Digital elevation model of Monte Pellegrino showed an altitude range of 1-600 m; sites n • 1 to n • 4 were located at low altitude (<100 m), whilst sites n • 5 and n • 6 were located at moderate altitudes         Table 1 shows for each tick species the number of specimens collected in the different months of the year.

Spatial Distribution
Among the different collection sites, the highest numbers of ticks were collected in sites n • 2 (n. 1522 ticks) and n • 5 (n. 1005 ticks) and the lowest number in site n • 4 (n. 34 ticks), as reported in Table 2. Percentage values of each tick species in all the collection sites are reported in Figure 7.
Site n • 2 was mainly characterized by a high presence of I. ventalloi (n. 853 ticks) and a comparable number of R. sanguineus and R. pusillus (n. 376 and n. 290, respectively); differently, site n • 5 showed a high presence of H. lusitanicum (n. 538), followed by I. ventalloi (n. 357). Data from site n • 3 showed a moderate presence of I. ventalloi, R. pusillus, and R. sanguineus (n. 115, n. 100, and n. 61, respectively), while a similar number of H. lusitanicum (n. 73) and I. ventalloi (n. 65) were observed in site n • 6. Ticks from sites n • 1 (total number of ticks: 82) and n • 4 (total number of ticks: 34), where the lowest number of vectors were collected, mainly belonged to the species R. pusillus, R. sanguineus, and I. ventalloi.
Monthly maps with circles proportional to the tick number were created using the geographical information systems (Figure 8A-L). In addition to the values for the two-year period, maps referring to each year of the study were created ( Figure 8A-L).

Discussion
In recent years, increasing attention has been directed to ticks and tick-borne pathogens and particularly to zoonotic agents. Many factors have, indeed, led to an increased contact between people and these arthropods, as for example climate changes [38], the increase of wild animals in rural and peri-urban areas [39], and the increased interest of people in outside activities [22].
The analysis reported in this study describes the results of a two-year survey conducted to investigate the presence of ticks in Monte Pellegrino Natural Reserve of Palermo (Italy), a peri-urban park attended by citizens for sportive and recreational activities.
The study provides information on tick distribution among collection sites with different environmental characteristics during each month and in correlation to the environmental characteristics of the territory (altimetry, land cover, and vegetation).
A great diversity of tick species was found in the Natural Reserve. Data analysis showed that sites having similar environmental features (1, 2, and 3 and 5 and 6) were characterized by similar patterns of tick species. In fact, sites n • 1, 2, and 3 shared the presence of I. ventalloi, R. pusillus, and R. sanguineus, although site-specific differences were present. Site n • 1 showed a low and comparable presence of the three species; site n • 2 was characterized by a dominance of I. ventalloi followed by similar numbers of R. pusillus and R. sanguineus. Finally, R. pusillus and I. ventalloi were equally present in site n • 3, followed by R. sanguineus. Of particular interest is the high presence of I. ventalloi in site n • 2 that may be related to different factors, such as a favourable natural (woodland) vegetation, evidenced by field observations and GIS analysis, as well as the presence of wild rabbits and cats. In fact, these animals are reported as preferential hosts for such tick species, which have been proposed as potential vectors for various pathogens (Rickettsia helvetica, Anaplasma phagocytophilum, Rickettsia monacensis, and Bartonella clarridgeiae (Lawson and Collins 1996)) and Eyach virus (Rehse-Küpper et al., 1976) [40,41]. Thus, site n • 2 may represent an important location for potential pathogens transmission from I. ventalloi, and monitoring activity should be enhanced to determine the effective public health risk in this area.
Sites n • 5 and n • 6 showed the greatest richness of tick species, in particular of H. lusitanicum and I. ventalloi. The relevant presence of H. lusitanicum in site n • 5, characterized by moderate altitude and artificial vegetation (Pine and Cypress), may require further attention given the vector role of such species for pathogens such as Theileria annulata (Dschunkowsky and Luhs, 1904) [42].
Considering all the sites, the most abundant tick species were I. ventalloi (n. 1425), H. lusitanicum (n. 618), R. sanguineus (n. 536), and R. pusillus (n. 498). All these tick species are proven or suspected vectors of animal and human pathogens (Table 3). Indeed, as aforementioned, I. ventalloi is a potential vector for several zoonotic bacteria, i.e., Rickettsia helvetica, Rickettsia monacensis, Anaplasma phagocytophilum, and Bartonella clarridgeiae [41]. R. sanguineus is a tick species of veterinary and public health significance as it is vector of Anaplasma marginale (Theiler, 1910), Babesia bigemina (Smith and Kilborne, 1893), and Babesia bovis (V. Babes, 1888). The tick, also known as the brown dog tick, is a parasite to a number of hosts, including humans, and it is a relevant vector of important zoonotic agents, such as Rickettsia spp. [13]. H. lusitanicum tick species are a frequent parasite of large mammals such as cattle, sheep, goats, and pigs, and it is mainly associated with the transmission of Anaplasma spp. and T. annulata [42]. Trans-ovarian transmission of Coxiella burnetii has also been documented for this tick.  Months in which the highest number of total ticks were collected were June 2012 and April and January 2013, while the lowest numbers of ticks were collected in August 2012, February 2013, and May 2014. The presence and/or absence of ticks during the year changed according to the life cycle of the different species. The graphs joined to the monthly maps ( Figure 8A-L) show the monthly trend of each identified tick species in each collection site, highlighting the differences in seasonality of each species due to its own peculiar biological cycle.
In fact, I. ventalloi was present mostly from autumn to spring, with the greatest abundance between October and January. On the contrary, H. lusitanicum was collected mainly from late spring to autumn. R. sanguineus and R. pusillus showed a similar seasonal trend, and their number was higher in spring and summer, while significantly decreasing to zero in winter. The few specimens of the ticks belonging to the other identified species (H. sulcata, D. marginatus, and R. turanicus) did not allow establishing the period of abundance/scarcity of these species.

Conclusions
To our knowledge, the area analysed in this study was not previously subjected to any entomological investigation concerning ticks. This study contributes to the understanding and mapping of the presence and distribution of ticks and it can lead to a powerful surveillance integrated strategy. These data also allow for the identification of periods of greater tick abundance in the study area, thus providing indications for precautions to be observed by the attenders during such periods.
The study constitutes a premise for additional research, including any correlation among pathogens in ticks, microclimate, and hosts distribution analysis. Various studies in northern and central Italy [22,54,55] analysed the presence of pathogens in ticks collected from urban parks, identifying different agents of zoonoses. In our study, almost all tick species found were already reported as potential or recognized vectors of a wide range of animal/human pathogens (i.e., Rickettsia spp., A. phagocytophilum, Coxiella burnetii, Bartonella clarridgeia, Babesia spp., and Theileria spp.), reinforcing the need for control programs within the area aiming to reduce both tick abundance and the impact of environmental factors favouring tick development and spread. The GIS-based ecological analysis allowed for the analysis of information on specific suitable habitats for such tick species in the area, providing a useful base for control interventions.
The comprehensive depiction of ticks and TBPs in the area would represent a useful tool for decision support for Health Authorities to define possible risk for specific TBDs and, thus, to choose adequate strategies of pest control for the preservation of public human health.